Computer simulations of direct DNA alkylation with acrylonitrile

V današnjem času smo povsod obdani s kancerogenimi snovmi, zato je naša naloga, da spoznamo, kako te snovi vplivajo na naše telo oz. kakšne spremembe povzročijo na dednem materialu DNK. Ena izmed teh snovi je akrilonitril, za katerega se je do sedaj predvidevalo, da se v telesu presnavlja predvsem po oksidativnem mehanizmu, saj se mehanizem direktne alkilacije oz. Michaelove adicije akrilonitrila na DNK še ni dovolj preučil. Preučevanja mehanizma direktne alkilacije akrilonitrila na DNK smo se posledično lotili v tem magistrskem delu. Mehanizem Michaelove adicije smo preučevali preko računalniških kvantnomehanskih simulacij na podlagi več različnih ab initio metod s programskim paketom Gaussian 09. Za realnejši izračun smo uporabili tudi implicitne modele vode, ki deluje kot topilo za reakcije v bioloških sistemih. Solvatacijske metode, ki smo jih uporabili, predstavljajo model polarizabilnega kontinuuma, ki spada v skupino metod samouglašenega reakcijskega polja, metoda Langevinovih dipolov in model AMSOL za semiempirične metode. Na podlagi aktivacijskih prostih energij smo ugotovili, da je reakcija direktne alkilacije z vidika energijske pregrade povsem mogoča. Rezultati so pokazali, da ima najnižjo aktivacijsko prosto energijo gvanin in je zatorej najbolj podvržen direktni alkilaciji s strani akrilonitrila, kar je tudi eksperimentalo potrjeno. Vendar pa so energijsko ugodne tudi reakcije adenina in citozina. Primerjava naših rezultatov z rezultati adicije cianoetilen oksida na DNK so razkrili, da je alkilacija akrilonitrila na DNK lahko celo energijsko ugodnejša, kar pomeni, da je mehanizem direktne alkilacije morda celo verjetnejši od oksidativnega mehanizma presnove akrilonitrila. Na podlagi dobljenih izsledkov smo torej pokazali, da je ključnega pomena nadaljevati z raziskavami mehanizma Michaelove adicije akrilonitrila na DNK, saj lahko le-ta predstavlja enega od ključnih mehanizmov kancerogeneze.Nowadays carcinogenic substances surround us at every step, so our goal should become to get acquainted with the effects they have on our bodies and with what kind of modifications they can cause to our DNA. Acrylonitrile is a known carcinogenic substances which can be metabolised in the body via two different pathways, and until now it was believed that the oxidative pathway represents the major one, mainly because direct alkylation - the Michael addition pathway - had not been studiedin sufficient detail. Studying the direct alkylation mechanism was therefore the objective of this master thesis. We have studied this mechanism through quantum mechanical simulations using several ab initio methods incorporated in the Gaussian 09 series of programs. To compare the theoretical results to real systems and thus incorporate the solvent effects we have used implicit water models: the polarisable continuum model that belongs to the group of self-consistent reaction field methods, the Langevine dipoles method, and the AMSOL model for semiempirical calculations. On the basis of activation free energy we have come to conclusion that direct alkylation of DNA by acrylonitrile is perfectly plausable. The results have in complience with experiment shown that among DNA bases guanine has the lowest activation free energy and is therefore the most reactive with acrylonitrile. However, the energy bariers for adenine and cytozine are very similar to those of guanine and are low enough to cause direct alkylation by acrylonitrile as well. The comparison of our results with the results of cyanoethylene oxide addition to DNA have shown that investigated mechanism might even be more energetically favorable than the oxidative mechanism, thus possibly making direct Michael addition more the plausable mechanism for the metabolism of acrylonitrile. With these results we have shown that further studies of the Michael addition of acrylonitrile to DNA are needed, as it may represent one of the carcinogenic mechanisms

THE ROLE OF IL10 AND TNFα GENES IN GLUCOCORTICOID TREATMENT OF CHILDHOOD ASTHMA

Diplomsko delo prikazuje študijo povezav genotipov in alelov polimorfizmov rs1800896 ob genu IL10 in rs1800629 ob genu TNFα z otroško astmo. V okviru raziskovanja problematike smo preučevali vplive posameznih genotipov ali alelov teh polimorfizmov na klinične in laboratorijske parametre pri astmatikih, ki so prejemali glukokortikoidno terapijo. Iskali smo tudi povezave med polimorfizmom rs1800629 ob genu TNFα in ekspresijo gena TNFα. V študiji je sodelovalo 355 astmatičnih otrok starih od 5-19 let med katerimi je bilo 169 (47,61 %) moških in 136 (38,31 %) žensk za 50 (14,08 %) pa ni bilo podatka o spolu. Atopijsko astmo je imelo 202 (56,90 %) otrok, neatopijsko 88 (24,79 %) otrok, za 65 (18,31 %) bolnikov podatek ni znan. Terapijo z glukokortikoidi je prejemalo 189 (53,24 %) bolnikov. Polimorfizem rs1800629 ob genu TNFα je razkril povezave na prav vseh področjih našega raziskovanja. Genotipske in alelne frekvence astmatikov v nasprotju s kontrolno skupino statistično značilno odstopajo od Hardy-Weinbergovega ravnovesja (p < 0.001). Ugotovili smo, da obstaja statistično značilna povezava SNP-ja rs1800629, ne zgolj z atopijsko astmo, temveč za astmo nasploh. Alel A je statistično značilno (p < 0,001) pogostejši pri skupinah astmatikov kot pri kontrolni skupini. Naša študija je pokazala, da imajo bolniki z A alelom polimorfizma rs1800629 statistično značilno (p = 0,037) boljši odziv na terapijo z glukokortikoidi, merjen z forsiranim ekspiratornim volumnom v prvi sekundi izdiha (FEV1), kot bolniki z alelom G. Polimorfizem rs1800629 ob genu TNFα je na različne načine povezan tudi s kliničnimi parametri PC20%, PEFR%, PC20% , FEV1. Mediane 2- ΔΔCt kažejo, da se gen TNFα značilno manj izraža pri posameznikih z alelom A za SNP rs1800629, kot pa z alelom G pri skupini astmatikov in kontrol in tudi pri posameznih skupinah astmatikov, atopikov ter neatopikov. Povezava se je pokazala tudi pri ekspresiji po glukokortikoidni terapiji skupine astmatikov, kjer se je gen TNFα statistično značilno manj izražal pri preizkovancih z alelom A SNP-ja rs1800629. Za polimorfizem rs1800896 ob genu IL10 pa smo ugotovili, da vpliva na posamezne klinične parametre povezane z manjšo hiperodzivnostjo in obstrukcijo dihal in dihalnih poti. Za več povezav pa je potrebno v prihodnje narediti še veliko raziskav. Rezultati naše študije so pokazali, da je polimorfizem rs1800629 z astmo povezan na številne načine, saj vpliva ne samo na odziv na zdravljenje, temveč tudi na resnost astme kot bolezni ter izražanje gena TNFα, ki povzroči vnetne procese in s tem simptome astme. V prihodnosti bi bilo zato smotrno ta polimorfizem še podrobneje preučiti, da lahko izboljšamo zdravje pacientov z astmo na način, da se zdravljenje prilagodi posamezniku glede na njegov genetski zapis. S tem bi pripomogli k boljšemu diagnosticiranju astme, učinkovitejši preventivi in nenazadnje tudi k boljšemu zdravljenju, ki lahko z individualnim pristopom postane bolj učinkovito in manj toksično.Graduation thesis represents the study of genotypes and alleles of polymorphisms rs1800896 near gene IL10 and rs1800629 near gene TNFα in regard with childhood asthma. Influences of genotypes and alleles on clinical and laboratory parameters were also studied in the group of pacients with childhood asthma that recieved glucocorticoid therapy. We have also been looking for significant connections between polymorphism rs1800629 near gene TNFα and the expression of gene TNFα. Our study subject were 355 astmatic children of age 5-19 among them there were 169 (47,61 %) males and 136 (38,31 %) females for 50 (14,08 %) the gender was not specified. Among asthmatic children there were 202 (56,90 %) with atopic asthma and 88 (24,79 %) with nonatopic asthma, for 65 (18,31 %) pacients this information were not given. Glucocoricoid treatment recieved 189 (53,24 %) patients. Polymorphism rs1800629 near gene TNFα revealed influences in all fields of our study. Frequencies of genotypes and alleles of asthmatic patients do not follow the Hardy-Weinberg principle in contrast with control group where they do. Significant tests have shown that there is a significant association of SNP rs1800629 with childhood asthma. Allele A is significantly (p < 0,001) more common in asthmatic groups of patients than in control group. Our study revealed that pacients with alelle A SNP rs1800629 have significantly (p = 0,037) better response to glucocorticoid threatment of atopic asthma, measured by forced expiratory volume in first second (FEV1) than pacients with alelle G. Polymorphism rs1800629 near gene TNFα is also associated with clinical parameters PEFR%, PC20% , FEV1. Medians of 2- ΔΔCt show that the expression of gene TNFα is significantly lower in individuals with allele A of SNP rs1800629 than in allele G in group of asthmatic pacients combined with control group and in all groups of asthmatic pacinets regardless of the phenotype. The same significant connection regarding the allele A of SNP rs1800629 and the expression holds even when measured after the glucocorticoid treatment of children with asthma. We have found that SNP rs1800896 near gene IL10 influences specific clinical parameters connected with lower hiperresponsiveness and obstruction of airways. There is a need for more torough studies of the influence of SNP rs1800896 on childhood asthma in the future in order to make stroger and clearer conclusions. The results of our study have shown that the polymorphism rs1800629 is associated with asthma in noumerous ways because it is associated not only with the response to the threatment but also with severity of the desease and expression of gene TNFα that causes inflamatory reactions and with this symptoms of asthma. That is why this polymorphism should be studied even more in the future so we can improve pacient\u27s health in a way that we adjust treatmen individualy according to patient\u27s genetic code. By doing so we could improve diagnostics, prevent more deseases and improve treatment by induvidual care so it becomes more efficient and less toxic

Polyphenols: extraction methods, antioxidative action, bioavailability and anticarcinogenic effects

Being secondary plant metabolites, polyphenols represent a large and diverse group of substances abundantly present in a majority of fruits, herbs and vegetables. The current contribution is focused on their bioavailability, antioxidative and anticarcinogenic properties. An overview of extraction methods is also given, with supercritical fluid extraction highlighted as a promising eco-friendly alternative providing exceptional separation and protection from degradation of unstable polyphenols. The protective role of polyphenols against reactive oxygen and nitrogen species, UV light, plant pathogens, parasites and predators results in several beneficial biological activities giving rise to prophylaxis or possibly even to a cure for several prevailing human diseases, especially various cancer types. Omnipresence, specificity of the response and the absence of or low toxicity are crucial advantages of polyphenols as anticancer agents. The main problem represents their low bioavailability and rapid metabolism. One of the promising solutions lies in nanoformulation of polyphenols that prevents their degradation and thus enables significantly higher concentrations to reach the target cells. Another, more practiced, solution is the use of mixtures of various polyphenols that bring synergistic effects, resulting in lowering of the required therapeutic dose and in multitargeted action. The combination of polyphenols with existing drugs and therapies also shows promising results and significantly reduces their toxicity