The H. Pylori strains demonstrate a high level of phenotypic and genotypic diversity, the expression of various genes, which confer greater pathogenicity to the bacteria. Most studies have found that the cagE gene is strongly associated with peptic ulcer disease and sites of tyrosine phosphorylation of CagA protei - EPIYA, has not been fully elucidated existing geographical differences. This study aimed to characterize the strains of H. pylori for the sites of tyrosine phosphorylation of CagA protein EPIYA and presence of genes cagE in patients with gastritis and peptic ulcer disease, and to correlate the presence of these genotypes with gastric disorder. Genotyping of H. pylori strains was performed by PCR and sequencing of phosphorylation sites EPIYA. 137 dyspeptic patients from H. pylori-positive, 72 with peptic ulcer and 65 with gastritis were evaluated. Of the 137 strains genotyped 68.6 % were cagA positive, strains cagA studied , 96.8 % had at least 1 C phosphorylation site EPIYA , while 53.2 % of these had only one site , 43.6 % had two or more sites C and 3.2 % had no place C. the mixed strains with two EPIYA patterns were present in 23.4 % of the studied samples . The group of patients with gastritis showed higher prevalence of genotype EPIYA ABCCC , the same was not observed in patients with peptic ulcer disease and this association with gastrics disorders studied significant ( p = 0.001 ). Of the 137 patients studied, 65 were positive for CAGE (47.4%). There was a significant inverse association between genotype and cage group of patients with gastritis (OR = 0.345, p = 0.002). There was a significant association between genotype cagE with peptic ulcer disease (OR = 2.898, p = 0.002) and the subgroup of patients with duodenal ulcer (OR = 3.839, p = 0.001). This study suggests that the population sample studied the presence of genotype Epiya three sites C was associated with gastritis and absent in patients with peptic ulcer. We found association of the cagE gene duodenal ulcer. / As cepas de H. pylori demonstram um alto nÃvel de diversidade fenotÃpica e genotÃpica, pela expressÃo de vÃrios genes, que conferem maior patogenicidade à bactÃria. A maioria dos estudos evidenciou forte associaÃÃo do gene cagE com doenÃa ulcerosa pÃptica e do aumento dos sÃtios de fosforilaÃÃo EPIYA C com doenÃas gÃstricas mais graves. No entanto, a associaÃÃo clÃnica e a funÃÃo dos sÃtios de fosforilaÃÃo de tirosina da proteÃna CagA â EPIYA e do gene cagE, ainda nÃo foi completamente elucidada, existindo diferenÃas geogrÃficas. O objetivo do presente estudo foi caracterizar as cepas de H. pylori quanto à presenÃa dos sÃtios de fosforilaÃÃo de tirosina da proteÃna CagA â EPIYA e do gene cagE em pacientes com gastrite e Ãlcera pÃptica, e correlacionar a presenÃa desses genÃtipos com as afecÃÃes gÃstricas. A genotipagem das cepas de H. pylori foi realizada atravÃs da tÃcnica de PCR e do sequenciamento dos sÃtios de fosforilaÃÃo EPIYA. Foram avaliados 137 pacientes dispÃpticos H. pylori positivo, 72 com Ãlcera pÃptica e 65 com gastrite. Das 137 cepas genotipadas 68,6% foram cagA positivas. Das cepas cagA estudadas, 96,8% apresentaram pelo menos 1 sÃtio C de fosforilaÃÃo EPIYA, sendo que 53,2% destas apresentaram somente um sitio, 43,6% tinham dois ou mais sÃtios C e 3,2% nÃo apresentaram sitio C. As cepas mistas com dois padrÃes EPIYA estavam presentes em 23,4% das amostras estudadas. O grupo de pacientes com gastrite apresentou maior prevalÃncia do genÃtipo EPIYA ABCCC; o mesmo nÃo foi observado no grupo de pacientes com Ãlcera pÃptica, sendo essa associaÃÃo com as afecÃÃes gÃstricas estudadas significante (p= 0,001). Dos 137 pacientes estudados, 65 foram positivos para cagE (47,4%). Houve associaÃÃo inversa significativa entre o genÃtipo cagE e o grupo de pacientes com gastrite (O.R=0,345; p=0,002). Houve associaÃÃo significativa entre o genÃtipo cagE com doenÃa ulcerosa pÃptica (O.R=2,898; p=0,002) e com o subgrupo de pacientes com Ãlcera duodenal (O.R=3,839; p=0,001). Esse estudo sugere que na amostra populacional estudada a presenÃa do genÃtipo EPIYA com trÃs sÃtios C estava associada à gastrite e ausente nos pacientes com ulcera pÃptica. Encontrou-se associaÃÃo do gene cagE a Ãlcera duodenal.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Gastric cancer is associated with H. pylori infection, environmental factors and genetic predisposition. Relatives of gastric cancer patients are at increased risk to develop cancer, possibly, due to infection by more virulent H. pylori strains and genetic predisposition associated with pro-inflammatory cytokines, which may, in turn, result in an exaggerated inflammatory response and oxidative stress. The aim of this study was to: 1) evaluate the oxidative stress in dispeptic patients without family history of gastric cancer (NRC), patients with gastric cancer (GC) and relatives of gastric cancer (RC); 2) evaluate the prevalence of the H. pylori genotypes homA, homB and cagE in RC and NRC and determine if the cagE gene is associated with GC as well as the pattern of cagA EPYIA phosphorylation in GC. This study was performed at the Hospital UniversitÃrio Walter CantÃdeo (HUWC) in Fortaleza. Blood samples were collected and centrifuged to obtain serum, in order to measure the concentration of reduced glutathione (GSH), an anti-oxidant, and Malondealdehyde (MDA), a product of lipid peroxidation. DNA extraction was performed in gastric biopsies, followed by PCR and electrophoresis for ureA, vacA, homA, homB, cagA e cagE genes. The concentrations of MDA and GSH were evaluated in 213 patients (54 CG, 91 RC e 68 NRC). The group of CG had higher MDA and lower GSH concentrations when compared to the groups without cancer (p=0.001 and p=0.005, respectively). The H. pylori genotypes were evaluated in 186 patients (48 CG, 74 RC and 64 NRC). The overall cagE prevalence was 54.3% (101/186), 70% (52/74) in the RC group, 52% (25/48) in the GC group and 36% (24/87) in the NRC group. A significant and independent association was found between the cagE genotype and the RC group (OR 3,357; IC 2,026 - 9,476 95%: p=0,001) and between the homB genotype and the RC group (OR (OR 2,357; IC 95%: 1,401 â 3,961; p=0,001). Forty six cagA positive samples from the GC group were included for gene sequencing to evaluate the EPIYA phosphorylation sites of the CagA protein. All of the strains studied had an EPIYA-C pattern and approximately 50% of the strains were mixed. This study demonstrates that patients with gastric cancer are under higher levels of oxidative stress. The group of RC have a higher prevalence of cagE and homB. The group of GC patients had a high prevalence of mixed strains regarding CagA EPIYA-C phosphorylation pattern. Decreasing the lipid peroxidation, increasing the antioxidant reserve and identifying groups at risk of infection with more virulent strains of H. pylori may be important to prevent gastric cancer. / O cÃncer gÃstrico està relacionado a infecÃÃo por H. pylori, fatores ambientais e predisposiÃÃo genÃtica. Familiares de pacientes com cÃncer gÃstrico (FC) tÃm maior risco de desenvolvimento da doenÃa, possivelmente, em decorrÃncia da infecÃÃo por cepas mais virulentas de H. pylori e predisposiÃÃo genÃtica relacionado a citocinas prÃ-inflamatÃriase o stress oxidativo. O objetivo do estudo foi: 1) avaliar o estresse oxidativo em pacientes portadores de cÃncer gÃstrico (CG), dispÃpticos sem histÃria familiar de cÃncer gÃstrico (NFC), e familiares de cÃncer gÃstrico de primeiro grau (FC); 2) averiguar em FC e NFC a prevalÃncia dos genÃtipos de H. pylori homA, homB, cagE e, em pacientes portadores de CG, avaliar se o gene cagE està associado a essa neoplasia e o padrÃo dos sÃtios de fosforilaÃÃo EPYIA da proteÃna cagA. Estudo foi realizado no Hospital UniversitÃrio Walter CantÃdio. O estresse oxidativo foi avaliado no soro atravÃs da dosagem de glutationa reduzida (GSH), antioxidante, e malondialdeÃdo (MDA), produto da peroxidaÃÃo lipÃdica. Realizou-se extraÃÃo de DNA da biÃpsia gÃstrica, seguida de PCR e eletroforese para os genes ureA, vacA, homA, homB, cagA e cagE. Foi avaliado a concentraÃÃo sÃrica de MDA e GSH em 213 indivÃduos (54 CG, 91 FC e 68 NFC). O grupo CG apresentou concentraÃÃes de MDA mais elevado e de GSH mais baixo quando comparado com o grupo sem neoplasia (p=0.001 e p=0.005, respectivamente). Os genÃtipos de H. pylori foram avaliados em 186 pacientes (48 CG, 74 FC e 64 NFC). A prevalÃncia geral de cagE na amostra foi de 54,3% (101/186), sendo 70% (52/74) no grupo FC, 52% (25/48) no grupo de CG e 36% (24/87) no grupo NFC. Houve associaÃÃo significante, mesmo apÃs anÃlise multivariada, entre o genÃtipo cagE e o grupo de FC (OR 3,357; IC 2,026 - 9,476 95%: p=0,001) e entre homB e o grupo FC (OR 2,357; IC 95%: 1,401 â 3,961; p=0,001). Foram incluÃdas 46 amostras de CG cagA positivas para avaliaÃÃo dos sÃtios EPIYA da proteÃna CagA. Todas as cepas avaliadas apresentaram padrÃo EPYIA-C, e aproximadamente 50% de cepas mistas. O presente estudo mostra que pacientes portadores de cÃncer gÃstrico possuem estresse oxidativo aumentado. Os indivÃduos FC estÃo infectados com maior prevalÃncia por cepas virulentas de H. pylori, cagE e homB. AlÃm disso, os pacientes portadores de cÃncer gÃstrico apresentaram prevalÃncia elevada de cepas mistas de H. pylori quanto ao segmentos EPIYA C da proteÃna CagA. Possivelmente, diminuir a peroxidaÃÃo lipÃdica, aumentar a reserva de antioxidantes e identificar grupos de maior risco quanto ao perfil de cepas de H. pylori poderà ser importante para a prevenÃÃo do cÃncer gÃstrico.

Orientadora: ProfS DrS Iara José de Messias-Reason / Co-orientadora: ProfS DrS Lilian Pereira-Ferrari / Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciencias da Saúde, Programa de Pós-Graduaçao em Medicina Interna. Defesa : Curitiba, 2008 / Inclui bibliografia

La prima parte del nostro studio riguarda la tecnica LAMP (Loop-mediated isothermal amplification), una tecnica di amplificazione isotermica recentemente inventata (Notomi et al., 2000). Essa presenta notevoli vantaggi rispetto alle tradizionali PCR: non necessita di strumentazioni sofisticate come i termociclatori, può essere eseguita da personale non specializzato, è una tecnica altamente sensibile e specifica ed è molto tollerante agli inibitori. Tutte queste caratteristiche fanno sì che essa possa essere utilizzata al di fuori dei laboratori diagnostici, come POCT (Point of care testing), con il vantaggio di non dover gestire la spedizione del campione e di avere in tempi molto brevi risultati paragonabili a quelli ottenuti con la tradizionale PCR. Sono state prese in considerazione malattie infettive sostenute da batteri che richiedono tempi molto lunghi per la coltivazione o che non sono addirittura coltivabili. Sono stati disegnati dei saggi per la diagnosi di patologie virali che necessitano di diagnosi tempestiva. Altri test messi a punto riguardano malattie genetiche del cane e due batteri d’interesse agro-alimentare. Tutte le prove sono state condotte con tecnica real-time per diminuire il rischio di cross-contaminazione pur riuscendo a comprendere in maniera approfondita l’andamento delle reazioni. Infine è stato messo a punto un metodo di visualizzazione colorimetrico utilizzabile con tutti i saggi messi a punto, che svincola completamente la reazione LAMP dall’esecuzione in un laboratorio specializzato. Il secondo capitolo riguarda lo studio dal punto di vista molecolare di un soggetto che presenza totale assenza di attività mieloperossidasica all’analisi di citochimica automatica (ADVIA® 2120 Hematology System). Lo studio è stato condotto attraverso amplificazione e confronto dei prodotti di PCR ottenuti sul soggetto patologico e su due soggetti con fenotipo wild-type. Si è poi provveduto al sequenziamento dei prodotti di PCR su sequenziatore automatico al fine di ricercare la mutazione responsabile della carenza di MPO nel soggetto indicato. / The first part of the present study concerns the LAMP technique (Loop-Mediated Isothermal Amplification), an isothermal amplification technique recently developed (Notomi et al., 2000). LAMP has many advantages over traditional PCR: it doesn’t require sophisticated instruments like thermal cyclers, it can be performed by unskilled staff, it is a highly sensitive and specific technique and it is very tolerant to inhibitors. All these characteristics make it suitable to be used outside diagnostic laboratories, as POCT (Point-of-care testing), with the advantage of not having to send the sample and obtaining results as accurate as PCR tests and in very short times. We designed and optimized assays to detect bacteria that require a very long time for cultivation or that are not even cultivable. We drew assays for the diagnosis of viral diseases that require to be diagnosed as soon as possible. We developed a test to assess two genetic diseases of the dog and two food contaminating bacteria. All tests were carried out using real-time technique to decrease the risk of cross-contamination. Finally, we developed a colorimetric method for showing results which can be applied to all of the assays we optimized. The second section presents the molecular study of a subject who had myeloperoxidase deficiency at the automated cytochemistry analysis (ADVIA ® 2120 Hematology System). The study was conducted through amplification and comparison of the PCR products obtained from the pathological subject and on two subjects with wild-type phenotype. The products were sequenced using an automated sequencer in order to find the responsible mutation for the MPO deficiency in the indicated subject.

Disorders of the somatosensory system causing neuropathic pain (NP) are common in people. In small animals medicine, this condition remains underestimated probably due to the lack of standardized assessment methods and studies focused on the description of structures playing a key role in developing and maintaining neuropathic pain, such as dorsal root ganglia (DRG). Hence, the aims of the present research project, structured in three different studies, were: a) investigate the possible effects of the reduction in radicular blood flow on the vascular density, ganglionic and endoneurial ischemia and shift in neuronal metabolism, in fifteen L7-DRG from a total of eight dogs suffering from painful compressed spinal L7 nerve roots (NRs) and DRG; b) perform a preliminary characterization of the expression of substance P, calcitonin gene-related peptide, calbindin D-28k, neuronal nitric oxide synthase and the colocalization of those molecules in lumbosacral DRG neurons of three neurologically normal dogs; c) Investigate the prevalence, risk factors and owners’ perception of their pets quality of life of the Phantom Complex (PC) in a client-owned population of 107 dogs with limb amputation through an online survey. Main results were: a) Our findings highlight a significant neuronal distress in compressed DRG. Despite the similarity of vascular density between controls and compressed L7-DRG, we found a significant increased in neuronal immunoreactivity for hypoxia-related markers and for markers related to glycolytic cellular metabolism. b) The wide variability of CGRP-, SP-, CALB-, and nNOS-immunoreactivity among different species was confirmed. Further studies are required in order to better define the characterization of those markers in canine DRG. c) Our study demonstrates for the first time the presence of PC.Significant risk factors associated with the frequency of post-amputation pain episodes are duration of pain before amputation and time between diagnosis and amputation.

Negli ultimi anni si è assistito ad un miglioramento della qualità di vita dei piccoli animali che, oltre ad aumentarne l'aspettativa di vita, ha determinato un aumento della frequenza di patologie associate all'età medio-avanzata, quali le patologie renali croniche. Il presente studio si fonda sulla necessità, sempre più sentita nella pratica clinica veterinaria, di poter fornire al proprietario del paziente affetto da CKD, una serie di parametri che, oltre a fungere da target terapeutico, possano aiutare a comprenderne la prognosi. Lo studio ha valutato una popolazione di cani affetti da CKD e ne ha seguito o ricostruito il follow-up, per tutto il periodo di sopravvivenza fino al momento dell’exitus. Di tali soggetti sono stati raccolti dati relativi ad anamnesi, esame clinico, misurazione della pressione arteriosa, diagnostica per immagini, esami ematochimici, analisi delle urine ed eventuale esame istologico renale. È stato possibile individuare alcuni importanti fattori prognostici per la sopravvivenza in pazienti con CKD. Oltre a fattori ben noti in letteratura, come ad esempio elevati valori di creatinina e fosforo, o la presenza di proteinuria, è stato possibile anche evidenziare il ruolo prognostico negativo di alcuni parametri meno noti, ed in particolare delle proteine di fase acuta positive e negative, e del rapporto albumina/globuline. Una possibile spiegazione del valore prognostico di tali parametri risiede nel ruolo prognostico negativo dell’infiammazione nel paziente con CKD: tale ruolo è stato suggerito e dimostrato nell’uomo e avrebbe alla base numerosi possibili meccanismi (sviluppo di anemia, complicazioni gastroenteriche, neoplasie, etc.), ma dati analoghi sono mancanti in medicina veterinaria. Una seconda possibile spiegazione risiede nel fatto che potenzialmente i livelli delle proteine di fase acuta possono essere influenzati dalla presenza di proteinuria nel paziente con CKD e di conseguenza potrebbero essere una conferma di come la proteinuria influenzi negativamente l'outcome. / In recent years there has been an improvement in the quality of life of small animals. In addition of increasing life expectancy, this has led to an increase in the frequency of diseases associated with elderly, such as chronic kidney disease. The present study is based on the need, in the veterinary practice, to be able to provide the owner of a patient with CKD some parameters which can help to understand the prognosis, besides being useful as a therapeutic target. The study evaluated a population of dogs with CKD and followed these dogs or rebuilt the follow-up, until the exitus. We collected data on medical history, clinical examination, blood pressure measurement, diagnostic imaging, blood tests, urinalysis and, when possible, renal histology. It was possible to identify important prognostic factors for survival in patients with CKD. The prognostic role of high levels of creatinine and phosphorus, or the presence of proteinuria is well known in the literature. It was also possible to emphasize the negative prognostic role of some less known parameters, such as acute phase proteins and albumin to globulin ratio. One possible explanation of the prognostic value of these parameters lies in the negative prognostic role of inflammation in patients with CKD: this role has been suggested and demonstrated in humans and has many possible underlying mechanisms (development of anemia, gastrointestinal complications, cancer, etc.), but similar data are missing in veterinary medicine. A second possible explanation lies in the fact that the levels of acute phase proteins may be influenced by the presence of proteinuria in the patient with CKD and therefore may be a confirmation of how proteinuria negatively affects the outcome.

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