Pomen proteinov (in encimov) v medicinski diagnostiki in pri zdravljenju Tumorski markerji so različni proteini
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1 Pomen proteinov (in encimov) v medicinski diagnostiki in pri zdravljenju Tumorski markerji so različni proteini
2 Encimi v medicinski praksi Encimi kot diagnostični indikatorji (markerji) - zmanjšana aktivnost holinesteraz pri zastrupitvi z organofosfati (pesticidi, bojni strupi...) - ala-transaminaza (ALT) in asp-transaminaza (AST) razpad jeter (ciroza, rak...) - alkalna fosfataza v krvi kostne in jetrne bolezni - kisla fosfataza v krvi rak prostate - specifični srčni encimi (LDH, CPK, AST) infarkt - lipaza in amilaza pankreatitis - Etc., etc.. Encimi kot zdravila - streptokinaza, aktivator plazminogena razgradnja strdkov v krvi - asparaginaza razgrajuje Asp, ki ga nujno rabi levkemično tkivo za rast - Dodajanje encimov zaradi pomanjkanja (genske napake, itd.) Encimi kot tarča zdravil
3 Določanje kreatinske kinaze (CK), ala- in asp-transaminaz (ALT, AST) in laktatne dehidrogenaze (LH) v serumu po infarktu Časovni potek encimske aktivnosti po infarktu Razpolovne dobe izoencimov mitochondrijska AST 1 h LDH-5 (M4) 12 h CK 18 h citoplazemska AST 1 dni ALT 2 dni LDH-1 (H4) 5 dni
4 Določanja serumskih CK in LDH z elektroforezo infarkt infarkt tehnična kontrola Obolenje jeter 2 dni po infarktu 1 dan po infarktu Zastoj srca, poškodba normalni vzorec vzorec CK pro2services.com/.
5 Encimi kot zdravila: trombolitična (fibrinolitična) sredstva razgradni produkti Tkivni plazminogenski aktivator tpa (serinska proteinaza) Streptokinaza (SK), and Urokinaza (UK). Streptokinaza je hemolitični encim bakterije iz rodu Streptococcus Je aktivator plazminogena in sodeluje v razgradnji strdkov v krvi Medicinska uporaba - razgradnja krvnih strdkov, npr. pri akutnem miokardnem infarktu Stranski učinki notranje krvavitve zaradi proteolize.
6 Terapija raka z encimsko aktiviranim pro-zdravilom
7 Terapija kroničnih vnetij inhibicija proteolitičnih encimov z anti-proteinazami
8 Primeri proteinov, ki se uporabljajo v zdravljenju Lehninger, 2008
9 21. Stoletje - od gena preko proteina do označevalcev bolezni in zdravil 19. Stoletje G. Mendel odkrije vzorce dedovanja 20. Stoletje J. Watson in F. Crick leta 1953 razkrijeta strukturo DNA 21. Stoletje Era funkcijske genomike, celostnih raziskav genoma in njegovih funkcij
10 Pristopi funkcijske genomike za odkrivanje bolezenskih markerjev in tarč za zdravila transkriptom proteom metabolom -Ekspresijske mikromreže -RT-PCR -sirna - 2D proteinske mape /MS -LC/MS(n) - metabolično profiliranje -Metabolični pretoki (fluks) Genomika, proteomika in druge omike so v medicinskih in farmacevtskih raziskavah močno pospešile razvoj novih zdravil (z manj stranskimi učinki) in odkrivanje novih bolezenskih markerjev.
11 Genomika -Obravnava sestavo genomov in njihovih genov -Dele DNA genomov lahko organiziramo v knjižnice (genomske ali cdna) - Z reakcijo verižnega pomnoževanja PCR lahko pomnožimo katerikoli del genoma (potebujemo začetne oligonukleotide) -Mednarodno povezovanje je omogočilo določitev zaporedja človeškega genoma in drugih genomov. Informacije so zdaj dostopne v javnih podatkovnih zbirkah Science, 2001
12 Reakcija verižnega pomnoževanja PCR revolucija v raziskavah genoma (faza 1) Polymerase Chain Reaction Lehninger, str. 315
13 Odkritje PCR je bilo ključno za pospešene raziskave genomov
14 Časovna skala določitve nukleotidnega zaporedja (sekvenciranja) genomov
15 Le dober procent človeškega genoma se prevede do proteinov Lehinger, 2008
16 Kaj pravzaprav pomeni sekvenciranje genoma, če pa ima vsak človek drugačen genom? Med ljudmi je cca 1% razlike v nukleotidnem zaporedju (polimorfizmi) Razlike v eni sami bazi (SNPs), Mikrosateliti Microsatellites (short sequence repeats), Minisateliti (long sequence repeats), Delecije Duplikacije
17
18 Funkcijska genomika pri odkrivanju bolezenskih markerjev education.scientity.com/bioinformatics
19 Tehnologija DNA čipov in nova (naslednja, druga) generacija sekvenciranja Analiza genomske DNA (genotpizacija enojnih nukleotidnih polimorfizmov SNP, analiza variance števila kopij CNV oz. CGH, resekvenciranje). Analiza izražanja genov (ekspresijsko profiliranje: 3' ekspresijski, eksonski ali genski čipi, čipi za sledenje izražanja mirna). Študije uravnavanja izražanja genov (kromatinska imunoprecipitacija na čipu ChIP-on-Chip, mapiranje prepisov)..
20 Uporaba DNA mikromrež Bazične (osnovne) raziskave globalni pogled na izbrani biološki proces Farmacevtska industrija - globalni pristop k testiranju potencialnih zdravilnih učinkovin - kreiranja tematskih DNA mikormrež za diagnosticiranje kompleksnih obolenj Klinika genotipizacija, odkrivanje normalnih in okvarjenih alelo - določanje enojnih nukleotidnih polimorfizmov (SNP) razvoj osebne medicine - odkrivanje genov, vključenih v bolezenski fenotip večfaktorskih obolenj
21 Ekspresijsko profiliranje z mikromrežami transkriptov genov
22 Iskanje novih bolezenskih označevalcev - ekspresijsko profiliranje in pregled genoma z mikromrežami
23 DNA čipi - povzetek DNA čipi so zbirka mikroskopskih DNA točk, cdna ali oligonukleotidov, pritrjenih na trdo podlago. Omogočajo nam celostni vpogled v genom in transkriptom. Uporabljajo se za: Analizo genomske DNA (genotipizacija, SNP analiza, CHG, sekvenciranje). Analizo izražanja genov (ekspresijsko profiliranje), kjer probe lahko predstavljajo 3' konce genov, eksone ali različne dele genov. Posebni čipi pa so za sledenje izražanja mirna. Študije uravnavanja izražanja genov (določanje vezavnih mest transkripcijskih faktorjev, metilacija kromatina), kjer probe predstavljajo 5 - neprevedene dele in nerepetitivne dele genov. Bistveno pripomogli k odkrivanju novih bolezenskih označevalcev, posebno pri različih vrstah raka.
24 Nova (naslednja, druga) generacija sekvenciranja
25 Nova generacija sekvenciranja - Sekvenciranje človeškega genoma je trajalo več let, z uporabo cca 20 kb BAC klonov, ki so vsebovali cca 100 kb dolge tarčne fragmente, in 8-kratnega pokrivanja vsakega dela tarče. Analiza s kapilarno elektroforezo. -Nadaljnji razvoj sekvenciranja je temeljil na sočasnem sekvenciranju celotnega genoma, ki je bil vstavljen v vektorje. Metoda je hitrejša, pušča pa velike praznine v zelo polimorfnih ali repetitivnih genomih. Analiza s kapilarno elektroforezo. -Naslednja generacija sekvenciranja (2004) visokozmogljivostno paralelno čitanje odsekov DNA na ravni celega genoma preko PCR pomnoževanja enoverižnih fragmentov genomske knjižnice.
26 Za čitanje zaporedja celotnega genoma posameznika danes potrebujemo le nekaj dni
27 Naslednja generacije sekvenciranja povzetek Nova generacija visokozmogljivostnega sekvenciranje omogoča razpoznavanje zaporedij DNA na ravni celega genoma, z resolucijo posameznega baznega para. Iz vsakega vzorca se pripravi knjižnica, ki vsebuje vse v vzorcu prisotne fragmente DNA ali RNA (cdna). Vse platforme bazirajo na PCR pomnoževanju, imajo pa različne pristope sekvenciranja: Razvijajo se tudi metode, ki pred sekvenciranjem ne potrebujejo pomnoževanja (tretja generacija) Uporaba je podobna, kot pri klasičnih mikromrežah, prednost je v preprosti pripravi vzorca in zmožnosti procesiranja velikega števila vzorcev v kratkem času. Procesiranje velikega števila vzorcev na eni/več aparaturah lahko upravlja 1 človek. Analiza celotnega genoma traja nekaj dni. Cena analize posameznikovega genoma naj bi v nekaj letih padla pod 500 eur nov pristop k osebni medicini V Sloveniji (2010) tovrstne aparature še nimamo
28 Proteomika -Proteom je zbirka proteinov, ki jih proizvede celica na podlagi zapisa v genomu. -Proteomika obravnava strukturo, funkcijo in izražanje teh proteinov v celici. -Integrirane raziskave, kjer poleg genoma, transkriptoma, proteoma preiskujemo še druge ome in skušamo celostno razumeti dogajanje v celici, opišemo z izrazom sistemska biologija -Celični proteom lahko preiskujemo z 2D gelsko elektroforezo ali različnimi večdimenzionalnimi tekočinskimi kormatografijami, v povezavi z masno spektrometrijo. - Poleg DNA čipov poznamo tudi proteinske čipe, s katerimi preiskujemo izražanje celičnega proteoma.
29 Pomen bionformatika pri iskanju bolezenskih markerjev (in tarč za zdravila)
30 Izzivi povezovanja podatkov
31 Primer projekta sistemske biologije za odkrivanje bolezenskih markerjev nealkoholne bolezni jeter
32 Bioinformatics Making sense of the huge amounts of DNA data produced by gene sequencing projects. Bioinformatics and computational biology involve the use of techniques from applied mathematics, informatics, statistics, and computer science to solve biological problems. Research in computational biology often overlaps with systems biology. Major research efforts in the field include sequence alignment, gene finding, genome assembly, protein structure alignment, protein structure prediction, prediction of gene expression and protein-protein interactions, and the modeling. The terms bioinformatics and computational biology are often used interchangeably, although the former typically focuses on algorithm development and specific computational methods, while the latter focuses more on hypothesis testing and discovery in the biological domain. Wikipedia
33 Funkcijska genomika pri razvoju zdravil - Farmakologija veda, ki se ukvarja z odkrivanjem, kemijo, sestavo, razpoznavanjem, biološkimi in fiziološkimi efekti, uporabo in proizvodnjo zdravil - Kandidati za zdravila morajo učinkovito modulirati svoje tarče (proteine, encime) - Morajo imeti primerne lastnosti, da dosežejo tarče Spojina Fiziološki efekt Molekularna tarča Molekularna tarča Spojina Fiziološki efekt
34 Molekularna tarča protein (encim), z znano 1D - 3D strukturo Rognan D. J Pharmacol. 2007, 152: Epub 2007 May 29. Review.
35 Prostor proteinske tarče in liganda molekulrani prstni odtisi (fingerprint) Primerjamo serijo kompleksov med n ligandi in enim proteinov ali med enim ligandom in n sorodnimi proteini Rognan D. J Pharmacol. 2007, 152: Epub 2007 May 29. Review.
36 Analize SAR povezava med strukturo molekule in njeno aktivnostjo Pomembne proteinske družine, ki so tarča zdravil: - Z G-proteini povezani receptorji - Jedrni receptorji -Kinaze - Proteinaze - Fosfodiesteraze Analitske metoda za določevanje aktivnosti Iskanje struktrnih značilnosti aktivnih spojin s SAR Knjižnice (zbirke) kemijskih spojin z znanimi biološkimi profili (aktivnostjo)
37 In silico ribarjenje pri iskanju ligandov in proteinskih tarč Uporaba algoritmov strojnega učenja, Bayesove statistike, itd. za iskanje prstnih odtisov molekul v biološko dobro anotiranih podatkovnih zbirkah.
38 Genom, ki ga lahko zdravimo Zbirka človeških proteinov, ki vežejo spojine vodnice, imenujeno genom, ki ga lahko zdravimo (druggable genome).
39 Obstoječi izzivi hitrejšega razvoja zdravil -Ključ za izboljšanje odkrivanja zdravil je v organizaciji verige funkcijskih raziskav - Informacije se morajo pretakati iz osnovnih raziskav analize genoma, proteinov, metabolitov, kemijskih spojin, itd., do stopnje kliničnih raziskav in sprostitve zdravila na trg. - Združevanje tako različnih področjih naravoslovnih in matematično-informatičnih znanosti predstavlja še vedno velik izziv. - Potrebni so mostovi, ki pomagajo raziskovalcem enega področja pri komunikaciji z drugimi raziskovalci interdisciplinarnih skupin.
40 Optimizem pri razvoju novih zdravil -Znano je zaporedje človeškega genoma -Genomske in proteomske tehnologije se hitro razvijajo -Pristopi sistemske biologije so vse bolj napredni -Razvijajo se napredne tehnike medicinske vizualizacije -Napredek je v nanotehnologiji in tkivnem inženirstvu -Napredek v kombinatorni kemiji in rešetanju spojin na mikro skali - Itd.
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