Medical biology

guide for the Oral Final exam 2021

written by
Maxim Komyshan and Nutsa Zviadadze

may the odds be ever in your favor

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 Cell and molecular biology
1. Medical biology as a science, hallmarks in the history of
biology and genetics
Medical Biology is a theoretical field which is based on the cell and molecular
biology and which is specifically focused on their aspects relevant for human
medicine.

Milestones in history of biology

Rudolf Virchow 1858

1. All living things are composed of one or more cells.

2. The cell is the basic unit of life in all living things.

3. New cells are produced from existing cells

CHARLES DARWIN – EVOLUTION THEORY

● 1859 On the Origin of Species (by means of natural selection).The

concept is simple but powerful: individuals best adapted to their
environments are more likely to survive and reproduce.

GREGOR JOHANN MENDEL– ESTABLISHED THE LAWS OF

MENDELIAN INHERITANCE; FATHER OF MODERN GENETICS

● 1866 Experiments on Plant Hybridiza<on (in German Versuche über

Pflanzen-Hybriden)

1953 James D. Watson, Francis H. C. Crick (1962 Nobel Prize)

● Watson and Crick published the double-helix structure of DNA in

2-pages article in the Nature, and embraced the idea that genes contained
a code that expresses information

1938-1945 George W. Beadle, Edward L. Tatum (1958 Nobel Prize) showed

the direct link between genes and enzymatic reactions and proposed the
hypothesis known as the One gene-one enzyme hypothesis (DNA->protein)

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1988-2001 Human Genome Organisation (HUGO) and Human Genome Project
(HGP)

2. Chemical composition of the cell and the human body.

Chemical bonds in biomolecules
The chemistry of life has several specific features

- is based mainly on carbon compounds

- is highly complex
- controlled by huge polymer molecules

The smallest particle of an element are atoms joined by chemical bonds to

form molecules. Atoms consist of a positively charged nucleus with neutrons
and protons and an envelope with negatively charged electrons. Outer electrons
determine the reactivity of atoms. A chemical bond is the stabilization of the
outer atomic shell by interaction with another atom.

Covalent bond

Occurs when the electron is almost equally shared. There are 2 types of covalent
bonds - polar and nonpolar. A nonpolar bond occurs between the same atoms -
H2, I2. A polar bond is when the electron is closer to one one of the atom’s
shells, but not significantly - H2O.

Molecule of ethane – single bond between the two carbon atoms; allows for
rotation of the carbons/CH3 groups

Molecule of ethene – double bond between the two carbon atoms; alters the
bond geometry and stabilizes all the atoms into the same plane, preventing the
rotation of the carbon atoms/CH3 groups.

Ionic bond

The electron is transferred to one of the atoms because it is more

electronegative - NaCl.

Hydrogen bond

Molecules of H2O are highly polar; they form a hydrogen bond. Two atoms are
connected by a covalent bond, where one end is slightly electronegative and the

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other - electropositive. The water molecule is overall neutral, the electrons are
asymmetrically distributed.

3. Biopolymers, general structure, lipids, polysaccharides

Saccharides

Monosaccharides have a general formula (CH2O)n where n can be 3-8 and have
2 or more hydroxyl groups. Aldoses - contain an aldehyde group(glucose);
Ketoses - a ketone group(fructose). In an aqueous solution aldehyde or a ketone
group of a sugar molecule tend to react with the hydroxyl group of the same
molecule, creating a ring. Many monosaccharides differ only in the spatial
arrangement of atoms - isomers. For example, glucose, galactose and mannose
have the same formula - C6H12O6, but differ in the arrangement of groups one
or two around carbon atoms.

Disaccharides

The carbon that carries the aldehyde or ketone group can react with a hydroxyl
group of another saccharide forming a disaccharide. Examples - maltose
(glucose+glucose); lactose (galactose+glucose); sucrose (glucose+fructose).

Oligosaccharides and Polysaccharides

Large linear and branched molecules can be made from simple repeating sugar
units. Short chains are called oligosaccharides, long chains - polysaccharides
(glycogen made out of glucose).

Lipids

The fatty acid (FA) molecule consists of two parts, hydrophobic (hydrocarbon
tail) and hydrophilic (carboxylic head) components, non-reactive and reactive,
hydrocarbon chain, carboxyl group. Lipids are esters of higher fatty acids. The
most important function of FA in the cell is the construction of cell membranes
(cytoplasmic and membrane organelles). The properties of fats depend on the
length and saturation of the fatty acid chains they carry. Phospholipids are the
main component of the cell membrane. In phospholipids two -OH groups are
linked to fatty acids while the third -OH group is linked to phosphoric acid.

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 Biopolymers

Biopolymers are natural polymers produced by the cells of living organisms.

Biopolymers consist of monomeric units that are covalently bonded to form
larger molecules. General forming of a macromolecule – in a condensation
reaction, a subunit is added to the growing end and a molecule of water is lost.
The reverse reaction (the breakdown) occurs by the addition of water.

4. Protein structure
Proteins make up the majority of a cell's dry weight. The simplest level of
protein structure, primary structure, is simply the sequence of amino acids in a
polypeptide chain. The sequence of a protein is determined by the DNA of the
gene that encodes the protein. The next level of protein structure, secondary
structure, refers to local folded structures that form within a polypeptide due to
interactions between atoms of the backbone. (The backbone just refers to the
polypeptide chain apart from the R groups - An abbreviation for any group in
which a carbon or hydrogen atom is attached to the rest of the molecule). The
most common types of secondary structures are the α helix and the β folded
sheet. Alpha-helix - the polypeptide chain wraps around itself and forms a rigid
cylinder. A hydrogen bond is formed between every fourth peptide bond and
connects the N-H of one and the C = O of the other peptide bond. Beta-folded
sheets arise from either adjacent polypeptides chains with the same orientation
or from strings composed of paper folded into an accordion, each section having
an opposite orientation to its neighbors. The overall three-dimensional structure
of a polypeptide is called its tertiary structure. The tertiary structure is
primarily due to interactions between the R groups of the amino acids that make
up the protein. Some proteins are made up of multiple polypeptide chains, also
known as subunits. When these subunits come together, they give the protein its
quaternary structure (hemoglobin).

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 5. Protein functions

Function Description Example

Antibody Antibodies bind to specific foreign particles, Immunoglo

such as viruses and bacteria, to help protect bulin G
the body. (IgG)

Enzyme Enzymes carry out almost all of the Phenylalani

thousands of chemical reactions that take ne
place in cells. They also assist with the hydroxylase
formation of new molecules by reading the
genetic information stored in DNA.

Messenger Messenger proteins, such as some types of Growth

hormones, transmit signals to coordinate hormone
biological processes between different cells,
tissues, and organs.

Structural These proteins provide structure and support Actin

component for cells. On a larger scale, they also allow
the body to move.

Transport/s These proteins bind and carry atoms and Ferritin

torage small molecules within cells and throughout
the body.

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 6. Architecture of prokaryotic and eukaryotic cell

Prokaryotic cell Eukaryotic cell

Nucleus
Nucleoid Eukaryotic nucleus

Not a real nucleus, Nuclear envelope, 1 or more

nucleoli
No nuclear envelope, no
nucleoli and histons Histones bound to DNA

DNA
7,5.105–5.106 bp 1.107–1,5.1011 bp Genes with
introns
Genes without introns
2 or more chromosome
1 mostly circular
chromosome

Cellular
organelles No membrane organelles Real membrane organelles –
mitochondria, endoplasmic
No sterols in membranes reticulum, Golgi apparatus
Sterols in membranes

Cytoskelet
on No true cytoskeleton, but Microtubules, microfilaments,
found similar structures intermediate filaments

Ribosomes 70S 80S

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 7. Biomembranes (structure, function)

Phospholipids:

- form the double-layer of the membrane

- are responsible for the fluidity of membranes

- allow passing of hydrophobic and small molecules (water, O2, CO2...)

• Cholesterol:

- maintains the integrity (stability) of membranes

- decreases the fluidity of membranes and permeability for water

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Proteins:

- Transport of molecules across the membrane (membrane transport)

- Cell signaling - receptors - signal receiving by a cell, its transduction to the

cell

- Glycoproteins - cell adhesions - organs and tissue integrity

- cell surface antigens – role in immune system

Function

- Separation of the cell from outer environment

- Separation of individual organelles in the cell
- Regulation of income and output of substances
- Role in signals detection – membrane receptors
- Participation in cellular adhesion
- Immune system – membrane antigen

8. Membrane proteins and membrane transport

-occupy about 50% of mass of the membrane

Functional Class Protein example Specific function

Transporters Sodium pump Actively pumps Na+ out of cells and
(Na+/K+ - ATPase) K+ into cells
Anchors integrins Link intracellular actin filaments to
extracellular matrix proteins
Receptors Platelet-derived Binds extracellular PDGF and
growth factors generates intracellular signals that
(PDGF detectors) cause the cell to grow and divide
Enzymes Adenylyl

An integral membrane protein is a type of membrane protein that is

permanently attached to the biological membrane. All transmembrane proteins
are integral. They extend across the bilayer as a single α-helix, a group of

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α-helixes, or as a β-sheet. Lipid-linked (anchored) proteins are located on the
surface of the cell membrane that are covalently attached to lipids embedded
within the cell membrane; the lipid serves to anchor the protein to the cell
membrane. They are also integral. Some are attached to the membrane by weak
interactions with other membrane proteins (protein attached, non-integral).

Membrane transport

Passive - without energy consumption, down the concentration gradient

- simple diffusion
- facilitated diffusion
- osmosis

Active - ATP consumption, against the concentration gradient

- always through membrane proteins - channels, transporters

- endocytosis (absorption of membrane-coated vesicles by the cell)

Two components of an
electrochemical gradient
– the driving force
(electrochemical
gradient)and the membrane
potential. The membrane
gradient reinforces the
membrane transport when
the two work in the same
direction.

Two main ways of active transport:

1. Primary active transport - The Na+ pump

a. a transporter protein
b. uses the energy of
ATP hydrolysis to pump Na+ out
of the animal cells and K+ in
against the concentration gradient

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 c. Ouabain drug inhibits its activity – interferes with K+ binding

2. Secondary active transport

a. does not use ATP directly, but
takes advantage of a separately existing
concentration gradient
b. the action of Na+/K+ ATPase
establishes K+ and Na+ concentration
gradients across the membrane

Two types of diffusion:

1. Passive (simple) diffusion

a. free movement of molecules across the membrane
b. no energy is required
c. continues until equilibrium is reached
d. consists of small non-polar molecules (O2; CO2) and uncharged
polar molecules (urea)
2. Facilitated diffusion
a. is the transport of charged molecules by a protein receptor down a
concentration gradient
b. proteins may be channels or carrier proteins

9. Cellular organelles (overview, structure, function)

- microenvironment for enzymes, cofactors
and substrates

- suitable ionic environment (pH, redox

potential,...)

- separation of "dangerous" reactions

(proteolytic enzymes, oxidative enzymes,...)

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 -They provide structure for the body, take in nutrients from food, convert those
nutrients into energy, and carry out specialized functions. Cells also contain the
body's hereditary material and can make copies of themselves.

Compartment Main function Percentage of Total Approximate N

Cell Volume per Cell

Cytosol Metabolic pathways, protein synthesis 54 1

Nucleus Main genome, DNA and RNA synthesis 6 1

Endoplasmic Synthesis of most lipids, synthesis of 12 1

proteins for distribution to many
reticulum
organelles and to the plasma membrane

Golgi apparatus Modification, sorting, and packaging of 3 1

proteins and lipids for either secretion or
delivery to another organelle

Lysosomes Intracellular degradation 1 300

Endosomes Sorting of endocytosed material 1 200

Mitochondria ATP synthesis by oxidative 22 1700

phosphorylation

Chloroplasts ATP synthesis and photosynthesis - -

(in plant cells)

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Peroxisomes Oxidation of toxic molecules 1 400

10. Cytoskeletal system - overview, intermediate filaments

- Network of protein fibers inside the cell
- Participates in:
● Spatial architecture of cells
● Ability of cells to take various shapes
● Integrity of tissues and organs
● Movement of organelles and
vesicles inside the cell
● Movement of cells
● Division of cells
● Cell signaling

There are three types of cytoskeletal fibres in animal cells - microfilaments

(actin filaments), which are helical polymers made of actin, they are most
highly concentrated in the layer of cytoplasm just beneath the plasma
membrane, their functions are cell movement, muscle contraction, and cell
division; intermediate filaments; microtubules - hollow cylinders made of the
protein tubulin, typically having one end attached to a centrosome, their
function includes mechanical support, organization of the cytoplasm, transport,
motility and chromosome segregation.

Intermediate filaments

- are important for cells integrity and

mechanical resistance
- more stable than microfilaments and
microtubules

Types of intermediate filaments

1. Keratins - can be divided into hard keratins

giving rise to hair and nails and cytokeratins found
in epithelia

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 2. Lamins - found in the nuclear lamina and the nucleoplasmic veil
3. Neurofilaments - found in neuron axons
4. Glial fibrillary acidic protein (GFAP) - found in glial cells
5. Vimentin - in mesenchymal cells, such as fibroblasts, and in endothelial
cells
6. Desmin - found in muscle cells

Structure:

Monomer – α-helical central rod domain and globular ends, form dimers.
Dimers line up to form a staggered, antiparallel tetramer. Tetramers pack
together into a helical array containing eight tetramer strands and into the final
ropelike intermediate filament.

11. Cytoskeletal system - microtubules, microfilaments

Microtubules

​ - A crucial organizing role

​ - rapidly disassemble in one location and reassemble where needed
​ - Grow out of the centrosome outward to the cell periphery
Function
- Location and movement of organelles within the cell - association with
motor proteins (kinesin and dynein)
​ - Cilia, flagella – movement of cells
- Division of genetic material (separation of the chromosomes during
mitosis and meiosis)

Microtubules can be either dynamic or stable, so they can either get shorter or
longer as necessary, or they can remain of the same length.

Structure

Microtubules are polymers of tubulin dimers (alpha and beta dimer) and they
extend from microtubule organising centres, such as centrosomes, which
stabilize the negative pole
of the extending polymers.
There are normally 12-13

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tubulin units per turn in the assembled microtubule.

How can we use microtubules in anti-cancer therapy?

The formation of microtubules spindles is essential for cell division.

Nocodazole, taxol and vinblastine are antimitotic cancer chemotherapy drugs
that interfere with the exchange of tubulin subunits between the microtubules
and free tubulin pool.

Microfilaments

- Composed of 2 intertwined chains of F-actin

- Monomers - G-actin
- Have (+) and (-) polar ends

ATP hydrolysis decreases the stability of the

actin polymer. ATP is attached to each actin
monomer – upon polymerization, ATP is
hydrolyzed to ADP, which remains trapped
within the actin filament. Exchange of ADP
to ATP is possible only after the dissociation
of the actin monomer from the filament

Cell crawling depends on cortical actin

​ - Polymerization of actin at the

leading edge pushes the plasma membrane
forward
​ - New points of anchorage are formed
between the bottom of the cell and the
surface
​ - Contraction at the rear of the cell
draws the body of the cell forward

12. Experiments leading to the discovery of DNA as a carrier

of genetic information
- Early 20th century - genes are found on chromosomes, which consist of
DNA and proteins.

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 - Griffith's transformation experiment - A heat- inactivated infectious
strain can transform a harmless strain of live bacteria into a pathogenic
one. Experiment with the bacterium Streptococcus pneumoniae - two
forms (R - rough - is harmless, S - smooth - is pathogenic; designation
according to the appearance of the colonies of the form). Griffith
observed that strain S contained a substance that could permanently
change the behavior of a harmless strain R.
- Transformation principle published in 1944; Avery, MacLeod, and
McCarthy have provided evidence that DNA is the genetic material.
Extract from pathogenic strain S divided into RNA, proteins, DNA, lipids
and carbohydrates, all applied separately to cultures of strain R
(non-pathogenic). Only part of the extract containing DNA was able to
transform strain R into strain S.
- In 1953 Watson and Crick published the double-helix structure of DNA
in an article, and embraced the idea that genes contained a code that
expresses information and thereby changed our view of life. Their DNA
model was enabled by X-ray diffraction analysis of DNA performed
byMaurice Wilkins and Rosalind Franklin.

13. Nucleic acids structure

Nucleotides are the building blocks of DNA and RNA. They are joined together
by phosphodiester bonds between 5’ and 3’ carbon atoms of the sugar ring, via a
phosphate group, to form nucleic acids. Two types of nucleic acids according to
the type of sugar in the sugar- phosphate backbone:

- Ribonucleic acid (RNA) - contains ribose and bases A, G, C, U In cells as a

small polynucleotide chain

- Deoxyribonucleic acid (DNA) - contains deoxyribose and bases A, G, C, T

Polymer of deoxyribonucleotides bound by phosphodiester bond = DNA

PRIMARY STRUCTURE. Adenine pairs with Thymine, while Guanine pairs
with Cytosine via hydrogen bonds.

DNA structure
DNA consists of two complementary chains of nucleotides; sugar-phosphate
backbone + protruding base (adenine, guanine, cytosine, thymine). Two

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antiparallel chains are connected via hydrogen bonds. Paired bases are always
complementary - purine and pyrimidine. Antiparallel arrangement of strings -
the polarity of one strand is opposite to the polarity of the other strand - crucial
for copying and repairing of the DNA. - SECONDARY STRUCTURE

Non-specific DNA-binding
proteins bind to a small groove,
which is stabilized by water and
small ions, of DNA by β-sheets.
B-DNA - proteins bind to specific
sequences of the large groove,
especially by inserting an α-helix
into the groove. A-DNA - small
and large grooves are almost the
same size. Z-DNA - the small
groove is deep and narrow, the large groove almost disappears.

Comparison:

DNA tertiary structure

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Supercoiling refers to the additional twisting of a DNA strand and is an
expression of the strain on that strand. DNA can be overwound (positive
supercoiling) or underwound (negative supercoiling) – most DNA is negatively
supercoiled

Supercoiling functions to reduce the space required for DNA packaging,

allowing for more efficient storage of DNA. DNA will form positive supercoils
when unwound by helicase and requires an enzyme (DNA gyrase) to reduce the
strain.

The structure of DNA provides hereditary mechanisms. Genes are carriers of

genetic information and they are passed down from one generation to another
via the division of cells. The genetic information is encoded in the chemical
structure and accurately copied. Organisms differ from each other because they
encode a different sequence of nucleotides therefore carrying different
biological messages.

14. Prokaryotic and eukaryotic genomes (characteristics and

differences)
Eukaryotic genomes

- Eukaryotic DNA is packed into chromosomes. Chromosome - one

molecule of DNA + bound proteins = chromatin (Greek chroma - color)
- two copies of each chromosome, one from each parent -paternal,
maternal.
- Chromosomes of one pair – homologous
- In a somatic cell, each chromosome 2x plus two sex chromosomes (XX
or XY)

Genome - total genetic information contained in one cell on all chromosomes.

C value - total quantitative DNA in the genome (haploid set). Correlation
between organism complexity and genome size -paradox of C value (in
eukaryotes) - there is no such thing in prokaryotes.

Eukaryotic chromosomes contain:

- the centromere, that allows duplicated chromosomes to be separated

during M phase.
- the telomeres at each of the two ends of a chromosome - repeated
nucleotide sequences that are required for the ends of chromosomes to be
replicated.

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 Prokaryotic genomes

Most genomes are:

- One circular chromosome (nucleoid)

- Double-stranded DNA
- Small extrachromosomal circular DNA (plasmids)
- The genome is looser and not enclosed in a membrane structure (unlike a
eukaryotic cell) The loops help to pack the DNA
- DNA is subject to supercoiling

15. Structure of human genome (histones, nucleosomes,

chromatin)
-Genetic information of humans is encoded in two genomes: nuclear and
mitochondrial.

Mitochondrial genome

- Contains 37 genes in total

- 24 out of them represent genes for various non-coding RNAs
- 13 genes encode their own mitochondrial polypeptides involved in the
enzymatic equipment of mitochondria.

Nuclear genome

- Contains 24 chromosomes consisting of 22 autosomes and 2 sex

chromosomes - X and Y (or XX)

Chromatin proteins are divided into histone and non-histone chromosomal

proteins (e.g. HMG, high-mobility group proteins).

- There are enormous amounts of histones in chromatin, a mass

comparable to DNA content.
- The complex of DNA and proteins is chromatin.
- Histones are responsible for the basic packaging of chromatin into its
basic units, which are nucleosomes.

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 16. Mitochondrial genome
- Human mitochondrial DNA is 16,569 base pairs in size,
- It contains a total of 37 genes, of which 24 represent genes for various
non-coding RNAs (2 genes for 16S and 23S rRNA and 22 genes for
tRNA).
- The remaining 13 genes encode their own mitochondrial polypeptides
involved in the enzymatic equipment of mitochondria.
- These proteins are utilized during oxidative phosphorylation.

17. DNA replication

Three models of DNA replication allow different predictions.

- Semiconservative model - each strand serves as a template for daughter

strand synthesis.
- Dispersion model - each generation of DNA will contain a mixture of
daughter and parent strands.
- Conservative model - the parent molecules remain intact after replication
and a whole new DNA composed of two daughter strands is formed.

DNA replication begins at replication origins -

places where DNA is divided. The

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double-stranded DNA structure loosens at the beginning of replication, allowing
single stranded DNA to serve as a template for self-duplication. Replication
forks are Y-shaped features typical for the beginning of replication. One origin
of replications contains two forks (one in each direction). Movement apart -
bidirectional replication- the movement of DNA relative to the replication
complexes through which the DNA is stretched.

DNA Synthesis.

Addition of a nucleotide to the 3́-hydroxyl end of a polynucleotide chain -

synthesis in the 5́-> 3́ direction. Base pairing between the free nucleotide and
the nucleotide in the template strand ensures that the correct base is included.

DNA polymerase – enzyme catalyzing the formation of a phosphodiester bond

between a 3́-OH group and a 5́-phosphate group of two nucleotides. Nucleotides
enter the reaction as triphosphates, by breaking the phosphoanhydride bond, the
release of diphosphate and energy - used for polymerization. The DNA
polymerase self-correcting function prevents the accumulation of mutations in
the genome - proofreading - before adding a new nucleotide to the chain, the
polymerase checks the correct pairing of the previous nucleotide.

The replication of forks during synthesis is asymmetrical, creating a leading

chain going in the natural direction 5->3, and the “lagging” chain going from 3
to 5. Leading strand - only the RNA primer at the origin of replication.
Lagging strand - continuous generation of RNA primers by the enzyme
primase for each new synthesized section (Okazaki fragment). DNA
polymerase adds nucleotides until it encounters another primer

- three enzymes are needed - nuclease (recognition and removal of

RNA-primer in duplex with DNA), DNA-polymerase (replacement of
DNA primer, correction),
DNA-ligase(connection of fragments
by phosphodiester bond)
- RNA primers are synthesized by an
RNA polymerase called primase,
which uses a DNA strand as a
template.
- Sliding clamp - DNA polymerase
connecting to a template

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 - DNA topoisomerases release the tension that arises in front of the
replication fork.

Topoisomerases are able to cleave a strand of DNA, unwind 1 or 2 turns of

a double helix, and reassemble the disrupted strand into a single form.
Topoisomerases can generally be divided into two basic groups:

- Topoisomerase I - cleaves only one strand of the double helix, the other
slips through the free space thus created;
- Topoisomerase II - cleaves both strands of a double helix;

18. Comparison of DNA replication in prokaryotes and

eukaryotes

Property Prokaryotes Eukaryotes

Origin of replications Single Multiple

Rate of replications 100 nucleotides/s 50-100 nucleotides/s

Types of DNA polymerase 5 14

Telomerase Not present Present

RNA primer removal DNA pol I RNase H

Strand elongation DNA pol III Pol δ (delta), pol ε (epsilon)

Sliding clamp Sliding clamp PCNA

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 19. Types of DNA damages and their causes
There are 2 principle types of DNA damage:

- Internal factors: reactive oxygen species (ROS) and other endogenous

molecules capable of interaction with DNA originating as products of
cellular metabolism causing DNA replication errors
- External factors:
- Physical factors - UV a ionizing radiation (thymine dimers, single-
and double-strand breaks in DNA)
- Chemical mutagens - chemical modification of bases- deamination,
alkylation, oxidation, and methylation, disruption of the
phosphodiester bonds in DNA, formation of DNA cross-links.
- Biological mutagens -
insertion of viral DNA

Depurination - the formation of

gaps in the nucleotide sequence,
phosphodiester binding is usually
unaffected. Deamination -
spontaneous loss of the amino
group of cytosine. These are the
two most common causes of DNA
damage. Depurination releases
adenine and guanine, deamination
usually converts cytosine to uracil.

Chemical modification of nucleotides causes mutations if the errors are not

eliminated in time. Deamination of cytosine leads to base substitution for
another pairing of uracil with adenine. Depurination leads to loss of nucleo:de
pair - the replication apparatus skips the missing base.

Oxidative DNA damage

Free oxygen radicals are intermediates

of electron transport during oxidative
phosphorylation in mitochondria and

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metabolism in peroxisomes (superoxide, hydrogen peroxide, especially
hydroxyl radical). They oxidize bases, disrupt the sugar phosphate backbone
and cause single- chain breaks.

UV radiation

- Ionizing radiation (X-ray, gamma, cosmic): causes DNA breaks

- Non-ionizing radiation (UV): formation of thymine dimers; the degree
of DNA damage corresponds to the dose of radiation absorbed.
- Formation of a covalent bond between two adjacent thymines causes the
fork replication to stop which increases the probability of a shift in DNA
replication.
- 80% of UVB induced mutations are pyrimidine dimers. This type of
DNA lesion is repaired by nucleotide excision repair (NER). Errors in
this system cause hereditary disease xeroderma pigmentosum, linked
extreme photosensitivity and higher cancer predisposition.

Chemical mutagens

- Chemical compounds with a

similar structure to nucleic acid
bases
- Chemically modified bases
(alkylating agents,
deaminating or oxidizing
agents)

Interaction of a chemical with DNA

and formation of a bond within one
strand or between two strands

- Leads to blockage of DNA

metabolic activity
(transcription, replication)
- Use for example in chemotherapy (cis-platinum)

DNA breaks

- Mainly caused by ionising radiation and oxidative DNA damage

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 - Several phosphodiester bonds close to each other are damaged, which
leads to the formation of double-strand break

20. Mechanisms of DNA repair (NER, BER, mismatch repair)

DNA repair pathways

1. Direct reversal (photoreactivation, dealkylation)

2. Mismatch repair (MMR)
3. Excision repair
a. Base excision repair (BER)
b. Nucleotide excision repair (NER)
4. Single-strand breaks repair
5. Double-strand breaks repair
a. Homologous recombination
b. Non-homologous end joining (NHEJ)
6. Tolerance repair mechanism (lesion bypass)

NER

- Cutting out a larger section of nucleotides

- In bacteria - the multi enzymatic complex recognises lesion; cleaving
takes place on the both sides of lesion; DNA helicase in the complex
removes part of the strand including dimers (approx.12 nucleotides)
- In humans - upon recognition of the damaged strand, the DNA helicase
locally unwinds the DNA duplex and the excision nuclease cleaves a
region of approximately 30 nucleotides, including the dimer.
- Distorts the normal structure of the double helix caused by UV
radiation.

There are two subsystems:

- Global genome repair (GGR) - a slow continuous repair of the

entire genome. Significant role of seven proteins called XPA-XPG
(discovered in the patients with Xeroderma pigmentosum).
- Transcription coupled repair (TCR) - repair of defects on the
template DNA strand; is activated when RNA polymerase II stops
at the site of injury - CSB protein (Cockayne syndrome B) binds -
binding of transcription factor TFIIH (among other subunits XPB,
XPD) and XPG protein.

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 Diseases associated with defective NER

- occurrence - 1-4/million
- all are autosomal recessive
- Xeroderma pigmentosum - 7 different genes (XPA-XPG). Disorders of
DNA damage repair after UV radiation
- Skin damage, frequent skin tumors, neurological abnormalities,
visual disturbances
- Cockayn syndrome - several genes (XPA, B, D, G). Nervous system
development and growth disorders, sensitivity to light, visual
disturbances, premature aging; there is no increased risk of developing
tumors.
- Trichothiodystrophy - several different genes (TTDA, XPB)
- Fine and brittle hair, mental and growth retardation, receding loin,
hardened skin, sensitivity to light; there is no increased risk of
tumor development.

BER

- Based on excision of the wrong section (only one base or nucleotide) of

DNA and its replacement by the correct section.
- DNA glycosylase enzymes - recognize a specific type of modified base
and catalyze its hydrolithic removal.
- At least six types of glycosylases. They test each base separately
by turning it out of the chain
- AP endonucleases - detect blank space after base removal by glycosylase
(AP site)
- It cleaves the phosphodiester bond and corrects the gap
- Repair of minor damage - oxidative damage, deamination and alkylation
of the base, AP site (abasic site - site where the base was removed).
- Hereditary syndromes or diseases caused by mutations in individual
genes of the BER system have not been described

Mismatch repair

- Ensures the correction of errors caused during DNA replication

- The MMR pathway is highly evolutionarily conserved.
- In humans, MMRs occur simultaneously with replication to ensure that
a newly emerging DNA strand with a mismatched nucleotide is repaired.

26
 - Congenital defects in the genes of the MMR system are the basis of a
hereditary tumor syndrome called hereditary non-polyposis colorectal
cancer (HNPCC, Lynch's syndrome),which accounts for 2–5% of all
cases of colorectal cancer.
- A characteristic feature of the impaired function of the MMR system is
the so-called microsatellite instability (MIN) - tandem repeats of one to
six nucleotides, the length of which remains stable in healthy individuals.

21. DNA double-strand breaks repair

DNA repair triggered by ATM kinases (DSB response) and ATR (SSB response
at the site of replication arrest)

Mutations in ATM can cause:

● ataxia telangiectasia disease

● DSB recognition disorder
● neurodegenerative diseases
● increased risk of acute lymphoblastic leukemia
● lymphoma, radiosensitivity

Mutations in CHK2 can cause:

● Li-Fraumeni syndrome,
● familial breast cancer
● sporadic tumors

Double-strand breaks (DSB) can be repaired by homologous recombination -

according to the sister chromatid as a template. This can only happen during
the G2 phase of the cell cycle.

22. Chromosomal instability and aneuploidy

Aneuploidy is an unbalanced change in chromosome number on a cellular or
organismal level, when certain chromosomes no longer come in pairs. In an
aneuploid cell the total chromosome number is increased or reduced compared
to the normal diploid genome of a given biological species.

27
Chromosome instability refers to the lack of capacity to maintain the same
chromosome number from one cell generation to the next.

While aneuploidy, especially in cancer, is often a product of chromosomal

instability, these two concepts are not equivalent. If a cell is aneuploid it does
not necessarily imply that it is also chromosomally unstable and will not pass on
its exact chromosome number to its offspring. However, aneuploidy and
chromosomal instability frequently coexist, which may underlie very complex
and diverse aneuploid karyotypes in high grade tumors.

one of the most

common examples
on aneuploidy -
Down Syndrome

23. Central dogma of molecular biology, eukaryotic and

prokaryotic gene
Flow of genetic information according to the central dogma of molecular
biology:

Replication, transcription and translation take place in all organisms;

reverse transcription is only possible in cells infected with RNA viruses.

In the broadest sense, the term gene expression refers to the process of
translating genetic information encoded by a DNA sequence into a product
with a particular effect. If the expression product is a protein, transcription and
translation will occur. If it is an RNA product, only transcription will occur

28
 24. Types of RNA molecules and general features of
transcription

General features of transcription:

- Transcription ≠ replication
- Transcription produces
RNA that is
complementary to one
of the strands of DNA
- DNA is transcribed by
the enzyme RNA
polymerase - catalysis
of the formation of a
phosphodiester bond.
- The mRNA does not
remain attached to the template strand by hydrogen bonds - just behind
the nucleotide addition to the RNA, the DNA double helix is renewed and
the RNA strand is displaced. Only a small part of DNA is transcribed -
RNA is much shorter than DNA.

29
 25. Transcription in prokaryotes
Transcription unit - promoter, RNA coding sequences and gene terminator
regions.

The coding sequence starts at the start codon. By convention, the transcription
unit is written „left to right“ in the 5´ to 3´ direction of the sense strand.

Bacterial RNA polymerase - used in the beginning of translation. Weakly bind

to DNA and slide along
it to the promoter
sequence.

Structure of a typical
bacterial promoter:
RNA polymerase binds
to the -35 promoter
sequence and initiates
DNA strand unwinding in the AT-rich region in the -10 sequence. Transcription
begins with a transcription bubble in the region of 5-9 bases after -10 sequences.
The -35 and -10 sequences are slightly variable, but some positions are strongly
conservative - consensus sequences. -10 consensus sequence - TATAAT -35
consensus sequence - TTGACA

Elongation:

Transcription of a bacterial gene by RNA polymerase.

Signals in the nucleotide sequence – start and stop transcription. Sigma-factor -

a subunit of RNA polymerase responsible for the recognition of the promoter
and the synthesis of the first 10 nucleotides - sigma factor is subsequently
released and transcription continues. After release of the polymerase from the
DNA, it re-associate with the free sigma factor and search for another promoter

Termination:

● Rho protein dependent (helicase)

- Binding to a 70-100 nucleotide long region upstream of the terminator
sequence
- rho utilization site, cytosine-rich and guanine-poor

30
 ● Independent of Rho protein. It does not require a Rho protein, but two
structural elements - a GC-rich hairpin structure on transcribed RNA
followed by a long section of T or A on the template.

26. Transcription in eukaryotes

● More complex transcription initiation.
- Transcription creates a primary transcript of pre-mRNA
(precursor)
● 3 types of chemical modifications:
- Adding a cap at the 5 ‘end
- Adding a poly (A) tail end to a 3 ‘end
- Splicing (removal of introns)

3 types of RNA polymerases

- RNA polymerase I - transcribes 18S, 5.8S and 28S RNA sequences -

components of ribosomes.
- RNA polymerase II - transcribes genes encoding proteins (into hnRNA -
mRNA precursors)m snoRNAs, miRNAs, siRNAs, lncRNAs and most
snRNA genes
- RNA polymerase III - transcribes tRNA genes, 5S rRNA genes and
some small RNAs.

Short explanation:

1) initiation and regulation of initiation - Initiation of eukaryotic gene

transcription by RNA polymerase II
2) elongation (addition of 5‘cap) - Elongation occurs by the same
mechanism as in prokaryotes.
3) termination (addition of
3‘poly(A) tail) - The 3 'ends of
RNA transcripts synthesized
by RNA polymerase II are
formed by endonuclease
digestion of primary transcript
instead of termination.
4) *(check topic 27) splicing -
Mechanism of RNA splicing:
catalysis using several snRNPs
and other proteins (splicing
complex called spliceosome),
brings both ends of introns
together and attack of specific

31
 adenine in the intron sequence - cleavage of the 5´-splicing site, and its
covalent attachment to adenine - formation of a lariat structure.

27. Post-transcriptional modifications in eukaryotes

The process that takes place after an RNA molecule has been transcribed, but
before it is translated into a protein, is called post-transcriptional
modification.

Post-transcriptional modifications OF RNA accomplish two things:

1) Modifications help the RNA molecule to be recognized by molecules that

mediate RNA translation into proteins;

2) During post-transcriptional processing, portions of the RNA chain that are

not supposed to be translated into proteins are cut out of the sequence.

In this way, post-transcriptional processing helps increase the efficiency of

protein synthesis by allowing only specific protein- coding RNA to go on to
be translated.

28. RNA editing and reverse transcription

RNA editing is a process of post-transcriptional modification of RNA by which
some cells induce specific changes in the nucleotide sequence of RNA molecule

32
after being synthesized by RNA polymerase II, so the sequence of the edited
RNA does not match the sequence of the gene encoding this RNA.

It is considered a relatively rare process. Addition of a 5 'cap, polyadenylation,

or splicing, are generally not referred to as "RNA editing". Main types of RNA
editing are deaminations, conversion of cytidine to uridine and adenosine to
inosine + addition or removal of nucleotides independent of the template.

Reverse transcription

Reverse transcription is the enzyme-mediated synthesis of a DNA molecule

from an RNA template. The resulting DNA, known as cDNA, can be used as a
template for PCR amplification.

Reverse transcriptases are used by certain viruses such as HIV and hepatitis B to
replicate their genomes

29. Genetic code

Genetic code -

- rules for the transfer of nucleotide sequence DNA / RNA to amino acid
- discovery of genetic code in the 1970s
- reading in triplets - four RNA subunits can create 4x4x4 = 64
combinations (theoretically encode up to 64 different amino acids)
- codon - a nucleotide triplet encoding a particular amino acid

Most AAs are encoded by more than one codon, the codons of a given amino
acid have a certain regularity - the difference especially in the third position.
Stop (termination) codons are not coding any amino acids but instead terminate
translation.

Overview:

1. The genetic code is composed of nucleotide triplets. The three nucleotides of

the mRNA identify one amino acid in the polypeptide chain, so each codon
contains three nucleotides.

33
2. The genetic code does not overlap. Each nucleotide of the mRNA belongs to
only one codon, with rare exceptions where the genes overlap and the
nucleotide sequence is read in two reading frames.

3. The genetic code does not contain commas. There is no punctuation in the
coding region of the mRNA, the codons are read one after the other.

4. The genetic code is degenerate. All but two amino acids can be encoded by
more than one codon.

5. The genetic code is ordered. Several codons for the same amino acid and
codons of amino acids with similar chemical properties are related, usually
differing by only one nucleotide.

6. The genetic code contains start and stop codons. Specific codons are used to
initiate and terminate translation of a polypeptide chain.

7. The genetic code is almost universal. With rare exceptions, the same codons
encode the same amino acids in all living organisms, or viruses except humans.

30. tRNA and aminoacyl-tRNA synthetases, ribosome

structure
Aminoacyl tRNA synthetase - an enzyme that catalyzes the recognition and
attachment of the correct AA to tRNA - Each cell has 20 different tRNA
synthetases - one for each AA

Amino acid activation by synthetase - The two-step reaction, the use of energy
from ATP to tRNA by high-energy binding, the whole process takes place on
synthetase,

1 - activation of AA by binding to AMP via carboxyl group, energy from ATP

- adenylated amino acid,

2 - the adenylated carboxyl group of AA is transferred to the hydroxyl group of

the sugar at the 3´ end of the tRNA molecule - phosphodiester bond

Ribosome structure

Codon recognition in the mRNA and assignment of the appropriate tRNAs take
place on ribosomes - a multiprotein complex that moves along the mRNA,

34
captures the tRNA, holds it in the correct position, and connects the AAs to
form the polypeptide chain.

The eukaryotic ribosome

is a large complex of four
rRNAs and more than 80
small proteins and
rRNAs.

Prokaryotic ribosomes are

similar – both are formed
from a large and small
subunit, which only come
together aIer the small
subunit has bound mRNA.

31. Translation
Initiation

Translation begins at the initiation codon (AUG) - a special tRNA with bound
methionine (in bacteria formylmethionine) - each newly synthesized protein
begins with (formyl) methionine, which is usually removed later.

Binding of the initiation tRNA to the small ribosomal subunit, assistance of

translation initiation factors - the only Met-tRNA that can bind tightly to the
ribosome.

Subsequently, the small subunit recognizes the 5´-cap of the mRNA, binds it,
begins to move along the

strand and searches for the start codon AUG

35
 Elongation

After starting the protein synthesis, each additional AA is

added in the same way.

The tRNA with bound chain is in the P-site, the new tRNA
with bound AA is in the A-site - the carboxyl terminus of
the polypeptide chain is cleaved from the tRNA in the
P-site - linking the polypeptide to the free amino group of
AA at A-site - probably catalyzed by some RNA subunit of
the ribosome).

The peptidyltransferase reaction is accompanied by a shift

of the large subunit with bound mRNA relative to the small
subunit - the shift moves the tRNA from the P- to E-site
and the tRNA from the A-site to the P-site, now with the
bound polypeptide chain. Step 3 - moving the small subunit
exactly three nucleotides forward, to the original position
with the large subunit, the tRNA is released from the
E-site. The whole cycle is repeated each time a new AA is
added up to the stop codon.

Termination
Incorporation of an amino acid into a polypeptide chain.
The polypeptide chain grows in several steps by adding an
amino acid to the C-terminus of the resulting peptide. The
formation of a peptide bond is energetically favourable
because the growing C-terminus has been activated by
covalent binding of the tRNA molecule. The
peptidyl-tRNA complex is regenerated aIer each addition of AA.

End of protein - stop codon (one of the three termination codons - UAA, UAG,
UGA). - no AAs are recognized, except for UGA - selenocysteine if it is
adjacent to certain nucleotides, - instead of tRNA, termination factors bind to
the stop codon at the A-site - alter peptidyltransferase activity

36
 32. Post-translational modifications
Post-translational modifications are responsible for diversity of the proteins

N-glycosylation of proteins - an important function of the ER, a signal for

distinguishing correctly folded/assembled proteins and also for the immune
system.

It occurs almost immediately after the protein enters the ER - the addition of a
branched oligosaccharide side chain from dolichol (lipid) by
oligosaccharyltransferase.

33. Protein folding and protein degradation, protein sorting

Protein folding is the physical process by which a protein chain is translated to
its native three-dimensional structure, typically a "folded" conformation by
which the protein becomes biologically functional.

Protein degradation

Protein degradation (proteolysis) - Proteins are marked for degradation

by the attachment of ubiquitin to the amino group of the side chain of a lysine
residue. Additional ubiquitins are then added to form a multiubiquitin chain.
Such polyubiquitinated proteins are recognized and degraded by a large,
multisubunit protease complex, called the proteasome.

Protein sorting

Protein sorting - is the biological mechanism by. which proteins are transported
to their appropriate destinations in the cell or outside it.

Proteins can be targeted to the inner space of an organelle, different.

intracellular membranes, plasma membrane, or to exterior of the cell via
secretion.

34. Regulation of gene expression in prokaryotes - operon

model, examples
Gene expression - the process of turning on a gene to produce RNA and
protein.

37
Regulation of gene expression allows differentiation into different cell types
and adaptation to changes in environmental conditions.

Transcription in prokaryotes – initiation

• Prokaryotic promoters are not equal

• Some of them have lower affinity to RNA polymerase

• They are regulated (positive – ACTIVATOR or negative – REPRESSOR)

• Regulators turn on/off transcription in response to environmental stimuli

• There is no need to synthesize all proteins at once

• Cells do not waste the energy

Operon model of gene expression

Operon genes - a group of genes controlled by a promoter and operator

Operon - a transcription unit that contains the "Promoter" and "Operator"
regulatory sites in front of the structural genes.

A typical operon comprises of several types of genes:

Structural genes (S1–Sn) which code for the primary structures of enzyme
proteins involved in a metabolic pathway, such as the biosynthesis of an amino
acid.

The promoter (P), a short sequence of DNA acting as the start point, and to
which RNA polymerase binds. The promoter is controlled by various regulatory
elements that respond to environmental stimuli.

The operator (O), comprising a short segment of DNA found adjacent to the
promoter is a control element which binds a regulator protein that can either
repress or activate transcription.

Tryptophan operon

The trp operon is an operon—a group of genes that is used, or transcribed,

together—that codes for the components for production of tryptophan.

● Contains 5 structural genes (trpE, trpD, trpC, trpB and trpA), promoter
(P), operator (O) and leading sequence (L)

38
Repressor - represses gene transcription

Activator - activates transcription - for promoters that bind only weakly to

RNA polymerase

Lac operon

Lac operon - the genes in the operon encode proteins that allow the bacteria to
use lactose as an energy source. LacZ, the first gene in the operon, encodes the
enzyme β-galactosidase, which breaks down lactose into glucose and galactose.

Regulation of gene expression was first studied in 1961 In 1965. Jacques

Monod and Francois Jacob discovered Lac operon (Nobel prize 1968)

35. Regulation of gene expression in eukaryotes

Levels (steps):

Chromatin remodeling –chromatin has to be relaxed (decondensed) to enable

access of transcription factors to gene promoter (0)

Transcriptional control – the most common type of regulation – turn on/off the
transcription (1)

Post-transcriptional control - pre-mRNA processing - RNA interference

(RNAi, microRNA) (2)

mRNA transport from nucleus (3)

mRNA degradation control (4)

Translation control – regulation of the initiation of translation (5)

Post-translational processes – protein modification and degradation (6,7)

36. Regulation at the transcriptional level, transcription

factors
Transcription factors:

1. Basal transcription factors (TFs) – in present in all eukaryotic cells –

mandatory for transcription, but do not enable regulation of the transcription
-TFIIA, TFIID, TFIIB, TFIIE, TFIIH, TFIIF - the most important is TFIID and

39
its subunit TBP (TATA binding protein) interact through TATA box with
promoter.

2. Regulatory transcription factors (TFs) – regulators of the transcription -

present the main regulatory mechanism of gene expression in eukaryotes -
proteins bind to
a) enhancers (enhancing transcription)
b) silencers
c) proximal promoter elements - are specific for concrete genes or gene
families

Initiation of eukaryotic gene transcription by RNA polymerase II. RNA

polymerase requires several basal transcription factors to initiate transcription.

Regulatory TF, distant control.

Mediator - a protein complex (approximately 20 subunits) mediating

interaction of the regulatory transcription factors with RNA polymerase

Enhancers – DNA sites to which eukaryotic activators bind, their presence

increases transcription efficiency

Regulation of expression at the post-transcriptional level (export from the

nucleus, +39mRNA degradation, non-coding RNA)

37. Regulation of expression at the post-transcriptional level

(export from the nucleus, mRNA degradation, non-coding
RNA)
The different RNA species that are produced in the nucleus are exported
through the nuclear pore complexes via mobile export receptors. Small
RNAs (such as tRNAs and microRNAs) follow relatively simple export routes
by binding directly to export receptors.

mRNA degradation is a process to eliminate mRNA that is either no longer

required in the cell or has aberrant features. Three subcategories of enzymes
prevail in the cells that mediate mRNA degradation; these enzymes are
categorized according to the localization at which they cut RNA
endonucleases, which cleave RNA internally, 5′ exonucleases, which degrade

40
RNA from the 5′ end, and 3′ exonucleases, which promote hydrolysis at the 3′
end.

The term non-coding RNA (ncRNA) is commonly employed for RNA that
does not encode a protein, but this does not mean that such RNAs do not
contain information nor have function. These RNAs (including those derived
from introns) appear to comprise a hidden layer of internal signals that control
various levels of gene expression in physiology and development, including
chromatin architecture/epigenetic memory, transcription, RNA splicing,
editing, translation and turnover.

38. Regulation of expression by chromatin remodeling

Chromosomes have to be opened (decondensed) to enable the access of
transcription factors (TF) and enzymes to the genes.

Chromatin-remodeling complex– group of the proteins changing the structure

of chromatin - condensation and decondensation of chromatin is carried out by
enzymes

1. Histone acetyl transferases (HAT) - opening (decondensing) chromatin to

euchromatin

2. Histone deacetylases (HDAC) - packing (condensing chromatin) to

heterochromatin

3. DNMT (DNA methyltransferases) - methylation of DNA (Cytosine) -

condensing chromatin

39. General principles of cell signalling

- Signal transduction - conversion of one form of signal to another
- Cell signaling - the signaling cell produces a specific type of molecules
that are transmitted to the target cell through receptors that are
responsible for recognition of the molecule and a specific response to it
- Can act over a shot or a long range

41
 1. Endocrine - hormones
produced in the endocrine glands
are secreted into the bloodstream -
distant action.
2. Paracrine - signals
released by cells into the extracellular
space - local action.
3. Synaptic - signals
transmitted along the axon of the
nerve cell.
4. Contact-dependent -
contact dependent signaling;
requires the physical contact of
neighboring cells.

Different forms of signal differ in the speed and selectivity of signal

transmission.

- Contact-dependent signaling controls the production of nerve cells. The

nervous system in the embryo comes from the epithelial cell layer, where
isolated cells begin to specialize as neurons and send an inhibitory
signal to the surrounding cells (the delta signal molecule binds to the
Notch receptor), preventing them from specializing as well - the
surrounding cells maintain the epithelial structure of the layer. The same
mechanism governs the detailed design of differentiated cell types in
various other tissues in vertebrates and invertebrates.
- Each cell responds to a limited set of extracellular signals depending on
its history and its current state.
- Different cells may respond differently to the same signal.
- Number of receptors determines the number of signals.

Different cell types

respond to the neural
mediator acetylcholine
according to their
specialization despite a
similar cell surface
receptor molecule.

42
 - An animal cell depends on multiple external signals, ensuring sensitivity
to many extracellular signals.
- If a cell is deprived of appropriate signals for survival, differentiation or
proliferation, it normally commits cell suicide, or programmed cell death
- apoptosis.

Speed of cellular response

- Slow reaction - for changes of cellular behaviour that require a change in

gene expression and synthesis of new proteins
(proliferation or cell growth).
- Fast reaction - for changes that don’t need the
nuclear apparatus (movement, secretion, metabolism).

Types of signaling molecules:

- large / hydrophilic molecules - cannot cross the

membrane - receptors must lie in the plasma membrane.
- small / hydrophobic molecules - can diffuse across
the membrane - the receptor can be in the cytosol or in the
nucleus, generally either regulatory proteins or enzymes.

40. Intracellular (nuclear) receptors

Some hormones cross the plasma
membrane and bind connectly to
intracellular receptors. For example -
cortisol - the steroid hormone that activates
the regulatory protein of various genes. It
causes a large conformational change to the
cytosolic receptor. Both cytosolic and
nuclear intracellular receptors are
referred to as nuclear - when activated,
they act as transcription regulators of gene
expressions.

Some dissolved gases can cross the

membrane as well. An example is the nitric
oxide (NO) that causes relaxation of
smooth muscle in the walls of blood vessels, therefore increase in blood volume

43
in blood vessels (use in treatment - rapid decrease in blood pressure).
Nitroglycerin is metabolized in the human body to nitric oxide, which then
causes dilation of blood vessels(treatment of hypertension) and reduces the
burden on the heart muscle (treatment of angina pectoris).

Intracellular signaling proteins act as molecular switches - receiving a signal

converts them from an inactive state (OFF)
to an active state (ON), where they remain
until another process switches them back to
OFF. Therefore, there must be an
inactivation mechanism for each activation
mechanism so that the signal can be
regulated and received again.

Intracellular signaling cascade.

- Many steps of such a cascade may be

affected by other processes in the cell.
- Feedback regulation within an
intracellular signaling pathway can adjust
the response to an extracellular signal.

Basic functions:

1. The relay of a signal to the cellular apparatus that

generates a response.
2. Transduction of the signal into a molecular form and its amplification.
3. Signal integration.
4. Distribution of the signal so that it affects several events
at the same time - branching the flow of information.
5. Signal modulation - according to the conditions that prevail inside the
cell.

41. Cell surface receptors (classification, general description)

44
- Many extracellular signals act via cell-surface receptors to change the
behaviour of the target cell.
- The receptor activates one or more intracellular signalling pathways,
which interact with specific effector proteins, altering them to change the
behaviour of the cell.
- Extracellular signal - binding of a signal molecule to a receptor protein,
capture of the signal and
generation of a response - a
new signal to the cell.

Three main classes of cell

surface receptors

- Receptors associated
with ion channels - ion flux,
leading to electrical
phenomena; transmitter-gated
ion-channels.
- G-protein-coupled
receptors - the activated form
of the membrane G-protein
moves in the plasma
membrane and triggers a
cascade of other signalling
events.
- Enzyme-associated receptors - change in enzyme activity in the
cytoplasmic domain of the receptor.

42. Ion-channel-coupled receptors (function, examples)

- The simplest type,
- Conduct fast signal
transmissions through
synapses into the
nervous system,
- Change the membrane
potential and create
action potential,

45
 - Convert chemical signals into electrical,
- Used by electrically stimulated cells, for example muscle cells,
- Examples: nicotine, acetylcholine, serotonin.

43. G-protein-coupled receptors (function, examples)

Summary

Structure:

- Composed of three subunits - α, β, γ,

- The alpha subunit has GPTase activity, which determines the time of
dissociation,
- GDP is in the inactive state,
- Binding of an activated receptors enables the GDP/GTP exchange
reaction

Functions:

- Direct regulation of ion channels

- Activation of membrane-bound enzymes
- Regulation of transporter proteins

Examples - hormones, local and nerve mediators, etc.

Dissociation

A - the inactive state, the receptor

and G-protein are not in contact

B - activation of the receptor and

binding of the G-protein

C - the binding to the receptor

triggers GDP/GTP exchange in the
alpha subunit; G-protein dissociates
into α and βγ subunits

46
The alpha subunit deactivates itself by GTP hydrolysis

GTP -> GDP - separation from the target protein -> reassembling into an
inactive G-protein

Regulation of
ion-channels

- Upon receptor
activation G-protein
associates with the
potassium channel in the
plasma membrane of
cardiac muscle cells. For
example - control of heart
rate.
- Receptor ligand -
acetylcholine - is
produced by nerve
fibers as a signal of
slowing the heart rate.
The activated βγ-subunit
binds to the K + channel
in the plasma membrane and opens it -> a change in the
electrochemical gradient-> less frequent contractions. Inactivation of
the α-subunit by GTP hydrolysis causes the reassembly of all G-protein
subunits and the closure of the potassium channel.

Activation of membrane-bound enzymes

Interaction of G-proteins with

enzymes makes them produse

47
messenger molecules: second messenger (receptor ligand); adenylate cyclase
(formation of cyclic AMP); phospholipase C (formation of inositol triphosphate
and diacylglycerol). The second messengers quickly diffuse from their source
and spread the signal in the cell. Coupling can be either activating or
inhibitory.

G-protein phospholipase C-pathway

- acts on the membrane inositol phospholipid in the inner part of the

plasma membrane (PM) - inositol phospholipid pathway.
- formation of two mediator molecules (second messengers) - inositol-
1,4,5-triphosphate (IP3) and diacylglycerol (DAG)
- IP3 diffuses through the cytosol and releases Ca2+ ions from
the ER by opening channels - a large electrochemical gradient
causes ions to flow into the cytosol.
- DAG remains in PM and together with Ca2+ activate the
enzyme protein kinase C.

44. Receptors with enzyme activity (function, examples)

- Response to growth factors
- Rapid effect on cytoskeleton
- Special importance on the development of cancer
- Ligand-binding domain on the cell surface, enzymatic domain in the
cytosol or in complex with enzymatically active protein

Tyrosine kinases

- Cytosolic domain, phosphorylation of tyrosine side chains of target

proteins
- Activation of the receptor tyrosine kinase leads to the formation of an
intracellular signaling complex

48
 - Binding of a signaling molecule to the extracellular domain of a receptor
tyrosine kinase causes two receptor molecules to associate to form a
dimer
- The kinase domains of the receptor come into contact -> mutual
phosphorylation of tyrosines -> the binding site for intracellular signaling
molecules.

45. Cell cycle

The cell cycle is the complete series of events from one cell division to the next.

Goal of the cell cycle regulation:

​ - The production of two genetically identical cells

from one precursor cell.
​ - The regulation of tissue growth and repair
The cell cycle is usually divided into several phases: the phase when
DNA is replicated (S phase), the phase when the cell actually divides into
two cells (M phase - mitosis), the two intervening gap phases (G1 and
G2), and a non-dividing state called "quiescence" (G0).

49
In the G1 phase, the cell grows in size, checks the status of its internal systems
and evaluates signals from the outer environment. If everything is functioning
normally, and potential damage to the DNA has been corrected, the cell moves
on through the cycle to the following phase. If the cell is obtaining signals from
outsides that it should not divide, or something is wrong and cannot be
corrected, the cell halts its progression through the cycle, or programmed cell
death – apoptosis – is initiated.
In the following synthetic (S) phase, the cell replicates its genetic information
stored in the chromosomes. Following this is a period of preparation for division
called the G2 phase. Then the cell divides into two daughter cells – the mitotic
(M) phase. The two new daughter cells then enter the G1 phase of their own cell
cycle. The average length of the cell cycle in mammalian cells is
approximately 24 hours. The cell spends most of this time in the interphase.

Cyclins and cyclin-dependent kinases

- MPF is a complex of cyclin B and CDK1. MPF promotes the transition

into mitosis from the G2 phase by phosphorylating multiple proteins
needed during mitosis. MPF is activated at the end of G2 by a
phosphatase
- Cyclins activate cyclin-dependent kinases; they are not constantly
present in cells; they are produced and degraded according to the cell’s
needs
- CDKs are serine/threonine kinases – enzymes that phosphorylate target
proteins, such as signalling molecules and transcription factors that
trigger the expression of
genes, on serine or
threonine amino acid
residues

The fluctuations of most cyclin

levels are tightly coordinated with
the progress through the cell
cycle, and are not influenced by
external factors. The levels of
D-type cyclins are induced by
extracellular signals (growth factors). In cancer cells the production of cyclins
is increased and the cell-cycle is deregulated. But the cyclin binding is not

50
enough to activate CDKs, inhibitory phosphates also must be removed. As soon
as the M cyclin-CDK complex is formed it is phosphorylated by an inhibitory
protein kinase (Wee1). This keeps the complex inactive until these phosphates
are removed by an activating protein phosphatase (Cdc25).

Cell cycle progression is also negatively regulated by the inhibitors of

cyclin-dependent kinases. CDK inhibitors are proteins that can bind CDKs and
inhibit their kinase activity. CDK inhibitors can also arrest the cell cycle
progression when necessary.

Ubiquitin-regulated protein degradation

The ubiquitin-proteasome pathway is responsible for the rapid and highly

specific degradation of selected proteins in mammalian cells (within minutes)

- Allows the cell to fine tune the levels of critical regulatory proteins
- Specific – a small subset of proteins can be degraded without any effect
on other proteins

46. Checkpoints and regulation of the cell

cycle
Checkpoints

Late in the G1 phase, the G1 checkpoint (or restriction

point) determines whether the cell will enter the

51
following S phase. In animal cells, the G1 checkpoint is largely controlled by
growth factors. The G2 checkpoint determines whether the cell will enter the
M phase. The most important criterion is the proper completion of DNA
synthesis. The third cell cycle checkpoint is the spindle assembly checkpoint
between metaphase and anaphase and requires the proper attachment of all
the chromosomes to the spindle apparatus. In the M-phase checkpoint the
mitotic cyclin has to be degraded to allow for the completion of mitosis and
cytokines.

Mitotic spindle checkpoint controls the activity of APC (anaphase-promoting

complex). APC targets securin and mitotic cyclin for degradation. The
destruction of securin allows separase to cleave the cohesins that hold sister
chromatids together, thereby initiating anaphase. The degradation of mitotic
cyclin decreases the mitotic CDK activity, leading to cytokinesis, chromosome
decondensation and nuclear envelope reassembly.

52
 47. Cell cycle deregulation and its consequences
Defects in mitotic checkpoint signalling are one of several pathways by which
cells might gain or lose chromosomes in somatic cells during mitosis. A
weakened mitotic checkpoint might allow cells to enter anaphase in the
presence of chromosomes unattached to the spindle or of misaligned
chromosomes. As a consequence, both copies of one chromosome might be
deposited into a single daughter cell. Both aneuploidy and mutations of genes
involved in the mitotic spindle checkpoint are common in cancer cells!

Genes commonly altered in cancer

1. Proto-oncogenes / oncogenes – they get activated in cancer cells

- Code for proteins that are usually involved in signal transduction and
regulate cell proliferation or differentiation
- Mutations or increased expression (production) of these proteins lead to
the increase in their activity in cell – a proto-oncogene becomes an
oncogene – a tumour-inducing agent
- Examples: Myc, Ras, Src, HER2, cyclin D, cyclin E - patients suffering
from breast carcinoma, who had increased level of cyclin E in cancer
cells, had more aggressive disease and responded less well to the therapy

2. Tumour suppressors – their function is commonly lost in cancer cells

- Prevent growth and survival of aberrantly dividing or damaged cells

- Some tumour suppressors take part in DNA repair and prevent the
accumulation of potentially oncogenic mutations
- Examples: p53, p16, p15, Rb, APC, MLH1, MSH2, BRCA1

TGF-β

Upon exposure to TGF-β, the cell increases the mRNA amount of one of the
CDK inhibitors, therefore the activity of the corresponding complex of cyclin
and CDK (CDK4/6 and cyclin D) is inhibited, and the targets of this CDK are
not phosphorylated – the cell does not progress through the cell cycle.

53
A deletion of a portion of the short arm of chromosome 9 is common in
glioblastoma multiforme and malignant melanoma. In most cases, it leads to a
concomitant loss of the CDKN2A (encoding the CDK inhibitor p16) and
CDKN2B (coding for the CDK inhibitor p15) genes. The cancer cells become
unresponsive to TGF-β and cannot go into senescence.

Rb protein

Retinoblastoma protein is a tumor suppressor protein that is dysfunctional in

several major cancers. One function of pRb is to prevent excessive cell growth
by inhibiting cell cycle progression until a cell is ready to divide. The control of
restriction point transition by mitogens is mediated by Rb phosphorylation.

1. Unphosphorylated
Rb - As cells pass
through the M/G1
transition, Rb is
dephosphorylated
2. Hypophosphorylated
Rb - As cells
progress through G1,
relatively small
numbers of
phosphate groups are
added by cyclin
D-CDK4/6 complexes
3. Hyperphosphorylated Rb – When cells pass through the restriction point,
cyclin E-CDK2 complexes heavily phosphorylate Rb and its
phosphorylation continues to increase throughout the remainder of the
cell cycle

Loss of Rb function confers:

1. Insensitivity to anti-growth signals (TGFβ)

2. Inhibition of senescence

54
 48. Types of cell division and their significance
There are three major types of cell division, which are:

● Binary fission
● Mitosis
● Meiosis

Whereas binary fission takes place in prokaryotic cells of simple single-celled

organisms such as bacteria. Mitosis and meiosis take place in eukaryotic cells
and are more advanced.

Although there are differences between prokaryotes and eukaryotes, there are a
number of features that are common during their processes of cell division.

These include:

● The replication of DNA,

● The segregation of the original and the replica,
● Cytokinesis

Binary fission

Genetic material (nucleoid) in these cells is arranged in a single circular

chromosome of the DNA.

In a similar fashion to eukaryotes, the genetic material of these cells is

duplicated before division.

As the prokaryote elongates, the two chromosomes attached to the plasma

membrane also move apart, and once the two copies have separated (the original
and replicate chromosomes), the cell divides, a process referred to as
cytokinesis.

55
 49. Mitotic cell division
Mitosis is division of the cell nucleus

Cytokinesis is division of the cytoplasm

Significance of mitosis:

- Even distribution of genetic material into daughter cells

- Daughter cells are genetically identical
- Distribution of centrosomes

Significance of cytokinesis:

- Distribution of cytoplasm into daughter cells

- Distribution of organelles into daughter cells

Before entering mitosis, the genome has to be fully replicated, sister chromatids
must be paired by cohesins during S-phase and finally centrosomes have to be
duplicated.

Mitosis - even segregation of sister chromatids into the daughter cells. Mitosis
is a type of cell division in which one cell (the mother) divides to produce two
new cells (the daughters) that are genetically identical to itself.

Key processes during mitosis include:

- weak metabolic activity

- nuclear membrane disintegration - spindle formation
- chromatin condensation
- kinetochore formation
- sister chromatid segregation
- spindle degradation
- chromatin decondensation
- nuclear envelope formation

56
 Phases

Cell cycle regulation *more information in question 45

Active MPF phosphorylates/activates:

1. Chromatin condensation
2. Spindle forming proteins (Exportin=Crm1=Xpo1)
3. Boosts its own activation and prevents its own deactivation
4. Nuclear lamin – nuclear envelope degradation
5. Activates APC complex

50. Mitotic apparatus (centrosomes and mitotic spindle),

chromatid separation
Centrosomes

The centrosome is the major microtubule-organizing center in the cell. During

interphase the centrosome is responsible for creating a microtubule array and
during mitosis it assists in bipolar spindle assembly. Tissue development and
homeostasis depend on the polarizing activity of the centrosome. Centrosomes
duplicated during S phase must be segregated by cytokinesis for daughter cells
to maintain the normal number of 1 during G1, and 2 at mitosis.

Movement of mitotic spindle

57
Molecular motors: transport cargo along microtubules and actin fibers

- kinesins
- dyneins
- myosins

Types of movements mediated by motors:

- Pushing polar microtubules apart (kinesin

5, 14)
- Centering spindle by astral microtubules
- Moving the kinetochore with chromatids
along the kinetochore microtubules toward
the spindle pole (kinesin 7, 13 and ?)
- Treadmill movement of kinetochore MTs
- Contraction of contractile ring in anaphase and further (myosin 2)

Chromatid separation

During anaphase each chromosome's sister chromatids separate and move to

opposite poles of the cell. Enzymatic breakdown of cohesin — which linked the
sister chromatids together during prophase — causes this separation to occur.
Upon separation, every chromatid becomes an independent chromosome.

51. Errors in mitosis and

their consequences
Mitotic defects have several
potential outcomes. Failed
alignment of chromosomes leads

58
to mitotic arrest/delay enforced by the spindle checkpoint. If the failed
alignment is not corrected, cells can follow several fates. They can undergo cell
death directly from mitotic arrest. Cells may also suffer various kinds of
abnormalities during mitotic exit, leading to the formation of aneuploid
progeny. Alternatively, cells may exit mitosis without proper chromosome
segregation and cytokinesis, resulting in a formation of a single tetraploid cell.
Aneuploid or polyploid daughter cells may undergo cell death, cessation of
proliferation and senescence, or continued proliferation.

Ploidy is the number of sets of chromosomes in a cell or in an organism.

Polyploid denotes a cell with more than two sets of chromosomes. Aneuploidy
produces changes in mRNA dosage which lead to changes in protein dosage for
genes on the gained or lost chromosome(s). Changed protein levels can have
direct effects on biological processes in which they are involved, or change the
stoichiometry of protein complexes of which they are components, causing
changes in their function. Changes in gene dosage of regulatory proteins like
transcription factors may also exert indirect effects on biological processes by
altering expression of their target genes on other chromosomes.

Hutchinson-Gilford progeria

Mutations in the LMNA gene cause Hutchinson-Gilford progeria syndrome.

The LMNA gene provides instructions for making a protein called lamin A.
This protein plays an important role in determining the shape of the nucleus
within cells.Progeroid syndromes are a group of diseases with premature aging.

59
 52. Meiosis and gametogenesis
Meiosis is a process where a single cell divides twice to produce four cells
containing half the original amount of genetic information. These cells are our
sex cells – sperm in males, eggs in females.

Consists of two subsequent cell

divisions:

- First meiotic
(heterotypic) division
- Second meiotic
(homotypic) division

During the first meiotic division

homologous chromosome segregation
takes place. During the second
meiotic division sister chromatid
segregation occurs.

Stages of prophase 1

- During leptotene individual

chromosomes begin to condense into
long strands within the nucleus.
- The zygotene stage occurs as
the chromosomes approximately line
up with each other into homologous
chromosomes. The combined
homologous chromosomes are said to
be bivalent. They may also be
referred to as a tetrad, a reference to
the four sister chromatids.
The two non-sister
chromatids become
"zipped" together, forming
the synaptonemal
complex, in a process
known as synapsis.

60
 - During pachytene stage chromosomal crossing over takes place, where
non-sister chromatids exchange segments. Exchange takes place at sites
where recombination nodules have formed.
- During the diplotene stage the synaptonemal complex degrades and
homologous chromosomes separate from one another a little.
- Chromosomes condense further during the diakinesis stage. This is the
first point in meiosis where the four parts of the tetrads are actually
visible; the nucleus disappears, the nuclear membrane disintegrates into
vesicles, and the meiotic spindle begins to form.

Gametogenesis

Gametogenesis is a biological process by which diploid or haploid precursor

cells undergo cell division and differentiation to form mature haploid gametes.
Depending on the biological life cycle of the organism, gametogenesis occurs
by meiotic division of diploid gametocytes into various gametes, or by mitosis.

53. Genetic impact of meiosis - crossing-over and its

significance
Genetic impact of meiosis:

- resulting haploid cells contain just one chromosome set (1n)

- there are 2n (223) possible chromosome combinations (increased
by crossing-over) in gametes
- homologous chromosome pairing makes possible gene
recombination by crossing-over

Significance of crossing over

1. Crossing over leads to the production of new combinations of genes and
provides a basis for obtaining new varieties of plants.
2. It plays an important role in the process of evolution.
3. The crossing over frequency helps in the construction of genetic maps of
the chromosomes.
4. It gives us the evidence for linear arrangement of linked genes in a
chromosome.

61
 54. Errors in meiosis and their consequences
Meiotic recombination mechanism

- Active
formation of double
stranded breaks and
D-loop formation
- Holliday
junction
movement,synthesis
and ligation, chi
structure formation
- DNA sequence
homology is key for
synapsis
- It is searched
for by means of
meiotic recombination

In a process called
meiotic
recombination, the
paternal and maternal
chromosomes are
replicated, pair, and
exchange parts of
their DNA by forming
double-strand breaks,
which are later
repaired.

The synaptonemal
complex (SC) is a
protein structure that
forms between homologous chromosomes (two pairs of sister chromatids)
during meiosis and is thought to mediate synapsis and recombination during
meiosis I in eukaryotes. SPO11 defect is associated with male infertility.

62
Absence of active DNA break formation leads to failure of synaptonemal
complex formation and failure to form gametes.

Cohesion of chromosomes

Replicated sister chromatids are kept connected to one another, from the time of
their synthesis onwards, by the chromosomal protein complex known as
cohesin. Sister chromatid cohesion is the basis for the pairwise alignment of
chromosomes on the spindle apparatus during mitosis, making possible the
segregation of chromatids at anaphase. In higher eukaryotes, a significant
portion of cohesin is removed from chromosomes as they condense in prophase.
This is important, allowing much of the sister sequences along chromosome
arms to separate so as to form the distinct sister chromatid axes characteristic of
metaphase chromosomes.

Shugoshin

- protects proximal cohesins from degradation by separase

- plays a central role in the cohesion of chromosomes during cell division

Errors and consequences

- Absence of crossing over blocks synaptic progression and causes

desynapsis
- Mutation of one allele of shugoshin causes a premature ovarian failure
- Unequal crossing over - can happen in prophase I, leads to deletion in the
first chromatid and duplication in the second chromatid; Example -
Hemoglobin Lepore syndrome.
- Nondisjunction - failure of homologous chromosomes in anaphase I or
sister chromatids in anaphase II to separate properly during cell division;
caused by centromeric defect or defect in mitotic spindle; leads to
aneuploidy.

63
55. Gametogenesis, differences in gametogenesis in women
and men
Male

- is called spermatogenesis in male

- takes 74 days
- start producing sperm when they reach puberty
- Meiosis I produces two haploid cells, known as
secondary spermatocytes. Meiosis II produces four
haploid cells, known as spermatids.
- After differentiation spermaids create
spermatozoa (mature)

Female

- In females is called oogenesis

- Begins in the fetus prior to birth

- Meiosis I begins before

birth and creates
primary oocytes;
flattened epithelial cells
surrounding them create
the primary follicle.
- When puberty starts,
primary oocytes mature
each month, but only
one reaches full
maturation and becomes
an oocyte
- Pre-ovulatory state -
One of the daughter
cells receives far less
cytoplasm than the other

64
 and forms the first polar body, which will not go on to form an ovum.
Another haploid cell is also formed, known as the secondary oocyte. Both
daughter cells then undergo meiosis II.
- As the oocytes develop, they get enclosed in structures known as
follicles.

56. Basic types of cell death and their significance

Three basic types of cell deaths:

- apoptosis (type I)
- autophagy (type II)
- necrosis (type III)

Apoptosis “suicide” - programmed cell death

- activation of caspases, chromatin condensation and fragmentation, cell

shrinkage, plasma membrane blebbing, formation of apoptotic bodies. In the
human body about 100 000 cells are produced every second, and approximately
the same number of cells dies due to apoptosis - a natural process of removing
unwanted cells. Allows the formation of hollow organs, reproductive organs,
massive cell death occurs during the early stage of development of the nervous
system (Development and Morphogenesis). Deletion of severely damaged or
dangerous cells (Homeostasis).

Autophagy

- massive accumulation of double- membrane vacuoles – autophagosomes,

which encapsulate cytoplasmic material. The autophagosomes subsequently
fuse with lysosomes –autolysosomes - degradation of the contents.
Self-digesting mechanism for cellular components. Recycling of cellular
material during starvation. Mostly cell-survival mechanisms. Death
accompanied with autophagy. It was discovered by Yoshinori Ohsumi and he won a
Nobel prize for it in 2016 in Physiology or Medicine.

Necrosis “murder” – accidental cell death

- cytoplasmic swelling, dilatation of organelles, which causes cellular

vacuolation and rupture of the plasma membrane, resulting in the

65
pro-inflammatory leakage of the intracellular content. Necroptosis –
programmed form of necrosis (Death receptors, RIP1, RIP3 kinases)

57. Apoptosis - extrinsic pathway of apoptosis, death

receptors
- Controls the cell proliferation during development
- Controls the cell homeostasis of multicellular organism
- Elimination of aged, damaged or genetically aberrant cells

Steps

1. Identify the victim (the decision to activate the pathway)

2. Kill

66
 3. Get rid of the corpse (engulfment of the cell remain by immune cells -
phagocytes)
4. Destroy the evidence (degradation of the engulfed cell)

Programmed cell death is a genetically controlled process that requires

receptors. There are two types of signals - recognising the cell “find me” (low
levels of nucleotides ATP and UTP, fractalkine, lysophosphatidylcholine, or
sphingosine 1-phosphate) and “eat me” (phosphatidyl serines, anti-cancer
immunity)

Extrinsic pathway

- is receptor mediated
- death receptors are located on the
cell surface
- begins outside a cell, when
conditions in the extracellular environment
determine that a cell must die

Death receptors

- transmembrane proteins that are able

to trimerise and contain the "death
domain" which bind cytosolic signaling
proteins (TRADD, FADD/MORT-1)
- bind and activate FADD in the
cytoplasm (FAS-associated death domain
protein)
- form a death inducing signalling complex

Name Ligand
Fas FasL
TNFR-1 TNF-alpha
APO-3 APO3L
DR4/TRAIL-R1 APO2L/TRAIL
DR5/TRAIL-R2/KILLER APO2L/TRAIL

67
 58. Apoptosis - intrinsic pathway of apoptosis, the role of
mitochondria
Intrinsic pathway

- Initiated in response from features within the cell – DNA damage, cell
cycle defects, detachment from extracellular matrix, hypoxia
- Releasing of pro apoptotic factors from mitochondria - cytochrome c,
which binds to and causes the aggregation of the adaptor
proteinApaf-1(apoptotic protease-activating factor).
- Apaf- 1 binds and aggregates procaspase-9 molecules, which leads to the

cleavage of these molecules and the triggering of a caspase cascade.

The role of mitochondria in apoptosis

- opening channels in outer mitochondrial membrane, releasing of

the factors from intermembrane space
- releasing of proapoptotic proteins in cytoplasm Bcl-2 family
proteins, which form channels in mitochondria through which
cytochrome c and other factors are released
- releasing of apoptosis mediators

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 59. Regulation of apoptosis, errors of the regulation
Apoptosis regulators

Mitochondria

- Cytochrome c – activation of caspase-9 –formation of apoptosome

- Smac/DIABLO – inactivation of IAPs (inhibitors of apoptosis – caspase
inhibitors) lead to caspase activation
- endoG–mitochondrial nuclease, translocation into the nucleus – DNA
cleavage
- AIF – translocation into the nucleus initiation of chromatin condensation

Bcl-2 protein family

- have either pro-apoptotic or anti-apoptotic activity

- the presence of so-called BH domains (Bcl-2 homology), which mediate
interactions with their binding partners. BH1, BH2 and BH3 sequences
affect the ability to dimerise.
- interaction of pro/anti-apoptotic proteins takes place on the mitochondrial
membrane
- Proapoptotic factors are released from the intermembrane space, the
apoptosome is formed and caspase cascade is activated.

Anti-apoptotic proteins - Bcl -2, Bcl-XL, Mcl-1 (four BH domains) - integral

membrane proteins of mitochondria, endoplasmic reticulum, nucleus

69
Pro-apoptotic proteins - Bax, Bak (three BH domains) - cytosol, cytoskeleton

Examples

BCL-2

- Importance in the regulation of mitochondrial membrane permeability -

protect against cytochrome c and other mediators releasing from
mitochondria
- Plays a role in cancer transformation of the cells
- Doesn’t activate cell proliferation, but blocks cell death

Caspases

- Cysteine-dependent aspartate-specific proteases

- Cleave their substrate after Asp residues
- Initiator caspases - procaspases 2,8,9,10 - long prodomains, containing
death effector domains, cleave and activate executioner caspases
- Executioner caspases - caspase 3,6,7 - short prodomain, cleave cellular
structures
- Cleave key cellular structures and organelles

Regulation of apoptosis

- FLIPs („FADD-like ICE inhibitory proteins“) – have sequences similar

to procaspase-8, but do not have catalytic sites. Similarly works also
ARC („apoptosis repressor with caspase recruitment domain“; CARD)
- IAP („inhibitor-of-apoptosis protein“) family proteins: cIAP1, cIAP2,
XIAP, surviving bind procaspases and caspases and inhibit their activity.
- Diablo/Smac proteins bind IAP proteins and inhibit their activity.
- Smac mimetics - drugs inducing apoptosis by inhibition of inhibitors of
IAP family

The balance between proliferation and apoptosis is essential for homeostasis.

Imbalance leads to various diseases.

Insufficient apoptosis

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Leads to:

- Cancer
- Multidrug resistance
- Autoimmunity (rheumatoid arthritis)
- Persistent infections

Excessive apoptosis

Leads to:

- Neurodegeneration (Alzheimers’ disease, Parkinson’s disease,

Huntington’s disease, amyotrophic lateral sclerosis)
- Ischaemia (stroke, myocardial infarction) - necroptosis

60. Principle of tissue arrangement of cells (cytoskeleton and

extracellular matrix)
The attachment of cells is accomplished by adhesion molecules and cellular
junctions. Some tissues consist mostly of cells, which are closely bound
together; other tissues contain fewer cells which are sparsely dispersed in the
extracellular matrix - a complex network of protein and polysaccharide chains.

Extracellular matrix

- Epithelial tissue - limited ECM

- Connective - plentiful ECM

Functions:

- Determines spatial architecture of organs and their mechanical properties

- Represents the external structural support to the cells
- Segregates tissues from one another
- Regulates intercellular communication
- Essential for processes like growth, differentiation and development,
wound healing, etc.

Main molecules:

71
 - Collagen
- Elastine
- Proteoglycans - polysaccharide chains and
protein-polysaccharide complexes – space filling and embedding the
structural proteins
- Adhesive glycoproteins – attach cells to matrix (e.g. laminin, fibronectin)

Cytoskeleton

- Network of filaments and tubules that extends throughout a cell, through

the cytoplasm, which is all of the material within a cell except for the
nucleus
- Composed of actin filaments, intermediate filaments and microtubules

61. Connective tissues and extracellular matrix

Is the tissue that connects, separates and supports all other types of tissues in
the body. Like all tissue types, it consists of cells surrounded by a compartment
of fluid called the extracellular matrix (ECM). There are three types of
connective tissue: loose, dense and reticular.

The nature of ECM determines the type of connective tissues

- Tough and flexible (tendons, dermis of the skin)

- Hard and dense (bone, tooth)
- Resilient (elastic) and shock-absorbing (cartilage)
- Soft and transparent (vitreous body of the eye)

Collagen

- the major proteins of ECM (the most abundant class of proteins in the
human body – 25% of the total protein mass)
- tensile strength in
connective tissues
(resistance to breaking
under tension)
- rod shaped helix of three
polypeptides (α chains)
- self-associate to form
banded fibrils which are

72
 packed together into still thicker collagen fibers

Mutations in collagen genes lead to various diseases, for example Brittle bones
(osteogenesis imperfecta) or Ehlers-Danlos syndrome

Elastine

- one of the components of elastic fibres

- stretchable
- prominent in skin and walls of arteries

Diseases:

- Marfan syndrome–dominant mutation in a gene

coding for one component of elastic fibers -
fibrillin
- Artery wall is overly flexible causing aneurysm.
Other symptoms include bad eyesight, abnormal
chest, long fingers and extremities

73
 62. Epithelia and intercellular junctions
Overview of intercellular junctions

Tight junctions are especially important in epithelial cells. They have a fence
and a barrier function. Fence function - preventing intermixing of molecules
between the apical and the lateral membrane. Barrier function - regulating
diffusion of solutes through paracellular spaces (paracellular transport). Some
enteric bacteria can change the junction’s permeability.

Adherens junctions are protein complexes that occur at cell–cell junctions in

epithelial and endothelial tissues, usually more basal than tight junctions. The
Adherens junction performs multiple functions including initiation and
stabilization of cell-cell adhesion, regulation of the actin cytoskeleton,
intracellular signaling and transcriptional regulation.

Desmosomes are specialized and highly ordered membrane domains that

mediate cell-cell contact and strong adhesion. By mediating both cell–cell

74
adhesion and cytoskeletal linkages, desmosomes mechanically integrate cells
within tissues and thereby function to resist mechanical stress.

Gap junctions are important in cardiac muscle; for maintenance of ionic and
metabolic homeostasis in the inner ear; for metabolic homeostasis and
transparency of the lens; for maturation of the egg follicles. They allow direct
sharing of small molecules by adjacent cells – ions, metabolism products,
signaling molecules.

There are four major classes of cell adhesion receptors: Cadherins,

Ig-superfamily, Integrins, Selectins

Жирная жопа — я мама на массе

Моя motorola все связи на связи

63. Transient intercellular interactions

Transient interactions, which involve protein interactions that are formed and
broken easily, are important in many aspects of cellular function. Transient
complexes can be further subdivided into weak and strong. Weak transient
complexes show a dynamic mixture of different oligomeric states in vivo,

75
whereas strong transient complexes change their quaternary state only when
triggered by, for example, ligand binding. Weak transient interactions are
characterized by a dissociation constant (KD) in the micromolar range and
lifetimes of seconds. Strong transient interactions, stabilized by binding of an
effector molecule, may last longer and have a lower KD in the nanomolar range.
White blood cells have the ability of transient interactions. The interactions
between cells of the immune system and infected cells or antigen- presenting
cells is also an example of transient interactions.

64. Disorders of cell to cell interactions and cell to

extracellular matrix interactions
- Selectins are important initiators of leukocyte adhesion to the blood
vessel wall (endothelium). An absence of selectins or selectin ligands has
serious consequences, leading to recurrent bacterial infections and
persistent disease
- Epidermolysis bullosa - butterfly disease - skin blisters in response to
even very slight mechanical stress (epidermis - dermis junction). Can be
caused by mutations of collagen VII, but also keratins and other
proteins involved in the whole attachment complex – as a result,
attachment of epidermis to dermis is impaired.
- Duchenne and Becker muscular dystrophies. Dystrophin gene located in
X chromosome (boys are affected)

76
 ​ mutations in the gene for dystrophin; muscle cells cannot properly attach
to ECM causing progressive muscle degeneration and weakness
​ ‒ Duchenne muscular dystrophy – absence of dystrophin
​ ‒ Becker muscular dystrophy – changed structure of dystrophin
(milder)

Genetics
Clinical genetics is a medical field dealing with diagnosis, treatment and
comprehensive care of patients with hereditary diseases.
Gene - part of a DNA molecule carrying genetic information for the synthesis
of a specific protein (structural gene)
Genotype - a summary of all genetic characteristics of an individual.
Allele - a specific form of a gene, in a diallelic system only two situations can
occur in a given individual genotype AA or aa - homozygous, genotype Aa -
heterozygous
Phenotype - a set of all characteristics of an individual; in the narrower sense -
a specific form of a trait, determined by concrete genotype, eventually modified
by the external environment
A zygote is a cell with a complete set of chromosomes

65. Mendel’s laws. Monohybridism. Dihybridism.

Mendel performed experiments with pea (Pisum sativum)

The flowers have male (anthers forming pollen grains) and female organs
(ovary with eggs)
The crown petals are closed down and also prevent pollen from getting out or
in, thus ensuring self-fertilization, during which both male and female gametes
from the same flower unite to produce the seeds.
If we study only a single trait, it is a monohybrid cross or monohybridisme
P generation - parental generation, always two different homozygotes
F1 - generation - the first generation of offspring (first filial generation) is
created by crossing two individuals from the P generation to form heterozygotes
F2 - generation - the second generation of offspring (second filial generation) is
created by crossing two individuals from the F1 generation

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 Mendel’s laws

1. LAW - LAW OF UNIFORMITY OF THE F1 GENERATION

- when 2 homozygotes are crossed, their offsprings have identical
genotypes and phenotypes
- when 2 different homozygotes are crossed, their offspring will be
heterozygous hybrids
- in a heterozygote one allele that is dominant may conceal the second
allele which is recessive
2. LAW OF ALLELE SEGREGATION INTO GAMETES
- during gamete formation, two alleles separate and segregate
- the law of genotypic ratio in F2 generation (punnett’s square)
3. LAW OF INDEPENDENT ASSORTMENT OF ALLELES
- alleles of different genes combine with each other independently

If a trait is determined by one gene (locus), we speak of monohybridisme or

monohybrid crossing
If we observe two such phenotypic traits at the same time, it is dihybridisme

66. Interactions of non-allelic genes

Interactions between two genes (alleles of two different genes - non-allelic gene
interactions) which are involved in the phenotypic expression of one trait.
Epistasis is like a dominance in the situation of allelic interactions.
Types of interactions
DOMINANT EPISTASIS (12: 3: 1) - only if both alleles of the first locus are
recessive (aa--), alleles of the second locus can be expressed phenotypically.
RECESSIVE EPISTASIS (9: 3: 4) - only if at least one allele of the first locus
is dominant (A ---), alleles of the second locus can also be phenotypically
manifested (albinism).
COMPLEMENTARY GENES (9: 7) - when recessive homozygosity in each
of the loci leads to the same phenotype, so that individuals aaB-, A-bb and aabb
will be the same. If the dominant alleles of both loci are present at the same
time, their effects complement each other and result in a different phenotype.

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DUPLICATED GENES WITH CUMULATIVE EFFECT (9: 6: 1) - occurs
when each of the dominant genotypes (homozygote or heterozygote) is
responsible for the production of the same trait (eg formation of a certain
amount of pigment) - then in the example of aabb pigment they produce none,
A-bb and aaB- produce a unit amount of pigment and in individuals with
genotype AB- the effect of dominant alleles accumulates and the largest amount
of pigment is formed (two units).
DUPLICATED GENES WITHOUT CUMULATIVE EFFECT (15: 1) - a
condition in which each of the dominant genotypes lead to the manifestation of
the same trait without their effect accumulating in any way - only double
recessive homozygotes will differ phenotypically, which do not carry even one
of the four possible dominant alleles.

67. Genealogical method (principles and examples)

Family history is consulted in a genetic counseling center with a clinical

geneticist
Data is recorded graphically in the form of a genealogical scheme (pedigree)
and supplemented by a so-called legend.

● standardized symbols are used to compose the pedigrees

● it is possible to graphically capture one, at most a few traits, in order not
to violate the clarity of this method

=========================================================
Autosomes – 22 pairs of homologous chromosomes in human
- men and women have the same probability of showing the traits and
diseases determined by genes located on autosomes
Homologous chromosomes – chromosomes of the same chromosome pair in a
diploid cell
Due to the random segregation of homologous chromosomes into gametes, the
total composition of the 23 chromosomes in a gamete is random.

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 68. Autosomal recessive inheritance (principles, examples)
- the pathological, mutated allele is recessive
- the trait or disease appears only in patients, who have both alleles of the
respective gene mutated (recessive)

Principles
- the gene change usually leads to production of protein (or RNA)
with no function
50% protein in the body is functional
– the function is retained in heterozygotes
a healthy couple (Aa x Aa) can have an affected child (aa)

- a recessive trait or disease does not necessarily occur in each generation -

might appear in multiple siblings, whose parents are healthy
- appear with a higher probability in children from a consanguineous
mating (mating between relatives)

Risk of disease
- probability 1/4 for siblings of a sick individual
- the probability of a heterozygous condition for healthy siblings of a sick
individual is ⅔
- men and women - the same probability of disease
AR inherited diseases occur with a lower frequency than autosomal dominant
diseases. Most AR diseases arise on the basis of a relationship between two
random carriers, in particular if the frequency of the recessive trait is high in the
population, such as cystic fibrosis, phenylketonuria, sickle-cell disease,
albinism, etc.

Cystic fibrosis
Incidence 1: 2500
Mutations in the CFTR gene - regulation of the function of ion channels of the
epithelial cell membrane

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transmembrane transfer of chloride ions (Cl-) increased concentration of Na +
and Cl- in sweat
Most affected: lungs - recurrent lung infections, pancreas - lack of pancreatic
enzymes Death usually around 30 years of age

Phenylketonuria
Incidence 1: 10,000
metabolic disease caused by a mutation in the gene for the liver enzyme
phenylalanine hydroxylase (PAH)
inability to digest the amino acid phenylalanine (convert phenylalanine to
tyrosine) - accumulation of phenylalanine in the body - convulsions, liver
failure, brain damage
strict low-phenylalanine diet required

69. Autosomal dominant inheritance (principles, examples)

- the pathological, mutated allele is dominant

presence of a single mutated allele (dominant) is sufficient to manifestation of

the disease

A sick individual has a one of the parents diseased (with exception of rare de
novo mutation)

One mutated allele → disease

Risk of disease for each offspring of a sick parent → 50% Men and women - the
same risk of disease

• two affected partners (Aa x Aa) can have a healthy child (aa)
• affected individuals frequently have newly emerged mutations (de novo
mutations), less frequently they are inherited – then the patent has at least one of
the parents affected, too
• in a dominant homozygous state they usually lead to aberrant development and
to spontaneous abortions of such foetuses, or death of the child at a very young
age (Huntington disease, for example, is an exception)

Principles

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 I. In a heterozygote one of two alleles is with increased expression, it
produces a permanently dimerized and active receptor, which then can
cause for examples permanent cell division
II. In a heterozygote one of two alleles is mutated, creating a permanently
active receptor or signal transducer causing permanent activation of gene
transcription, permanent cell division
III.In a heterozygote there is one allele that remains unchanged creating a
functional protein, while the second mutated one creates a non-functional
protein damaging the cell
IV. In a heterozygote one of the alleles is mutated and creates a
non-functional protein, the other allele creates a functional unit which is
not sufficient to maintain the function needed (haploinsufficiency)
V. In a heterozygote two alleles, one of which is mutated, create a functional
and non-functional proteins. The non-functional protein blocks the
second one.

Examples
POLYCYSTIC KIDNEY DISEASE
Incidence: 1/600 -1/1000
Polycystic kidney disease is the most common AD disease. It leads to cysts in
the kidneys, liver, pancreas and spleen. Cysts in the kidney are usually
asymptomatic until renal failure or the onset of hypertension (usually in the
fourth decade of life).
Mutations in the PKD1 or PKD2 gene (encodes the polycystin protein)
By adulthood mild or no symptoms
1/3 of patients - cysts in the liver
10% of patients have dilated veins in the brain → stroke

HUNTINGTON'S DISEASE
Incidence is 1 / 10,000–20,000 individuals.
Huntington's chorea is a neurodegenerative disease.
It is characterized by ineffective (involuntary) movements and progressive
dementia.
The onset of the disease is variable.
A mutation in the gene that encodes the protein huntingtin manifests as an
expansion of triplet nucleotide 5′-CAG-3 ′ at the 5 'end of the first exon of the
gene

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10 - 34 repeats 5'-CAG-3 ‘(in healthy individuals)
Patients with severe disabilities 42 - 100 5'-CAG-3 'repeats
If the symptoms appear in childhood, more than 60 repeats are usually present
in the gene.

ACHONDROPLASIA
Point mutation (nucleotide change) in the FGFR 3 (fibroblast growth factor
receptor) gene
Short limbs → during embryonic development and in childhood the bones of
the limbs grow more slowly; body size is average
Difficulty in children: speech, hearing, breathing

POLYDACTYLY

● non-functional transcription factor Gli3 regulating the production of

proteins involved in determining the anteroposterior polarity of
developing limbs

=========================================================
====
Gonosomal inheritance

● inheritance of traits determined by genes localized on sex chromosomes

● Gonosomes (heterochromosomes, sex chromosomes) - determine the
gender (among other characteristics) – X and Y in men, XX pair in
women
● Homologous chromosomes – chromosomes of one chromosomal pair in
diploid cells (one inherited from the father and one from the mother) -
carry the same genes (for the same traits), each carrying one allele
● Hemizygote – an individual with only one allele of a given gene (men for
X- and Y-linked genes)

70. Gonosomal recessive inheritance (principles, examples)

PRINCIPLES

● There is no transfer from father to son

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 ● Men are affected more often than women (one X chromosome)
● Diseased son - a mutated allele from the mother
● Sons of heterozygous mothers - 50% risk of gaining a mutated allele
● Daughters of a heterozygous mother - carriers - risk 50%
● Daughters of disabled men - 100% carrier
● X - linked - mostly men are affected, women can be affected only if
recessive homozygous; heterozygous women are carriers to their sons;
two healthy partners can have an affected child; manifesting with an
increased probability in children of consanguineous marriage

EXAMPLES

HEMOPHILIA
Hemophilia A 1/10000 affected men - mutations in the gene for the production
of coagulation factor VIII
Hemophilia B - mutation of the coagulation factor IX gene

DUCHENNE'S MUSCULAR DYSTROPHY (DMS)

The incidence of DMS is 1/3000 of boys born.
DMS is caused by a mutation in the gene encoding the dystrophin protein,
which affects the function of all muscle types.
The onset of the disease is in childhood.
The first symptom is swaying when walking, difficulty walking up the stairs,
progressive weakness. Around 10 years, the affected individuals become
immobile.
Muscle weakness gradually continues, death occurs around the age of 20 due to
heart and respiratory failure.
Although boys die before they reach reproductive age, the recessive allele in
the population is maintained in the genome of healthy heterozygous
women.
One third of the affected boys developed a de novo mutation in the dystrophin
gene. The gene encoding dystrophin is large and has a higher frequency of
spontaneous mutations.

DALTONISM
One of the congenital causes of color blindness. Affected people lack or have
limited ability to distinguish between red and green.

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 71. Gonosomal dominant inheritance

PRINCIPLES

● X linked
● two affected partners (XAXa and XAY) can have a healthy son (XaY)
● the mutation associated with the disorder can occur newly (de novo
mutation) or it is inherited – then at least one of the patient’s parents is
also affected
● disorders usually have more severe progression in men
● in some of these disorders, the absence of the healthy allele (and,
therefore, the absence of the functional protein) can lead to
developmental impairment and to abortion of such a foetus
● the transmission of the mutation is not from father to son, affected men
pass on the mutated allele to all daughters
● female heterozygous → offspring of mutated allele with 50% probability
● women are more likely to have GD disease

EXAMPLES

Hypophosphatemic rickets
(vitamin D-resistant rickets, phosphate diabetes) – failure of phosphate
reabsorption in kidney tubules → excessive loss of phosphate in urine and its
lack in the body (osteoporosis, pain of bones, spine and joints – most of the
clinical symptoms vary with the patient’s age and the degree of phosphate loss)

Tooth enamel dysplasia

(amelogenesis imperfecta) – several forms, the hereditary form is caused by
defect of dental enamel formation (the enamel is not thick enough, the teeth
appear smaller)

Incontinentia pigmenti
(Bloch-Sulzberger syndrome) – abnormalities in development of the skin, hair,
nails, teeth, eyes, and the central nervous system, occurs only in women-

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heterozygotes (in male embryos and women-dominant homozygote embryos
usually leads to spontaneous abortions)

72. Dominance and codominance, penetrance and expressivity

PENETRANCE OF THE GIVEN FORM

if individuals with the same genotype show that the phenotype is manifested
only in some of them, we are talking about incomplete penetration
- the relative number of those individuals in whom the phenotypic trait is
present; for example, in an autosomal dominant hereditary polydactyly, only 80
are found in humans between 100 Aa individuals with this disease
- the penetrance of the responsible allele is 80% - sometimes penetrance can be
dependent on the age of the individual - late onset disease e.g. clinically
manifested in adult age (Huntington's chorea)

● accumulation of toxic metabolites or reduced ability of the organism to

compensate for damage

EXPRESSIVITY OF THE GIVEN FORM

hereditary traits manifest themselves unequally in individuals of a given
genotype, we speak of different expressivity
- expressivity is an extend of the expression of a given genotype in the
phenotype of an individual
- depends on many factors (other genes (modifying genes), environmental
factors, etc.)

● for allele carriers e.g. for polydactyly there are various morphological
deviations (from skin algae to fully functional extra fingers)

COMPLETE DOMINANCE

● the phenotype of heterozygote Aa is the same as the phenotype of

dominant homozygote AA
● the phenotypic ratio in F2 in this case is 3⁄4 of dominant individuals (AA
/ Aa) and 1⁄4 of recessive individuals (aa)
● dominance and recessivity is a designation of the relationship between
two alleles, but also a description of the dominant or recessive trait

INCOMPLETE DOMINANCE

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The heterozygote phenotype is somewhere between the phenotypes of both
parents
Heterozygote differs phenotypically from both homozygotes, it is a transitional
form of both extreme alternatives.

CODOMINANCE
in situations where the heterozygote phenotype has the characteristics of both
parents in them, both alleles encode functional forms of the protein, which are
the essence of a certain trait (eg the AB blood group system).

73. Chromosomal disorders (overview)

Chromosomal disorders fall into two general categories: those involving an

incorrect chromosome number, called aneuploidy, and those that result from
large chromosomal mutations, as described earlier. Aneuploidy is the result of
nondisjunction during meiosis, in which both members of a homologous pair
of chromosomes move to the same daughter cell. As a result of nondisjunction,
the fertilized egg receives either one or three copies of the chromosome instead
of the usual two. Because they involve numerous genes, with disturbance in the
normal genomic balance, most disorders affecting chromosome number are
embryonic lethal, particularly if the defect is loss of a chromosome. Disorders
that are not lethal usually result in sterility, because they prevent meiosis from
proceeding normally. The best-known and most common chromosomal
disorder is Down syndrome, which generally results from trisomy of
chromosome 21 but also can be due to a duplication or translocation of a
specific region of chromosome 21. Trisomies of chromosome 13 or 18 also
occur but are much less common in live born infants than is Down syndrome.
Turner syndrome occurs in women who receive only a single X
chromosome, whereas Klinefelter syndrome occurs in men who receive two
X chromosomes in addition to the Y chromosome.

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 74. Chromosomal aneuploidy (numerical chromosomal
aberrations)
- arise mainly as a consequence of errors in nuclear division
- changes in the number of chromosomes in a cell
- numerical aberrations usually lead to serious problems already during
embryonic development - abortions or developmental defects

Organisms that contain more than two sets of chromosomes - POLYPLOIDY

(triploid, tetrapoloid, hexaploid, octaploid, etc. cells). In animals, this condition
is incompatible with life. The change in the number of chromosomes is called
ANEUPLOIDIA. The whole set does not multiply, but a specific chromosome
is extra or is missing. The incorrect gene dosage leads to an incorrect dose of a
specific protein or RNA causing a failure in regulatory processes.

CAUSES
Unequal distribution of chromosomes (non-disjunction)
• during meiosis - numerical aberrations in gametes - by dividing the zygote it
spreads to the whole resulting individual
• during mitosis - numerical aberrations in somatic cells
- by division it spreads to a part of the cells of the organism
- contributes to the development of cancer or leads to milder forms of
developmental syndromes (less severe symptoms than if aneuploidy is present
in all somatic cells)
Only some cells in the body will be aneuploid (chromosomal mosaicism).

EXAMPLES

Human monosomy
The absence of a chromosome with the all respective genes is usually critical -
the human embryos fail to develop successfully, will die
The only exception is the lack of one copy of the X chromosome in women
(Turner syndrome) - 1 in 5000 females are born with it, ovaries do not develop
properly → sterility, without hormonal therapy with estrogen there is no

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development of secondary sexual characteristics, reduced sensitivity to growth
hormone somatotropin
X chromosome inactivation
- extra copy of the X chromosome is inactivated in women - one of the X
chromosomes condenses and its genes are not transcribed→ Barr's body
- inactivation occurs during embryogenesis
Trisomy in humans
- chromosome redundancy with all contained genes is usually critically
harmful
- overdose of some proteins leads to deregulation of regulatory processes in
the human body
- the human embryo usually fail to develop successfully and die

Exceptions

Down syndrome
TRISOMY 21

- 1 in 700 to 800 live births

- heart development disorders, slower growth, slower mental development,
slower motor development, weaker muscle tone, need for physiotherapy,
increased risk of leukemia, average life expectancy with modern
treatment is 60 years

Aneuploidy may occur during meiosis (nondisjunction - incorrect separation of

chromosomes or chromatids to cell poles) or during mitotic division.

Edwards syndrome
TRISOMY 18

- 1 in 6,000 to 7,000 live births (over 50% of pregnancies end in

miscarriage)
- severe developmental disorders
- congenital malformations of the heart, lungs, digestive and urogenital
tract - frequent clefts of the lip or palate
- average life expectancy in the order of months (only 5% of children live
to be 1 year old)

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 Patau syndrome
TRISOMY 13

- 1 in 14,000 or more live births

- very severe developmental disorders
- congenital malformations of the heart, central nervous system, lungs,
urogenital tract, polycystic kidneys, frequent facial clefts and
polydactyly; deafness, damage to eyesight
- average life expectancy in the order of weeks (only 5-10% of children
live to be 1 year old)

Klinefelter syndrome
TRISOMY OF GONOSOMES - XXY

- 1 in 500 to 1000 people

- usually male, but sometimes slightly feminized phenotype or intersex
without testosterone replacement therapy
- sometimes there is no proper testicular development and testosterone
production → fertility disorders (for some patients the possibility of
having a child due to surgical collection of sperm)

Trisomy X
TRISOMY OF CHROMOSOME X

- - 1 in 1,000 women (estimate)

- often taller
- usually slightly delayed development and development of speech, but
some patients don’t experience problems
- sometimes weaker muscle tone or scoliosis - the need for physiotherapy
- many women with karyotype XXX do not know about their X
chromosome at all

XYY syndrome ("supermale")

TRISOMY OF GONOSOMES – XYY

- 1 in 1,000 men (estimate)

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 - high stature
- standard testosterone production, normal fertility, healthy offspring →
most men with karyotype XYY do not know about their Y chromosome
at all

75. Structural aberrations of chromosomes

- changes in chromosome structure

- the DNA sequence of the affected chromosomes changes:
part of the chromosome may be deleted or amplified
• the number of copies of genes located in a given (missing or redundant)
region changes
• incorrect gene doses of these genes and their products
• changes in large regions of DNA containing many genes usually lead to
miscarriages or developmental defects
• chromosome rearrangements can also lead to the development of
cancer
or part of a chromosome is translocated (to another chromosome)

● near a strong promoter can lead to altered gene expression or the

joining of two genes and the formation of a fusion product with an
undesired function
● may occur during sperm and oocyte development, during early
fetal development, or during later life in any cell
● the effect depends on the size and location of the structural
reconstruction

Causes
1. incorrect chromosome pairing / crossing-over during meiosis and subsequent
gamete formation with incorrect structure of some chromosomes
- deletion, duplication (in case of incorrect pairing of homologous
chromosomes)
- translocation (in case of incorrect pairing of non- homologous
chromosomes)
- whole embryo is impacted
2. DNA breaks and their incorrect repair
- deletions, duplications, translocations, inversions, ring chromosomes …

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 - impact depending on whether the break occurs in gametes or in
somatic cells
Deletions
- part of the chromosome is missing
- the more genes are missing, and the more important are the genes, the
more severe the consequences
- usually disorders of development or cell division
Examples:
1. DiGeorge syndrome
a small part of chromosome 22 is deleted
2. Cat’s Cry syndrome
part of chromosome 5 is deleted

Balanced translocation

exchange of the parts between two different chromosomes genes are relocated,
which can cause:

- change in expression (eg when they come close to a strong promoter)

- the fusion of two genes and formation of a fusion product with an
undesired function problems with chromosome pairing during gamete
formation (meiosis)

76. Multifactorial inheritance (multifactorial trait

determination, heritability, threshold effect model)

Multifactorial inheritance

- For disorders that show non- Mendelian familial aggregation

- Determined by genes+environment
- Quantitative traits- determined by 2 or more genes - skin colour, height,
intellect
- Is evaluated statistically, graphically can be expressed by a normal
frequency distribution
- Inheritance patterns are often more complex than predicted by simple
Mendelian genetics

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 Inheritance of complex (polygenic) disorders

- Although these disorders tend to cluster in individual families, they do

not have a clear-cut pattern of inheritance.
- It is difficult to determine a person’s risk of inheriting or passing on these
disorders.
- Strong influence of environmental factors and lifestyle.

Threshold traits
In the case of some traits (diseases, cleft lip) the genetic contribution involves
many loci, but the phenotype does not have a continuous distribution (it is either
present or absent). The risk factors contribute to a variable called the liability –
includes all factors that contribute to the cause of the condition.
Threshold trait - a trait that appears only when liability exceeds a certain level
(threshold).
Heritability

- summarises the relative contributions of genetic and environmental

factors to the observed variability in a population.

Heritability ( H2 ) is the proportion of a trait that is due to genetic variation

divided by the total variation in the trait in a given population. Total variation =
genetic+environmental.
H2 = Vg:Vp (G - genetic; P - environmental)
H2 = 0 – a completely environmentally dependent phenotype
H2 = 1 – a completely gene dependent phenotype

77. Twin method, concordance

The study of twins (twin method) is essential for determining the role of genes
in phenotype in multifactorial traits.
Concordance rate - the fraction of twin pairs in which both twins show the trait
among pairs in which at least one of them does.
The much greater concordance rate for monozygotic twins strongly suggests
that genetic factors influence the trait.
Examples

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Obesity and insulin resistance
60-90% variability in BMI (body mass index)
BMI – at least 32 known locuses
+ inveronmental factors– imbalance between energy intake and expenditure,
lack of physical activity
Type I Diabetes
The concordance rates in MZ and DZ twins are approximately 50% and 12%,
respectively.
Known environmental factors include diet, viral exposure in early childhood,
and certain drugs. The disease process involves irreversible destruction of
insulin producing islet β cells in the pancreas by the body’s own immune
system.
Heart disease
Many factors predispose an individual to develop heart disease: body weight,
amount of exercise, diet, blood cholesterol level, whether or not the individual
smokes, and the presence of heart disease in close relatives such as parents or
siblings.
78. Gene linkage, gene mapping, LOD score

Two loci positioned adjacent, or close to each other on the same chromosome,
will tend to be inherited together and are linked. When genes are close together
on the same chromosome, they are said to be linked. Genes on different
(nonhomologous) chromosomes are not linked. They assort independently
during meiosis and have a 50 % chance of ending up in different gametes.
Frequencies of crossing-over can be used to construct a linkage map.
Linked genes (linkage group) - a set of genes on one chromosome = number of
linkage groups corresponds to a number of chromosome in a haploid nucleus
and to a number of homologous chromosome pairs in a diploid nucleus. Genes
located on a single chromosome during meiosis segregate together and cannot
undergo independent assortment – known as haplotype = haploid genotype
(deviation from 3rd Mendel ́s law - Law of Independent Assortment)

- Complete linkage - no crossing-over during meiosis

- Incomplete linkage - there is crossing-over during meiosis, non-sister
chromatids exchange and recombination occurs

Gene mapping

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Assessment of distance between two genes can serve for construction of gene
maps which illustrate the order of individual genes and their relative distance on
the chromosome.
Morgan ́s number expresses the probability of recombinant gametes; unit -
centimorgan - - a measure of the genetic, or linkage, distance between two loci
The recombination fraction (θ; theta) - number of recombinants/number of all
individuals - indicates how often two genes located on one chromosome will be
separated (recombine) at meiosis.
LOD score
The logarithm of the odds (LOD) score is a mathematical indication of the
probability that two loci are linked. A LOD score of +3 or greater is taken as
confirmation of linkage. It distinguishes whether the observed deviation from
dihybridisme is statistically significant. The logarithm to the base 10 of this
ratio is known as the LOD score (Z)—that is, LOD (θ) = log10 [Lθ/L(0.5)].
Logarithms are used because they allow results from different families to be
added together.
79. Genome-wide association studies (GWAS), SNPs,
examples of diseases

Association studies are undertaken by comparing the frequency of a particular

variant in affected patients with its frequency in a control group, a case control
study - identifying genes that cause multifactorial diseases. The allele encoded
by the marker is either directly involved in causing the disease or the marker is
in linkage with a closely linked susceptibility variant.
Single nucleotide polymorphisms (SNP)
• The most frequent type of a genetic variation in the human population
• Occur with a very high frequency (with estimates ranging from about 1 in
1000 bases to 1 in 100 bases) – about 10 million single nucleotide
polymorphisms are present in the entire human genome.
• Found in both coding and non-coding regions of the human genome.
Diseases
Alzheimer disease – mutations in four genes, located on chromosomes 1, 14,
19, and 21.
PS1 (presenilin or AD3) on chromosome 14 and PS2 (or AD4) on chromosome
1.
Lesions formed of fragmented brain cells surrounded by amyloid-family
proteins – similar structures found in Down syndrome.

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Chromosome 19 – gene APOE (apolipoprotein E, ε4) – risk of Alzheimer
disease up to 80% (normal population 10%, at 85 years of age)

Parkinson disease a candidate gene on chromosome 4

α-synuclein – fragments of this protein are found in patients of both Alzheimer
and Parkinson disease (constituents of Alzheimer's disease plaques)
80. Population genetics, allele frequency theory
(Hardy-Weinberg equilibrium)
According to the Hardy-Weinberg principle, the relative proportions of the
possible genotypes at a particular locus remain constant from one generation to
the next. Factors that may disturb Hardy-Weinberg equilibrium are non-random
mating, mutation, selection for or against a particular genotype, small
population size, and migration.
Population genetics – key terms:

- Studies the genetic structure of populations, the frequencies of alleles,

genotypes and phenotypes and factors that influence these frequencies
- Population - collection of organisms of a particular species living in a
given geographic area, who can mate and have fertile offspring
- Genetic structure of population is determined by specific allelic and
genotypic frequencies; allelic frequency - relative frequency of a
particular allele in a population; genotypic frequency - relative
frequency of particular genotype in a population
- Gene pool - collection of all alleles of all genes in the population
- Over generations, the genetic structure of population can exhibit stability
- no changes in allelic and genotypic frequencies
- Non-evolutionary changes - changes in genotypic frequencies, allelic
frequencies remain unchanged
- Evolutionary changes - changes in both genotypic and allelic
frequencies

Hardy-Weinberg law:
p – frequency of dominant allele (A)
q – frequency of recesive allele (a)
p2 - frequency of dominant homozygotes (AA)
q2 - frequency of recessive homozygotes (aa)
2pq- frequency of heterozygotes (Aa)

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p+q=1
p2+2pq+q2=1

for autosomal genes in human populations:

If the population is in genetic equilibrium, the genetic structure of a population
(allelic and genotypic frequencies) will remain constant from generation to
generation.
for X-linked genes in human populations:
Upon fulfilling the conditions, the genetic equilibrium is set after several
generations only.
conditions of genetic equilibrium:

1) Population is big enough

2) Population is panmictic
3) There is no selection present
4) There are no de novo mutations
5) There is no migration
81. Factors affecting a population gene pool (inbred,
assortative pairing, selection, drift, migration)
Inbreeding (breeding between close relatives)
- human population - relational marriages
- lowers genetic heterogeneity of the population - population homogenization
Consanguinity - childbearing between blood relatives who have at least one
common ancestor no more remote than a great-great-grandparent.
Assortative mating - non-random mating –– the tendency of humans to choose
partners who share characteristics such as height, intelligence. Limits of the
partner selection due to cultural habits enables occurrence and maintenance of
rare hereditary disorders specific for a certain cultures.
Selection
Decline in frequency of some alleles caused by negative selection. Sickle-cell
disease -affected recessive homozygotes have severe anemia. Heterozygotes are
relatively resistant to infection with Plasmodium falciparum (malaria) because
their red blood cells undergo sickling and are rapidly destroyed when
invaded by the parasite. For some AR disorders there is evidence that
heterozygotes show a slight increase in biological fitness compared with
unaffected homozygotes—referred to as heterozygote advantage.
Heterozygote advantage - fixing of some alleles by a selection pressure -

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malaria (Plasmodium) is not manifested so severely in heterozygotes for
sickle-cell disease and thus heterozygotes are preferred by a selection pressure
to survive and have offspring in areas with presence of this parasites.
Drift
One allele may be lost altogether, and the other ‘fixed’
Migration
If new alleles are introduced into a population through migration and
intermarriage - change in the relevant allele frequencies. Gene flow - slow
diffusion of alleles across racial or geographical boundaries
82. The human genome project, information content of the
human genome
In 1988-1990 was founded the Human Genome Project that included 20
different laboratories from USA, Britain, Germany, France, Japan and China.
Objectives:
- genetic map of the human genome
- physical map: marker every 100 kbp
- sequencing of model organisms (E. coli, S. cerevisiae, C. elegans,
Drosophila, mouse)
- discover all human genes (assumed 60-80 thousand)
- sequencing of the whole human genome (3000 Mbp) by 2005 with a
budget of 3 bil. USD
In may 1998 HGP sequenced about 4% of the human genome. Craig Venter
founded CELERA GENOMICS, Inc. Then Celera Genomics & acad.
collaborators publish a draft genome Drosophila melanogaster (approx. 2/3 of
180 Mbp).
Information content of the human genome
- 127 books
- 1000 pages per book
- about 25000 characters per page
- From largest to smallest (Genome - Exome - Interindividual variability -
500-gene panel - One gene)
- is approximately 3,200 Mb in size
- is divided into 22 pairs of autosomes and one pair of sex chromosomes
- contains approximately 20,000 genes
- identification is still ongoing and the number of genes is being refined

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 83. Architecture of the human genome (coding and
non-coding genome, repetitive sequences)
Only about 1 percent of DNA is made up of protein-coding genes; the other 99
percent is noncoding. Noncoding DNA does not provide instructions for making
proteins, but at least some of it is integral to the function of cells, particularly
the control of gene activity. For example, noncoding DNA contains sequences
that act as regulatory elements, determining when and where genes are turned
on and off.

Other regions of noncoding DNA provide instructions for the formation of

certain kinds of RNA molecules. Examples of specialized RNA molecules
produced from noncoding DNA include transfer RNAs (tRNAs) and ribosomal
RNAs (rRNAs), which help assemble protein building blocks (amino acids) into
a chain that forms a protein; microRNAs (miRNAs), which are short lengths of
RNA that block the process of protein production; and long noncoding RNAs
(lncRNAs), which are longer lengths of RNA that have diverse roles in
regulating gene activity.
Repetitive sequences
Most of the human genome consists of repetitive sequences in the form of:

- tandem repeats - are divided into satellites, minisatellites and

microsatellites
- dispersed repeats (mobile elements)

84. Tandem repeats (classification, significance)

Tandem repeats - are divided into satellites, minisatellites and microsatellites

Satellite DNA - consists of sections hundreds of kilobases to several megabases

long. Due to the different content of bases compared to other DNAs, satellite
DNA forms an additional band, so-called satellite, after density gradient
centrifugation (α satellite).

Minisatellite DNA
- forms blocks of hundreds of bases to hundreds of kilobases in length
- in total, there are over 1000 blocks of minisatellite DNA in the genome

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 - these sequences are highly polymorphic (recombination hotspots), which
is used in genetic mapping
- minisatellites are the basis of the "DNA fingerprinting" method, which
enables the identification of individuals, eg in forensic medicine
- VNTR (variable number tandem repeat), a type of minisatellite used
historically in DNA fingerprinting

Microsatellite DNA
- sometimes also short tandem repeats short tandem repeats, STR
- short sections of simple sequences scattered across the genome
- some microsatellites, such as CA, are very common and may even form
unusual DNA conformations
- although mostly located outside genes, some microsatellites lie in genes
where their expansion can lead to a number of diseases such as
Huntington's disease (CAG expansion), fragile X chromosome (CGG
expansion), and many more
- microsatellite instability is an accompanying phenomenon of Lynch
syndrome hereditary form of colorectal cancer
85. Dispersed repeats: mobile genetic elements
These are genetic entities of hundreds of bases to tens of kilobases long that
jump across the genome from place to place.These are either so-called
retroelements, spreading through the RNA intermediate (copy and paste
mechanism) or so-called DNA transposons, which are excised from one place
and inserted elsewhere (cut and paste mechanism). Mobile elements make up
approximately 50% of the genome and are a key factor in its dynamics, their
share in our genome is up to 70%.
Transposons were found in all genome-sequenced organisms. Historically, two
views on the function of transposons
• Non-functional DNA, parasitic sections of DNA („selfish genes“) that
replicate whether or not it is useful for the "host" organism
• repeats have a hitherto unknown function, e.g. in chromatin structure or other
regulatory functions, and represent key players in evolution, without which
genomes could not function as they do.
Human dispersed repeats are divided into LINE, SINE, retrovirus-like elements,
and DNA transposons.
Representative of SINE elements are Alu elements (primates only), which with
their more than 1 million copies are the most abundant human sequence,

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occurring on average every 3 kb. They do not encode any proteins and can be
immobilized by adjacent LINE sequences.
The main LINE element is LINE1 (L1). There are > half a million copies in the
human genome found in euchromatin. The basic element is 6.1 kb long and
contains two genes, one of which encodes a reverse transcriptase, integrating
into AT-rich regions.
Endogenous retroviruses are sequences similar to retroviruses, they are not
infectious because they are limited to the genome of their host. They encode
reverse transcriptase and contain long terminal repeats.
86. Variability of the genome (polymorphisms and gene
variants)
Gene variants (mutations) are changes in genotype leading to the emergence of
new gene forms, so-called alleles, which ultimately may have no or minimal
effect on the phenotype, or, conversely, can seriously disrupt cell function.
Variants occur spontaneously at a low frequency or can be induced by various
factors - mutagens. According to the general consensus, in the case of the
occurrence of more than two alternative allelic variants of one gene in a
population with a frequency of ≥ 1%, it is a so-called genetic polymorphism.
Polymorphisms define interindividual variability and polygenic inheritance.
Types of genomic variants
There are germline and somatic mutations. Germline mutations are inheritable,
occur in the egg or sperm, always occur in tumor DNA as well. Somatic
mutations occur in non-germline tissues and cannot be inherited.
Types of genomic variants according to the nature of DNA change

● Base substitution (change of one base in DNA)

● Insertions and deletions
● Copy number alterations (amplifications or homozygous deletions
pf a single gene or chromosomal region)
● Rearrangements (gene fusions) - chromosome breaking resulting in
juxtaposition of 2 genes

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 87. Epigenetics (genetic and epigenetic code)

Epigenetics is the study of how your behaviors and environment can cause
changes that affect the way your genes work. Unlike genetic changes, epigenetic
changes are reversible and do not change your DNA sequence, but they can
change how your body reads a DNA sequence.

The epigenetic code is hypothesised to be a defining code in every eukaryotic

cell consisting of the specific epigenetic modification in each cell. It consists of
histone modifications defined by the histone code and additional epigenetic
modifications such as DNA methylation.
88. DNA methylation

DNA methylation is a chemical modification by a methyl group on cytosines at

the CpG sequences of DNA. When a region that regulates gene expression (a
promoter region) is methylated, the gene cannot be expressed.

DNA methylation is essential for development in higher organisms such as

humans. In addition to specification of cellular types, DNA methylation is
deeply involved in other biological phenomena, such as differential use of genes
in genomic imprinting and inactivation of one of the two X chromosomes in
females (X chromosome inactivation). Furthermore, aberrant patterns of DNA
methylation are observed in human diseases such as cancer.

When cells divide, nuclear DNA is replicated. Although the newly synthesized
DNA strands are unmethylated, DNA methyltransferase 1 (DNMT1)
precisely maintains the DNA methylation status.

89. Histone modifications

A histone modification includes methylation, phosphorylation, acetylation and
others. The PTMs (post-translational modification) made to histones can impact
gene expression by altering chromatin structure or recruiting histone modifiers.

There are 8 histones in each of the nucleosomes and all of them can be modified
in several positions (in different amino acids) and in different ways (by various
enzymes).

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At least nine different types of histone modifications have been discovered.
Acetylation, methylation, phosphorylation, and ubiquitylation are the most
well-understood, while GlcNAcylation, citrullination, crotonylation, and
isomerization are more recent discoveries that have yet to be thoroughly
investigated.
90. Genomic imprinting, uniparental disomy

Imprinting is considered a specific type of methylation.

For most genes, we inherit two working copies - one from mom and one from
dad. But with imprinted genes, we inherit only one working copy. Depending
on the gene, either the copy from mom or the copy from dad is epigenetically
silenced.
The epigenetic tags on imprinted genes usually stay put for the life of the
organism. They are reset during egg and sperm formation, but certain
genes are always silenced in the egg, and others are always silenced in the
sperm.
Paternal and maternal gametes imprint distinct regions from one another, but
all paternal gametes of all human males imprint the same regions (paternal
imprinting) and all maternal gametes of all human females imprint the same
regions (maternal imprinting). When two gametes combine to form a diploid
(2n) zygote, the imprinting from maternal and paternal chromosomes
remains the same.
Every somatic cell of the offspring keeps the same imprinting as the original
zygote. This means that genes that are paternally imprinted are only expressed
on the maternal chromosome and vice versa.
The classic example of disorders involving imprinting are the Prader-Willi
syndrome (PWS) and the Angelman syndrome (AS), two syndromes that
present very differently but have similar cytogenetics.
➔ Prader–Willi syndrome (PWS)
PWS is a genetic disorder caused by a loss of function of specific genes on
chromosome 15. If a paternal chromosome is paired with maternally
imprinted chromosome 15, it results in a formation of a zygote carrying PWS.
➔ Angelman syndrome (AS)
AS is also considered a genetic disorder caused by a loss of function of specific genes
on chromosome 15. When a maternal chromosome 15 is paired with a
paternally imprinted chromosome 15, the AS is observed.

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About 70% of PWS and AS patients have the same 3-4 million base pair
deletion in the long arm of chromosome 15 (most often through chance
mutation).
Beckwith-Wiedemann Syndrome
A syndrome that is usually caused by the activation of the lfg2 gene, which is
normally maternally imprinted.
• occurs approximately in 1/15000 births
• 85% of the patients have no family history linked to that disease
Uniparental disomy (UPD)
UPD happens in a situation in which a child inherits both copies of a
chromosome from one parent and none from the other. Uniparental disomy
usually arises due to an error in meiosis. Two chromosomes in either the egg
or sperm cell fail to separate and both get passed to the fetus. If a child inherits
both of the imprinted chromosomes, PWS and AS can appear.
91. Non-coding RNAs and X-chromosome inactivation
- Non-coding RNA is a molecule that is not translated into a protein
(usually microRNA and miRNA)
- Inactivation of chromosome X (lyomization) - a condition when one of
the female X chromosomes is non-active.
- XIST (X-inactive specific transcript) - RNA gene on the X chromosome
which is a major effector of the X-chromosome inactivation process. It is
processed similarly to mRNA by splicing and polyadenylation but it’s
untranslated.

92. Immune response. Innate and adaptive immunity

The immune response is triggered by the action of an antigen and represents a

complex network of interconnected reactions mediated by immunocytes, which
communicate with each other either directly or through signaling molecules
called cytokines.

Non-specific immune response is considered phylogenetically older, response

speed is within several minutes and it does not contain immunological memory,
whilst specific immunity if phylogenetically younger, takes from several hour to
several days to respond and has immunological memory.

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Innate (non-specific) immune
response
1) Cellular components
•phagocytic cells (monocytes,
neutrophils and macrophages) that
digest foreign cells.
•cytotoxic NK (natural killer cells)
2) Humoral component
•It includes components of
complement,
•interferons and other proteins.
•Non-specific components of
immunity respond quickly but do not
have immunological memory.
3) PAMP
• (Pathogen-Associated Molecular
Patterns) - molecular patterns
associated with pathogens - their
bacterial walls, DNA and dsRNA
(viruses only)
• These patterns are recognized by
Pathogen Pattern Receptor (PPR) =
Pathogen Recognition Receptor
(PRR). The subdivision is: secreted,
endocytic and signaling.
Adaptive (specific) immune
response
1) Cellular components
•lymphocytes and their
antigen-specific receptors (TCR
and BCR)
2) Humoral component
•mainly consists of antibodies
(formed by B lymphocytes)
3) Lymphocytes:
• B Lymphocytes produce antibodies
in defense against extracellular
pathogens/their products.
•T lymphocytes divide further into
several subpopulations

93. Components of the immune system

Basic components of the immune system comprise of

• lymphatic tissues and organs
• cells of the immune system, so-called immunocytes (leukocytes, dendritic
cells, etc.)
• immune system molecules (e.g. antibodies, cytokines, receptors, adhesion
molecules, etc.)
Lymphatic organs are further divided into 2 groups:
• Primary (central) lymphatic organs are the site of lymphocyte formation and
maturation. These include the bone marrow and thymus.

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• Secondary (peripheral) lymphatic organs are primarily the site of initiation
and other phases of a specific immune response. These include the spleen,
lymph nodes, tonsils, Peyer's patches, appendix, and mucosal lymphatic
tissue.

Immune cells:
All types of blood and immune cells are produced in the bone marrow.
While B-lymphocytes also mature in blood marrow, T-lymphocytes leave the
bone marrow as unfinished precursors and complete their development in the
thymus

A granulocyte is a type of leukocyte that contains granules in the cytoplasm of

its cell. Their nucleus is polymorphic, so they are called polymorphonuclear
leukocytes. Granulocytes are formed in the bone marrow. They are able to
produce proteolytic enzymes (granules). They have the phagocytic ability. There
are three main groups of granulocytes. Commonly, however, a granulocyte is
primarily a neutrophilic granulocyte, which is the most common of them.
White blood cells that are not granulocytes are called agranulocytes (these
include monocytes and lymphocytes).

T lymphocytes named after the thymus, where the main part of their maturation
and development takes place Two major T cell subpopulations leave the
thymus:
• Th lymphocyte (T helper) precursors
• Tc lymphocyte (cytotoxic) precursors
On the surface of their membranes there are receptors (T-cell receptor - TCR)
for antigen recognition and other differentiation molecules (CD molecules):
CD4 in Th lymphocytes CD8 in Tc lymphocytes
B lymphocytes are formed in the bone marrow and maturation is completed
after encountering the antigen in secondary lymphatic organs – lymph nodes,
spleen, Peyer's patches. B lymphocytes have membrane immunoglobulins

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serving as receptors (B-cell receptor - BCR) Upon contact with a specific
antigen, they induce clonal expansion-> clonal proliferation of B cell and the
process by which B lymphocytes differentiate into plasma cells that produces
antibodies. Some B cells do not differentiate and stay in circulation as memory
cells.

94. Immunoglobulins, antibody diversity, V-D-J

recombination

Immunoglobulins are glycoproteins occurring in two forms:

• anchored in the plasma membrane of B lymphocyte as antigen specific B cell
receptor (BCR, IgM, IgD)
• circulating in the blood and lymph and other body fluids, therefore as
antibodies.

V(D)J recombination is the mechanism of somatic recombination that occurs

only in developing lymphocytes during the stages of T and B cell maturation. It
results in the highly diverse number of antibodies/immunoglobulins and T cell
receptors (TCRs) found in B cells and T cells, respectively.

Immunoglobulins according to heavy chain and function can be divided into:

• IgM - forms a cellular receptor (BCR) on the surface of B lymphocytes.
Secreted IgM (found in serum) is the first type of antibody that is formed
upon encounter with an antigen (primary response).
• IgD - is found in serum and on the surface of B lymphocytes, where it forms
BCR.
• IgG - is the most abundant class of circulating (serum) immunoglobulins.
It is involved in secondary immune responses. It crosses the placenta.
• IgA - occurs in two forms - mucosal form (protection against
microorganisms) and serum.
• IgE - is in serum only in low concentrations in healthy people. It is the main
cause of allergic reactions.

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 95. T cell and B cell receptors (TCR, BCR)
During the differentiation of T and B lymphocytes, there is a random
rearrangement of segments of supergenes, which encode the variable regions
of their antigen-specific receptors (TCR, BCR)

A huge variety of TCRs and BCRs are created by the mechanism of gene
rearrangement and somatic hypermutation to react to various antigens. The
variety is estimated to reach 1018 for TCR and 1014 for BCR.

● TCR and BCR receptors responsible for reacting with a wide range
of antigens in the future.

● BCR and TCR have similar immunoglobulin antigen recognition

receptors but the types of antigens they recognise are very different. BCR
can recognise naïve (as a whole) antigens and TCR can only
recognise a single antigen peptide sequence presented onto cell
surfaces by MHC (Major histocompatibility complex) molecules.

96. Major histocompatibility complex, compatibility,

transplantation

Major histocompatibility complex:

• Found only in higher animals
• They are essential for the specific response of immune system cells to antigen.
Molecules encoded by the genes of the major histocompatibility complex (HLA
- Human Leukocyte Antigens) are present in cell membranes

Physiological functions of the molecules of the histocompatibility system:

● the presentation of antigens (respectively epitopes) to the cells of the
immune system and
● the recognition of foreign and own components of the organism.

The genes of the main histocompatibility complex are characterized by a high

degree of polymorphism (multiple alleles), the relationship of alleles is
codominant.

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 Compatibility:
The HLA complex (HLA locus) is located on the short arm of chromosome 6
(6p2). The HLA complex is 2-3x106 bp long The HLA complex is a supergene
that consists of three subregions.

● Class I molecules: HLA class I molecules (antigens) are present on the

plasma membrane of all cells (although in quite different amounts)
excepting erythrocytes (classical A, B, C and non-classical E, F, G)

● Class II molecules lie closest to the centromere in a series of D genes,

they are expressed only in the membranes of B lymphocytes, activated T
lymphocytes and macrophages

● Class III molecules are not involved in encoding of HLA antigens. These
are the genes for complement components C2 and C4, cytochrome P450,
tumor necrosis factor - TNF, the gene encoding the enzyme
21-hydroxylase and several other genes.

★ TCR of Tc lymphocytes recognize foreign antigen in complex

with HLA class I molecules and TCR of Th lymphocytes
recognize the antigen in complex with HLA class II molecules

Transplantation:
● Transplantation is the transfer of tissues and organs between
individuals. Because the transplanted organ (tissue, cells) may present an
encounter with foreign antigens to the recipient, it may be rejected by the
immune mechanisms.

● Transplants in humans are, with the exception of monozygotic twins,

allogeneic transplants (donor and recipient are not the same). An
individual's genetic polymorphism causes the immune system to respond
to the presence of foreign antigens in another individual's tissues and
induce transplant rejection. It is mainly affected by the antigens in the
major histocompatibility complex (HLA in humans)

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 ● When transplanting, it is necessary to pay attention not only to
compatibility in the AB0 system but also the maximum similarity of
the donor and recipient antigenic equipment in the HLA system
(HLA haplotype).
Transplantation rules:
1) Transplantation is successful among members of the same inbred strain, we
call this transplantation syngeneic. This situation in the human population
only exists in transplantation between monozygotic twins.
2) Transplantation is unsuccessful between members of different inbred strains
A and B. It is an allogeneic transplant in which the donor and recipient of the
transplant are not genetically identical.
3) By crossing the parental (P) generation (AA x BB) we get a uniform F1
generation (heterozygotes AB). Transplantation from both parental strains into
F1 individuals is successful. The recipient F1 does not recognize antigen A or
B as foreign.
4) Another situation occurs when transplanting tissue from a heterozygous F1
generation to a homozygous P generation. The transplant is rejected. This is a
situation where the antigen present in the donor (B) is not present in the
recipient's tissues.

97. Genetic determination of ABO blood group system

• The blood group of each individual depends on the presence of specific
antigens.
• More than 20 blood group systems are known in humans. Among the most
important are AB0, Rh and MN, Ss systems. According to the presence of
antigens A and B on the plasma membrane of erythrocytes, 4 basic blood
groups can be distinguished A, B, AB and 0. A

The AB0 system:

The expression of AB0 system antigens requires the interaction of several genes
that have different loci at different sites chromosomes. In particular, they are
two independent loci, the AB0 locus (chromosome 9) and the H locus
(chromosome 19). The primary products of both genes are enzymes -
glycosyltransferases.

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 Rh system:
The Rh system was discovered by Wiener, who after immunizing the
rabbit with the blood of the monkey Maccacus rhesus found that rabbit
immune serum agglutinated not only monkey blood cells but also human
blood cells. On this basis, humans were originally divided into two
phenotypic groups: to Rh positive (Rh +) and Rh negative (Rh−)
individuals.
Currently, a model is used that assumes 2 genes in a very close linkage
defining the Rh system. The genes are called RHD and RHCE. Rh
negative people are recessive homozygotes. Antigens are present only
on erythrocytes, no natural antibodies are present.

Fetal erythroblastosis:
Incompatibilities in the RH system cause a serious neonatal disease called fetal
erythroblastosis. The frequency of fetal erythroblastosis is approximately
possible in 1/200 births. The mother Rh negative (dd) and the child inherits
from the father the allele D and is Rh+ (genotype Dd) Only a low number of
erythrocytes passes through the placenta. The first pregnancy is going without
problems Followed by incompatible pregnancies, even a small number of fetal
erythrocytes that have crossed the placenta are sufficient to elicit a memory cell
response.

98. Genetic disorders of the immune system

Some HLA antigens are more common in patients with a disease than in the
general population. This phenomenon is called association. We observe only an
inherited tendency to disease in association with this allele. The manifestation

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of the disease is therefore conditioned by genetic factors and environmental
influences Association with certain alleles, especially of the main HLA class II
subgenes, were found in many autoimmune diseases.

Immunodeficiency:

Immunodeficiencies cause an increased susceptibility to infections.

Primary immunodeficiencies are caused by germline mutations in genes that

encode proteins involved in immune responses. These include disorders of
antibody production, disorders of T cell function, phagocytic system,
complement production. They affect males more often because many of the
genes involved in immune system functioning are located on the X
chromosome.

Secondary immunodeficiency is acquired by the individual during life. The

cause is, for example, malnutrition, HIV retrovirus infection (AIDS),
metabolic disorders, cytostatic therapy, treatment of immunosuppressive
drugs in patients after transplantations, chronic infection, chronic stress,
severe injuries.

Allergies:
Allergy is a pathological reaction of the immune system to external stimuli -
allergens, which are a common part of our environment. The allergic reaction
can take place locally or systemically.
Local reaction - allergic rhinitis, conjunctivitis, bronchial asthma, atopic
dermatitis.
Systemic reaction - anaphylactic shock in which an allergen enters the
bloodstream of a sensitized individual. It increases the permeability of blood
vessels and lowers blood pressure, which can lead to multiorgan failure.

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 99. The origin of life on Earth and genetic evolution
mechanisms
Genetic evolution mechanisms:
Mechanisms of evolution correspond to violations of different Hardy-Weinberg
assumptions. They are: mutation, non-random mating, gene flow, genetic
drift and natural selection.
Biological evolution - the emergence of living forms from inanimate matter - an
ongoing process of accumulation of gradual changes in the properties of
populations of organisms based on changes in their gene pool - necessary
genetic variability of populations (source - mutations, recombination)
Necessary evolutionary mechanism - natural selection (advantage of
individuals) and gradual adaptation to abiotic and biotic conditions

The view that life emerged through a long process of chemical evolution was
set forth by the Russian biochemist Alexander Oparin in 1924.
In the 1920s, A. I. Oparin and J. B. S. Haldane hypothesized that the early
atmosphere was a reducing environment
In 1953, Stanley Miller and Harold Urey conducted lab experiments that
showed that the abiotic synthesis of organic molecules in a reducing atmosphere
is possible. Demonstrate that in an electric shock environment it is possible to
form amino acids and carbohydrates from a mixture of water, methane,
ammonia and hydrogen

100. Evolution of genes and genomes

Genome evolution is the process by which a genome changes in structure or
size over time. The study of genome evolution involves multiple fields such as
structural analysis of the genome,

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 101. Species and speciation, human evolution

Speciation - splitting of developmental lines leading to the emergence of new

species
Phylogeny = phylogenetic development - historical development of species of
organisms
Phylogenetics - an effort to classify organisms into the system and clarify the
interrelationship of species (genetic relation of DNA)
Phylogenetic trees - graphical representation of mutual relations between
groups of organisms originating from one ancestor

Human evolution:
1.9 - 1.5 million years ago - Homo ergaster, had a body shape and limb
proportions like those of modern humans, and its teeth and jaws were also
human in structure. Thus, H. ergaster is the first hominin th. Šmajsat can
confidently be placed within the genus Homo.
The first hominin species to produce fossils outside of Africa was Homo
erectus. H. erectus was widespread and probably gave rise to archaic
populations of humans in Europe, Asia, and Africa. Modern humans may have
evolved simultaneously in Europe, Asia, and Africa from the archaic human
populations that existed on each of those continents, or they may have evolved
on one continent—for instance, Africa—and subsequently spread to the others.

Selected applications of medical biology

102. Cancer classifications systems, tumour as complex tissue
Tumor classification
Tumor classification 1: according to the ability to infiltrate other tissues
Benign tumors remain at the site of their origin, they are bounded, do not
invade other tissues, migrate or establish metastases (polyps, lipomas,
chondromas, leiomyoma, fibroma,…)
Malignant tumors infiltrate and destroy surrounding tissues and through the
blood and lymphatic system it spreads throughout the body, in new tissues they
cause the formation of secondary tumors (metastasis) X secondary tumors.

Tumor classification 2: according to the type of cells (tissues) from which the
tumor cell originates
● carcinomas epithelial cell tumors (about 80-90% of human tumors)

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 ● sarcomas - solid tumors of connective tissues - muscles, bones, cartilage,
make up about 1% of the tumor, from mesenchymal cells
● leukemias and lymphomas - derived from hematopoietic cells and cells
of the immune system neuroectodermal - tumors derived from nervous
tissue (gliomas, neuroblastomas, medulloblastomas,…)
● germinal tumors - derived from totipotent germ cells
● mixed tumors

Classification of tumors 3- according to the affected organ or tissue (lung

cancer, colorectal cancer, breast cancer, etc.)

103. Carcinogenesis
Carcinogenesis is a multi-stage process of tumor formation and
development; T
The essence of carcinogenesis if the gradual accumulation of genetic
(and epigenetic) changes.
Malignant transformation – is the transformation of somatic cell
into a tumor cell;

Types of genetic changes in tumor cells:

1. Minor changes in the DNA sequence - mutations, minor deletions and
insertions
2. Changes in the number of chromosomes - losses or gains of whole
chromosomes
3. Chromosomal translocation - the fusion of parts of different chromosomes
or normally unrelated parts of the same chromosome, on the molecular level
may be accompanied by fusions of two different genes
4. Gene amplification

Physical: Ultraviolet radiation induces p53 mutations and skin tumors

Classical epidemiology has long associated sunlight and skin tumors, molecular
epidemiology has identified specific wavelengths of sunlight that are
carcinogenic (290-320 nm - UVB) and lead to mutations in the tumor
suppressor p53

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Viral DNA: tumor viruses do not contain oncogenes, but encode proteins that
interact with the tumor suppressors (RB, p53, p300 / CBP) of the host cell and
thus push the host cell into the S phase: HPV-16, HPV-18 papillomaviruses: E6
interacts with p53, p300 / CBP; E7 interacts with RB

104. Oncogenes and tumor suppressors

Mutations in proto-oncogenes are:
activating
dominant - occur in somatic cells and only exceptionally in germ cells

Tumor suppressors
Gene products for tumor suppressors in normal cells do not induce
proliferation, but instead suppress it and keep the cells at rest (G0). Their loss is
manifested by unregulated proliferation.
Knudson's "two hit hypothesis" (RB)
Tumor suppressor mutations are:
● inactivating
● recessive (associated with LOH) ("recessive oncogenes")
● occur in somatic as well as germline cells

105. Hereditary cancer syndromes

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 106. Hallmarks of cancer (Weinberg and Hanahan model)

Six acquired features of malignant tumors

(proposed by Robert A. Weinberg in 2000)

Genomic instability is a
pre-condition for the accumulation
of all necessary changes.

Douglas Hanahan and Robert A. Weinberg (Cell, 2011)

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 107. Basic principles of cancer therapy
Molecularly controlled combinatorial approaches
The tumor consists of genetically distinct subpopulations of tumor cells, each
showing a characteristic sensitivity / resistance profile to therapy
• Each treatment regimen then represent a filter that removes only the
population of tumor cells showing molecular characteristics associated with
sensitivity to the therapy
• Combination therapy leads to long-term remission, tumor as a chronic disease

108. Molecular classification of cancer and therapy

personalization
Tumor stroma
• mesenchymal tissue components: vascular components,
supporting mesenchymal tissues, lymphocytes,
macrophages, ..
• it serves mainly to nourish the parenchyma and as a
skeleton for the arrangement of the parenchyma
• transport role (signal transmission, reservoir of growth
factors…)
• key role in tumor angiogenesis and metastasis
Tumor parenchyma
• cancer cells
Personalized medicine or personalized care. Precision medicine looks at how
a specific gene change (gene mutation) might affect a person's risk of getting a
certain cancer or, if they already have cancer, how their genes (or genes in their
cancer cells) might affect treatment.

109. Cancer genome, variant classification, precision

oncology
WGS (whole genome sequencing) always provides only a snapshot of cancer
genome
• Certain mutations reflect paternal and/or maternal germline variation
• Additional somatic mutations accumulate through life
• “Driver” mutations cause cancer, “passenger” mutations are carried along
• Additional drivers evolve and diversify the cancer

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• Some alter aggressiveness; these may alter treatment response, leading to
relapse

Precision oncology, defined as molecular profiling of tumors to identify

targetable alterations, is rapidly developing and has entered the mainstream of
clinical practice.

110. Bacterial genome, transcriptome and proteome

Transcriptome is the full range of messenger RNA, or mRNA, molecules

expressed by an organism. The term "transcriptome" can also be used to
describe the array of mRNA transcripts produced in a particular cell or tissue
type.

Proteome is the entire set of proteins that is, or can be, expressed by a genome,
cell, tissue, or organism at a certain time.

111. Phylogenetic relationship, genetic diversity and plasticity

of bacterial genomes

Enabling characteristics - genomic instability

Enabling characteristics - tumor-associated inflammation Emerging
hallmarks - deregulation of cellular energetics (Warburg ef.)
Emerging hallmarks – evading immune surveillance

Prokaryotic cells have developed a number of methods for recombining their

genetic material, which, in turn, contributes to their genetic diversity. The three
most common ways that bacteria diversify their DNA are transformation,
conjugation, and transduction.

Genome plasticity, that can be considered a measure of evolvability, reflects

both the availability of the genes of a given functional class in the external gene
pool that is accessible to the evolving microbial population, and the ability of
microbial genomes to accommodate these genes.

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 112. Mechanisms of genetic recombination in bacteria
DNA Fragmentation

The purified DNA is digested using restriction endonucleases. The recognition

sites of these enzymes are generally 4 to 6 base pairs in length. The shorter the
sequence recognized, the greater the number of fragments generated from
digestion.

For example, if there is a short sequence of GAGC that occurs repeatedly in a

sample of DNA. The restriction endonuclease that recognizes the GAGC
sequence cuts the DNA at every repetition of the GAGC pattern.

If one sample repeats the GAGC sequence 4 times whilst another sample
repeats it 2 times, the length of the fragments generated by the enzyme for the
two samples will be different.

Gel Electrophoresis

The restriction fragments produced during DNA fragmentation are analyzed

using gel electrophoresis.

The fragments are negatively charged and can be easily separated by

electrophoresis, which separates molecules based on their size and charge. The
fragmented DNA samples are placed in the chamber containing the
electrophoretic gel and two electrodes.

When an electric field is applied, the fragments migrate towards the positive
electrode. Smaller fragments move faster through the gel leaving the larger ones
behind and thus the DNA samples are separated into distinct bands on the gel.

Visualization of Bands

The gel is treated with luminescent dyes in order to make the DNA bands
visible.

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113. Regulation of bacterial genome transcription, variation of
antigens in the proteome

● Bacterial genes are often found in operons. Genes in an operon are

transcribed as a group and have a single promoter.
● Each operon contains regulatory DNA sequences, which act as binding
sites for regulatory proteins that promote or inhibit transcription.
● Regulatory proteins often bind to small molecules, which can make the
protein active or inactive by changing its ability to bind DNA.
● Some operons are inducible, meaning that they can be turned on by the
presence of a particular small molecule. Others are repressible, meaning
that they are on by default but can be turned off by a small molecule.

114. Structure, reproduction and recombination of viruses

● The simplest virions consist of two basic components:

nucleic acid (single- or double-stranded RNA or DNA)
and a protein coat, the capsid, which functions as a shell
to protect the viral genome from nucleases and which
during infection attaches the virion to specific receptors
exposed on the prospective host cell.
● Capsid proteins are coded for by the virus genome.
Because of its limited size the genome codes for only a
few structural proteins (besides non-structural
regulatory proteins involved in virus replication).
Capsids are formed as single or double protein shells
and consist of only one or a few structural protein
species.

Reproduction of viruses
Some viruses reproduce using different methods, while others only use
the lytic cycle. In the lytic cycle, the virus attaches to the host cell and

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 injects its DNA. Using the host’s cellular metabolism, the viral DNA
begins to replicate and form proteins. Then fully formed viruses
assemble. These viruses break, or lyse, the cell and spread to other cells
to continue the cycle.

In the lysogenic cycle the virus attaches to the host cell and injects its
DNA. From there, the viral DNA gets incorporated into the host’s DNA
and the host’s cells. Each time the host’s cells go through replication, the
virus’s DNA gets replicated as well, spreading its genetic information
throughout the host without having to lyse the infected cells.

Recombination of viruses

Viral recombination occurs when viruses of two different parent strains infect the
same host cell and interact during replication to generate virus progeny that have
some genes from both parents.

115. Replication and pathogenesis of viruses

Viral replication is the formation of biological viruses during the infection

process in the target host cells. Viruses must first get into the cell before viral
replication can occur. Through the generation of abundant copies of its genome
and packaging these copies, the virus continues infecting new hosts.

Viruses cause disease when they breach the host's primary physical and natural
protective barriers; evade local, tissue, and immune defenses; spread in the
body; and destroy cells either directly or via bystander immune and
inflammatory responses. Viral pathogenesis comprises of several stages,
including:
(1) transmission and entry of the virus into the host,
(2) spread in the host,
(3) tropism, (a biological phenomenon, indicating growth or turning movement of a
biological organism)
(4) virulence,
(5) patterns of viral infection and disease,
(6) host factors,
(7) host defense.

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 116. Transduction and development of viral vectors for gene
therapy
Viruses have evolved specialized molecular mechanisms to efficiently transport
their genomes inside the cells they infect. The term "transduction" is used to
describe a virus-mediated transfer of nucleic acids into cells. In contrast to
transfection of cells with foreign DNA or RNA, no transfection reagent is
needed here.

The viral vector, itself, also called virion, is able to infect cells and transport
the DNA directly into the nucleus, independent of further actions. After the
release of the DNA into the nucleus, the protein of interest is produced using the
cells’ own machineries.

RETROVIRUSES

● are able to attack only dividing cells (does not pass through a compact
nuclear membrane)
● they attack mainly T-lymphocytes
● ex-vivo therapy only and random integration into the host genome - risk
of insertional mutagenesis
● maximum transgene size up to 7.5 kbp
● retroviruses do not cause cell lysis

LENTIVIRUSES they can replace classical retroviruses, as they are able to

infect and integrate their genome into non-dividing cells e.g. neurons,
macrophages, hematopoietic stem cells and muscle and liver cells

● deletion in the LTR, is unable to produce infectious particles, but is

capable of integration

ADENOVIRUSES

● dsDNA viruses
● they attack all types of cells viral DNA
● does not integrate into the host genome expression is strong but only
temporary (max. month)
● cause a strong immune response (expression of wild-type proteins of the
viral particle)
● a new generation of adenovectors not containing natural genes they are
not suitable for the treatment of tumor cells because they
● do not have receptors for them

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 ● most people have antibodies, 90% of the vectors are degraded within the
first 24 hours after administration

ADENO-ASSOCIATED VIRUSES (AAV)

● dsDNA viruses
● Most derived from serotype 2 (AAV2) never associated with human
disease they attack all types of cells viral DNA integrates into the host
genome at a specific site (in humans at the end of chromosome 19)
● There is no risk of insertional mutagenesis however, expression is weak
and the vectors have a small capacity of 4 kb

117. Forward and reverse genetics, from affected tissue to its

function and therapy
Forward genetics is the examination of the genetic cause of an altered or
abnormal phenotype introduced by a chemical mutagenesis or mutation by
irradiation (e.g., phenotype → genotype).

In reverse genetics, a particular gene is altered and the phenotype is

investigated (e.g., genotype → phenotype).

118. Basic methods of molecular biology (biological material,

restriction enzymes, electrophoretic methods)
Biological materials are natural biocompatible materials that comprise a whole
or a part of a living structure or biomedical device that performs, augments, or
replaces a natural function.

Restriction enzymes are DNA-cutting enzymes. Each enzyme recognizes one

or a few target sequences and cuts DNA at or near those sequences.
● Many restriction enzymes make staggered cuts, producing ends with
single-stranded DNA overhangs. However, some produce blunt ends.
● DNA ligase is a DNA-joining enzyme. If two pieces of DNA have
matching ends, ligase can link them to form a single, unbroken molecule
of DNA.
● In DNA cloning, restriction enzymes and DNA ligase are used to insert
genes and other pieces of DNA into plasmids.

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The electrophoresis technique describes migration of charged particles under
the influence of an electric field. The rate of migration depends upon various
factors like charge of the particle, applied electric field, and temperature and
nature of the suspended medium.

119. PCR and quantitative PCR

PCR = polymerase chain reaction; principle - use of DNA polymerase for
repeated copying of a template DNA molecule

DNA amplification by PCR

​ - The region of interest is delimited by two oligonucleotides - primers

for DNA synthesis
​ - At the beginning of each cycle, the separation of DNA strands by high
temperature - denaturation
​ - Cooling of the solution, large excess of primers - annealing of primers
and synthesis of new chains
​ - Cyclic repetition of denaturation and synthesis, use of thermostable
DNA polymerase from thermophilic
bacteria
​ - In each cycle, all DNA in solution serves as templates, including the
strands synthesized in the previous step
​ - After a few cycles, the amplified molecules begin to predominate
​ - Usually 20-30 cycles, in each the number of molecules is doubled

Quantitative PCR/ Real-Time PCR

- is based on monitoring the progress of the PCR directly during the

reaction using fluorescent probes or dyes that detect the amount of PCR
product during the reaction by increasing its fluorescence activity.
- the fluorescence increases exponentially along with the amplification
product. However, along with continued amplification, PCR products
ratio also decreases.

Use of PCR

- detection of the viral genome in a blood sample (HIV for example)

- obtaining a genomic or cDNA clone

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 120. Methods for identification of mutations (RFLP, Sanger
sequencing)
DNA sequencing is the process of determining the sequence of nucleotide bases
(As, Ts, Cs, and Gs) in a piece of DNA.

Sanger sequencing: The chain termination method

- Was developed by the British biochemist Fred Sanger and his colleagues
in 1977.
- Requires the same things as PCR or DNA replication: DNA polymerase
enzyme, primer, DNA template, four DNA nucleotides. Unique
component - Dideoxy, or chain-terminating, versions of all four
nucleotides (ddATP, ddTTP, ddCTP, ddGTP, they lack a hydroxyl group
on the 3’ carbon of the sugar ring)

Method

- The DNA sample is combined in a tube with primer, DNA polymerase,

and DNA nucleotides (dATP, dTTP, dGTP, and dCTP), dideoxy
nucleotides are added as well, but in much smaller amounts
- The mixture is first heated to denature the template DNA (separate the
strands), then cooled so that the primer can bind to the single-stranded
template. Once the primer has bound, the temperature is raised again,
allowing DNA polymerase to synthesize new DNA starting from the
primer. DNA polymerase happens to add a dideoxy nucleotide instead of
a normal one, ending the strand.
- This process is repeated in a number of cycles. By the time the cycling is
complete, a dideoxy nucleotide will have been incorporated at every
position of the target DNA. The ends of the fragments will be labeled
with dyes that indicate their final nucleotide.
- After the reaction is done, the fragments are run through a long, thin tube
containing a gel matrix in a process called capillary gel electrophoresis.
Short fragments move quicker than the long ones. The dye is detected by
a laser at the end of the tube
- The data is recorded by a detector, the results can be read on a
chromatogram.

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 RFLP

- Restriction fragment length polymorphism (RFLP) is a technique

invented in 1984 by the English scientist Alec Jeffreys
- Used for analysis of unique patterns in DNA fragments in order to
genetically differentiate between organisms

Principle

Restriction endonucleases are enzymes that cut lengthy DNA into short pieces.
Each restriction endonuclease targets different nucleotide sequences in a DNA
strand and therefore cuts at different sites. The distance between the cleavage
sites of a certain restriction endonuclease differs between individuals. Hence,
the length of the DNA fragments produced by a restriction endonuclease will
differ across both individual organisms and species.

Method

1. DNA extraction (from blood, saliva or other)

2. DNA Fragmentation - the purified DNA is digested using restriction
endonucleases. The shorter the sequence recognized, the greater the
number of fragments generated from digestion.
3. Gel Electrophoresis - the restriction fragments produced during DNA
fragmentation are analyzed using gel electrophoresis. The fragments are
negatively charged and can be easily separated by electrophoresis, which
separates molecules based on their size and charge. When an electric field
is applied, the fragments migrate towards the positive electrode. Smaller
fragments move faster through the gel leaving the larger ones behind and
thus the DNA samples are separated into distinct bands on the gel.
4. Visualization of Bands - the gel is treated with luminescent dyes in
order to make the DNA bands visible.

Use of RFLP

- To determine the status of genetic diseases such as Cystic Fibrosis in an

individual.
- To determine or confirm the source of a DNA sample such as in paternity
tests or criminal investigations.

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 - In genetic mapping to determine recombination rates that show the
genetic distance between the loci.
- To identify a carrier of a disease-causing mutation in a family.

121. DNA microarrays

- Are used for analysis of the production of thousands of different mRNAs

in a single experiment
- Microscope slides that are printed with thousands of tiny spots in defined
positions, with each spot containing a known DNA sequence or gene.
- DNA molecules attached to each slide act as probes to detect gene
expression, which is also known as the transcriptome or the set of
messenger RNA (mRNA) transcripts expressed by a group of genes.
- The data gathered through microarrays can be used to create gene
expression profiles, which show simultaneous changes in the expression
of many genes in response to a particular condition or treatment.

Method

To perform a microarray analysis, mRNA molecules are typically collected

from both an experimental sample and a reference sample. For example, the
reference sample could be collected from a healthy individual, and the
experimental sample could be collected from an individual with a disease like
cancer. The two mRNA samples are then converted into complementary DNA
(cDNA), and each sample is labeled with a fluorescent probe of a different
color. The two samples are then mixed together and allowed to bind to the
microarray slide. The microarray is scanned to measure the expression of each
gene printed on the slide. If the expression of a particular gene is higher in the
experimental sample than in the reference sample, then the corresponding spot
on the microarray appears red. In contrast, if the expression in the experimental
sample is lower than in the reference sample, then the spot appears green.
Finally, if there is equal expression in the two samples, then the spot appears
yellow.

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 122. Next-generation sequencing
There are a variety of next-generation sequencing techniques that use different
technologies. However, most share a common set of features that distinguish
them from Sanger sequencing:

- Highly parallel: many sequencing reactions take place at the same time
- Micro scale: reactions are tiny and many can be done at once on a chip
- Fast: because reactions are done in parallel, results are ready much faster
- Low-cost: sequencing a genome is cheaper than with Sanger sequencing
- Shorter length: reads typically range from 50-799 nucleotides in length

123. Gene cloning and recombinant DNA

DNA cloning is the process of making multiple, identical copies of a particular
piece of DNA. In a typical DNA cloning procedure, the gene or other DNA
fragment of interest is first inserted into a circular piece of DNA called a
plasmid. The insertion is done using restriction enzymes, and it produces a
molecule of recombinant DNA, or DNA assembled out of fragments from
multiple sources. Next, the recombinant plasmid is introduced into bacteria.
Bacteria carrying the plasmid are selected and grown up. As they reproduce,
they replicate the plasmid and pass it on to their offspring, making copies of the
DNA it contains.
A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target
sequence and cuts DNA into two pieces at or near that site.
Method
1. Cut open the plasmid and "paste" in the gene. This process relies on
restriction enzymes (which cut DNA) and DNA ligase (which joins
DNA).
2. Insert the plasmid into bacteria. During transformation, specially prepared
bacterial cells are given a shock (such as high temperature) that
encourages them to take up foreign DNA. A plasmid typically contains an
antibiotic resistance gene, which allows bacteria to survive in the
presence of a specific antibiotic. Use antibiotic selection to identify the
bacteria that took up the plasmid.

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 3. Grow up lots of plasmid-carrying bacteria and use them as "factories" to
make the protein. Harvest the protein from the bacteria and purify it.

Uses of DNA cloning

- Biopharmaceuticals
- Gene therapy
- Gene analysis

124. Animal models in biomedicine

- Animal models provide precise genetic and experimental control, and they
also provide a route to more rapid discovery and validation of disease
prevention and treatment.

Transgenic organisms contain genetically modified genes

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Changes in haploid organisms (bacteria, yeast) are easy - a system of
homologous recombination between mutated DNA and chromosomal DNA
easily introduces a mutated form into the genome.

Changes in diploid organisms more complex - two copies of the gene

- necessary introduction into germ cells

- technically possible also in humans - illegal, ethically controversial,
only modifications in human somatic cells - gene therapy
- Mouse knockout - a laboratory mouse in which researchers have
inactivated an existing gene by replacing it or disrupting it

125. Gene therapy (history, types)

- The insertion of usually genetically altered genes into cells especially to
replace defective genes in the treatment of genetic disorders or to provide
a specialized disease fighting function

Milestones

1990 - A four-year-old girl with severe immunodeficiency became the first

patient to undergo gene therapy in the United States.

1999 -
American
patient Jesse
Gelsinger dies
following a
gene therapy
experiment,
setting the
field back
several years
as U.S.
regulators put
some key
experiments
on hold.

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2002-03 - Cases of leukaemia are diagnosed in French children undergoing gene
therapy for genetic immunodeficiency in a further blow to the field.

2016 - The first gene therapy for children has been approved in Europe

Types

Ex vivo: delivered to the person's cells

outside the body, which are then
returned to them.

In vivo: delivered directly to the

person’s cells inside the body

126. Basic strategies of gene therapy

Gene augmentation is the development of a new protein-encoding gene to treat
a disease. It is used to treat diseases caused by a mutation that stops a gene from
producing a functioning product, such as a protein.

Gene suppression is the regulation of gene expression in a cell to prevent the

expression of a certain gene. It can occur during either transcription or
translation and is often used in research.

Genome editing based therapy can be achieved through a number of approaches

including correction
or inactivation of
deleterious mutations,
introduction of
protective mutations,
addition of
therapeutic
transgenes, or
disruption of viral
DNA.

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 127. Methods of DNA delivery to target tissues
There are two general categories of gene delivery systems, or vectors as they are
commonly called: viral and nonviral vectors. Mostly viral vectors are utilized.
Virally derived vectors have many of the characteristics of the parent viruses
themselves, and some of these viruses may be pathogens in their normal state.
One of the most important characteristics is the ability of the virus to assure
transport of its genomic DNA to the nucleus of the host cell without degradation
by lysosomes. Included among the nonviral vector systems are direct DNA
delivery, liposomes, and DNA–protein complexes.

128. Gene-editing approaches

Genome-editing technologies.

1. Restriction enzymes - the original method that made genome-editing a

reality in 1970. Not commonly used nowadays.
2. Zinc Finger Nucleases (ZFNs) - discovered in 1980s. Consist of two parts
- an engineered nuclease (Fokl) fused to zinc finger DNA-binding
domains. The zinc-finger DNA-binding domain recognizes a 3-base pair
site on DNA and can be combined to recognize longer sequences;

133
 function as dimers, increasing the length of the DNA recognition site and
consequently increasing specificity. Can be used for treatment of
malignant melanoma.
3. TALENs Gene Editing - the structure is similar to ZFNs, both methods
use the Fokl nuclease to cut DNA and require dimerization to function,
however, the DNA binding domains differ.
4. CRISPR-Cas9 Gene Editing - discovered in 2012, the most common and
efficient method. The standard Cas9 protein cuts the DNA at the target -
this is the most common approach to genome editing. When the cut is
repaired, mutations are introduced that usually disable a gene. The Cas9
nuclease is targeted to DNA sequences complementary to the targeting
sequence within the single guide RNA (gRNA) located immediately
upstream of a compatible protospacer adjacent motif (PAM).

134
 129. Medicinal gene therapy products
List of products

Gendicine

- the first approved gene therapy in the world

- was approved in 2003 by the China Food and Drug Administration
(CFDA) as a first-in-class gene therapy product to treat head and neck
cancer
- entered the commercial market in 2004
- a biological therapy that is delivered via minimally invasive intratumoral
injection, as well as by intracavity or intravascular infusion
- the wild-type (wt) p53 protein expressed by Gendicine-transduced cells is
a tumor suppressor that is activated by cellular stress, and mediates
cell-cycle arrest and DNA repair, or induces apoptosis, senescence, and/or
autophagy, depending upon cellular stress conditions

Glybera

- gene therapy for the lipoprotein lipase deficit (AAV)

- the first approved gene therapy in Europe in 2012

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 - AAV serotype 1 (tropism to muscle cell) carrying a copy of human
lipoprotein lipase

Strimvelis

- drug for ADA-SCID - incidence in Europe about 15 cases per year, 12 in

the US
- ex vivo hematopoietic gene therapy stem cells (HSCs) using retroviral
vector with human ADA
- the price was set at € 594,000 (twice the annual price of enzyme therapy)
- approved by the EC in May 2016

130. Antibodies - their production and use in medicine

Production
Antibody production is a main function of the immune system and is carried out
by B cells. Antibodies act as an important part of the immune response by
specifically recognizing and binding to particular antigens, such as bacteria or
viruses, and aiding in their destruction. Antibodies are produced either to
neutralize or to eliminate antigens or pathogens. Antibodies are produced by
B-lymphocytes after differentiation of B-lymphocytes into plasma cells.
Antibodies are naturally produced by the immune system. However, scientists
can produce antibodies in the lab that mimic the action of the immune system.
These man-made (synthetic) antibodies act against proteins that attack normal
tissues in people with autoimmune disorders. The term monoclonal antibody
means that the man-made antibody is synthesized from cloned immune cells,
and the identical monoclonal antibody produced binds to one type of antigen.

Medical application
The use of monoclonal antibodies to treat diseases is called immunotherapy
therapy because each type of monoclonal antibody will target a specific targeted
antigen in the body.
It is used for treatment of different diseases, such as:
- Cancer
- Rheumatoid arthritis
- Multiple sclerosis, etc
In these conditions the monoclonal antibody targets and interferes with the
action of a chemical or receptor that is involved in the development of the
condition that is being treated.

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 131. Microbiome, symbiosis of the human body with
microorganisms

Microbiome
- the collection of all microorganisms that reside on the surface and inside
of the human body – in deep layers of skin, in the saliva and oral mucosa,
conjunctiva, gastrointestinal tract, urogenital tract, etc. – and genes of
these microorganisms
- microorganisms participate in processing the nutrients, vitamin synthesis,
metabolism of introduced substances (production of enzymes), they
stimulate renewal of the intestinal epithelium, stimulate the immune
system, etc
- diet has crucial influence on intestinal microbiome composition
- each healthy person has a unique microbiome composition
Symbiosis of the human body with microorganisms
There are 100 trillion bacteria in the microbial ecosystem of the human body.
Interactions with these microorganisms take place daily at the level of the skin,
in the urogenital tract, mouth, pharynx, and respiratory system, and the digestive
tract which contains by far, the largest density and diversity of microorganisms.
The microbiota contributes to trophic functions, metabolism, barrier function,
immune stimulation, and signalization to virtually all organs of the body. The

137
maintenance of symbiosis between the human organism and microbiota is
essential for health.
132. Formation of the human microbiome during life and in
disease
Human microbiome project
- Started in 2007, lasted 5 years
- Objectives: determining the microbiomes stability, the microbiome
common for all people, factors that influence the composition of the
microbiome; characterizing of the microbe communities at various sites
of the human body
- Results: each healthy person has a unique microbiome; microbiomes
from different body sites differ; there is a high microbial diversity in the
intestine and in oral cavity and a low microbial diversity in the vagina

Microbiome during disease

Characterizing the microbiome of healthy individuals via the Human
microbiome project opens a possibility of studying the relations between
microbiome and diseases. The following diseases are associated with
microbiomes.

Carcinogenesis
- Human
oncoviruses
can drive
carcinogenesis
by integrating
their oncogenes
into host cell
genomes or by
blocking
human proteins
encoded by tumour suppressors from function
- Microbes can affect genome stability of human cells, their resistance to
cell death and proliferative signalling
- Some microbial defence factors can lead to mutations in human cells’
DNA, that subsequently contribute to carcinogenesis – for example
colibactin expressed by B2 group of Escherichia coli (as well as by other

138
 Enterobacteriaceae) and cytolethal distending toxin (CDT) produced by
several Escherichia coli and other ε- and γ-proteobacteria both can cause
double-stranded DNA damage in mammalian cells
Obesity
- Research has shown that in contrast to healthier people the microbiomes
of obese individuals are structurally and functionally distinct
- People with intestinal microbiome containing low number of genes (and
therefore low number of microorganism species) are more frequently
obese, resistant to insulin, and have dyslipidemia (abnormal amounts of
lipids in the blood)
Formation of microbiome

In health
Commensal bacteria colonize the host shortly after birth. Over time,
host-bacterial associations have developed into beneficial relationships.
Symbiotic bacteria metabolize indigestible compounds, supply essential
nutrients, defend against colonization by opportunistic pathogens, and
contribute to the formation of intestinal architecture
In disease (infections)
Infection is one of the most common diseases caused by dysbiosis of the
microbiota. Importantly, infectious disease and its treatment have a
profound impact on the human microbiota, which in turn determines the
outcome of the infectious disease in the human host.

139
 133. Principles and methods of classification of prokaryotic
organisms based on their genome and RNA
- Prokaryotic organisms can be classified according to 16S RNA -
ribosomal RNA specific for prokaryotes
- As with the 16S rRNA gene, genome sequences can be used to construct
a robust phylogenetic framework on which to base a systematic
classification. Genome-based classification affords greater resolution
than the 16S rRNA gene for both the most ancient and most recent
relationships due to a larger fraction of the genome being used in the
comparison, which provides an improved phylogenetic signal

134. Basic characteristics of stem cells

Stem cells have the ability to differentiate into specific cell types. The two defining
characteristics of a stem cell are perpetual self-renewal and the ability to differentiate
into a specialized adult cell type.
Totipotent stem cells – They can differentiate into any cell type of the embryo,
fetus, developing organism including embryonic components of trophoblast and
placenta (e.g. Zygote, early 2-cell stage embryo)
Pluripotent stem cells – They can differentiate into any cell type of the
embryo, fetus, developing organism, but they do not participate on formation of
embryonic components of trophoblast and placenta (e.g. Embryonic stem cells;
inner cell mass in blastocyst)
Multipotent stem cells – they can differentiate into many cell types usually
associated with tissue, organ or physiological system (e.g. Hematopoietic stem
cells)
Oligopotent cells – can differentiate into several cell types (often progenitors;
example: neural stem cells versus myeloid progenitor cells)
Progenitor cells (often confused with stem cells) – early descendants of stem
cells, more differentiated compared to stem cells . Progenitors can differentiate,
but don’t not self-renew.

140
 135. Structure and function of the stem cells "niche"

Microenvironment - niche is a microenvironment in particular anatomical

location where the stem cell is located and which interacts with the stem cell.

Niche includes:
- Physical interaction of the cells surfaces
- Autocrine and paracrine signaling
- Neural signals
- Products of metabolic activity of the tissue

Examples of niches and their effect on stem cell differentiation

● Proliferation of hematopoietic stem cells is determined by their
interaction with mesenchymal cells
● The HSC niche is anatomically located at bone endosteum
● Based on anatomical location HSC can differentiate into oocytes,
chondrocytes, adipocytes or short lived stromal cells

136. Stem cells and cell therapy

Cell therapy aims to introduce new, healthy cells into a patient’s body, to
replace the diseased or missing ones. A challenge for this type of therapy is
having enough cells for transplantation into a patient. This is because
specialised cells, such as brain cells, are difficult to obtain from the human
body. Also, specialised cells typically have a limited ability to multiply, making
it difficult to produce sufficient numbers of cells required for certain cell
therapies. Some of these issues can be overcome through the use of stem cells.

Two main types of stem cells are being explored in the context of cell therapy:
pluripotent stem cells and tissue-specific (also referred to as adult) stem cells.

To obtain specialised cell types, either pluripotent or tissue-specific stem cells

are grown in a laboratory and treated with cocktails of molecules, which
provide signals for their development into functional cells.

141
 137. Principles and methods of tissue engineering
Tissue engineering is a biomedical engineering discipline that uses a
combination of cells, engineering, materials methods, and suitable biochemical
and physicochemical factors to restore, maintain, improve, or replace different
types of biological tissues. Tissue engineering often involves the use of cells
placed on tissue scaffolds in the formation of new viable tissue for a medical
purpose but is not limited to applications involving cells and tissue scaffolds.

Tissue engineering strategies generally fall into two categories: the use of
acellular scaffolds, which depend on the body's natural ability to
regenerate for proper orientation and direction of new tissue growth, and
the use of scaffolds seeded with cells.

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