Module 5

Nervous System
Nervous system can have either:

1. Neurons(nerve cells)
These are excitable meaning they can convey information through action potentials
from one to the other part of the body. Part of the distal signalling system.

2. Glial cells
Support neurons physically, immunologically (defend from pathogens), and
metabolically(supply nourishment and remove the metabolic waste from brain)

A nerve is a bundle of axons that come from may different neurons.

Dendrites collect information from the surrounding axon terminals or take in the nerve
impulse initated by the central nervous system itself.
Neurone features:
Cell body: Contains nucleous and most of the cell organelles. Integrate incoming signal
and generate outgoing signal to axon.
Dendrites: Shrub like projections that extend from the cell body. Collect electrical
signals.
Axon: A projection that is longer than dendrites. Carry action potential
away from the cell body.Pass electric signals to dendrites of another cell or to an

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 effector cell.
Axon terminals: Swollen tips of the nerve ending at the end of axons. Places from where
impulse passes from one neuron to the other.

Differential gene expression allows the body to form different types of specialized
neurons.

More dendrites = more inputs

Longer axons = longer distance communication

Brain and spinal cord → Central nervous system (CNS), Driving all sort of
processes like driving, movement, etc. Responsible for all voluntary actions (some
involuntary too).

Neurons extending or residing out the brain and spinal cord and their supporting
cells → Peripheral nervous system (PNS)

Sensory neurons = Afferent neurons → carry sensory information into nervous system.
Sense information and carry it to CNS.

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 Motor neurons = Efferent neurons → Carry commands from CNS to physiological and
behavioral effectors such as muscles and glands.
Interneurons → Integrate and store information and communicate between afferent and
efferent neurons.

Glial Cells

Do not generate or transmit electric signals but can release neurotransmitters.

Important
Neurotransmitters are chemicals that can pass on certain chemical signals . That said,
Glial signals generate or transmit electric signals.
Support neurons physically, immunologically (defend from pathogens), and
metabolically(supply nourishment and remove the metabolic waste from brain)
Types

1. Oligodendrocytes:
Insulate axons of neurons in CNS

2. Schwann cells
Insulate axons in PNS through myelination

3. Astrocytes
Contribute to blood brain barrier through myelination

4. Microglia
Can invite macrophages and are involved in the immunological defence of the brain

Neurons insulated by myelin sheaths can conduct action potential at high rates. They
also ensure that the action potential is not impacted by the touching of any external
neurons.

Multiple Sclerosis → Demyelinating disease

In which system destroys myelin sheaths in some regions of CNS, which significantly
decreases the rate of propogation of nerve impulse

Nodes of Ranvier provide a boost to the action potential.

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 Astrocytes (Learn these properties)

a. Contribute to forming the blood brain barrier which ensures no harmful chemical
reaches the brain. (btw antibodies dont go into the brain)

b. Surrounded the smallest, most permable blood vessels in the brain

c. Permeable to fat soluble substances such as anesthetics and alcohol, that’s how
they put your brain to sleep

d. Take up neurotransmitters released into the synapse

e. Supply neurons with nutrients

f. Signalling properties, can release neurotransmitters

g. Aid in repair and regeneration of neurons

h. Can change the composition of the blood

Microglia

a. Act as macrophages i.e. macrophages can go to a pathogen, eat it, and take it
away so it doesn’t impact neurons.

b. Mediators of inflammatory responses → A way that our immune system provides

support to the nervous system when things get out of macrophages’ hands.

c. Immune defense for the nervous system

Electric Signals
Membrane potential:
the difference in electrical charge between the inside and the outside of a cell (across a
membrane)

Resting potential:
Represent the normal resting state of the cell until when the action potential has not
arrived.

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 1. Intracellular conditions are negative due to the presence of large anions, such as
proteins inside the cell. ( NOT due to chlorine ions, as in fact, they are high in
concentration outside the cell )

2. There’s a higher concentration of potassium ions, and lower concentration of

sodium ions inside the cell than outside.

Receptors working at the resting potential:

1. Potassium leak channels.

Send small amount of K+ down the concentration gradient outside of the cell.

2. Sodium leak channels

Send small amounts of Na+ down the concntration gradient inside the cell

3. Sodium-Potassium pump
Primarily responsible for mainting the potentials. Sends 3 Na+ outside and 2K+
inside the cell using ATP by active transport.

Negative molecules attract K+ from moving outside. When this factor equates that of K+
leaking outside through electrochemical gradient, we achieve the resting potential

Resting potential is -60mV to-70mV.

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 Action potential

Sudden, large, transiet reversal in restig potential generated by sudden oepning and
rapid closing of ion channels.

Its value is 50mV

Once an action potential from nearby cells arrives, the Na+ voltage-gated channels
open. Note that there needs to be a certain threshold level of this potential around
these channels for them to open. If the threshold is surpassed, the voltage-gated Na+
channels open, allowing more Na+ to move inside down the potential gradient. This
rapidly depolarizes the cell as more and more Na+ voltage gated channels not start
having experiencing enough voltage to surpass the threshold level and open.
This causes a spike in the membrane potential, with it rising up to 50mV from about -60
to -70mV

Repolarization and Hyperpolarization

One the potential has reached 50mV, the voltage-gated Na+ channels close, and the
voltage-gated K+ channels open. These channels now allow K+ to move down the
concentration outside the cell, making the inside of the cell negative again, and this is
called repolarization . This channel allows even more K+ to move outside than the Na+
that entered the cell, taking the cell into a potential even below that of the resting. This
phenomenon is called hyperpolarization.

Restoring resting potential

Once hyperpolarization occurs, voltage-gated K+ too close. With this, the Na-K pump
still working restores the resting potential.

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 Types of Ion transporters and Channels

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 1. Na-K pump (described above)

2. K+ leak channel

3. Na+ lead channel

4. Voltage-gated Na+ channel → Only open and allow Na+ to move inside down the
potential gradient when an action potential arrives.

5. Voltage-gated K+ channels → Only open after Na+ voltage-gated channels have

closed and cause the cell to go into hyperpolarization.

Different mechanisms for the working of gated ion channels →

1. Voltage gated → changes in voltage around the channel can allow it to open

2. Ligand gated → Attatchment of a ligand extracellular or intracellular can allow it to

open

3. Mechanically gated → Open due to some physical influence e.g. the Venus Flytrap
plant uses its mechanisms

In short
Resting potential is maintained by → Na+ leaky, K+ leaky, and Na-K pump
Action potential occurs when → Na+ voltage gated channels open causing
depolarization i.e. the potential becomes less negative
Hyperpolarization → K+ voltage gated channels open, and K+ moves outside. In fact,

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 even more K+ moves outside than Na+ had entered, momentarily leading to a potential
even lower than the resting.

Na+ voltage gated channels go into a refractory phase once they close i.e. they can not
be opened again until a short period of time. This is essential to ensure that the action
potential travels in a single pathway.

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 How do neurons communicate with other cells?
We use neurotransmitters → chemicals or molecules which can transfer a signal from
one neuron to the other.

Synapse → A specific type of junction where a neuron meets its target cell and
information in the form of a neurotransmitter molecule is exchanged.

When an action potential arrives in the neuromuscular junction/neuron-neuron junction

1. The depolarization of the presynaptic membrane causes the voltage-gated calcium

channels to open, so Ca2+ moves inside the neuron

2. Ca2+ pushes the neurotransmitter vesicles containing acetylcholine outside the cell

3. Acetylcholine travels across the junction to the acetylcholine receptors on the post-
synaptic membrane.

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 4. Activation of these receptors causes chemically-gated Na+ channels to open so
Na+ rushes inside the post-synaptic membrane, making it depolarized as well. So
by now, the action potential has been transferred across the neuron.

Neuromuscular junction:

Neuron-Neuron junction:

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 There can be different types of neurotransmitters:

1. Excitatory

Open cation channels

Cause influx of Na+ that depolarizes te postsynaptic membrane
e.g. Acetylcholine, Glutamate, Serotonin

2. Inhibitory

Open either Cl- ir K+ channels (More chlorine inside the cell, or less K+ outside the
cell, both ensure the cell remains polarized and negative)
Suppresses firing→ Less depolarization
e.g. GABA, Glysin

It is also necessary to turn of the responses once they’ve occurred, so responses could
be generated again if need be.

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 So, to remove the neurotransmitter from the synaptic cleft:

1. Enzymes such as Acetylcholinesterase (AChE) destroy the neurotransmitters in the

synaptic cleft
(If this enzyme was inhibited, and acetylcholine is not removed, spastic muscle
paralysis will occur as Na+ will keep moving inside the post-synaptic membrane
until equillibrium is reached whereby the potential inside and outside the post-
synaptic membrane becomes equal)

2. They can also be taken up by active transporters on the membrane by Glial cells
(we can change this uptake through drugs)

Microbiology

Similarities b/w Prokaryotes and Eukaryotes:

1. Plasma membrane and Ribosomes → present in both

2. Common sets of metabolic pathay

3. Replicate their DNA semiconservatively

4. Use DNA to encode protein → similar genetic codon

Differences:

1. Prokaryotic cells use binary fission to divide. Eukaryots use mitosis and meiosis

2. Organization of genetic material and content is different (Prokaryos don’t have

histones and introns and much more. Also prokayotic chromosomes are present in
a region called a nucleoid, while eukaryotes have them in the nucleus)

3. Prokaryots don’t have any membrane bound organelled

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 Most of the eukaryotes don’t have cellulase to digest cellulose but use indigenous
bacteria to do so.
Nitrogen fixation in plants is dependent on bacteria
Particular strains of the microbes can induce autoimmune diseases
They can also produce essential metabolic products

Uncle Robert about microbes (not that important)

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 Microbes also provide Cofactors → Certain small chemicals which are required for
functioning of enzymes. Like DNA polymerase requires Magnesium as a cofactor to
work.
They can also metabolize food, or stimulate immune responses when attacking other
organisms
Eating antibiotics can disrupt microbial populations in our body
Fungi
More complex than bacteria, and is a eukaryote since it contains membrane-bound
organelles

1. Yeast → Unicellular, can replicate primarily asexually but also sexually

2. Mold → Filamentous, replicates both sexually and asexually

Bacteria
Has much more RNA than DNA. (That’s why we added RNAase in bio100 when
isolating plasmids from a bacterial culture)
Bacteria can come in different sizes and shapes
They can be motile only if they have flagella
Classifying bacteria:

1.Size and Shape

2.Characterized on the basis of genes - genotypic properties
3.On the basis of observable characteristics - phenotypic properties (i.e. their
colony the colour, shape, and structure of the colonies)
4.Microscopic, macroscopic, and biochemical characteristics (e.g. certain
bacteria utilize certain metabolites for growth. We can test them with these
metabolites to classify them)

Gram Staining → Differentiating bacteria on the basis of differences in cell wall

Bacterial Cell wall

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 It acts as a barrier to prevent osmotic pressure from building up and bursting the
bacteria if the water potential is too high outside of the bacteria.

Composed of highly cross-linked peptidoglycan

Many antibiotics(e.g. β -lactam antibiotics) impact the bacterial cell wall to inhibit its
functioning and kill the bacteria. Note that these antibiotics don’t impact any of our cells
since they don’t have any cell wall to begin with.

N-acetyl glucosamine(NAM) and N-acetyl muramic acid(NAM) linked together with

peptides to make the cell wall. (so peptido= peptide bonds and glycan chains from these
two molceules make the peptidoglycan cell wall)
Cross linking only occurs b/w NAM btw

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 Gram-Negative cell wall

1. Has a thin peptidoglycan

2. Has lipopolysaccharides

There are three layers:

Outer membrane of cell envelope
Middle peptidoglycan layer (very thin)
Plasma membrane towards inside of the cell

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 Gram-positive cell wall:

1. Thick peptidoglycan layer

2. No lipopolysachaide

Just two layers:

Outer thick peptidoglycan
Plasma membrane towards the inside

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 Gram-positive cells retain the dye due to this thick PG and No lipopoly

Glycocalyx: Capsule Slime layer and S-layer (Present outside the cell wall)
→ Layer of polysaccharide extending from the surface. It can either be in the form of a
i. Capsule→ Can not be washed
ii. Slime layer → Can be wahsed (imp)
→ Made of sugars
→ Protects against drying i.e. even if the water starts moving out of the cell, there will
always be some of it left due to a protective mechanism by glycocalyx
→Protects against immune response (the layer contains certain sets of glycoprotein
which help the bacteria to mimic as a resident glycoprotein, so our immune system
sometimes fails to recognize this bacteria as a pathogen)
→ An important virulence factor i.e. increases bacteria’s pathogenicity

Bacterium motility-Flagella

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 Pili →Allow bacteria to attatch to each other to share genetic material b/w them
Fimbriae → Slightly shorter than pili and allow bacteria to attatch to a substrate

Viruses
Obligate intracellular species i.e. will always be infectious if they attack, and infect
only if they reach the inside of the cell.
They are acellular i.e. they can’t make ATP

Can’t grow on media since they need to be inside cells to grow

Just replicate and assemble the machinery
EITHER DNA or RNA
No protein synthesis machinery→ no ribosomes
Not sensitive to antibiotics→ viruses are inside the cell, and they don’t have the
structures that antibiotics target
Structure
Nucleocapsid → Nucleic acid+protein shell
Contain structural and non-structural proteins

1. Matrix proteins

2. Replicase and transcription factors (replicase replicates the viral genome)

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 (Shape of capsid depends on nucleic acids inside the capsid)

Envelope

1. Lipid bilayer external to capsid

2. Driven from host (made of host cell membrane)

3. Contains viral transmembrane proteins (important to facilitate the entry process of

viruses into cells)

Not every virus has an envelope (Important)

Budding:

So you can see from here how the envelope of a virus is that of a membrane
Envelope also allows the virus to ext the cell without killing it
(Lytic cycle of a virus → Kill the host cell as it leaves
Lysogeny cycle → Virus stays inside the host cell and uses its machinery
So having an envelope can prevent the lysis from happening)
Contains atleast one virally encoded protein

Viruses are very small, and can be visualized usually under electron microscopes only.

Hosts for viruses:

1. Bacteria (e.g. bacteriophages)

2. Anthropods

3. Animals

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 Can be differentiated based on their genome
DNA viruses
RNA viruses→ further into→ +Strand RNA viruses(running 5’→3’,can be converted into
a protein directly) or -Strand RNA viruses(running 3’→5’, needs to be first converted to
apositive-strandd before into a protein)

Viral Transmission routes:

Steps in viral infection

1. First of all virus needs to bind to a receptor to on our cell.

2. Once attatched, it moves inside by a process endocytosis

3. Virus will now uncoat (the capsid will wear off and the nucleic acids, transcription
factor, replicases etc will move out)

4. The biosynthesis → virus will hijack the host’s protein translational machinery

5. Assembly →New phage particles will be assembled in the cell

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 6. New viral particles made and released into the extracellular fluid. (It won’t kill the
cell while moving outside only if the virus has an envelope)

Influenza Virus

1. Enveloped (so doesn’t kill our cell as it leaves it)

2. Contains a segmented RNA genome ( so RNA is present in different fragments

instead of a single strand)

3. Undergoes extensive genetic variation (makes sense why we keep getting flu)
(Imp)

Genetic Shift: e.g. the Influenza virus from a human and a duck combines in
another specie to form a new reassorted form of this virus.

Genetic Drift: Point mutations can occur in the hemagglutinin(HA) and

neuraminidase(NA) genes making proteins on its surface, leading to variation in the
virus.
(Viruses have a very high mutation rate since replicases don’t have proofreading
mechanisms)

Human pathogens → Bacteria, vius, fungi, prions(not properly folded proteins),

and parasites

Treatment and prevention modalities

(imp) Antimicrobials → Bactericidal(end up killing bacteria) or Bacteriostatic(inhibit

bacterial growth. The β -lactam as we dicussed before also inhibits the bacterial growth)
Vaccines → Prevent both bacterial and Viral infections

Immunology
Study of the cells and tissues that mediate immunity and the investigating of genes and
proteins underlying their functions

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 Two types of immune systems:

1. Innate
ALWAYS present and ready inside our bodies to defend against pathogen
Antigen non-specific → there for and attacks all types of pathogens without fully
differentiating the pathogens. That said, they can detect some specific patterns.
Rapid response
No memory → have the same response even if the same pathogen attacks the 1st
or the 100th time(primary response)
Present from birth

2. Adaptive
Antigen specific → very specific in detecting pathogens
Slow in response → takes some time to kick in this immune system after the
pathogen attacks (is a secondary response)
Memory → once you’ve been infected by some pathogen, the body will be ready to
counter this pathogen in a better way if it attacks in.

Origin of immune cells

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 All these different cell types come from totipotent stem cells, being specialized at each
stage.
Cells of the Two Immune Systems

1. Macrophages and Neutrophils are phagocytes that can engulf foreign cells by
phagocytosis. Form part of the innate immunity

2. Dendritic cells → Antigen presenting cells(APC) → innate

3. T and Natural killer T cells → both innate and adaptive

Immune System Proteins

1. Antibodies → Proteins produced by adaptive immune system that bind specifically

to certain substances identified by the immune system as non-self -antigens

2. Major Histocompatibility Complex (MHC)

Proteins used to display antigens on the surface of cells. MHC-I detects self-cells
and MHC-II detect non-self cells (Imp)

3. T Cell receptors
Recognize and bind to antigen presented by the MHC proteins on the surface of

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 other cells. Usually the first step to an immune response

4. Cytokines
Soluble signalling proteins that bind to cell surface receptor and alter their behavior

Components of Innate Immunity

Surface Barriers → Physical (skin) and chemical (saltiness, dryness, presence of
normal flora preventing development of serious colonies on the skin, enzymes,
bactericidial proteins, tear, saliva, antibacterial peptides)
Internal Barriers → Mucus, Lysozyme(attacks bacterial cell wall), Defensins (short
peptides or other kinds of proteins toxic to pathogens that kill after inserting themselves
in invaders)

Pathogen recognition by cells of innate immunity

Although innate immunity is non-specific, they can still detect some patterns

Pattern Recognition Receptors (PRRs)

> Present on ALL innate cells
> Intracellular(e.g. viruses) and Extracellular
> Include Toll like Receptors (TLR) or Nod like Receptors (NLR)

Pathogen Associated Molecular Patterns (PAMPS)

>Cell surface antigens
>Lipopolysaccharide (on gram -ve cell wall)
>Lipoteichoic acid (on gram +ve cell wall)
>Nucleic Acid (double stranded RNA triggers interferron response)

So PRRs detect PAMPs

Function of innate Immune cells

1. Phagocyte adheres to pathogens or debris

2. Endocytosis of phagocyte into the cell

3. Lysosome containing acid hydrolase enzymes attatches to the phagocyte

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 4. Lysosomal enzymes digest the particles, leaving a residual body

5. Exocytosis of vesicle removes indigestable and residual material

Specialized proteins and cells participate in the Innate Immune system

>Complement proteins
i)Part of the innate immune system ii) Can be activated by both adaptive and innate
responses iii)Lyse the invading cells by literally punching holes in them, or increase the
recognition of the pathogen by innate cells
>Interferons
i) Molecules including double stranded RNA (dsRNA) induce production of interferons

Specialized proteins and cells in the Innate Immune system

1. Phagocytes
>Circulate freely or adhere to certain tissue
>Infected cells, viruses, or their fragments recognized by phagocytes
>Chemicals present in phagocytes → Defensins, nitric oxide and reactive oxygen
intermediates kill pathogens

2. Natural Killer Cells

>Distinguish virus-infected cells from normal cells

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 >Initiate apoptosis (programmed cell death in target cells

3. Dendritic Cells (like a bridge b/w the innate and adaptive immune system)
>Present antigenic fragments on their surface. This allows the information to be
conveyed to the adaptive immune system for the secondary response to now take
place
>Activate the adaptive immune system

Inflammation
Involves the recruitment of cells and molecules, primarily pro-inflammatory cytokines
The body Isolates the damaged area to stop the spread

Killing by natural killer cells (Non-phagocytic) (imp)

1. Non-phagocytic and generally non specific

2. Their cytoplasmic granules contain Granzyme and Perforin which are delivered
directly into the target cells to kill it

3. NK and N T cells don’t proliferate upon activation

4. So, Tumor or virus infected cells can be detected as well.

So a natural killer cell has both an activation receptor and an inhibitory receptor.
Whenever it attacks a target cell, if its inhibitory receptor detects an MHC I present on
the target cell, it means it’s a self cell so NK calms down and does nothing. If however, it

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 detects a target cell without the MHC I, the NK cells release perforin and granzymes
into this target cell to kill it as it believes it has intracellular toxic parasites(e.g. viruses).
This is how it differentiates between an infected and a healthy cell.

Antigen
A substance that induces an immune response by stimulating cell immune cells (mostly
detected due to their conformational or sequential form)
> They can be large complexes
> Presented by specific molecules on the surface of antigen-presentinge cells called
MHC

Antigens → Presented on MHC → Presented on Antigen-presenting cells(Dendritic or B

cells)

The Adaptive Immune Response

Antigen-presenting cells can either engulf free antigens and present them on their
surface, or directly detect a pathogenic cell’s antigen
T-helper cells can then detect antigens presented by MHC on antigen presenting cells,
after which they can either

i. Activate B cells to produce antibodies, which act on the free antigens or on the
infected/pathogenic cell directly. (Note that these B cells could have directly
recognized the free antigens with their antibody receptors on their surface)

ii. Activate T killer cells to bind and kill the pathogenic cell by apoptosis by releasing
perforin or granzyme into the cell

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 The pathogenic cell as well as a dendritic cell displaying antigens of a non-self cell does
it through MHC II
Inductive Adaptive Immunity
Induction can occur by two overlapping mechanisms (imp)

1. Cellular
>Based on cell-mediated responses from T-lymphocytes to kill cells. Requires a
direc cell-to-cell contact as the T cells need to travel to the infected cell/pathogen
>Detect mainly intracellular pathogens

2. Humoral
>Based upon antibodies from B lymphocytes. No direct cell-to-cell contact needed
>Detect mainly extracellular pathogens

Cells of adaptive immunity B and T lymphocytes

B cells
>Develop and mature in (B)one marrow
>Also antigen-presenting cells
>Express cell-surface receptors → B cell receptor(BCR) that binds to antigen
>Can and only B cells produce antibodies
>Can store memory of which antibody was made against which pathogen

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 T cells
>Develop and mature in (T)hymus
>Express T cell receptors on their surface
>Upon activation differentiate into T-helper and T-cytotoxic cells

Clonal Selection
The different types of B cells are there for different types of antigens, but once the
antigen reinfects you, it will lead towards proliferation of certain types of B cells i.e. B
cells are specfic for different types of antigens. Once a certain antigen is detected by
the B cell, that specific B cell will proliferate so there are multiple B cells against that
type of antigen
Clonal Deletion
Any immature B or T cell that shows the potential to mount immune response against
self antigens is eliminated

Cancer
Cancer is a large group of diseases characterized by the uncontrolled growth and
spread of abnormal cells
Abnormal cells can grow into disorganized mass of cells → Tumour
Tumour can either be

1. Non-cancerous (Benign)
Cancer is localized, and the uncontrolled division stops after some period

2. Cancerous (Malignant)
Cancer can spread from one part of the body to the other, and the cells keep
dividing abnormally.

Normally, each cell keeps receiving a signal which tells it to divide, and if something
goes wrong the cell receives an apoptotic signal telling it to die.
But tumour cells have an abnormal growth. They don’t need any growth signal to
survive so can undergo rapid and uncontrolled growth.

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 Carcinoma → epithelial cancer
Sarcomas→ Bone and muscle connective tissue cancer
Leukemia → Blood cancer
Lymphoma → Lymphocytes cancer

Cancer can come from just a single cell that underwent abnormal procedures which
then divides into a lump and spread if malignant. The cancer mass can also form
different layers.

When a malignant cancer starts to spread through the bloodstream, T killer cells detect
and kill many of these cancer cells. However, often some of the cancer cells survive and
reach some other part of the body whereby they start dividing again. This transfer of
cancer cells from one region to the other is called
metastases

Not all cancers are equally lethal, equally treatable

Most of the cancers are environmentally linked, only few happen due to a genetic basis
Carcinogen is any chemical that may cause cancer

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 Somatic cell mutations actually result in carcinomas, germ cell mutations are dangerous
for your offsprings, but not you.

Not all carcinogens are mutagens → i.e. it is not necessary for cancer to occur only
through a mutation; rather, there can be other reasons as well.
Hallmarks of cancer

1. Sustaining proliferative signalling → only one signal is enough for a cell to keep
dividing

2. Evading growth repressors → They will continue to grow even in the presence of
growth repressors

3. Activativating invasion and metastasis → It is the metastasis property of tumours

which makes them cancerous

4. Enabling replicative immortality → they are immortal, they’ve solved the telomer
problem

5. Inducing angiogenesis → A process by which cancer cells develop blood vessels

between them so when the mass grows, the inner cancer can be provided with
nutrients and their waste products be removed efficiently

6. Resisting cell dealth → cell doesn’t respond to apoptotic signals

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 How do mutations cause tumourigenesisns? (imp)

1. Gain of function mutations

>Proto-oncogenes→ genes that are always on (constitutively active)
>A type of mutation that occurs when a cell literally gains a function. For instance
there might be a transcription factor that’s normally repressed, but now it’s turned
on and it causes uncontrolled proliferation of the cell.
>These are dominant mutations. Only a single chromosome from the mother or
father undergone mutation is enough.
>In short, gains the ability to proliferate without any signals

2. Loss of function mutation

>Occurs when tumour suppressor genes(which are normally monitoring if the cell
goes abnormal, in which case they cause apoptosis of the cell) lose their ability to
work due to mutations.
>Tumour suppressor mutations are recessive. Both the chromosomes from the
mother or father have to undergo mutation.

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 > In short, failure of apoptotic signals to stop the cell growth

Normally, a ligand attatching to a receptor would activate the cascade leading to further
actions. In cancer, whether the ligand is attatched or not, the receptor stays active (i.e.
constitutively active → oncogene)

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 Inactivation of tumour suppressor gene
Can either be due to

1. A genetic change i.e. a change in codon of the tumour suppressor gene

2. An epigenetic change i.e. methylation on a region of the tumour suppressor gene

which silences it.

provided that both changes are in the homozygous formation

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 The p53 alarm system
p53 is called the guardian of the genome!

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 Hyerproliferative signals, DNA damage, telomere shortening, and hypoxia(shortening of
oxygen) can all activate the p53 which then causes the cell cycle to i) be arrested so no
faulty DNA is passed on, ii) or senescence i.e. the cell becomes a terminally
differentiated cell to prevent further divisions when telomere has greatly shortened iii) or
apopotosis if hypoxia or DNA damage has become too severe

p53 is impacted in almost all of the cancer cells

In 50% it has undergone a mutation,
In the other 50% it has undergone a functional inactivation

‫کوئی راہ میں جچا ہی نہیں‬ ‫مقام فیؔض‬

‫( جو کوئے یار سے نکلے تو سوئے دار چل‬qtd. in AJ tut6 46:20-46:35)

So once the cell loses p53, it is bound to become cancerous.

Oncogene activation

1. Deletion or point mutation in the coding sequence →a hyperactive protein is made

in normal amounts

2. Regulatory mutation → normal protein greatly overproduced

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 3. Gene amplification → gene copy number increases and a normal protein is greatly
produced

4. Chromosome rearrangement
i) Nearby regulatory sequence causes normal protein to be overproduced (e.g. a
constitutively active promoter region)
ii)Fusion to actively transcribed gene produces hyperactive protein (e.g. the

In short, a mutation in the coding region or chromosome rearrangement can lead to a

hyperactive protein being produced in normal amounts
If a mutation occurs in regulatory sequence, or there’s a gene amplification, a normal
protein is produced at higher levels.
Greater amounts of a protein present in cells can greatly increase the cell division.
Recall the RAS stuff

GTP isn’t hydrolysed anymore, and RAS is constitutively active causing cell divisions.

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 Self-explanatory

Mutations accumulate over-time. There can be multiple mutations in a step-wise manner

which eventually lead to invasion and metastases
Cancer cells grow initally as benign, but can eventually become metastatic if they start
invading other parts of the body.

Altered Sugar Metabolism in Cancer (Warburg Effect)

Tumour cells have a different metabolism system for cancers. They use glucose
primarily as building blocks (e.g. DNA, lipids, sugars etc.), and use anaerobic
respiration which produces lactate as the main by-product producing less amounts of
energy

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 Drug development

Treatment of cancers:
Surgery → physically remove the tumour before it becomes malignant
Radio and Chemotherapy → uses medicines and radiation to stop cells having very
rapid cell divisions (so often hairfall occurs as cells around hair follicles are rapidly
dividing). Most drugs work by not allowing microtubules to perform in anaphase to stop
cell cycles.
Targeted/Personalized Therapy → Every person has a different mutation, so we can
pinpoint mutations and design drugs specifically for that person. With this we can
prevent side-effects and make our drug more effective overall

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 Target Identification → Find a molecule that is integral to to the cell. Like inhibiting
which protein in a cancer cell will lead to decreased tumour growth etc. We can find
such targets by comparing sequences of healthy and cancer cells; if all cancer cells
seem to be expressing a gene while the healthy ones don’t, that one protein is definitely
doing something to support the cancer growth

Target Validation → Take that specific sequence and express that protein etc. we
found above (which is as we are saying only active in cancer cells) in multiple healthy
cells. If the protein causes the healthy cells to become cancerous, the protein was
definitely a good target.

Assay Development (testing the drug) → Let’s say we have about a thousand drugs to
test against this protein, which is a kinase. What we can do is to test our drugs and
monitor ATP levels. If increasing our drug concentration causes the concentration of
ATP to decline at a decreasing rate, it means the protein is being inhibited as it is a

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 kinase and kinases use ATP to work.
We can screen large amounts of drugs at the same time.

Ex-vivo testing (living cells but outside the organism) → What phenotypes can you
expect in your cells upon the addition of drug. In our case of cancer, we will expect
slower cell division and increased apoptosis.

In-vivo testing (testing living cells inside the organism) → We never know the cells
might behave differently inside the conditions of an organism

Clinical trials → Has different phases we will discuss later

Target Identification

Detecting Infectius diseases

>Identify molecules that are conserved across bacterial kingdom are indispensable for
the bacterial growth (e.g. β -lactam which targeted bacterial cell wall)
>Viruses (they synthesize all their proteins all altogether and then cleave it later).
HIV virus encodes for a protease which is responsible for cleaving the peptides of its
total protein when translated. So by targeting this protease, we can signifcantly impact
HIV’s function.

Detecting Non-infectious diseases (cancer, diabetes etc.)

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 >Genechips/Microarrays → help in identify genes being under or overexpressed
>Proteomics → Which protein is being expressed more (or less)
>Karyotype → Checks chromosomal abnormalities (e.g. cancer cells often increase
their chromosome number)
>Detect mutations
>Check aberrant DNA methylation patterns → tumour suppressor genes could be
silenced by methylation
All this can help us identify possible disease causing molecules.

The slide shows there can be multiple levels in a pathway that could have become
constitutively active, so we can inhibit any one of them to stop the pathway.
Protein kinases as drug targets for cancer
>Good drug targets for cancer since they are often mutated in cancers, and they have a
certain ATP binding pocket which can be inhibited.

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 Types as mentioned in the slide
i)Tyrosine specific → only phosphorylate tyrosine specific kinases
ii)Serine/Threonine
iii)Dual specific → can phosphorylate either Kinase and serine or kinase and threonine
residues

Oncogene activation via chromosomal translocation

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 So Bcr gene from chromosome 22, and AbI gene from chromosome 9 can both
translocate and form a philadelphia chromosome containing fused Bcr/Abi gene which
when transcripted and translated into a protein, forms a constitutively active kinase→so
keeps phosphorylation its residues which can lead to increased cell proliferation

Target Validation
Selection for cancer

i)Deregulating the target linked to a clinical outcome i.e. impacting the target alters does
alter outcomes in the best possible way
ii) There should preferably be present a suitable drug/complementary structure that
could detect and inhibit the target
iii) Preferable if you can inhibit that target’s interaction with DNA or protein which leads
to cancer or some other disease

iv) Manipulation of target in a living model system causes malignant phenotype (imp)

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 Assay Development
(For a kinase target, if adding the drug increases ADP concentration it means it is
increasing the drug’s ATP)
Testing of drugs can either be

Biochemical Assay (in vitro) i.e. testing in living cells but outside organisms e.g.
in test tubes
Advantages:
>Well defined and easy to control environmental
>Clean environment as external proteins, conditions, solution etc. is under our control.
No random molecule is playing its part
Issues:
>Solubility of proteins often not possible → can have hydrophobic exterior
>Not physiological i.e. you never know how all the external conditions in a real organism
are playing their part.

Cell based Assay

Advantages:
>Physiological → if the drug is working over here, that’s a very good sign since it is
doing it despite all the external real conditions inside an organism
>Can express any protein (ofcourse, the target protein will be present in the organism)

Issues:
>Messy → alot of other proteins, and extrnal influences are playing their part as well
>Low signal in some cases due to noise since our drug might also be interacting with
alot of other proteins

High throughput screening

A technique through which we can screen alot of compounds as potential drugs against
are drugs

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 Generic.

Lead generation and optimization

>Identify lead compounds that show promise
>Play around with their functional groups to increase their activity
>Check activity of modified compounds in assays e.g. the drug could better fit and
interact in the target’s attatchment site
IC50→ concentration of the drug required to inhibit 50% of the target protein
So a drug with a lower value of IC50 is more potent.
Lipinski aunty’s rule of five to correlate with a good drug (imp)

1. Fewer than five hydrogen bond donors (estimate by counting the total number of
OH and NH groups in the molecule)

2. Fewer than 10 hydrogen bond acceptors (estimated by the total of N and O atoms)

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 Like this molecule has 2 hydrogen bond donors, and 3 hydrogen bond acceptors

3. Molecular weight of less than 500

4. A partioning coefficient (logP) of less than 5

Structural base drug design

Takeaway point → In silico models can be used to check the interaction of the drug with
the target protein

Pharmacokinetics and Pharmacodynamics(imp)

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 Kinetics part → What your body is doing to the drug
Dynamics part → What drug does to your body
ADMETox properties

Absorption → Passes the drug from Gastro-Intestinal track into the blood stream →
should be higher (Kinetics, body is absorbing)

Distribution →Gets to target tissue (blood brain bariier) → should be higher

(kinetics?)

Metabolism → should not be readily metabolized (but not too slow that it starts
accumulating) (kinetics)

Excretion → should not be readily secreted (kinetics)

Toxicity → should not be toxic to other cells or tissues(dynamics)

Clinical Trials
Pre-clinical testing → before entering a living organism (or at most in the mouse)
Go to FDA

Phase I → prefer on doing patients on life sentences → determining safety and dosage

Phase II → Effectiveness and side effects

Phase III → Verify effectiveness and monitor long-term use side effects
(The sample size of patients keeps increasing along the phases)
Now the FDA might approve one out of the millions of compounds evaluated.

Mode of action of Gleevec→ a successful drug produced for treatment of cancer

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 Gleevec works against the Bcr-Abi constitutively active protein kinase, by attaching itself
on the ATP pocket of the kinase, inhibiting it from phosphorylating residues and the cell
from proliferating

Cancer drug Resistance

>Cancer cells can respond and develop resistance against such drugs by
i) changing protein structures ii)start expression of transmembrane proteins that pump
the drug out of the cell
etc.

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Module 5 52