Des études d'associations pangénomiques ont démontré une relation entre le taux plasmatique de la vitamine B12 et le polymorphisme du gène FUT2 (fucosyltransferase 2). Dans des modèles expérimentaux, le statut sécréteur pour FUT2 a été impliqué dans la susceptibilité à l'infection par Helicobacter pylori (H. pylori). Nous avons évalué l'influence du polymorphisme FUT2 461 G>A sur les marqueurs du métabolisme des monocarbones dans deux populations ambulatoires en Europe et en Afrique de l'Ouest ainsi que la possible association entre l'infection par H. pylori et le polymorphisme de FUT2. Nous avons mis en évidence une influence de FUT2 461 G>A sur le taux plasmatique de la vitamine B12 mais n'avons pas retrouvé d'influence du statut sérologique pour H. pylori sur cette association, du moins chez les sujets ambulatoires en Europe et en Afrique de l'Ouest. L'hyperhomocystéinémie est un marqueur de carence en donneurs de méthyle. Plusieurs travaux ont évalué le taux plasmatique de l'homocystéine au cours des maladies inflammatoires chroniques de l'intestin (MICI) et ont abouti à des résultats mitigés. Par ailleurs, l'ampleur de l'association entre le métabolisme de l'homocystéine et les MICI reste méconnue. Nous avons réalisé une méta-analyse afin : (i) d'évaluer l'association entre le métabolisme de l'homocystéine et les MICI et (ii) d'étudier le risque de thrombose lié à l'hyperhomocystéinémie au cours des MICI. Le risque d'hyperhomocystéinémie était significativement plus élevé chez les patients avec une MICI en comparaison aux sujets contrôles. L'évaluation du risque de thrombose associé à l'hyperhomocystéinémie au cours des MICI requiert des études complémentaires. Un statut carencé en folates était associé à un impact plus fort du polymorphisme MTHFR C677T sur le risque primaire de MICI. L'hyperhomocystéinémie et plusieurs polymorphismes sur les gènes du métabolisme des monocarbones sont associés au risque primaire et à la sévérité de la maladie de Crohn (MC). L'hyperhomocystéinémie augmente l'activité de la superoxyde dismutase (SOD), un marqueur fiable et validé du stress oxydatif. A l'aide d'un SNP array Illumina exhaustif du métabolisme des monocarbones, nous avons (i) étudié les déterminants génétiques (single nucleotide polymorphisms, SNPs) associés au taux plasmatique de l'homocystéine et de la SOD chez des patients suivis pour une MC et (ii) recherché les SNPs associés à l'âge du diagnostic de la MC. Deux SNPs étaient indépendamment associés au taux plasmatique de l'homocystéine (MTHFR, AHCY). Cinq SNPs étaient indépendamment associés au taux plasmatique de la SOD. Parmi ces cinq SNPs, trois sont liés à la vitamine B12 (FUT2, CUBN, et TCN2), un aux folates (GGH), et un dernier à la synthèse cellulaire de l'homocystéine (AHCY). Par ailleurs, nous avons mis en évidence deux SNPs associés à un âge précoce du diagnostic de la MC (CHDH, ABCB1). / Genome wide association studies demonstrated an association between plasma vitamin B12 and FUT2 (fucosyltransferase 2). It has been suggested that the association between FUT2 and low plasma vitamin B12 level may be the consequence of an increased susceptibility to Helicobacter pylori (H. pylori) infection. We evaluated the association between FUT2 461G>A polymorphism and vitamin B12 and investigated whether the influence of FUT2 on H. pylori serology is part of the mechanisms that underlie this association, in two populations from Europe and West Africa. In this study we confirmed the influence of FUT2 461 G>A polymorphism on plasma vitamin B12 level and found no influence of H. pylori serological status on this association, at least in ambulatory subjects from Europe and West Africa. The magnitude of the association between homocysteine metabolism and inflammatory bowel diseases (IBD) is unknown while the association between hyperhomocysteinemia and thrombosis remains controversial in IBD. We conducted a systematic review of the literature and performed a meta-analysis to examine these issues. The risk of hyperhomocysteinemia is significantly higher in IBD patients when compared to controls. The risk assessment of hyperhomocysteinemia-related thrombosis in IBD requires further investigation. Deficient folate status is associated with a higher impact of MTHFR C677T polymorphism on IBD risk. Hyperhomocysteinemia and several gene variants of one-carbon metabolism are associated with the occurrence and severity of Crohn's disease (CD). Hyperhomocysteinemia results in part from methyl donors deficiency - which is frequent in patients with CD - and increases the activity of superoxide dismutase (SOD), a validated and reliable marker of oxidative stress. We designed a 384-plex GoldenGate oligo pool assay for the comprehensive one-carbon metabolism genotyping using Illumina platform. The aims of this study were (i) to assess genetic determinants of plasma homocysteine and superoxide dismutase (SOD) levels in patients with IBD and (ii) to look for single nucleotide polymorphisms (SNPs) associated with age at CD onset. Two SNPs were associated with plasma homocysteine level (MTHFR, AHCY). Five SNPs were independently associated with plasma SOD level. Of these five SNPs, three are related to vitamin B12 (FUT2, CUBN, and TCN2), one is related to folate (GGH), and the last one to homocysteine (AHCY). In addition, we identified two SNPs associated with early CD onset (CHDH, ABCB1)

Submitted by JACIARA CRISTINA ALMEIDA DO AMARAL (jaciaramaral@ufpa.br) on 2018-03-14T18:08:28Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_PrevalenciaAssociacaoInfeccao.pdf: 914836 bytes, checksum: 230ad3b3857c71456bdb6ad0d9a3fde0 (MD5) / Approved for entry into archive by JACIARA CRISTINA ALMEIDA DO AMARAL (jaciaramaral@ufpa.br) on 2018-03-14T18:13:01Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_PrevalenciaAssociacaoInfeccao.pdf: 914836 bytes, checksum: 230ad3b3857c71456bdb6ad0d9a3fde0 (MD5) / Made available in DSpace on 2018-03-14T18:13:01Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_PrevalenciaAssociacaoInfeccao.pdf: 914836 bytes, checksum: 230ad3b3857c71456bdb6ad0d9a3fde0 (MD5) Previous issue date: 2017-11-03 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A epidemiologia da HP e do vírus Epstein-Barr (EBV) é mundial. A prevalência de ambos os agentes carcinogênicos na população humana mundial é de cerca de 45%. Um estudo recente sugere que coinfecção de EBV com HP cagA positiva, aumenta o potencial oncogênico desta bactéria. O objetivo deste trabalho foi identificar a prevalência da bactéria HP e do EBV e a associação desses patógenos e do gene cagA em pacientes com gastrite na população do Amapá. Foi realizado um estudo descritivo, do tipo transversal, onde foram analisadas 292 amostras de mucosa gástrica de pacientes com gastrite submetidos a endoscopia, com faixa etária entre 14 e 83 anos de idade. Para detecção da HP foi utilizado o teste da Urease e a Reação em Cadeia da Polimerase, esta metodologia também serviu para revelar as cepas cagA positivas da bactéria. Adicionalmente, foi utilizada a técnica de hibridação in situ para detecção do EBV e a análise microscópica que determinou as características histopatológicas da mucosa gástrica. Resultados: Nosso estudo mostrou alta prevalência de casos de HP em pacientes com gastrite com uma frequência relativa de 87,67% dos 292 casos analisados, sendo maior incidência, dos casos positivos para HP, no sexo feminino, 88,27%. A incidência do gene cagA em amostras de pacientes positivos para HP foi de 72,66%, com maior prevalência no sexo feminino, 75,32%. No presente estudo foram encontrados 8,59% dos pacientes com infecção viral causada por EBV em amostras positivas para HP com maior prevalência no sexo masculino, 9,18%. De acordo com a faixa etária nosso estudo mostrou maior prevalência do gene cagA e do EBV em pacientes positivos para HP no segmento entre 44 e 54 anos, com 23,12% e 36,37%, respectivamente. A maioria dos achados deste estudo assemelha-se aos relatos da literatura, contudo, evidenciou-se a necessidade de estudos com maior casuística a fim de melhor esclarecer se há ou não há correlação entre a infecção por HP e EBV no norte do Brasil. / The epidemiology of HP and of the Epstein-Barr virus (EBV) is worldwide. The prevalence of both carcinogenic agents, in the world human population is about 45%. A recent study suggests that EBV coinfection with HP cagA positive increases the oncogenic potential of this bacterium. The objective of this study was to identify the prevalence of the bacterium HP and of the virus EBV and the association of those pathogens and of the cagA gene in patients with gastritis in the population of Amapá. A descriptive study was accomplished, of the traversal type, where 292 samples of gastric mucous of the patients were analyzed with gastritis submitted to the endoscopy, age group between 14 and 83 years. For detection of HP, Urease test and Polymerase Chain reaction were used; this methodology was also useful to reveal the positive cagA of the bacterium. Additionally, the technique of in situ hybridization was used for detection of EBV and the microscopic analysis that determined the histopathological characteristics of the gastric mucous. Results: The study showed high prevalence of cases of HP in patients with gastritis with a relative frequency of 87,67% of the 292 analyzed cases, a higher incidence, of HP positive cases, in female, 88,27%. The incidence of the cagA gene in samples of positive patients for HP was 72,66%, higher prevalence in female, 75,32%. In the present study 8,59% of the patients were found with viral infection caused by EBV in positive samples for HP with bigger prevalence in male, 9,18%. According to the age group, the study showed higher prevalence of the gene cagA and of EBV in positive patient for HP in the age group between 44 and 54 years, with 23,12% and 36,37%, respectively. Conclusion: Most of the findings of this study are similar to the reports from the literature, however, it is necessary other studies in order to explain if there is or there is no correlation between the infection for HP and EBV in the north of Brazil.

Helicobacter pylori est associée à des pathologies gastro-duodénales sévères mais est également un marqueur bactérien de migrations humaines. Nous avons montré que des populations génétiques distinctes de H. pylori ont accompagné au moins quatre migrations en Asie du sud-est et en Océanie : i) une expansion des ancêtres des austronésiens il y a 5000 ans à partir de Taiwan en Océanie, ii) une migration d’Inde en Asie du sud-est depuis 2000 ans,iii) une migration des ancêtres des locuteurs des langues austro-asiatiques au Vietnam et auCambodge il y a 4000 ans, i) une migration des ancêtres des Thaïs du sud de la Chine vers l’actuelle Thaïlande au début du second millénaire. Ces données confirment la résolution plus élevée de la diversité génétique de H. pylori pour retracer les anciennes migrations humaines par comparaison aux marqueurs génétiques humains traditionnels. Nous avons ensuite investigué les facteurs de virulence de souches isolées de patients présentant des symptômes gastriques au Sénégal et au Cambodge. Au Sénégal, une association significative a été observée entre le cancer gastrique et le gène cagA, deux motifs EPIYA-C et l’allèle vacA s1. De multiples segments EPIYA-C étaient observés moins fréquemment que dans les autres régions du monde, contribuant probablement à la faible incidence du cancer gastrique. Au Cambodge, une introgression fréquente d’allèles cagA et vacA européens dans des souches d’Asie de l’est a été observée. CagA et VacA ayant des effets antagonistes, cette expansion pourrait entraîner la rupture de l’équilibre entre les effets biologiques de ces deux protéines et être responsable de conséquences graves sur l’évolution de la maladie. / Helicobacter pylori is associated with severe gastroduodenal disorders but is also a bacterial genetic marker of human migrations. First, we provide evidence that distinct H. pylori genetic populations accompanied at least four ancient human migrations into Oceania and Southeast Asia: i) an expansion of Austronesian speaking people about 5000 years ago from Taiwan into Oceania, ii) a migration from India into Southeast Asia within the last 2000 years, iii) a migration of Austro-Asiatic speaking people into Vietnam and Cambodia about 4000 years ago, and iv) a migration of the ancestors of the Thai people from Southern China into Thailand during the early second millennium AD. These findings demonstrate that H. pylori genetic diversity has more discriminatory power than traditional human genetic markers for tracing old human migrations. Second, we investigated virulence factors of H. pylori strains isolated from patients with gastric symptoms in Senegal and Cambodia. In Senegal, a significant association was observed between gastric cancer and the cagA gene, two EPIYA-C segments, and the s1 vacA allele in Senegal. Multiple EPIYA-C segments were less frequent than reported in other countries, possibly contributing to the low incidence of gastric cancer. In Cambodia, the frequent introgression of European vacA and cagA alleles into East Asian H. pylori strains was observed. As VacA and CagA have opposing effects, this expansion may disrupt the balancing activities on epithelial cells which might result in severe consequences for individual disease outcome.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-09-29T15:40:45Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ComparacapCepasHelicobacter.pdf: 553158 bytes, checksum: aaa0b8cac9880eb73c7991f3ccb45cfb (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-10-02T15:07:25Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ComparacapCepasHelicobacter.pdf: 553158 bytes, checksum: aaa0b8cac9880eb73c7991f3ccb45cfb (MD5) / Made available in DSpace on 2017-10-02T15:07:26Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ComparacapCepasHelicobacter.pdf: 553158 bytes, checksum: aaa0b8cac9880eb73c7991f3ccb45cfb (MD5) Previous issue date: 2006 / A infecção pela Helicobacter pylori, é extremamente frequente em todo o mundo, com prevalência maior em países em desenvolvimento. Está associada à gastrite crônica, úlcera gástrica e duodenal e é considerada um fator de risco para câncer gástrico e linfoma MALT do estômago. As rotas de transmissão desta bactéria ainda não estão completamente esclarecidas, sendo as mais prováveis a oral-oral e fecal-oral. A importância da presença da bactéria da placa dental permanece obscura, podendo constituir-se em fonte de infecção gástrica. Objetivando identificar e correlacionar as cepas da H. pylori presentes em biopsias gástricas e em amostras de placa dental, foram avaliados 99 pacientes adultos dispépticos, submetidos a endoscopia digestiva alta no Hospital Universitário João de Barros Barreto, da Universidade Federal do Pará, no ano de 2005. Amostras da placa dental foram obtidas utilizando coletores esterilizados, e analisadas pelo teste da urease e pela Reação em cadeia da polimerase (PCR). Durante a endoscopia, seis biopsias foram retiradas do antro gástrico e analisadas pelo teste da urease, exame histopatológico e PCR, após consentimento informado e aprovação pelo Comitê de Ética do Hospital Universitário João de Barros Barreto. Os resultados obtidos foram analisados utilizando-se o programa BioEstat 3.0. A bactéria foi identificada em 96% das biopsias gástricas e em 72% das amostras de placas dentais, diferença estatisticamente significante (p<0,001). Em relação à idade e sexo, não houve diferença estatística. Todos os pacientes apresentaram alterações gástricas, sendo que 18% apresentaram grau maior de severidade, com lesões ulceradas à endoscopia e/ou lesão pré-maligna do tipo metaplasia intestinal à histopatologia, nestes houve concomitância de infecção na placa dental e mucosa gástrica em 82,4%. Dentre os testes utilizados o de maior sensibilidade foi a PCR, tanto em amostras gástricas como na placa dental. Nos casos de positividade para a bactéria na placa dental (71 casos), houve coincidência entre as cepas da mucosa gástrica e placa dental em 89%. O genótipo mais frequentemente identificado foi s1bm1 cagA positivo, tanto na mucosa gástrica, quando na placa dental. As cepas tipo 1, consideradas as mais patogênicas foram encontradas em 63 pacientes na mucosa gástrica e em 58 pacientes na placa dental. A frequência elevada da H. pylori na placa dental, pode ser um indicador de que a cavidade oral seria um sítio de colonização deste micro-organismo, a partir do qual poderia ser diretamente disseminada, constituindo-se em um fator de risco para infecção gástrica. / Helicobacter pylori infection is extremely frequent over the world, mainly in development countries, including Brazil. It’s associated to chronic gastritis, duodenal and gastric ulcer, and is considered as an important risk factor for gastric cancer and gastric MALT lymphoma. The transmission routes remain unclear, but oral-oral and fecal-oral routs seem to be the most probable ones. The value of the presence of the bacteria on the dental plaque also remains unclear, and it maybe a source for gastric infection. Aiming in identifying and correlating the H. pylori stains found in gastric mucosa and dental plaque of 99 adult dyspeptic patients submitted to upper digestive endoscopies at Hospital Universitário João de Barros Barreto, in 2005 were evaluated. Samples from dental plaque were collected by sterile sticks and urease test and polymerase (PCR) chain reaction were undertaken. During the endoscopic procedure 6 pieces were collected from antrun and investigated by urease test, histopathology and PCR, after obtaining informed consent. The results were analyzed using BioEstat 3.0 package. The bacteria was found in 96% of gastric samples and in 72% of dental plaque samples, and this difference was statistically significant (p<0,001). There weren’t statistically significant differences related to age or gender. Every patient presented gastric diseases. In 18% of the cases lessons considered as of higher severity such as ulcers or pre-malignant lesions, as intestinal metaplasia, were found, and, among these, there were 82.4% of cases with both gastric and dental plaque infection. PCR was the most efficient test either on dental plaque and gastric mucosa samples. Among the 71 cases where the dental plaque samples were positive for the presence of the bacteria, the stains were identical to the gastric mucosa H pylori stains in 89%. The most common genotype was s1bm1cagA positive, either at dental plaque and gastric mucosa. The type 1 strains, considered the most pathogenic ones, were found in 63 patients on gastric mucosa and in 58 patients on dental plaque. The high frequency of H. pylori found on dental plaque might indicate the oral cavity as a colonizing locus for this bacteria and a risk factor for gastric infection.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-09-29T13:49:53Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_InfeccaoHelicobacterpylori.pdf: 1425946 bytes, checksum: 07cb26ccb9b82ee590d083e7377e566e (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-10-06T14:53:43Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_InfeccaoHelicobacterpylori.pdf: 1425946 bytes, checksum: 07cb26ccb9b82ee590d083e7377e566e (MD5) / Made available in DSpace on 2017-10-06T14:53:43Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_InfeccaoHelicobacterpylori.pdf: 1425946 bytes, checksum: 07cb26ccb9b82ee590d083e7377e566e (MD5) Previous issue date: 2012-09-13 / O presente estudo teve como objetivo analisar a relação entre a infecção pela Helicobacter pylori em crianças e seus respectivos pais, mediante informações diagnósticas laboratoriais e epidemiológicas, contribuindo para esclarecer os possíveis fatores etiológicos desta infecção. Foi realizado um estudo descritivo e analítico do tipo transversal, nos períodos de março a junho de 2012. A população de estudo incluiu 48 famílias residentes às margens do rio Tocantins no município de Imperatriz-Maranhão, cadastradas e assistidas pela equipe de saúde da família Beira Rio. Foi aplicado formulário epidemiológico e coletado material biológico das crianças menores de 12 anos que corresponderam a amostras de fezes e saliva, enquanto que dos pais ou responsáveis e filhos a partir de 12 anos corresponderam a amostras de sangue e saliva. Nas amostras de soro foi pesquisada a presença de anticorpos IgG anti-H. pylori através de ensaios imunoenzimáticos (ELISA), na saliva foi utilizada a técnica de DOT-ELISA em membranas de nitrocelulose para identificação de fenótipos ABH e Lewis, as fezes foram usadas para a pesquisa de antígenos da H.pylori através de ensaio imunocromatográfico qualitativo. A prevalência global da infecção nas crianças menores de 12 anos foi de 69,23%, tendo início antes do primeiro ano de vida. A prevalência da infecção nas mães e nos pais foi de 76,60% e 59,09% respectivamente, entre as mães infectadas 77,27% dos filhos estavam também infectados. A prevalência da infecção pela H. pylori, entre os membros das famílias estudadas não mostrou associações com os fenótipos de grupos sanguíneos ABO, Lewis e estado secretor. Os aspectos socioeconômicos são sugestivos de que a transmissão intrafamiliar pode ser facilitada pelas precárias condições socioambientais, com ausência de saneamento, higiene e pobreza. / The present study aimed to analyze the relationship between infection by Helicobacter pylori in children and their parents through diagnostic laboratory and epidemiological information, helping to clarify the possible etiological factors of this infection. A descriptive and analytical cross-sectional was conducted, from March to June 2012. The study population included 48 families living on the river in areas of the river Tocantins, in the municipal of Imperatriz-Maranhão, registered and assisted by the family health team operating in that area. Form epidemiological applied and biological material collected from children under 12 years corresponded to samples of feces and saliva, while the parents or guardians and children from 12 years corresponded to samples of blood and saliva. Serum samples were screened for the presence of anti-H. pylori by immunoassays (ELISA) was used in saliva DOT-ELISA technique on nitrocellulose membranes for identifying phenotypes ABH and Lewis, feces were used for the detection of H. pylori antigens using immune chromatographic assay qualitatively. The overall prevalence of infection in children under 12 years was 69,23%, with onset before the first year of life. The prevalence of infection in mothers and fathers was 76.60% and 59.09% respectively; between infected mothers 77,27% of the children were also infected. The prevalence of infection by H. pylori, among the members of the families studied showed no associations with the phenotypes of blood groups ABO, Lewis and secretor status. Socioeconomic aspects are suggestive of interfamilial transmission that can be facilitated by poor environmental conditions, with lack of sanitation, hygiene and poverty.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-16T16:24:16Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AssociacaoVariantesRegiao.pdf: 1409940 bytes, checksum: 8e34e8ff0f8dd9a4764a50dee1d96ab2 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-10-18T14:50:30Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AssociacaoVariantesRegiao.pdf: 1409940 bytes, checksum: 8e34e8ff0f8dd9a4764a50dee1d96ab2 (MD5) / Made available in DSpace on 2017-10-18T14:50:30Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AssociacaoVariantesRegiao.pdf: 1409940 bytes, checksum: 8e34e8ff0f8dd9a4764a50dee1d96ab2 (MD5) Previous issue date: 2012 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A citotoxina CagA do Helicobacter pylori, codificada pelo gene cagA, é associada ao aumento da resposta inflamatória do tecido gástrico e a alteração do controle do crescimento e proliferação celular. A ativação desta citotoxina ocorre pela fosforilação em resíduos específicos de tirosina dentro de uma sequência de aminoácidos denominada motif EPIYA, sendo quatro os tipos de motifs descritos na literatura (EPIYA-A,-B,-C e-D). Contudo, o sítio EPIYA-C constitui o local mais comum de fosforilação de proteínas CagA das cepas bacterianas isoladas nos países ocidentais, podendo ainda ser encontrado em repetições. Assim, este estudo teve como objetivo determinar os tipos de motifs EPIYA de CagA presentes em pacientes com gastrite e adenocarcinoma gástrico e sua associação com estas doenças. Foram coletadas amostras de biópsias gástricas de 384 pacientes infectados por H. pylori, dos quais 194 apresentavam gastrite crônica e 190 adenocarcinoma gástrico. As biópsia gástrica foram utilizadas para análise histológica, extração de DNA bacteriano e análise do gene cagA por PCR. Houve predomínio de adenocarcinoma gástrico no sexo masculino, com média de idade de 58 anos. O gene cagA foi mais prevalente nos pacientes com câncer gástrico, apresentando associação com maior grau de inflamação, atividade neutrofílica e desenvolvimento de metaplasia intestinal (OR = 4,31, IC 95% = 2,71-6,87, p <10-3; OR = 3,57, IC 95% = 2,18 – 5,84, p <10-3; OR = 11,11, IC 95% = 5,48 – 22,30, p <10-3; OR = 3,65, IC 95% = 1,50-8,88, p=0,004, respectivamente). O número de repetições do sítio EPIYA C foi significativamente associada com o aumento do risco de carcinoma gástrico (OR = 2,99, IC 95% = 1,53-5,82, p <10-3) e o maior número de motifs EPIYA C também foi associado com metaplasia intestinal (p = 0,02). Neste estudo a infecção por cepas de H. pylori portadoras do gene cagA com mais de um motif EPIYA-C demonstrou associação com o desenvolvimento de metaplasia intestinal e adenocarcinoma gástrico, entretanto, não apresentou associação com a atividade neutrofílica e grau de inflamação. / Helicobacter pylori CagA cytotoxin, encoded by the cagA gene, has been associated with increased inflammatory response in gastric tissue and the change in control of cell growth and proliferation. Activation of this cytotoxin occurs by phosphorylation in specific tyrosine residues within an amino acid sequence termed motif EPIYA, four types of motifs are described in the literature (EPIYA-A,-B-C and D). However, the site EPIYA-C is the most common site of phosphorylation of CagA protein of the bacterial strains isolated in Western countries, may still be found in repetitions. This study aimed to determine the types of motifs EPIYA of CagA present in patients with gastritis and gastric cancer and its association with these diseases. Were collected samples from gastric biopsies of 384 patients infected with H. pylori, of this 194 presented chronic gastritis and 190 had gastric cancer. The gastric biopsy was used for bacterial DNA extraction and analysis of the cagA gene by PCR. The prevalence of gastric cancer occurs in males, mean age 58 years. The cagA gene was more prevalent in patients with gastric cancer, showing association with a higher degree of inflammation, neutrophil activity and development of intestinal metaplasia (OR = 4,31, IC 95% = 2,71-6,87, p <10-3; OR = 3,57, IC 95% = 2,18 – 5,84, p <10-3; OR = 11,11, IC 95% = 5,48 – 22,30, p <10-3; OR = 3,65, IC 95% = 1,50-8,88, p=0,004, respectively). The number of repetitions EPIYA-C site was significantly associated with increased risk of gastric cancer (OR = 2,99, IC 95% = 1,53-5,82, p <10-3). The higher number of motifs EPIYA-C was also associated with intestinal metaplasia (p = 0,02). In this study the infection by strains of H. pylori carriers cagA gene with more than one motif EPIYA-C shown to be associated with the development of intestinal metaplasia and gastric cancer, but without an association to neutrophil activity and degree of inflammation.

<p>The stomach is frequently exposed to hazardous agents and to resist this harsh environment, several protective mechanisms exist. Of special interest is the gastric pathogen <i>Helicobacter pylori </i>which causes gastritis, ulcers and cancer but the mechanism leading to these diseases are still unclear. However it is very likely that <i>H. pylori </i>negatively influence the protection mechanisms that exist in the stomach. </p><p>The aims of the present investigation were first to develop an in vivo mouse model in which different protection mechanisms could be studied, and second to investigate the influence of <i>H. pylori</i> on these mechanisms. </p><p>An in vivo preparation of the gastric mucosa in mice was developed. This preparation allows studies of different gastric mucosal variables and can also be applied for studies in other gastro-intestinal organs. </p><p>Mice chronically infected with <i>H. pylori</i>, were shown to have a reduced ability of the mucosa to maintain a neutral pH at the epithelial cell surface. This could be due to the thinner inner, firmly adherent mucus gel layer, and/or to defective bicarbonate transport across the epithelium. The Cl^-/HCO₃^- exchanger SLC26A9 was inhibited by NH₄⁺, which also is produced by <i>H. pylori</i>. The mRNA levels of SLC26A9 were upregulated in infected mice, suggesting a way to overcome the inhibition of the transporter. Furthermore, the hyperemic response to acid pH 2 and 1.5 was abolished in these mice. The mechanisms by which the bacteria could alter the blood flow response might involve inhibition of the epithelial iNOS.</p><p>Water extracts of <i>H. pylori </i>(HPE) reduces the blood flow acutely through an iNOS and nerve-mediated pathway, possibly through the endogenous iNOS inhibitor ADMA. Furthermore, HPE alters the blood flow response to acid as the hyperemic response to acid pH 0.8 is accentuated in mice treated with HPE. </p>

The stomach is frequently exposed to hazardous agents and to resist this harsh environment, several protective mechanisms exist. Of special interest is the gastric pathogen Helicobacter pylori which causes gastritis, ulcers and cancer but the mechanism leading to these diseases are still unclear. However it is very likely that H. pylori negatively influence the protection mechanisms that exist in the stomach. The aims of the present investigation were first to develop an in vivo mouse model in which different protection mechanisms could be studied, and second to investigate the influence of H. pylori on these mechanisms. An in vivo preparation of the gastric mucosa in mice was developed. This preparation allows studies of different gastric mucosal variables and can also be applied for studies in other gastro-intestinal organs. Mice chronically infected with H. pylori, were shown to have a reduced ability of the mucosa to maintain a neutral pH at the epithelial cell surface. This could be due to the thinner inner, firmly adherent mucus gel layer, and/or to defective bicarbonate transport across the epithelium. The Cl-/HCO3- exchanger SLC26A9 was inhibited by NH4+, which also is produced by H. pylori. The mRNA levels of SLC26A9 were upregulated in infected mice, suggesting a way to overcome the inhibition of the transporter. Furthermore, the hyperemic response to acid pH 2 and 1.5 was abolished in these mice. The mechanisms by which the bacteria could alter the blood flow response might involve inhibition of the epithelial iNOS. Water extracts of H. pylori (HPE) reduces the blood flow acutely through an iNOS and nerve-mediated pathway, possibly through the endogenous iNOS inhibitor ADMA. Furthermore, HPE alters the blood flow response to acid as the hyperemic response to acid pH 0.8 is accentuated in mice treated with HPE.

The occupational exposures of sewage workers are complex and variable, and include a great variety of biological and chemical agents. Previous research has focused mostly on infections and various symptoms among sewage workers, e.g. abdominal and respiratory symptoms. At several sewage plants in Sweden, concern arose about occupational cancer, specifically cancer of the stomach, the kidney, and the lung. The aim of this study was to study the cancer incidence among municipal sewage workers, some exposures that might be connected with cancer risk, and self reported abdominal and respiratory symptoms. In a cohort of municipal sewage workers there was no increase in the overall incidence of cancer when compared with the general population. However, there was a slight increase in the incidence of prostate cancer, but not in the sites of original concern among the workers. Infection by the gastric carcinogen Helicobacter pylori (determined from the presence of IgG antibodies in serum against H pylori) was no more prevalent in sewage workers than in comparable referents. Neither were sewage workers more exposed to genotoxic agents than comparable referents, as measured by the alkaline single cell gel electrophoresis (SCG) assay performed on peripheral lymphocytes. There was no increase in the three-month prevalence of abdominal symptoms when compared with other municipal workers. Specifically, there was no difference in prevalence of the common disorders dyspepsia and irritable bowel syndrome. Sewage workers reported adult bronchial asthma significantly more than the referents.

The availability of completely sequenced genomes for a number of organisms provides an opportunity to understand the molecular basis of physiology, metabolism, regulation and evolution of these organisms. Significant understanding of the complexity of organisms can be obtained from the functional characterization of repertoire of proteins encoded in their genomes. Computational approaches for recognition of function of proteins of unknown function encoded in genomes often rely on ability to detect well characterized homologues. Homology searches based on pair-wise sequence comparisons can reliably detect homologues with sequence identity more than 30%. However, detecting homologues characterized by sequence identity below 30% is difficult using these methods. Distant homology relationship can be established using profiles or position specific scoring matrices, which encapsulate information about structurally and functionally conserved residues. These conserved residues imply high constraints at a particular amino acid residue site due to their involvement in structural stability, enzymatic activity, ligand binding, protein folding or protein–protein interactions. In addition, information on three dimensional structures of proteins also aid in detection of remote homologues, as tertiary structures of proteins are conserved better than the primary structures of proteins. The gross objective of the work reported in this thesis is to employ various sensitive remote homology detection methods to recognize relevant functional information of proteins encoded mainly in pathogenic organisms. Since proteins do not work in isolation in a cell, it has become essential to understand the in vivo context of functions of proteins. For this purpose, it is essential to have an understanding of all molecules that interact with a particular protein. Thus, another major area of bioinformatics has been to integrate protein-protein interaction information to enable better understanding of context of functional events. Protein-protein interaction analysis for host-pathogen can lead to useful insight into mode of pathogenesis and subsequent consequences in host cell. Chapters 2-6 of the thesis discuss the sequence and structural characteristics along with remote evolutionary relationships and functional implications of uncharacterized proteins encoded in genomes of following pathogens: Helicobacter pylori, Plasmodium falciparum and Leishmania donovani. The Chapters 6-8 discuss mainly various sequence, structural and functional aspects of protein kinases encoded in genomes of various prokaryotes and viruses. Chapter 1 discusses background information and literature survey in the areas of homology detection and prediction of protein-protein interactions. The growth of genomic data and need for processing genomic data to infer context of various functional events have been highlighted. Different approaches to recognize functions of proteins (experimental as well as computational) have been discussed. Various experimental and computational approaches to detect/predict protein-protein interactions have been mentioned. Chapter 2 discusses recognition of non-trivial remote homology relationships involving proteins of Helicobacter pylori and their implications for function recognition. H. pylori is microaerophilic, Gram negative bacterial pathogen. It colonizes human gastric mucosa and is a causative agent of gastroduodenal disease. The pathogen infects about 50% of the human population. It can lead to development of Mucosa-associated lymphoid tissue lymphoma. About 10% of the infected population develop gastric or duodenal ulcer and approximately 1% develop gastric cancer. H. pylori has been classified as class I carcinogen by WHO. Pathogen is characterized by type IV secretion system. The complete genomic sequences of three widely studied strains including 26695, J99 and HPAG1 of Helicobacter pylori are available. According to the genome analysis, the number of predicted open reading frames in strain 26695, J99 and HPAG1 are 1590, 1495 and 1536 respectively. Out of predicted H. pylori proteins from 26695, J99 and HPAG1 strains, numbers of proteins with no functional domain assignments in Pfam database (Protein family database) are 453, 357 and 400 respectively. There are proteins in different strains of H. pylori genomes where one part of the protein is associated with at least one protein domain of known function and hence preliminary indication of their functions is available whereas rest of the region is not associated with any function. There are 772, 803 and 790 such segments in proteins from strains 26695, J99 and HPAG1 respectively with at least 45 residues with no functional assignment currently available. Sensitive remote homology detection methods have been employed to establish relationships for 294 amino acid sequences and results have been grouped into 4 categories. Results of homology detection have been further confirmed by studying conservation of amino acid residues which are important for functioning of the proteins concerned. (i) Remote relationship has been established involving protein domain families for which no bonafide member is currently known in H. pylori. For example: DNA binding protein domain (Kor_B) has been assigned to a H. pylori protein at sequence identity of 20%. Study involving secondary structure prediction and conservation of amino acid residues confirms the results of homology detection methods. (ii) Remote relationship has been established involving H. pylori hypothetical proteins and protein domain families, for which paralogous members are present in Helicobacter pylori. For example, Cytochrome_C, an electron transfer protein domain could be associated with a Helicobacter pylori protein sequence which shows a sequence identity of 14% with sequences of bonafide cytochrome C. (iii) “Missing” metabolic proteins of H. pylori have also been recognized. For example, Aspartoacylase (EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartic acid to produce acetate and L-aspartate. This enzyme in aspartate metabolism pathway has not been reported so far from H. pylori. A remote evolutionary relationship between a H. pylori protein and Aspartoacylase domain has been established at sequence identity of 17% thus filling the gap in this metabolic pathway in the pathogen. (iv) New functional assignments for domains in H. pylori sequences with prior assignment of domains for the rest of the sequences have been made. For example, DNA methylase domain has been assigned to C-terminal region of H. pylori protein which already had Helicase domain assigned to the N-terminal region of the protein. All these information should open avenues for further probing by carrying out experiments which will impact the design of inhibitor against this pathogen and will result in better understanding of pathogenesis of this organism in human. Chapter 3 describes prediction of protein–protein interactions between Helicobacter pylori and the human host. A lack of information on protein-protein interactions at the host-pathogen interface is impeding the understanding of the pathogenesis process. A recently developed, homology search-based method to predict protein-protein interactions is applied to the gastric pathogen, Helicobacter pylori to predict the interactions between proteins of H. pylori and human proteins in vitro. Many of the predicted interactions could potentially occur between the pathogen and its human host during pathogenesis as we focused mainly on the H. pylori proteins that have a transmembrane region or are encoded in the pathogenic island and those which are known to be secreted into the human host. By applying the homology search approach to protein-protein interaction databases DIP and iPfam, in vitro interactions for a total of 623 H. pylori proteins with 6559 human proteins could be predicted. The predicted interactions include 549 hypothetical proteins of as yet unknown function encoded in the H. pylori genome and 13 experimentally verified secreted proteins. A total of 833 interactions involving the extracellular domains of transmembrane proteins of H. pylori could be predicted. Structural analysis of some of the examples reveals that the predicted interactions are consistent with the structural compatibility of binding partners. Various probable interactions with discernible biological relevance are discussed in this chapter. For example, interaction between CFTR protein (NP_000483) and multidrug resistance protein (HP1206) has been predicted. The structure of the CFTR intracellular domain is known in the homomeric form and consists of five AAA transport domains in tandem (PDB code 1XMI). Out of the five identical subunits, two subunits (the B chain and the E chain in the PDB structure) have been selected. The structure of multidrug resistance protein of the pathogen based on the B chain (sequence identity 32%) of the template has been modeled. This exercise suggests that interface residues in the model are congenial for interaction. This makes the structural complex feasible in in vitro conditions and suggests that the pathogen protein may compete for occupancy with the host protein. Chapter 4 describes recognition of Plasmodium-specific protein domain families and their roles in Plasmodium falciparum life cycle. Malaria in humans is caused by the parasites of intracellular, eukaryotic protozoan of apicomplexan nature belonging to the genus Plasmodium. Out of five species of Plasmodium, namely, P. falciparum, P. ovale, P. vivax, P. malariae and P. knowlesi which infects human, P. falciparum causes lethal infection. P. falciparum proteins have diverged extensively during the course of evolution. Pathogen genome is rich in A+T composition which larger than the homologous proteins from other organisms due to presence of low complexity regions. Organism specific families are important as they play roles in peculiar life style of an organism. If the organism is a pathogen, then these family members may play roles in pathogenesis. Inhibiting these specific proteins is unlikely to interfere with host system as no homolog may be present in host. In the present work we identify Plasmodium specific protein families and their role in different stages of life cycle of the pathogen. A total of 5086 amino acid sequences (full length sequences/fragments of proteins) show homology only with amino acid sequences from Plasmodium organisms and hence are Plasmodium-specific. These Plasmodium-specific amino acid sequences cluster into 106 Plasmodium-specific families (≥2 members per family). 14 Plasmodium-specific protein domain families with known physico-chemical properties are observed. These Plasmodium-specific protein domain families are involved in various important functions such as rosetting and sequestering of infected erythrocytes, binding to surface of host cell and invasion process in life cycle of pathogen. Also, 89 new Plasmodium-specific protein domain families have been recognized. Analysis of various aspects of members of Plasmodium-specific proteins domain families such as their potential to target apicoplast, protein-protein interaction, expression profile and domain organization has been performed to derive relevant information about function. New Plasmodium specific domain families for which no function can be associated could provide some insight into much diverged Plasmodium species. These proteins may play role in parasite-specific life style. Experimental work on these Plasmodium-specific proteins might fill the gaps of less understood physiology of this parasite. Chapter 5 presents genome-wide compilation of low complexity regions (LCR) in proteins. An indepth analysis of the nature, structure, and functional role of the proteins containing low complexity regions in Plasmodium falciparum, was undertaken given the high prevalence of LCRs in the proteome of this organism. Low complexity regions and repeat patterns have been recognized in proteins encoded in 986 genomes (68 archaea, 896 prokaryotes and 22 eukaryotes). Low complexity regions have been classified into following three categories: a) Composition of LCRs: (i) LCRs can be stretches of homo amino acid residues (ii) LCRs can be stretches of more than one amino acid residue type b) Periodicity of amino acids in LCRs: Certain amino acid residues can be observed at certain specific periodicity in proteins. c) Repeat patterns: Certain motif of amino acid residues are repeated in protein. 850 Plasmodium falciparum proteins are observed to have at least one repeat pattern where the repeating unit is at least 5 amino acid residues long. Statistical analysis on single amino acid residue repeats indicate that occurrence of stretches of homo amino acid residues is not a random event. Studies on recognition of functions, protein protein interactions and organization of tethered domain(s) in proteins containing LCR suggest that these proteins are part of variety of functional events such as signal transduction, enzymatic processes, cell differentiation, pyrimidine biosynthesis, fatty acid biosynthesis and chromosomal replication. Representations of low complexity regions of Plasmodium falciparum in protein data bank suggest that LCRs can take conformation of regular secondary structure (apart from disordered regions) in 3-D structures of proteins. Chapter 6 describes sequence analysis, structural modeling and evolutionary studies of Leishmania donovani hypusine pathway enzymes. Leishmania is an eukaryotic kinetoplastid protozoan parasite which causes leishmaniasis in humans. Hypusine is a non standard polyaminederived amino acid Nε-(4-amino-2-hydroxybutyl) lysine and is named after its two structural components, hydroxyputrescine and lysine. The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein containing hypusine. Synthesis of hypusine is critical for the function of elF5A and is essential for eukaryotic cell proliferation and survival. Formation of hypusine is the result of a two step post-translational modification process involving enzymes (i) deoxyhypusine synthase (DHS) (ii) deoxyhypusine hydroxylase (DOHH). DHS, the first enzyme involved in hypusine pathway catalyzes the NAD-dependent transfer of the butylamino moiety of spermidine (substrate) to the ε-amino group of a specific lysine residue of eIF5A precursor and generates deoxyhypusine containing intermediate. DOHH, the second enzyme in same pathway catalyzes the hydroxylation of deoxyhypusine-containing intermediate, generating hypusine-containing mature eIF5A. Two putative deoxyhypusine synthase (DHS) sequences DHS34 and DHS20 have been identified in Leishmania donovani, by Professor Madhubala and coworkers (Jawaharlal Nehru University, New Delhi) with whom the work embodied in this chapter was done in collaboration. Detailed comparison of DHS34 sequence from Leishmania with human DHS protein indicated conservation of functionally important residues. 3D structural modeling studies of protein suggested that residues around the active site were absolutely conserved. NAD binding regions are located spatially closer, however, one NAD binding region was observed in a large (225 amino acid residues long) insertion. Based on these observations, DHS34 was predicted to have enzymatic activity. Experimental studies done by our collaborators confirmed preliminary results of computational analysis. Based on sequence and structural analysis of DHS20 and DOHH proteins, DHS20 and DOHH were proposed to be catalytically inactive and active respectively. Experimental studies on these proteins supported results of computational analysis. Deoxyhypusine synthase (DHS) and Deoxyhypusine hydroxylase (DOHH) are key proteins conserved in the hypusine synthesis pathways of eukaryotes. Because they are highly conserved, they could be coevolving. Comparison of the genetic distance matrices of DHS and DOHH proteins reveals that their evolutionary rates are better correlated when compared to the rate of an unrelated protein such as Cytochrome C. This indicates that they are coevolving, further serving as an indicator that, even non-interacting proteins that are functionally coupled, experience correlated evolution. However, this correlation does not extend to their tree topologies. Chapter 7 provides a classification scheme for protein kinases encoded in genomes of prokaryotic organisms. Overwhelming majority of the Ser/Thr protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Ser/Thr protein kinases prokaryotic Ser/Thr protein kinases. Traditional sequence alignment and phylogenetic approaches have been used to identify and classify prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence databases, it is recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly, subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, it was also possible to identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular organization which indicates a degree of complexity in protein-protein interactions and the signaling pathways in these microbes. Chapter 8 focuses on recognition, classification of protein kinases encoded in genomes of viruses and their implications in various functions and diseases. Protein kinases encoded by viral genomes play a major role in infection, replication and survival of viruses. Using traditional sequence homology detection tools, sequence alignment methods and phylogenetic approaches, protein kinases were recognized. 646123 protein sequences from 35799 viral genomes (including strains) have been used in this analysis. Protein kinases are identified using a combination of profile-based search methods such as PSI-BLAST, RPS-BLAST and HMMER approaches. Based upon sequence similarity over the length of catalytic kinase domains, 479 protein kinase domains recognized in 244 viral genomes have been clustered into 46 subfamilies with minimum sequence identity of 35% within a subfamily. Viral protein kinases are encoded in genomes of retro-transcribing viruses or viruses which possess double stranded DNA as genetic material. Based on the available functional information present for one or more members of a subfamily, a putative function has been assigned to other members of the subfamily. Information regarding interaction of viral protein kinases with viral/host protein has also been considered for enhancing understanding of function of kinases in a subfamily. Out of 46 subfamilies, 14 subfamilies are characterized by various functions. Kinases belonging to UL97, US69, UL13 and BGLF subfamilies are virus specific. For 7 subfamilies, nearest neighbors are from well characterized eukaryotic protein kinase groups such as AGC, CAMK and CDK. Out of 25 new uncharacterized subfamilies observed in this analysis, 13 subfamilies are virus specific. Different subfamilies have been characterized by various functions which are crucial for viral infection such as synthesis of structural unit, replication of genetic material, modification of cellular components, alteration in host immune system, competing with cellular protein for efficient usage of host machinery. Also, many viral kinases share very high sequence identity (~97%) with their eukaryotic counterpart and represent disease state. For example, a protein kinase encoded in Avian erythroblastosis virus shares 97% sequence identity with catalytic domain of human epidermal growth factor receptor tyrosine kinase. Leucine at position 861 in human protein is substituted by Gln in cancer conditions; the viral protein kinase sequence possesses Gln at corresponding position and thus represents disease state. Chapter 9 provides study of dependency on the ability of 3-D structural features of comparative models and crystal structures of inactive forms of enzymes to predict enzymes by considering protein kinases as case study. With the advent of structural genomics initiatives, there is a surge in the number of proteins with 3-D structural information even before functional features are understood on many of these proteins. One of the useful annotations of a protein is the demarcation of a protein into an enzyme or non-enzyme solely from the knowledge of 3-D structure. This is facilitated by the identification of active sites and ligand binding sites in a protein. In this work, which was carried out in collaboration with Dr Jim Warwicker of Manchester University, UK, an approach developed by Warwicker and coworkers has been used. In the 3D structure of proteins, the largest clefts are generally considered to be ligand binding sites. This feature along with other sequence alignment independent properties such as residue preferences, fraction of surface residues and secondary structure elements have been considered to differentiate enzymes from non-enzymes. Electrostatic potential at the active site is one of the key properties utilized in this respect. Active sites in enzymes are generally associated with ionizable groups which can take part in catalysis. In addition to the feature of large clefts in enzymes, active site residues are in buried environments and show larger deviation in pKa values than surface residues. The method proposed by Warwicker and co-workers distinguish proteins in to enzymes and non-enzymes considering the electrostatic features at clefts along with the sequence profile of the protein concerned. Conformation of the inactive state of an enzyme is not congenial to the catalytic function. In an ideal situation, a method should be capable of predicting an enzyme irrespective of whether determined structure corresponds to active or inactive state. Peak potential values have been calculated by using Warwicker program for a set of 15 protein kinases for which 3-D structures are present in active as well in inactive conformations. Comparison of peak potential values calculated for active and inactive conformations suggests that algorithm can differentiate between active and inactive conformations as value for active conformations are generally higher than corresponding values for inactive conformations. However, the peak potential values are high enough for even the inactive conformations to be predicted as enzyme. Peak potential values calculated for generated homology models of protein kinases (for which crystal structures are already available) at different sequence identities with template sequences predict protein kinases as enzymes and their peak potential values are comparable to corresponding values for X-ray structures. This suggests that proteins for which there are no crystal or NMR structures yet available and no good template with high sequence identity are present, peak potential values for models generated at low sequence identity can still give insight into probable function of protein as an enzyme. The enzyme/non-enzyme prediction algorithm was also found to be useful in confirming enzyme functionality using 3-D models of putative viral kinases. Initially, putative function of kinase has been assigned to these viral proteins based solely upon their sequence characteristics such as presence of residues/motifs which are important for activity of the protein. The enzyme recognition method which is not directly sensitive to these motifs confirmed that all the analyzed putative viral kinases are enzymes. Chapter 10 presents conclusions of work embodied in the entire thesis. Very briefly, various computational approaches have been used to analyze and understand structural and functional properties of repertoire of proteins of pathogenic organisms. Analysis of uncharacterized protein domain families has helped to understand the functional implications of constituent proteins. Experimental validation of these results can further facilitate unraveling of functional aspects of proteins encoded in various pathogenic organisms. Apart from studies embodied in the thesis, author has been involved in two other studies, which are provided as appendices. Appendix 1 describes comparison of substitution pattern of amino acid residues of protein encoded in P. falciparum genome with substitution pattern of corresponding homologous proteins from non-Plasmodium organisms. Salient differences have been highlighted. Appendix 2 discusses study of bacterial tyrosine kinases with an objective of recognition of all putative protein tyrosine kinases in E. coli. Computational study suggests that protein SopA can be a potential tyrosine kinase and this conclusion is being tested experimentally in collaborator’s laboratory.