Authorial Note:

Notes for weeks 11,13 and 14 are not

included (cuz I didnt make any). I
strongly recommend that you study
from the word documents uploaded in
Coospace and use this as a side dish. An
appetizer at most.

Good Luck. May the force be with you.

 Week 1:
G protein coupled receptors control:
cAMP 2nd messenger system
Phophonositol 2nd messegenr system

G proteins are active when bound to GDP.

cAMP means cyclic AMP.

Ligand is a signal molecule. Ligand binds to receptors and this

causes a cascade of events in the cell.

Adenylate cyclase is membrane bound. Uses ATP to catalyze

production of cAMP.
cAMP activates protein kinase.

Regulatory subunits are chaperone molecules.

cAMP is a 2nd messenger.

Activated protein kinases (by cAMP) cause voltage gated ion

channels to open.

Enzyyme coupled receptors. 2 types.

1) Enzyme is a part of the receptor. Intercellular
2) Enzyme is a separate molecule. Bound by H bonds.

Tyrosine is an amino acid.

Activation of receptor tyrosine kinase (RTK) by cross
phosphorylation.
Phosphate groups act as a signal for other intracellular
signalling molecules.
Adaptor and Ras activating proteins attach to RTK and this
activates Ras proteins.

Ras proteins are used in osmotic and heat control, mitosis,

apoptosis, inflammation, cell differentiation.

Activated Ras protein also activates MAP kinase for

proliferation.

G protein coupled and enzyme coupled pathways can overlap.

JAK/STAT pathway
1) JAK cross phosphorylate each other.
2) Activated JAKS phosphorylate receptors on tyrosine.
3) JAKS phosphorylate STATs
4) STAT dissociate from receptors and attach to DNA.
STAT (Signal Transducers and Activators of Transcription) are
involved in transcription and defence against viruses.

TGF (transforming growth factor).

TGF - Beta/SMAD pathway functions in:
apoptosis, cell proliferation, cell differentiation.

SMAD I binds to SMAD II and this binds to and DNA. Carries out
above.

TNF - tumor necrosis factor. Used in immune defense &

inflammation.
Hedgehog signalling pathways control early embryonic
development .

Morphogene: signal molecules whose activity depends on

concentration and duration of signal exposure.

Insulin reduces blood glucose level when it gets too high.

Glucose becomes ATP due to glycolysis and Kreb Cycle.

Insulin binds to insulin receptors and induce the production of
new glucose transporters and mobilization of existing ones.

Glucose metabolism involves liver cells, beta cells, insulin target

cells.

Apoptosis is programmed cell death. Before it, cellular content

exits in apoptotic bodies which can be ingested by phagocytic
bodies.

Necrosis is the sudden, accidental death of the cell.

Autophagy is another way the cell can die.

Cellular stress (extreme temperature, pH, excess Ca2+ ions,

irradiation) can lead to apoptosis.

Apoptotic pathways can be extrinsic or intrinsic.

Extrinsic: ligands/receptors
Intrinsic: withdrawal of survival factors, mitochondria controls
apoptotic events)
Week 2:
Local signalling:
- paracrine signalling
- synaptic signalling

Hormonal signalling is a form of distant communication.

Nervous sytem can be considered as as long and short range.
The response of a neurone to a stimulus depends on its internal
state.

Neurons can be sensory or motor.

Sensory neurons transport information to the brain
Motor neurons carry out effector

Interneurons can be local or project

The brain is made up of nerve and glial cells.

Charged particles move due to either concentration or electric

gradient. - Passive movement.
Ion pumps are active movement.

Cell membrane is semi permeable.

In the resting potential:

Lots of Na+ outside the cell and lots of K+ inside the cell.
Overall voltage is negative (-70mV).
K+ naturally (during resting potential) flows into the cell due to
an ion pump (active) and they also leave due to concentration
gradient (passive).
Resting potential - voltage across a plasma membrane of an
excitable unstimulated cell.

Ion channels (passive transport) can be ion specific channel (Eg:

specific to K+, Na+, Ca2+, Cl-)
or they can be cation selective channels. Any cation can go
through.

Ion channels can also be:

a) Leaky channels - non regulated
b) Gated channels - regulated (voltage, ligand gated, gap
junction, strech/pressure, phosphorylation gated) These open
to certain signals.

Ion pumps are forms of active transport. Thus they use ATP
In the Sodium/Potassium pump, 1 rotation means expelling 3
Na+ ions and taking in 2 K+ ions into the cell. uses ATP.

Sodium ion channels have 4 domains.

For voltage gated ion channels: activation gate has to be

opened (conformational change). This happens after voltage
sensors sense a voltage

The inputs to nerve cells can be excitory or inhibitory. Inhibitory

inputs open K+ channels.

Action potential only takes place if threshold (-50 mV) is

reached.
In action potential, there is (1) depolarisation. Na+ voltage gated
channels open and Na+ ions flood into the cell.
This makes the cell voltage positive (+30-40mV)
There is then (2) repolarisation. Closure of Na+ and opening of
K+ voltage gated channels. This causes the mV to become
negative again.
Cell then goes back to resting potential and equilibrium with
the help of sodium potassium pump.

Action potentials are passed along axons in waves.

Axons have myelin sheaths.
Neuron inputs can be from dendrites and synapses (electric or
chemical)

Synapses can be axosynapse or axodendrites

How synapses work

1) Action potential arrives at the synapse
2) Ca2+ ions enter the cells
3) Vesicles containing neurotransmitters fuse with plasma
membrane
4) Neurotransmitters are released into synaptic cleft
5) Neurotransmitters bind to receptors on other side of the cleft
6) Neurotransmitters are broken down and released.

Types of receptors:
- Iootropic: Fast effect, direct gating of ion flow
- MEtabotropic: Slow effect, indirect gating of ion flow

PCR - polymerase chain reaction &

DNA cloning (recombinant DNA technology)
are 2 important methods for DNA amplification.

PCR is in vitro (in test tubes and PCR machines)

DNA cloning is in vivo (i different bacterial host cells)

In PCR, parts of the genome (certain genes only) are amplified.

Taq polymerase. This is a DNA polymerase used in the PCR
process. It is thermophilic and can withstand very high
temperatures. its optimal temperature is 72 degrees.

For PCR, you need:

Taq polymerase,
template DNA,
dNTPs (building blocks of DNA)
Primers

PCR is artificial DNA replication and causes massive

amplification of DNA quantity.

DNA polymerase binds to primers and then starts DNA

synthesis. In DNA replication, primers are made by RNA primase.
In PCR, primers are made by DNA primase.

Steps of PCR:
1) Denaturation - Heat up to 95 degrees. This separates the 2
DNA strands and creates template strands.

2) Annealing - Cooled down to 54 degrees. Primers attached to

separated DNAs.

3) Extension - Heat up to 72 degrees. Taq binds to primers and

starts DNA synthesis.

30-40 cycles in 2 hours

35 cycles of PCR will give 34 billion copies. Thus its called
exponetial amplification.

Verification of PCR can be done on agarose gel.

PCR is used for diagnosis of infections (bacterial and viral)
PCR cant amplify RNA so to diagnose RNa viruses, there is an
extra step at the beginning which converts viral RNA to cDNA
using reverse transcriptase.

PCR can also be used to diagnose genetically inherited diseases

Monogenic diseases: cystic fibrosis, sickle cell anaemia, color

blindess, Huntingtons disease.
Such diseases are rare, but most are lethal and uncurable.

Cancer is a polygenic disease.

There are 23,000 human genes which code for 400,000 proteins.

Prenatal diagnosis can be:

- amniocentesis
- chorionic villus sampling
- preimplantation genetic diagnosis.

PCR us also used in forensic medicine to identify criminals.

in recombinant DNA technology: DNA fragments from different

sources are joined together to create human proteins (eg:
insulin).

Steps:
1) Bacterial plasmid and desired human gene is isolated.
2) Both molecules are exposed to the same restriction
endonucleases which cut/digest the molecule and creates
complementary sticky ends.
3) The human gene is inserted into the bacterial plasmid and is
sealed with DNA ligase to form recombinant DNA.
4) The recombinant plasmid is then inserted into a bacterial cell
5) Bacterial cell multiplies and express human protein.
Recombinant DNA technology produces:
insulin
blood factors
monoclonal antibodies
vaccines
anticoagulants
growth factors
interferons
colony stimulating factors
erythroproteins
human growth hormones
interleukins

Restriction endonucleases are DNA cutting enzyme only at

specific recognition sites.

For recombinant DNA technology you need:

Host bacterial cell
DNA ligase
Restriction endonucleases
plasmids
Vectors

Plasmids can replicate on their own.

Plasmids contain selectable genetic markers (eg antibody
resistance). They also have bacterial promoter and operator
sequences. In front of these sites are polylinker sites where the
human gene attaches.

To insert a gene into a plasmid, need to insert its cDNA

Sometimes, functional human proteins just cant be made in
bacterial cells due to needed post translational process and
bacteria don't have these facilities.
In such cases, eukaryotic cells are used instead which is a much
harder process.

Insulin is produced in E.Coli.

Week 3:
Immune system: defense against pathogenic organisms (fungi,
bacteria, viral) and against tumor cells..

Autoimmune disease is a malfunction of this system.

Immune system is decided into 2 parts:

Innate (non specific) - Action is not specific to invading organism
Adaptive (specific) - Action is specific to invading organism

Both have cellular and hormonal components.

Innate immune system:

- Response is antigen independent
- Immediate maximal response
- Not antigen specific.
- There is no immunologic memory

Adaptive immune system:

- Response is antigen dependent
- There is lag time between exposure and maximal response
- Antigen specific
- There is immunologic memory
Cells of immune system:
Lymphoid cells:
T cells lead to suppressor, cytotoxic and helper cells.
B cells lead to plasma and memory cells.
NK cells

Myeloid cells:
Granulocytic which includes neutrophils, basophils, eosinophils,
Monocytic which includes macrophages.

Barriers in the innate immune system:

A) Anatomical barriers
Mechanical: skin, cilia, mucus
Chemical: low pH in stomach
Biological: natural flora of intestines

B) Hormonal barriers
Complement system
Coagulation system
Interferon/Defensin.

C) Cellular barriers
Macrophage
Natural Killer cells
Neutrophil, BAsophil, Eosinophil

Inflammation is also an immune response.

After injury and invasion by foreign bacteria:
- Mast cells gather at site of injury
- Mast cells release histamine
- Histamine diffuses into nearby blood vessels and other
capillaries
- This leads to the dilation of capillaries
- This allows more phagocytes to easily travel to the site of
injury and kill invading organisms.

Antibodies work against extracellular parasites.

They block the ability of viruses to bind to receptors.
They can block the effect of toxins by masking them
They can mark pathogens for phagocytosis.

T cells work against intracellular parasites.

1) Induce apoptosis (cytotoxic T cells)
2) Activate macrophages
3) Activate B cells ----> leads to antibody production.

Antigen is a part of the pathogen outer surface which is

recognized by host cells.
Pathogens have certain molecules on them (PAMPs). These are
recognized by pattern recognition patterns.

Hormonal immune response:

(activation phase)
1) Antigen is taken up by phagocytosis and degraded in a
lysosome
2) Helper T cell is activated
3) T cell receptor recognizes the antigen fragment bound to a
class II MHC protein on the macrophage
4) T helper cell proliferates and forms clones containing
antigenic fragments
(effector phase)
5) T helper cells presents the antigenic fragment it has to B cells
and
6) B cells proliferate and differentiate into plasma cells and
memory cells.
7) Plasma cells produce antibodies.

Cellular immune response:

(activation phase - is the same)
1) Antigen is taken up by phagocytosis and degraded in a
lysosome
2) Helper T cell is activated
3) T cell receptor recognizes the antigen fragment bound to a
class II MHC protein on the macrophage
4) T helper cell proliferates and forms clones containing
antigenic fragments
(effector phase)
5) T cell releases perforin
6) This lyses the infected cell before the virus can multiply.

B cells become memory and plasma cells. Plasma cells produce

antibodies.
B cells can act over long distances by secreating antibodies into
the bloodstream.
T cells can migrate long distances but once there, only act
locally.

1) cytotoxic T cells: directly kill infected host cells

2) Helper T cells: Activate macrophages, B cells and cytotoxic T
cells.
3) Regulatory T cells: Inhibit the function of helper and cytotoxic
T cells.

Different resting B cells can differentiate into antibody

secreating effective beta b cells.

Epitopes: Those parts of an antigen that bind to the antigen

binding site on the antibody molecule or lymphocyte receptor.
B cells can turn into memory cells or effector cells.

Autoimmune disease: immune reaction against the khud ka

cells.
Eg: Diabetes Type I: immune response against insulin secreating
cells in the pancreas.

Antibodies are produced by effector plasma cells which come

from B cells.
Antibodies have highly variable antigen binding sites

Classes of antibodies:
IgG
IgM (pentamer)
IgA (dimer)
IgD
IgE

B cell development:
common lymphoid progenitor cell ---> pro B-cell ----> pre B cell
---->immature naive B cell ----> mature naive B cell.
Until and including the immature naive B cell stage, the
development takes place in the bone marrow. It then circulates
through the peripheral lymphoid organs.

Allelic exclusion: selection of antibody loci during B cell

development in the bone marrow.

AID can cause mutation of antibody genes during somatic

hypermutation.
In class switch recombination, there is DNA rearrangment.
Antibody diversification:
joining of gene segments
junctional diversification during gene segment joining
combinatral joining of joining of L and H chains.
Somatic hypermutation + class switch recombination.

T cell receptor has 2 different chains (alpha and beta chain)

There are 3 types of proteins on the surface of an activated

dendritic cell involved in activating a T cell:
1) MHC protein - displays antigenic fragments on cell surface.
MHC class I: is antiviral. Produced in every cell type
MHC class II: antigen presenting cells. Antibacterial.
2) Cell-cell adhesion proteins
3) Co-stimulatory proteins

How do effector cytotoxic T cells kill their target cells?

1) Perforin-dependent killing
2) Fas-dependent killing

Both induce apoptosis

T cells can diversify (using T cell receptor diversification) into

Negative Selection (Signalled Death)
Positive Selection (Signalled survival) ---> Survival and
Maturation.
Apoptotic cells
Week 4:
In monogenic diseases, the presence of a single mutant allele is
decisive in the development of the disease.

Genetic diseases can be monogenic, chromosomal or epigentic.

Mixed diseases include cardiovascular, autoimmune, metabolic
and cancerous diseases.

In complex diseases the "appropriate environment and other

alleles of different genes are also necessary for the
development of the disease.

Monogenic diseases can be autosome linked, chromosome

linked, dominant, recessive or due to incomplete dominance.

People heterozygous for a disease tend to have an advantage

as they are only carriers and not too severely affected.
Eg: heterozygous for sickle cell anemia are resistant against
malaria and don't suffer too much from sickle cell anaemia.

Elimination of mutant alleles takes time especially if they have

benefits.

X linked diseases are frequent in males and rare in females as

males have only one X chromosome and females have 2 X
chromosomes.

Y linked diseases ae rare due to few genes present on the Y

chromosome.

Examples of monogenic diseases.

1) Cystic Fibrosis
A normal functioning CFTR channel moves chloride ions outside
the cell while a mutant CFTR channel does not causing sticky
mucus to build up on the outside of the cell.

2) Sickle Cell Anemia

Healthy RBC have glutamine while sickle cell anemia is caused
due to the glutamine being replaced by valine. Normal RBC are
compact and flexible, enabling them to squeeze through small
capillaries. Sickle shaped RBC are stiff and inflexible causing
them to get stuck in small capillaries and cause blockages.

3) PKU (phenylketonuria)
People with PKU have a defective PAH enzyme
Normal PAH enzyme breaks down phenylalanine.
Due to defective PAH enzyme, toxic levels of phenylalanine
builds up in the body.

4) SCID (Severe combined immunodeficiency)

There are 2 types: X linked SCID and ADA deficiency.
Defects in the ILR2G protein can cause immune cells to
malfunction leading to SCID.
SCID gene therapy involves the transplantation of own, better
stem cells into the individuals bone marrow.

Deoxyadenosine is a natural compound found in the body. It is

an intermediate product made during the breakdown and
synthesis of DNA. ADA binds to deoxyadenosine and converts it
into deoxyinosine which is not toxic.
In ADA deficiency, deoxyadenosine is not broken down and thus
high levels of deoxyadenosine kill T and B cells of the immune
system leading to SCID.

5) Duchene Muscular Dystrophy is due to a mutation in the

dystrophin gene which is present on the X chromosome.
6) Thalassemias
Anemia is a characteristic feature of thalassemias.
It is caused due to:
decrease in number or size of RBCs
decrease in the number or O2 binding capacity of hemoglobin
molecules.
Genetic causes:
Mutation in the coding region for of haemoglobin: leads to
abnormal protein structure.
Mutation in the regulatory region of hemoglobin gene: leads to
few hemoglobin molecules

7) Galactosemia
In normal individuals: GALT binds to galactose and converts it
to glucose which is then used for energy.
In galactosemia, there is no GALT present which leads to
galactose concentration rising to toxic levels causing kidney
failure, enlarged liver and brain damage.

8) Hemophilia
Is X chromosome linked.
Those with hemophilia have no fibrin generation and thus there
is an inability for blood clotting to take place.

9) Albinism - melanin formation problem.

90% of the time, the cause is tyrosinase gene albinism.
Other 10%, the cause is P gene albinism.

10) Color blindness

X chromosome linked.
Males are effected more than females.
Can be: achromacy, monochromacy, dichromacy, anomalous
trichromacy.

Cancer causes are:

infection (20%)
inherited (10%)
sporadic (70%

Cancer tissues:
Carcinoma (epithelial cells - 65%)
Sacroma (cells of mesodermal origins)
Leukemia (WBC)

Tumors can be:

Benign
Pre malignant
Malignant.

Tumors are intelligent. They:

- Divide in situations in which normal cells would normally wait
(activation of oncogenes)
- Ignore 'stop dividing' comments (inactivation of tumor
suppressor genes)
- Lose the ability to commit suicide (ignore death and lack of
survival signals)
- Persuade nearby blood vessels to build the infrastructure they
need to thrive.
- Accquire immortality. (telomere regeneration)
- Invasion (activation of metastasis genes)
- Evasion of immune system detectors (activation or blocking of
appropriate genes. (activation or blocking of appropriate
genes)

2 important genes in cancer development: proto-onco gene

(stimulates cell proliferation) and tumor suppressor gene
(inhibits cell proliferation).
Due to the normal interpllay of both genes, there is regular cell
division.

Too much of proto onco gene or too little of tumour suppressor

gene leads to cancer. This is all due to mutations.
In tumor suppressor genes, 2 mutations or loss of an allele lead
to cancer.

When there is too much proto onco gene it is referred to as

oncogene.

In colon cancer, there is multiple hit hypothesis. This means

multiple mutations needed for cancer development.

STR - Short tandem repeat

VNTR - variable number tandem repeat.

Genome of each individual is unique and is inherited from both

parents.

In DNA profiling, genetic variation is probed in order to

differentiate between individuals.

STRs are found surrounding the chromosomal centromere.

Y-STR. These STRs are found on the Y chromosome and are used
in identifying males.

Mitochondrial DNA is found in non nucleic cells such as hair

shafts. It is circular and inherited through female lineage but is
found in both genders. Used for difficult samples like hair, bone,
teeth.

PCR
1) Denaturation: separating DNA strands
2) Annealing: addition of primers
3) Elongation: using Taq polymerase to create DNA strands.
Taq polymerase works best at high temperatures.

More the GC content, more the H bonds present (as there are 3
between guanine and cytosine), higher the melting point.

RFLP needs large, long amounts of DNA

DNA sources for DNA prolifing: blood, saliva, semen, teeth, hair,
epithelial cells.

Some genes wake up after death which can be used.

Week 5:
How to map bicoid locus:
1) Linkage mapping (finds which chromosome has the bicoid
locus)
2) Genetic mapping (Where is the bicoid locus on the
chromosome)
3) Molecular cloning

Hybridization: the joining of complementary nucleic acids.

Bicoid protein:
- it is localized on the nucleus.
- It is likely to be a transcription factor - a DNA binding protein
that regulates gene expression)
- Is a morphogene. This means it determines cell fate directly.
Bicoid protein binds to both RNA and DNA using its
homeodomain to regulate transcription and translation.

Maternal effect is determined by the mothers genetic material.

This genetic material comes from mRNA and protein molecules
that are deposited into the egg cytoplasm during egg cell
formation

Ways to help infertile women:

1) Cytoplasm injection to replace the missing component in egg
cells.
2) Transplantation of zygote nucleus into an enucleated egg.
Called 3 parent child as DNA comes from 3 sources: DNA of
maternally, paternally derived chromosomes in the nucleus and
mitochondrial DNA molecules in the egg cytoplasm
3) Fusion of the zygote with an enucleated egg.

Perfect loading of the egg cells is a necessary condition for the

development of a healthy child.

Genetic barrier: mutations causing infertility

Environmental barriers for proper fetal development.

1) Insufficient nutrition. To combat this folic acid is consumed in
pill form
2) Smoking: Hinders proper loading of the egg cell and is
harmful for the developing fetus.

Zygote lethal mutations: zygote dies because of its own faulty

genetic content.
Gap genes encode transcription factors.
Pair rule genes regulate the formation of segment pairs

Bicoid protein controls the expression of the Kruppel and

hunchback gene. Both are gap genes.
Bicoid protein is a negative regulator of Kruppel gene and
positive regulator of hunchback gene
Negative regulator means: It is the process of regulating gene
expression by inhibiting the expression of that specific gene.

Atavism: the recapitulation of an evolutionary ancient trait due

to mutation.

Uterusbicornis: Uterus splits into 2. 2 Uterine horns are present.

Homeobox: highly conserved sequence in the homeotic genes.

Homeodomain: homeobox encoded part of the homeotic
protein.
A section of the homeodomain is the helix turn helix motif, one
of the DNA binding motifs.

Homologus recombination: recombination between homologous

sequences.

FGF10 gene is necessary for limb formation.

Hox-D 9--13 genes are necessary for the formation of limb parts
D9 - Scapula
D10 - Humerus
D11 - ulna and radius
D12 - metacarpals
D13 - digits
Limb development in the anterior & posterior direction is
regulated by ZPA.

Lack of apoptosis leads to webbed digits as cells remain

between fingers.

Polydactyly (multiple digits) is a dominating inherited trait. 6

genes are involved in in the build up to it.

Sources of embryonic DNA:

- cells in amniotic fluid
- Chorionic villa
- Blood of umbilical cord
- Fetal cells collected from the blood of the mother.

Week 6:
Human genome has 23 pairs of chromosomes and a bit of
mitochondrial DNA.
First 22 are called autosomes and the 23rd chromosome is
called sex chromosome.

Centromere: connecting point of the 2 arms of a chromosome.

After DNA replication, there are 2 identical sister chromatids for

every chromosome.

Cancer cell karyogram shows much more chromosomes than

normal. More than 23 pairs (46) present. There are also
structural chromosomal mutations.
All cancer cells have chromosomal number abnormalities.

Chromosomal disorders can be structural or numerical.

Structural disorders (problem with chromosome structure):
- Insertions
- Deletion
- Duplication
- Inversions
- Translocations

Numerical disorders (problem in number of chromosomes):

- Polyploidy (3n, 4n... from each chromosome)
- Aneuploidy (extra one/two or one/two less of just one
chromosome)
Eg: Trisomy (2n+1), Tetrasomy (2n+2), Monosomy (2n-1)
Aneuploidy happens because of non disjunction.

In non disjunction, monosomy and trisomy take place together.

In meiosis I, homologous chromosomes get separated.

In meiosis II, sister chromatids get separated

Non disjunction could happen at meiosis I (homologous

chromosomes doesn't get separated) or it could happen at
meiosis II (sister chromatids don't get separated).

Mitotic non disjunction affects a few cells in the body.

If meiotic non disjunction take place and the affected gamete
gets fertilized, every cell in the new organism is affected.

Spindle Assembly Checkpoint (SAC) is a metaphase checkpoint.

It prevents non disjunctions and therefore prevents aneuploidy.

Numerical Aberrations (in autosomes):

- Patau syndrome (13-trisomy)
- Edwards Syndrome (18-trisomy)
- Down syndrome (21-syndrome)
Any other autosomal trisomy or any monosomy at all is lethal to
the feetus.

Numerical aberrations (in sex chromosomes):

- Turner Syndrome (XO)
- Triple X syndrome (XXX)
- Klinefelter syndrome (XXY)
- Double Y syndrome (XYY)

Patau syndrome (13-trisomy):

Brain, spinal cord abnormalities. Heart, eye defects.
Only 5-10% of affected individuals live past the first year.
1/16,000

Edwards syndrome (18-trisomy):

Heart defects. Small head and jaw. Intellectual disabilities.
1/5,000

Down syndrome (21-trisomy)

Cognitive delays. Increased risk of hearing and vision problems.
Mild to moderate intellectual disabilities. Half of all cases are
born with a heart defect.
1/8,000

As age of woman increases chances of her offspring being born

with Down Syndrome increases.

Turner Syndrome (XO):

Short stature. Affected girls don't undergo puberty. Infertility.
1/2500

Triple X syndrome (XXX):

increased risk of learning disabilities. Delayed development of of
speech and language skills. Taller than normal.
1/1000

Klinefelter syndrome (XXY):

Small testes. Do not produce as much testosterone. LEads to
enlarged breasts, reduced body and facial hair. Infertility,
1/1000

Double Y syndrome (XYY)

Taller than normal. Increased risk of learning disabilities.
Delayed development of speech and language skills.
1/1000.

Structural abnormalities (single chromosomal mutations):

- Deletion of a segment
- Duplication of a segment
- Inversion of a segment
(2 chromosome mutation)
- insertional reciprocal
- Translocation

If telomeres break off, chromosomes could become a ring

structure.

p arm (short arm) above the centromere.

q arm (long arm) below centromere.

- Isochrome: loss of one arm of a chromosome and duplication

of the other to replace it.

Structural chromosomal abnormalities are due to double

stranded DNA breaks.
double stranded DNA breaks due to:
- ionizing radiation
- replication errors
- mechanical stress on chromosomes
- retroviruses, retrotransposons, DNA transposons
- Mutagenic chemicals
- Heavy metal ions

Double stranded DNA breaks lead to:

- inhibition of cell cycle progression
- DNA repair
- Apoptosis
- Cellular senescence (cell stops replicating. Just exists)
If the above 2 don't happen then genomic instability happens:
oncogenesis.

Repairing DNA breaks via:

NHEJ (non homologous end joining)
HDR (Homologus directed repair)

Telomere is made up of repeated sequences. They get shorter

and shorter after every replication. This causes aging.

Telomeres prevent fusion of the chromosomes by noon

homologous end joining repair (NHEJ) system.

Once telomeres get too short, cellular senescence or apoptosis

happens.

Chromosomal deletion syndromes:

- Wolf Hirschhorn syndrome
- Chri du Chat syndrome

Chromosomal duplication syndromes:

- Charcot-Marie-Tooth disease.
Week 7:
Cancer immunotherapy can be via:
- Monoclonal antibodies targeting cancer associate proteins.
- Immune Checkpoint Therapy (Antibodies that prevent T
lymphocyte inactivation aka. checkpoint blockade)
- Adoptive immunotherapy
- Cancer Vaccine Therapy

In monoclonal antibodies targeting cancer associate proteins:

monoclonal antibodies bind to cancer specific antigens.
Effector cells recognize the antibody, come close to the tumor
cells and kill them. (Tumor specific IgG)

Angiogenesis inhibition: Prevents growth factor working in a

capillary. Due to this, tumor doesn't get nutrients and oxygen.

Antibodies can attach radioactive isotopes to cancer cells

(radioimmunotherapy)

(Bispecific Antibody Therapy) Uses artificial T cell molecules that

goes near the cancer cells.

Can attach to the tumor cells and kill them: Killer cells,
Liposomes, special enzymes, immunotoxin, immunocytokines.

Antigen -------> Plasma Cell + Tumor Cells -----> Hybridoma ------>

Monoclonal Antibodies
100 % human origin antibodies have low potential for
immunogenicity. (Low chance of getting rejected by the human
immune system.

Chimeric antibodies (65% human): suffix is -ximab

Humanized antibodies (90%): suffix is -zumab

Cetuximab/ERBITUX (anti-EGFR) is for colorectal cancer

Trastuzumab/HERCEPTIN is for breast cancer

ADCC - Antibody dependent cellular cytotoxicity

In tumor specific IgG:

Antibodies bind Natural Killer cells, monocytes, granulocytes
and they induce apoptosis or kill cancer cells.

Dendritic Cell Activation can be induced by antibodies. It leads

to T cell activation.

Complement-mediated cytotoxicity results in target cells death

through the development of MAC (membrane attack complex).

Antibodies can block ligands, inhibit receptor dimerization and

indcue apoptotic signalling

ADCP - antibody dependent cell mediated phagocytosis

PCD - programmed cell death
CDC - complement dependent cytotoxicity
ADC - antibody drug conjugate

Modifying monoclonal antibody structure:

- Alter amino acids in a constant region
- Glycomodified mAb
- Using different human mAb isotype
- Link radioactive isotype to mAb with a stable linker
- Ling drug ti mAb with cleavable linker
- Crosslink regions from 2 mAbs
- Insert DNA for mAb variable region fused to signalling peptide
into T cells to induce expression of CAR.

Immune Checkpoint Therapy:

Antibodies do not attack cancer cells but stimulate T cells
(which kill cancer cells)
Thus it is not tumor specific but is universal

After a successful immune response, T cell must be inactivated.

T cell inactivation can be doe by dendritic cells.

PD1 is very important in T cell inactivation. PD1/CTLAM signalling

pathway leads to decreased proliferation, decreased cytokine
production.

Blocking this pathway is an important part of immune

checkpoint therapy. (inhibiting the inhibitors)

Antibodies can also stimulate T cell activator receptors (Eg: Oxo

40)

Thus immune checkpoint therapy uses combined use of

stimulatory and inhibitory antibodies on T cells.

Genetic engineering used in all of the below:

- Adoptive Cell Transfer Therapy

- Tumor infiltrating lymphocyte therapy
- Antigen specific T cell therapy
- T cell receptor therapy
- Chimeric antigen receptor therapy

Antigen specific T cell therapy:

Using endogenous T cells sourced from peripheral blood. Fully
personalized. DNA is extracted from the patient. Mutatiosn are
found and an artificial structure is created which is mixed with T
cells of patients.

Cancer Vaccine Therapy:

Cytotoxic T cells release perforin and granzymes to induce
apoptosis in target cells.
Providing dendritic cells with tumor specific antigens is crucial.
Dendritic cells can inudce T cell differentiation, T cell activation,
T cell inactivation.

Tumor specific activation of dendritic cells can be done with

nanocarriers carrying tumour antigene coding RNAs.

Nanomedicie:
- improved drug delivery system
- New therapies
- In vivo imaging
- Nanosensors

Nanocarriers can be taken up by receptor mediated

endocytosis.
Advantages of nanocarriers:
- Higher stability
- Higher solubility
- Higher permeability
- Less side effects
- Less toxicity

Week 8:
Obesity: Accumulation of excess body fat.
Intake of energy exceeds expenditure.
It is a disease with no universally effective medicine against it.

Evolutionary theories for obesity:

- Thrifty genotype
- Gluttonous genotype
- Slothy genotype

BMI = mass (kg)/height^2 (m)

Effects of obesity:
- increased chances of stroke
- increased chances of breathing problems
- increased chances of diabetes
- increased chances of cardiovascular diseases
- increased chances of arthiritis
- increased chances of chronic inflammation
- increased chances of early death

With a BMI over 30:

Cancer risk increased 64% in males
Cancer risk increased 48% in females.

Genetic component of obesity is 40-70%.

Weight and height is a polygenic determinants.

Polygenic obesity: 36 genes have been found so far which

contribute to obesity.

Allelic variants of FTO gene results in the expression of certain

transcription factors.

3 types of fat cells:

Brown fat cell
Beige fat cell
White fat cell

Brown fat cells convert chemical energy to heat to protect

against cold weather.
White fat cells are the most common fat cell. They are used to
store fat and found beneath the skin and abdomen.
Beige fat cells are immature cells in white fat tissue which
matures to burn fat.

Brown fat cells are similar to muscle cells. Is smaller lipid

droplets.

When protons go through UCP 1 (uncoupling protein 1),

thermogenesis (heat production) happens.
UCP1 is present in the mitochondria membrane.

Activation of brown adipose tissue is a powerful therapeutic

avenue to treat obesity.
Brown fat cells use fat to create heat (thermogenesis) via UCP 1.

F10 allele variants affects the differentiation of adipocyte

precursor cells.
Hypothalamus controls hunger and appetite.

Monogeneic obesity: Obesity caused due to mutation in only

one gene.

orexigen: increasing food intake/appetite

Anorexigen: decreasing food intake/appetite.

Leptin decreases appetite and increases fat metabolism

Leptin therapy/injections can be used to treat obesity.
Loss of leptin or leptin receptors leads to obesity.
Antibody has:
- 2 heavy chains
- 2 light chains
Held together by disulfide bonds. Is a quartnery structure.
Antibody aka immunoglobulin.

In variable region of antibody there is J,D,V parts.

Antibody detects epitopes of antigenes. One antibody can bind

to 2 epitopes at the same time.

First antibody class created is IgM. Then IgD, IgG, IgE, IgA.

Class Switching: B cells switches from producing one class of

antibody to another class.

IgM, IgD are primary antibody repertoire. They are made before
the antibody comes in contact with the antigen.
IgG, IgE, IgA are secondary antibody repertoire. They are made
after the antibody has come in contact with the antigene.

IgD is never secreted out of the B cell.

For protein detection: it is possible to detect antibodies

produced against a pathogen, not the pathogen proteins
themselves.

Polyclonal antibodies recognize the same antigene but different

epitopes.
Monoclonal antibodies recognize the same antigene and same
epitope.

Growth Factor Receptor initiates map kinase cascades in cells

after the receptor dimerizes.
Immunohistochemistry: Determination of the localization of
proteins in cells and tissues.

Antibodies are used in diagnosis and therapy.

Western blotting also detects proteins using electric current

and is based on molecular weight. Lyme disease is detected by
western blotting.

ELISA:
Specific and quantitative detection of hormones, cytokines,
immunoglobulins, viral and bacterial antigenes in the blood.

Protein chips separate labelled proteins into healthy and

diseased ones.
Red labelled proteins expressed in healthy cell/tissue,
Green labelled proteins expressed in cancerous/diseased
cells/tissue.

Week 9:
Restriction enzymes aka restriction endonucleases.
Can cut specific sequences of phage DNA.
Bacteria prevents the effect of restriction enzymes on its self by
methylating restriction sites.

DNA ligase joins 2 sequences.

Plasmids - Small, circular DNA bacterial fragments present in

the cytoplasm.
They have polycloning site and antibiotic resistance gene can
be added to it.
Recombinat plasmids have foreign DNA in them.
Molecular cloning:
Generation of recombinant plasmids by DNA ligation using
plasmid vectors.
Delivery of plasmid vector to cells.
Multiplication of plasmids within cells.
Multiplication of bacterial cells
Colony arises

Generation of recombinat plasmid is because of restriction

enzymes and DNA ligase.

PCR
Start with a single DNA molecule. PCR produces millions of
copies. Useful in amplifying DNA.
Steps of PCR:
- Denaturation - 2 DNA strands are separated using high
temperatures.
- Annealing - Using 2 DNA primers. Primers recognize the
templates to be amplified,
- Synthesis - Recognize primers and makes new strands.
This is one cycle. Many cycles are done to get millions of copies.
Natural DNA replication uses RNA primers.
Requirements foe PCR: DNA Polymerase, DNA template, 2 DNA
primers, Buffer, DnTPs.

Gel electrophoresis:
Detetction of DNA sequences and proteins.
Migration of DNA sequence on the basis of their molecular
weight.
DNA molecules move from negative pole to positive pole
because DNA is negatively charged.
SDS-PAGE used to detect proteins.
Proteins are heated with SDS and mercaptoethanol. Proteins
lose 3D structure, form negatively charged SDS molecule.

Detection of macromolecules:
Southern Blot (detection of separated DNA that is radiolabeled)

Detection of RNA:
Northern Blot (using autoradiography)

Detection of proteins:
Western Blot (using primary and secondary antibodies.)

Gel retardation analysis detects DNA/protein interaction.

Immunohistochemistry detects proteins on plasma membrane

or in the cytoplasm. Uses antibodies connected to a fluroscent
dye.

In situ hybridization - detection of DNA/RNA.

Labelling of probe can be radioactive or non radioactive
(fluorescent/enzymatic)

FRET - Fluorescense Resonance Energy Transfer

Week 10:
Biotechnology can be genetic or non genetic.

Cloning can be reproductive or therapeutic.

If nucleus removed from egg cell - called enucleated egg cell.

Cloning:
Normal cell fused with enucleated egg cell to give a cloned
embryo.
Or the nucleus of the normal cell is transferred to the
enucleated egg cell. to give a cloned embryo.

Genetic material comes from the normal cell (eg: skin cell).

The cloned embryo can be:

inserted into a surrogate mother for reproductive cloning. The
offspring has the exact same characteristics as the organism
which provides the normal cell.

For therapeutic cloning, the cloned embryo becomes embryonic

stem cells.

For creating hybrids:

1) The nucleus of a single human cell - containing all of its DNA -

is transferred into an animal egg.
2) The female animal egg has all of its genetic information
removed.
3) The resulting chimera embryo is 99% human
4) The embryo grows in a lab via cell division.
5) It is harvested for stem cells. These embryonic stem cells can
become any type of tissue and are used for research

Human hybrid involves 3 parents:

Woman 1: Healthy nuclear DNA goes into
Woman 2 (surrogate mother): Healthy donor egg
Man 1: Provides sperm which ferilizes the reconstructed egg cell.

GMO - genetically modified organism.

PG gene in tomato causes ripening.

The tomato is genetically modified to inhibit PG gene and thus
increase storage
In 3rd generation GM plants/animals:
gene injection to zygote. Integration of gene to the genome.
Transgenic gene develops in surrogate mother. Offspring
expresses the gene.

Genetic recombination

Homologus recombination: targeted DNA insertion.

Can be knock in, knock out.

Cultured cell types:

Primary cell culture
Tumor cell culture
Immortalized cell culture.

Gene delivery can be via:

Transfection
Virus Vector
Liposomes

Foreign gene (transgene)

To acquire immunity in bacteria:

adaptation
crRNA biogenesis
Interference

CRISPR/Cas9 system used in gene editing.

Recombinant genomic DNA uses foreign DNA with homologous

flanking sequences.
Week 12:
Stem cell - A cell that can differentiate to specialized cells.

Totipotent - gives rise to any cell type (zygote, 8 cell embryo)

Pluripotent - Gives rise to any cell type except tropoblast cells
(blastocyst inner cell mass)
Multipotent - Gives rise to limited type of cells (adult stem cells)
Unipotent - Gives rise to identical cells. (skin cells)

Non dividing cells include neurons, skeletal/cardiac muscle

cells.

Adult stem cells found in bone marrow

Cystic Fibrosis is due to faulty CFTR gene.

Neural stem cells can form:

Glial cells
Neurons

Hematopoietic stem cells:

Platelets, RBC, WBC, mesenchymal cells

Adult stem cells ----> Progenitor cells ----> Differentiated cells

Human embryonic stem cells from:

in vitro fertilisation (replace egg cell nucleus with the nucleus of
a differentiated cell) aka Nuclear transfer.
From inner cell mass cells

Inner layer of skin contains stem cells.

Induced pluripotent stem cells done by reprogramming body
cells.
They are reprogrammed to form embryonic steam cells.

Blastocyst can give embryonic stem cells.

Gene therapy:
Germline gene therapy
Somatic gene therapy

Isolate embryo and find faulty gene. Genetically modify nucleus

using vector and enucleated egg.

In vivo gene therapy:

Using viral vector/liposome to deliver gene to body.
Ex vivo means cell based gene delivery.

Gene therapy:
1) Delivery of foreign genes to cells
2) Integration of foreign DNA into host genome

Repeated use of viral vectors may cause an immunological

response .

Gene therapy can be used for: cystic fibrosis, spinal muscular

atrophy, Parkinsons disease

Live vaccines use attenuated (weakened) viruses.

Inaactive vaccines use dead viruses.