Emerging evidence suggests the immune system has a role in preventing cancer, and in advanced cancer evidence of immune dysfunction is widespread. This project focused on cytotoxic T lymphocyte antigen 4 (CTLA4), a key negative regulator of T cell activation found on dedicated regulatory T cells (Treg) and activated T lymphocytes, and asked whether modulation of immune control with anti-CTLA4 blockade led to significant anti-tumour activity. Clinical and laboratory investigation of anti-CTLA4 blockade using tremelimumab in a phase II trial of second-line therapy in advanced oesophageal and gastric adenocarcinomas was combined with an attempt to establish a suitable pre-clinical model based on therapeutic vaccination against the tumour associated antigen (TAA) 5T4.Eighteen patients received tremelimumab. Most drug-related toxicity was mild but there was a single death due to bowel perforation. Four patients had stable disease with clinical benefit; one achieved a partial response after eight cycles (25.4 months) and remains well on study after four years. Markers of regulatory phenotype, forkhead box protein 3 (FoxP3) and CTLA4, doubled transiently in CD4+CD25high lymphocytes in the first month after tremelimumab before returning to baseline. In contrast, CTLA4 increased in CD4+CD25low/negative lymphocytes throughout the cycle of treatment. Post-treatment expanded Treg expressed FoxP3 without interleukin-2 and their defining suppressive function was not abolished despite prolonged anti-CTLA4 blockade. De novo proliferative responses to TAA 5T4 (8 of 18 patients) and carcinoembryonic antigen (CEA; 5 of 15) were detected. Patients with a post-treatment CEA proliferative response had median survival of 17.1 months compared to 4.6 months for non-responders (p=0.002). Baseline interleukin-2 release after T lymphocyte activation was higher in patients with clinical benefit and toxicity. Heterologous mouse 5T4 (m5T4) vaccination showed some evidence of weak therapeutic benefit, but all tumour models investigated had rapidly lethal kinetics. Specific m5T4 immune responses could be detected by serum antibody ELISA and IFN-gamma ELISPOT assays in naive animals but were lower frequency than published responses to h5T4, and were further attenuated in tumour-bearing animals. The addition of anti-CTLA4 blockade did not result in significant augmentation of m5T4 specific immunity after vaccination in non tumour-bearing animals and combination treatment was ineffective as therapy in this autologous model. Results are discussed in the context of emerging immunotherapeutics in melanoma and prostate cancer. In the absence of supportive data from the model system it would not be appropriate to pursue combination heterologous 5T4 vaccine with anti-CTLA4 blockade, but in view of the unusual durability of the best response to tremelimumab, and in vitro evidence of enhanced proliferative responses to relevant TAA, further investigation of drug activity may be warranted in metastatic gastric and oesophageal second-line treatment.

Introdução: Apesar de não ser puramente disabsortiva, o Bypass Gástrico em Y de Roux pode provocar alteração da absorção de muitas vitaminas e minerais. Considerando ainda o fato de existirem poucos estudos que relatem o estado geral destes indivíduos, sobretudo com relação aos parâmetros nutricionais em longo prazo, observou-se à necessidade do desenvolvimento de um estudo que verifique a prevalência das carências nutricionais e a efetividade da técnica empregada, bem como o estado geral destes pacientes e suas inter-relações com outros fatores, para especialmente direcionar com maior efetividade as condutas a serem empregadas no pós-operatório pela equipe multidisciplinar. Métodos: 8 homens e 67 mulheres de uma amostra inicial de 130 pacientes compareceram a entrevista. Estes pacientes foram operados pela técnica de Bypass Gástrico em Y de Roux, possuíam entre 18-65 anos e tinham mais que 5 anos de pós-operatório. Foram coletados os seguintes dados: IMC pré e pós-operatório, perda do excesso de peso, queixa de sintomas gastrointestinais, além de dados referente a deficiências nutricionais através da análise de sangue pelos métodos padrões. Resultados: O IMC inicial foi de 56,5 +/- 10 Kg/m2. Após 2 anos, o IMC médio havia caído para 29,4 +/-6 e após 87 meses após a cirurgia, este era de 34,3 +/-10 Kg/m². Uma associação inversa entre perda do excesso de peso (PEP) e tempo de pósoperatório foi observada (P= 0,27; p=0,0183). Após 2 anos apenas 1,33 % (n=1) não atingiu a PEP esperada de 50% do excesso de peso e no momento da entrevista, 30,6% (n=23) não haviam conseguido mantê-la. As deficiências mais comumente verificadas foram as deficiências de vitamina B12 (61,82%) e D (60,53%). Baixos níveis de hemoglobina também foram verificados (50,82%). Vômitos e Síndrome de Dumping foram às queixas gastrointestinais mais observadas 66,19% e 56,76%. Verificou-se correlações significantes entre baixos níveis de hemoglobina e o sexo feminino (p=0,011), % de PEP e ocorrência de vômito com deficiência de vitamina B12 (p=0,028) e (p=0,022). Conclusão: O BGYR é eficiente na promoção e manutenção de perda de peso em longo prazo. Especial atenção deve ser dada aos grupos de maior risco para desenvolvimento de deficiências nutricionais, mulheres em idade fértil, perda de peso excessiva e naqueles que apresentam vômitos freqüentes. / Background: The goal of this study is determining both the efficacy of the surgery and the prevalence of nutritional deficiencies in the long term after the Roux-en Y Gastric Bypass and search for relations of theses deficiencies with other factors. Methods: 8 men and 67 women consecutives patients, who had 5 years or more after the surgery were assessed during regular visits. Pre and Post-operative BMI, excess weight loss and gastrointestinal symptoms were registered. Nutritional deficiencies were accessed by standard laboratory assays. Result: The initial BMI was 56,5 +/- 10 Kg/m2. After 2 years, the mean BMI had dropped to 29,4 +/-6 and by and average of 87 months it was 34,3 +/-10 Kg/m². A inverse association beetwen Excess Weight Loss (EWL) and time of postoperative was verified (P= 0,27; p=0,0183). After 2 years only 1,33 % (n=1) had not achieve a EWL of at least 50%. At the end, 30,6% (n=23) could not maintain this EWL. The more commom nutricional deficiencies are vitamin B12 (61,82%) and D (60,53%). Low levels of hemoglobin (50,82%) was also verified. Vomiting and dumping syndrome was reffered in 66,19% and 56,76%. Significant correlation and with clinical signify was observed between low hemoglobin levels and femine sex (p=0,011), % of weight loss and the B12 deficiency (p=0,028) and vomiting and deficiency of B12 (p=0,022). Conclusion: The RYGB is efficient to promotes and maintain the weight. Special attention should be given to patients with massive weight loss, frequent vomiting and women in reprodutive age.

O Helicobacter pylori, é tido como o principal fator de risco para o carcinoma gástrico. Diferentes estudos experimentais em animais procuram relacionar essa carcinogênese a outros fatores carcinógenos sem sucesso. Neste estudo, procurou-se avaliar-se em ratos, se há correlação entre o refluxo duodenogástrico, o Helicobacter pylori e o desenvolvimento do câncer gástrico ou de seus precursores. Para tal, realizou-se nos três grupos de ratos (n de dez por grupo) as técnicas de: piloroplastia precedida de infecção, gastrectomia subtotal precedida de infecção e um grupo no qual foi praticada apenas a infecção. Apois seis meses, analisou-se as alterações da mucosa, comparando-se os três grupos. As alterações da mucosa pesquisadas foram as seguintes: gastrites, metaplasias, displasias e neoplasias epiteliais. Ao término do estudo, foi encontrado, no grupo submetido a piloroplastia precedida de infecção um alto percentual de alterações epiteliais. Conclui-se que, no rato, a operação de piloroplastia, levou ao maior desenvolvimento da população do Helicobacter pylori, que se relaciona com as lesões pré- malignas e o adenocarcinoma gástrico. / Helicobacter pylori is considered the main risk factor for gastric carcinoma. Different experimental studies in animals seek to relate this carcinogenesis to other carcinogenic factors without success. This study sought to evaluate in rats, if there was a correlation between duodenogastric reflux, Helicobacter pylori and the development of gastric cancer or its precursors. To this end, it was carried out in three groups of rats the techniques: pyloroplasty, subtotal gastrectomy and only infection. After six months, the changes in mucosa were analyzed comparing the three groups. The mucosal changes reseached were: gastritis, metaplasia, dysplasia and epithelial neoplasms. At the end of the study, was found in the group that underwent pyloroplasty a high percentage of epithelial alterations and these correlated with the population of Helicobacter pylori. It is concluded that in the rat, the operation of pyloroplasty led to increased colonization of the population of Helicobacter pylori and is related with the development of benign lesions and gastric cancer.

Made available in DSpace on 2014-06-11T19:30:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2007Bitstream added on 2014-06-13T20:01:07Z : No. of bitstreams: 1 ferrasi_ac_dr_rcla.pdf: 1030993 bytes, checksum: 9a0fbcc4634e2697322fd343cddaa074 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Gastric cancer (GC) is one of the most common cancer types and it is associated with high mortality frequencies. Although a decrease in the worldwide incidence is observed, the prognosis of this disease still remains poor, mainly when the diagnosis is carried out at advanced stages. Recent evidences have identified DNA methylation as an important mechanism for tumor suppressor gene inactivation. Helicobacter pylori infection is considered one of the most important etiological factors and the CagA gene is associated with more severe pathologies including cancer. Likewise, EBV is another infectious agent that has been associated with gastric carcinoma in at least 10% of the cases. In this study, we determined the promoter methylation status of the CDH1, DAPK, COX2, hMLH1 and CDKN2A and MSI frequency in 89 primary gastric carcinomas and correlated the findings with the presence of H. pylori and EBV infections and also with clinicopathological features of gastric carcinomas. COX2 was the most frequently hypermethylated gene (63.5%) in these patients, followed by DAPK (55.7%), CDH1 (51%), CDKN2A (36%) and hMLH1 (30.3%). In this study, MSI was correlated with hMLH1 methylation, as shown before, and there was an inverse correlation between DAPK hypermethylation and MSI. Also, MSI was inversely correlated with H. pylori CagA+, providing new evidence for the association of MSI and better prognosis.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-03-18T11:35:43Z No. of bitstreams: 1 PDF - Cynthia Layse Ferreira de Almeida.pdf: 3612014 bytes, checksum: ff23a88527b4e07c470c1b649ddb0716 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-22T15:01:26Z (GMT) No. of bitstreams: 1 PDF - Cynthia Layse Ferreira de Almeida.pdf: 3612014 bytes, checksum: ff23a88527b4e07c470c1b649ddb0716 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-22T15:01:35Z (GMT) No. of bitstreams: 1 PDF - Cynthia Layse Ferreira de Almeida.pdf: 3612014 bytes, checksum: ff23a88527b4e07c470c1b649ddb0716 (MD5) / Made available in DSpace on 2016-07-22T15:01:35Z (GMT). No. of bitstreams: 1 PDF - Cynthia Layse Ferreira de Almeida.pdf: 3612014 bytes, checksum: ff23a88527b4e07c470c1b649ddb0716 (MD5) Previous issue date: 2013-09-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Spondias purpurea L. (Anacardiaceae), popularly known as “siriguela”, is used in folk medicine for the relief of fever and pain, as diarrhea medication, antispasmodic, diuretic, anti-anemic and analgesic. Pharmacognostic studies with the species indicate the strong presence of tannins, flavonoids and triterpenes in their leaves, and roots, which are generally related to the antiulcer activity. The aim of this study was to evaluate the gastroprotective activity of the extract phase of S. purpurea in animal models. For this, the ethanolic extract (Sp-EtOHE) and the dichloromethane fraction (Sp-DCMF) of leaves of S. purpurea were obtained. Regarding the gastroprotective activity, doses of 125, 250 and 500 mg/kg (p.o.) were tested against different models of acute induction of ulcer by acidified ethanol, ethanol, stress immobilization and cold, and nonsteroidal anti-inflammatory drug (NSAID). In the model of acidified ethanol, there was a reduction in the ulcerative lesion index (ULI) for 61, 71 and 65% Sp-EtOHE and 29, 77 and 68% to Sp-DCMF. In ulceration caused by ethanol, Sp-EtOHE and Sp-DCMF in the same doses protect the gastric mucosa and were not altered parameters of the stomach contents of rats. The evaluation by the stress model, Sp-EtOHE and Sp-DCMF decreased the ULI in 23, 59 and 70% and 24, 61 and 72%, respectively. Previous models were made for the determination of C-Reactive Protein (CRP) in blood samples and were observed significant reductions of this parameter. Similarly, in the model of NSAID-induced gastric ulcers, there was inhibition of injuries to Sp-EtOHE and Sp-DCMF. In order to investigate the mechanisms of action related to gastroprotection promoted by SpEtOHE (250 mg/kg) and Sp-DCMF (250 mg/kg) was evaluated the involvement of nitric oxide and sulfhydryl compounds. Thus, it was verified that the gastroprotective effect of S. purpurea not involve the participation of nitric oxide. However, this effect is related to the participation of sulfhydryl compounds. Thus, these data suggest that S. purpurea presents gastroprotective activity, possibly related to mechanisms cytoprotectives. / Spondias purpurea L. (Anacardiaceae), popularmente conhecida como “siriguela”, é utilizada na medicina popular para o alívio de febre e dores, como antidiarréico, antiespasmódico, diurético, analgésico e antianêmico. Estudos farmacognósticos realizados com a espécie indicam a presença marcante de taninos, flavonoides e triterpenos em suas folhas, e raízes, os quais geralmente estão relacionados com a atividade antiulcerogênica. Assim, o objetivo deste trabalho foi avaliar a atividade gastroprotetora do extrato e fase de S. purpurea em modelos animais. Para isso foram obtidos o extrato etanólico (EEtOH-Sp) e a fase diclorometano (FaDCM-Sp) das folhas de S. purpurea. Com relação à atividade gastroprotetora, as doses de 125, 250 e 500 mg/kg (v.o.) do EEtOH e FaDCM foram testados frente a modelos de indução aguda de úlcera por etanol acidificado, etanol, estresse por imobilização e frio e anti-inflamatório não-esteroidal (AINE). No modelo de etanol acidificado, houve a redução do índice de lesão ulcerativo (ILU) em 61, 71 e 65% para EEtOH e 29, 77 e 68% para FaDCM. Nas ulcerações causadas por etanol, EEtOH-Sp e FaDCM-Sp nas mesmas doses protegeram a mucosa gástrica, bem como não foram alterados parâmetros do conteúdo estomacal dos ratos. Na avaliação pelo modelo do estresse, EEtOH-Sp e FaDCM-Sp diminuíram o ILU em 23, 59, 70 e 24, 61, 72%, respectivamente. Foram realizados para os modelos anteriores a dosagem da Proteína C reativa ultrassensível (PCR) nas amostras de sangue e foram observadas reduções significativas deste parâmetro. Da mesma maneira, no modelo de úlceras gástricas induzidas por AINE, houve inibição das lesões para EEtOH-Sp e FaDCM-Sp. No intuito de investigar os mecanismos de ação relacionados à gastroproteção promovida pelo EEtOH-Sp (250 mg/kg) e FaDCM-Sp (250 mg/kg), foi avaliado o envolvimento do óxido nítrico e dos grupamentos sulfidrílicos. Assim, foi observado que o efeito gastroprotetor de S. purpurea não envolve a participação do óxido nítrico. Entretanto, este efeito está relacionado à participação dos grupamentos sulfidrílicos, sugerindo que S. purpurea apresenta atividade gastroprotetora, possivelmente relacionada a mecanismos citoprotetores.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O cÃncer gÃstrico apresenta, mundialmente, uma elevada taxa de mortalidade, com alta incidÃncia no Brasil, sendo a infecÃÃo com Helicobacter pylori um fator de risco bem estabelecido. Dependendo da presenÃa de genes de virulÃncia como cagA, cagE, vacA e virB11, H. pylori pode causar respostas inflamatÃrias diferenciadas, apresentando grande quantidade de Ãxido nÃtrico (ON) gerado principalmente por iNOS. Quantidade elevada de ON resulta em acÃmulo de espÃcies reativas do oxigÃnio cuja instabilidade causa danos oxidativos no DNA. A integridade genÃmica à garantida por enzimas de reparo importantes como: APE-1, OGG-1, e PARP-1. Polimorfismos genÃticos que modifiquem a atividade dessas enzimas podem influenciar a capacidade de reparo e, portanto, a susceptibilidade do hospedeiro ao desenvolvimento do cÃncer gÃstrico associado à H. pylori. Assim o objetivo deste estudo foi avaliar a associaÃÃo dos polimorfismos C150T em iNOS, T2197G em APE-1, C1245G em OGG-1 e A40676G em PARP-1 com o genÃtipo de H. pylori em 109 amostra de pacientes diagnosticados com adenocarcinomas gÃstricos atendidos em hospitais de Fortaleza, CearÃ. A identificaÃÃo dos polimorfismos foi feita por PCR-RFLP e a detecÃÃo e genotipagem de H. pylori foram feitas por PCR. Os polimorfismos estudados apresentaram as seguintes freqÃÃncias: iNOS - 78% CC, 21,1% CT e 0,9% TT; PARP-1- 69,7% AA, 26,6% AG e 3,7% GG, para OGG-1 56% CC, 39,4% CG, e 4,6% GG e para APE-1 38,5%TT, 47,7%TG e 13,8% GG. Salienta-se a baixa freqÃÃncia do genÃtipo polimÃrfico (TT) de iNOS e alta frequÃncia do heterozigoto (TG) de APE. Os alelos variantes de iNOS e de PARP-1 foram correlacionadas com indivÃduos ≤55 anos, sugerindo que estes polimorfismos estariam associados ao desenvolvimento precoce da neoplasia. Os tumores intestinais localizados na regiÃo nÃo-antro correlacionaram-se com o genÃtipo OGG-1 CG; enquanto que os difusos, localizados no corpo com o genÃtipo AA de PARP-1. H. pylori foi detectada em 92,6% dos casos. Os genes cagA, cagE e virB11 foram detectados em 65,3%, 50,4% e 60,3% dos casos, respectivamente e vacAs1m1 detectado em 72,2%. Os casos foram agrupados considerando os alelos de vacA e a integridade da ilha de patogenicidade cag, sendo os grupos A1 e A2, composto por cepas mais patogÃnicas, o qual foi observado em 33,6% e 13,8% dos pacientes, respectivamente. Na anÃlise individual de cada enzima, observou-se que os indivÃduos portadores dos alelos variantes de APE-1 (TG+GG) estavam infectados com cepas pouco patogÃnicas (p=0,0422). Essas cepas pouco patogÃnicas tambÃm foram associadas aos pacientes portadores do genÃtipo selvagem (AA) de PARP-1 (p=0,0396). Esses dados foram confirmados quando os pacientes infectados por cepas mais virulentas foram comparadas aos infectados por cepas menos virulentas (p=0,046). Analisando apenas o grupo A1 observou-se tambÃm uma correlaÃÃo de APE-1 (TG) com OGG-1(CC). Quando os genÃtipos foram combinados considerando somente as enzimas de reparo estudadas ou duas a duas, verificou-se que parte dos pacientes infectados com o genÃtipo selvagem de PARP-1 eram portadores do alelo variante para pelo menos uma das enzimas e que parte dos pacientes infectados com cepas menos patogÃnicas possuÃam o alelo polimÃrfico de APE-1, independente do genÃtipo da enzima de reparo associada. Somados, esses dados indicam a relevÃncia do polimorfismo da APE-1 no desenvolvimento do cÃncer gÃstrico em indivÃduos infectados com cepas menos virulentas e corroboram com a importÃncia do genÃtipo bacteriano, uma vez que, de maneira geral, indivÃduos com genÃtipo selvagem para as enzimas de reparo estudadas desenvolveram cÃncer gÃstrico quando infectados por cepas virulentas. / Gastric cancer is the most deadly malignant neoplasia worldwide, with high incidence in Brazil and Helicobacter pylori infection is a well-established risk factor. Depending on the presence of virulence genes such as cagA, cagE, vacA and virB11, H. pylori can cause differentiated inflammatory responses, with large amounts of nitric oxide (NO) generated primarily by iNOS. High amount of NO resulting in accumulation of reactive oxygen species can cause DNA oxidative damage. The genomic integrity is guaranteed by important repair enzymes as: APE-1, OGG-1 and PARP-1. Genetic polymorphisms that modify the activity of these enzymes may influence the ability to repair and therefore the host susceptibility to the development of gastric cancer H.pylori associated. Therefore, the goal of this study was to evaluate the association of the C150T polymorphism in iNOS, T2197G in APE-1, C1245G in OGG -1 and A40676G in PARP-1 with H.pylori genotype in 109 cases of patients with gastric adenocarcinoma from hospitals in Fortaleza, CearÃ. The identification of polymorphisms was performed by PCR-RFLP and the detection and genotyping of H.pylori were performed by PCR. The studied polymorphisms showed the following frequencies: iNOS 78% CC, 21.1% CT and 0.9% TT; PARP-1 69.7% AA 26.6% AG and 3.7% GG to OGG -1 56% CC, 39.4% CG and 4.6% GG and APE-1 38.5% TT, 47.7% TG and 13.8% GG. Valuable to note the low frequency of the homozygous polymorphic (TT) of iNOS and the high frequency of heterozygous (TG) from APE-1. The variant alleles of iNOS and PARP-1 were correlated with subjects ≤ 55 years, suggesting that these polymorphisms were associated with early development of the neoplasia. Intestinal tumors located in the non-antrum were correlated with heterozygous genotype of OGG-1 (CG), while diffuse, located on the body with the AA genotype of PARP-1. H. pylori was detected in 92.6% of cases. The genes cagA, cagE and virB11 were detected in 65.3%, 50.4% and 60.3% of cases respectively and vacAs1m1 was detected in 72.2%. The cases were also grouped considering the alleles of vacA and the integrity of the cag-pathogenicity island. Thus, the groups A1 and A2, consist of more pathogenic strains, were observed in 33.6% and 13.8% of patients, respectively. In the individual analysis of each enzyme, we observed that individuals carrying the variant alleles of APE-1 (TG+GG) were infected with low pathogenic strains (p=0.0422). These low pathogenic strains were also associated with patients carrying the wild genotype (AA) of PARP-1 (p=0.0396). These data were confirmed when patients infected with more virulent strains were compared to those infected with less virulent strains (p = 0.046). Analyzing only the group A1, it was also observed a correlation of APE-1 (TG) with OGG-1 (CC). When genotypes were combined by considering only the repair enzymes studied or two by two, it was found that most patients infected with the wild-type of PARP-1 were carriers of the variant allele for at least one of the enzymes and that most patients infected with less pathogenic strains possess a polymorphic allele of APE-1, independent of the genotype associated with the repair enzyme. Taken together, these data indicate the relevance of the APE-1 polymorphism in the development of gastric cancer in individuals infected with less virulent strains and corroborate the importance of the bacterial genotype, since; in general, individuals with wild-type for enzymes repair studied developed gastric cancer when infected with virulent strains.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / INTRODUÃÃO: O papel do LPS na defesa da mucosa gÃstrica ainda nÃo està estabelecido. OBJETIVOS: 1-Verificar o efeito protetor do LPS na lesÃo gÃstrica (LG), na infiltraÃÃo de neutrÃfilos (IN), no aumento da adesÃo leucocitÃria, na diminuiÃÃo dos nÃveis de GSH induzidos por indometacina (INDO) em ratos; 2-Investigar o papel da COX-2, NOSi e dos canais de K sensÃveis ao ATP (KATP) na gastroproteÃÃo do LPS na gastropatia por INDO. MÃTODOS: Os ratos foram tratados com LPS da E. coli (30, 100 ou 300 g/Kg, e.v.). ApÃs 6 hs, foi administrado INDO (20mg/Kg, p.o.). Decorridas 3 hs, o sangue foi colhido para determinaÃÃo do leucograma. Posteriormente, os ratos foram sacrificados e a LG foi aferida. Fragmentos do estÃmago foram retirados para avaliaÃÃo da atividade de mieloperoxidase (MPO) e determinaÃÃo dos nÃveis de glutationa (GSH). A adesÃo e o rolling dos leucÃcitos foram avaliados por microscopia intravital. Diferentes grupos foram tratados com rofecoxib, L-NAME, aminoguanidina, dexametasona, glibenclamida, diazÃxido ou glibenclamida + diazÃxido. ApÃs 3 horas da administraÃÃo de INDO (20mg/Kg, p.o.), foram avaliadas a LG, a MPO e GSH. RESULTADOS: LPS reduziu a LG e o aumentou a MPO induzidas por INDO de forma dose-dependente, com o efeito mÃximo na dose de 300 g/Kg e no tempo de 6 hs. O prÃ-tratamento com LPS induziu uma neutrofilia na gastropatia induzida pela INDO. LPS reverteu à queda dos nÃveis de GSH no estÃmago com INDO. O tratamento com LPS diminui a adesÃo e aumentou o rolling dos leucÃcitos quando comparado com o tratado com INDO. Rofecoxib, L-NAME, aminoguanidina ou dexametasona nÃo reverteram o efeito protetor do LPS. Glibenclamida, mas nÃo diazÃxido, reverteu o efeito protetor do LPS na gastropatia induzida por INDO, aumentando de forma significativa a LG, MPO e diminuindo a GSH. A associaÃÃo de glibenclamida com diazÃxido nÃo reverteu o efeito protetor do LPS. CONCLUSÃES: LPS protege contra a LG por INDO, atravÃs da inibiÃÃo da IN por uma diminuiÃÃo da adesÃo de leucÃcitos ao endotÃlio e por um aumento dos nÃveis de GSH no estÃmago. Este evento dependente da abertura de KATP. Nossos dados tambÃm sugerem que a atividade de COX-2 e NOSi nÃo estÃo envolvidos no efeito protetor do LPS. / INTRODUCTION: The role of the LPS in the defense of the gastric mucosa is still not established. AIMS: To verify the protective effect of the LPS in the gastric damage (GD), in the neutrophil infiltration (NI), in the increase of the leukocyte of adhesion, in the reduction of the induced glutathione levels for indomethacin (INDO) in rats and to investigate the role of the COX-2, NOSi and of ATP-sensitive k channels (KATP) in the protective effect of LPS administration on INDO- induced gastropathy. METHODS: The rats were treated with LPS of E. coli (30, 100 or 300 mg/Kg, e.v.). After 6 hs, INDO was administrated (20mg/Kg, p.o.). 3 hs later, the blood was harvested for determination the total and differential number of white blood cell counts. Later, the rats had been sacrificed and the GD was surveyed. Piece of the stomach had been removed for evaluation of the MPOactivity and determination of the GSH levels. The adhesion and rolling of the leukocytes had been evaluated by intravital microscopy. Different groups were treated with rofecoxib, L-NAME, aminoguanidine, dexamethasone, glibenclamide, diazoxide or glibenclamide + diazoxide. After 3 hs of the administration of INDO (20mg/Kg, p.o.), had been evaluated the GD, MPO and GSH. RESULTS: LPS reduced dose- dependently INDO- induced GD and increase in MPO, with the maximal effect at the dose of 300 g/kg and in the time of 6 hs. The LPS treatment neutrophilia induced in INDO induced gastropathy. LPS reverted to the fall of the GSH levels in the stomach with INDO. The LPS treatment decreased the adhesion and increased rolling of the leukocytes when compared with the INDO treated. Rofecoxib, L-NAME, aminoguanidine or dexamethasona had not reverted the protective effect of the LPS. Glibenclamide, but not diazoxide, reverted the protective effect of the LPS in the induced gastropathy for INDO, increasing of significant form the GD, MPO and decreasing the GSH. The diazoxide + glibenclamide association of with did not revert the protective effect of the LPS. CONCLUSIONS: LPS protects against INDO induced GD, through the inhibition of the NI for a reduction of the adhesion of leukocytes to the endothelin and for an increase of the GSH levels in the stomach. This dependent event of the KATP opening. Our data also suggest that the activity of COX-2 and NOSi are not involved in the protective effect of the LPS.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / A hemorragia subaracnÃidea (HSA) pode advir de diversos agravos ao sistema nervoso central. A HSA pode aumentar a pressÃo intracraniana (PIC) a patamares de grande morbidade. AlÃm de sintomas neurolÃgicos e cardiovasculares, bem descritos na literatura; sintomas gastrintestinais como nÃuseas, vÃmitos e gastrenterites sÃo comuns, mas pouco estudados. O retardo do esvaziamento gÃstrico (EG) à uma das alteraÃÃes fisiolÃgicas encontrada em pacientes com hipertensÃo intracraniana (HIC). Tal distÃrbio reduz as respostas terapÃuticas tendo em vista a reduÃÃo na absorÃÃo dos nutrientes e fÃrmacos, alÃm de levar à aspiraÃÃo pulmonar, que pode levar a Ãbito. Avaliamos o efeito da HSA sobre as alteraÃÃes da motilidade gastrintestinal (Esvaziamento GÃstrico - EG; TrÃnsito Intestinal â TI e ComplacÃncia GÃstrica â CG), dos parÃmetros hemodinÃmicos (PressÃo Arterial MÃdia â PAM, FreqÃÃncia CardÃaca - FC) e sobre a PIC. Bem como os mecanismos neuro-humorais relacionados a essas alteraÃÃes. Utilizamos ratos Wistar (300-350g, N=113), sob auspÃcios do COBEA (CEUA/UFC- Protocolo 41/13). ApÃs anestesia (Ketamina/Xilasina 20-10mg/Kg-IP), os animais foram contidos por estereotÃxico sendo injetados 0,1; 0,2 ou 0,3ml na cisterna magna (sangue autÃlogo â grupo HSA ou Liquor sÃmile â grupo Sham). No momento da induÃÃo da HSA os animais foram submetidos à canulaÃÃo dos ventrÃculos laterais para monitoraÃÃo da PIC e dos vasos femorais Direito a fim de se obter dados hemodinÃmicos. ApÃs 72h da induÃÃo, e sob jejum (24h) com soluÃÃo de reidrataÃÃo oral ad libitum, procedemos aos estudos de EG, TI e CG. Para determinaÃÃo da taxa de EG, uma refeiÃÃo teste (1ml/100g) (vermelho-fenol 0,5mg/ml em glicose-5%) foi administrada via gavagem. Jà o TI foi determinado com administraÃÃo direta da refeiÃÃo no duodeno por meio de cÃnula previamente implantada. Jà a CG foi avaliada utilizando um sistema de barostato a 4, 8 e 12 cm de pressÃo de distensÃo gÃstrica. A PA, FC e PIC foram aferidas no momento da avaliaÃÃo da motilidade gastrintestinal. Os dados, mÃdiaÂEPM, foram analisados pelo teste âtâ de Student (P<0,05). A HSA retardou o EG (38,90Â2,73 vs 47,00Â0,72%; 52,85Â5,14 vs 31,12Â2,0% ou 22,89Â4,46 vs 46,24Â3,56%) e aumentou a PIC (3,33Â0,47 vs 16,10Â0,47cmH2O; 7,68Â0,650 vs 30,86Â0,82cmH2O ou 17,50Â1,29 vs 37,90Â1,38cmH2O) nos diferentes volumes de sangue, seja 0,1; 0,2 ou 0,3ml respectivamente, quando comparados ao controle. A HSA tambÃm promoveu retarde no TI e diminuiÃÃo da CG, aumento da PA e diminuiÃÃo da FC. A vagotomia subdiafragmÃtica, a esplancnotomia e o prÃ-tratamento com guanetidina reverteram o efeito da HSA sobre as alteraÃÃes do EG. Os resultados sugerem que os agravos gastrintestinais advindos da HSA refletem um padrÃo de dismotilidade secundÃrios a HIC, mostrando uma forte correlaÃÃo com os valores de PIC / Subarachnoid hemorrhage (SAH) can arise from various types of damage to the central nervous system. The HSA can increase intracranial pressure (ICP) to levels high morbidity. In addition to neurological and cardiovascular symptoms, well described in the literature; gastrointestinal symptoms such as nausea, vomiting and gastroenteritis are common but little studied. The delay gastric emptying (GE) is one of the physiological changes found in patients with intracranial hypertension (ICH). This disorder reduces therapeutic responses with a view to reducing the absorption of nutrients and drugs, and lead to lung aspiration, which can lead to death. We evaluated the effect of HSA on gastrointestinal motility disorders (Gastric Emptying - EG; Intestinal Transit â IT or Gastric Compliance - CG), hemodynamic parameters (Mean Arterial Pressure - MAP, Heart Rate - HR) and the PIC. As well as neurohumoral mechanisms related to these changes. We used Wistar rats (300 - 350g, N=113) under the auspices COBEA (CEUA/UFC- Protocol 41/13). After anesthesia (Ketamine/Xylazine 20-10mg/Kg-IP), the animals were restrained by stereotactic aparatus being injected 0.1, 0.2 or 0.3 ml in the cisterna magna (autologous blood - HSA group or Liquor simile - Control group). At the time of induction of HSA, the animals were subjected to cannulation of the lateral ventricles for measurement of ICP and rigth femoral vessels in order to obtain the hemodynamic data. After 72h of induction, and fasted (24h) animals with oral rehydration solution ad libitum, proceeded studies EG, IT and CG. To determine the rate of GE a test meal (1ml/100g - phenol red - 0.5 mg/ml in 5 % glucose) was administered by gavage. The IT was already determined on direct administration of the meal into the duodenum through previously implanted cannula. Since the CG was evaluated using a barostat system at 4, 8 and 12 cm of gastric pressure distension. The MAP, HR and ICP were measured when assessing gastrointestinal motility. Data, mean  SEM, were analyzed by the "t" Student test (p<0.05). The HSA delayed gastric emptying (38.90  2.73 vs 47.00  0.72 %; 52.85  5.14 vs 31.12  2.0 % and 22.89  4.46 vs 46,24  3.56 % ) and increased the ICP (3.33  0.47 vs 16.10  0.47 cmH2O; 7.68  0.650 vs 30.86Â0.82 cmH2O and 17.50  1.29 vs 37.90  1.38 cmH2O ) in different volumes of blood, either 0.1, 0.2 or 0.3 ml, respectively, when compared to control. The HSA also promoted delay in IT and GC, increased BP and decreased HR. The subdiaphragmatic vagotomy, the esplancnotomia and pretreatment with guanethidine reversed the effect of HSA on changes in EG. The results suggest that the SAH arising gastrointestinal diseases reflect a pattern of secondary gut dysmotility, showing a strong correlation with the values of ICP

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O cÃncer gÃstrico à a quarta neoplasia maligna mais freqÃente e a segunda causa de morte por cÃncer no mundo, registrando-se aproximadamente 934 mil novos casos e 700 mil mortes por ano. No Brasil, o cÃncer gÃstrico à o sexto tumor maligno mais freqÃente. Na regiÃo Nordeste à a segunda neoplasia mais freqÃente entre os homens e a quarta entre as mulheres, sendo que no estado do Cearà à a terceira neoplasia mais freqÃente. Dentre os agentes infecciosos, a bactÃria Helicobacter pylori (H. pylori), considerada pela OMS agente carcinogÃnico do grupo I, tem sido destacada, nas Ãltimas dÃcadas, visto sua relaÃÃo com a gastrite crÃnica ativa, com o desenvolvimento de Ãlceras gÃstricas e duodenais e aumento do risco de cÃncer gÃstrico. AlÃm da presenÃa, a variaÃÃo genÃtica das cepas de H. pylori parece influenciar na gravidade das doenÃas causadas pela infecÃÃo. O carÃter patogÃnico à dado pela presenÃa, em algumas cepas, da denominada ilha de patogenicidade, cag-PAI; um fragmento de DNA de 35-40 Kb cepa especÃfico, a qual possui uma sÃrie de genes associados a um virulento aparato secretÃrio. AlÃm disso, outro gene, denominado vacA, tambÃm apresenta-se como um importante fator de virulÃncia. Apesar dessas associaÃÃes nÃo hà ainda uma relaÃÃo direta entre a presenÃa desses genes com o processo tumorigÃnico. Uma das possÃveis explicaÃÃes à a presenÃa de outros genes que contribuam para o fenÃtipo mais grave ou maligno, principalmente associada a cag-PAI. Nesse contexto, o presente estudo objetivou investigar a freqÃÃncia de H. pylori genotipando quanto as variantes alÃlicas de vacA, a presenÃa dos genes cagA, cagE e virB11 e sua associaÃÃo com os dados clinico- patolÃgicos de adenocarcinoma gÃstricos de uma populaÃÃo do Estado do CearÃ. Para tanto, 101 casos de adenocarcinoma gÃstricos (68 homens e 33 mulheres), obtidos de dois hospitais de Fortaleza, foram analisados por PCR quanto à presenÃa de H. pylori e os genes estudados. A distribuiÃÃo do cÃncer gÃstrico por sexo, sÃtio anatÃmico do tumor e anÃlise histopatolÃgicas, de modo geral, reproduziram as tendÃncias da literatura mundial. A bactÃria esteve presente em 93% dos casos analisados. Os genes de H. pylori apresentaram as seguintes freqÃÃncias: vacAs1m1 (75,5%), vacAs1m2 (13,8%), vacAs2m1 (4,6%), vacA s2m2 (6,5%), cagA (64,9%), cagE (53,2%) e virB11 (60,6%). Esse dados sÃo os primeiros na literatura mundial citando a freqÃÃncia virB11 e o segundo para o gene cagE em cÃncer gÃstrico e indicam uma variaÃÃo de cepas circulantes quanto a presenÃa desses genes quando comparado esses dados com os de outras doenÃas gÃstricas. A combinaÃÃo mais freqÃente foi vacAs1m1cagA(+)cagE(+)virB11(+), encontrada em 36,2% dos casos analisados, sendo considerada a cepa mais patogÃnica. A integridade de cag-PAI foi verificada em 38,3% dos casos, quando considerados os trÃs marcadores estudados, entretanto, considerando-se pelo menos um marcador de lado direito (cagA e/ou cagE) e o marcador do lado esquerdo (virB11), a freqÃÃncia foi de 56,4%. A distribuiÃÃo dos genÃtipos de H. pylori em grupos demonstrou que a maior freqÃÃncia das cepas consideradas mais patogÃnicas foi em tumores do antro gÃstrico, nÃo houve predileÃÃo das variaÃÃes genotÃpicas por nenhum dos tipos histolÃgicos, alÃm de verificar-se a alta freqÃÃncia das cepas mais patogÃnicas nos estadiamentos II e IV, demonstrando a participaÃÃo de H. pylori na carcinogÃnese gÃstrica. / The gastric cancer is the fourth more frequent cancer, and the second cause of death for cancer in the world, recording approximately 934 thousand of new cases and 700 thousand of deaths a year. In Brazil, the gastric cancer is the sixth more frequent malignant tumor. In the Northeast area, it is the second more frequent cancer among the men and fourth among the women. In the state of Ceara, it is the third more frequent neoplasia. Among the infectious agents, the bacterium Helicobacter pylori (H. pylori), which is considered carcinogenic agent of the group I, has been pointed in the last decades because of the connection with activated chronic gastritis, with the development of peptic ulcers and duodenais, and the increase of the risk of gastric cancer. Besides the presence, genetic variation of the strains of H. pylori seems to influence in the seriousness of the disease caused by infection. The pathogenic character is given by the presence, in some cepas, of the called cag pathogenicity island, cag-PAI; one gene fragment of 35-40 Kb strain specific, which possess a series of genes associates to a virulent type IV secretion apparatus. Moreover, another gene, called vacA, it is presented as an important virulence factor. Despite these associations does not have still a direct relation enters the presence of these genes with the tumorigenic process. One of the possible explanations is the presence of other genes that contribute for fenotype more serious or malignant, mainly associates cag-PAI. In this context, the present study objectified to investigate the frequency of H. pylori genotyped how much the alelics variants of vacA the presence of the genes cagA, cagE and virB11 and its association with the clinic- pathological dates of gastric adenocarcinoma of one population of the Ceara State. For in such a way, 101 cases of gastric adenocarcinoma (68 men and 33 women), gotten of two hospitals of Fortaleza, had been analyzed by PCR how much to the presence of H. pylori and the studied genes. The distribution of the gastric cancer by sex, anatomical sites and the histopatologic analysis, in general way, had reproduced the trends of world-wide literature. The bacterium was present in 93% of the analyzed cases. The genes of H. pylori had presented the following frequencies: vacAs1m1 (75,5%), vacAs1m2 (13,8%), vacAs2m1 (4,6%), vacA s2m2 (6,5%), cagA (64,9%), cagE (53,2%) and virB11 (60,6%). These data are the first ones in world-wide literature citing the frequency of virB11 and as for the gene cagE in gastric cancer and indicate a circulating variation of strains how much the presence of these genes when compared these data with the ones of other gastric diseases. The most frequent combination was vacAs1m1cagA(+)cagE(+)virB11(+), found in 36,2% of the analyzed cases, being considered strain more pathogenic. The integrity of cag-PAI was verified in 38,3% of the cases, when considered the three studied markers, however, considering at least one marker of right side (cagA and/or cagE) and the marker of the left side (virB11), the frequency was of 56,4%. The distribution of the genotypes of H. pylori in groups demonstrated that the biggest frequency of strains considered more pathogenic was in tumors of the gastric antrum, did not have predilection of the genotypic variations for none of the histologic types, besides verifying it high frequency of more pathogenic strains in tumor stage II and IV, demonstrating to the participation of H. pylori in gastric carcinogenesis.

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.