Exam 5 Flashcards
Intrinsic apoptosis
Initiated by perturbations of the extracellular or intracellular microenvironment, demarcated by MOMP, and precipitated by executioner caspases, mainly C3 
Extrinsic apoptosis
Initiated by perturbations of the extra cellular microenvironment detected by plasma membrane receptors, propagated by C8, and precipitated by executioner caspases, mainly C3
Autophagy-dependent cell death
Mechanistically depends on the autophagic machinery
Necroptosis
Triggered by perturbations of extracellular or intracellular homeostasis that critically depends on MLKL, RIPK3, and kinase activity of RIPK1
Ferroptosis
Initiated by oxidative perturbations of the intracellular microenvironment that is under control by glutathione peroxidase (GPX4), can be inhibited by iron chelators or lipophilic antioxidants
Caspase target MST1
Chromatin condensation
Caspase target ICAD (inhibitor of a DNase)
DNA cleavage
Caspase target Lamins
Nuclear envelope breakdown
Caspase target Rho Kinase
Actin cytoskeleton disruption
Caspase target cell-cell and cell-ECM adhesion junctions
Cell rounding and deattachment (blebbing)
Caspase target golgi and ER proteins
Fragmentation of organelles
Caspase target eIFs
Translation arrest
Caspase triggering externalization of phosphatidyl serine
“Eat me” to phagocytic white blood cells
How does membrane blebbing occur?
Process of ROCK (Rho-associated kinase) leads to actin bundle contraction and membrane blebbing
How does chromatin condensation occur?
Proteolysis of the protein kinase MST1 releases an active fragment that phosphorylates histamine 2B and promotes condensation
Initiator Caspases
C 9, 2, 8, 10
Activated in protein complexes, the nature is site-dependent (Close together perpetuates signals Causing DISCs assemble a plasma membrane receptors, and PIDDosomes appear to interact with mitotic machinery)
Effector caspases
C 3, 7, 6
Apoptosome
A protein complex that activates C9
DISC
Death inducing signaling complex (activates C8 and C10) 
PIDDosome
A Caspase-activating protein complex that includes p53 induced death domain protein (activates C2)
Mitotic spindle problems involving kinetochores and spindle poles activate C2 which activates PIDDosomes —> hydrolyze Mdm2 —> inducing p53 activity 
Extrinsic pathway of Caspase activation
Death ligand binds to death receptor, FasL to Fas, Which creates the DISC Intracellularly: includes scaffolding which can then bind and activate C8  —> C3 —> apoptosis
Intrinsic pathway of Caspase activation
Involves damage or stress, which is transmitted to the mitochondria, resulting in MOMP and release of cytochrome C —> set a currency forms a complex with a PAF1 and C9 (apoptosome) —> Activates C9 —> C3
Inhibit MOMP and suppress apoptosis
• Bcl-2
• Bcl-xL
Promote MOMP and promote apoptosis
• Bax
• BAK
BH1,2,3 protein
Inhibit Bcl-2 and Bcl-xL and promote apoptosis
• Bid, Bad
• Puma, Noxa
BH3-only protein
IAPS: Inhibitor of apoptosis proteins
Bind to caspases and directly inhibit their proteolytic activity
Smac/Diablo
Released by mitochondria after MOMP, these proteins can inhibit IAPs 
AIF: Apoptosis inducing factor
A nucleus that is released by mitochondrial MOMP that can translocate the nucleus and cleave DNA, resulting in cell death, independent of caspases
ER stress transduced into apoptotic response
Activate C12 —> C9 —> Rest of Caspase cascade
DNA damage transduced into apoptotic response
• Damage detected by ATM/ATR kinases
•  activates Chk1/2 —> Stabilizes p53
• p53 Target Bax, which promotes MOMP, cytochrome C release, formation of apoptosomes leading to apoptosis
What keeps Bcl-2 free from inhibition by proapoptotic Bcl-2 family members?
PKB/Akt regulated by PI3K
Autophagy
Provides bulk degradation and recycling of cytoplasmic components in lysosomes promoting cell health and survival
When in excess, this compromises the cytoplasm and activates apoptosis 
• Beclin1, PI3K, Atg
AMPK responding to metabolic conditions
Promotes autophagy both by inhibiting mTOR and activating ULK1
Beclin-PI3K-Atg Complex
Receives input from mTOR (inhibits complex)
Receives input from AMPK (activates complex) 
Bcl-2 functioning as a switch between apoptosis and controlled autophagy
• binds and inhibits Beclin1, thereby restraining autophagy— if Phosphorylated by jnk, Beclin1 is free and autophagy occurs
• when bound to Beclin1, it is not bound to Bak/BAX, promoting apoptosis
Necroptosis
C8 inhibited by FLIP/pathogens -> increases RIPK1/3 -> increases MLKL (penetrates membrane w 4 alpha-helices, cell swelling) -> increases necroptosis
Necroptosis and PAMP/DAMP
Necroptosis releases PAMPs and DAMPs (Pathogen/damage associated molecular patterns), that active the inflammatory response
Ferroptosis
decrease GPX4, increase accumulation of ROS. Lipid peroxidation in the associated rupture of cellular membranes is a hallmark of ferroptosis
• occurs with age in Lipofuscin granules, Fenton rxns
(1) death receptor signaling at the plasma membrane
(2) Extensive DNA or organelle damage
(3) Malformed mitotic spindles was compromised function
(1) DISCs
(2) apoptosomes (cyt C, APAF1, C9)
(3) PIDDosomes
Increasing bax-mediated pore formation in mitochondrial outer membrane is most likely to result in the assembly of
Apoptosomes
A dying sell displaying an unusually large number of lysosomes is most likely undergoing:
Autophagy
Increasing the expression of which of the following proteins would be expected to make a cell more resistant to apoptosis?
Bcl-2
Cells can shift from an apoptotic response to a necroptotic response if _______ is inactivated by _________.
Caspase 8 , viral proteins (pathogens) 
What would be expected to facilitate MOMP and promote apoptosis?
Extrinsic pathway activation leading to the production of t-Bid from BID with the consequent activation of the intrinsic pathway
Protein that functions in transducing DNA damage into either cell cycle arrest or an apoptotic response, depending on the severity of the damage in the overall cell state
p53
Selection bias: Berkson Bias
Also known as collider bias, Study population selected from a hospital that can lead to spurious negative associations
Strategy to reduce bias: choice of comparison group
Selection bias: healthy worker effect
Study population is healthier than the general population
Strategy to reduce bias: study design, choice of comparison group
Selection bias: nonresponse bias
People who choose to participate are different in meaningful ways from people who choose to not participate
Strategy to reduce bias: study design
Recall bias
Awareness of disorder/disease causes people to recall potential exposures differently
Strategy to reduce bias: study design
Measurement bias
Differential versus non-differential
Hawthorne effect: participants change their behavior because they know they’re being observed
Strategies to reduce bias: choice of comparison group, use standard/validated tests/procedures, regular calibration of equipment, training of study staff
Procedure bias
Participants in different groups are not treated the same
Strategy to reduce bias: masked study design
Observer expectancy bias
Researchers preconceived ideas of results influence measurement, documentation, statisical analyses
Strategies to reduce bias: masked study design, independent statistical analyses
Confounding bias
Risk factor is related to both exposure and health outcomes, but is not casually related (Ex. Use of cod liver oil is positively associated with age related hearing loss)
Strategies to reduce bias call study design, replication studies
Lead-time bias
Early detection is confused with increased survival time
Strategies to reduce bias: study design
Survivor bias
A.k.a. incidence prevalence bias
Strategies to reduce bias: limit analysis to incident cases
Publication bias
Studies were statistically significant results are more likely to be pushed, as are those documenting a novel finding
Strategies to reduce bias: measure existence with Donald lots, include novel sources to identify data for meta-analyses 
Example of a common genetic polymorphism
Rh system: (+) and (-)
Alpha1-antitrypsin (ZZ)
Major serum protein that inhibits proteolytic enzymes, major target is leukocyte elastase which can damage lung connective tissue if not down regulated— Early onset emphysema 
Ecogenetics
Genetic variation in susceptibility to environmental agents
Examples: fair complexion and UV light, ADH deficiency and alcohol, G6PD deficiency and fava beans
Heterozygous advantage
A deleterious allele that is maintained in a population because it increases reproductive fitness when it is heterozygous — Ex. Sickle cell
 Hardy Weinberg law
1.) there is no appreciable rate of new mutation
2.) Individuals with all genotypes are equally capable of mating and passing on their genes— no selection against any particular genotype
3.) No significant immigration of individuals from a population with allele frequencies very different from the endogenous population
Hardy Weinberg equation
p^2 + 2pq + q^2
p^2: probability of AA genotype
q^2: probability of aa genotype
pq: heterozygous
p= 1-q
Importance of Hardy Weinberg disequilibrium
When alleles at a locus are not in HW equilibrium this can indicate that a particular allele is associated with a disease— fatal/ doesn’t show up as frequently in a community
Mendelian diseases
Primary single gene diseases, the disruption of a single gene
The most common functional protein change mutation
Loss of function— Miss sense, nonsense, frameshift, deletions, insertions
Gain of function mutations
Increase gene dosage/proteins function
Ex. Down syndrome, achondroplasia
Allelic heterogeneity
Different alleles of the same gene/locus causing very disease severity
— PKU
Locus heterogeneity 
Mutations in different genes/locus is can yield a similar clinical phenotype 
— hyperphenylalaninemia 
Modifier genes
People with the same mutation can present dramatically different phenotypes due to the presence of modifier genes
Ex: ApoE4 frequency increasing neurological and neurodegenerative disorders
Example of enzyme defect
PAH gene and the disease PKU— Also an example of defect that occurs in one tissue (liver, kidney) but where the phenotype is manifest elsewhere (brain)
Defects in lysosomal storage
Leads to increased tissue mass and is a common cause of neurodegeneration and CNS problems
Example: Tay-Sachs disease buildup of GM2 gangliosides sphingolipids in the retina lysosomes
Defects in protein trafficking
I-cell disease: autosomal recessive lysosomal storage disease caused by a defect in proteins trafficking, acid hydrolases which are required are not properly modified with glycoproteins
Acid hydrolases get sent out of the cell instead of to the lysosomes
Defect in cofactor metabolism example
Alpha 1-AT : Involved in the breakdown of various proteases that can damage lung tissue if not regulated. (Interaction with environmental factors like cigarette smoke) 
Defect in receptor proteins example
Hypercholesterolemia: LDLR defect, causes cardiovascular disease
Defect in transport examples
Cystic fibrosis, delta F508, Mutation of a chloride channel, affects lungs and exocrine pancreas
Defects in structural proteins example
DMD: X-linked recessive disease caused by a mutation in the dystrophin gene
Female carrier: elevated creatine kinase levels, no clinical manifestation
Triple repeat expansion disorders
Huntington’s, fragile X, Friedreich ataxia, myotonic dystrophy 1 and 2
Mitochondrial genetic bottleneck
The restriction and subsequent amplification of mtDNA during oogenesis: mosaicism/heteroplasmy
Mothers with a high proportion of mutant mtDNA are more likely to have clinically affected offspring
Complex genetic diseases
Do not demonstrate a simple Mendelian pattern of inheritance, often demonstrate familial aggregation, inheritance is more common among the close relatives of a proband, more likely in MZ versus DZ twins
Human characteristics which show a continuous normal distribution
• blood pressure
• dermatoglyphics
• head circumference
• height
• IQ
• skin color 
Four characteristics of inheritance of complex diseases
1.) no simple Mendelian pattern of inheritance
2.) Familial aggregation
3.) Environmental factors
4.) More common among close relatives of the proband 
Relative risk ratio
Prevalence of the disease in the relatives of an infected person
___________________________________
Prevalence of the disease in the general population
Odds ratio
OR=1 would show complete lack of association
OR>1 A higher risk of an outcome
OR<1 Lower risk of an outcome
Examples of recurrence risks and relative risk ratios
Schizophrenia, bipolar disorder, coronary artery disease, MI, Alzheimer’s disease
Approaches to identifying genes underlying complex diseases
1.) Test a candidate gene for variance in a disease population
2.) map a gene in a family/families with history of disease
Quantitative trait loci (QTLs) 
Complex disease genes identified often have quantitative affects
Physical mapping of disease genes
Actual sequence and physical location on a chromosome are being looked at
Genetic mapping for disease genes
Based on the following a phenotypic trait in the families, indicates the relative position of genes (as identified by their function) as shown by linkage analysis
Linkage analysis
Uses statistics to determine whether to genes, loci or the markers they are based on, are likely to live near one another. Estimated by the frequency that they are transmitted together
Two genetic loci are linked if they are transmitted together from parent to offspring more often than expected under independent inheritance
Recombination frequency
RF > 50% means two genes are unlinked
RF < 50% means two genes are linked
Mendel’s first law
The principle of segregation: the two members of a gene pair segregate from each other in the formation of gametes. Half of the gametes carry one allele, and the other half carry the other allele
Mendel’s second law
The principle of independent assortment: genes for different traits assort independently of one another in the formation of gametes. —unlinked 
A disease locus can be mapped by:
Following its co-transmission with known chromosomal markers (Without knowledge of the actual gene)
LOD scores
Logarithm of the odds: odds ratio is expressed as the log10 of this ratio
— this is the likelihood of data if loci are linked
LOD of 3+ : 1000:1 odds favor of linkage (not random)
Association analysis
Start with a candidate gene, suspect that a defect or polymorphism is responsible and then look into families and or population to determine if people with a disease are statistically more likely to carry a particular rotation or polymorphism
Steps in mapping a complex disease gene
• linkage analysis/prior knowledge
• Genomic analysis, association analysis to determine the correspondence
•  alternative strategy: SNP chips
• Follow up with bio chemical and molecular analysis/animal models
Calpain-10 (CAPN10)
A type two diabetes gene, found by linkage analysis of Mexican American family
— Is not a polymorphic marker, is actually the disease. Assisting protease that interferes with insulin secretion and glucose uptake
Sib-pair analysis
Uses small families and asks whether affected siblings share specific gene alleles had a frequency higher than expected by random chance
 Comparing identical by descent (IBD) to identical by state (IBS)
Identical by descent
Siblings have the same allele from the same parent
Identical by state
Siblings have the same allele, regardless of what parent it came from
Hirschsprung disease and Sib-Pairs
55/67 shared three polymorphic markers at three different genes
The other 12 shared at least two of the three polymorphic markers
— RET gene malfunction 
Sources of one-carbon units
Serine, glycine, histidine, formaldehyde, formate
Regulation of the one-carbon pool
FH4 delegating a C — formyl <—> methylene <—> methyl
Folate
• Vitamin precursor of FH4
• found in leafy green vegetables, liver, legumes, yeast, fortified flour
• has glutamic acid tail
Major form of folate in the blood
Reduced N5-methyl tetrahydrofolate from the intestinal epithelial cells
PCFT
Proton (H+) coupled folate transporter encoded on the SLC46A1 gene on enterocytes and hepatocytes
Hereditary folate malabsorption
An inherited mutation in the proton coupled folate transporter causing a functional folate deficiency despite adequate folate in the diet: no absorption 
What is a folate deficiency associated with before and during pregnancy?
Spina bifida, neural tube defects
Folate to FH4
Folate —dihydrofolate reductase—> dihydrofolate — dihydrofolate reductase—> tetrahydrofolate
What drugs target dihydrofolate reductase?
Methotrexate colon cancer, rheumatoid arthritis
Trimethoprim: antibacterial
Pyrimethamine: antimalarial
How is formate formed?
The degradation of tryptophan— Formate is one source of carbon for the “one carbon pool”
Redox of formate/tetrahydrofolate 
FH4 + formate —> N10- formyl <—> N5,N10-methenyl <—> N5,N10-methylene —> N5-methyl
Forminotransferase deficiency
FIGLU accumulation (from FH4 + histidine —> 5-formimino + glutamate) characterized by Megaloblastic anemia and mental retardation
Serine and the one carbon pool
Serine is the most important contributor to the one carbon pool. It forms N5,10- methylenetetrahydrofolate when it donates its carbon to FH4 using PLP as a cofactor
Sources of products of the one carbon pool
Serine, glycine, Choline, Histidine, tryptophan
Two forms of B12 in the body
Methylcobalamin
Adenosylcobalamin 
Methylcobalamin reaction:
Donation of a methyl to homocystine to make methionine
— Methylcobalamin is consumed in the process
Adenosylcobalamin reaction:
Catalyzes the isomerization of a methyl group in converting methylmalonyl CoA to succinylacetone CoA 
— catabolism of branched chain an odd chain length fatty acids, isoleucine, valine
— Adenosylcobalamin not consumed in the reaction
How B12 enters the body
1.) binds to R binder proteins in the stomach
2.) R binders get digested, B12 then binds to intrinsic factor
3.) Intrinsic factor B12 complex is taken up by intestinal epithelial cells and transported into the blood
— Transcobalamin II is the protein used
4.) B12 get stored in the liver complex with Cubillin
B12 deficiency
• Causes pernicious anemia, megaloblastic anemia plus neurological problems
• caused from dietary deficiency, loss of function of intrinsic factor, transcobalamin II, or cubillin 
What is S – adenosylhomocystine used for?
SAM — A methyl donor for many biosynthetic and regulatory enzymes, and it must be regenerated with carbon that comes from N5-methyltetrahydrofolate
Methotrexate
Targets dihydrofolate reductase,
FH2 —x—> FH4
Folate deficiency leads to decrease in nucleotide production. Use against cancer, because those cells require deoxynucleotides for rapid cell division
5–fluorouracil
A uracil analog that inhibits thymidylate synthase. Is a suicide inhibitor
dUMP—x—> dTMP
Folate deficiency leads to decrease in nucleotide production. Use against cancer, because those cells require deoxynucleotides for rapid cell division
Hyperhomocystinemia
Accumulation of homocystine because it cannot be converted to methionine by B12 (Methylcobalamin)
This can also be caused by a vitamin B6 deficiency, because the homocysteine cannot convert to cysteine 
Betaine
This is the bodies second pathway to turn homocysteine to methionine. It is a metal donor
The buildup of homocysteine affects: 
• PNS & CNS deficiencies
• atherosclerosis
• Osteoporosis
— Interferes with collagen maturation 
Methylmalonyl CoA mutase activity lost (Adenosylcobalamin def) causes:
Inappropriate synthesis causing branched chain fatty acids rather than unbranched. These branched fatty acids compromise the membrane structure
Methyl trap hypothesis
The only metabolic fate of N5-methyl FH4 is to lose its methyl to cobalamin. In a dietary or functional deficiency, folate becomes trapped as N5-methyl FH4, unable to participate in other one carbon transfers
Clinical features of cobalamin deficiency
Megaloblastic Anemia, sore tongue, auto immune gastritis, numbness and ataxia
Severe osteoporosis and young patient should always suggest the possibility of what?
Homocystinuria
The two main processes of tissue repair
1.) regeneration: restoration of normal cells
2.) Scarring/fibrosis: deposition of connective tissue
Ultimate repair is usually a combination of both
Mechanisms of regeneration of tissue
1.) proliferation of differentiated cells that survive injury and have ability to proliferate (ex. Liver)
2.) Tissue stem cells produce new and differentiated cells (ex. Skin, GI)
— Regeneration typically requires intact supporting structures
Mechanism of scarring/fibrosis of tissue
1.) injured area is patched with connective/fibrous tissue. Occurs when cells are not capable of regeneration, or supporting structures are too severely damaged
2.) functional cells are replaced by connective tissue, which provides structure but does not provide function
Three classes of tissues that can be repaired
1.) labile, constantly dividing tissue
2.) Stable tissue
3.) Permanent tissue
Labile, constantly dividing tissue
• Constant overturn fed by stem cells and proliferation of mature cells
• Occurs in the lung, skin, G.I. tract, and hematopoietic
• surface of the epithelia 
Types of labile cells
• Type two pneumocyte in the lung
• basal cell in the skin
• Crypt cells in the G.I. tract
• CD34+ cells
Stable tissue
• made of quiescent cells (G0)
• Capable of proliferating when tissue is injured or lost
— In solid tissues: liver, kidney pancreas
— in connective tissue: endothelium, smooth muscle, fibroblasts
Permanent tissue
• cells are terminally differentiated and cannot proliferate
• This includes most neurons, cardiac, and skeletal muscle
• Repair is almost entirely scarring/fibrosis
Regeneration after injury is driven by:
Growth factors derived from:
• Active macrophages at the side of injury (most common)
• Platelets
• Epithelial cells
• Stromal cells (Fibroblasts)
• Sequestered pool in extracellular matrix
Heparan sulfate
An extra cellular matrix signaler for tissue damage 
Phase 1 of scarring/fibrosis repair
Inflammatory (days 1-3)
• Cellular mediators: platelets, neutrophils, macrophages
• Actions: acute inflammatory responses
Phase 2 of scarring/fibrosis repair
Proliferative (day 3-weeks)
• Cellular mediators: fibroblasts, myofibroblasts, endothelial cells, epithelial cells, macrophages
• actions: establishment of granulation tissue, angiogenesis, epithelial cell proliferation, type III collagen deposition
Phase 3 of scarring/fibrosis repair
Remodeling (1 week- months)
• Cellular mediators: fibroblasts
• Actions: type III collagen deposition replaced by type I collagen (stiffer, less elastic) 
Granulation tissue
Many new blood vessels, fibroblasts, and chronic inflammation including numerous macrophages
Angiogenesis: action of VEGF
Vascular endothelial growth factor:
• Stimulates both migration and proliferation of endothelial cells
• Promotes vasodilation by stimulating the production of NO
• Contributes to the formation of vascular lumen
(Important for healing) 
Angiogenesis growth factors
FGF, TGF-beta, VEGF,
Fibrosis growth factors
TGF-beta, PDGF
Vascular remodeling and smooth muscle migration growth factor
PDGF
Cell proliferation and regeneration growth factor
EGF
Central role of the macrophage
M1: clearing offending agents and dead tissue
M2:
• Provide growth factors causing the proliferation of various cells
• Secrete cytokines that stimulate fibroblasts to proliferate and deposit connective tissue in ECM
Temporally regulated
Initially, the macrophages are classically activated (M1), but are gradually replaced by alternately activated type (M2)
TGF-beta
• produced by cells in granulation tissue
• Stimulus fibroblast migration and proliferation
• Increases synthesis of collagen and fibronectin
• decreases degradation of ECM
• Has anti-inflammatory properties
Clinical factors that can impede repair
INFECTION, diabetes, nutritional deficiencies, medication/steroids, mechanical stress, ischemia/poor perfusion, foreign material, injury type and extent, injury location
First intention wound
A suture surgical incision with tightly apposed edges
Second intention wound
A wide open wound, which leads to more inflammation, more granulation tissue, increased risk of infection, large scar
myofibroblastic contraction plays a major role in wound closing
Wound strength
At one week: wound is about 10% as strong as normal skin
After two months: maximum strength is achieved, still only 75% of the original tissue strength
Hypertrophic scar
Raised, limited to boundaries of original wound, tends to regress overtime, parallel collagen I bundles as in normal scar
Keloid scar
Markedly raised, expands beyond the boundaries of the original wound, contains bright eosinophillic collagen bundles that are disorganized
Scar contracture
Failure to make a scar, the fibroblasts pull the skin back together and it pulls too tightly
Leak channels:
Always open, help maintain resting membrane potential, permeate K more than Na
Voltage gated channels
Activated by membrane potential: Na, Ca, K, Cl channels 
Extracellular ligand activated channels
Regulated by ligands (neurotransmitters), glutamate, GABA, and glycine receptor channels
Intracellular ligand gated ion channels
Activated by Ca2+, ATP, cyclic AMP and GMP, often activated indirectly by GPCRs including CFTR and ABC transporters
Thermos sensitive channels
Regulated by temperature and pain, TRP channels
Mechano-sensory and volume regulated channels
Activated by touch, hearing, cardiovascular regulation, sensing of gravity, and osmotic stress
Light gated channels
Used for Optogenetics
Channelrhodopsin: blue light (Na+,K+)
Halorhodopsin: yellow light (Cl-)
Types of gated ion channels
1.) Confirmational change in one region
2.) General structural change
3.) Blocking particle (AA)
4.) ligand gated
5.) Phosphorylation gated
6.) Voltage gated
7.) Stress or pressure gated (Cytoskeleton)
Mechanisms for channel inactivation
1.) change in membrane potential
2.) Calcium binding
3.) Dephosphorylation
Which channel type is the most diverse?
K+
— they can be sensitive to depolarization, hyper polarization, pH, and change in intracellular Ca2+ 
Equilibrium of K+
-58mV
The action potential
Na channel
1.) resting potential, leak K channel, -60mV
2.) Rising phase, opening Na channels
3.) Overshoot phase, Na close, K open
4.) Falling phase, K opens maximally
5.) Under shoot phase: refractory because K is slow to close
Action potential of cardiac muscle
0.) Rapid influx Na+ through open fast Na+ channels
1.) Transient K channels open and K efflux returns TMP to 0mV
2.) inflow of Ca2+ through L-type Ca2+ channels is electrically balanced by K efflux through delayed rectifier K channels
3.) Ca2+ channels close but delayed rectifier K channels remain open and return TMP to -90mV
4.) Na+, Ca2+ channels closed, open K rectifier channels keep TMP stable at -90mV
Long QT syndrome
Loss of K channels, dangerous because no recovery before another spike
Symptoms: torsades de pointes, syncope, seizures, sudden death
Purines are constructed by:
Adding atoms from formyltetrahydrofolate, glutamine, glycine, aspartate, and CO2 sequentially to PRPP
Pyrimidines are constructed by:
Building the orotate base from aspartame, CO2, and glutamine. The base is then transferred to PRPP and further modified to cytosine, thymidine, or uracil
PRPP
An activated ribose sugar created by transfer of pyrophosphate to ribose-5-phosphate by PRPP synthetase (KEY REGULATORY STEP!)
PRPP synthetase is allosterically inhibited by purine diphosphonucleosides (GDP, ADP)
IMP is synthesized by
N10-formyl FH4 (twice)
IMP + aspartate + GTP
AMP (+ fumarate)—>ADP—>dADP—>dATP—>DNA
Or
AMP—>ADP—> ATP—> RNA
IMP + glutamine + ATP
XMP—> GMP—> GDP—> GTP —> RNA
or
XMP—> GMP—> GDP—> dGDP—> dGTP—> DNA
Ribonucleotide reductase uses what as substrates?
Nucleotide diphosphate (ADP, GDP)
Purine salvage: free bases to nucleotides and back
Converting free bases to nucleotides, using APRT & HGPRT (Phosphoribosyltransferases that add ribose form PRPP)
Hypoxanthine (HGPRT)—> IMP
Guanine (HGPRT)—> GMP
Adenine (APRT)—> AMP
Purine salvage: Nucleosides to nucleotides
Purine nucleoside phosphorylase removes ribose, leaving the free base
Inosine—> hypoxanthine
Guanosine—> guanine
Purine salvage: Adenosine kinase
Adenosine can be converted to AMP directly through phosphorylation by adenosine kinase 
Gout
Precipitation of uric acid in distal joints. Caused by purine degradation leading to hyperuricemia
CPS II
Uses glutamine as an amine donor to form carbamoyl phosphate (Same product as CPS I, doesn’t use ammonia) It is allosterically inhibited by UTP and activated by PRPP
Thymidine synthase
Uses dUMP as a substrate, so dUDP has to be the phosphorylated before it can be methylated to dTMP. dTMP Is then phosphorylated twice to make dTTP, a substrate for DNA synthesis
PRPP synthetase superactivity
Inhibited by ADP, and GDP normally. Super activity loses inhibition
X-linked condition seen only in males, symptoms are due to increased purine production and increase uric acid
— Crystalluria, urinary stones, gout

ADA-SCID
Severe combined immunodeficiency resulting from adenosine deaminase deficiency (ADA— Normally removes amine group from adenosine in salvage pathway)
Leads to an accumulation of adenosine and 2-deoxyadenosine in the blood. Toxic to developing lymphocytes
— Requires hemopoietic stem cell transfer
PNP-SCID
Combined immunodeficiency resulting from purine nucleotide phosphorylase deficiency
— Requires hemopoietic stem cell transfer
Lesch Nyhan Syndrome
X-linked, results in Deficiency in hypoxanthine- guanine phosphoribolsyltransferase
He’s Got Purine Recycling Trouble (HGPRT deficiency)
Symptoms: self injury, uric acid in the urine, mental retardation, dystonia, recurrent vomiting, renal failure 
Allopurinol
Drug aimed at reducing uric acid
Treatment for gout (Suicide inhibitor of xanthine oxidase), and renal failure

Capacitation
A set of biochemical changes in the sperm that occur after about 5 to 6 hours of residency in the female reproductive tract
Normal site of fertilization
Ampulla
What happens during capacitation?
• cholesterol efflux from plasma membrane
• Membrane hyperpolarization
• Increased cytosolic pH
• unmasking of cell surface receptors that bind the sperm to the egg
• Ca2+ and HCO3- enter the sperm and activate adenylyl cyclase —> increase cAMP —> tyrosine phosphorylation of many proteins 
Acrosomal reaction
Necessary for sperm egg fusion to occur
• Vigorous swimming leading to penetration of the follicle cell layer
• Sperm plasma member associated proteins binding to ZP3 —> Activating sperm plasma membrane Na/H transporters —> opens Ca2+ channels • The calcium influx triggers exocytosis of the acrosomal vesicle, liberating its contents (Hydrolytic enzymes, that digest an opening through the Zona) 
Sperm egg fusion
Izumo1 On sperm and Juno on egg
Once sperm egg fusion occurs:
PLC-zeta Enters the egg, cleaving PI(4,5)BP into IP3 and DAG, initiating a calcium spike in the egg cytoplasm 
What are the three reactions triggered in the egg once PLC-zeta enters?
1.) triggers cortical granule exocytosis
2.) Stimulates the completion of meiosis II (and 2nd polar body forms)
3.) Activates the first mitotic cell signal and development of the zygote 
The fertilization induced calcium signaling is:
A profoundly important event that triggers sets of events that will culminate into an activated zygote
The Zona reaction
The calcium induced cortical granules digest ZP2 and alter ZP3, there by inhibiting the passage of more sperm
Also sheds Juno
Metaphase II arrest
LH —> increased Emi2 —> inhibits APC/C —> M2 arrest
Completion of meiosis II
Increased Ca2+ —> inhibits Emi2 —> increases APC/C —> proteolysis of cyclin B and inhibition of M-CDK activity
Pronuclei
The nuclear envelopes form around each set of parental chromosomes
The zygote centrosome is formed from:
The sperm basal body and some maternal pericentriolar material
What degenerates and disappears from the sperm in the first cell cycle?
The sperm axoneme microtubules and mitochondria
Formation of the male pronucleus includes remodeling where
Protamines are exchanged for maternal histones
Causes of female infertility
• Physical damage to the uterus or fallopian tubes
• deficits in ovulation
Causes of male infertility
• Failure of emmission
• retrograde ejaculation
• varicocoele
• Oligospermia (reduced sperm #)
•Asthenospermia (reduced motility)
• tetrazoospermia (wrong morphology)
ART: assisted reproduction techniques
Eggs are obtained by hormone-based super ovulation protocols, and collected by physically rupturing the follicles with an ultrasound guided catheter and subsequent aspiration. Eggs are flushed into a Petri plate with appropriate buffer
GIFT: gamete intra-fallopian transfer
Collection of 1 to 4 eggs, and 100,000 sperm placed directly into an ovaduct
ZIFT: zygote intra-fallopian transfer
Collected eggs and sperm are combined in a petri dish and allowed to undergo fertilization, fertilized zygote are placed in the oviduct
IVF – ET: in vitro fertilization embryo transfer
Eggs and sperm are combined in a petri dish and allowed to undergo fertilization. Embryos then placed into the fallopian tube or uterus. Main difference from ZIFT is that the fertilized egg is allowed to develop for a longer period of time
SUZI: subzonal insemination
2 to 10 sperm are injected with a micro pipette directly into the peri-vitelline space of the oocyte. Fertilization is monitored and zygote/embryo replaced in uterus or fallopian tube
— sometimes uses zonal drilling: acid digestion of Zona to allow sperm access to egg surface
ICSI: Intracytoplasmic sperm injection
A single sperm is placed into a micro pipette and micro injected directly into the egg cytoplasm. Zygote/embryo transferred back to uterus/fallopian tube
— Most commonly performed procedure
Two fundamental differences of meiosis from mitosis
1.) One S-phase followed by two M-phases
2.) pairing of homologous chromosomes in prophase one: homologs find each other in prophase and form cohesins 
To mechanisms to promote genetic variability in meiosis
1.) homologous recombination (crossing over)
2.) Independent assortment
Homologous recombination
• occurs in prophase of meiosis I
• Synapsis: Homologous chromosome pairs find each other the other formation of synaptonemal complexes
• recombination nodules formed between chromatids of different homologs (2-3 spots)
Chiasma
Where reciprocal splicing occurs during crossover
Independent assortment
Which replicated homolog (M1) or daughter chromatid (M2) faces which spindle pole in meitotic cells is random
Pairing of homologous chromosomes in meiotic prophase I
• Dyein associate with cytoplasmic portion of KASH5 and binds to microtubules
• adapter proteins bind chromosome telomeres in the nuclear portion of SUN1
• Microtubule motor based motility pulls the telomeres together to form bouquets— Facilitating pairing of homologs 
Shugoshin
Protects the centromeric cohesions from being digested by separase in M1
Shugoshin is dislodged from daughters chromatids allowing separase to destroy cohesin, in anaphase II of M2
Nondisjunction
Failure of chromosomes to separate normally in anaphase. can occur in meiosis I or II
This can cause monosomies/trisomies (downs 21, Edward 18, and patau 13) 
XY nondisjunction
The most common type of paternal nondisjunction event
Progression of gametogenesis
Primordial germ cells—> Spermatogonia and oogonia—> primary spermatocytes and oocytes—> M1 —> secondary spermatocytes and oocytes —>
Males: M2 —> haploid spermatids —> spermatozoa
Females: arrest in M2 , ovum until fertilized
When does imprinting occur?
PGCs become detectable at the fourth week of embryonic development. As they differentiate into oogonia and spermatogonia, DNA methylation occurs: which is imprinting
Supporting cells of spermatogenesis
Sertoli cells
Transformation of spermatogonia to spermatids into spermatozoa
- production of haploid spermatids by meiosis
- Morphological transformation of spermatids into spermatozoa by Formation of an acrosomal vesicle, elaboration of a flagellum, replacement of histones in sperm chromatin with protamines (condense DNA into sperm head) 
Protamines
Small, arginine-rich proteins that replace histones late in spermatogenesis. This allows for greater compaction of DNA to form the small, dense head of the sperm. It also removes histone epigenetic marks 
Oogenesis
In fetal development: PGCs —> M1, arrest prophase I (primary oocytes) —> puberty, M1 resumed —> M2: arrested in metaphase II until fertilization
RA and Stra8 signaling
Females: retinoic acid + Stra8 in fetal ovary regulates a number of genes involved in driving oogonia into meiosis
Males: RA is rapidly metabolized in the testes until puberty, then RA and Stra8 rapidly accumulates driving spermatogonia into meiosis
Regulation of mitotic arrests during oogenesis meiosis I
Follicle gap junctions —> High cAMP —> PKA —> activating phosphorylation of Wee1 —> inhibitory phosphorylation of M-CDK —> arrests oocyte in prophase I
Mid cycle increase in LH —> decreases cGMP and cAMP —> increases Mos kinase —> increases M-CDK —> completion of M1
Regulation of mitotic arrests during oogenesis meiosis II
Increase LH —> increases Emi2 —> inhibits APC/C —> arrests in metaphase II
fertilization —> increased Ca2+ —> inhibits Emi2 —> increases APC/C —> proteolysis of Cyclin B and inhibition of M-CDK —> M2 completion
The main components of the cell complex released during ovulation
Secondary oocyte arrested in metaphase II, a daughter polar body, surrounding layers of ECM (zona pellucida) , and follicle cells (corona radiata)
 Early development events by week
Week 1: cleavage and implantation
Week 2: Bilaminar disc formation and delamination
Week 3: gastrulation, neurulation, somitogenesis
Week 4: embryo rolling and folding, establishment of the adult body plan
The four processes used to make multicellular organisms
1.) cell proliferation
2.) cell specialization
3.) cell interactions
4.) Cell movements
Cleavage
Cell divisions resulting in a loosely adherent ball of cells, called a morula
Compaction— Initial cell differentiation
Flattening of cells against each other, due to formation of cell-cell junctions
— as this occurs, the outer cells envelop a core of inner cells. The outer cells give rise to the trophoblast, inner cells give rise to the inner cell mass (embryo)
Hatching
Escape from the Zona Pellucida— The ZP serves as a barrier to implantation, and must be discarded prior to blastocyst attachment to the uterine epithelium
Normal implantation site
The superior and posterior wall of the uterus
Abnormal includes: abdominal cavity, ovary, uterine tube, or too close to the cervix
Placenta previa
Occurs when implantation occurs low in the uterus, and the placenta subsequently covers part or all of the cervix. It can result in bleeding in the second half of pregnancy, and growth restriction of the fetus
Syncytiotrophoblast
Expansion of the trophoblast, forms a multinucleated mass which overlays the cellular trophoblast
Cytotrophoblast
Once the cellular trophoblast is over laid by the syncytiotrophoblast, the inside becomes the cytotrophoblast
Hypoblast
Differentiates and separates the inner cell mass (embryoblast), from the blastocyst cavity
Primitive streak
Created from epiblast cells, it begins caudally, identifying cranial, coral, left, right embryo axes 
Downward dive: endoderm
Partially downward dive: Mesoderm
Top layer: ectoderm (overlying epiblast 
Notochord
Extends downward through the primitive node and then cranially in the mesoderm
Supporting structure for the embryo, a source of midline signals that pattern surrounding tissues
Ectoderm derivatives
Epidermis, hair, nails, cutaneous, memory glands, central and peripheral nervous system
Mesoderm derivatives
Paraxial: Muscles of the head, trunk, limbs, axial skeleton, dermis, connective tissue
Intermediate: urogenital system, including gonads
Lateral: serous membranes of pleura, pericardium, and peritoneum, connective tissue and muscle of viscera, heart, blood cells
Endoderm
Epithelium of lung, bladder and G.I. tract, glands associated with G.I. tract, including liver and pancreas
Sacrococcygeal teratoma
Develops at the base of the coccyx in the developing fetus, derives from pluripotent cells from the primitive streak
Neurulation in the third week
Ectoderm thickens to form neural plate —> fold to form neural groove —> Neural crest cells invade the underlying mesoderm —> folds fuse to form neural tube
Failure of the neural tube closure at the cranial and
Anencephaly
Failure of the neural tube closure at the caudal end
Rachischisis (spina bifida)
Neural crest derivatives
- spinal, autonomic, and cranial nerve ganglia
- Schwann cells
- meninges
- Melanocytes
- Adrenal medulla
- Craniofacial muscles and skeleton
Lateral plate mesoderm
Splanchnic: ventral wing
Somatic: dorsal wing
Extraembryonic: continuity of wings on yolk sac and amnion 
Intermediate mesoderm
Kidney, gonads
Paraxial mesoderm 
Head
Somite: sclerotome, myotome, dermatome 
Somite derivatives
Sclerotome: Vertebrae, ribs, rib cartilage
Myotome: musculature of the back, ribs, and limbs
Dermatome: dermis of the back
Spina bifida
Wnt and Shh signaling errors: failure of the bilateral dorsal sclerotome to completely encircle the spinal cord leaving a gap on the dorsal side
Blood and blood vessel formation
Blood islands + peripheral cells —> primitive vasculature and endothelium
Cardio genie mesoderm—> forms toward cranial end and rolls caudally during embryo folding
Transfers embryo rolling and cephalocaudal embryo folding
Converts the body plan from disc-like to tubular
Re-organize the cranial end, positions the heart, forms the foregut, midgut and hindgut
How does citrate leave the mitochondria?
Citrate/Malate antiporter to create either oxaloacetate or acetyl CoA
Glycolysis from citrate
Citrate lyase (forms OAA from citrate), malate dehydrogenase (forms Malate from OAA) , malic enzyme, NADP+ (pyruvate from Malate)
The pyruvate/Malate cycle has two functions in lipogenesis
1.) transports acetyl Coa away from the mitochondria to the cytosol
2.) Malic enzyme generates NADPH to power fatty acid synthesis
Fatty acid synthase (FAS) consumes what?
NADPH generated in the cytosol by the PPP and malic enzyme 
Acetyl CoA —> Malonyl CoA 
Acetyl CoA carboxylase + CO2 + ATP
Activated by citrate, xylulose-5-p, insulin
Inhibited by palmitoyl CoA, phosphorylation by AMPK/PKA 
Rate limiting step in fatty acids synthesis
Acetyl CoA carboxylase. It is regulated in multiple ways
Inhibition of CPT1
Malonyl CoA inhibits CPT1, this stops beta oxidation of fatty acids while fatty acid synthesis is occurring
Fatty acid synthesis reaction sequence
Bond formation, reduction, dehydration, reduction
Electron acceptor for the creation of carbon carbon double bonds by fatty acyl CoA desaturase
O2. Energy for this comes from NADH —> H2O
Precursor of prostaglandins
Arachidonic acid. 20 carbons with 4 points of unsaturation. Must be obtained from the diet because we cannot synthesize double bonds near the omega carbon like it has
Fatty acids are packaged as
Triacylglycerides, glycerophospholipids, ether phospholipids, sphingolipids
Glycerol 3-phosphate in the liver
Glycerol (from Lipolysis) + ATP—Glycerol kinase—> glycerol 3-phosphate
Only liver cells express glycerol kinase
Glycerol 3-phosphate in the adipose tissue
Glucose—>DHAP + NADH —> glycerol 3-phosphate
(Glycolysis coupling)
VLDL
Very low density lipoprotein, delivers fatty acids to adipose tissue to store fatty acids as triacylglycerol
Lipoprotein lipase, LPL, is an enzyme on endothelial cells activated by apoC2 that cleaves off FAs to reduce the VLDL —> IDL —> LDL
Glycerophospholipid
2 FAs and a phosphate head group on a glycerol backbone
Mainly used in cell membranes, but are also constituents of Lipoproteins, bile, and lung surfactant
Etherlipid (plasmogen)
1 FA, 1 phosphate head group, 1 ether-linked hydrocarbon tail on a glycerol backbone
Sphingomyelin: lung surfactant
1 FA, 1 phosphate choline group on a sphingosine backbone
Glycolipids
1 FA, 1 carbohydrate group on a sphingosine backbone
Common glycerophosphates
Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylserine
Phosphatidylinositol biphosphate
Glycerophospholipids formed similarly to triacylglycerols
Phosphatidic acid is dephosphorylated to diacylglycerol. Then the head group bound to a nucleotide (CDP) is added to glycerol backbone.
Ex. Phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine
Glycerophospholipids formed other ways:
Bonding phosphatidic acid to a nucleotide, then exchanging the nucleotide for a head group
Ex. Phosphatidylinositol, Cardiolipin, phosphatidylglycerol
Sphingolipids
Use ceramide instead of glycerol for their backbone. Ceramide is derived from serine and Palmitoyl CoA (sphingosine)
Cerebrosides
Ceramide plus either glucose or galactose
Globosides
Two or more sugar is attached to a ceramide
Gangliosides
Have two or more sugars, plus NANA
Major lung surfactant
Dipalmatoylphosphatidylcholine: The major component of lung surfactant, required to prevent alveolar collapse
Sphingomyelin is another lung surfactant. The ratio of sphingomyelin to phosphatidylcholine in amniotic fluid is an indicator of gestational progress
Totipotent
Able to give rise to all embryonic and extraembryonic (triphoblasts) cells
This includes the zygote and early blastomeres
Pluripotent
Able to give rise to cells representative of all three germ layers, but not trophoblast or other extra embryonic cells
This includes the inner cell mass cells
Multipotent
Can give rise to a number of different cell types, but not as many as pluripotent cells. Limited to one germ layer
Unipotent
Only able to give rise to a single type of differentiated cell
Cell fate of the embryo is not determined until:
After the inner mass cells and early epiblast stages
Committed cells
Cells gain a general type of identity, but the precise cell type is not yet defined, and can be modulated by environmental signals
Determined cells
The type of cell is defined, and further development is independent of environmental signals
Waddington’s model of self fate determination
Like a marble rolling down a landscape that separates into different grooves: initial major groups would be trophoblast versus inner cell mass, next would be epiblast versus hypoblast, and then ectoderm, mesoderm, or endoderm. Cells acquire specific fates as they roll down and enter different grooves
Asymmetrical cell division
Specific factors are partitioned unequally among daughter cells during cell division. This results in the assignment of different fates as daughter cells are produced
Mosaicism 
Induction
Cells are initially the same, but can be separately influenced by other cells or other types of environmental signals. Cells are not born different, but instead become different by receiving distinct environmental signals. Regulative pattern of development (most common) 
How do you cells become different from one another during early development?
1.) cell signaling, select activation of transcriptional regulators via cell signaling pathways
2.) Epigenetic modification of chromatin
Morphogens
Signals that alter a cell’s fate in the process of induction 
Instructive cell signaling behavior
Cell a gives A signal, causing specifications and differentiation of cell B
Permissive cell signaling behavior
Cell B already specified, but a signal from cell A allows differentiation to proceed
Most embryonic inductions are mediated by:
Secreted signaling factors. Example: inductive, gradient, antagonist, cascade, combinatorial, lateral signaling 
Gradient signaling
Different concentrations of a single morphogen can generate different cell types
An example of sequential induction
Eye development: optic vesicle —> FGF8 released to form lens (only ectoderm can respond due to expression of Pax6)
Morphogen: FGF8
Inducer: optic vesicle
Pax6: renders ectoderm in head “competent” to respond
Major signaling pathways involved in induction:
• RTKs
• TGF-beta
• Wnt
• Hedgehog
• Notch
Two mechanisms that direct Cell’s decisions and “lock” them in
- Stabilization of cell signaling pathways
- Epigenetic modification of gene expression
DNA methylation
• responsible for epigenetic regulation of chromatin activity
• DNMTs methylate DNA. DNMT1 copies methylation patterns during cell division, making epigenetic pattering heritable
• ex: imprinting in pre-primordial germ cells and X chromosome in activation 
Histone modifications
• responsible for epigenetic regulation of chromatin activity
• Activating modifications found at active promoters, and are negatively associated with DNA methylation
• ex. repressive modifications, trithorax complex proteins, Polycomb complex proteins
Hydatiform mole
Instead of forming a normal placenta, the trophoblast forms of mass of cysts. These are a manifestation of imprinting, caused by an under representation of the maternal genome
Maternal imprinting:
Favors the development of the embryo
Paternal imprinting
Favors the development of the trophoblast
Two global genomic DNA methylation events in the human life cycle
- Associated with the gain of totipotency by the zygote and blastomeres
- Associated with PGC‘s as they “wipe the slate clean” prior to the application of sex specific imprints 
Major early developmental sell differentiation events
- Trophoblast vs inner cell mass
- Formation of epiblast and hypoblast
- Formation of ectoderm, mesoderm, and endoderm
- Information of body axes (Dorsal/ventral, cranial/caudal, left/right)
Inner cell mass or trophoblast?
Asymmetric division —> increase CDX2 in outer cells —> Inhibition of hippo pathway —> Increasing yap —> Increasing CDX2 expression —> inhibits Oct4/NANOG/Sox2 which become restricted to inner cell mass—> inhibition of CDX2 in inner cell mass 
CDX2
Trophoblast differentiation, asymmetric cell divisions initially concentrate CDX2 in outer cells
Oct4/NANOG/Sox2
Define inner cells and are associated with the maintenance of pluripotency
Epiblast or hypoblast?
ICM Express both NANOG and Gata6—> ICM variably express FGFR2, lots of FGFR2 —> ERK signaling —> increases Gata6
Gata6: differentiation into hypoblast (primitive endoderm)
NANOG: maintains pluripotency and promotes epiblast
Creating the cranio-caudal axis
• Hox genes (A,B,C,D), Homeobox genes
• Retinoic acid regulates Hox expression
• RA in the primitive node, the longer cells stay there the more posterior they will become
• RA can be teratogenic in pregnancy
Dorsal ventral axis— Neural tube
• notochord—> ventral floor plate—> Shh
• dorsal roof plate —> BMP
• Neural cell differentiation in the neural tube is instructed by opposing gradient of BMP and Shh, which result in differential expression of transcription factors 
Left right asymmetry
Arises from the ability of the primitive node cilia to concentrate Nodal on the left side of the embryo resulting in the regional activation of transcription factors such as Pitx2
• driven by dynein 
Nodal—> Pitx2 and Lefty
Lefty—> inhibits Nodal
Humoral mediated immunity
• Antibody mediated
• B lymphocytes
• Antibodies circulating in serum
• Primary defense against extracellular pathogens, extracellular bacteria, circulating viruses
B cell maturation stages
1.) in the bone marrow, antigen independent, stem cell to pre-B cells, creates IgM
2.) in lymphoid tissue, antigen dependent, B cells migrate into follicles, form germinal centers (GC), IgM and IgD—> plasma cell 
Self tolerance of B cells
- Receptor editing: replacement of self reactive receptor with non-self reactive receptor
- Clonal deletion: elimination of self reactive B cell clones
- Clonal anergy: An antigen specific hyporesponsiveness, does not respond
Clonal selection
Epitopes: the specific molecular target of which a complementary antibody binds
Immunoglobins: B cells surface acting as antigen binding sites, binding causes proliferation of clone daughter cells
Mono clonal antibody response
One antibody binds to a singular, specific epitope on an antigen. This is the most specific antibody response
Polyclonal antibody response
A combination of more than one antibody, each specific for a different Epitope and varying affinities (group of things)
Germinal center zones
Dark zone: proliferating cells (centroblasts) undergo somatic hyper mutation
Light zone: Hypermutated resting B cells (centrocytes) and follicular dendritic cells
Mantle zone: mutated B cells and Tfh cells stimulation by Tfh —> plasma or memory cell transformation
Chemokines
Family of proteins orchestrating the migration of B cells from the dark zone to the light zone (reversible too)
The reason for so many mechanisms to prevent self reactive B cells 
B cells Express IgM, which can trigger non-specific antibody response such as TLRs and the complement cascade —> destroy all self cells
Ex. Lupus, celiac, rheumatoid arthritis
Structure of antibody
Y: heavy chain with a hinge region, and an FC region (constant/crystalline)
II: Attachments on the outside of the two Y arms, light chain, contain the Fab, and the antigen binding sites
Somatic hypermutation
The process of generating a larger repertoire of antibodies with diverse specificities via mutation of the V region DNA (not germline) 
Primary infection antibody response
- IgM: very rapid, short-lived
Isotype switching
- IgG: Longer, sustained, secondary recall
Antibody affinity
The strength of an interaction between a specific epitope and an individual antibody antigen binding site.
High affinity antibody will bind a greater amount of antigen in a shorter period of time
Antibody Avidity
The overall strength of an antibody antigen complex across all binding sites
Dependent on:
1.) Affinity Cohen of the antibody for an epitope
2.) Valency: number of binding sites of both antibody and antigen
3.) Structural arrangements: of interacting regions of antibody and antigen 
IgD
• no isotypes
• Monomer
• Antigen receptor on B cells not previously exposed to antigen
• Basophils and mast cells
IgE
• no isotypes
• Monomer
• Parasitic worm protection via activation of eosinophils — histamine
IgG
• IgG - 1,2,3,4 isotypes
• monomer
• Antibody-based immunity
• Opsonization, complement activation, antibody dependent cell cytotoxicity, neonatal immunity
• Passive immunity to baby from pregnant mom, short-lived
IgA
• IgA- 1,2 isotypes
• Dimer most commonly, can be monomer or trimer
• mucosal immunity— Good, respiratory tract, urogenital tract, saliva, tears
• breast milk —> passive to baby 
IgM
• no isotypes
• Pentamer
• Naïve B cell antigen receptors
• The first produced, complement activation
• associated with cold autoimmune hemolytic anemia
The diverse roles of natural IgM 
• pathogen neutralization
• complement activation
• Antigen recruitment to secondary lymphoid organs
• Antibody dependent cell mediated cytotoxicity
• Apoptotic cell phagocytosis
• controlling inflammation
• Prevent auto immunity
• Immune regulation and homeostasis
Neutralization: isotypes
IgM, IgA, IgG1,2,3,4
Opsonization: isotypes 
IgM, IgA, IgG1,3,4
Sensitization for killing by NK cells (ADCC) 
IgG1, 3
Sensitization of mast cells
IgE, IgG1,3
Activate complement system
IgM, IgA, IgG1,2,3
The goal of mechanisms are current and secondary lymphoid tissues
- Antibody secretion
- Isotype switching
- Affinity maturation
- Memory B cell 
B cell functions
- Antibody production
- Neuronal survival and differentiation
- Immunosuppression
- Inflammation
- Memory
- Antigen presenting
T cell independent mechanisms of B cell activation
• pattern recognition receptors
• Complement pathways
• neutralization
• Opsonization
• Antibody dependent cellular cytotoxicity (ADCC)
— Mainly IgM, low affinity antibodies, short-lived plasma cells
Opsonization
Antibodies coding the surface of a pathogen act as opsonins. These opsonins change the charge of the cell walls to help drive phagocytosis creates (+) infected cell to bind (-) phagocyte 
Where are complement receptors expressed for B cells
Follicular and marginal zone
T cell dependent B cell activation
• Activated helper T cell expresses CD40L, secretes cytokines
• B cells are activated by CD40 engagement, cytokines
• causes B cell proliferation and differentiation
Live attenuated vaccine
Pathogen rendered non-pathogenic with transient growth in host
Pros: cellular and humoral, strong, lifelong
Cons: no use for pregnancy and immuno compromised
Ex Adenovirus, polio, MMR, smallpox, InfluA 
Killed or inactivated vaccine
Epitope structure of antigen surface. inactivated via heat or chemicals
Pros: Safe
Cons: mostly humeral, boosters needed
Ex: Have a comma rabies, IM influenza, polio
Subunit, recombinant, polysaccharide, and conjugate vaccine
Use specific antigens to best stimulate response
Pros: target specific epitopes of antigen
Conns: expensive, weaker immune response
Ex: HPV, HPV, streptococcus pneumonia, herpes zoster
Toxoid vaccine
Denatured bacterial toxin with receptor binding site
Pros: protects against bacterial toxins, antibody response without causing disease
Cons: boosters needed
Ex: Diphtheria
MRNA vaccine
Lipid nano particle delivers mRNA, cells synthesize for proteins
Pros: cellular and humoral, high efficacy, safe in pregnancy
Cons: local transient systemic inflammation, myocarditis
Ex: Covid vaccine 
Risk factors for cancers
• 70% of cancers arise initially from environmental causes such as smoking, diet, obesity, viral infection
• Regardless of the initiating factor, all cancers involve multiple genetic and epigenetic changes that occur in a sequence overtime (aging = more cancer)
• Cancer arises from a clone of transformed cells, Gene-gene, gene-environment, modifier genes
Neoplasia
A disease process associated with uncontrolled cellular proliferation leading to a mass, or tumor (neoplasm)
The most common aggressive cancer, largely incurable
Metastatic solid tumors
— Defined by acquisition of genetic changes that permit invasion, evasion, and translocation
Carcinoma
Epithelial: intestine, breast, lungs
Hemopoietic and lymphoid
Leukemias and lymphomas
Sarcomas
Mesenchymal origin: bone, muscle, connective tissue
Most cancers are thought to arise in:
A stem cell tumor precursor. Cancer comes from transformed stem cell, differentiated cell does not have the potency
How neoplastic cells evade cellular constraints on growth and proliferation
• Independence from cell cycle regulation
• Independence from external growth signals
• Evasion of cell death
• Non-detection by the immune system
• avoidance of cellular senescence
• Disabling DNA repair mechanisms
• Capacity for angiogenesis

Clonal evolution hypothesis
Every tumor cell is equally capable of initiating neoplastic growth. Genetic and epigenetic changes occur over time, and with a selective advantage they will allow individual clones of cells to outcompete other cells and expand
The cancer stem cell hypothesis
Growth and progression of many cancers are driven by small sub populations of CSC‘s
Tumors contain a cell hierarchy in which a minority of SCs could self-renew and be able to regenerate a timer
Inherited cancer
Mendelian inherited: every cell in your body carries the mutation, you just need one hit/mutation
Results:
— Multiple tumors
— bilateral
— Early onset
Sporadic cancer
Most common, requires two hits/mutations to silence/mutate both alleles
Results:
— Single tumors
— Unilateral
— Later onset
Oncogene mutations
Turn on a stimulatory pathway and leave it stuck in the on position (gas— broken accelerator)
Examples: sis, Ret, Abl, K-Ras2, Myc, telomerase,Blc2
Tumor suppressor genes
Control aberrant cell growth, Considered the brakes
Examples: RB1, TP53, APC, VHL, BRCA1/BRCA2, MLH1/MSH2
Modes of action of the P 53 tumor suppressor
Activators: hyperproliferative signals, DNA damage, telomere shortening, hypoxia
Results: cell cycle arrest, senescence, apoptosis
What contributes to tumorigenesis?
Increased cell division, decreased apoptosis, and tumor microenvironment
Knudson’s two hit hypothesis and LOH
Almost all tumor suppressor genes act recessively at the cellular level: All function of the tumor suppressor gene must be lost
Loss of the second allele is called loss of heterozygosity
Inherited cancers considered autosomal dominant because the germline mutation is invariably followed by loss of the wild type of oil in a subset of cells
Dominant of the level of the organism but recessive at the level of the cell
Ways to lose the good copy of the tumor suppressor gene
(LOH: 2nd event)
Nondisjunction causing chromosome loss, chromosome loss then chromosome duplication, mitotic recombination, gene conversion, deletion, point mutation
Small scale: microinstability (MIN)
Arises from defects in mismatch repair or nuclear excision repair. Defects include MMR mutations that caused microsatellite expansions
Large scale: chromosomal instability (CIN) 
Arises downstream of defects in DNA damage checkpoints, genes that control chromatin condensation, chromatid separation, and genes that control mitotic events
Detection of a microunstable cancer is done by:
PCR amplification of select microsatellite regions in the genome. Tumors show extra PCR bands due to a failure to repair expansion or retraction of DNA repeat regions
A classic example of a tumor suppressor gene for colorectal cancer
APC, mutations of APC are rate limiting in about 85% of colorectal cancer. Two hits needed
APC regulates cellular level of beta-catenin, When not regulated it moves into the nucleus and uncontrolled expression of proliferation genes occurs
FAP: Familial adenomatous polyposis Inherited form of CRC, Mutant copy of APC —bad
Gleevec (imatinib)
Drug that targets the activity of the fusion of protein and halts CML (inhibits BRC 9-22 mutation activities)
—Must take the drug for life
Example of splitting diseases
Classification of diffuse large B cell lymphoma (DLBCL)
• consists of two distinct cancers, 40% of patients respond to chemotherapy R-CHOP
— Germinal Center B-cell DLBCL (good outcome) vs. activated B-cell-like DLBCL (poor outcome)
Cholesterol is synthesized:
From Acetyl CoA in the cytosol or taken up from the diet
Cholesterol functions:
- membranes
- lipoprotein particles
- bile acids
- steroid hormones
-vitamin D
Four stages of cholesterol synthesis
1.) Three Acetyl CoA make mevalonate (6C)
2.) mevalonate converted to isoprene (5C)
3.) six isoprenes condense to form squalene (30C)
4.) squalene is cyclized and converted to cholesterol ( 27C)
Key regulatory enzyme in cholesterol synthesis
beta-hydroxy-beta-methylglutaryl CoA reductase (HMG CoA reductase)
Statins target ____ for ______
HMG CoA reductase; managing high cholesterol
Transcriptional regulation of HMG CoA reductase
High cholesterol: SREBP sequestered in intracellular membranes with SCAP. Promotes proteolysis of HMG CoA reductase
Low cholesterol: SCAP leaves the DNA binding domain of SCREBP, which then translocates to the nucleus to increase activity of HMG CoA reductase
Regulatory phosphorylation of HMG CoA
Fasted/low energy: HMG CoA reductase is (P) by AMPK (AMPK activated by AMP and sterols)
Insulin/Fed: promotes cholesterol synthesis by activating phosphatases that dephosphorylate HMG CoA reductase
Farnesyl pyrophosphate
FP–> squalene –> cholesterol
FP –> ubiquinone
FP –> Dolichol (glycosylation in the ER)
The main source of cholesterol synthesis
Liver. Can export cholesterol in the form of: cholesterol esters in VLDL particles into the blood, and bile acids into the lumen of the gut
Glucose –> glycerol-3-P + FA CoA –> TG –> VLDL–> interacts with LPL –> TG –> FA
The rate limiting step in bile acid synthesis is:
7-alpha-hydroxylase (high levels of bile acids inhibit this step). This step also uses energy from NADPH
Bile acids as detergents
Bile acids are used to break down fats in the body. They become better detergents by oxidation of their side chains (3-alpha, 7-alpha, 12-alpha), and by forming conjugates with either taurine or glycine.
Secondary bile salts
Recycled bile acids that have lost their 7-alpha-hydroxyl group from gut bacteria breakdown. 95% of bile salts are recycled, they must have the 3-alpha-hydroxyl “ reuptake tag” otherwise they are excreted in feces
Nascent chylomicrons
Store dietary fat and cholesterol in an immature form with ApoB48, they enter the lymph and then the blood. In the blood they uptake ApoCII and ApoE to become mature chylomicrons.
ApoCII activates lipoprotein lipase (LPL)
The largest and least dense of the lipoprotein particles
Chylomicrons
Funtion of Bile salts in the gut
Gallbladder–> breakdown of TGs –> FA + 2-MG –> into nascent chylomicrons and reform into TG
Lecithin cholesterol acyl transferase
Within HDL particles, this enzyme allows cholesterol to be picked up by HDL and be trapped within the particle for reverse cholesterol transport to the liver from the blood.
Two functions of HDL particles:
1.) give ApoCII to nascent chylomicrons and immature VLDL particles
2.) Reverse cholesterol transport
VLDL contains
ApoB100, ApoE, and ApoCII
ApoB100 and ApoB48 are the same gene, changed by mRNA editing of a C–>U initiating a stop codon via APOBEC-1
VLDL and HDL swap:
HDL gives cholesterol esters to VLDL
VLDL gives TGs to HDL (return to liver)
Macrophages as scavengers
uptake oxidized LDL to create foamy cells when overabundance or when LDL receptors are mutated/inhibited
Steroid hormones derived from cholesterol
- glucocorticoids (cortisol, mobilize stored fuels)
- mineralcorticoids (Aldosterone, fluid + salt balance)
- androgens (testosterone, male dev)
- estrogens (estradiol, female dev)
- progestins (progesterone, pregnancy + female dev)
Difficulty with production of cortisol will create:
increased testosterone, and estradiol (slower)
Cortisol synthesis
Hypothalamus –> CRH –> ACTH in anterior pituitary –> cortisol synthesis from adrenal gland —> inhibits CRH and ACTH
Calcium homeostasis
Low Ca2+ –> PTH –> PTH1R –> increase CYP27B1 –> Ca2+ reabsorption from kidney
Low Ca2+ –> PTH –> PTH1R –> 25(OH)D –> calcitriol –> increased VDR –> Ca2+ reabsorption from the intestine
Low Ca2+ –> PTH –> PTH1R –> bone resorption and Ca2+ release
Three steps of vitamin D synthesis
1.) skin, sunlight (UV)
2.) liver, 25-hydroxylase
3.) Kidney, PTH-1-alpha-hydroxylase
Creates 1,25-dihyroxycholecalciferol (Calcitriol) - most active form of vitamin D
Mutation in the LDL receptor
Familial hypercholesterolemia– can lead to atherosclerotic plaques under the endothelial layer of blood vessels
Congenital adrenal hyperplasia
mutations in steroid hormone biosynthesis genes leading to increased androgens and decreased aldosterone and cortisol.
Symptoms: salt wasting, hirsutism, virilization, short stature, high androgens, high ACTH
Fungi types
1.) yeast, single cellular-budding/nuclear fission reproduction– psuedohyphae
2.) mold, chains growing together, hyphae with septates or smooth
Dimorphic fungal cell
can occur as mold OR yeast, can switch between the two
Fungi characteristics
ALL: heterotrophic: cannot produce food on their own
MOST: saprobes: obtain food from dead and decaying cells
SOME: obtain food from living plants and animals
Sporangiospore
Spore bearing sac, all spores are released at the same time–reproductive
Conidia
no sac-like structure around the spores, growing in cilia-like formations, one-a few spores pop off the top of the chain when ready–reproductive
Types of Conidiospores
1.) Coccidioides: release arthrospores
2.) Candid albicans: release Chlamydospores and/or blastospores
3.) Aspergillus: release phialospores
4.) Microsporum: release microconidia and macroconidia
5.) porospores
Superficial tissue involvement fungal infections
1.) Malassezia furfur
2.) Microsporum, trichophyton, epidermophyton
3.) Candida albicans
Systemic involvement fungal infections
Lung, lung and skin
1.) Coccidioides immitis dermatitidis, blastomyces, histoplasma capsulatum, cryptoccus neoformans
2.) paracoccidioides brasiliensis
Helminths
Roundworm: nematodes: ingestion, fecal pollution, close contact, meat, larva burrowing, fly bite, water containing cyclops
Flatworm: Trematodes/Cestodes: fresh water containing larva stage, pork, fish
Protozoa life cycle
Trophozyte (all)–> cyst (some, resting dormant phase)–> Trophozyte reformation
Pathogenic protozoa
Classified by how they move:
1.) Amoeboid (entamoeba, naegleria)
2.) ciliated (balantidium)
3.) flagellated (giardia, trichomonas vag, trypanosoma, leishmania)
4.) nonmotile (plasmodium, toxoplasma, cryptosporidium, cyclospora)
Reduviid bug
Example of infection cycle of Trypanosome: infects this bug which can infect humans and animals via bite
Type I hypersensitivity: immediate
IgE, CD4+, Th2 cells, histamine release and inflammation
Mech: mast cells, eosinophils leading to vasoactive amines, lipids, proteolytic enzymes, cytokines: IL-4, 5, 13
Type II hypersensitivity: Antibody mediated
IgM, IgG against cell surface or extracellular matrix proteins
Mech: Cell destruction (opsonization, complement, ADCC), and Inflammation (cellular dysfunction:antibodies block or activate signaling)
Type III hypersensitivity: Immune complex mediated
Circulating antigen:antibody complexes (large, hard to break down)- isotypes
Mech: Complement, neutrophils, lysozymes
Type IV hypersensitivity: Delayed, T-cell mediated
CD4+, Th1, 17, CD8+, cytotoxic T lymphocytes– 48-72hrs
Mech: CD4+ Th cytokine mediated inflammation, macrophage, neutrophil, direct target cell killing (CD8+ and CTLs)
Examples of Type I hypersensitivity
Atopy: atopic allergy to common environmental substances (dust mites, pollen, etc)
Anaphylaxis: Food, drugs, bee stings
Examples of Type II hypersensitivity
Blood transfusions (ABO and Rh +/-) including hemolytic anemia, thrombocytopenia, and hemolytic diseases of the newborn
Goodpasture syndrome (type II)
rare fetal autoimmune disease mediated by anti-glomerular basement membrane antibodies that target alpha-3 chains of type IV collagen in kidney and lungs
Acute rheumatic fever (type II)
Following strep infection, lectin complement pathway activated, binding antibody to heart cells causing rheumatic heart disease
Grave’s Diease (type II)
antibodies against thyrotropin receptor–hyperthyroidism (opthalmopathy)
Myasthenia Gravis (type II)
Antibodies attack acetylcholine receptor (AChr) affecting skeletal muscles and neuromuscular signal transmission
Serum sickness (type III)
antibodies to foreign antigens form complexes that travel around the body and deposit randomly: complement reaction
Systemic lupus erythematosus (type III)
autoantibodies to FNA, nucleoproteins, cytoplasmic antigens, leukocyte antigens, clotting factors, etc
Rheumatoid arthritis (type III)
autoantibodies to self IgG molecules
Examples of Type IV hypersensitivity
Delayed-type hypersensitivity: proteins (venom, TB)
Contact hypersensitivity: haptens (metal, poison ivy)
Gluten-sensitive enteropathy: Celiac, alpha-gliadin
Hallmark feature of Type IV hypersensitivity
Granulomas (macrophage accumulations, multinucleated giant cells, and lymphocytes)
The TB test is an example of:
Type IV hypersensitivity (T-cell mediated)
Contact dermatitis (type IV)
Urushiol + self antigens –> neoantigen that recruits cytokines, chemokines, leukocytes, Langerhans cells present to memory CTLS to result in direct killing, mast cells and basophils recruited
Multiple Sclerosis (type IV)
demyelinating disease of the CNS, forming demyelinating plaques
1.) initiating events
2.) Th1 (IFN-gamma), or Th17 (IL-17/IL-22)
3.) T cells, B cells, monocytes, and macrophages cross the BBB
4.) presentation of myelin antigen to immune cells
5.) CD4+, CD8+ destruction of myelin via complement system or ADCC
Type I Diabetes Mellitus (type IV)
destruction of beta-islets of Langerhans in the pancreas leading to insulin deficiency
Inflammatory Bowel Disease (type IV)
Ulcerative colitis or Chron’s
– Dysfunctional immune response to commensal organisms (Th1 and Th17)
Celiac Disease (type IV)
anti-gliadin response –> T-cell, plasma cell, macrophage infiltration –> villi destruction –> diarrhea, dehydration, malabsorption
Neoplasia
An uncontrolled, monoclonal proliferation of cells
Neoplasm
 A combination of neoplastic cells(Parenchyma) and supporting stroma (blood vessels and connective tissue, which are not neoplastic)
Benign neoplasm
Remains at the site of origin, usually amenable to surgical removal
Malignant neoplasm
Invades surrounding tissues and has the capacity to spread to distant sites (metastasis) 
-oma
Benign neoplasm of mesenchymal origin
Sarcoma
Malignant neoplasm of mesenchymal tissue origin
Benign neoplasm of epithelial origin
• adenoma: glandular tissue
• Papilloma: fingerlike or warty projections
• Cystadenoma: glandular tissue and forming large cysts
Carcinomas
Malignant tumors of epithelial origin
Leukemias/lymphomas
Malignant neoplasms of blood forming cells
Differentiation
How much a neoplasm resembles the tissue of origin
Anaplasia
Lack of differentiation, malignant neoplasms often are in a plastic, benign neoplasms are not
Pleomorphism
The degree to which cells within a neoplasm differ from each other
Malignancies are more pleomorphic then benign neoplasms 
Dysplasia
Refers to a pre-malignant pleomorphic state
Invasion
Ability of a neoplasm to breach normal barriers (penetrating basement membrane) and survive in new environments
Metastasis
Spreading to distance sites
Mechanisms:
1.) seeding of body cavities
2.) lymphatic (carcinomas)
3.) hematogenous/hepatocellular carcinoma/thyroid follicular carcinoma 
Synaptophysin and chromogranin stain
Target Neuroendocrine cells
Ex: Small cell carcinoma of the lung, carcinoid tumors
Cytokeratin stain
Targets epithelial cells
Ex: Carcinomas
Desmin stain
Targets muscles
Ex: leiomyoma, rhabdomyosarcoma
GFAP stains
Target neuroglia
Ex: astrocytoma, glioblastoma
Neurofilament stains
Targets neurons
Ex: neuroblastoma, neuroma
PSA stains
Target prostate epithelium
Ex: adenocarinoma of the prostate
S-100 stains
Targets neural crest cells
Ex: melanocytes and neural tumors
TRAP stains
Target tartrate-resistant acid phosphatase
Ex: hairy cell leukemia
Vimentin
Targets mesenchymal cells
Ex: sarcomas
Cachexia
A hyper catabolic state with muscle loss, often debilitating and fatal. Comes from the production of inflammatory mediators and hormones that can be produced/activated by tumors
Paraneoplastic syndrome
Tumors develop signs and symptoms that are not related to their anatomic location, and are due to the production of substances acting at a distance
Incidence versus deaths of cancer
Incidence: most common is CRC, breast
Deaths: most common is lungs and bronchus
Oncogenes
Mutated genes that cause excessive growth, independent of external cues. These are gain of function, behave in a dominant fashion (only one allele needs to be mutated) 
Tumor suppressor genes
Mutation of genes that inhibit cell proliferation normally, loss of growth inhibition. Loss of function, behaving in a recessive fashion (requiring mutation of both alleles)
Hallmarks of cancer
1.) self-sufficiency in growth signals
2.) insensitivity to growth inhibitory signals
3.) Altered cellular metabolism
4.) evasion of apoptosis
5.) Immortality, telomerase
6.) angiogenesis
7.) Invasion and metastasis
8.) avoiding host immune response
9.) Enabling of inflammation, prolong regeneration; more cellular division
Warburg effect
Cancer cells show market activation of glycolysis, with less use of mitochondrial oxidative phosphorylation— this helps create substrates for biosynthesis needed for growth and proliferation
Immune checkpoint inhibitors
Tumor and CTL binding at the TCR and peptide MHC leads to cytotoxic granules being released to kill the tumor if anti-PD1 and anti-PD1 ligand is available (The tumor can’t secondary bind to the CTL and inhibit it’s “not self” recognition)
 Grading of malignancies
Higher grades occur when the tumor lacks differentiation, have a high mitotic rate, and have more nuclear pleomorphism
Staging of malignancies
Measuring the degree of localization or spread, TNM (Tumor, lymph nodes status, metastasis) 
