Life is Dynamic Enthalpy vs Entrophy The information

Life is Dynamic Enthalpy vs Entrophy The information

Life is Dynamic Enthalpy vs Entrophy The information DNA Nucleotides: fancy sugars DNA Regulated by proteins Autosomes > 22 matched sets (44 total) > 100% identical in males and females All sequences for a particular mRNA are not identical Some sequences are defective, some are just a little different Males and Females Autosomes: 22 matched set Sex chromosomes: the difference Chromosome Squashes Human Chromosome Launchpad http://www.ornl.gov/hgmis/launchpad/ NCBI http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/LocusLink/list.cgi Genetics and Translation: Gene Expression There are 4 different RNA nucleotides that can pair with the 4 different DNA nucleotides Messanger RNA (mRNA) is a small sequence of RNA nucleotides that describes the DNA code for the protein. The rest of the small DNA sequences in the incredibly large DNA molecule we call a chromosome is WHEN to make the molecule.

Proteins Coded by DNA Every three nucleotides in a gene sequence code designates the code for one amino acid specific type of amino acid in a protein sequence A protein is a sequence of amino acids To Make a protein, a cell must transcribe (Copy) the DNA coding sequence into a portable message (mRNA) that can leave the nucleus of the cell and be used to make the protein. Transcription: DNA mRNA Nucleus Translation: mRNA Cytoplasm Protein Proteins are made of Amino acids Regulation of Gene Expression Transcription factors Measuring gene expression (mRNA) 1.Coding sequences are not entirely unique 2. Motifs 3. Hybridization 4. http://www.ncbi.nlm.nih.gov:80/blast Gene Expression GS mRNA TAT mRNA GS mRNA ( pmole/gm ) 0.5

Model A 0.4 Model B 0.3 0.2 0.1 TAT mRNA ( pmole/gm ) 4 TAT Activity ABS at 331 nm GS Activity 8 Glutamine Synthetase (pmole/g/hr) Model B 2 1 0 0.0 6 4 2 0 Model A 3 0 5 10 15

20 25 TAT Activity 3 2 1 0 30 Time ( Hour ) 0 4 8 12 16 20 Other Patterns IGF-I mRNA (fg/ng total RNA) GR mRNA GR mRNA (fmole/gm) 6 Model A 5

Model B 4 3 2 1 200 160 120 80 40 0 GR Density 80 Plasma IGF-I (ng/ml) GR Density (fmole/L/mg protein) 1600 60 40 1200 800 20 400 0 0 20 40 60

80 0 Time ( hr ) 20 40 60 80 Gene Arrays Two basic types 1. Spotted macro-arrays 2. Synthesized micro-arrays Spotted macro array Figure 4 Results: Liver, Control vs treated (5.5hr.) 51 identifiable genes Genes Involved in Acute phase/Immune Response Genes Involved in Energy Metabolism Genes Affecting Microsomal Enzymes Alterations of Other Genes eg protein synthesis, signal transduction Genes Involved in Acute phase/Immune Response

Pre-pro-complement C host immune response (22, 55) Parathymosin thymocyte maturation (56, 57) MHC class 1 presages immune-mediated damage (58-60) 2, 5, - OAS INF-induced antiviral response (23, 61) target of antiproliferative antibody regulation of mitotic activity (62, 63) anti-acetylcholine receptor antibody immunoglobulin expression thiol-specific antioxidant NFB signaling pathway (64, 65) Biglycan increased in inflammation (66-68) 2 - globulin acute phase protein (69) growth hormone regulated protein anti-protease (70, 71) thrombin blood clotting/pro-inflammatory (24, 72) 1 - macroglobulin inhibits inflammatory proteases (73) 2 - macroglobulin inhibits inflammatory proteases (25) 1 - acid glycoprotein inhibits microvascular permeability (26, 27, 74) serum amyloid P scavenges chromatin (75, 29) Vitamin D binding protein Vitamin D carrier/scavenges actin (28, 76) -fibrinogen blood clotting (30) fibrinogen -chain blood clotting (30) fibrinogen -chain blood clotting (30) proline rich protein blood clotting (77) metallothionein-2/-1 metal binding protein (32, 78)

metallothionein-I metal binding protein (32, 78) delta-aminolaevulinate synthase heme synthesis (31, 79) Experimental Design 50 mg/kg dose of methylprednisolone sodium succinate (MPL) in the right external jugular vein Rats were sacrificed at 17 time points after MPL administration (between 0 and 72 hours) 4 controls 3 rats per time point Liver and muscle flash frozen Trizol Eg. one hour Total RNA Total RNA cDNA biotin-labeled cRNA hybridize Total RNA cDNA biotin-labeled cRNA hybridize Total RNA

cDNA biotin-labeled cRNA hybridize Each chip contains cRNA from one rat 5.00 4.80 4.60 4.40 4.20 4.00 3.80 3.60 3.40 3.20 3.00 2.80 2.60 2.40 2.20 2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 TIME

0 2 4567 12 18 30 48 72 Selected Experiment:New Affy alogrhythm analysis of live...Colored by: New Affy alogrhythm analysis of liver time cou... Graphed by: New Affy alogrhythm analysis of live...Gene List: all genes (8799), M26161_at selected Trees of Normalized Liver and muscle ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...... ...... ... ... ... ......... ...... ... trypsin... ......

...... ...... Selected Gene Tree: Liver New Affy alogrhythm Colored by: New Affy alogrhythm analysis of liver time course Default Interpretation Gene List: all genes (8799), M26161_at selected Selected Gene Tree: Skeletal muscle normalized to controls R4Pstats Colored by: Skeletal muscle normalized to controls Default Interpretation Gene List: all genes (8799), X63369cds_at selected Filtering and Clustering The goal is to separate the regulated genes from the unregulated genes. Start with filtering to reduce the number of genes. Reducing the number of genes makes statistical approaches more powerful Muscle Eliminate genes not present on at least 4 chips ... Selected Gene Tree: Skeletal muscle normalized to controls R4Pstats Colored by: Skeletal muscle normalized to controls Default Interpretation Gene List: restrictions4P (4636), X63369cds_at selected Filter on response characteristics 0.65 two conditions ... 1.5 two conditions ... Selected Gene Tree: Copy of Copy of skeletal Muscle MPL 1 (Default Interpretation) Colored by:

Skeletal muscle normalized to controls Default Interpretation Gene List: muscle R4P max .65 two conditions (676) Selected Gene Tree: Copy of Copy of skeletal Muscle MPL 1 (Default Interpretation) Colored by: Skeletal muscle normalized to controls Default Interpretation Gene List: muscle R4P min1.5 two conditions (565) ANOVA Bonferroni post test ... ... Selected Gene Tree: Copy of Copy of skeletal Muscle MPL 1 (Default Interpretation) Colored by: Skeletal muscle normalized to controls Default Interpretation Gene List: muscle R4P max .65 2 conditions ANOVA bonferoni (126) Selected Gene Tree: Copy of Copy of skeletal Muscle MPL 1 (Default Interpretation) Colored by: Skeletal muscle normalized to controls Default Interpretation Gene List: muscle R4P i.5 fold two conditions bonferoni (141) Recombine lists for clustering ... Selected Gene Tree: Copy of Copy of skeletal Muscle MPL 1 (Default Interpretation) Colored by: Skeletal muscle normalized to controls Default Interpretation Gene List: muscle up and doun two conditions bonferoni (241) A Few Genes 9.0 8.0 7.0 6.0

C/EBP 5.0 4.0 3.0 2.0 1.0 Time (hours) 012 45678 12 18 30 48 72 3.0 MyoD 2.0 1.0 Time (hours) 0 2 4567 12 18 30 48 72

3.0 Glutamine Synthetase 2.0 1.0 Time (hours) 012 4 56 7 8 12 18 30 48 72 A Few More Genes 7.0 6.0 5.0 Mt1 4.0 3.0 2.0 1.0 Time (hours) 012 45678 12

18 30 48 72 2.0 EST AA859663 1.0 Time (hours) 0 2 4567 12 18 30 48 72 2.0 Cyclin D1 1.0 Time (hours) 0 2 4567 12 18 30 48 72

Mechanism of Action for Glucocorticoids (GC) EFFECTS (Direct) protein binding diffusion Mediated by Glucocorticoid Receptor Acute effects (e.g. adrenal suppression, lymphocyte trafficking) Gene-mediated effects via GRE (e.g. TAT induction, PEPCK induction) 90 56 90 70 binding 90 56 90 70 activation Cytosol P P

translocation dimerization P P P P P P P P GRE Nucleus Target DNA transcription Repressed mRNA Induced mRNA translocation Repressed mRNA Induced mRNA translation Repressed Protein Induced Protein EFFECTS (Gene-Mediated) Methylprednisolone PK/PD in Rats

MPL Plasma Conc (ng/ ml) Hepatic Cytosol GR Density (fmole/mg protein) Nuclear Receptors % Binding : DR (N) Time, hour TAT Activity (unit/mg protein) TAT mRNA (pmol/g liver) Time, hour Cluster 1 (23 probes, 21 genes) Cluster 4 (66 probes, 49 genes) After cluster analysis using GeneSpring Cluster 2 (9 probes, 6 genes) Cluster 5 (8 probes, 5 genes) Cluster 3 (21 probes, 18 genes) Cluster 6 (67 probes, 44 genes) Gene Array (cluster1 TAT Like mRNAs) [linear stimulation by DR(N) on mRNA production] I(t)Rm ks_Rm

mRNAR k d_Rm ks_R D + R DR kon kT S DR(N) Rf kre kd_R mRNA kd_m (1-Rf) kre dmRNA k s _ m 1 S DR( N ) k d _ m mRNA dt Baseline : ks_m 0 k s _ m k d _ m mRNAnormalized 0 I .C . mRNAnormalized 1 Mechanistic Modeling of Cluster 1 and 2 S ks_m DR(N)

mRNA kd_m Cluster 1 Cluster 2 10 30 Ornithine Decarboxylase (EC 4.1.1.17) X07944exon#1-12_s_at (#1) 8 alpha-2-macroglobulin X13983mRNA_at (#1) 20 6 10 2 mRNA (normalized to time zero level) mRNA (normalized to time zero level) 4 0 10 microtubule-associated proteins U05784_s_at (#2) 8 6 4 2 0

10 Ornithine Decarboxylase J04792_at (#10) 8 0 30 alpha-2-macroglobulin M22670cds_g_at (#2) 20 10 0 5 3 6 4 2 2 1 0 Novel members of the Vesl/Homer family of PDZ proteins AB007690_s_at (#9) 4 0 12 24

36 Time (hr) 48 60 72 0 0 12 24 36 Time (hr) 48 60 72 Inhibition of Expression 2.0 A 1.5 1.0 D R (N ) I(t) m R

N A k s_m k d _m mRNA (normalized to time zero level) 0.5 0.0 2 .0 B 1 .5 1 .0 0 .5 0 .0 2.0 C 1.5 dmRNA DR(N) ks_m mRNA 1 IC50 kd_m

1.0 dt DR(N) 0 I.C. mRNA 1 normalized 0 normalized Baseline : ks_m kd_m mRNA 0.5 0.0 0 1 2 2 4 3 6 T im e ,h o u

r 4 8 6 0 7 2 Inhibition Followed by Stimulation S m BS_s k s_m D R (N ) BS m R N A BS k d_m B S I(t)m _s S k s_ m dmRNA BS dt dBS dt k k

s _ BS dmRNA dt k Baseline : Fixed Parameters 1 S s _ mBS mRNA s_ m k BS 1 IC k s _ mBS : mBS k

50 _ s d _ BS DR ( N ) k d _ mBS BS I . C . BS DR ( N ) S m _ s DR ( N ) d _ mBS mRNA ( assu min m _ s 0 BS g mRNA mRNA k BS is a Cluster d _ m

s _ BS m _s BS k s_ B S m R N A k d_B S k d_m 0 BS _ normalized I . C . mRNA BS 0 normalized BS k 1 1 mRNA k 0 BS d _ BS

2 gene 0 normalized I . C . mRNA ) k /( mRNA d _ mBS 0 BS 1 ) 0 . 04 hr k 1 , S mBS _ s s_ m k d _ m 0 . 2 ( fmol

0 mRNA / mg protein /( 1 S )1, m _s BS 1 0 ) Fit of Data to Previous Set of Equations 2 2 C 4 B A 2 1 0 0 12 24

Time,hour 36 48 1 0 60 72 mRNA(normalized) mRNA(normalized) mRNA(normalized) 3 0 12 24 Time,hour 36 48 1 0 60 72 0 12

24 Time,hour 36 48 60 72 Pathway Modeling Kyoto Encyclopedia of Genes and Genomes http://www. genome.ad.jp/ kegg/metabolism.html International Union of Biochemistry and Molecular Biology http://www.chem.qmul.ac.uk/ iubmb/enzyme/ Transcription Factor Modeling D R k S T k D R (N ) m B S_s m R N A s_ m B S k S BS dt

k 1 S s _ mBS dBS k s _ BS mRNA BS dt dmRNA k s _ m (1 S dt Baseline : Fixed Parameters k k s _ mBS : mBS k m _ s _ s d _ BS DR ( N ) k

d _ mBS BS I . C . BS BS ) k d _ mBS mRNA ( assu min d _ m 1 S m _ d mRNA 0 BS g mRNA k BS is Cluster s _ BS B S k s_B S

S m R N A k 0 BS _ normaliz ed I . C . mRNA d_B S m _d d_m 1 1 mRNA DR k s_m BS 0 normalized d_m B S m _s k dmRNA B S

0 normalized I . C . mRNA k 1 gene d _ BS ) BS k 0 d _ mBS /( mRNA 0 . 3 hr 0 BS 1 ) 1 k , S mBS _ s 0 . 03 s _ m

k ( fmol d _ m / mg mRNA protein 0 /( 1 S )1, m _ s BS 1 0 ) S i m u l a t i o n (cluster3-A rgiase) 6 600 BS mRNA

BS mRNA DR DR(N) 400 4 200 2 0 0 0 12 24 36 Time (hr) 48 60 72 DR or DR(N) (fmol/mg protein)-1 BS mRNA, BS or mRNA (normalized to time zero level) Model for C/EBP Induction of Arginase

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