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No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoPolimorfismoIndel.pdf: 1495935 bytes, checksum: 9ccc7fbfe35c473aadbc780a1ecae2c2 (MD5) Previous issue date: 2014 / Introdução: O câncer consiste em um problema de saúde publica mundial, com estimativa de 27 milhões de casos novos e 17 milhões de mortes por câncer no ano de 2030. No Brasil, as estimativas para o câncer no ano de 2014, apontam a ocorrência de aproximadamente 580 mil casos novos. As fluoropirimidinas são usadas nos principais esquemas quimioterápicos direcionados a tumores do trato gastrointestinal. Nos últimos anos muito se tem investigado sobre causas de respostas individuais diferenciadas em relação ao tratamento quimioterápico; dessa forma têm-se buscado uma terapia individualizada que possa maximizar a eficácia dos medicamentos e minimizar os efeitos adversos associados aos fármacos. Objetivamos buscar a associação do INDEL (rs16430) do gene TYMS com o padrão de resposta ao tratamento oncológico de fármacos com base em fluoropirimidinas, de maneira a contribuir para o desenvolvimento da medicina personalizada. Material: Estudadas 151 amostras de sangue periférico de pacientes oncológicos tratados com fluoropirimidinas, da população da região amazônica brasileira com elevado grau de mistura interétinica. Foi genotipado um polimorfismo INDEL (rs16430) no gene TYMS envolvido na resposta ao tratamento com uso de fluoropirimidinas. Resultados: A investigação relatou que a maioria dos pacientes tinha doença avançada no momento do diagnóstico; 32,7% receberam tratamento com intenção paliativa; e 22,8% tratamento neoadjuvante. Nossos resultados evidenciam que o polimorfismo INDEL no gene TYMS demonstrou ter um efeito de proteção à progressão tumoral (p=0,033). Pacientes tratados com fluoropirimidinas que eram homozigotos selvagens (INS/INS) apresentaram uma proteção à progressão tumoral de 24% comparado com outros genótipos desse polimorfismo. As estimativas de ancestralidade genômica global da amostra investigada foram: 62,4% europeia; 25,2% nativo americana e 12,4% africana. Foi possível estabelecer uma correlação inversa entre o aumento da ancestralidade ameríndia e a presença de metástase (p=0,024). Conclusão: Estudos farmacogenéticos podem proporcionar uma terapia personalizada reduzindo mortalidade por toxicidades e aumentando a eficácia terapêutica, desta forma proporcionando um tratamento oncológico com melhores resultados clínicos. / Cancer is a public health problem worldwide, with an estimated of 27 million new cases and 17 million cancer deaths in 2030. In Brazil, estimates for cancer in 2014, indicate the occurrence of approximately 580 000 new cases. Fluoropyrimidines are used in the main chemotherapy regimens targeted to tumors of the gastrointestinal tract. Recently, much has been investigated on causes of different individual responses to chemotherapy.Thus, it has been sought an individualized therapy that can maximize drug efficacy and minimize adverse effects associated with drugs. We aimed to seek the association of an INDEL polymorphism (rs16430) in TYMS gene with the pattern of response to chemotherapy drugs based on fluoropyrimidines, in order to contribute to the development of personalized medicine. We studied 151 samples of cancer patients treated with fluoropyrimidine, from a population of the Brazilian Amazon region with high interethnic admixture. An INDEL polymorphism (rs16430) was genotyped in TYMS gene that is involved in the response to treatment using fluoropyrimidines. The research reported that most patients had advanced disease at diagnosis, of which 32.7% were treated with palliative intent, and 22.8% neoadjuvant treatment. Our results show that the INDEL polymorphism in the TYMS gene appears to have a protective effect on tumor progression (p = 0.033). Patients treated with fluoropyrimidine who were wild homozygous (INS / INS) had a 24% protection to tumor progression compared to other genotypes of this polymorphism. Estimates of global genetic ancestry of the sample investigated were: 62.4% European, 25.2% Amerindian and 12.4% African. It was possible to establish an inverse correlation between the increase of Amerindian ancestry and metastasis (p = 0.024). Pharmacogenetic studies can provide a personalized therapy toxicity reducing mortality and improving therapeutic efficacy, thereby providing a cancer therapy with better clinical results.

<p>Drugs and other foreign compounds must often be metabolised before they can be excreted from the body. One enzyme system that is responsible for this is the cytochrome P450 gene family (CYP). In this thesis, new sensitive molecular techniques have been used to study the human gene expression of some CYP enzymes, as well as the P-glycoprotein transporter (P-gp). The aim was to evaluate whether tissues other than the liver, e.g. the blood, could be used to assess an individual's drug metabolic capacity. Another aim was to investigate the gene expression in relation to the liver transplant process and a third aim was to evaluate the expression in gastrointestinal mucosa in both normal and inflamed mucosa.</p><p>We evaluated the CYP gene expression in paired specimens of liver and blood but found no correlation in the expression patterns of these two tissues. Instead, we found the opposite pattern, where, for example, CYP1B1 had the highest expression in the blood but the lowest in the liver and CYP2E1 was the enzyme with the highest expression in the liver. In an investigation of the expression of four different CYP enzymes and P-gp in liver transplants before and during the first year after transplantation, we found that the levels of all the CYP enzymes but not P-gp increased with time. We also found that the expression of CYP3A4 was inversely related to the normalised plasma levels of the immunosuppressive drugs cyclosporine and tacrolimus.</p><p>In the gastrointestinal tract, CYP2E1 was the enzyme with the highest mRNA expression compared with CYP3A4, CYP3A5 and the transporter P-gp. CYP3A4 has its highest expression in the duodenum compared with the expression in the stomach and the colon. CYP3A5 is expressed at a higher level than CYP3A4 in the colon. P-gp expression levels increase through the gastrointestinal tract to the left colon. Gene expression levels of CYP2E1 and CYP3A4 decrease in severely inflamed rectal mucosa. </p><p>In conclusion, this is a sensitive method for studying gene activity in a clinical situation, even though at this point we are not able to use blood or gastrointestinal mucosa as “surrogate” tissue to estimate an individual’s drug metabolic capacity. The studies in liver transplants and gastrointestinal mucosa are unique in that the gene expression is investigated during a clinical course of events.</p>

Drugs and other foreign compounds must often be metabolised before they can be excreted from the body. One enzyme system that is responsible for this is the cytochrome P450 gene family (CYP). In this thesis, new sensitive molecular techniques have been used to study the human gene expression of some CYP enzymes, as well as the P-glycoprotein transporter (P-gp). The aim was to evaluate whether tissues other than the liver, e.g. the blood, could be used to assess an individual's drug metabolic capacity. Another aim was to investigate the gene expression in relation to the liver transplant process and a third aim was to evaluate the expression in gastrointestinal mucosa in both normal and inflamed mucosa. We evaluated the CYP gene expression in paired specimens of liver and blood but found no correlation in the expression patterns of these two tissues. Instead, we found the opposite pattern, where, for example, CYP1B1 had the highest expression in the blood but the lowest in the liver and CYP2E1 was the enzyme with the highest expression in the liver. In an investigation of the expression of four different CYP enzymes and P-gp in liver transplants before and during the first year after transplantation, we found that the levels of all the CYP enzymes but not P-gp increased with time. We also found that the expression of CYP3A4 was inversely related to the normalised plasma levels of the immunosuppressive drugs cyclosporine and tacrolimus. In the gastrointestinal tract, CYP2E1 was the enzyme with the highest mRNA expression compared with CYP3A4, CYP3A5 and the transporter P-gp. CYP3A4 has its highest expression in the duodenum compared with the expression in the stomach and the colon. CYP3A5 is expressed at a higher level than CYP3A4 in the colon. P-gp expression levels increase through the gastrointestinal tract to the left colon. Gene expression levels of CYP2E1 and CYP3A4 decrease in severely inflamed rectal mucosa. In conclusion, this is a sensitive method for studying gene activity in a clinical situation, even though at this point we are not able to use blood or gastrointestinal mucosa as “surrogate” tissue to estimate an individual’s drug metabolic capacity. The studies in liver transplants and gastrointestinal mucosa are unique in that the gene expression is investigated during a clinical course of events.

Pancreatic endocrine tumors and gastrointestinal stromal tumors are rare. Evidence regarding prognostic factors, and in the former also treatment, is scarce. We evaluated the survival and prognostic factors in a consecutive series of 324 patients with pancreatic endocrine tumors treated at a single institution. Radical surgery, WHO classification, TNM stage, age and Ki67 ≥2% emerged as independent prognostic factors. Having a non-functioning tumor was not an independent prognostic marker, and neither was hereditary tumor disease. We present the first evaluation of the newly proposed TNM staging system for these patients. A separate analysis of well-differentiated neuroendocrine carcinomas is reported, suggesting tumor size ≥5cm and Ki67 ≥2% as negative prognostic markers in this group. The first 36 patients with advanced neuroendocrine tumors treated with temozolomide at our clinic were evaluated. The median time to progression was seven months. Fourteen percent showed partial regression and 53% stabilization of disease. Side effects were generally mild. Investigation of O6-methylguanine DNA methyltransferase revealed a low expression in a subset of tumors. Four out of five patients responding to treatment had tumors with low expression. Concomitant expression of the orexigen ghrelin and its receptor in pancreatic endocrine tumors is demonstrated. No significant difference in mean plasma ghrelin between patients and controls were found, but elevated plasma ghrelin was seen in five patients. We provide the first report of expression of ghrelin and its receptor in gastrointestinal stromal tumors. Concomitant expression was frequent, indicating the presence of an autocrine loop. The tumors also expressed the neuroendocrine marker synaptic vesicle protein 2. Together, these findings are suggestive of neuroendocrine features.

Gastrointestinal microflora has been shown to have a bi-directional relationship with the host immune system. A variety of fermentable carbohydrate polymers largely pass through the small intestine, providing fermentable substrates for gut microflora. Dietary fibre supplementation may provide a strategy for manipulating the intestinal bacterial profile, changing the interaction with the mucosal immune system, thereby modulating the host immune system. We used a BBc rat animal model to evaluate the effects of oat bran and wheat bran dietary fibre on the immune system. Previous collaborative efforts have shown that these dietary fibres can change the intestinal microflora, with wheat bran fibre showing a greater ability to influence colonic microbial community diversity. We have shown that dietary wheat bran fibre led to reduced IL-4 levels in the liver and T lymphocyte numbers in the Mesenteric Lymph Node and may be involved in reduced IgA levels in the cecal contents. In addition, IgA in the cecal contents was decreased while MLN B cell numbers increased in response to dietary wheat bran fibre. It was observed that neither wheat bran or oat bran treatments exerted any pro-inflammatory effects, with oat bran actually improving antioxidant status. These results suggest that both oat and wheat bran fibre treatments induce changes in the intestinal microflora, and that the microflora changes due to wheat fibre are associated with immunomodulatory effects on the host. This type of dietary fibre supplementation could ultimately provide a potential strategy for promoting health through microflora-associated effects on the immune system.

Thesis (Ph. D.)--University of Oklahoma. / Includes bibliographical references (leaves 148-162).

Background Transthyretin amyloid (ATTR) amyloidosis is a systemic disorder caused by amyloid deposits formed by misfolded transthyretin (TTR) monomers. Two main forms exist – wild-type and hereditary ATTR amyloidosis, the latter associated with TTR gene mutations. Wild-type ATTR amyloidosis has a late onset and primarily cardiac manifestations, whereas hereditary ATTR amyloidosis is a rare autosomal dominant condition with a considerable phenotypic diversity. Both disorders are present all over the world, but endemic areas of the hereditary form are found in Sweden, Portugal, Brazil and Japan. Gastrointestinal (GI) complications are common in hereditary ATTR amyloidosis and play an important role in the patients’ morbidity and mortality. Malfunction of the autonomic and enteric nervous systems has been proposed to contribute to the GI disturbances, but the underlying mechanisms have not been fully elucidated. The aims of this thesis were to assess the prevalence of GI disturbances for different subtypes of ATTR amyloidosis, to further explore the mechanisms behind these disturbances, and to evaluate the outcome of the patients’ GI function after liver transplantation, which currently is the standard treatment for hereditary ATTR amyloidosis. Methods The Transthyretin Amyloidosis Outcomes Survey (THAOS) is the first global, multicenter, longitudinal, observational survey that collects data on patients with ATTR amyloidosis. THAOS enrollment data were used to assess the prevalence of GI symptoms and to evaluate their impact on nutritional status (mBMI) and health-related quality of life (EQ-5D Index Score). Data from routine investigations of heart-rate variability and cardio-vascular response to tilt tests were utilized to evaluate the impact of autonomic neuropathy on the scintigraphically measured gastric emptying half-times in Swedish patients with hereditary ATTR amyloidosis. Gastric wall autopsy specimens from Japanese patients with hereditary ATTR amyloidosis and Japanese non-amyloidosis controls were analyzed with immunohistochemistry and computerized image analysis to assess the densities of interstitial cells of Cajal (ICC) and nervous tissue. Data from gastric emptying scintigraphies and validated questionnaires were used to evaluate the outcome of Swedish patients’ GI function after liver transplantation for hereditary ATTR amyloidosis. Results Sixty-three percent of the patients with TTR mutations and 15 % of those with wild-type ATTR amyloidosis reported GI symptoms at enrollment into THAOS. Subsequent analyses focused on patients with TTR mutations and, among them, unintentional weight loss was the most frequent symptom (32 %) followed by early satiety (26 %). Early-onset patients (&lt;50 years of age) reported GI symptoms more frequently than late-onset cases (70 % vs. 50 %, p &lt;0.01), and GI symptoms were more common in patients with the V30M mutation than in those with non-V30M mutations (69 % vs. 56 %, p &lt;0.01). Both upper and lower GI symptoms were significant negative predictors of nutritional status and health-related quality of life (p &lt;0.01 for both). Weak but significant correlations were found between gastric emptying half-times and the function of both the sympathetic (rs = -0.4, p &lt;0.01) and parasympathetic (rs = -0.3, p &lt;0.01) nervous systems. The densities of c-Kit-immunoreactive ICC were significantly lower in the circular (median density 0.0 vs. 2.6, p &lt;0.01) and longitudinal (median density 0.0 vs. 1.8, p &lt;0.01) muscle layers of the gastric wall in patients compared to controls. Yet, no significant differences in protein gene product 9.5-immunoreactive nervous cells were found between patients and controls either in the circular (median density 3.0 vs. 6.8, p = 0.17) or longitudinal (median density 1.4 vs. 2.5, p = 0.10) muscle layers. Lastly, the patients’ GI symptoms scores had increased slightly from before liver transplantation to the follow-ups performed in median two and nine years after transplantation (median score 7 vs. 10 vs. 13, p &lt;0.01). However, their gastric emptying half-times (median half-time 137 vs. 132 vs. 125 min, p = 0.52) and nutritional statuses (median mBMI 975 vs. 991 vs. 973, p = 0.75) were maintained at follow-ups in median two and five years after transplantation. Conclusion GI disturbances are common in hereditary ATTR amyloidosis and have a negative impact on the patients’ nutritional status and health-related quality of life. Fortunately, a liver transplantation appears to halt the progressive GI involvement of the disease, although the patients’ GI symptoms tend to increase after transplantation. An autonomic neuropathy and a depletion of gastrointestinal ICC seem to contribute to the GI disturbances, but additional factors must be involved.

No description available.

Current and potential future trends in the burden of acute gastrointestinal illness (AGI) in Rigolet, Nunatsiavut and Iqaluit, Nunavut, Canada were investigated in the context of climate change. A concurrent mixed methods design was used in which quantitative and qualitative data were concurrently collected and analyzed and then combined to better understand the burden of AGI. In-depth interviews with government stakeholders (n=11), PhotoVoice workshops (n=11), and two community surveys (n=185) were conducted to identify and characterize climate-sensitive health priorities in the Nunatsiavut region. Then, four cross-sectional retrospective surveys in Rigolet (two community censuses, n=462) and Iqaluit (two surveys with randomly selected households, n=1,055), as well as in-depth interviews with cases (n=9) were conducted to examine the incidence, risk factors, and healthcare seeking behavior of AGI. Finally, a scenario planning approach was used to identify and rank trends and conditions driving changes in future waterborne disease in Nunatsiavut. This involved in-depth interviews with national and international experts (n=20) and community focus group discussions (n=29). Climate-sensitive health priorities identified in Nunatsiavut included food security, water security, mental health, new hazards and safety concerns, and health services and delivery. The annual estimated incidence of self-reported AGI ranged from 2.9-3.9 cases/person/year in Rigolet and Iqaluit, which are the highest published estimates globally. Significant risk factors for AGI included food, water, animal exposure, and socio-economic conditions; while community interviewees perceived hygiene, retail food, tap water, boil water advisories, and personal stress to be important risk factors. The proportion of AGI cases seeking medical services ranged from 3-19%, which are among the lowest published rates globally. In the scenario planning process, critical drivers of AGI included ‘extreme weather events’; ‘technology development’; and ‘global interest in Northern resources’. These results provided information about AGI-related exposures and sensitivities to climate change, which can be used to provide information for public health planning, prioritization, and programming in Inuit regions. The improved understanding of AGI in two Canadian Aboriginal communities sheds light on the need to better understand the burden in sub-sets of the population that might be at higher risk, including Aboriginal populations in the context of climate change. / Vanier Canada Graduate Scholarship (CIHR); Public Health Agency of Canada; IRIACC initiative (CIHR, NSERC, SSHRC, IDRC); Nasivvik Centre for Inuit Health and Changing Environments

The aim of this study was to determine firstly if there exist variations in fibrolysis among herbivore microbial ecosystems and secondly, the effect on fibre hydrolysis of compositing the most active systems with ruminal microbial ecosystem harvested from a Jersey cow. A literature review pointed to the complexity of carbohydrate (fibre) and how the physical and chemical nature of the forage carbohydrate can present barriers that hinder digestion in the rumen, especially its association with hemicelluloses, pectin, lignin and tannins. Fresh rumen fluid was collected from fistulated herbivores (Jersey cow and sheep) and faecal samples from non-fistulated herbivores (buffalo, horse, impala, camel, elephant, llama, sheep, wildebeest and elephant). Crude protein samples were precipitated with 60% ammonium sulfate. Sample activities were monitored and optimised by incubating with carboxymethyl cellulose (CMC) for 2 h at 39°C. The crude protein samples precipitated from the 11 herbivore microbial ecosystems were active. This was confirmed by an increase in enzyme specific activity with a decrease in total crude protein concentration. In vitro pH optimisation showed a broad range of activity for all ecosystems (4.5-8.0) but for the zebra, horse and elephant which peaked at pH 5. In experiment two (Chapter 4), seasonal variation of the enzymes (exocellulase, endocellulase, cellobiase and xylanase) were monitored through winter and summer. Enzyme specific activity of exocellulase, endocellulase, cellobiase and xylanase were determined by incubation with the specific substrates, crystalline cellulose, CMC, pNPG and xylan, respectively. The amount of reducing sugar released was used to determine the enzyme specific activity. Exocellulase analysis was suitable in winter while summer was preferred for carboxymethyl cellulase and xylanase due to their relative abundance. Cellobiase analysis did not depend on any particular season. Eleven herbivore microbial ecosystems were characterised according to their fibrolytic enzyme specific activities. Enzyme catalytic activities were calculated from kinetic parameters (Km and Vmax) obtained from Eisenthal and Cornish-Bowded plots (Chapter 5). Fibrolytic enzyme expression as well as their activities differed among the 11 ecosystems (P<O.OOOI). They were classified into three groups based on fibrolytic enzyme concentrations; group A with high enzyme concentrations (horse, impala, zebra, wildebeest and the elephant), group B with intermediate (cow, llama, camel, buffalo and giraffe) and group C with low enzyme concentrations (sheep). Exocellulase activity was reasonably correlated with endocellulase activity (r = 0.8978). Xylanase activity was also correlated with carboxymethyl cellulase actvity (r = 0.7104). Enzyme kinetic studies revealed that crude protein samples from the horse, zebra, wildebeest and elephant had the highest enzyme catalytic activities. Microbial or enzyme composite systems were created from the most active ecosystems (horse, wildebeest and zebra) in an attempt to improve the Jersey cow system. These systems were B (cow and horse), C (cow and wildebeest), D (cow and zebra) and E (cow, horse, zebra and wildebeest). The specific activities and enzyme efficiencies of these new systems were determined and compared with system A (cow). Microbial synergism of these systems was also investigated by measuring the amount of gas produced and true degradability (TD) after 72 h of incubation. The composite systems Band E were the most active fibrolytic enzyme systems while C and D were intermediate when compared to that of A. In vitro microbial synergism assays showed that systems B, D, and E had the highest potential of improving milky maize stover (MM) and nutral detergent fibre (NDF) fermentation and degradability in Jersey cows. It was concluded that: (i) fibrolytic and hemicellulolytic enzyme concentrations vary from one season to another with the changing forages; (ii) microbial fibrolytic activities vary among animals grazing on the same field or different geographical regions; and (iii) lastly microbial synergisms of active ecosystems have the potential of improving fibre hydrolysis. However, there is a need to conduct in vivo experimentation to determine the real potential of these in vitro observations. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.