Submitted by Diogo Misoguti (diogo.misoguti@gmail.com) on 2016-06-14T14:37:43Z No. of bitstreams: 1 Publico-39222.pdf: 1977138 bytes, checksum: fc8aa8b1fdd51076eef8be3cb204c292 (MD5) / Approved for entry into archive by Diogo Misoguti (diogo.misoguti@gmail.com) on 2016-06-14T14:39:39Z (GMT) No. of bitstreams: 1 Publico-39222.pdf: 1977138 bytes, checksum: fc8aa8b1fdd51076eef8be3cb204c292 (MD5) / Made available in DSpace on 2016-06-14T14:39:39Z (GMT). No. of bitstreams: 1 Publico-39222.pdf: 1977138 bytes, checksum: fc8aa8b1fdd51076eef8be3cb204c292 (MD5) Previous issue date: 2007 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introdução: Muitos pacientes ainda morrem devido a infecções. Crescentes relatos da literatura atual têm atribuído ao intestino o papel de agravamento de doenças graves, e/ou a sua participação na gênese da sepse por mecanismo de translocação bacteriana (TB). Objetivo: Avaliar de forma cinética a TB e suas repercussões microcirculatórias e imunológicas. Método: Ratos Wistar-EPM (n=162) foram aleatoriamente distribuídos em grupo Sham (n=72) e grupo TB (n=90), e avaliados nos períodos de 2h, 6h, 24h, 72h, 7 e 14 dias, em relação a índice de translocação bacteriana; microscopia intravital; perfusão tecidual; e componentes celulares e humorais da linfa mesentérica por linfograma, citometria de fluxo e CBA-Flex. Resultado: A TB ocorreu somente no grupo TB e foi expressiva nas primeiras 24 horas tornando-se negativa somente com 7 dias. A conseqüência de um episódio de TB repercutiu na celularidade e citocinas pró e antiinflamatórias da linfa mesentérica associado a lesões da microcirculação e hipoperfusão tecidual de forma local e sistêmica. A citometria de fluxo mostrou que a linfa mesentérica eferente pós TB difere significativamente do grupo Sham quanto a número e subpopulação de linfócitos. Conclusão: Um episódio agudo de TB determinou uma recuperação máxima bacteriana com 6 horas e sua completa depuração entre 3 e 7 dias, além de provocar alterações da microcirculação intestinal e sistêmica associadas à ativação do GALT, principalmente no período de permanência das bactérias translocadas no hospedeiro, sendo a via linfática mesenterial uma importante rota na intercomunicação imunológica entre o ambiente intestinal e sistêmico. / Introduction: Many patients still die from infection. Currently, growing evidences have pointed out the role of the gut in the worsening of the critical illness, and/or its participation in the genesis of the bacterial translocation. Objective: Evaluate the bacterial translocation (BT) kinetics and its repercussion on microcirculation and immune response. Method: Wistar-EPM rats (n=162) were randomly distributed in Sham group (n=72) and BT group (n=90), and were monitored at 2h, 6h, 24h, 72h, 7 and 14 days periods in relation to bacterial translocation index, intravital microscopy, tissue perfusion index, and cellular and humoral components of the mesenteric lymph by lymphogram, flow cytometry and CBA-Flex. Results: Bacterial recovery was positive only in BT-group and it was expressive in the first 24 hours, becoming negative only after seven days. The consequences of one episode of BT could be seen in the cellularity and proinflammatory and antiinflammatory citokines of the efferent mesenteric lymph associated to local and systemic microcirculation and tissue hypoperfusion. The flow cytometry showed that efferent mesenteric lymph after BT was significantly different as compared to Sham group in relation to lymphocites number and their subtype. Conclusion: An acute episode of BT determined maximal bacterial recovery at 6h and its complete clearance occurred between 3 and 7 days, in addition to the gut and systemic microcirculation injuries due to the GALT activation, specially at the presence of translocated bacteria in the host, demonstrating the importance of the lymphatic route in the immune crosstalk between the gut and systemic enviroment. / FAPESP: 05/53826-7

This study is an outgrowth of a long felt need to make a survey of the existing district organization of t h e Gait Area to determine the educational effectiveness of the organizational pattern now in use. If the quality of educational opportunity offered by a given area is dependent upon the number of children plus taxable resources, does the present organizational pattern provide an equal opportunity to all children of the area?

No description available.

The swine whipworm, Trichuris suis, is a parasite present throughout the United States and is of concern to the swine industry worldwide because it is very pathogenic to growing pigs. The economic threat posed by T. suis and other intestinal parasite infections has created a strong interest in the development of parasite vaccines for the swine industry. Use of a vaccine either alone or with anthelmintics should reduce the economic losses. However, before effective parasite vaccines can be created, the swine gastrointestinal immune response to parasite antigens must be understood. In this study, an enzyme-linked immunospot (ELISPOT) assay was developed to measure total and antigen-specific IgG and IgA antibody secreting cells (ASC) from gut-associated lymphoid tissues (GALT) [mesenteric lymph node explants from jejunal region of small intestine (SI-MLN) and cecum in large intestine (C-MLN); and ileocecal Peyer's patches (IC-PP)] and lamina propria from the proximal colon removed from T. suis infected pigs. Tbe local antibody responses were compared to peripheral antibody responses found in the spleen and submandibular lymph nodes. The hypotheses to be tested was that parasite antigen-specific antibody secreting cells would be greatest in lymphoid tissue draining the site of infection compared to peripheral lymphoid tissues and that 19A ASC would predominate over IgG ASC in the lamina propria of T. suis infected pigs. The total IgG and IgA ASC frequencies for the spleen, SI-MLN, and ICPP did not significantly change (P> 0.05) over time. For C-MLN, there was a significant increase (p< 0.05) of total IgG ASC during a primary infection with T. suis. Antigen-specific IgG ASC were greatest at the GALT site closest to the infection, CMLN, whereas, antigen-specific IgA ASC predominated in the proximal colonic: lamina propria. Host protection to T. suis develops after anthelmintic: treatment of a primary exposure to parasite. The ELISPOT assay provided valuable information on the localization and compartmentalization of the swine gastrointestinal immune response to T. suis which resides in the cecum and proximal colon. In the future, this technique may be useful for monitoring gastrointestinal immune parameters of pigs exposed to a T. sllis vaccine. / Ph. D.

A galactosemia clássica ou tipo I (GC) é um erro inato do metabolismo da galactose causada pela deficiência da enzima galactose-1-fosfato uridiltransferase (GALT). É transmitida como uma doença autossômica recessiva e é tipicamente caracterizada pela intolerância neonatal a galactose, com complicações que vão desde icterícia, para os casos mais leves, à insuficiência hepática nos mais graves, e às complicações tardias, como disfunções motoras e reprodutivas. A galctosemia também é heterogénea do ponto de vista molecular, com 266 mutações diferentes descritas no gene GALT, algumas específicas para certas populações, refletindo o que se espera de alguns eventos de efeito fundador. O objetivo deste trabalho foi avaliar o perfil de mutações no gene GALT, dos pacientes brasileiros com galactosemia clássica e fazer um estudo da correlação do genótipo com o fenótipo, uma vez que se sabe que parte da variação observada na evolução clínica está relacionada com o nível de atividade residual da enzima e do genótipo. Para tanto, foram incluídos no estudo 31 pacientes com o diagnóstico bioquímico de galactosemia de diversas regiões do Brasil, que tiveram seus dados clínicos obtidos a partir de revisão de prontuários médicos e preenchimento de ficha clínica. Foi realizado o sequenciamento genético direto bidirecional do gene GALT e também estudos adicionais, como genotipagem do gene GALK1 de um paciente, estudo de ancestralidade de sete pacientes, além de simulações de patogenicidade in silico das novas mutações identificadas. Os principais achados clínicos dos pacientes que participaram deste estudo foram hepatomegalia, icterícia, baixo ganho pondero-estatural, vômito recorrente, anemia e catarata. As principais mutações que causam GC descritas na literatura foram identificadas neste estudo, como por exemplo, a p.Q188R, p.S135L e p.K285N, bem como o alelo Duarte 2 e seis mutações novas, p.M1T, p.R33S, p.P73S, IVS3+1G>A, IVS4+4A>C e p.Q169P. Este resultado era esperado, dada a elevada miscigenação da população brasileira. Alguns indivíduos foram diagnosticados através do teste de triagem neonatal expandido, que não está disponível rotineiramente a todos os recém-nascidos, portanto, começaram o tratamento dietético antes de desenvolverem os sinais e sintomas da doença. Para estes indivíduos não foi possível fazer uma análise da relação genótipo-fenótipo. Para os demais indivíduos esta relação foi consistente com o que é descrito na literatura, com os indivíduos homozigotos para a mutação p.Q188R com uma evolução mais grave do que os indivíduos que tinham pelo menos uma mutação p.S135L. Para os indivíduos com as mutações novas, foi observado um amplo espectro de fenótipos, como de pacientes que foi a óbito por insuficiência hepática e sepse à um caso assintomático. Este estudo amplia o espectro de mutação no gene GALT descrito na literatura e reforça a importância tanto do diagnóstico precoce quanto da introdução do tratamento dietético; também acrescenta mais evidências para a discussão sobre a introdução da galactosemia no programa de triagem neonatal do Brasil, onde a incidência da doença é estimada em cerca de 1:20.000. / Classical galactosemia (CG) or type I galactosemia is an inborn error of galactose metabolism caused by the deficiency of the galactose-1-phosphate uridyltransferase enzyme (GALT). It is transmitted as an autosomal recessive disease and is typically characterized by neonatal galactose intolerance, with complications ranging from neonatal jaundice and liver failure to late complications, such as motor and reproductive dysfunctions. Galactosemia is also heterogeneous from a molecular standpoint, with 266 mutations described to date in the GALT gene, some of them specific to certain populations, reflecting what is expected as some events of founder effect. The objective of this study was to evaluate the profile of mutations in the GALT gene of Brazilian patients with classical galactosemia and perform a genotype-phenotype correlation study, since it is known that part of the observed variation in clinical outcome is related to the level of residual enzyme activity and genotype. Therefore, this study included 31 patients with biochemical diagnosis of galactosemia from different regions of Brazil, who had their clinical data obtained from review of medical records and from a standardized case report form. We conducted a direct bidirectional sequencing of the GALT gene and also additional studies, as GALK1 genotyping for a patient, ancestrality study of seven patients and in silico simulation of pathogenicity for the new mutations identified. The main clinical features of the patients in this study were hepatomegaly, jaundice, low weight and height gain, recurrent vomiting, anemia and cataract. The major CG causing mutations described in the literature have been identified in this study, for example, p.Q188R, p.S135L and p.K285N, as well as the Duarte 2 allele and six novel mutations: p.M1T; p.R33S; p.P73S; IVS3+1G>A; IVS4+4A>C and p.Q169P. This result was expected, given the high miscegenation of the Brazilian population. Some individuals were diagnosed through expanded newborn screening test, which is not available routinely to all newborns, and so began dietary treatment before they develop signs and symptoms of the disease. For these individuals, was not possible to analyze the genotype-phenotype correlation. For other individuals this relationship was consistent with what is described in the literature, with the homozygous for p.Q188R mutation presenting a more severe phenotype than individuals who had at least one p.S135L mutation. For individuals with new mutations, was observed a wide range of phenotypes, from patients who died due to liver failure and sepsis to an asymptomatic case. This study expands the spectrum of mutations in the GALT gene described in the literature and reinforces the importance of early diagnosis and the introduction of dietary treatment; also adds more evidence to the discussion on the introduction of galactosemia in the neonatal screening program of Brazil, where the incidence of the disease is estimated at about 1:20,000.

A galactosemia clássica ou tipo I (GC) é um erro inato do metabolismo da galactose causada pela deficiência da enzima galactose-1-fosfato uridiltransferase (GALT). É transmitida como uma doença autossômica recessiva e é tipicamente caracterizada pela intolerância neonatal a galactose, com complicações que vão desde icterícia, para os casos mais leves, à insuficiência hepática nos mais graves, e às complicações tardias, como disfunções motoras e reprodutivas. A galctosemia também é heterogénea do ponto de vista molecular, com 266 mutações diferentes descritas no gene GALT, algumas específicas para certas populações, refletindo o que se espera de alguns eventos de efeito fundador. O objetivo deste trabalho foi avaliar o perfil de mutações no gene GALT, dos pacientes brasileiros com galactosemia clássica e fazer um estudo da correlação do genótipo com o fenótipo, uma vez que se sabe que parte da variação observada na evolução clínica está relacionada com o nível de atividade residual da enzima e do genótipo. Para tanto, foram incluídos no estudo 31 pacientes com o diagnóstico bioquímico de galactosemia de diversas regiões do Brasil, que tiveram seus dados clínicos obtidos a partir de revisão de prontuários médicos e preenchimento de ficha clínica. Foi realizado o sequenciamento genético direto bidirecional do gene GALT e também estudos adicionais, como genotipagem do gene GALK1 de um paciente, estudo de ancestralidade de sete pacientes, além de simulações de patogenicidade in silico das novas mutações identificadas. Os principais achados clínicos dos pacientes que participaram deste estudo foram hepatomegalia, icterícia, baixo ganho pondero-estatural, vômito recorrente, anemia e catarata. As principais mutações que causam GC descritas na literatura foram identificadas neste estudo, como por exemplo, a p.Q188R, p.S135L e p.K285N, bem como o alelo Duarte 2 e seis mutações novas, p.M1T, p.R33S, p.P73S, IVS3+1G>A, IVS4+4A>C e p.Q169P. Este resultado era esperado, dada a elevada miscigenação da população brasileira. Alguns indivíduos foram diagnosticados através do teste de triagem neonatal expandido, que não está disponível rotineiramente a todos os recém-nascidos, portanto, começaram o tratamento dietético antes de desenvolverem os sinais e sintomas da doença. Para estes indivíduos não foi possível fazer uma análise da relação genótipo-fenótipo. Para os demais indivíduos esta relação foi consistente com o que é descrito na literatura, com os indivíduos homozigotos para a mutação p.Q188R com uma evolução mais grave do que os indivíduos que tinham pelo menos uma mutação p.S135L. Para os indivíduos com as mutações novas, foi observado um amplo espectro de fenótipos, como de pacientes que foi a óbito por insuficiência hepática e sepse à um caso assintomático. Este estudo amplia o espectro de mutação no gene GALT descrito na literatura e reforça a importância tanto do diagnóstico precoce quanto da introdução do tratamento dietético; também acrescenta mais evidências para a discussão sobre a introdução da galactosemia no programa de triagem neonatal do Brasil, onde a incidência da doença é estimada em cerca de 1:20.000. / Classical galactosemia (CG) or type I galactosemia is an inborn error of galactose metabolism caused by the deficiency of the galactose-1-phosphate uridyltransferase enzyme (GALT). It is transmitted as an autosomal recessive disease and is typically characterized by neonatal galactose intolerance, with complications ranging from neonatal jaundice and liver failure to late complications, such as motor and reproductive dysfunctions. Galactosemia is also heterogeneous from a molecular standpoint, with 266 mutations described to date in the GALT gene, some of them specific to certain populations, reflecting what is expected as some events of founder effect. The objective of this study was to evaluate the profile of mutations in the GALT gene of Brazilian patients with classical galactosemia and perform a genotype-phenotype correlation study, since it is known that part of the observed variation in clinical outcome is related to the level of residual enzyme activity and genotype. Therefore, this study included 31 patients with biochemical diagnosis of galactosemia from different regions of Brazil, who had their clinical data obtained from review of medical records and from a standardized case report form. We conducted a direct bidirectional sequencing of the GALT gene and also additional studies, as GALK1 genotyping for a patient, ancestrality study of seven patients and in silico simulation of pathogenicity for the new mutations identified. The main clinical features of the patients in this study were hepatomegaly, jaundice, low weight and height gain, recurrent vomiting, anemia and cataract. The major CG causing mutations described in the literature have been identified in this study, for example, p.Q188R, p.S135L and p.K285N, as well as the Duarte 2 allele and six novel mutations: p.M1T; p.R33S; p.P73S; IVS3+1G>A; IVS4+4A>C and p.Q169P. This result was expected, given the high miscegenation of the Brazilian population. Some individuals were diagnosed through expanded newborn screening test, which is not available routinely to all newborns, and so began dietary treatment before they develop signs and symptoms of the disease. For these individuals, was not possible to analyze the genotype-phenotype correlation. For other individuals this relationship was consistent with what is described in the literature, with the homozygous for p.Q188R mutation presenting a more severe phenotype than individuals who had at least one p.S135L mutation. For individuals with new mutations, was observed a wide range of phenotypes, from patients who died due to liver failure and sepsis to an asymptomatic case. This study expands the spectrum of mutations in the GALT gene described in the literature and reinforces the importance of early diagnosis and the introduction of dietary treatment; also adds more evidence to the discussion on the introduction of galactosemia in the neonatal screening program of Brazil, where the incidence of the disease is estimated at about 1:20,000.

God-concepts of 24 third to sixth grade evangelical Christian children were compared with the children‘s logical thinking abilities in a mixed-method study. Measurements included the Children‘s Interview and the Group Assessment of Logical Thinking (GALT). God-concepts among the children were Biblical, comforter, communicates, creator, empowering, protector, provider, purposeful, human characteristics, lives in heaven, male, counselor, God is Jesus, all-knowing, loving, perfect, powerful, real, and parental. The majority of concrete thinkers conceptualized God as a gracious guide. The majority of transitional thinkers viewed God also as a gracious guide as well as a distant divinity. Implications were given for religious educators to develop a model for age-appropriate instruction and curriculum and to equip parents to promote spiritual development with children at home.

Transmissible spongiform encephalopathies (TSEs) or prion diseases, are subacute neurodegenerative diseases that infect humans and animals. Many of these diseases are acquired by peripheral exposure (e.g. orally). After oral exposure prion replication within the Peyer's patches (PP) in the small intestine is necessary for the efficient spread of the disease to the brain. Within the intestine, bacteria and pathogenic microorganisms can affect the status of the gut associated lymphoid tissue (GALT). GALT consists of PP and isolated lymphoid follicles (ILF) that maintain homeostasis and protect from infections. Therefore, factors which modify GALT status, might dramatically affect oral prion disease pathogenesis by influencing the uptake of prions from the gut lumen or expanding their distribution within the host. Chronic intestinal helminth infections are common in animals and in man, and can cause significant pathology within the intestine. Little is known of the effects that intestinal helminth infections may have on oral prion diseases susceptibility. Therefore, in this study the influence that co-infection with Heligmosomoides polygyrus (a natural pathogen of the mouse small intestine) may have on oral prion disease pathogenesis and susceptibility was determined. The studies consisted of groups of 4 (for H. polygyrus characterization and for early prion detection) and 8 (for H. polygyrus-prion co-infection to terminal stage) mice infected with H. polygyrus (orally) alone or subsequently infected with ME7 scrapie prions (orally) at different time-points after parasitic infection. The effects of the H. polygyrus infection alone, and on oral prion disease pathogenesis and susceptibility were then determined. Initially the characterization of H. polygyrus infection on the host intestine revealed that this parasite caused significant pathology in the small intestine and affected the GALT microarchitecture. In the PP follicles, H. polygyrus infection increased the area of follicular dendritic cell expression, altered the positioning of mononuclear phagocytes and increased M cell density. H. polygyrus infection also reduced the number of ILF in both the small and large intestines. Additional studies in mice co-infected with a low dose of prions, revealed that these pathological changes affected the survival time and disease susceptibility. Data also show that the extent of the effects on prion disease pathogenesis and susceptibility were dependent on the stage of the helminth infection at which the mice were orally-exposed to prions. Data demonstrate that co-infection with the gastrointestinal helminth H. polygyrus can influence oral prion disease pathogenesis and susceptibility. Helminth infections can significantly modify the microarchitecture of the gut and the GALT. Data presented suggest the pathological changes that pathogens such as small intestinal helminths cause, may also influence the uptake of prions from the gut lumen after oral exposure.

M. Tech. (Biotechnology, Department of Biosciences, Faculty of Computer and Applied Sciences) Vaal University of Technology / This study seeks to design and test specific short hairpin RNA (pshRNA) for their efficiency in knocking down the GALT gene RNA products thereby limiting the resultant enzyme activity. The following objectives were followed in designing the current study: 1. Designing a shorthairpin RNA (pshRNA) to target different regions of the coding sequence of the target GALT gene. 2. Propagating the pshRNAs in Escherichia coli (E.coli) and subsequently isolation of the respective plasmids for transfection. 3. Transfection of HeLa cells to test the efficiency of relevant pshRNAs in knocking down the GALT gene expression. 4. Transfection was followed by extraction of total mRNA, purification and quantification of total mRNA. 5. The GALT gene expression was qualitatively quantified against a house-keeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH) to evaluate efficiency of knockdown using real time PCR. The three newly designed pshRNA (pshRNA2, pshRNA3 and pshRNA4) targeting the GALT gene expression showed a knockdown efficiency of 171 %, 48 % and 200 %, respectively. The results of this study will be useful for future evaluation of the possible long term glycosylation patterns under proper UDP glucose/UDP galactose levels compared with variable defective GALT gene levels.

Certaines fibres alimentaires réduiraient l'apparition des cancers colorectaux mais l'hypothèse reste contreversée. Un des mécanismes fait intervenir la production par fermentation de butyrate. Nous avons testé deux fibres butyrogènes chimiquement différentes (fructo-oligosaccharides à chaîne courte scFOS, et amidon résistant) versus une fibre faiblement productrice de butyrate (son de blé désamidonné) chez la souris Min, un modèle de carcinogenèse intestinale spontanée. Seul le régime scFOS réduisait le nombre de tumeurs coliques et stimulait l'immunité locale. La nature chimique de la fibre ou bien les propriétés prébiotiques des scFOS (ils favorisent la croissance de bactéries lactiques qui agiraient sur la réaction immunitaire) sont donc impliquées. Deux autres types d'amidons résistants réduisaient le nombre de petites tumeurs. L'addition de bifidobactéries à l'un de ces deux régimes réduisait le nombre de petites tumeurs, mais augmentait celui de grosses tumeurs illustrant le concept de modulation par l'immunogénicité de la dualité immunofacilitation/surveillance. La déplétion en lymphocytes T associée au régime scFOS doublait le nombre de tumeurs par rapport aux souris immunocompétentes. Le régime scFOS diminuait l'expression du récepteur à l'IL2 à la surface des lymphocytes intra-épithéliaux (LIE) suggérant la mise en anergie temporaire du système immunitaire, mais pouvant aussi signifier l'implication d'une autre voie d'activation des LIE. Enfin, pour mettre en place une modélisation des intéractions lympho-épithéliales, nous avons croisé des souris C57BL6 et Min avec la souris transgénique Immortomouse et isolé à partir de primo-cultures de muqueuses des lignées cellulaires conditionnellement immortalisées saines et mutées sur Apc. Ce modèle permettra d'étudier en co-culture sur filtre, l'influence des glucides indigestibles et de leurs produits de fermentation sur les interactions lympho-épithéliales au cours de la carcinogenèse.