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Estudo clínico, epidemiológico, histológico de papilomas de mucosa oral e sua relação com Papilomavírushumano (HPV) através das técnicas de hibridização in situ e PCR / Clinic, epidemiologic, histologic study of oral papillomas and its relation to Humanpapillomavirus (HPV) by In Situ Hybridization and PCRTancredi, Angelo Rafael Calabria 07 December 2007 (has links)
O Papilomavírushumano (HPV) é um DNA vírus do grupo papovavírus, que é altamente transmissível sexualmente, sendo bastante encontrado na região anogenital e mucosa oral. A sua implantação oral pode ser por auto-inoculação ou pelo contato oro-sexual. As principais manifestações orais associadas ao HPV são: papiloma, condiloma acuminado, verruga vulgar e hiperplasia epitelial focal. O papiloma é uma lesão epitelial associada ao HPV e pode ocorrer em vários locais da mucosa oral. Histopatologicamente, o papiloma oral é caracterizado por coilocitose, disceratose, papilomatose, hiperceratose e acantose. A literatura ressalta a importância do estudo dessas lesões, uma vez que estudos demonstram que mesmo com os achados macroscópicos e histológicos serem compatíveis com a presença do vírus, somente técnicas de detecção podem comprovar a presença viral.O objetivo deste estudo foi verificar a presença do HPV, por meio das técnicas de hibridização in situ e PCR, em lesões diagnosticadas histopatologicamente como papilomas e comparar esses resultados com características clínicas e histológicas. Cinqüenta casos foram selecionados da Disciplina de Patologia Bucal e da Disciplina de Estomatologia Clínica da FOUSP. Esta seleção de 50 casos foi submetida à reação de hibridização in situ, e 10 casos dentre os mesmos por técnica da PCR e 100% casos foram negativos. Nenhuma das características histológicas previamente analisadas puderam formar estreita relação com a hibridização in situ e PCR. Conclui-se que a análise histopatológica ao HE não se correlaciona com os resultados das técnicas de hibridização in situ e PCR, porém importante ressaltar para detecção do HPV nas lesões estudadas é imprescindível o uso de técnicas de Biologia Molecular otimizadas como a hibridização in situ e PCR realizadas nesse estudo. / The Humanpapillomavirus (HPV) is a DNA virus from the group of papovavirus, which is highly sexually transmitted and it can be often found in the anogenital area and in the oral mucosal. The oral implantation can be by self-inoculation or by the oral sexual contact. The main oral appearances associated to HPV are: papilloma, condylomata acuminata, verruca vulgaris and focal epithelial hyperplasia. Papilloma is an epithelial lesion that is associated to HPV and can occurs in many places of the oral mucosal. Histopathologically, the oral papilloma is characterized by koylocitosis, dyskeratosis, papillomatosis, hyperkeratosis and acanthosis. The literature shows the importance of these lesions once studies show that macroscopic and histologic founds suggest the presence of the virus, only detection technics can prove the viral presence. The target of this study is to check the presence of HPV, by means of In situ hybridization and PCR, in lesions diagnosed histopathologically as papillomas and compare these results with clinic and histologic features. Fifty cases were selected from the Discipline of Oral Pathology and the Discipline of Oral Diagnose of FOUSP. This selection of 50 cases were submitted to the reaction of In situ hybridization, and 10 cases among the same by PCR and 100% of the cases were negative. None of the histologic features were previously analyzed could form a narrow relation with the In situ hybridization and PCR. Therefore it is concluded that the histopathologic analysis to HE do not correlate with the results of the In situ hybridization and PCR, however the detection of the HPV in the studied lesions it is absolutely necessary the use of Molecular Biology techniques as the In situ hybridization and PCR done in this study.
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Diversidade de bactérias em amostras de água do mar no canal de São Sebastião / Diversity of bacteria in seawater samples at São Sebastião ChannelAlmeida, Bianca Caetano de 24 September 2009 (has links)
A diversidade bacteriana pode ser estudada, combinando técnicas convencionais e técnicas que empreguem tecnologias modernas para sua melhor compreensão. O objetivo do trabalho foi analisar a diversidade de bactérias cultiváveis e não cultiváveis em amostras de água do mar coletadas no Canal de São Sebastião no período de agosto/2005 a março/2007. As bactérias marinhas foram quantificadas em Agar marinho e identificadas por seqüenciamento do gene 16S rDNA. A concentração dos grupos a-, b-, g- e s-proteobacteria foi verificada através da técnica de FISH. A comunidade total foi analisada através da construção de três bibliotecas mensais (novembro/2006, fevereiro/2006, fevereiro/2007). O seqüenciamento identificou 87% das bactérias marinhas como Vibrio sp. A técnica de FISH detectou maior concentração de b-proteobacteria (10,2%), em relação ao número de células totais (DAPI) que variou de 7,0x106 a 2,3x107 céls/mL. As bibliotecas de clones foram compostas pelos filos Proteobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Fusobacteria, Verrucomicrobia e Chloroflexi. / Microbial diversity can be studied by a combination of techniques of both conventional and modern approaches for better understanding. The aim of this study was analyze marine bacteria culturable and nonculturable diversity from seawater samples collected at São Sebastião Channel during August 2005 to March 2007. Marine bacteria were quantified using Marine Agar and identified by 16S rRNA sequencing. Concentration of a-, b-, g- e s-proteobacteria group was verified through three clones library monthly (November 2006, February 2006, February 2007). The sequencing identified 87% of marine bacteria such as Vibrio sp. The FISH technique to detect higher concentration of b-proteobacteria (10.2%), compared to number total cells (DAPI) which range from 7.0 x 106 to 2.3 x 107 cells/mL. Clones library were composed of the phylum Proteobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Fusobacteria, Verrucomicrobia e Chloroflexi.
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Identificação de deleções do gene SHOX: comparação das técnicas de FISH, análise de microssatélites e MLPA / Identification of SHOX gene deletions: comparison of FISH technique, microsatellites analysis and MLPAFunari, Mariana Ferreira de Assis 02 October 2009 (has links)
O gene SHOX (short stature homeobox containing gene), expresso em altos níveis nas células osteogênicas, é fundamental para o desenvolvimento ósseo e para a determinação da altura. Haploinsuficiência do SHOX é responsável por vários fenótipos que envolvem a baixa estatura, como a síndrome de Turner, a discondrosteose de Léri-Weill e a baixa estatura idiopática. Cerca de dois terços das haploinsuficiências são causados por deleções. Neste trabalho, foi realizada uma comparação entre três técnicas para detecção de deleções do SHOX: a hibridação in situ com fluorescência (FISH), o estudo de microssatélites e o multiplex ligationdependent probe amplification (MLPA). Nos pacientes sem deleção do SHOX, foi realizado um rastreamento para identificação de mutações de ponto no gene que levassem à sua haploinsuficiência. Foram analisados seis pacientes com discondrosteose de Léri-Weill (DLW) e 20 com baixa estatura desproporcionada (BED). Na técnica de FISH, os cromossomos metafásicos obtidos a partir de cultura de linfócitos foram hibridados com o cosmídio LLNOYCO3M34F5. DNA genômico extraído a partir de leucócitos de sangue periférico foi submetido à análise de microssatélites e MLPA. Foram amplificados seis marcadores de microssatélites (repetições CA, DYS290, DXYS10093, DXYS10096, DXYS233 e DXYS234) e o MLPA foi realizado de acordo com as instruções dos kits SALSA MLPA P018-C1 e P018-D1 SHOX. Estes kits contêm oito sondas específicas para o gene SHOX e 13 para a área do SHOX, localizada a jusante do gene. O seqüenciamento direto da região codificadora do gene foi realizado nos pacientes sem deleção. Todos os pacientes com DLW apresentaram deleções envolvendo todo o gene. Entre os pacientes com BED, apenas um (5,0%) apresentou uma deleção intragênica envolvendo os exons 4, 5 e 6a. Os resultados das três metodologias foram concordantes na maioria dos casos, exceto em dois casos. No primeiro caso, inicialmente o FISH não identificou uma deleção envolvendo todos os éxons em um paciente com DLW. No segundo, uma deleção envolvendo os exons 4, 5 e 6a, identificada em uma paciente com BED, foi detectada apenas pelo MLPA. Ainda entre os pacientes com BED, três (15%) apresentaram deleção da região do marcador DXYS10096, a 3 do gene. Outros três (15%) pacientes apresentaram mutações de ponto identificadas pelo seqüenciamento direto: a mutação p.Tyr35X, que resulta na substituição de uma tirosina por um códon de parada prematuro; a p.Arg147His localizada na região do homeodomínio e a NM_000451:c.1236 -10T>C que se encontra a 10 nucleotídeos antes do início do éxon 5. Em uma comparação das três metodologias, o FISH foi considerado a técnica mais trabalhosa e com menor sensibilidade, levando até oito dias para sua realização. A análise por microssatélites requer o estudo dos progenitores, além de um grande número de marcadores para a análise de regiões extensas. O MLPA detectou todas as deleções, sendo considerada a metodologia mais sensível. Ele apresentou também menor custo e tempo de execução, além de possibilitar a estimativa do tamanho da deleção. Desta forma, o MLPA foi considerado a melhor metodologia para investigação inicial dos pacientes com DLW e BED. / The SHOX gene (short stature homeobox containing gene), expressed at high levels in osteogenic cells, is essential for bone development and growth process. SHOX haploinsufficiency is responsible for several phenotypes involving short stature, such as Turner syndrome, Léri-Weill dyschondrosteosis (LWD) and idiopathic short stature. Deletions are responsible for 2/3 of SHOX haploinsufficiency. In this study, a comparison among three techniques for detection of SHOX deletions: fluorescence in situ hybridization (FISH), microsatellites analysis and multiplex ligationdependent probe amplification (MLPA) was performed. A screening for point mutations that could lead to haploinsufficiency was performed in patients without SHOX deletion. Six patients with Léri-Weill dyschondrosteosis (LWD) and 20 with disproportionate short stature (DSS) were analyzed. FISH analysis was performed using the cosmid LLNOYCO3\"M\"34F5 and metaphase spreads obtained from lymphocytes culture. Genomic DNA extracted from peripheral blood leukocytes was used to microsatellite and MLPA analysis. Six microsatellite markers (CA repeats, DYS290, DXYS10093, DXYS10096, DXYS233 and DXYS234) were amplified by PCR and MLPA was performed according to the manufacturers instructions for SALSA MLPA P018 and P018-C1-D1 SHOX kits. These kits contain 8 specific probes for SHOX gene and 13 for \"SHOX area, which is located downstream of the gene. The direct sequencing of entire encoding region was performed in patients with no SHOX deletions. All patients with LWD presented deletions involving the entire gene. One (5.0%) patient with DSS, presented an intragenic deletion involving exons 4, 5 and 6a. The results of the three methods were concordant in most cases, except in two cases. In the first case, a patient with DLW, the FISH did not identify a deletion involving all SHOX exons. In the second case, a deletion of exons 4, 5 and 6a in a patient with BED was identified only by MLPA. Other 3 (15%) DSS patients had deletion in SHOX area, in the DXYS10096 marker. Other three (15%) patients presented a point mutation identified by direct sequencing: p.Tyr35X, which replaces a tyrosine for a premature stop codon, p.Arg147His located in the homeodomain region and NM_000451: c.1236-10T> C which is 10 nucleotides before the exon 5. In a comparison of three methods, the FISH technique was considered the more laborious and less sensitive, taking until eight days to obtain the results. The microsatellite analysis requires the parents DNA study. In addition, several markers are essential for the analysis of extensive regions. The MLPA was considered the most sensitive methodology since it detected all deletions. It also presented lower cost and execution time, and allowed the estimation of the size of the deletion. Thus, the MLPA was considered the best approach for initial investigation of LWD and DSS patients.
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Detecção do vírus Epstein-Barr (EBV) por meio da técnica de hibridização in situ em lesões sugestivas de leucoplasia pilosa / Detection of Epstein-Barr virus (EBV) by in situ hibridization in lesions like oral hairy leukoplakiaSilva, Paulo Henrique Braz da 19 December 2005 (has links)
O vírus Epstein-Barr (EBV) é um herpes vírus humano que estabelece infecção persistente e está associado com várias doenças, como mononucleose infecciosa, linfomas, carcinoma de nasofaringe e leucoplasia pilosa, afetando principalmente pacientes imunossuprimidos. Leucoplasia pilosa é uma lesão epitelial não maligna associada ao EBV que ocorre principalmente nas bordas laterais de língua. É comum em indivíduos infectados pelo HIV e em pacientes que recebem medicações imunossupressoras. Histopatologicamente, a leucoplasia pilosa é caracterizada por hiperparaqueratose, acantose, células semelhantes a coilócitos ou células balonizantes, e discreto ou nenhum infiltrado inflamatório. As características histopatológicas da lesão não são patognomônicas, sendo necessária a detecção do EBV para o diagnóstico final de acordo com vários autores. O objetivo desse estudo foi verificar a presença do EBV, por meio da técnica de hibridização in situ, em lesões diagnosticadas histológicamente como sugestivas de leucoplasia pilosa e comparar esses resultados com algumas características histopatológicas.Trinta e seis espécimes foram selecionados do Serviço de Patologia Cirúrgica da Disciplina de Patologia Bucal. Todos foram submetidos à reação de hibridização in situ, e 27 casos (75%) foram positivos, confirmando o diagnóstico anterior. Nenhuma das características histológicas analisadas puderam se correlacionar com a hibridização in situ. Pudemos concluir que a análise histopatológica ao H&E não pode substituir a hibridização in situ no diagnóstico final da leucoplasia pilosa / Epstein-Barr virus (EBV) is a human herpesvirus that estabilishes persistent infection and is associated with many diseases, including infectious mononucleosis syndrome, lymphomas, nasopharyngeal carcinoma, and oral hairy leukoplakia, affecting principaly immunocompromised patients. Oral hairy leukoplakia is a non malignant, EBV-associated, epithelial disease that typically occurs on the lateral tongue borders. It is common in individuals with HIV infection and in patients receiving iatrogenic immunossupression. Histologically, hairy leukoplakia is characterized by shaggy hyperparakeratosis, acanthosis, koilocyte"-like or ballon cells, and a paucity of inflamation. The histologically features of hairy leukoplakia are not patognomonic, and for the many authors definitive diagnosis requires demonstration of EBV. The aim of this study were to verify the presence of EBV, by in situ hibridization, in lesions diagnosed histologically suggestive of hairy leukoplakia and compare this results with histologically features. Thirty six biopsy specimens from lesions histologically suggestive of hairy leukoplakia were selected from the Department of Stomatologys Oral Pathology Service archives. EBV in situ hibridization was performed on all 36 cases, and 27 cases (75%) were positive, confirmed the diagnose of oral hairy leukoplakia. Histopatologically features did not agree well with EBV in situ hibridization. We concluded that H&E histopathology should not be used as a substitute for in situ hibridization in the definitive diagnosis of hairy leukoplakia
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Análise da expressão do fator de crescimento HER-2 e do fator de transcrição FOXO3a em sarcomas e carcinossarcomas uterinos / Evaluation of the HER-2 growth factor receptor and FOXO3a transcriptional factor in uterine sarcomas and carcinossarcomasAlmeida, Thaís Gomes de 27 October 2015 (has links)
Sarcomas uterinos são tumores mesodérmicos raros que compreendem, aproximadamente, 3% de todos os cânceres uterinos. Até o momento não há consenso quanto os fatores de risco para determinar um pior prognóstico e tratamento mais adequado. A radioterapia adjuvante não tem impacto na sobrevida e o papel da quimioterapia ainda é limitado. Nesse contexto, a busca por marcadores moleculares mostra-se importante tanto para a individualização do tratamento, quanto para o diagnóstico e prognóstico desses tumores. O objetivo deste estudo foi avaliar a expressão de HER-2 e do fator de transcrição FOXO3a nos sarcomas e carcinossarcomas uterinos. Para isso, foram avaliadas 100 amostras incluindo: 56 leiomiossarcomas (LMS), 24 carcinossarcomas (CS), 18 sarcomas do estroma endometrial (SEE) e 2 adenossarcomas (AS). A expressão das proteínas foi avaliada por imunoistoquímica e a presença de alterações no número de cópias do gene HER-2 foi analisada por FISH e SISH. A amplificação de HER-2, resultando na hiperexpressão da proteína, foi observada no componente epitelial de uma única amostra de carcinossarcoma. Não foi observada deleção do gene. Os dados de SISH mostraram maior correlação com os de imunoistoquímica. A expressão de FOXO3a foi significativamente maior em todos os tumores avaliados em comparação ao miométrio, e mostrou associação significativamente com maior sobrevida livre de doença nos casos de LMS. Maior expressão dessa proteína também foi observada nos SEE de alto grau. Mulheres com mais de 50 anos, portadoras de LMS, apresentaram menor expressão de FOXO3a. Em conjunto, os resultados sugerem ser o FOXO3a potencial marcador para o risco de malignização e prognóstico para pacientes com LMS. Além disso, permitem indicar a utilização da técnica de SISH para melhor correlacionar a amplificação de HER-2 com os dados imunoistoquímicos / Uterine sarcomas are rare mesodermal tumors that comprise approximately 3% of all uterine cancers. To date, there is no consensus related to risk factors for poor prognosis and appropriate treatment. Adjuvant radiotherapy has no impact on survival of the patients and chemotherapy has limited role. In this context, the search for molecular markers is important in a search for individualization of treatment, for the diagnosis and prognosis of these tumors. The objective of this study was to evaluate the expression of HER-2 and FOXO3a transcription factor in uterine sarcomas and carcinosarcomas. For this, we evaluated a total of 100 samples including: 56 leiomyosarcomas (LMS), 24 carcinosarcomas (CS), 18 endometrial stromal sarcoma (ESS) and 2 adenosarcomas (AS). The protein expression were assessed by immunohistochemistry and copy number of the HER-2 was performed by FISH and SISH. HER-2 gene amplification, resulting in the protein overexpression was found in the epithelial component of an only one case of carcinosarcoma. SISH data showed higher relationship with protein expression than FISH. FOXO3a expression was significantly higher in all tumors than normal myometrium and it showed association with lower disease free survival, in LMSs patients. Protein overexpression was observed in the high grade SEE samples. LMS women´s with > 50 years old showed lower FOXO3a protein expression. Our results suggest that FOXO3a is involved in leiomyosarcoma risk of malignancy and might become a potential prognostic marker to these patients. Moreover, they allow us to indicate the SISH analysis as a better correlation method with the immunohistochemical data for HER-2 amplification, in the future
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Análise da expressão do fator de crescimento HER-2 e do fator de transcrição FOXO3a em sarcomas e carcinossarcomas uterinos / Evaluation of the HER-2 growth factor receptor and FOXO3a transcriptional factor in uterine sarcomas and carcinossarcomasThaís Gomes de Almeida 27 October 2015 (has links)
Sarcomas uterinos são tumores mesodérmicos raros que compreendem, aproximadamente, 3% de todos os cânceres uterinos. Até o momento não há consenso quanto os fatores de risco para determinar um pior prognóstico e tratamento mais adequado. A radioterapia adjuvante não tem impacto na sobrevida e o papel da quimioterapia ainda é limitado. Nesse contexto, a busca por marcadores moleculares mostra-se importante tanto para a individualização do tratamento, quanto para o diagnóstico e prognóstico desses tumores. O objetivo deste estudo foi avaliar a expressão de HER-2 e do fator de transcrição FOXO3a nos sarcomas e carcinossarcomas uterinos. Para isso, foram avaliadas 100 amostras incluindo: 56 leiomiossarcomas (LMS), 24 carcinossarcomas (CS), 18 sarcomas do estroma endometrial (SEE) e 2 adenossarcomas (AS). A expressão das proteínas foi avaliada por imunoistoquímica e a presença de alterações no número de cópias do gene HER-2 foi analisada por FISH e SISH. A amplificação de HER-2, resultando na hiperexpressão da proteína, foi observada no componente epitelial de uma única amostra de carcinossarcoma. Não foi observada deleção do gene. Os dados de SISH mostraram maior correlação com os de imunoistoquímica. A expressão de FOXO3a foi significativamente maior em todos os tumores avaliados em comparação ao miométrio, e mostrou associação significativamente com maior sobrevida livre de doença nos casos de LMS. Maior expressão dessa proteína também foi observada nos SEE de alto grau. Mulheres com mais de 50 anos, portadoras de LMS, apresentaram menor expressão de FOXO3a. Em conjunto, os resultados sugerem ser o FOXO3a potencial marcador para o risco de malignização e prognóstico para pacientes com LMS. Além disso, permitem indicar a utilização da técnica de SISH para melhor correlacionar a amplificação de HER-2 com os dados imunoistoquímicos / Uterine sarcomas are rare mesodermal tumors that comprise approximately 3% of all uterine cancers. To date, there is no consensus related to risk factors for poor prognosis and appropriate treatment. Adjuvant radiotherapy has no impact on survival of the patients and chemotherapy has limited role. In this context, the search for molecular markers is important in a search for individualization of treatment, for the diagnosis and prognosis of these tumors. The objective of this study was to evaluate the expression of HER-2 and FOXO3a transcription factor in uterine sarcomas and carcinosarcomas. For this, we evaluated a total of 100 samples including: 56 leiomyosarcomas (LMS), 24 carcinosarcomas (CS), 18 endometrial stromal sarcoma (ESS) and 2 adenosarcomas (AS). The protein expression were assessed by immunohistochemistry and copy number of the HER-2 was performed by FISH and SISH. HER-2 gene amplification, resulting in the protein overexpression was found in the epithelial component of an only one case of carcinosarcoma. SISH data showed higher relationship with protein expression than FISH. FOXO3a expression was significantly higher in all tumors than normal myometrium and it showed association with lower disease free survival, in LMSs patients. Protein overexpression was observed in the high grade SEE samples. LMS women´s with > 50 years old showed lower FOXO3a protein expression. Our results suggest that FOXO3a is involved in leiomyosarcoma risk of malignancy and might become a potential prognostic marker to these patients. Moreover, they allow us to indicate the SISH analysis as a better correlation method with the immunohistochemical data for HER-2 amplification, in the future
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Avaliação do desgaste superficial do esmalte e da dentina radicular submetidos ao tratamento clareador: in vitro e in situ / Evaluation of enamel and root dentin surface wear submitted to bleaching treatment: in vitro and in situJuliana Jendiroba Faraoni Romano 16 May 2008 (has links)
Devido a alterações químico-estruturais causadas pelo clareamento, os substratos dentais poderiam tornar-se mais susceptíveis a perda tecidual, principalmente se expostos a desafios erosivo/abrasivos. Desta forma, o presente estudo teve como objetivos: 1) analisar in vitro, se o esmalte e a radicular dentina clareada com diferentes agentes e concentrações, apresenta uma maior susceptibilidade ao desgaste, quando submetido a ciclos de erosão e abrasão; 2) comparar o efeito da aplicação de um agente clareador a base de peróxido de carbamida a 10% a um placebo no desgaste do esmalte e da dentina radicular, através de um modelo in situ. Os resultados do estudo in vitro mostraram que, independentemente do agente usado, o clareamento não aumentou o desgaste do esmalte frente a episódios erosivo-abrasivos. Na dentina, o desgaste foi dependente do agente clareador aplicado. Baseado no protocolo in situ adotado, o peróxido de carbamida a 10% não causou maior desgaste superficial no esmalte, mas aumentou a perda de tecido dentinário comparado ao placebo. Pode-se concluir que, em termos de desgaste superficial, o esmalte não foi afetado pelo tratamento clareador, enquanto a dentina mostrou-se mais susceptível. Assim, sugerem-se cuidados adicionais na seleção do agente clareador em situações clínicas que apresentam dentina radicular exposta. / Due to the chemical and microstructural alterations caused by bleaching, the dental substrates can become more susceptible to tissue loss, mainly if exposed to erosive/abrasive challenges. Therefore, the present study had the following objectives: 1) to analyze in vitro, if enamel and root dentin that had been bleached with different agents and concentrations, were at increased risk of wear when submitted to cycles of erosion and abrasion; 2) to compare the effect of the application of a 10% carbamide peroxide bleaching agent to a placebo on wear of enamel and root dentin, through an in situ model. The results of the in vitro study showed that independent of agent used, the bleaching demonstrated no increase in the wear of enamel when exposed to the erosive-abrasive episodes. In dentin, the wear was dependent on the bleaching agent applied. Based on the in situ protocol adopted, the 10% carbamide peroxide did not cause higher wear on the enamel, but increased the wear of the root dentin compared to the placebo. It could be concluded that in terms of superficial wear, enamel was not affected by bleaching treatment, while dentin showed to be more susceptible. Thus, additional caution is suggested in the choice of the bleaching agent when root dentin is exposed.
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Importance of intracellular Mitochondria-Associated endoplasmic reticulum Membranes (MAM) in insulin-resistance / Importance des interactions intracellulaires entre la mitochondrie et le réticulum endoplasmique dans l'insulino-résistanceTubbs, Emily 17 October 2014 (has links)
Les mitochondries et le réticulum endoplasmique (RE) interagissent au niveau de points de contacts appelés « Mitochondria-Associated ER Membranes » (MAM), afin d'échanger du Ca2+ via le complexe TP3Rl/Grp75/VDACl et maintenir l'homéostasie énergétique. Bien que des dysfonctions mitochondriales, un stress du RE et des altérations de l'homéostasie du Ca2+ participent au développement de l'insulino-résistance, on ne sait pas si ce sont des facteurs indépendants ou s'ils sont inter-reliés par une altération des MAM. Mes travaux de thèse ont permis de mettre en évidence un nouveau rôle des MAM dans l'insulino-résistance hépatique. J'ai mis au point et validé la technique d'in situ PLA pour visualiser et quantifier les interactions mitochondrie-RE dans les cellules. J'ai montré que l'intégrité des MAM était nécessaire pour la signalisation de l'insuline dans le foie, et qu'un défaut d'intégrité des MAM était impliqué dans l'insulino-résistance hépatique. Des données préliminaires suggèrent qu'une altération des MAM est également associée à l'insulino-résistance musculaire. J'ai ensuite mis en évidence la présence de la protéine kinase B, une protéine clé de la signalisation de l'insuline, dans les MAM, et démontré que sa phosphorylation par l'insuline est altérée dans cette fraction dans le foie de souris diabétique. Enfin, j'ai participé à la mise en évidence l) de la présence de la cyclophilin D à l'interface des MAM régulant les échanges calciques entre les deux organites dans les cardiomyocytes et les hépatocytes, et 2) d'une régulation des MAM par le glucose dans le foie qui permet un contrôle de la dynamique et de la fonction mitochondriale au cours des transitions nutritionnelles. Par conséquent, mes travaux ont permis d'améliorer les connaissances actuelles sur les partenaires, la fonction et la régulation des MAM et de dévoiler les MAM comme une nouvelle cible pour moduler la signalisation de l'insuline et le métabolisme hépatique / Mitochondria-associated endoplasmic reticulum membranes (MAM) are functional domains between both organelles involved in Ca2+ exchange, through the voltage-dependent anion channel (VDAC)-1/glucose regulated protein 75 (Grp75)/inositol 1,4,5-triphosphate receptor (TP3R)-1 complex, and regulating energy metabolism. Whereas mitochondrial dysfunction, ER stress, and altered Ca2+ homeostasis are associated with altered insulin signalling, the implication of MAM dysfunctions in insulin resistance is unknown. During my PhD, my work has underlined a new role of MAM in hepatic insulin- resistance. T have developed a quantitative method called in situ Proximity Ligation Assay to visualise and quantify the interactions between ER and mitochondria. T have shown that MAM integrity is required for insulin signalling and that disruption of MAM is implicated in hepatic insulin resistance. Preliminary data also suggest that MAM alterations are also associated with muscle insulin resistance. T have also identified the presence of the protein kinase B (PKB), a key protein involved in metabolic effects of insulin, at the MAM interface, and demonstrated that its phosphorylation by insulin is altered in this fraction in liver of diabetic mice. Lastly, T have also participated to the identification of: 1) the presence of cyclophilin D (CypD) at MAM interface which regulates calcium transfer from ER to mitochondria in both cardiomyocytes and hepatocytes, and 2) a regulation of MAM by glucose in liver, which is involved in the regulation of mitochondria dynamics and function during nutritional transitions. Consequently, my work improved the knowledge on the composition, function and regulation of MAM, and highlighted MAM as a potential new target for the modulation of hepatic insulin action and metabolism
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Méthode Hydro-Géomécanique de caractérisation de la susceptibilité des sols à l'érosion interne / Hydro-Geomechanics method of characterizing the susceptibility of soils to internal erosionNguyen, Duc Manh 16 December 2013 (has links)
L'érosion interne est le résultat de l'arrachement des particules d'un sol sous l'action d'un écoulement hydraulique. Ce phénomène est à l'origine des ruptures hydrauliques des barrages et des digues. Sur le plan mondial, 46% des désordres observés, sur les ouvrages en terre, ont pour origine l'érosion interne. En France, 70 cas critiques ont déjà été détectés. Malheureusement, à cause du phénomène d'érosion lui-même, la prévision du risque reste délicate et les autorités ont du mal à mettre en place des plans d'urgence. Compte tenu du risque potentiel, la compréhension des phénomènes à l'origine de l'érosion interne apparaît comme un enjeu scientifique majeur. L'objectif du travail de thèse est de mettre au point une méthode de diagnostic de l'érosion interne dans les digues et autres ouvrages en terre. Nous cherchons à établir un protocole qui nous guidera sur les essais à faire et qui nous aidera à interpréter les résultats. Cette méthode permettra d'expertiser les risques d'érosion interne. (i) Dans un premier temps, les principaux résultats des reconnaissances géomécaniques permettent d'identifier des couches géotechniques de sol de caractéristiques homogènes dans lesquelles sont réalisés les essais pressiométriques. L'analyse cluster des résultats des essais pressiométriques permet de déterminer le type, la nature de sol, ainsi que les caractéristiques géotechniques moyennes. (ii) Les résultats obtenus servent ensuite à déterminer si le sol est instable et quel est son seuil de sensibilité à l'aide du logiciel expert. Ce logiciel permet de calculer les critères d'érosion interne et de qualifier la sensibilité du sol à l'érosion en déterminant son seuil érosif. (iii) Un nouveau dispositif expérimental appelé l'Essai d'Erosion Transverse (Cross Erosion Test - CET) permet de caractériser expérimentalement le sol par rapport au risque d'érosion interne. Cet essai consiste à injecter de l'eau dans un forage, puis à pomper cette eau dans un forage parallèle au premier. L'eau extraite est chargée en particules produites par l'érosion interne du sol. L'érosion est estimée en se basant sur la mesure de l'évolution de la masse des particules recueillies au cours du temps. Les résultats montrent que cette expérience permet de caractériser l'érosion interne dans un sol spécifique. L'avantage de cette technique c'est qu'elle est transposable in situ et qu'elle peut être utilisée pour prédire le risque de suffusion dans les barrages et les digues. (iv) La validation de l'expérience est faite par un modèle numérique 3D réalisé avec le logiciel Comsol Multiphysics 4.3b. Ce modèle permet de montrer que sous les conditions de perméabilité observées, le gradient hydraulique se concentre autour des points d'injection et de pompage. Une modélisation de base en 2D est développée. Cette première approche permet de décrire le phénomène d'érosion et le transport des particules fines dans le milieu poreux du sol. / Internal erosion is the displacement of the fine particles of a soil under the action of an internal flow. This mechanism could be the origin of the damage on embankments and earth dams. In the word, 46% of failure observed on earthwork, are caused by internal erosion. In France, 70 critical cases have been detected. Unfortunately, due to internal erosion, the prediction of risk remains difficult. The understanding of this phenomenon of internal erosion appears as a major scientific challenge. The aim of this thesis is to develop a diagnostic method of internal erosion in dams and other earthworks. We seek to establish a protocol that will guide us to the test and help us to interpret the results. This method allows concluding about the risk of internal erosion appearance. (i) Initially, the main results of geotechnical survey allow identifying the layers of homogeneous characteristics of soil which are measured by the pressuremeter test. The results of cluster analysis of pressuremeter tests allow determining the localisation, the nature and the mean geotechnical characteristics of the soil. (ii) The results are used to determine the threshold of sensibility to internal erosion with the help of expert software. This software allows to calculate the criteria of internal erosion and to describe the sensitivity of the soil to internal erosion. (iii) A new experimental device is developed in our laboratory. This experiment called “Cross Erosion Test" (CET) allows determining the experimental resistance of the soil to the risk of internal erosion. The test consists of the injection, in a first drilling, of clear water and of the recovery, in another drilling, of water charged with eroded particles. For different initial state of the soils, it is possible to measure and to characterize the internal erosion by visualisation of the water flow and the measurement of the weight of the extracted eroded particles. The results show that this experience allows characterizing the internal erosion in a specific soil. The advantage of this technique is that it can be used in situ to predict the risk of suffusion in dams and dikes. (iv) A validation of experiences, with a 3D finite element method is carried out with the help of the Comsol Multiphysics 4.3b software. This model shows that under experiment hydraulic conditions, the hydraulic gradient is concentrated around the injection and the pumping. A finite element 2D model is developed to simulate erosion process. This approach describes the phenomenon of internal erosion and transport of fines particles in the porous medium of soils tested.
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Caractérisation par cytogénétique moléculaire des chromosomes marqueurs surnuméraires et étude de leur implication dans le développement et la reproduction humaine / Molecular cytogenetic characterization of small supernumerary marker chromosomes and study of their implication in human development and reproductive functionGuediche, Narjes 12 June 2012 (has links)
Les chromosomes marqueurs surnuméraires (CMS) sont définis comme des chromosomes de structure anormale qui ne peuvent pas être identifiés ni caractérisés de façon non ambigüe par les techniques de cytogénétique conventionnelle seules et qui sont de taille égale ou plus petits qu’un chromosome 20 de la même métaphase. La prévalence de cette anomalie chromosomique est estimée à 0,071% en post-natal, 0,075% en diagnostic prénatal et 0,288% chez les patients atteints de retard mental et/ou du développement. Chez les patients infertiles, la fréquence des CMS est estimée à 0,122% et varie selon le sexe. Les CMS sont sans conséquence clinique dans 70% des cas. Dans un tiers des cas, ils peuvent être responsables de nombreuses anomalies du développement et de la reproduction humaine. A ce jour, il existe très peu d’études de caractérisation des CMS permettant une cartographie précise des gènes présents. Dans ce travail, nous avons étudié une série de huit CMS par cytogénétique conventionnelle, FISH (fluorescent in situ hybridization) et CGH array (array comparative genomic hybridization). Nous avons établi une cartographie des gènes présents dans ces CMS. L’étude de la relation génotype-phénotype des patients nous a permis de proposer l’implication de certains gènes candidats dans des anomalies du développement et de la reproduction humaine. Notre étude de l’implication des CMS dans les anomalies du développement humain s’est basée sur l’étude cytogénétique de trois fœtus. Les deux premiers fœtus étaient porteurs d’un CMS(20) en anneau. Le sujet 1 présentait un retard de croissance intra-utérin (RCIU) et une dysmorphie cranio-faciale. Le sujet 2 n’avait pas d’anomalies particulières à part une obésité diagnostiquée à l’âge de quatre mois. La taille de ces CMS(20) était de 13,6 Mb pour le sujet 1 et 4,8 Mb pour le sujet 2. Le gène SSTR4 présent sur le CMS(20) du sujet 2 code pour un récepteur de la somatostatine. Cette hormone joue un rôle dans le comportement alimentaire. Le troisième fœtus présentait un hygroma kystique et un RCIU associé à un CMS(13) néocentromérique. Les explorations par CGH array ont révélé un gain chromosomique de la région 13q21.1qter de 39 Mb contenant 80 gènes dont GPC5, GPC6, SPRY2, EFNB2, SOX1 et DZIP1. La modification d’expression de ces gènes est susceptible d’être responsable du phénotype des sujets étudiés.Notre étude de l’implication des CMS dans les anomalies de la reproduction humaine s’est basée sur l’étude cytogénétique de cinq patients qui présentaient des troubles de la fertilité (anomalies de la spermatogenèse, insuffisance ovarienne prématurée, syndrome des ovaires polykystiques et fausses couches spontanées). Les CMS explorés par CGH array correspondaient aux régions chromosomiques 15q11.2 (3,6 Mb), 21p11.2 (0,266 Mb), 6p11.2q12 (9 Mb) et 20p11.21 (3,3 Mb). Le CMS d’une des patientes ne contenait pas d’euchromatine et une autre patiente était porteuse de deux CMS d’origines chromosomiques différentes. Plusieurs gènes candidats (POTE B, BAGE et THBD) ont pu être identifiés. La modification de leur expression ainsi que des effets mécaniques ou biochimiques perturbant la méiose et la maturation des gamètes pourraient être responsables des troubles de la fertilité observés chez ces patients. L’étude des CMS par CGH array nous a permis de caractériser précisément les points de cassure des CMS, leur taille et leur composition génétique afin de cartographier les gènes présents dans les CMS et d’établir des relations entre le génotype et le phénotype des patients. / Small supernumerary marker chromosomes (sSMC) are defined as structurally abnormal chromosomes which cannot be unambiguously identified or characterized by conventional banding cytogenetic techniques alone and are generally equal in size or smaller than a chromosome 20 of the same metaphase spread. sSMC frequency is estimated at 0.071% in postnatal cases, 0.075% in prenatal cases, and 0.288% for mentally and/or development retarded patients. In infertile patients cases, sSMC frequency is estimated at 0.122% and is different in male (0.165%) and female infertility (0.022%). sSMC have no clinical consequences in 70% of the cases. In one third of the cases, they can be responsible for various human development and reproduction anomalies. To date, only a few studies precisely characterizing the sSMC contents have been performed.In this study, we used conventional cytogenetics, FISH (fluorescent in situ hybridization) and array CGH (array comparative genomic hybridization) to characterize eight sSMC and to precisely localize the genes included. The study of the genotype-phenotype correlations of the patients led us to suppose the implication of some candidate genes in human development and reproduction anomalies.Our study of the implication of sSMC in human development anomalies was based on the cytogenetic study of three fetuses. The first two fetuses carried a ring sSMC(20). Case 1 presented with intrauterine growth retardation and craniofacial dysmorphism. Case 2 had a normal phenotype except for obesity diagnosed at the age of four months. The size of these sSMC(20) was approximately 13,6 Mb for case 1 and 4,8 Mb for case 2. The SSTR4 gene located on the case 2 sSMC(20) is coding for one of the somatostatin receptor. This hormone has multiple effects on variable cells and is implicated in the regulation of food behavior, which could explain the obesity of case 2. Case 3 presented with intrauterine growth retardation and a cystic hygroma associated with a neocentric sSMC(13). Array CGH investigations showed a 32.9 Mb gain from 13q31.1 to 13qter region containing 80 genes. Among these genes, six genes could be involved in the phenotype of the proband (GPC5, GPC6, SPRY2, EFNB2, SOX1 and DZIP1). The expression modification of these genes could be responsible for the phenotype observed.Our study of the implication of sSMC in human reproduction anomalies was based on the cytogenetic study of five patients presenting fertility troubles (spermatogenesis impairment, ovarian insufficiency, polycystic ovary syndrome and repeated abortions). The sSMC explored by array CGH corresponded to the 15q11.2 region (3.6 Mb), the 21p11.2 region (0.266 Mb), the 6p11.2q12 region (9 Mb) and 20p11.21 region (3.3 Mb). The sSMC of one of the patients did not contain euchromatin and one patient carried two sSMC derived from two different chromosomes. Among the genes present on the sSMC, some candidate genes (POTE B, BAGE and THBD) have been identified. The modification of their expression and mechanical or biochemical effects of the sSMC impeding meiosis could be directly responsible for the fertility trouble observed in these patients. A detailed molecular cytogenetic investigation using array CGH allowed us to precisely characterize the chromosomal breakpoints, the size and genomic constitution of sSMC. This study may be helpful to address genotype–phenotype correlations and for medical and genetic counseling.
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