Far

 Eastern  University  –  Nicanor  Reyes  Medical  Foundation   Most  Common  forms  of  DNA  Variations  
Pathology  A  –    The  Cell  as  a  Unit  of  Health  and  Disease   1. Single  nucleotide  polymorphisms  (SNPs)  
J.Q.  Gacasan,  M.D.   2. Copy  number  variants  (CNVs)  
  SNPs  
Pathology   − variants  at  SINGLE  nucleotide  positions  
− study  of  suffering   − 1%  occurs  in  coding  regions  
− study  of  disease   − w/in  exons,  introns,  intergenic  regions,  coding  regions  
− study  of  cellular  abnormalities   *may  serve  as  a  marker  for  multigenic  diseases  (e.g.  Diabetes,  
  Hypertension)  
Genome    
− a  full  set  of  chromosomes   CNVs  
− all  the  inheritable  traits  of  an  organism   − different  numbers  of  large  continuous  stretches  of  DNA  
− Human  Genome  Project  (completed  in  April,  2003)   − 50%  involve  gene-­‐coding  sequences  
  • phenotypic  diversity  
DNA    
− Deoxyribonucleotide   Epigenetic  Factors  
− hereditary  material  in  humans  and  almost  other  organisms   − affect   cell   type-­‐specific   differences   in   DNA   transcription   and  
− most  located  in  nucleus  (nuclear  DNA)   translation  
− some  in  mitochondria  (mitochondrial  DNA)    
− made  up  of  4  bases:   Histone  Organization  
• Adenine,  Guanine,  Cytosine,  Thymine  
 
Human  Genome  
− 3.2  billion  DNA  base  pairs  
− 1.5%  (20,00)  –  protein  coding  (enzymes,  structural,  signaling)  
− 98.5%  do  not  encode  proteins  
• 80%  regulation  of  gene  expression  
 
Major  Classes  of  Functional  Non-­‐protein  Coding  Sequences  
1. Promoter  and  enhancer  regions  
2. Binding  sites  
3. Non-­‐coding  regulatory  RNAs    
 
a. micro  RNAs   DNA  +  histones   Nucleosome   Chroma4n  
 
b. long  non-­‐coding  RNAs  
 
4. Mobile  genetic  elements  (transposons)  
Two  Basic  Forms  of  Nuclear  Chromatin:  
5. Special  DNA  structural  regions  
1. Heterochromatin  
a. Telomeres  
b. Centromeres   − transcriptionally  inactive  
  − cytochemically  dense  
Organization  of  Nuclear  DNA   2. Euchromatin  
− transcriptionally  active  
− cytochemically  dispersed  
 
Histone  Methylation  
− Lysine  and  Arginine  
− Transcriptional  activation  or  repression  
 
Histone  Acetylation  
  − Lysine  
  − Histone  acetyl  transferase  (HAT)  
Genetic  Variations  
• open  up  chromatin  
− Polymorphisms  
− Histone  deacetylation  (HDAC)  
− located  in  non-­‐protein  coding  regions  
• condensation  of  chromatin  
− humans  share  >99.5%  DNA  sequences    
− variation  encoded  in  <0.5%  of  our  DNA  (15million  bp)    

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Chromatin  organizing  factors   B. Gene  suppression  
− bind  non-­‐coding  regions  
− control  long  range  DNA  looping  
 
Histone  Phosphorylation    
B:    Conversely,    lncRNAs    can    preemptively    bind    transcription    factors  and  thus  
− Serine  
prevent  gene  transcription.  
− may  open  DNA  or  condense  it    
  C. Promote  chromatin  modification  
DNA  Methylation  

High  levels   Silencing  

 
Epigenetic  Alterations  
− heritable  up  to  a  certain  number  of  generation    
C:  Histone  and  DNA  modification    by    acetylases    or    methylases    (or    deacetylases    
− reversible   and    demethylases)    may    be    directed  by  the  binding  of  lncRNAs.  
 
− amenable  to  treatment  
D. Assembly  of  protein  complexes  
 
Non-­‐coding  RNAs  
− encoded  but  not  transcribed  
− micro-­‐RNA  (miRNA)  
− long  non-­‐coding  RNA  (lncRNA)    
  D:  In  other  instances,  lncRNAs  may  act    as    scaffolding    to    stabilize    secondary    or    
tertiary     structures     and/or     multi-­‐   subunit     complexes     that     influence     general    
miRNA   chromatin    architecture    or    gene    activity.  
− modulates  translation  of  mRNA    
− post  transcriptional  silencing   The  Cell  as  a  “city”  
− about  22  nucleotides   *Each  organelle  has  a  job-­‐-­‐-­‐your  cells  run  like  a  little  city  with  many  
  jobs  to  be  done  
  • workers  à  proteins  
• power  plant  à  mitochondria  
• roads  à  actin  fibers,  microtubules  
• trucks  à  kinesin,  dynein,  myosin  
• factories  à  ribosomes  
• library  à  genome  
• recycling  centers  à  lysosomes  
 
• police  à  chaperones  
 
Small  interferring  RNAs  (siRNAs)   • post  office  à  golgi  apparatus  
− can  be  synthetically  produced   • comunicators  à  signalling  networks  
 
− mimics  miRNAs  
CELLULAR  HOUSEKEEPING:  
− used  in  knockdown  technology  
− Plasma  membrane  
− possible  therapeutic  agents  
− Cytoskeleton  and  cell-­‐cell  interactions  
 
lncRNAs   − Biosynthitic  machineries:  ER  and  Golgi  
− modulate  gene  expression   − Waste  disposal:  Lysosomes  and  Proteasomes  
   
A. Gene  activation   Housekeeping  functions  
− Protection  
− Nutrient  acquisition  
− Communication  
  − Movement  
A:    Long    non-­‐coding    RNAs    (lncRNAs)    can    facilitate    transcription    factor    binding    
and    thus    promote    gene    activation.   − Renewal  of  senescent  molecule  
  − Catabolism  
 
  − Energy  generation  
   
 
 
 

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− Compartmentalized   Extracellular  part  of  plasma  membrane  
− w/  carbohydrates  
− oligosaccharides  (glycolipids,  glycoprotein)  
− polysaccharides  (proteoglycans)  
− chemical  and  mechanical  barrier  
− cell-­‐cell,  cell-­‐matrix  interaction  
 
Plasma  membrane  diffusion  
− small,  non-­‐polar  molecules  (e.g.  O2,  CO2)  
− hydrophobic  molecules  
− polar  molecule,  <  75daltons  
− aquaporins  augment  passive  water  transport  
 
Carriers  and  channels  
− for  low  MW  species  (ions  and  up  to  1000  daltons  small  
molecules)  
− Channel  proteins  
  − Carrier  proteins  
A.  Plasma  membrane    
− Protection  and  nutrient  acquisition   Receptor  mediated  and  fluid  phase  uptake  
− Bilayers  of  amphipathic  phospholipids   1. Endocytosis  
− Hydrophilic  heads,  hydrophobic  tails   − uptake  of  fluid  or  macromolecules  
− With  heterogenous  phospholipids  assymetrically  partitioned   − caveolae  
− clathrin  
2. Exocytosis  
− export  of  large  molecules  
3. Transcytosis  
− between  apical  and  basolateral  compartments  

 
 
Phospholipids  
1.  Phosphatidylinositol  
− electrostatic  scaffold  for  intracellular  proteins  
nd
− generate  intracellular  2  signals  
1.  Phosphatidylserine  
− electrostatic  CHON  interactions  
− apoptosis  “eat  me”  signal  
 
2.  Glycolipids  &  Sphingomyelin    
− cell-­‐cell  &  cell-­‐matrix  interactions   B.  Cytoskeleton  
− inflammatory  cell  recruitment   − shape,  polarity,  organize  relationship  of  intracellular  organelles  
− sperm-­‐egg  interactions    
   Major  classes  of  cytoskeletal  proteins:  
“LIPID  RAFTS”   1.  Actin  microfilaments  
− for  horizontal  interactions   2.  Intermediate  filaments  
  3.  Microtubules  
Proteins  and  Glycoproteins    
1. Ion  and  metabolite  transport   Eukaryotic  cytoskeleton:  
2. Fluid-­‐phase  and  receptor  mediated  uptake  of   − Actin  
macromolecules     − Microtubules  
3. Cell-­‐ligand,  cell-­‐matrix,  cell-­‐cell  interactions   − Nuclei  
*function  together  as  large  complexes    
   

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Actin  
•  5-­‐9  nm  
•  G-­‐actin  (globular  protein  actin)  non-­‐covalently  polymerized  into      
F-­‐actin  (long  filaments)  
•  F-­‐actin  intertwine  to  form  double  stranded-­‐helices  
•  determine  shape  of  cell’s  surface  
•  necessary  for  whole  cell’s  locomotion  
 
Intermediate  filaments  
•  10  nm    
•  large,  heterogeneous  family   2. Anchoring  junctions/  desmosomes  
•  for  tensile  strength   •  mechanically  attach  cells  (and  their  cytoskeletons)  to  
•  provide  mechanical  strength   other  cells  or  to  the  ECM  
•  resistance  to  shear  stress   •  spot  desmosome/macula  adherens  
•  nuclear  morphology  and  transcription*   → (small)  between  cells  
•  major  structural  proteins  of  hair  and  skin   •  belt  desmosome  
•  lamin  (A,B,C)   → (broad)  between  cells  
•  vimentin   •  hemidesmosome  
•  desmin   → cell-­‐ECM  
•  neurofilaments   Cadherins  
•  glial  fibrillary  acidic  protein  (GFAP)   •  spot  desmosomes:  desmogleins,  desmocollins  
•  cytokeratins   •  belt  desmosomes:  E-­‐cadherins  
• Disease  associated:   Integrins  
  muscular  dystrophy  in  Progeria  due  to  lamin  mutation   •  transmembrane  connector  in  hemidesmosomes  
  Focal  adhesion  complex  
Microtubules   •  large  
•  25  nm   •  located  at  hemidesmosomes  
•  dimers  of  α-­‐  and  β-­‐  tubulin  with  “+”  and  “-­‐”  ends   •  generate  intracellular  signals  with  increased  shear  stress*  
•  determines  the  positions  of  membrane  enclosed  organelles  and   3. Communicating/  gap  junctions  
direct  cellulartransports   •  for  passage  of  chemical  or  electrical  signals  between  
•  microtubule  organizing  center  (MTOC  or  centrosome)  where  the     cells  
“-­‐”  end  is  embedded   •  connexons  (hexamers  of  connexins)  
•  “long  highways”   → pores  
•  preferred  track  for  long  distance  transport   → permit  passage  of  ions,  nucleotides,  amino  acids,  
•  plus  ends  us.  pointed  out  to  the  periphery  and  the  minus  ends   vitamins,  other  small  molecules*  
towards  the  center  of  the  cell    
  D.  Endoplasmic  Reticulum  
C.  Cell-­‐cell  interactions   − site  of  synthesis  of  transmembrane  proteins  and  lipids  for  
plasma  membranes  and  cellular  organelles  
− protein  modifications  occurs  here:  
• folding,  formation  of  polypeptide  complexes,  disulfide  
bonds  formation  
− chaperone  proteins  
• retain  unmodified  proteins  
****failure  to  modify  leads  to  protein  degradation  
− in  cystic  fibrosis:  
  (+)  CFTR  protein  mutation,  misfolding  occurs  >>>  
    ER  retention  and  degradation  >>>  capacity  exceeded  >>>  
    ER  stress  response  >>>  apoptosis  
1. Occluding/  tight  junctions     E.  Golgi  Apparatus  
•  seal  adjacent  cells  together   − stacked  cisternae  
•  a  continuous  barrier   − progressive  CHON  modifications  from  cis  (near  ER)  to  trans  (near  
•  restricts  paracellular/  cell  to  cell  movement  of  ions   PM)  
•  sets  boundary  between  apical  and  basolateral  cell   − recycling  and  dispatching*  
domains    

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F.  Lysosomes   C.  Cell  death  
− membrane  bound  with  about  40  acid  hydrolases*   − regulate  balance  of  cell  survival  and  death  
− intracellular  catabolism   − 2  major  pathways:  
− pinocytosis  or  endocytosis*       1.  Necrosis    
− autophagy   • external  cell  injury  
− phagocytosis   (  Toxin,  ischemia,  trauma)  
    2.  Apoptosis  (programmed  cell  death)  
 
 
 

 
 
G.  Proteasome  
− degrades  cytosolic  proteins  (denatured/  misfolded)  bound  to  
ubiquitin  

 
 
CELLULAR  ACTIVATION  
Cell  signaling  
− to  differentiate  
− to  proliferate  
− to  perform  special  functions  
− to  continue  living  
*no  signal,  cell  die  by  apoptosis  
  Signals:  
 
1.  Damage  to  neighboring  cells  and  pathogens  (danger  signals)  
CELLULAR  METABOLISM  AND  MITOCHONDRIAL  FUNCTION:  
2.  Contact  with  neighboring  cells  (gap  junction  signals)  
− energy  generation   3.  Contact  with  ECM  
− intermediate  metabolism   4.  Secreted  molecules  (GFs,  cytokines)  
− cell  death   Signaling  based  on  distance  
  1. Paracrine    
Mitochondria   2. Autocrine  
− have  small  genome   3. Synaptic  
− capable  of  replication,  transcription  and  translation   4. Endocrine  
− mtDNA  –  maternally  inherited    
  Receptors  
A.  Energy  generation   − Intracellular  receptors  
• transcription  factors  activated  by  lipid-­‐soluble  ligands  
− Cell-­‐surface  receptors  transmembrane  proteins  w/  
• extracellular  domains  that  bind  soluble  secreted  ligands  
 
SIGNAL  TRANSDUCTION  PATHWAYS  
− cellular  receptors  
− modular  signaling  proteins,  hubs,  and  nodes  
 
  − •  transcription  factors  (TF)  
B.  Intermediate  metabolism    
− intermediates  are  used  to  make  lipids,  nucleic  acids,  and    
proteins    
   

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Cellular  receptors   Key  functions  of  ECM  
1.  Receptors  associated  with  kinase  activity  (RTKs)*   1.  Mechanical  support  
2.  Non  receptor  tyrosine  kinase  based  receptor   2.  Cell  proliferation  control  
3.  G-­‐protein  coupled  receptors   3.  Scaffolding  for  tissue  renewal  
4.  Nuclear  receptors   4.  Establishment  of  tissue  microenvironment  
5.  Other  receptors    
Two  basic  forms  of  ECM  
1. Interstitial  matrix  
2. Basement  membrane  
 
Components  of  ECM  
1.  Fibrous  structural  CHONs  –  collagen  and  elastin  
2.  Proteoglycans  and  hyaluronan  
  3.  Adhesive  glycoproteins  –  fibronectin,  laminin,  integrins  
Receptor  activation    
− leads  to  orderly  sequence  of  biochemical  intermediates  that   Collagen  
leads  to  changes  in  gene  expression   − 3  polypeptide  chains  
  − ropelike  
Results  in  multiple  effects   − fibrillar,  non-­‐fibrillar  
− enzyme  activation/inactivation   − diseases  assoc’d:  
− TF  localization  (nuclear/cytoplasmic)       •Osteogenesis  imperfecta  
− TF  activation/inactivation       •Ehlers-­‐Danlos  syndrome  
− actin  polymerization/depolymerization    
− CHON  degradation/stabilization   Elastin  
− activation  of  feedback  mech.  (inhibitory/stimulatory)   − gives  ability  to  recoil  and  recover  shape  
  − in  cardiac  valves  and  large  blood  vessels  
Transcription  factors   − associated  with  fibrillin  
− activation  and  nuclear  localization  of  TFs  modulates  gene   − disease  assoc’d:  Marfan  syndrome  
transcription    
− MYC,  JUN,  p53   Proteoglycans  
  − resistance  to  compressive  forces  
Growth  factors  and  receptors   − lubrication  in  joint  cartilage  
GF  role  –  to  stimulate  activity  of  genes  for  cell  growth  and  cell   − consists  of  long  polysaccharides  
division     •  Glycosaminoglycans  (keratan  sulfate  and  chondroitin  sulfate)  
  − reservoir  for  growth  factors  •  associated  with  hyaluronan  
   
  Fibronectin  
  − provide  scaffolding  for  ECM  deposition,  angiogenesis,  and  
  reepithelialization  in  healing  wounds  
  − a  large  disulfide  linked  heterodimer  in  tissue  and  plasma  forms  
   
  Laminin  
  − most  abundant  glycoprotein  in  BM  
  − 820  kD  cross-­‐shaped  heterotrimer  
  − connects  cells  to  underlying  ECM  components*  
  − also  modulate  cell  proliferation,  differentiation,  and  motility  
   
  Integrins  
  − transmembrane  heterodimeric  glycoproteins  
 
− with  α  and  β  subunits  
 
− allow  cells  attach  to  ECM  components  (laminin  and  fibronectin)  
Interaction  with  Extracellular  matrix  
− on  WBC  –  adhesion,  transmigration  
− cell  interactions  with  ECM  are  critical  for  development  and  
− platelet  aggregation  
healing,  and  maintaining  normal  tissue  architecture  
 

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−  trigger  signaling  cascades  in  locomotion,  proliferation,  shape,   Good  detection  of  DNA  irregularities  
differentiation   − delays  cell  cycle  progression  
  − triggers  repair  mechanisms  
Cell  population  maintenace   − if  cannot  be  repaired,  apoptosis  is  activated  or  senescence  (p53)  
1. Proliferation  and  cell  cycle    
2. Stem  cells   Defective  CDKI  checkpoint  proteins  
− allows  division  of  damaged  cells  
− potential  for  malignant  tumor  formation  
 
Warburg  effect  
− example  of  an  event  that  promote  changes  in  cellular  
metabolism  that  support  growth  
− marked  by  increased  cellular  uptake  of  glucose  and  glutamine,  
increased  glycolysis,  and  decreased  oxidative  phosphorylation  
 
Stem  cells  

 
 
Cell  cycle  sequence  
1.  G1  –  presynthetic  growth  
2.  S  –  DNA  synthesis  
3.  G2  –  premitotic  growth  
4.  M  –  mitotic  growth  
G0  state  –  where  quiescent  cells  are  
   
Cell  cycle  activators  and  inhibitors   − give  rise  to  all  the  various  differentiated  cells  
− cyclins  (>  15)   − in  adult  organisms:  replace  damaged  cells,  maintain  tissue  
− cyclin  dependent  kinases  (CDKs)   populations  
− CDK  inhibitors    
Important  characteristics  of  stem  cells  
1. Self-­‐renewal  
2. Asymmetric  division  
• one  daughter  cell  differentiates,  other  remains  
undifferentiated  and  retains  self-­‐renewal  capacity  
 
Stem  cell  varieties  
1.  Embryonic  stem  cells  (ES  cells)  
2.  Tissue  stem  cells/Adult  stem  cells  
 
Embryonal  stem  cells  
− most  undifferentiated  
− in  the  inner  cell  mass  of  blastocyst  
− limitless  cell  renewal  capacity  
− can  give  rise  to  every  body  cell  
− totipotent  
 
 
Cell  cycle  checkpoints  
− replication  of  cells  with  genetic  imperfections  is  hindered  
− done  by  CDK  inhibitors  
1.  G1-­‐S  –  monitors  DNA  integrity  
2.  G2-­‐M  –  ensures  accurate  genetic  replication  
 
   
 
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Tissue  stem  cells    
− admixed  intimately  with  differentiated  cells  
− protected  within  stem  cell  niches  
− limited  
− only  produce  cells  of  a  particular  tissue  

 
STEM  CELL  NICHES  
 
Regenerative  medicine  
− theoretically:  stem  cells  can  be  used  to  repopulate  damaged  
tissues  or  construct  an  entire  organ  
− (+)  problems:  integration,  immunogenecity  
 
Induced  pluripotent  SC  (iPS  cells)  
− derived  from  patients  themselves  
− (-­‐)  rejection  reaction  

 
 
Cas  9  technology  
− with  guide  RNAs  (CRISPRs)  
− selectively  alter  or  correct  DNA  sequences  

 
 
Trans  DB  Link:  
https://www.dropbox.com/sh/xmcmo6n7ecpwxqz/AAAay5-­‐
hcrZrkQc76PWv-­‐G9Pa?dl=0  
 
Yne  sytivilibagon,  Drogon.    
“Fight  for  me  Drogon.”  

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