The development of whole exome sequencing has transformed the study of disease predisposition. The sequencing of both large disease sets and smaller rare disease families enables the identification of new predisposition variants and potentially provide clinical insight into disease management. There is no standard protocol for analysing exome sequencing data. Outside of extremely large sequencing studies including thousands of individuals, statistical approaches are often underpowered to detect rare disease associated variants. Aggregation of variants into functionally related regions, including genes, gene clusters, and pathways, allows for the detection of biological processes that, when interrupted, may impact disease risk. In silico functional studies can also be utilised to further understand how variants disrupt biological processes and identify genotype-phenotype relationships. This study describes the exploration of sequencing datasets from cancers and benign tumour diseases including: i) hereditary diffuse gastric cancer, ii) sweat duct proliferation tumours, iii) adrenocortical carcinoma, and iv) breast cancer. Each set underwent germline whole exome sequencing followed by additional tumour or targeted sequencing to identify associated predisposition genes. Variants within a cluster of risk genes that are involved in double strand break repair were identified as associated with hereditary diffuse gastric cancer risk via gene ontology enrichment analysis. This cluster included PALB2 within which, using externally collated data, loss of function variants were identified as significantly associated with hereditary diffuse gastric cancer risk. Germline protein-affecting variants in the myosin gene MYH9 were identified in all individuals with a rare sweat duct proliferative syndrome, suggesting a role for MYH9 in skin development, regulation and tumorigenesis. These MYH9 variants were analysed in silico to identify a genotype-phenotype relationship between the clinical presentation and variants in the ATP binding pocket of the protein. Tumour matched normal sequence data from adrenocortical carcinoma cases was used to elucidate the role of Lynch syndrome genes in disease pathogenesis. Within the breast cancer set, candidate genes were selected to undergo targeted sequencing in a larger set of cases to further explore their role in breast cancer risk. Risk associated genes identified within this study may ultimately aid in diagnosis and management of disease. This thesis has also generated multiple novel tools and sequencing analysis techniques that may be of use for further studies by aiding in the prioritisation of candidate variants. The described techniques will provide support to researchers working on rare, statistically underpowered datasets and to provide standard analysis pipelines for a range of dataset sizes and types, including familial data and unrelated individuals.

No description available.

Environmental chemicals may affect human health by disrupting endocrine function. Many of the endocrine disrupting chemicals (EDCs) are estrogens or estrogen-like molecules that have been classified as environmental estrogens or xenoestrogens (XEs). XEs include endosulfan, chlordance, nonylphenol, bisphenol A, octylphenol, and coumestrol, etc. Although these compounds have wide structural diversity, but all have in common the and/or other hydrophobic components. Many studies have shown that XEs affect cell viability. For instance, Nonylphenol is used in surfactants or plasticizers and bisphenol A (4, 4¡¦-isopropylidene-2-diphenol) is used as protective coatings on food containers and for composites and sealants in dentistry. Most previous studies have focused on the toxicity of XEs on development process and reproductive system, especially in aquatic ecosystems. Thus, the effects of these two environmental chemicals on the toxicological effect are still controversial. The aim of this study is to investigate the molecular mechanisms of nonylphenol and bisphenol A in induction of cell death in human gastric cancer (SCM-1) cells and Madin Darby canine renal tubular (MDCK) cells. First, WST-1 reduction assays and propidium iodide-staining assay were used to determine cell viability and apoptosis in the present of nonylphenol and bisphenol A. Furthermore, we will use immunoblotting to measure the activity of apoptotic markers caspase-3, mitogen-activated protein kinases (MAPKs) to survey how nonylphenol affects apoptotic pathways. Besides, I will explore bisphenol A whether induces cell death and the mechanisms underlying the [Ca2+]i rise in MDCK cells. The results may be helpful for understanding the pharmacological and toxicological effects of these two environmental chemicals in cells from important organs. Results showed that nonylphenol caused apoptosis via the activation of caspase-3 in cultured human gastric cancer (SCM-1) cells. Although nonylphenol could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Nonylphenol was also found to induce [Ca2+]i increases and pretreatment with BAPTA/AM, a Ca2+ chelator, prevented nonylphenol-induced [Ca2+]i increases, and protect cells from death. These results suggest that nonylphenol induced apoptosis via a Ca2+- and p38 MAPK-dependent pathway. On the other hand, the effect of the environmental contaminant bisphenol A on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Bisphenol A at concentrations between 50-300 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Bisphenol A induced Mn2+ influx, leading to quench of fura-2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholiapase A2 inhibitor aristolochic acid, store-operated Ca2+ channel blockers nifedipine and SK&F96365; and protein kinase C inhibitor GF109203X. In Ca2+-free medium, pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) and the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited bisphenol A-induced Ca2+ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca2+]i rise. Bisphenol A caused concentration-dependent decrease in cell viability via apoptosis in a Ca2+-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca2+]i rises by causing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and mitochondria and Ca2+ influx via phospholipase A2-protein kinase C-sensitive store-operated Ca2+ channels. Key words: calcium, apoptosis, human gastric cancer cells (SCM-1), Madin Darby canine kidney (MDCK), nonylphenol, bisphenol A.

No description available.

Interleukin-1 (IL-1) is a prototypic multifunctional cytokine. IL-1 family include interleukin-1 a (IL-1 a), interleukin-1b (IL-1 b) and interleukin-1 receptor antagonist (IL-1 Ra). IL-1 b is the archetypeal pleiotropic cytokine which have been produced by many cells and exerting its biological effects on almost all cell types. IL-1 b is the most potent of known agents that are gastric cytoprotective, antiulcer, antisecretory and an inhibitor of gastric emptying. IL-1 Ra competes with IL-1 b for cell surface receptor occupancy. Host genetic factors that affect interleukin-1 (IL-1) have been reported to influence the susceptibility of Caucasians to gastric cancer. Whether Asians have the same genetic susceptibility remains unclear. In this study, the genetic associations of IL-1B and IL-1RN polymorphisms with gastric cancer and duodenal ulcer in Taiwan were evaluated. Genomic DNA from 140 unrelated Taiwanese patients with gastric adenocarcinoma, 94 with duodenal ulcer and 165 ethically matched healthy controls was typed for polymorphisms at positions ¡V31, -511, and +3954 in the IL-1B gene, and the variable number of tandem repeats polymorphisms in intron 2 of the IL-1RN gene. The allele frequencies of IL-1RN 2R in gastric cancer cases were much higher than those in healthy controls (9% vs. 3%, p = 0.781). The allele frequencies of IL-1B ¡V31, IL-1B ¡V511 and IL-1B +3954 did not differ. An increased risk of the development of intestinal type gastric carcinoma was found in IL-1RN 2R carriers with an odds ratio (OR) of 4.06 (95% confidence interval [CI]: 1.68 ¡V 9.79, p-value=0.085). And another increased risk of the development of diffuse type gastric carcinoma was found in IL-1RN 2R carriers with an odds ratio (OR) of 3.15 (95% confidence interval [CI]: 1.16 ¡V 8.56, p-value=0.061). A significant association was found in IL-1RN 2R/4R genotype and the risk of the development of duodenal ulcer, with an odds ratio (OR) of 2.57 (95% CI: 1.03 ¡V 6.38, p = 0.292). No significant relationship was noted in duodenal ulcer patients with IL-1B genotype examed in this study. Additionally, a synergistic interaction between blood type A and IL-1 RN 2R carriers existed in gastric cancer patients (OR= 4.51; 95% CI: 1.20 ¡V 16.88, p-value=0.516). The synergistic interaction was even stronger between blood type O and IL-1 RN 2R carriers of duodenal ulcer patients (OR= 10.3; 95% CI: 2.10 ¡V 50.61, p-value=0.160). In conclusion, the genetic polymorphisms of IL-1RN 2R and blood type A are associated with the development of gastric cancer. The genetic polymorphisms of IL-1RN 2R and blood type O are associated with the development of duodenal ulcer.

Background: Gastric cancer is often diagnosed in the metastatic stage, and only 10% of patients survive for as long as 2 years. Current chemotherapy regimens show significant treatment-related toxicities. It is crucial to identify the patients that will benefit most from certain chemotherapy regimens in order to avoid unnecessary side effects. Patients and Methods: 2 patients with advanced gastric cancer repeatedly received 5-FU/cisplatin combination chemotherapy. Genomic DNA was extracted from tumor tissue and mononuclear blood cells. Genotype analysis of genes of metabolizing and DNA repair enzymes was carried out using a PCR-RFLP technique. Direct sequencing was used to identify mutations of the gene dihydropyrimidine dehydrogenase (DPD). Results: Prolonged survival of 51 and 29 months, respectively were observed in our 2 patients. Both patients were positive for genotypes of thymidylate synthase - the target enzyme of 5-FU - that are associated with improved drug response. DPD variants connected with increased toxicity were not observed. However, both patients also showed genotypes in cisplatin metabolizing enzymes which enhance the effect of the drug. Conclusion: Genotype analysis in drug metabolizing enzymes of 5-FU and cisplatin provide a possible explanation for extraordinary therapy effects observed in 2 patients with advanced gastric cancer. / Hintergrund: Das Magenkarzinom wird häufig im fortgeschrittenen Stadium diagnostiziert, und nur etwa 10% der Patienten überleben 2 Jahre. Aktuelle Chemotherapien zeigen eine hohe therapiebedingte Toxizität. Es ist daher von großer Bedeutung, diejenigen Patienten zu identifizieren, die von einer bestimmten Therapie profitieren, um anderen Patienten die Nebenwirkungen einer solchen Therapie zu ersparen. Patienten und Methoden: 2 Patienten mit fortgeschrittenem Magenkarzinom erhielten wiederholt eine Kombinationschemotherapie aus 5-FU/Cisplatin. Genomische DNS wurde aus Tumorgewebe und Leukozyten isoliert. Genotypanalysen von Genen, die am Metabolismus der Substanzen und am DNS-Reparaturprozess beteiligt sind, wurden mithilfe einer PCRRFLP-Methode durchgeführt. Das Gen der Dihydropyrimidindehydrogenase (DPD) wurde direkt sequenziert. Ergebnisse: Beide Patienten zeigten ein deutlich verlängertes Überleben von 51 bzw. 29 Monaten. Genotypen des 5-FU-Zielenzyms Thymidylatsynthase, die mit einem verbesserten Ansprechen assoziiert sind, konnten in beiden Patienten nachgewiesen werden. DPD-Varianten, die mit einer erhöhten Toxizität verbunden sind, wurden nicht beobachtet. Zusätzlich konnten bei beiden Patienten Genotypen in Cisplatin metabolisierenden Genen gefunden werden, die eine prolongierte Wirkung der Substanz bedingen. Schlussfolgerungen: Durch Genotypanalysen in Genen des 5-FU- und Cisplatin-Metabolismus konnte ein spezifisches pharmakogenetisches Profil identifiziert werden, das möglicherweise die Ursache eines außergewöhnlich guten Therapieeffektes in 2 Patienten mit fortgeschrittenem Magenkarzinom ist. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

DNR pažaidų atitaisymas ir apoptozė - dvi pagrindinės grandys, palaikančios žmogaus genomo vientisumą. Sutrikus šiems procesams, ląstelė išgyvena, nepaisant susikaupusių DNR pažaidų ir sudaromas pagrindas tolesnei transformacijai. Tyrimu įvertinome DNR pažaidų atitaisymo funkcijoje dalyvaujančių (hMLH1, MGMT) ir su apoptoze susijusių (DAPK-1, CASP8) genų epigenetinio reguliavimo - metilinimo aspektus pakopiniame skrandžio onkogenezės procese. Šio mokslinio tyrimo metu pirmą kartą buvo nustatytas skirtingas hMLH1 geno metilinimo dažnis atskirose skrandžio anatominėse dalyse atrofiniu pangastritu sergančiųjų audinyje. Įvertinta, kad hMLH1 geno metilinimas sergančiųjų skrandžio vėžiu aplinkiniame nenavikiniame audinyje sietinas su pacientų amžiumi. Išgyvenamumo analizės rezultatai parodė, kad MGMT geno metilinimas agresyvios skrandžio vėžio histologinės formos atveju yra geresnės prognozės rodiklis. Tyrimo metu nustatėme mokslinėje periodikoje neaprašytų tirtųjų genų metilinimo derinių sąsajų su klinikiniais, morfologiniais ir prognoziniais onkologinės ligos ypatumais. / DNA repair and apoptosis are two main pathways supporting the integrity of human genome. After the disturbance of these processes the cell survives, despite the accumulation of DNA lesions, and in this way a basis for a subsequent transformation is formed. In our research we evaluated the epigenetic regulation - methylation - aspects of genes participating in DNA repair function (hMLH1 and MGMT) and also of apoptosis-related genes (DAPK-1, CASP8) in relation to a stepwise gastric oncogenesis process. During this investigation a different hMLH1 gene methylation observation frequency in tissues obtained from separate anatomical parts of the stomach in atrophic pangastritis patients was determined for the first time. It was estimated, that hMLH1 gene methylation in tumor-surrounding non-cancerous tissue in gastric cancer patients could be associated with patient age. Results of survival analysis indicated that MGMT gene methylation is an indicator of better prognosis in case of diffuse form of gastric cancer. During the study we determined some additional associations (not described in previous publications) between methylation combinations of analyzed genes and clinical, morphological and prognostic features of oncological illness.

Predictive and prognostic factors in gastric cancer Šmíd D. Surgical clinic of University Hospital in Pilsen and Faculty of Medicine in Pilsen, Charles University in Prague. Introduction: Gastric cancer is one of malignant diseases which have the worst prognosis. Unfortunately, there are most patients with advanced-stage disease who have to be treated in a palliative way. Patients suffered from the same type of tumor, being at the same stage of disease and treated with the same chemotherapy have various rates of survival, which can be caused by diverse expression of selected genes impacting on the mechanism of cytostatic effects. The determination of these genes or microRNAs which regulate these genes could be used as a predictive factor for prediction of effects of administered chemotherapy. The determination of some microRNAs, or in the combination with suitable plasmatic factors, could be used as a prognostic factor for patients with gastric cancer. It is also possible to use this combination for early diagnosis of cancerogenesis Object: The aim is to verify the possibility to use expression of selected genes and some microRNAs in tumor tissue as a prognostic factor or a predictor for therapeutic effects of chemotherapy in patients with gastric cancer. Methodology: We retrospectively evaluated the group...

O câncer gástrico está entre as neoplasias com a mais alta mortalidade no mundo, sendo que as modalidades de tratamento envolvem a cirurgia, a quimioterapia e também a radioterapia. Na quimioterapia, o uso de drogas isoladas ou em conjunto enfrenta um dilema frequente nesta patologia: a quimiorresistência. Assim, torna-se essencial, na clínica, a necessidade de encontrar novos compostos ou alvos protéicos que sejam capazes de manter uma resposta por longos períodos de tempo de tratamento. Dessa forma, este estudo utilizou ferramentas de farmacologia de sistemas, avaliando as interações entre proteínas e pequenos compostos a partir de dados proteômicos já existentes para a espécie humana. Neste contexto, as drogas estudadas foram o 5-fluorouracil, a Capecitabina, a Oxaliplatina, o Irinotecan e o Docetaxel. Uma rede de interações entre proteínas (physical protein-protein interactions; PPPI) e proteínas-quimioterápico (physical compound-protein interaction; PCPI) foi obtida, seguida da definição de cinco sub-redes, representando os diferentes processos biológicos observados. Além disso, foram empregadas análises de centralidade para buscar os principais componentes da rede e as suas interações, denominados de gargalos (bottlenecks), os quais regulam o fluxo de informação dentro da rede. Assim, o estudo demonstrou que as drogas atualmente em uso clínico convergem para processos biológicos semelhantes, o que explicaria o desenvolvimento de quimiorresistência a médio e longo prazo. Além disso, foram identificados sete novos gargalos, que representam novos alvos de proteínas e pequenos compostos capazes de interferir no controle e na comunicação entre outros vértices na própria rede. Esses dados poderão ser utilizados no desenvolvimento de novas combinações de drogas com o objetivo de melhorar os protocolos utilizados no tratamento quimioterápico do câncer gástrico. / Gastric cancer is among the cancers with the highest mortality in the world, and treatment modalities involve surgery, chemotherapy and radiotherapy. In chemotherapy, the drugs alone or in combination face a dilemma common in this disease: the chemoresistance. Thus, it becomes essential in the clinic, the need to find new protein target or compounds that are capable of maintaining a response for long periods of treatment. Thus, this studied used tools of systems pharmacology, evaluating the interactions between proteins and small compounds from existing proteomic data for the human species. In this context, the study drugs were 5-fluorouracil, capecitabine, oxaliplatin, irinotecan and docetaxel. A network of protein interactions and protein-chemotherapy (PPPI-PCPI, respectively) was obtained, followed by the definition of five sub-networks, representing different biological processes observed. Moreover, centrality analysis were used to search the main network components and their interactions, called bottlenecks , which regulate the flow of information within the network. Thus, the study showed that the drugs currently in clinical use converge to similar biological processes, which would explain the development of chemoresistance in the medium and long term. Furthermore, we identified seven new bottlenecks that represent new target proteins and small compounds able to interfere in the control and communication between other nodes in the network itself. These data could be used to develop new combinations of drugs with the aim of improving the protocols used in chemotherapy of gastric cancer.

Made available in DSpace on 2015-07-22T20:50:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Em casos de carcinoma gástrico, para contribuir ao conhecimento do processo de carcinogênese: Objetivo: Estudar a expressão das proteínas da família Bcl-2 (BcI-2, Bcl-xl, Bak, Bad, Bax). Correlacionar a expressão destas proteínas com 0 índice apoptótico mediante a expressão da proteína Caspase 3 clivada, com 0 índice mit6tico mediante a expressão da proteína Ki-67 e com a expressão da proteína p53. Método: Técnica de arranjo em matriz de amostras teciduais (TMA): em 87 amostras de adenocarcinomas gástricos (grupo teste) e de mucosa gástrica não tumoral (grupo controle) foi avaliada a imuno-expressão das proteínas da família BcI-2 (BeI-2, Bcl-xl, Bak, Bad, Bax), da proteína p53, da proteína caspase 3 e da proteína Ki-67. Resultados: Todas as proteínas examinadas foram observadas nos adenocarcinomas e mucosa não tumoral, porem com diferenças de expressão em relação à porcentagem de positividade e intensidade. Observamos: i) Houve associação entre 0 tamanho do tumor e a proteína p53. ii) Houve associação da proteína Bad no adenocarcinoma com a idade dos pacientes. iii) Associação das proteínas Bax, Bad e Ki-67 com 0 adenocarcinoma de tipo intestinal. iv) As proteínas Bcl-xl, Bak, Bad, p53 e Ki-67 apresentaram diferenças estatisticamente significantes entre a imuno-expressão no tumor e na mucosa não tumoral. v) Associação das proteínas Bax, Bak e Bad na mucosa não tumoral. vi) Não houve correlação da imunoexpressão das proteínas com a sobrevida dos pacientes. Conclusão: A expressão aumentada da proteína Bcl-xl nos adenocarcinomas, com evidente diferença de expressão entre 0 grupo teste e 0 grupo controle, esta relacionada com 0 efeito anti-apoptótico da proteína. A expressão reduzida das proteínas Bak e Bad e a expressão aumentada das proteínas p53 e Ki-67 nos adenocarcinomas demonstram 0 desequilíbrio entre morte e proliferação celular, permitindo 0 crescimento descontrolado das células neoplásicas. / Purpose: To study the immunoexpression of Bcl-2 family proteins (Bcl-2, Bcl-xl, Bax, Bak, Bad) and to evaluate the correlation between the immunoexpression of these proteins with the cleaved caspases 3, Ki-67 and p53 immuno-expression. Methods: A TMA paraffin block was constructed with gastric carcinoma tissue (test group) and normal gastric adjoining mucosa (control group) of 87 patients. The TMA block was submitted to immunohistochemistry for Bcl-2, Bcl-xl, Bax, Bak, Bad, p53 and-cleaved Caspase 3. Results: All studied proteins were present in tumor and normal gastric adjoining mucosa, but with different intensity and amount of positive cells. i) There was an association between tumor size and p53 expression. ii) association between Bad expression in the tumor and patient’s age. iii) Intestinal type adenocarcinoma was positively correlated with the expression of Bax, Bad and Ki-67. iv) The protein Bcl-xl, Bak, Bad, p53 and Ki-67 showed statistically significant differences between the immuno-expression in tumor and normal gastric adjoining mucosa. v) There was an association between the proteins Bax, Bak and Bad expression in the normal gastric adjoining mucosa. vi) No correlation between patient’s survival rates and the expression of the proteins was observed. Conclusions: The higher expression of Bcl-xl protein in adenocarcinoma, the difference of Bcl-xl expression between test group and control group, might be related with the anti-apoptotic effect of this protein. The lower expression of Bak and Bad and the increased expression of p53 protein and Ki-67 protein in adenocarcinomas demonstrate the imbalance between death and cellular proliferation, which allows the uncontrolled tumor cell proliferation. / FAPESP: 04/09932-4 / FAPESP: 06/54187-0 / TEDE