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Heterozygous mutations and deletions of the gene that encodes the transcription factor hepatocyte nuclear factor 1β (HNF1B) are the commonest known monogenic cause of developmental kidney disease. However, diagnosis remains challenging due to phenotypic variability and frequent absence of a family history. There is also no consensus as to when HNF1B genetic testing should be performed. This thesis includes work looking at the identification of HNF1B-associated disease. An HNF1B score was developed in 2014 to help select appropriate patients for genetic testing. The aim in chapter 2 was to test the clinical utility of this score in a large number of referrals for HNF1B genetic testing to the UK diagnostic testing service for the HNF1B gene. An HNF1B score was assigned for 686 referrals using clinical information available at the time of testing; performance of the score was evaluated by receiver-operating characteristic curve analysis. Although the HNF1B score discriminated between patients with and without a mutation/deletion reasonably well, the negative predictive value of 85% reduces its clinical utility. HNF1B-associated disease is due to an approximate 1.3 Mb deletion of chromosome 17q12 in about 50% of individuals. This deletion includes HNF1B plus 14 additional genes and has been linked to an increased risk of neurodevelopmental disorders, such as autism. The aim in chapter 3 was to compare the neurodevelopmental phenotype of patients with either an HNF1B intragenic mutation or 17q12 deletion to determine whether haploinsufficiency of the HNF1B gene is responsible for this aspect of the phenotype. Brief behavioural screening showed high levels of psychopathology and impact in children with a deletion. 8/20 (40%) patients with a deletion had a clinical diagnosis of a neurodevelopmental disorder compared to 0/18 with a mutation, P=0.004. 17q12 deletions were also associated with more autistic traits. Two independent clinical geneticists were able to predict the presence of a deletion with a sensitivity of 83% and specificity of 79% when assessing facial dysmorphic features as a whole. These results demonstrate that the 17q12 deletion but not HNF1B intragenic mutations are associated with neurodevelopmental disorders; we conclude that the HNF1B gene is not involved in the neurodevelopmental phenotype of these patients. Extra-renal phenotypes frequently occur in HNF1B-associated disease, including diabetes mellitus and pancreatic hypoplasia. Faecal elastase-1 levels have only been reported in a small number of individuals, the majority of which have diabetes. In chapter 4 we measured faecal elastase-1 in patients with an HNF1B mutation or deletion regardless of diabetes status and assessed the degree of symptoms associated with pancreatic exocrine deficiency. We found that faecal elastase-1 deficiency is a common feature of HNF1B-associated renal disease even when diabetes is not present and pancreatic exocrine deficiency may be more symptomatic than previously suggested. Faecal elastase-1 should be measured in all patients with a known HNF1B molecular abnormality complaining of chronic abdominal pain, loose stools or unintentional weight loss. Hypomagnesaemia is a common feature of HNF1B-associated disease and is due to renal magnesium wasting. The aim in chapter 5 was to measure both serum and urine magnesium and calcium levels in individuals with an HNF1B molecular defect and compare to a cohort of patients followed up in a general nephrology clinic in order to assess their potential as biomarkers for HNF1B-associated disease. The results of this pilot study show that using a cut-off for serum magnesium of ≤0.75 mmol/L was 100% sensitive and 87.5% specific for the presence of an HNF1B mutation/deletion. All individuals in the HNF1B cohort had hypermagnesuria with fractional excretion of magnesium >4%; a cut-off of ≥4.1% was 100% sensitive and 71% specific. This suggests serum magnesium levels and fractional excretion of magnesium are highly sensitive biomarkers for HNF1B-associated renal disease; if these results are confirmed in a larger study of patients with congenital anomalies of the kidneys or urinary tract they could be implemented as cheap screening tests for HNF1B genetic testing in routine clinical care.

A capacidade de adquirir, com facilidade, resistência à terapia antimicrobiana torna-se uma das características mais estudadas em Acinetobacter sp., mundialmente conhecido por estar relacionado a surtos de infecções associadas à assistência a saúde (IAAS) e por ser apontado como um grave problema de saúde pública. Embora isolados clínicos desse gênero sejam extensivamente estudados quanto à presença e a diversidade de genes do tipo OXA-carbapenemases,existem poucos estudos sobre essa relação em isolados ambientais. Este trabalho teve como objetivo determinar a presença de carbapenemases do tipo OXA (e sua diversidade), de ISAba1 e de sua relação com as carbapenemases em isolados clínicos e de esgoto hospitalar de Acinetobacter sp. na cidade de Porto Alegre, Rio Grande do Sul. Um total de 310 isolados (145 clínicos e 165 ambientais) foram submetidos a análise por PCR das regiões genéticas de interesse. A diversidade dos genes do tipo blaOXA-51 foi realizada por DGGE. Os resultados indicam a presença do gene blaOXA-58 em um isolado, pela primeira vez no Brasil. A sequência ISAba1 foi frequentemente encontrada (92,9%), porém, a sua associação com blaOXA-51 foi baixa e encontrada em 9,8% dos isolados clínicos e em 6,5% dos isolados ambientais. A análise de diversidade revelou uma baixa freqüência de alelos de OXA-51 entre os isolados estudados, sendo blaOXA-65 a variante mais encontrada. Contudo, podemos afirmar que o esgoto hospitalar analisado constitui-se de uma fonte de contaminação de bactérias patogênicas e que as precárias políticas de saneamento podem proporcionar a disseminação de multirresistência para o meio ambiente. / The ability of easily acquiring resistance to antimicrobial therapy becomes one of the most studied features in Acinetobacter sp., world renowned for being related to outbreaks of infection associated with health care and for being appointed as a serious public health problem. Although clinical isolates of this genus are extensively studied for the presence and diversity of OXAcarbapenemase genes, there are few studies about this relationship in environmental isolates. This study aimed to determine the presence of OXAtype carbapenemases (and yours diversity), of ISAba1 and its relationship with carbapenemases in clinical and hospital sewage isolates of Acinetobacter sp. in Porto Alegre, Rio Grande do Sul. A total of 310 strains (145 of clinical origin and 165 of environmental origin) were subjected to PCR analysis of genetic regions of interest. The diversity of blaOXA-51-like genes was performed by DGGE. The results indicate the presence of the gene blaOXA-58 in an isolated, for the first time in Brazil. ISAba1 was frequently found (92.9%) but its association with blaOXA-51-like was low and was found in 9.8% of clinical isolates and in 6.5% of environmental isolates. The diversity analysis showed that there is a low frequency of alleles of OXA-51 among the studied isolates being blaOXA-65 variant the most often found. However, we can state that the hospital sewage is considered to be a source of pathogenic bacteria contamination and that the poor sanitation politics contributes to the spread of multidrug resistance in the environment.

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Made available in DSpace on 2015-12-06T23:05:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2005 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / CAPES: BEX0612/02-2 / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2015-12-06T23:05:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2005 / As infecções causadas por P. aeruginosa são freqüentemente de difícil tratamento devido à virulência e a resistência a vários antimicrobianos apresentados por este patógeno. Os carbapenens são geralmente o tratamento de escolha para infecções causadas por P. aeruginosa multirresistentes, porém, a resistência a estes compostos tem aumentado rapidamente, principalmente, devido ao surgimento de amostras produtoras de metalo-ß-lactamases. Os objetivos deste estudo foram: i) avaliar o perfil de sensibilidade a antimicrobianos das amostras de P. aeruginosa resistentes aos carbapenens isoladas de hospitais brasileiros; ii) avaliar a freqüência da produção de MPL entre as amostras de P. aeruginosa resistentes aos carbapenens; iii) avaliar o perfil de sensibilidade a antimicrobianos das amostras de P. aeruginosa produtoras de MβL pela técnica de microdiluição em caldo; iv) detectara presença dos genes biasSPM-1, blaIMP-1, blaVIM-1, e blaVIM-2 entre as amostras de P. aeruginosa produtoras de MßL; v) avaliar a presença de variantes alélicos do gene blasIPM-1, entre as amostras de P. aeruginosa produtoras da MPL do tipo SPM-1, e vi) avaliar a similaridade genética entre as amostras de P. aeruginosa resistentes aos carbapenens produtoras de MßL. Foram avaliadas 206 amostras de P. aeruginosa resistentes aos carbapenens isoladas entre 2001 e 2003, de 25 centros médicos. As 206 amostras foram testadas pelo método de disco-difusão. Os testes de sensibilidade foram realizados de acordo com a padronização do "National Committee for Clinical Laboratory Standards" (NCCLS). Os isolados resistentes aos carbapenens foram triados para a produção de metalo-ß-lactamases pelo teste de aproximação de discos utilizando dois inibidores enzimáticos (EDTA e ácido 2mercaptopropiônico). A produção de MßL foi confirmada pela metodologia do Etest® (fita Etest©MBL). Em seguida foi realizada a técnica de PCR para a pesquisa dos genes blaIMP-1, blaVIM-1, bIaVIM-2 e blaSPM-1. A avaliação da similaridade genética entre as amostras produtoras de MßL foi realizada pela técnica da ribotipagem automatizada e PFGE. Para fins comparativos, também foram selecionadas para análise da similaridade genéticaamostras não produtoras de MßL isoladas em centros médicos, que apresentavam amostras de P. aeruginosa produtoras de MPL. Altas taxas de resistência a maioria dos antimicrobianos foram observadas entre as amostras de P. aeruginosa resistentes aos carbapenes…(au). / Infections caused by Pseudomonas aeruginosa are often difficult to treat because of its virulence and antimicrobial resistance. Carbapenems are usually active against multiresistant P. aeruginosa, but resistant to these compounds has been increasing rapidly. High-level resistance to carbapenems is still uncommon in P. aeruginosa and is usually indicative of metallo-β-lactamases (MβL). The purpose of the study were to evaluate the antimicrobial susceptibility profiles of carbapenems resistant P. aeruginosa isolated from Brazilian Hospitals, were to detect the presence of gene blaSPM-1, blaIMP-1, blaVIM-1 e blaVIM-2 and to evaluate the genetic similarity of P. aeruginosa resistant to carbapenems. Were evaluated 206 P. aeruginosa resistant to carbapenems collected during 2001 – 2003 from 25 medical centers. Antimicrobial susceptibilities testing to selected agents were determined by disk diffusion to 206 strains. The suscepptibility testing were to using the reference “National Committee for Clinical Laboratory Standards” (NCCLS). Carbapenems resistant isolates were screened for MβL production by disk approximation test using 2 enzyme inhibitors (EDTA and 2-mercaptopropionic acid). The procedure was confirmed by Etest (Etest MBL strip) and also by PCR assays using the following primers blaIMP-1, blaVIM-1, blaVIM-2 e blaSPM-1. The genetic similarity was performed in MβL producers strains and one negative-MβL strains for medical centers by automated ribotyping and PFGE. High resistance rates to majority of the antimicrobial agents were demonstrated. The polymyxins were the most active antimicrobial agents evaluated with 100,0% of susceptibility rate to all strains. The MβL production was detected in 98 (47,6%) of 206 strains. The blaSPM-1 gene was detected in 82 strains (83,7%) and blaIPM-1 gene in 3 strains (16,3%). Thirteen isolates did not show PCR amplification for none of the primers used. The analysis of genetic similarity showed a clonal diversity among the 98 carbapenems-resistant strains (46 ribogroups). CONCLUSION: This study indicates high prevalence of MβL producing in carbapenems-resistant P. aeruginosa and clonal dissemination these microrganism to a wide geographic area of Brazil. / BV UNIFESP: Teses e dissertações

The goal of this research project is to understand how a high sugar diet may affect pancreatic beta cell function. High glucose concentrations lead to an increase in lipid droplets and TORC1 in beta cells, which promote high basal secretion of insulin (Erion K.A. et al., JBC, 2015). SREBP is a key regulator of cholesterol and lipid synthesis and depends on TORC1 activity. The active form of SREBP is located in the nucleus. Does glucose-induced lipid synthesis in beta cells increase via SREBP? To answer this question, we propose: 1) To test the effect of high glucose (11mM) on nuclear SREBP in INS-1 cells in comparison to physiological glucose (4mM). 2) To determine if nuclear SREBP is affected when PIP4Kgamma (a regulator of TORC1) is suppressed. SREBP translocation from the cytosol to the nucleus was measured by immunofluorescence. SREBP processing was measured by western blot. SREBP1 activation increased in response to prolonged exposure to excess glucose after at least 48hrs. Both translocation and processing increased in 11mM glucose compared to 4mM glucose. When PIP4Kgamma was suppressed in INS-1 cells, SREBP translocation was inhibited. Lipid droplet accumulation was measured by nile red staining and it was found that de novo lipid synthesis only contributes to a small fraction of total lipid droplets. In conclusion, SREBP is activated in beta cells when in excess glucose. This may allow for lipid accumulation and basal hypersecretion of insulin due to over nutrition.

Diabetes is a metabolic disease characterized by the loss of functional pancreatic beta cells. The incidence of diabetes has risen rapidly in recent decades, which has been attributed at least partially to alterations in host-associated microbial communities, or microbiota. It is hypothesized that the loss of important microbial functions from the microbiota of affected host populations plays a role in the mechanism of disease onset. Because the immune system also plays a causative role in diabetes progression, and it is well documented that immune cell development and function are regulated by the microbiota, most diabetes microbiota research has focused on the immune system. However, microbial regulation is also required for the development of many other important tissues, including stimulating differentiation and proliferation. We therefore explored the possibility that the microbiota plays a role in host beta cell development. Using the larval zebrafish as a model, we discovered that sterile or germ free (GF) larvae have a depleted beta cell mass compared to their siblings raised in the presence of bacteria and other microbes. This dissertation describes the discovery and characterization of a rare and novel bacterial gene, whose protein product is sufficient to rescue this beta cell developmental defect in the GF larvae. Importantly, these findings suggest a possible role for the microbiota in preventing or prolonging the eventual onset of diabetes through induction of robust beta cell development. Furthermore, the loss of rare bacterial products such as the one described herein could help to explain why low diversity microbial communities are correlated with diabetes.

The DNA sequence of the gene coding for the OXA-2 beta-lactamase has been completed. The primary amino acid sequence deduced from the DNA sequence was used to study homologies with other beta-lactamases; no good homologies were observed with any class of beta-lactamase. A more detailed analysis has revealed from comparison of both primary and predicted secondary structure, that the OXA-2 beta-lactamase may be more closely related to class A enzymes. Confirmation that the OXA-2 beta-lactamase is a serine enzyme has come from the DNA sequence and the interaction with mechanism based inactivators. Clavulanic acid and a novel beta-lactamase inhibitor, BRL36148, both interact specifically with the OXA-2 beta-lactamase by branched pathway mechanisms. Analysis of inactivated enzymes by peptide mapping and isoelcetric focusing have revealed that more than one inactive enzyme species is formed with each inactivator. A specific DNA probe was designed to come entirely from within the coding sequence of the OXA-2 beta-lactamase gene. This probe was found to interact only with plasmids specifying the OXA-2 or OXA-3 type enzymes. These results confirm earlier observations that OXA-2 and OXA-3 beta-lactamases are related. Another probe which comprised the whole of the OXA-2 beta-lactamase gene as well as segments of DNA on either side of the gene, was found to hybridise with a number of resistance plasmids. This interaction suggests that multi resistance plasmids carry common segments of DNA. Characterisation of the physical properties of the OXA-2 enzyme by analytical ultracentrifugation have not only confirmed the dimeric nature of this beta-lactamase but have also shown that this enzyme forms aggregates at high protein concentrations. Probing of the enzyme structure with trypsin, strongly points to the OXA-2 beta-lactamase having a domain structure.

O crescimento da expectativa de vida média da população mundial tem sido acompanhado do aumento na prevalência de doenças neurodegenerativas, como a doença de Parkinson, isquemia cerebral e, especialmente, a doença de Alzheimer. A doença de Alzheimer (DA) caracteriza-se por um crescente declínio na função mental e memória do paciente. Estes sintomas são explicados por uma profunda perda neuronal e pela presença de alterações estruturais no tecido cerebral: as placas senis, extracelulares e os emaranhados neurofibrilares, intracelulares. O passo que desencadeia a neurodegeneração na DA é ainda objeto de estudo, mas a hipótese mais aceita atualmente é a de que a secreção anormal do peptídeo β amilóide (Aβ), principal componente das placas senis, dê início ao processo. O presente trabalho teve como objetivos investigar a toxicidade induzida pelo peptídeo Aβ1-42 e seu fragmento, o peptídeo Aβ25-35 em culturas organotípicas de hipocampo de ratos. Na primeira parte do trabalho, investigamos a toxicidade induzida pela exposição de culturas organotípicas de hipocampo de ratos a 10 ou 20 μM do peptídeo Aβ1-42, por 24 ou 72 h. Além disso, investigamos o envolvimento das proteínas iNOS e GSK-3β no mecanismo de morte celular induzida pelo peptídeo Aβ1-42. Na segunda parte do trabalho, investigamos a toxicidade induzida pelo fragmento Aβ25-35 na concentração de 25 μM, nos tempos de 1, 3, 6, 12, 24 ou 48 h de exposição às culturas organotípicas, e seu efeito sobre as proteínas Akt, GSK-3β e PTEN. A morte celular foi quantificada pela incorporação do iodeto de propídeo, corante marcador excluído de células sadias, e as proteínas foram quantificadas por imunodetecção com o uso de anticorpos específicos. Nossos resultados mostraram que o peptídeo Aβ1-42 apresentou toxicidade nas concentrações de 10 e 20 μM, induzindo a uma considerável morte celular após 72 h de exposição. A análise do imunoconteúdo da proteína iNOS mostrou um aumento significativo em relação ao controle, após 72 h de exposição ao peptídeo e na concentração de 20 μM. Os resultados obtidos na segunda parte do trabalho mostraram uma morte celular significativa apenas após 48 h de exposição ao peptídeo Aβ25- 35 na concentração de 25 μM. O tratamento com peptídeo Aβ25-35 levou ao aumento no estado de fosforilação da proteína Akt após 6 h de exposição e também da proteína GSK-3β, um potencial substrato da Akt. Após 12 h de exposição ao peptídeo, observamos uma diminuição de ambas as proteínas (Akt e GSK-3β). Porém, após 24 h de exposição ao peptídeo, a proteína Akt continua menos fosforilada enquanto que a proteína GSK-3β apresenta um novo pico de fosforilação. O imunoconteúdo da proteína fosfatase PTEN apresentou um aumento significativo em 24 h e 48 h. Os resultados do presente estudo mostraram que o tratamento das culturas organotípicas de hipocampo de ratos com o peptídeo Aβ1-42 como com o fragmento Aβ25-35 apresentou toxicidade após um período de aproximadamente 48 h de tratamento, e mostrou ser um bom modelo para o estudo de sua toxicidade. Com relação ao mecanismo investigado, os dados sugerem que a proteína iNOS pode estar envolvida na toxicidade induzida pelo peptídeo Aβ1-42. Além disso, sugerem que o fragmento Aβ25-35 possa exercer sua toxicidade através da inibição da via de sobrevivência celular PI3-K, por diminuir a fosforilação/ativação da proteína Akt, parecendo envolver a proteína fosfatase PTEN, principal regulador negativo da via PI3-K/Akt.