Exam 4 Flashcards
Locus
A segment of DNA at a specific location
Alleles
Alternative possible versions of a gene
Wild type
Single prevailing allele, present in the majority of individuals in a population
Variants or mutants
The other versions of alleles that are not wild type
Polymorphic alleles or polymorphisms
Variant alleles are said to show polymorphism which affect disease susceptibility 
Genotype
An entire set of alleles in a genome, or the set of alleles at a specific locus
Phenotype
Observable expression of a genotype as a morphological, clinical, cellular, biochemical, or other trait 
Homozygous and heterozygous
Homozygous: An individuals two alleles are functionally identical at a locus
Heterozygous: two alleles are functionally different add a locus 
Hemizygous
When an individual only has one allele of a gene
Compound heterozygotes
Individuals with two heterogeneous recessive alleles at a particular locus that can cause genetic disease
Pedigree
Graphical representation of the family tree
Kindred
Extended family
Proband vs consultand
The first affected person who is brought to clinical attention
Vs
The person who brings the phenotype to clinical attention 
 Mosaicism
 Phenotype may only be expressed in a subset of cells, typically 50-50
Ex: muscular dystrophy
Pure dominant
When both homozygous and heterozygous shown identical severity of phenotype
Semidominance/ Incomplete dominance
A disease is more severe in homozygotes compared to heterozygotes
Codominance
Two different variant alleles are expressed together
 Penetrance
The probability that a mutant gene will have any phenotypic expression— Anything less than 100% is reduced penetrance
Expressivity
The severity of expression of the phenotype among individuals with the same disease causing phenotype— Usually variable expressivity 
Modifier genes
Segregating variant alleles, distinct from the disease causing genes, can also influence penetrance and variable expressivity 
Neurofibromatosis (NF1)
NF1 is an autosomal dominant disease and is a common disease of the nervous system/eyes
 Always exert some kind of disease phenotype in heterozygotes (100% penetrance) But the severity varies greatly (Variable expressivity due to different mutations) 
Allelic heterogeneity
The occurrence of more than one allele at a locus— ex. Thalassemia
Different mutations at the same gene making it more or less severe
Locus heterogeneity
The association of more than one locus with a clinical phenotype— ex. Thalassemia from a-globin or b-globin

Different genes give the same phenotype
Clinical or phenotypic heterogeneity
The association of more than one phenotype with mutations at a single locus — ex. B-Thalassemia and sickle cell result from the same b-globin gene mutation
Examples for allelic heterogeneity
CTFR
PKU
a/b-thalassemia
Examples of locus heterogeneity
Hyperphenylalaninemias
rentinitis pigmentosa
Familial hypercholesterolemia
Examples of phenotypic/clinical heterogeneity
RET gene — Encoding receptor tyrosine kinase
• colonic ganglia, Hirschsprung disease
• Cancer of thyroid and adrenal glands
• both 
Hemachromatosis
• mutation in the HFE gene
• Sex influenced autosomal recessive disorder
• Iron overload and damage to the heart, liver, pancreas
• Reduced penetrance in women, because low iron levels, menstruation, and lower alc intake
Consanguinity 
When parents are closely related (second cousins or closer) causing autosomal recessive mutant alleles to be more prominent
Ex. Xeroderma pigmentosum
Inbreeding
Consanguinity at population-level. Individuals from a small population tend to choose their mates from within the same population— shared gene alleles from ancestors
Ex. Tay-Sachs, Ashkenazi Jews 
Autosomal dominant inheritance
• phenotype usually appears in every generation, any child of an affected parent has a 50% risk for inheriting the trait 
• exceptions include fresh mutations in a gamete of a phenotypically normal parent
Incomplete dominant inheritance example
 Achondroplasia— (dwarfism) Homozygotes tend to show a more severe phenotype
Familial hypercholesterolemia
New mutations in autosomal dominant disorders
Mutations most commonly arise in the gametes of the parents (Sperm, eggs) And the likelihood of new mutations rises dramatically with the age of the parents 
Male limited precocious puberty
Mutation in the LCGR which becomes constitutively active in the absence of its hormone ligand: puberty around age 4 
• on an autosomal chromosome
 Manifesting heterozygotes
When female heterozygotes for an X-linked recessive disease demonstrate a disease phenotype
Unbalanced X inactivation
the proportion of mutant genes expressed is significantly different than 50% (not following typical mosaicism)
X-linked dominant inheritance
 all of the daughters but none of the sons of an affected male will have the disease
 typically semi dominant— Different levels of severity
Male lethality can occur— never affected male in pedigree 
Y-linked dominant disorders
• SRY genes are important— sex determination genes
• Y-linked disorders involve infertility/reproductive abnormalities
• there is one form of male deafness associated with Y chromosome
Unstable repeat expansion disorders and pre-mutation alleles
Huntington disease, fragile X, myotonic muscular dystrophy, and Friedreich ataxia 
Transgenerational epigenetic inheritance
Non-gene based inheritance, environmental such as diet. May involve small non-coding RNAs
Changes in metabolism, susceptibility to diseases such as type two diabetes
Gluconeogenesis
The liver uses amino acids, lactate, and glycerol to produce glucose, which it exports to the blood
Gluconeogenesis substrates come from
• Anaerobic glycolysis using lactate
• muscle protein degradation (Ser, Ala)
• Lipolysis leading to glycerol
The key regulatory steps of glycolysis enzymes
Glucokinase, PFK1, pyruvate kinase (PK-L)
The key regulatory steps of gluconeogenesis
Glucose-6-phosphatase, fructose-1,6-bisphosphate, PEP carboxylase
Oxaloacetate and gluconeogenesis 
OAA —> Malate
OAA —> Asparate
^ Used to remove OAA from the mitochondria to Create PEP and CO2 from carboxykinase and GTP (PEP—> glucose after that)
When is malate-OAA transfer used vs Aspartate-OAA transfer?
Malate-OAA requires an abundance of reduced NADH (Asp-OAA doesn’t require anything) so it is used when there is a lot of NADH 
Oxaloacetate —PEP-CK—> PEP
Phosphoenolpyruvate carboxykinase (PEP-CK) is a key regulatory step in gluconeogenesis, regulated by transcription
Transcriptional regulation of PEP-CK
- Insulin response element (IRE)
- Glucocorticoid response element (GRE)
- Thyroid response element (TRE)
- Two cAMP response elements (CREI and CREII)
- TATA box
^^^ in that order
PEP-CK and steroid hormones binding its transcriptional receptors
1. Cortisol bunds to the GRE (agonist)
- Glucagon -> cAMP -> PKA -> binds CREII (agonist)
- Insulin prevents FOX01 binding to IRE (antagonist—inhibits PEP-CK expression)
 insulin and PEP-CK 
Insulin —> INS1 —> PI3K —> Akt (PKB) —> phosphorylates FOX01 preventing it from binding to IRE
TORC2 and CREB
when they bind they create PGC1alpha and PEP-CK expression to increase gluconeogenesis
Regulation of TORC2
AMPK (Energy sensor, AMP kinase) gets phosphorylated by LKB1, which in turn phosphorylates TORC2, preventing its nuclear localization. This prevents transcription of gluconeogenesis genes and decreases hepatic glucose production
PKA phosphorylation in the liver
- Inhibits pyruvate kinase in the fasted state
- PK phosphorylation of CREB activates transcription of PEP-CK

Fructose 2,6- bisphosphate as an activator/inhibitor
Allosterically activates PFK1, and allosterically inhibits fructose 1,6-bisphosphate (drives towards glycolysis)
glucose 6-phosphatase vs Glucokinase
g-6-p always outcompetes and sends glucose out to cells with hexokinase
Energetics of gluconeogenesis
11 high energy phosphate bonds are consumed (a lot of energy), but this is required for RBC‘s 
High-protein meal with no carbs
Insulin and glucagon both increase. Insulin promotes storage of dietary amino acids as proteins, and glucagon promotes the conversion of dietary amino acids into glucose. Blood glucose levels stay the same after eating
PEP-CK disorder
Inherited loss of function mutations that are rare. Early death, fatty liver, FTT 
Functions of the cell membrane
- Mechanical structure
- Selective permeability
- Transport
- Markers and signaling
Electrochemical gradient of the lipid bilayer
- e- gradient, charge not even
- Chemical gradient, pH not even
Simple diffusion
No ATP required, gases, hydrophobic molecules, small polar molecules
Ex. CO2, 02, Benzene, H2O, ethanol
Aquaporin
Water channel that allows the movement of larger amounts of water
They also facilitate the reabsorption of water in the kidney collecting ducts 
Human AQP2 and nephrogenic diabetes insipidus (NDI)
Disease due to kidney pathology, where APQ2 doesn’t work
APQ2 normally is single file and diffusion limited, governed by water-protein interactions
Facilitative transporter
Moves molecules from higher concentration to lower concentration by binding the desired molecule to be moved 
Transport proteins can be saturated and have a Vmax
Gated channels
• Opens both sides of the membrane simultaneously to move substrate down electrochemical gradient
• can occur selectively (Ex. Cations or anions), or gated by voltage, ligand, light, temperature
ligand gated Cl- channel
ABD and R domains—> PKA Phosphorylates R group causing confirmational change—> opening of channel and influx of Cl- out of the cell
Dysfunction of CFTR (Cl- ligand gated channel) leading to cystic fibrosis
• CFTR mutation of F508
• Secretion of Cl- in sweat is very high in CF patients  because reabsorption of Cl- is low
Typical ion distributions across the plasma membrane
• Na+ high in extracellular
• Ca2+ high in extracellular
• Cl- high in extracellular
• K+ high in cytoplasm
Classes of ATPases
P class: located on plasma membranes, or auto phosphorylated during catalysis, ex: sodium potassium pump
V class: Located in secretory vesicles like synaptosomes, Transport H+ into vesicle 
F class: located in mitochondria, ATP is formed here (think: F0 and F1 from skildum) 12 H+ —> 3 ATP
Sodium potassium pump
3 Na+ out
2 K+ in
— Regulated by ATP, phosphorylation causes confirmational change on pump
Secondary active transport of glucose
Symporter: sodium goes passively down gradient, creating energy to move glucose from low concentration—> high concentration
HCO3- and Cl-
Anti-porter: bicarbonate-chloride via AE1 pushing chlorine into the cell and bicarb out of the cell
Digoxin
Plant used to treat heart failure by inhibiting sodium potassium pump
— Causes dysfunction in the sodium potassium pump: too much intracellular Na+ leads to Na/Ca exchanger malfunction, leading to too much Ca2+ in the cell causing contractions
ATP binding cassette transporters (ABC transporters)
Increased efflux and decreased influx caused by MDR1 transport protein-1
— Commonly and cancer cells to get rid of anticancer drugs
Types of endocytosis
- Phagocytosis (cell eating)
- Pinocytosis (cell drinking)
- Receptor mediated endocytosis (Receptor binding to molecule you want to trigger endocytosis) 
Cholesterol uptake by receptor mediated endocytosis
ApoB-100 on LDL particle binds to receptor triggering endocytosis, LDL receptor is maintained while endosome gets degraded
Duchennes Muscular Dystrophy
Mosaic expression of x-inactivation gene
Hemophilia A
X-linked recessive disease: females are carriers, males cannot pass to sons
Rett Syndrome
X-linked dominant syndrome — male lethality (homozygous female lethality)
Symptoms: neurological, 6-18mo, spastic, ataxic, autism, seizures
Mutational mosaicism
Can occur in germlines or somatic cells. One cell mutates and spreads, not ALL cells mutated
Childhood cancers and OI
Mitochondrial DNA
• Maternally passed down
• demonstrate mosaicism by having a wide range of severity based on how many mutant mitochondria end up in the germ cells
• There is a threshold for phenotypic expression between normal and diseased
What type of SNP’s are important for precision medicine?
• actionable genetic variants
• Genomics diversity in three main categories: efficacy disrupters
1.)  polymorphisms in enzymes
2.) Drug transporters
3.) Drug targets
Benefits of precision medicine
- Determine individuals risks of developing certain diseases
- Find biological markers to aid in prevention and diagnosis
 - Find the most effective therapy for different people
- Identify solutions to health disparities
Clinically actionable SNP
Testing for an SNP to determine drug dosing and safety
Example: 6-mercaptopurine in Acute lymphoblastic leukemia and NUDT15/TMPT mutated SNPs 
Homozygous deficiency in TPMT or NUDT15 change of treatment
Typically require 10% or less of the standard PURIXAN dose (mercaptopurine)
(ALL)
Heterozygous deficiency in TPMT and or NUDT15
Reduce the PURIXAN dose based on tolerability. Most patients with heterozygous deficiency tolerate recommended mercaptopurine doses 
(ALL)
Example of functional testing
Prior to treatment with methylene blue, patient must be tested for G6PD deficiency (more susceptible to oxidative stress, acute hemolytic anemia)
Pharmacogenomics
The study of how an individuals genetic inheritance affects the body‘s response to drugs
Benefits of pharmacogenomics 
• reduce or eliminate side effects
•  Access to targeted therapies
• Increase effectiveness of treatments
• Tailored to the individual
Types of genetic testing
- Functional tests (phenotype)
- Direct sequencing (genotype)
- PCR, quantitative RT-PCR, digital droplet PCR
- Deep sequencing (NGS) (need to confirm disease relevance with functional studies) 
Actionable SNP’s
Have actionable genetic variants, including polymorphisms in enzymes, drug transporters, and drug targets
Types of metabolizers
- Poor metabolizer (too slow/not at all)
- Extensive metabolizer (just right)
- Ultra rapid metabolizer (too fast)
Poor metabolizer
Has a genetic predisposition or polymorphism that blocks the metabolism of certain drugs. They may overdose on less because it cannot be metabolized
Ultra rapid metabolizer
(Rare, less than 10% of the population) they metabolize the drug too fast to gain any benefit from the medication
Erroneously labeled “drug addicts”
Important cytochrome P450
CYP3A4 metabolizes many drugs/detoxification. Located in liver and intestines
Genotype versus phenotype in the context of precision medicine
Genotype: PCR, SNP’s, Genomic variation
Phenotype: blood tests, functional tests, enzymatic expression
Etiology 
Initial causes of a disease (Genetic, environmental, chance)
Pathogenesis
How do the etiologies produce the disease? (Sequence of events)
Morphologic changes
Observable structural alterations that are characteristic of a disease (diagnostic) 
Clinical manifestations
Functional abnormalities that determine signs, symptoms, clinical course, and outcome of a disease
Four aspects of disease constitute the core of pathology
1. Etiology
2. Pathogenesis
3. Morphologic changes
4. Clinical manifestations
Cellular adaption
- Hypertrophy: increase in cell size
- Hyperplasia: increase in cell number
- Atrophy: decrease in cell size and/or number
- Metaplasia: change from one cell type to another (Particularly in endothelial cells)
Hypertrophy
Driven by increased workload, typically due to hypertension in heart, can be due to hormones (uterus)
Increase in cytoplasm size and occasionally nuclear enlargement
Hyperplasia
Driven by hormones and growth factors
Common example is endometrial hyperplasia with a larger epithelial area with more glands 
Metaplasia
Occurs as a response to chronic stress and irritation from an altered environment
• Squamous epithelium replacing columnar ciliated epithelium (smoker’s airway)
• Metaplastic bone formation in soft tissue after trauma (connective tissue)
Reason for metaplasia?
Protective in the short term, metaplasia is often at risk of development into malignancy in long-term (cancer) 
Atrophy
Decrease in both size and number of cells due to:
- Decreased work load
- Denervation
- Nutritional deprivation
- Decreased blood supply
- Pressure (chronic, ulcers)
- Loss of endocrine stimulation 
Hypoxia— Decreased ATP
Loss of activity of Ca2+ and sodium potassium pumps 
• cells swell, plasma membrane breaks
• Ca2+ Activates proteases, possible lipases, endonucleases, and DNAases
• Switch to anaerobic metabolism— lactic acid 
Free radical (ROS) generation
Causes: in redox reactions, UV light, radiation, metals, chemicals, inflammation
Results: Lipid peroxidation, DNA fragmentation, protein cross-linking 
Chemical injury
Drug or other chemical, sometimes via toxic metabolite of that drug (CYP)
ER UPR and DNA damage
Both can lead to apoptosis if severe
Mitochondrial dysfunction in tissue
Failure/abnormal oxidative phosphorylation: Depletion of ATP, generating ROS
Membrane barrier damage releases cytochrome C into cytoplasm and triggers apoptosis
Membrane defects
Mitochondrial: loss of ATP production, release of cytochrome C
Plasma membrane: influx of fluid ions, lots of critical metabolites
Lysosomal membrane: leak of lysosomal hydrolyzes in the cytoplasm and digestion of cellular components
Reversible cell injury
• Mitochondrial/cell swelling
• plasma membrane blebs (Areas pinching off)
• Nuclear chromatin clumps
• Myelin figures (Phospholipid aggregates) 
Irreversible cell injury
• plasma membrane breakdown
• Autolysis from lysosome rupture
• nuclear breakdown: Pyknosis, Karyorrhexis, karyolysis
Pyknosis
Condensation of nucleus, appears smaller and darker
Karyorrhexis
Fragmentation of the nucleus
Karyolysis
Dissolution of the nucleus. Fades and goes away, not able to see with hematoxylin stain
Apoptosis
ATP dependent cell program to death
• Minimal surrounding tissue reaction
• caspases activated (cytosolic proteases)
Histologically: deeply is eosinophilic cytoplasm and basophilic nucleus
Intrinsic (mitochondrial) pathway and apoptosis
BAX & BAK are proapoptotic and activate when p53 activates due to DNA damage. they regulate initiator caspases to kill the cell
Bcl-2 and Bcl-xL are anti-apoptotic and try to prevent cell death 
Extrinsic (death receptor) pathway
Ligand receptor interactions
• FasL binding to Fas (CD95)
• TNF binding to it’s receptor
^^ both activate caspases
• cytotoxic T cell T cell releases granzyme B and perforin into the cell 
Necrosis
Extrinsic injury causing plasma membrane damage, with leakage of cellular components
Local inflammatory tissue response (not silent to other cells like apoptosis)
Hypoxia versus ischemia
Hypoxia is decreased supply of oxygen, while ischemia is decreased blood supply which leads to hypoxia, loss of nutrients and accumulation of toxic metabolite waste
Reperfusion injury
Exacerbate injury, ROS, calcium overload, inflammation, activation of the complement system
Lipofuscin accumulation 
Wear and tear pigment, product of lipid peroxidation, seen in heart and liver, yellow brown find granules often perinuclear
Protein accumulations
Renal tubes, accumulations of fragments in cytoskeleton, defective intracellular transport, wrestle bodies
All of these look hypereosinophilic
Fat accumulations (steatosis)
Primarily liver, heart, skeletal muscle, kidney
Iron accumulation (hemosiderin)
Local excess in iron, usually hemorrhage
Systemic excess in iron, hemachromatosis
Chunky, yellow brown granules
Cholesterol accumulation in macrophages
Atherosclerosis, xanthomas, cholesterolosis, foamy cells
Dystrophic calcification
Seen in areas of necrosis, atherosclerotic plaques, aging, and damaged cardiac valves
 Metastatic calcification
Calcium deposition in normal tissue and systemic calcium is elevated
Ex. Hyperparathyroidism, renal failure, Vit D, increased bone resorption
Polyol pathway
Glucose > aldehyde > alcohol > ketone > fructose
Occurs in the eye, can increase intraocular pressure and cause Cataracts 
Sucrose goes to:
Glucose and fructose via sucrase isolmaltase
How is dietary fructose taken up?
GLUT5 in ilium
GLUT2 sometimes in liver, pancreas, and jejunum
Process of fructose to energy
Fructokinase creates Fruc-1-P
Aldolase B cleaves it into dihydroxyacetone phosphate and glyceraldehyde
Triose kinase creates glyceraldehyde 3- phosphate : glycolysis
How is excess fructose stored?
Fatty acids via pyruvate/TCA cycle/citrate
Glycogen via gluconeogenesis > glycogen synth
UDP-glucose
A common intermediate, helps with glycosylation and can be turned into UDP-galactose
How is galactose transported from lumen to blood?
GLUT2 and SGLT1 (Na+ and sugar) transporters 
Brush border enzymes lactase can also convert lactose to glucose and galactose
Process of galactose to energy
Galactokinase creating galac-1-P
galac-1-P uridyl transferase with UDP-glucose creating UDP-galactose and gluc-1-P
phosphoglucomutase creating gluc-6-P 
How does UDP-glucose become UDP-galactose?
Epimerase 
Nonclassical galactosemia
Inhibition of galactokinase.
Galactose accumulates and is converted to galactitol through polyol pathway in eyes: cataracts
Treatment: eliminate lactose from diet
Classical galactosemia
Inhibition of epimerase or galactose-1-P uridylyltransferase (serious) : FTT, jaundice, hypoglycemia
Treatment: eliminate galactose from diet, prognosis is poor
Products of the pentose phosphate pathway
Reduced NADPH and a five carbon sugar. Used for antioxidant defense, and nucleotide biosynthesis
The oxidative phase of the pentose phosphate pathway
Creating 2 NADPH, CO2, and ribulose 5-phosphate
The nonoxidative/regenerative  phase of the pentose phosphate pathway
Creating xylulose 5-phosphate —> ribulose 5-phosphate —> ribose 5-P —> nucleotide biosynthesis 
Glutathione
Tripeptide: glutamate, cysteine, glycine

Purpose: Neutralizes ROS by creating disulfide bonds
NADPH maintains glutathione in the reduced to state (reduced= GSH, oxidized= GSSG)
Transketolase
Transfers two carbon groups
Transaldolase
Transfers three carbon groups (6–>3+3)
ChREBP
Carbohydrate response element binding protein
A transcription factor which is inhibited by phosphorylation by PKA & AMPK 
Xylulose 5-P and ChREBP
X5P acts as an allosteric activator of PP2A, which removes the inhibitory phosphate allowing for a translocation of ChREBP to nucleus 
Essentially, X5P promotes transcription of genes that convert carbs to fat
Genes that are upregulated by ChREBP
— pyruvate kinase
— malic enzyme
— Citrate lyase
— Acetyl-CoA carboxylase
— Fatty acid synthase
Essential fructosuria
Inherited loss of function mutations in fructokinase. Benign, may cause false positive dipstick urine tests for diabetes
Hereditary fructose intolerance
Inherited mutations in aldolase B. Much more serious, build up of fruc-1-P with no metabolic fate
This traps all of the cells phosphates, and ATP synthesis is impaired
Lactase deficiency
Dietary lactose is not broken down to monosaccharides in the small intestine. Gut bacteria ferments lactose into lactic acid and water enters the lumen of the gut to offset the increase lactate and proton concentration.
Three types of lactase deficiency
Primary: autosomal recessive, lactase activity declines over many years
Secondary: damage of brush border of intestinal enterocytes due to intestinal disease
Congenital lactase deficiency: complete absence of lactase
Uridine diphosphate galactose 4-epimerase deficiency 
The treatment is to restrict, but not eliminate galactose from the diet because we need UDP galactose for glycosylation reactions and can only get it from the diet
G6PD deficiency
X-linked trait, their capacity to regenerate NADPH through the pentose phosphate pathway is limited causing severe ROS rxn.
Acute hemolytic anemia (think: sulfonamides)
Warfarin
Rate of elimination is independent of dose, zero order/nonlinear kinetics, Low therapeutic index, causes many adverse drug reactions, weak acid
Oral anticoagulant that decreases concentrations of vitamin K-dependent clotting factors
Two enantiomers of warfarin
S: Active, CYP2C9
R: Less active, CYP3A4, CYP2C19, CYP1A2
VKORC1 polymorphism and warfarin
PD: A mutation (G WT)
Reduction vitamin K —> increase in warfarin activity
CYP2C9 and warfarin
PK: *2, *3 mutations
Lowered metabolism elimination —> increase in warfarin activity
Antacids and warfarin
PK
Less absorption (ion trapping) —> less warfarin activity
Salads (greens) and warfarin
PD:
increase/decrease absorption Vit K, —> increase/decrease warfarin activity
Aspirin and warfarin
PK: lowered plasma binding protein —> increased warfarin
PD: antiplatlet —> increased warfarin activity
Glipizide and warfarin
PK:
Substrate of CYP2C9 lowers metabolism elimination —> increased warfarin
Decreased PPB —> increased warfarin
Cimetidine and warfarin
PK: inhibitor CYP2C9 decreasing metabolism elimination —> increase warfarin activity
Rifampin and warfarin
PK: inducer of CYP2C9 increasing metabolism elimination —> decreased warfarin activity
Four types of enzyme-linked receptors
- Receptors that are Tyrosine kinases
2. receptors that recruit tyrosine kinases
- Receptors that are serine-threonine kinases
- Receptor guanylyl cyclases 
Activation of RTKs (receptor tyrosine kinases)
Ligand binding dimerizes or oligomerizes RTKs
Relayed along:
1. PLC — Ca2+/PKC
2. Ras/Rho — MAPK
Ligands can be what?
- Monomers or multimers
- Arrayed on proteoglycans of the ECM or other transmembrane proteins
3. Other transmembrane proteins (like ephrins)
RTKs do what after binding ligands?
Autophosphorylate themselves In order to further increase their Kinase activity, and serve as docking sites
Which phosphatidylinositol cannot be cleaved into IP3 and DAG by PLC?
PI(3,4,5)P3
It instead serves as an anchor point for the assembly of other signaling platforms
PTEN
Phosphatase that converts PI(3,4,5)P3 back into PI(4,5)P2
it is an important inhibitor of PI(3,4,5)P3 associated signaling platforms
RTK and Ras—MAPK
RTK -> GEFs of Ras -> Ras activation -> MAPK pathway (serine threonine kinase cascade) -> cell proliferation or differentiation gene expression (important for cancer)
ERK1/2 and ERK5
Growth factors, hormones, Ras
Growth, survival, differentiation, development
ERK5 is similar, can be acted on by stress
p38
Inflammatory cytokines and stress (Rho)
Inflammation, apoptosis, growth, differentiation 
JNK
Cell and environmental stress (Rac)
Inflammation, apoptosis, growth, differentiation
RTK and PI 3-kinase — PKB/Akt
Binding of growth and survival factors to RTK‘s can turn on PI3K which can lead to the phosphorylation and activation of PKB/Akt which can activate a number of targets with pro-survival and growth functions
mTOR signaling pathway integrates:
A serine threonine kinase that functions in dual complexes, and integrates: metabolic status and growth control
• Growth factors
• Energy status
• Oxygen
• Amino acids
mTOR signaling pathway regulates:
Cell survival and growth processes:
• angiogenesis
• Cell growth
• nutrient uptake and utilization
• Metabolism
• Cytoskeletal organization
GPCR signaling:
• adenylate cyclase (cAMP/PKA)
• Phospholipase C (Ca2+ and PKC)
• Rho monomeric G proteins (cytoskeleton & MAPK)
RTK signaling:
• PI3-K (PKB/Akt)
• Phospholipase C (Ca2+ and PKC)
• Ras and Rho g-proteins (MAPK and cytoskeleton) 
Receptors that recruit tyrosine kinases
Integrins: FAK and Src
Many: Src
Cytokine receptors: Jak
Integrins, FAK, and Src 
Associated with focal adhesions to modulate numerous signaling pathways
Src tyrosine kinase activity activated by:
It’s unfolding after the binding of a ligand to its SH2 and or SH3 domain
SH2: Autophosphorylation ligand
SH3: Proline motif on beta-arrestin bound to the receptor
Cytokine receptors and Jak-STAT
• Responds to cytokines and hormones promoting Jak, which auto phosphorylates to increase its activity and then phosphorylate the receptor
• STAT proteins bind to the receptors, and are also phosphorylated by Jak
• STAT moves into the nucleus where it can turn on target genes 
TGF-beta: serine-threonine kinases
Transforming growth factor beta: Super family of signaling molecules (TGF-b, activins, BMPs)
They regulate the number of processes required for cell function by phosphorylating SMAD
Receptors that are guanylyl cyclases 
Single pass transmembrane proteins with extracellular binding site for signal molecule and an intracellular guanylyl cyclase catalytic domain
• bind ANP/AMP ligands-> cyclase domain -> cGMP-dependent serine-threonine kinases and PKG
• intracellular mediator
NPR-A
ANP/BNP
• regulate salt and water balance, stimulates the kidneys to secrete sodium and water, induces the smooth muscle cells in the blood vessel walls to relax
^^^ lower blood pressure 
Four major signaling pathways are associated with proteolytic events:
- Notch
- Wnt
- Hedgehog
- NF-kB
Notch
• signaling pathway in early development
• Delta and jagged ligands bind and trigger proteolysis of notch on both sides of the plasma membrane (let’s tail enter cytosol)
^^^ transcription regulator
Wnt of Frizzled family
• secreted signal molecules that act as a local mediators to control development
• Associated with colorectal cancer
• They signal through Dvl (disheveled)

Frizzled family
Seven pass transmembrane proteins, which are atypical members of the GPCR family
In the absence of Wnt
Protein degradation of beta-catenin
In the presence of Wnt
Wnt binding couples and activates frizzled-LRP Which binds and activates disheveled (Dvl)
• this inhibits the degradation complex of beta-catenins, so they can move into the nucleus and displace repressor proteins to activate Wnt via TCF/Lef1 
Hedgehog signaling
Patched(Ptch1) and smoothened(SMO) release signals from hedgehog to GLI to influence cell proliferation

Dysfunction of hedgehog signaling can give rise to:
Cyclopia
Absence of hedgehog
Patched inhibits smoothened, GLI is phosphorylated and partially cleaved, fragment moves into nucleus and access repressor of hedgehog
Presence of hedgehog
HH binding to patched blocks inhibition of SMO, allowing full length GLI to move into nucleus and activate hedgehog target gene transcription
Occurs near primary cilium 
NF-kB
Gene regulatory proteins that play major roles in stress and inflammation
IkB: Inhibitory protein
Nuclear hormone receptor parts
A/B: Active domain
C: DNA binding domain
D: Hinge domain
E/F: Ligand binding domain
Broad behaviors of NRs
Steroid hormone receptors in the cytoplasm
Retinoid/thyroid/vitamin D receptors in nuclei
Orphan receptors: unknown
NO mediated vasodilation
Cooperative event involving endothelial cells and the surrounding smooth muscle cells
IP3 -> Ca2+ -> calmodulin -> NO synthase -> NO -> guanylyl cyclase -> cyclic AMP -> PKG -> muscle relaxation
Neuronal nitric oxide synthase
• Long-term potentiation
• Cardiac function
• Peristalsis
• Sexual arousal
Endothelial nitric oxide synthase
• vascular tone
• Insulin secretion
• Airway tone
• Regulation of cardiac function and angiogenesis
• Embryonic heart development
Inducible nitric oxide synthase
• in response to attack by parasites, bacterial infection, tumor growth
• Causes septic shock, autoimmune conditions
Nitrodilators
Drugs that mimic the actions of endogenous NO by releasing NO or forming it within tissues. These drugs act on the vascular smooth muscle to cause relaxation
Endothelial independent vasodilators
Gap junctions
Connexons Which are hexameric hemi-channels composed of protein connexin
Regulated to open or close the passage between cells
Malfunction in gap junctions
Heart arrhythmias
Pleiotropy
Phenotypic expression can create a wide range of issues, differentiation 
Fructose 1,6-bisphosphatase deficiency:
Autosomal recessive disorder exacerbated by metabolic stress, injury, and infection
Liver reverts to glycogenolysis for energy
PEP carboxykinase deficiency
RARE. Early death, fatty liver, infant death
Transcriptional regulation of lipogenesis
- ChoRE: bunds ChREBP from glucose
- LXRE
- SRE: binds SREBP1C from insulin
- E-Box: binds BAF60C from insulin
= lipogenesis (in that order)
Polymorphic chromosomal markers
• markers that can distinguish between individuals and between carriers and non-carriers of a disease gene
• Used to detect, map, clone
• Most common markers are “DNA sequence variants”
Using polymorphisms in human and medical genetics
Researching a particular region of a chromosome, prenatal diagnosis of genetic disease, forensic applications, and genomics-based personalized medicine
Indel
And insertion or deletion of a nucleotide base
Restriction fragment length polymorphism (RFLP) 
• allelic variant that abolishes or generates a restriction endonuclease recognition site or changes the size of an RFLP
• biomarker, not the cause of a dysfunctional gene
Variable number of tandem repeats VNTR/ Simple sequence length polymorphism’s SSLP’s
Tandem repeats, polymorphic in size between chromosomes and individuals, can be used as genomic markers
• Analyzed by PCR
Single nucleotide polymorphisms (SNPs)
• Single base substitution, insertion, deletion
• Detected by sequencing a particular region from different individuals who may have polymorphisms
• True SNP must have at least 1% frequency 
Haplotypes
Haploid genotypes, can be any combination of alleles, loci, or markers on the same chromosome but commonly refers to groups of nearby alleles or markers on a chromosome that are inherited together
• Haplotype blocks are large sets of SNPs that are co-segregating in the human population (created haplotype maps) 
Haplotype size determining ancestry
A larger haplotype block suggests that the particular alleles arose relatively recently in human history
Prader Willi syndrome
• PW gene is maternally imprinted. When a deletion or other mutation occurs in the expressed allele no PW gene product is made and thus the result is the syndrome
• snoRNA (SNORD116) mutation
Angelman syndrome
• AS gene is paternally imprinted. When a deletion or other mutation occurs in the expressed allele, no AS gene product is made and thus the result is the syndrome
• UBE3A mutation
Three molecular genetics subtypes of PWS
— 65-75% of PWS is caused by paternal 15q11-q13 deletions
— 20-30% of PWS is caused by maternal uniparental disomy 15
— 1-3% of PWS is caused by an imprinting defect
Phenotype is likely due to Hypothalamic dysfunction
Uniparental disomy (UPD)
Occurs when a person receives two copies of a chromosome, or part of a chromosome, from one parent and no copies from the other parent
Clinical cytogenics
The study of chromosomes, their structure and their inheritance, as applied to medical genetics
Karyotyping
To detect a chromosomal abnormality, one can start with large defects visible on whole chromosomes :all 46 chromosomes 
How do we identify chromosomes?
- Banding
- Centromeric position
- Size
- Morphology
- Chromosome markers
Metacentric
Central centromere and equal size arms (normal) 
Submetacentric
Off-center centromere and different size arms
Acrocentric
Centromere near one end, creates satellites connected to the centromere by a stalk 
Telocentric
Single arm, only in mice
FISH
• molecular cytogenetics
• Multi-chromatic fluorescent probes can target chromosomes, chromosome regions, or genes 
• combinations of FISH probes= Spectral karyotyping/SKY 
The four phases of the cell cycle
G1, S (DNA synth), G2, M (mitosis)
Mitosis: prophase
• Replicated daughter centrosomes move apart
• Chromosomes begin to condense
• Nuclear envelope breaks down
Mitosis: prometaphase
• bipolar spindle becomes apparent
• Microtubules attach to kinetochores
• Chromosome/nuclear events continue
Mitosis: metaphase
• Maximally condensed chromosomes align on a disk like plate
Mitosis: anaphase
• daughter chromosomes separate and begin to move apart, toward opposite spindle poles
Mitosis: telophase
• plasma membrane begins to constrict around the spindle midline via contractile actin and myosin pinching
• Chromosomes begin to decondense
• Nuclear envelope begins to reform
Mitosis: cytokinesis
•Cell pinches into two daughter cells
Condensin and cohesin complexes
Related complex is composed of SMC and associated proteins
Cohesins: Encompass adjacent daughter chromatids so that they remain attached to each other throughout G2, prophase, prometaphase, and metaphase (proteolyzed at the onset of anaphase)
Condensin: Activate it in prophase, causes chromosome compaction
The centrosome cycle
- Grow in G2 — daughter centrioles
- Split and move apart to form spindle poles in M-phase
^^ microtubule driven, Kinesins and Dyneins
Nuclear envelope breakdown and reformation
• Lamin filaments phosphorylated by CDK1, causing them to depolarize— nuclear envelope vesiculates and scatters throughout cytoplasm
• Dephosphorylation of lamin, Envelope vesicles and Lamins aggregate around the chromatin of each daughter cell. Begin fusion and reform nuclear envelope
Spindle microtubules include:
• kinetochore microtubules: opposite directions
• interpolation microtubules: overlap and interact, affects spindle pole distances
• astral microtubules: migrate away from spindles
Kinetochore
A complex of proteins that assembles on centromeric DNA
Functions:
• attachment points for spindle microtubules
• Sensing alignment of chromosomes in metaphase
• Separation of daughter chromosomes in anaphase
What is a kinetochore made of?
• CENP-A: A specialized histone that defines centromeric chromatin and mediates binding of kinetochores to DNA
• linkers to microtubules
• Microtubule motor proteins
• Sensors and signaling proteins— responsive to binding + tension 
Chromosomes aligning on a metaphase plate
• kinesins push chromosomes away from spindle poles
• microtubule depolymerization at the kinetochore pulls chromosomes toward poles
• Microtubule flux, tubulin subunits are removed at the (-) end, pulls microtubules and attached chromosomes toward poles
Separation of daughter chromatids in anaphase
APC/C Induces proteolysis of securin, activating separase, which proteolyzes shugoshin and cohesin at the centromere region, allowing the pulling forces of the spindle to begin separation of daughter chromatids
How is APC/C activated?
At the metaphase-anaphase transition by association with Cdc20 — Tags securin for destruction
Anaphase A/anaphase B
A: Pulling force is separating sister chromatids (towards separate poles)
B: pushing of spindle poles farther apart by interpolar microtubules
Cytokinesis
• A central spindle assembly forms at the overlap of interpolar and astral microtubules (Recruits and activates Ect2, a GEF for RhoA)
• Activated RhoA reorganizes actin microfilaments to form a contractile ring— pinching cell into two daughter cells
Monopolar spindle assembly (defects)
Causes defects in centrosomal proteins (tubulin), microtubule motor proteins, kinase dependent effects
Multipolar spindle assembly— Centrosome amplification
• cytokinesis failure from previous division cycle
• Mitotic slippage— checkpoint bypass
• centrioles over duplication, fragmentation, or fusion
• cell-cell fusion 
Major cell cycle driver reactions
- Phosphorylation/dephosphorylation
- Regulated proteolysis— Irreversible
Cyclin dependent kinases (CDKs)
Kinase family that phosphorylates numerous cell targets— Denoted by numbers
Regulated by:
• Cyclins
• Activating and inhibitory kinases and phosphatases
• CKIs 
Cyclins
A family of regulatory proteins that help control CDK activity— Denoted by letters
• Different Cyclins function in different stages of the cell cycle
• influence CDK substrate specificity
Cyclin-dependent kinase inhibitors (CKIs)
”brakes” for CDK
• proteins that can inhibit CDK activity
• Can have complex regulatory roles
Different cyclins/CDKs and cell cycle stages 
G1: cyclin D, CDK4/6
G1/S: cyclin E, CDK2
S: cyclin A, CDK2/1
M: cyclin B, CDK1
Cell division decisions based off of environmental effectors
• growth factors (Enzyme linked receptors)
• Hormones (G protein coupled receptors)
• Contact signaling (integrins, integrin-linked kinases)
• Stress (DNA damage, p53)
The targets of M-Cdk include:
- Condensins
- Centrosomal proteins
- Nuclear pore complexes and lamins
- Proteins associated with Golgi and ER, promoting their fragmentation
- APC/C: a ubiquitin ligase
Two crucial proteosomal targets of APC/C
- Securin : Initiate anaphase
- Mitotic cyclins : Negative feedback loop where M-Cdk leads to its own inactivation via activation of APC/C
How to achieve mitotic exit:
Destruction of cyclins abolishes CDK activity, this is necessary to reset the cell so another cycle can be initiated if conditions are appropriate
— Low cyclin needed for helicase loading in order to lyse another round of division
Why does proteolysis provide directionality?
It is irreversible, so it prevents the cycle from going backwards.
Proteolysis facilitates:
1.) entry into the S phase by destruction of CKI
2.) onset of anaphase by destruction of securins
3.) mitotic exit by destruction of cyclin B
p53
• heavily involved in cell checkpoints throughout the cycle, tumor supressor gene — p53 limits its own quantity by upregulating Mdm2: ubiquination ligase that targets p53 (neg feedback)
1.) Upregulates CKI to hold the cycle progression to allow DNA repair
2.) Upregulates Bax (proapoptotic) if DNA damage unrepairable
DNA damage leading to p53 activation and p21 inhibition of CDK activity
Damage -> ATM/ATRK -> Chk1/2 -> phosphorylates p53 increasing free amount (not bound to Mdm2) -> increase CKIs -> cell arrest to fix damage -> BAX -> apoptosis
Bub/MAD2: Spindle assembly checkpoint
Inhibits APC/C blocking anaphase initiation and mitotic exit, so cohesions cannot release from one another, makes the cell “stuck” until alignment is completed
Oncogenes
“Gas”
stimulate proliferation of cell division
Tumor suppressor genes
“Brakes”
Inhibits proliferation in cell division
Growth factor signaling
Growth factor, RTK, G proteins, ERK MAPK —> signals immediate/early response genes
Second wave: delayed response genes, Cyclone D, CDK4/6, pRb/EZF, DNA proteins made (cyclin E, cyclin A), S phase and division begins
What drives cells into M phase?
Cyclin B binding to CDK1, but it is phosphorylated at both activating and inhibitory sites by CAK and Wee1 — low activity
Removal of inhibitory phosphate by Cdc25 activates CDK1 
CAK activates, Wee1 inhibits M phase
Three key cofactors for enzymes in amino acid metabolism:
- Pyridoxal phosphate (PLP) B6
— Transamination, deamination, carbon chain transfers - Tetrahydrofolate (FH4) folic acid
— One carbon transfers - Tetrahydrobiopterin (BH4)
— Ring hydroxylation’s, redox cofactor, used for Phe —> Tyr 
Essential amino acids, required from the diet:
M. V. Pitthall
Methionine, valine, phenylalanine, isoleucyl, tryptophan, threonine, histidine, arginine*, leucine, lysine
Amino acids produced from glycolytic intermediates
Alanine via pyruvate
Serine via 3-phosphoglycerate
Cysteine and glycine via serine 
Amino acids derived from the TCA cycle intermediate
From alpha-ketoglutarate:
1. Glutamate
2. Glutamine
3. Arginine
4. Proline
From oxaloacetate:
1. Aspartate
2. Asparagine
Two ways glutamate can be produced from alpha-ketoglutarate:
1.) oxidative deamination by glutamate dehydrogenase using NADP and creating NADPH and ammonium
2.) Transamination by aspartate aminotransferase
Amino acids can be degraded into TCA cycle intermediate and ketone bodies 
K. L. WIFTY
G. SCAWT
Branched chain amino acids
Valine, isoleucine, leucine 
Rate limiting step in branched chain amino acid degradation
Branched chain alpha-keto acid dehydrogenase
This enzyme has a similar organization to PDH:
E1: decarboxylation, TPP
E2: acyl transferase, Lipoate & CoA
E3: redox FAD and NAD+
Degradation of phenylalanine
The first step is synthesis of tyrosine, phenylalanine hydroxylase hydroxylates the ring of Phe, which requires BH4 as redox cofactor
Cysteine vs. cystine
Cystine is two cysteines creating a disulfide bond— this is the oxidized form of cysteine and is hydrophobic 
Cystinuria
Inherited autosomal recessive disorder in the amino acid carrier for cysteine and basic amino acids (Lys, Arg, Orthinine)
• Causes cysteine kidney stones
• Type A transporter: SLC3A1
• Type B transporter: SLC7A9
Maple syrup urine disease MSUD
Loss of function in the E1 subunits of branched chain alpha-keto acid dehydrogenase— no oxidative decarboxylation
 symptoms: convulsions, vomiting, maple syrup odor in urine
Labs: elevated plasma and urine Val, iso, leu, and keto acids
Treatment: BCAA diet
Classic MSUD
Complete loss of enzymatic activity: need branched chain AA free diet
Intermittent and mild MSUD
Some residual enzymatic activity, treated with oral thiamine (B1) to have cofactor in surplus
Familial autism and seizures
Unregulated catabolism of branched chain AAs, mutations were found in BCKDK
Treatment: high BCAA diet — However, not the best treatment because BCAA metabolism causes ROS (shouldn’t oversupply the problem just to maintain BCAA synthesis in body)
Phenylketonuria PKU
Defect in phenylalanine hydroxylase, prevents tyrosine biosynthesis. Instead, creates phenyllactate (toxic intermediate)
Symptoms: seizures, cognitive delay, light complexion, mousy odor
Diagnosis: newborn screening
Treatment: Phe restricted diet
What does excess phenylalanine do to large neutral amino acid transporters?
Acts as a competitive inhibitor, decreasing tyrosine and tryptophan intake and use
• impaired melanin/myelin/protein synthesis
• Impaired glucose metabolism
• Amyloid- like plaque formation
• oxidative stress damage
• Epigenetic alterations
Nonclassical PKU
Defects in tetrahydrobiopterin Metabolism, dihydropyridine reductase can mimic PKU
Phenylalanine restricted diet— restrict toxicity, but you need Phe for protein synthesis
Tyrosinemia type I
Loss of function mutation of fumaryloacetoacetate hydrolase (FAH) Resulting in accumulation of succinylacetone leading to liver failure. Most severe

Diagnosis: succinylacetone in blood and urine
Treatment: Nitosinone: slows catabolism of tyrosine
Tyrosinemia type II
Increased levels of Tyrosine, mutation of tyrosine aminotransferase (Normally clips off amine group)
Diagnosis: patient develops plaques on the hands and feet, corneal ulcers, and mental delays
Treatment: synthetic diet low in Phe and Tyr
Tyrosinemia type III
Loss of function mutation in the 4-hydroxyphenylpyruvate dioxygenase
Symptoms: intellectual disability, seizures, intermittent ataxia. Rarest and severe
Alcaptonuria
Deficiency in homogentisate oxidase Leading to dark colored urine, ochronosis, and homogentistic acid in urine
Diagnosis: Patients are asymptomatic until middle age, when they develop arthritis, back pain, renal calculi
Uses of serine
1.) donation of one carbon to tetrahydrofolate (FH4)
2.) converted to pyruvate
3.) protein synthesis (storage)
Excess glycine is converted to:
Glyoxylate through transaminatjon or oxidative deamination. Metabolites of this can be oxidized to CO2 or excreted in urine
What pathway involves tandem receptors associated with primary cilia?
Hedgehog signaling
What pathway involves the proteolytic release of the cytoplasmic tail of a transmembrane receptor tail, which then migrates into the nucleus to regulate genes involved with lateral inhibition or cooperatively between neighboring cells?
Notch signaling
What pathway is associated with small, hydrophobic signaling molecules that can trigger multiple waves of gene expression responses?
Nuclear hormone signaling
What pathway is associated with enhancing protein kinase G activity through phosphodiesterase inhibition?
NO signaling
What signaling pathway leads to polyp formation and increased risk of colorectal cancer when the function of a proteolysis inducing complex that includes the APC protein is compromised?
Wnt signaling
What pathway is associated with inflammation and stress, and is activated by TNF signaling?
NF-kB signaling
The two positive feedback loops that result in a switch-like activation of cyclin B — CDK1 (M-cyclin/CDK) involve:
Activation of CDC25 phosphatase and inhibition of Wee1 kinase
What stage of cell cycle (decision of a cell to commit to division or leave the cycle) impacted by growth factors, contact signaling, and stress?
G1
What situation would lead to active Bub/MAD signaling metaphase arrest?
Kinetochores unattached to microtubules in M-phase
Which protein is upregulated by p53 and binds to cyclin-CDK complexes to inhibit their activity?
The CKI p21
A loss of function mutation in which protein could potentially lead to unregulated cell division and cancer promotion?
p53
Specialized chromosomal region required for the accurate segregation of a replicated pair of chromatids among daughter cells
Kintechores (line up on metaphase plate)
What pathway is an important transducer of cytokine signaling, and involves the recruitment of tyrosine kinases when activated?
JAK-STAT pathway
What pathway involves phosphorylation of an inositol phospholipid followed by activation of kinases that promote self survival?
PI3K-protein kinase B/AKT pathway
What pathway includes a large family of ligands that bind and activate receptors with serine- threonine kinase activity?
TGF-beta / SMAD pathway
What pathway is known to encompass multiple parallel pathways, each consisting of three kinase cascades that are important regulators of cell proliferation, differentiation, and stress response?
MAP kinase pathway
What pathway regulates salt and water balance through the activation of protein kinase G?
Natriuretic peptide/ cGMP pathway
What pathway functions to integrate metabolic status with growth control?
mTOR pathway
Spectral (waveform) Doppler
Used to measure velocity of blood flow by frequency shift of the echoes
• Pulse wave: Used for cardiac, exact spot sampling is viewed
• continuous wave: all RBCs in entire spectrum viewed (mitral valve stenosis) 
Doppler
Shows estimate blood flow moving through veins and arteries using sound waves
Remember: cos (90)=0, hold parallel
Colorful Doppler: blue is flow away from the probe, red/orange is flow towards the probe
M-mode
Time motion display of ultrasound wave along Chodron ultrasound line. Mono dimensional view displayed along an axis (heart)
B – mode
Two dimensional image display composed of bright dots representing the ultrasound echoes
• Perpendicular probe is best
15-60fps, 60=cardiac, 15=abdominal
Piezoelectric crystals
Converts kinetic/mechanical energy into electrical energy which could be interpreted by the ultrasound machine and appear as light in a picture
Temporal resolution
The frame rate fps, Anything >40 is not distinguishable by the human eye
When using a Doppler:
High PRF: talks more, listens less: use in a high flow areas such as the carotid where delicate hearing is not necessary
Low PRF: Listens more, more sensitive: use in low flow areas such as the testicles
Artifacts in ultrasound
1.) High attenuation
2.) low attenuation
3.) gas scatter
4.) refraction
5.)  reverb
6.) Mirror image
High attenuation
dense objects produce white picture, with a shadow
Low attenuation
creates echo enhancement posteriorly, used as a window to visualize anatomy
Gas scatter
bowel gas, must push through it to overcome it
Refraction
sound gets redirected creating an edge artifact/lateral cystic shattering
Reverb
equidistant arcs that come from the top of the transducer
Mirror image
sound glances off diaphragm, returning to probe with a longer flight time. Typically interpreted as more liver seen in the chest (good, means chest is dry)
Echogenicity of the body
• Cortex of the kidney is slightly less echogenic than the liver
• diaphragm is most echogenic, Followed by the kidney/renal pelvis, liver parenchymal, renal cortex, and hepatic vein (Basically anechoic from blood) 
Adjusting depth and gain
Always adjust depth first— CCW is shallower, CW is deeper
Gain is adjusted with an AO knob and sliders
Different arrays
1.) convex array: sometimes called large footprint curve
2.) Phased array: also known as cardiac transducer, single crystal, wide array however what is in the center is seen the best
3.) Linear array: no splaying, good for superficial what you see is what you get, also called the vascular probe
Change in chromosome structure
Balanced: results in the same amount of genetic material, may or may not result in phenotype
Unbalanced: usually leads to a clinical phenotype due to inappropriate gene dosage
BCR – ABL Philadelphia chromosome
Chronic Myelogenous leukemia CML, caused by 9 and 22 reciprocal translocation. This translocation is specific for hematopoietic cancers because expression is driven by B-cell receptor promoters
Abnormal chromosome segregation caused by nondisjunction
Aneuploidy (down syndrome, Klinefelter syndrome), Uniparental disomy 
Down syndrome
Trisomy 21, most common chromosomal birth defect, increased risk to 1 out of 15 in women over 45, eightfold risk of recurrence if you have a downs child already, disease likely caused by increased gene dosage
Sex linked disorders
• Trisomy X (females)
• Klinefelter syndrome (second x in males)
• Turner syndrome (x deletion— hemizygous)
Amniocentesis
Invasive technique, removal of amniotic fluid transabdominally by syringe, fetal cells are cultured for diagnostic tests
Most common in the United States
Cordocentesis
Invasive technique, removal of fetal blood from the umbilical cord, more often used when the other methods have failed or are ambiguous
Chorionic villus sampling (CVS)
Invasive technique, biopsy of tissue from the villous area of the chorion, can occur 4 to 5 weeks before amniocentesis— earlier read out
More common in Europe
Noninvasive techniques
• maternal serum screening
• Ultrasound
• Radiography
• MRI
• NSG/deep sequencing
First and second trimester screening for trisomy 21
• increased nuchal translucency
• decreased PAPP-A
• Increased free beta hCG
• decreased uE3
• Decreased AFP
• Increased hCG
• Increased inhibin A
First and second trimester screening for neural tube defects
Significant increase in AFP in second trimester
Ultrasound is used to determine:
Fetal age, sex, gross abnormalities, and viability
Ultrasonic indicator of trisomy 21
Nuchal translucency (neck thickness) is increased greatly and can be detected at 10 to 14 weeks
Ultrasonic detection of neural tube defect such as spina bifida
Meningomyelocele sac protruding through the skin
Genetic testing: carrier detection
Heterozygote screening
tested by clinical manifestation in the carrier, biochemical abnormality, and DNA analysis
Categories of genetic tests
- Carrier detection
- Pre-symptomatic diagnosis
- Prenatal and newborn testing and diagnosis
Examples of biochemical abnormalities in heterozygous carrier detection
Tay-Sachs: hydrolase enzyme activity
DMD female carriers: Serum creatine kinase levels
Examples of using pre-symptomatic genetic testing
- Huntington disease
- Tuberous sclerosis
- Familial hypercholesterolemia
- Familial adenomatous polyposis (ACP gene)
- Breast cancer (BRCA 1/2) 
Adaptive immune response
Antigen specific response, long-term protective immunity
What cells are included in adaptive immunity?
Natural killer T cells, CD4 T cells, CD8 Tcells, B cells
Cellular versus humoral adaptive immunity
Cellular is the T cells, humoral is the B cells—humoral=fluid 
Which cells are antigen presenting?
- DC cells
- Macrophages
- Thymic epithelial
- B cells
Dendritic cells (DCs) 
Long motile protrusions resemble dendrites of nerve cells, critical for the initiation of the immune response, capture antigen by phagocytosis and endocytosis, present antigen and active naïve T cell 
Express high levels of peptide: major histocompatibility complexes, co-stimulatory molecules, and produce cytokines 
DCs vs macrophages
Macrophages are resident tissue APCs (stay in their tissue), and DCs are in tissue but home to the lymph node (roam)
Major histocompatibility complex (MHC)
Tells a T cell whether a cell/tissue is self versus non-self (recognition)
The MHC is a group of genes that code for proteins on the surface of cells to facilitate immune system recognition of foreign materials
Polygenic MHC’s
Encoded by multiple genes: isotopes
MHC class I: HLA-A, -B, -C (one long chain, one short chain)
MHC class II: HLA-DP, -DQ, -DR (two long chains)
(Class one has one letter, class two has two letters) 
Polymorphic MHC’s
Genes can have various alleles. Includes polymorphism (one gene with different alleles) and polgeny (multiple genes without alleles)
MHC class and cell type
T cells: MHC class I
B cells: MHC class I and II
Macrophages: MHC class I and II
Thymic convolution
The thymus shrinks with age, and the T cells mature here. Capacity to produce T cells decreases with age which is why we vaccinate young children 
T cell maturation
Positive selection: recognizes the antigen
Negative selection: recognizes self — self tolerance (too strong = autoimmune) 
T cell development: rule of eight
MHC II x CD4+ (and T helper cells) = 8
MHC I x CD8+ (and cytotoxic T lymphocytes) = 8
The two signals of T cell activation
- MHC signal
- Co-stimulation/inhibition
If only one signal, T cell becomes anergic (non-responsive to that antigen)
- Cytokines to program T cells even further
Effector T cell Th1
IFN-gamma, macrophages, macrophage activation, autoimmunity, chronic inflammation
Effector T cell Th2
IL4,5,13, eosinophils, allergy
Effector T cell Th17
IL17, 22, Neutrophils, extracellular bacteria and fungi, Large contributor to auto immunity and inflammation
Effector T cell Tfh
IL21, IFN-gamma, IL4, B cells, antibody production, auto antibodies
CD8+ cytotoxic T cell lymphocytes (CTLs) cause apoptosis of cells via:
Cytoplasmic granules: granzyme B, perforin —> protease, cell death
Fas:FasL: FasL (CD95L) Expressed on surface of T cells, binds Fas on antigen —> apoptosis 
Natural killer cells
• major role in protection against infected, stressed, and cancer cells
•  Cell killing initiated by absence of self (kill or don’t kill)
• NK cells stimulated by innate cytokines (IFN-alpha, beta, and IL-12)
• Potent producer of IFN-gamma by driving CD4+ cells to become TH1s to make more IFN-gamma
Receptors expressed by natural killer cells
- Activating receptors
- Inhibitory receptors
Both expressed simultaneously. Inhibitory (recognizing self) always works over an activating signal (recognizing antigen)
Natural killer cells kill via what two mechanisms?
- Cytoplasmic granule mediated apoptosis (granzyme B, perforin)
- Fas:FasL
Same mechanism as CD8 however NK cells know the “self” and do not kill, unlike T cells
What is a virus?
•  obligate intracellular parasite
• Can infect all types of cellular life
• Nucleic acid surrounded by a protein shell
• Ultramicroscopic in size
• defined host range of cell types that can support its viral lifecycle, tropism
• Replicate in stepwise fashion, rather than binary fission
Tropism
Defines the range of cell types that can be infected by a specific virus
Virus sizes
Mimivirus, herpes Symplex virus are the biggest, smallest includes yellow fever and polio virus
Viral structure
• Naked or enveloped
• capsid surrounding nucleic acid always
• Icosahedral (box around) or helical nucleocapsid (capsid right on genome)
Enveloped virus
Has a tegument, an envelope, and spike proteins
Coronavirus is a:
Non-segmented, enveloped, RNA single strand, helical nucleocapsid, with spike proteins that are large
Influenza is a: 
Single stranded RNA, segmented, enveloped, helical capsid, with H1N1 spike proteins
Structure of bacterial virus
Head, collar, tail used for injection of nucleic acid, end plate, tail pins, tail fibers 
General viral replication cycle
1.) attachment/adsorption
2.) Penetration/injection
3.) Synthesis of nucleic acids and proteins
4.) Assembly and packaging
5.) Released by lysis
Antigenic shift
Occurs when major changes in antigens occur due to gene reassortment in influenza virus
— Common host infects the same cell at the same time and they recombine with each other 
Antigenic drift
Occurs when minor changes in antigens occur due to gene mutation in influenza virus
— Point mutations change the virus just enough to make vaccines ineffective 
Exponential growth versus one step growth
Bacterial: exponential growth from replication per cell
The viral: eclipse and latent period, maturation, assembly and release— big burst of growth
Titer
Number of infectious units per volume of fluid
Plaque assay
Analogous to the bacterial colony, one of the most accurate ways to measure virus infectivity and infectious viral particle numbers
Plaques: are clear zones that develop on lines of host cells, the plaque is the absence of cells
Effects that animal viruses can have on cells
1.) transformation into tumor cell
2.) Death of cell and release of virus
3.) Slow release of virus without death, persistent infection
4.) virus present but not replicating, latent infection (may be lyric eventually)
5.) cell fusion
BCS— Class I 
 double stranded DNA (+/-) virus
Transcription of minus strand
• classical semiconservative 
BCS— Class VII
double stranded DNA (+/-) virus
Transcription of minus strand
• transcription followed by reverse transcription 
BCS— Class II
Single-stranded DNA (+) virus
Synthesis of other strand, double strand of DNA intermediate, transcription of (-) strand
• Classical semi conservative, discard the (-) strand
BCS— Class III
Double-stranded RNA (+/-)
Transcription of minus strand
• makes ssRNA (+) and transcribe from this to give ssRNA (-) partner
BCS— Class IV
Single-stranded RNA (+) virus
Used directly as mRNA (no further steps) 
• makes ssRNA (-) and transcribe from this to give ssRNA (+) genome
BCS— Class V
Single-stranded RNA (-) virus
Transcription of the minus strand
• makes ssRNA (+) and transcribe from this to give ssRNA (-) genome
BCS— Class VI
Single-stranded RNA (+) retrovirus
Reverse transcription, double-stranded DNA intermediate, transcription of (-) strand
• makes ssRNA (+) genome by transcription of (-) strand to dsDNA
Causes of inflammation
1.) infection
2.) tissue necrosis
3.) Foreign bodies
4.) immune reaction/hypersensitivity
Five Rs of inflammation
Recognition: receptors that recognize microbes, and sensors of cell damage, leukocytes/sentinel cells, antibody and complement receptors
Recruitment: of leukocytes
Removal: of the microbes/damaged tissue
Regulation: of the inflammatory response
Repair: of the site of damage
 Three major components of the acute inflammatory response
1.) vascular changes
2.) Leukocyte recruitment
3.) Leukocyte activation and removal of the offending agent 
Signs of inflammation
Rubor (redness), Calor (warmth), tumor (swelling) , dolor (pain), Functio laesa (loss of fxn)
Vascular changes in acute inflammation
Vasodilation: histamine, erythema, increased blood flow
Increased vascular permeability: gaps in between endothelial cells, leakage of fluid causing edema, movement of plasma proteins and inflammatory cells from vascular space to the site of injury an infection
Key steps in leukocyte recruitment and migration to the site of injury
• margination
• Rolling— selections
• Adhesion— integrins (ICAM & VCAM)
• Migration— PECAM-1 (CD31)
“SIP”
Removal of the offending agent
• activation of recruited leukocytes
• Phagocytosis
• Destruction of phagocytosed material
• granule enzymes (proteases)
Chronic inflammation
A response of prolonged duration in which inflammation, tissue injury, and attempts at repair coexist in varying combinations
Caused by persistent infections, hypersensitivity diseases, prolonged exposure to toxic agents in foreign material
The dominant cell in most chronic inflammatory reactions
Macrophages/Monocytes
Lymphocytes
• amplify and propagate chronic inflammation
• Generate memory cells, allowing for persistent and severe reactions
CD4+ T lymphocytes promote and influence the nature of the inflammatory reaction
Granulomatous inflammation 
A pattern of chronic inflammation induced by persistent T cell response in some infections, immune mediated diseases, and foreign bodies
TNF plays a crucial role 
Conditions with granulomas
Infections: bacterial (TB, bartonella hens., listeria, tertiary syphillis), fungal, and parasitic
Non-infections: immune mediated, vasculitis, foreign bodies, and chronic granulomatous disease
Transport of amino acids from the gut into the intestinal epithelial cells is by:
Secondary active transport with sodium
Key amino acids for protein digestion
• glutamate -> alpha ketoglutarate
— The amino group pool of the cell
• Aspartate -> oxaloacetate
— donates nitrogen to the urea cycle
• Alanine -> pyruvate
— A key role in gluconeogenesis, transport nitrogen to liver in the form of alanine
• Glutamine -> glutamate
— transports nitrogen to liver for urea cycle
Urea is specific to the breakdown of:
Amino acids for energy, diverting to carbs (CO2+H2O) for energy and urea cycle to get rid of the amine groups
What promotes mobilization of stored fuels?
Glucagon, cortisol, epinephrine, norepinephrine
Major transporter of amino acids from muscle to liver
Alanine: directly to liver to be used for many different pathways
Glutamine: either straight to the liver, or deposits ammonia in the kidney and leaves as alanine to the liver
Alanine glucose cycle
Alpha KG and glutamate are common amine exceptors and donors causing transamination reactions
Alanine broken down to carbon for glucose and nitrogen for urea in the liver. Glucose goes back to muscle to create pyruvate to amino transfer to get alanine back
three enzymes that can “fix” free ammonium: require energy
IN MUSCLE:
1.) Glutamate dehydrogenase (GDH)
2.) glutamine synthase
IN LIVER
3.) Carbamoyl phosphate synthetase I (CPS I) 
Two ways alanine enters the urea cycle:
Firstly, alanine aminotransferase converts alpha-KG to glutamate. Then:
- Glutamate dehydrogenase makes ammonium (NH4+)
- Aspartate aminotransferase makes aspartate
Order of enzymes in the urea cycle
1.) CPS I
2.) Ornithine transcarbamoylase
3.) Citrulline ornithine antiporter
4.) Argininosuccinate synthetase
5.) argininosuccinate lyase
6.) Arginase
7.) NAG synthetase (Activates CPSI)
Urea cycle disorders manifest as:
— hyperammonaemia
— hyperglutaminaemia
— neural disorders (Seizures, irritability, lethargy, ataxia, FTT, refusal to eat protein)
Arginine increases what?
Synthesis of N acetyl glutamate (NAG).
NAG acts as an allosteric activator of CPS I 
Because arginine is a major regulator of the urea cycle, when arginine builds up:
1.) it increases the synthesis of NAG, which activates CPS I
2.) it increases arginase activity (to create urea and ornithine)
If ornithine transcarbamoylase (OTC) is inactivated: 
This causes leakage to pyrimidine synthesis, which elevates urinary orotic acid
HHH syndrome
hyperammonaemia, hyperornithaemia, homocitrullinaemia (Comes from carbamoyl phosphate reacting with lysine)
The ornithine/citrulline anti-porter SLC25A15 is defective
Nitrogen scavengers
1.) arginine —arginosuccinate (2N per)
2.) Benzoic acid (1N per)
3.) Phenylbutyrate (2N per)
These are used to make amino acids as excretable in the urine (more soluble)
What is used to treat NAG synthase deficiency?
N-carbamoyl glutamate which is an analog of N-acetyl glutamate
Using arginine to treat HHH syndrome
It generates spermine and creatine, which can be urinated out to eliminate nitrogen (like N scavengering)
Rubor, calor, vasodilation
Main mediators: Histamine, prostaglandins
Tumor, Increase vascular permeability
Main mediators: Histamine, serotonin, C3a, C5a, leukotrienes C4 D4 E4
Leukocyte recruitment and activation
TNF, IL-1, chemokines, C3a, C5a, leukotriene B4
Fever
IL-1, TNF, prostaglandins
dolor/ pain
Prostaglandins, bradykinin, substance P
Loss of function (functio laesa), Tissue damage
Lysosomal enzymes, ROS
