Section 3: Special Topics Flashcards
Second law of thermodynamics
Entropy will increase over time
Early human dev. - three main techniques
Ability to dissociate multi-cellular organisms into single cells
Ability to barcode those cells
Sequence every cell - make a map of where those cells are being derived from
What happens in embryogenesis
Patterning
Major axis laid down
3 germ layers form
Rudiments of major organs
Embryogenesis - patterning
Development process where cells acquire diff identities depending on their relative positions in their embryos
Pattern laid down on a small scale, typically <1cm
Lay down the 3 main germ layers to allow further differentiation to take place
Embryogenesis - major axis
Anterior - head
Posterior - tail
Dorsal - back
Ventral - tummy
Embryogenesis - 3 germ layers
Broad brushstrokes of development
Further differentiation takes place within those germ layers
Ectodermal layer
Mesodermal layer
Endodermal layer
Fate map
Tells you what a cell is likely to become if development continues normally
Ectodermal layer (outside, blue) Mesodermal layer (between, red) Endodermal layer (inside, yellow)
Sperm vs egg size
Sperm quite small in size compared to egg
Sperm - major components
Genetic material
Tail to help it swim towards egg
Sac of enzymes (acrosome) on head - helps the sperm burrow through the layers surrounding the egg (corona radiata)
When is meiosis completed
Post-fertilisation, where 2 nuclei become pronuclei
Placenta is a combination of…
The maternal tissues and tissues from the embryo itself
Types of proteins
Digestive enzyme/catalytic - break down nutrients in food into small pieces that can be readily absorbed
Transport - carry substances throughout the body in blood or lymph
Structural - build diff structures, e.g. cytoskeleton
Hormone signalling - co-ordinate activity of diff body systems
Immunological - protect body from foreign pathogens
Contractile - muscle contraction
Storage - provide food for early development of embryo
Toxins - used by pathogens or other organisms to cause disease
Peptides
Short polypeptides (~less than 50 amino acids) Very short peptides can be referred to as dipeptides, tripeptides or tetrapeptides
Residues
Individual amino acids in a polypeptide/protein
Forms of amino acids
Un-ionised/deionised form
Zwitterionic (doubly ionised) form - dominant form, at physiological pH ~7.4, +ve and -ve charge on either side)
Peptide (amide) bond formation
Condensation / dehydration synthesis reaction
Peptide bond properties
Bonds are rigid and can’t rotate due to resonance
O-C-N-H of peptide bonds are essentially co-planar
Rotation can occur at the single bonds between the α-carbon and its neighbouring atoms
R amino acid side chains can be cis (same side) or trans (one up one down) - typically trans as cis is less stable due to steric repulsion
N and C terminus
N (amino) terminus
C (carboxyl) terminus
Proteins always drawn N to C i.e. the direction they come off the ribosome
Protein structures - complex
To facilitate all varied functions proteins provide, they can adopt complex structures
Shape and function of proteins
Shape of a protein is critical to its function
Shape is driven by chemical properties and sequences of amino acids in the protein
Binding of substrates to an active site can cause…
Conformation changes, which provide a function or strengthen the interaction
Proteins - primary structure
The unique sequence of amino acids of a protein
Entirely driven by DNA sequence of gene encoding protein - can deduce the primary structure of a protein by the DNA sequence of a gene
Proteins - secondary structure
Localised folding of the polypeptide driven by H bonding interactions within the polypeptide backbone
Two common types: β (pleated) sheet, α helix
Diff amino acids have a tendency to favour structures
Can fairly accurately predict regions of secondary structure in a protein by the sequence
β sheets
Can be parallel or anti-parallel
Driven by H bonding between a backbone amine (N-H) group on one strand and a backbone carbonyl (C=O) group on other strand
Large aromatic residues and β-branched amino acids are favoured in β strands
α helices
Right-handed helix
Normally each turn is 3.6 amino acids with a pitch of 5.4Å
Driven by H bonding between a backbone amine or backbone carbonyl group 3 or 4 residues earlier
Tightly packed with almost no free space within the helix
Side chains protrude from helix
Helices - amino acid examples
Methionine, alanine, leucine, glutamate and lysine like to form helices
Proline and glycine don’t
Proline may create unique conformations in polypeptide, and often referred to as a helix breaker as it’s always at the end of a helix
Proteins - tertiary structure
Where secondary structures fold in on themselves
The 3D shape of a protein - primarily driven by the chemistry of side chains and interactions between them
Range of non-covalent interactions - H bonding, ionic bonding, d-d interactions, Van der Walls forces
Ionic: opposite charged R groups attract and like charges can repel
Tertiary structures - hydrophobic interactions
R groups of non-polar amino acids orient themselves towards the center of the polypeptide and cluster to avoid water
In membrane spanning proteins, hydrophobic R groups may be outside interacting with lipid tails
Tertiary structures - disulphide bridge
Amino acid cysteine forms a covalent bond with another cysteine through its R group –> disulfide bond
Thiol (S-H) groups are oxidised, removing the H and forming a covalent linkage between the 2 sulfur atoms
Tertiary structures - H bonds
Polar ‘R’ groups on the amino acids form bonds with other polar R groups
Tertiary structures - hydrophilic interactions
R groups of amino acids orient themselves outward to interact with water and maintain solubility of protein
Tertiary structures - ionic bonds
Positively charged R groups bond together
Relative strength of bonds
Disulfide > Ionic > Hydrogen > Van der Waals
Tertiary structure - co-factors
Some proteins (particularly enzymes) can co-ordinate a co-factor or 'prosthetic groups' within the protein using R groups May be essential for function/structure of protein Metal ions (Mg, Mn, Zn, Fe, Ca), organic molecules (heme) or vitamins
Proteins - quaternary structure
Multiple folded protein subunits
Driven by ionic interactions, H bonding and hydrophobic interactions
Often dynamic - may have one protein coming on and off another protein or moving around
Homooligomers or heterooligomers
Not all proteins form quaternary structures
Types of proteins
Globular
Fibrous
Membrane proteins
Globular proteins
Typically soluble in water Often enzymes, transport, immune Often irregular sequence and secondary structure Moderate or no quaternary structure Lower stability
Fibrous proteins
Typically insoluble in water Often structural Often repetitive primary and secondary structure High level of quaternary structure Highly stable Keratin, actin, collagen, silk
Membrane proteins
Transverse through a lipid bilayer (membrane)
Transport, receptors, signalling, adhesion
Transmembrane region - single α-helix or a α-helical bundle
Generally high degree of non-polar (hydrophobic) amino acids, which face membrane
Polar (hydrophilic) side chains face inwards
Quite abundant
Levinthal’s paradox
Very large number of degrees of freedom in an unfolded polypeptide chain
100 amino acid proteins will have 3^198 diff conformations
Most protein correctly fold in the ms - μs time scale
Protein folding
Need help to fold correctly - correct environment: solute, salt conc, pH, temp, macromolecular crowding etc
Temporal - co-translational folding as the polypeptide is coming off the ribosome i.e. N folds before C terminus
Chaperones
Enzymes involved in disulfide bond formation
Methods for structure determination
X-ray crystallography
NMR
Cryo-electron microscopy
Resolution
The distance corresponding to the smallest observable feature - if two objects are closer than this distance, they appear as one combined blob rather than two separate objects
Units: 1 Å (angstrom) = 0.1nm
Protein structure representations
Backbone model
Ribbon model
Wire model
Space-filling model
Homooligomers
A protein where there are two or more subunits of the same protein
Heterooligomers
A protein where there are multiple polypeptides coming together to form one functional group
Abbe’s diffraction limit
If we have a perfect microscope, we can still only resolve objects sized half the wavelength of the imaging light - can’t see viruses or proteins
Chaperones
Dedicated proteins which bind to folding proteins, e.g. binding to a patch that is particularly prone to misfolding, or encircling the whole protein to create a localised environment that favours a particular type of folding
Skin - total body surface area and body weight
Average 2 m^2
7 - 16% of total body weight
Skin - thinnest and thickest
Thinnest: eyelids - 0.5mm
Thickest - palms and soles of feet - 4mm or more
Human skin functions
Protection/barrier, e.g. from pathogens and UV
Blood reservoir - can hold 8-10% of total blood volume
Thermoregulation - sweat glands and blood vessels
Sensation - touch/pressure, pain, temp
Vitamin D synthesis - Vit D precursor require modification by UV before active form can be made in liver
Thermoregulation - blood vessels
Vessel constriction in dermis reduces blood flow –> reduced heat loss
Vasodilation in dermis increases blood flow –> increased heat loss
Epidermis
Provides a barrier and continued renewal
No structural strength
Mainly consists of layers of keratinocytes
Epidermis - layers of thin and thick skin
Thin skin has 4 layers of keratinocytes
Thick skin has 5 layers; 5th layer is stratum lucidum
Epidermis - vasculature
No vasculature - all nutrient supply and waste removal occur by diffusion to vascular system of dermis
Stratification
Crucial for barrier function and continued renewal of epidermis
Stratum basale Stratum spinosum Stratum granulosum Stratum lucidum Stratum corneum
Stratification process
Proliferating keratinocytes on bottom of epidermis push cells up and away from dermis
Undergo programmed cell death
Complete epidermal turnover approx once a month
Basement membrane
Interface between dermis and epidermis
Important for epidermal attachment to dermis
Basement membrane contains…
Collagen IV
Perlecan
Nidogen
Laminin 332
Mutation in basement membrane proteins can result in…
Epidermolysis Bullosa (epidermis easily detached by shear forces)
Rete ridges
AKA Dermal papillae
Contour provides resistance to shear forces
Wave-like pattern strengthens attachment between epidermis and dermis
Pigmentation - Melanocytes
Reside at epidermal side of BM - spaced out as their dendrites allow single melanocytes to contact and transfer melanosomes to an average of 36 diff keratinocytes
Make melanosomes which contain melanin
Pigmentation - Melanin
Pigment that gives skin its colour Pheomelanin - red Eumelanin - brown/black Protects from UV Nuclear cap protects keratinocyte DNA
Langerhan’s cells
Immune cells
Surveil the epidermis for foreign organisms - if barrier if broken, Langerhan cells move into dermis and go find help from immune system to destroy bacteria in epidermis
Dermis
Dense matrix made up of collagen and elastin fibres
Strong and supple - provides structural strength
Thickness varies - thickest on soles and palms
Very stable, cellular turnover minimal
Dermis - fibroblasts
Produce collagen and elastin
Collagens - strength
Elastin - elasticity
Dermis layers
Papillary dermis - high cell density, loose CT
Reticular dermis - low cell density, dense CT
Dermis - vasculature
Supply nutrients and remove waste for both dermis and epidermis
Laminin 1+2 lines vessels of vascular system in dermis
Alpha SMA - contractile protein
Classifications of wound types
Superficial
Partial thickness
Full thickness
Superficial wounds
Damage to epidermis only
Superficial wound healing
Healing occurs by migration of keratinocyte from wound edges and dermal appendages (sweat glands, hair follicles, sebaceous glands)
Once all keratinocytes are in contact on all sides, stratification can occur to reform epidermis
Partial thickness wounds
All epidermis and some of dermis is destroyed
Partial thickness wound healing (phases)
Inflammatory phase - immune cells migrate into clot and clean up the wound/pathogens
Migratory phase - keratinocytes migrate from wound edge and appendages, and fibroblasts migrate into the clot to make collagen fibres
Proliferative phase - keratinocytes proliferate
Maturation phase - epidermal stratification, scab falls off
Full thickness wounds
All of epidermis and dermis is destroyed
Hypodermis can be destroyed too, exposing bone and muscle
Wound repair is difficult since all reservoirs of epidermal stem cells have been destroyed
Keratinocytes have to migrate from wound edges
Heals as scar tissue
Intervention required to improve patient outcomes
Full thickness wound treatment
Split thickness skin graft:
Removes all of epidermis, part of dermis
Donor site - patient’s own undamaged skin so it doesn’t get rejected
Covers wound
Donor site becomes a partial thickness wound and heals in 10-14 days
Burn wound treatment - engineered skin
Reduces time to complete wound coverage
Start with a small sample of undamaged patient skin
Isolate and expand skin cells in laboratory
Grow enough skin to cover all wounds
Digest sample of patient skin
Isolate and grow fibroblasts and keratinocytes
Grow large sheets of autologous, full thickness skin
Permanent wound coverage solution
Engineered skin limitations
No pigmentation No hair follicles No sweat glands No sebaceous glands Still a long way to go before being able to grow fully functional skin in laboratory
Hypodermis generally in contact with…
Muscle and bone
Keratinocytes produce…
Produce keratin
Transit amplifying keratinocyte - division
Can divide rapidly to make layers of epidermis above stratum basale
Only capable of a limited number of cell divisions before they die
Primitive endoderm and ectoderm tissues
Endoderm: hypoblast
Ectoderm: epiblast
Purpose of extra-embryonic structures
Transport of nutrients and waste to and from embryo
Zygote created by process of…
Syngamy
Stomoderm gives rise to the…
Oral cavity
