Made available in DSpace on 2014-06-11T19:29:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-06-21Bitstream added on 2014-06-13T18:59:02Z : No. of bitstreams: 1 araujo_db_me_botfmvz.pdf: 249258 bytes, checksum: 539ad5602bdd6df582591ea9174f378e (MD5) / Fundação para o Desenvolvimento da UNESP (FUNDUNESP) / A utilização de métodos sensíveis e específicos para o diagnóstico da raiva constitui uma importante ferramenta para o controle e profilaxia dessa enfermidade. A Reação em Cadeia pela Polimerase através de Transcriptase-Reversa (RT-PCR) tem sido utilizada com bons resultados no diagnóstico do vírus rábico, mesmo quando as amostras estão em estágio de decomposição. Adicionalmente as técnicas moleculares têm sido utilizadas para estudos epidemiológicos possibilitando um melhor conhecimento da epidemiologia viral. O presente trabalho teve como objetivo avaliar as técnicas de RT-PCR e hnRT-PCR para a detecção do vírus rábico em estudos retrospectivos, para isso foram avaliadas 101 amostras cerebrais de diferentes espécies animais armazenadas por diferentes períodos, recém-descongeladas e mantidas em temperatura ambiente por 72 horas para decomposição. Os resultados das técnicas de RT-PCR e hnRT-PCR foram comparados com resultados prévios da Imunofluorescência Direta (IFD) e Inoculação Intracerebral em Camundongos (IC). Das 50 amostras positivas testadas, 26 (52%) apresentaram resultados positivos para a RT-PCR e 45 (90%) com a associação da hnRT-PCR quando realizadas em amostras recentemente descongeladas. Das amostras em decomposição foram analisadas 48 previamente positivas; onde 17 (34,3%) apresentaram resultado positivo para a RT-PCR e 36 (75%) com a associação da hnRT-PCR. Não foram encontrados resultados falso-positivos nas amostras negativas submetidas às técnicas de biologia molecular. A hnRT-PCR apresentou maior sensibilidade em relação à RT-PCR nas amostras recém-descongeladas e em decomposição. Os resultados sugerem a viabilidade de sua aplicação em estudos retrospectivos em materiais descongelados e decompostos. / The use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. Reverse-Transcriptase Polymerse Chain Reaction (RT-PCR) has been used in rabies diagnosis with good results, even in decomposed materials. Additionally, molecular techniques have been used for epidemiological studies and better knowledge of viral epidemiology. The aim of this work was to evaluate the RT-PCR and hnRT-PCR in rabies virus detection in retrospectives studies. RT-PCR and hnRT-PCR were evaluated in 151 brain samples from different animal species, thawed and left at room temperature for 72 hours for decomposition. The RT-PCR and hnRT-PCR results were compared with preview results from Fluorescent Antibody Test and Mouse Inoculation Test. From the 50 positive fresh samples, 26 (52%) were positive for RT-PCR and 45 (90%) for hnRT-PCR. From the 48 positive decomposed samples, 17 (34, 3%) were positive for RT-PCR and 36 (75%) for hnRT-PCR. No false-positives results were found in the negatives samples submitted to the molecular techniques. These results show that the hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples. The hnRT-PCR presented greater sensibility than the standards techniques (IFD e IC) in decomposed materials for rabies diagnosis. These results suggest the viability of the application of molecular techniques in thawed and decomposed materials for retrospective studies.

Submitted by Lucelia Lucena (lucelia.lucena@ufpe.br) on 2015-03-13T17:37:58Z No. of bitstreams: 2 dissertacao Cristiane Araujo Simoes.pdf: 819117 bytes, checksum: 0a57c0b1fda36c45fbb123fbb3de1e94 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T17:37:58Z (GMT). No. of bitstreams: 2 dissertacao Cristiane Araujo Simoes.pdf: 819117 bytes, checksum: 0a57c0b1fda36c45fbb123fbb3de1e94 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012-05-02 / A pesquisa do vírus da Hepatite C (HCV) em outros fluidos corporais além do sangue é importante quando se avalia a existência de outras possíveis vias de transmissão, afinal cerca de 40% dos pacientes contaminados não apresentam história de exposição por via parenteral. A presença do RNA do HCV (RNA-HCV) na saliva de indivíduos infectados foi observada em alguns trabalhos e esta vem sendo sugerida como uma possível via de transmissão. No entanto, o papel dos fluidos orais na transmissão do HCV permanece controverso. O objetivo deste trabalho foi identificar o HCV-RNA na saliva e no soro de pacientes anti-HCV positivos. Foi realizado um estudo transversal com uma amostra de conveniência composta por 50 pacientes atendidos no Setor de Gastroenterologia do Hospital das Clínicas de Pernambuco (HC-UFPE) no período de junho a setembro de 2011. Amostras de sangue e saliva foram coletadas e processadas. O HCV-RNA foi extraído e uma alíquota utilizada na RT-PCR. O HCV-RNA foi detectado em 82% (41/50) das amostras de soro e em 0% das amostras de saliva. Pela metodologia empregada neste trabalho, não houve presença do HCV-RNA em saliva.

The diagnosis of dengue cases in Central Laboratories of Public Health (LACEN) of the country includes the research of NS1 protein and only in positive cases, diagnostic confirmation occurs by identifying the serotype. Data provided by the Epidemiological Surveillance of Sergipe revealed that the first half of 2016 all suspected cases that reached the LACEN / SE were negative on screening tests, even in the thirty-first epidemiological week of the same year, Sergipe has reached the seventh place in the racking of dengue cases in the Northeast region and the third in the Quick Survey Infestation Index by Aedes aegypti. This study aimed to identify the serotypes and the epidemiological profile of suspected dengue cases in the first half of 2016. The 437 suspected dengue patients from January to June of this year were registered in LACEN / SE. After exclusion criteria, 382 serum samples of these patients diagnosed and negative for dengue by ELISA NS1 Antigen Platelia kit method were analyzed by RT-PCR in real time on the multiplex system for confirmation of suspected cases of dengue. The distribution of patients according to epidemiological and demographic variables revealed that there was a predominance of cases in the south central region of the state and large Aracaju (53.5%) inferring the ease in access to outpatient and laboratory service in the region the most affected gender was female (62.8%) and the age group of highest incidence was 20-59 years (59.3%). The highest rate was in urban areas (84.9%) and the first three days of symptoms (63.9%) with higher sampling rate. The prevalence of dengue in cases initially presented as a suspect was 22.5% (86) distributed among serotypes DENV4 82.5% (71), DENV1 9.3% (8) and DENV3 8.1% (7). Compared to official data reported that there were no cases of dengue in the period, this study reveals the positive for dengue with cocirculation of three distinct serotypes being DEV4 the predominant. As also, reports the first evidence of the last ten years of DENV3 serotype circulating in the state. Data analysis enabled to view an underreporting of cases screened and thus suggest a reassessment of the methods used as diagnostic screening so that they can take control measures to the potential for severe disease in a population. / O diagnóstico de casos de dengue nos Laboratórios Centrais de Saúde Pública (LACEN) do país contempla a pesquisa da proteína NS1 e, somente em casos positivos, a confirmação diagnóstica ocorre pela identificação do sorotipo. Dados disponibilizados pela Vigilância epidemiológica de Sergipe revelaram que no primeiro semestre de 2016 todos os casos suspeitos que chegaram ao LACEN/SE foram considerados negativos nos testes de triagem, ainda que na trigésima primeira semana epidemiológica do mesmo ano, Sergipe tenha alcançado o sétimo lugar no racking de casos de dengue da região Nordeste e o terceiro no Levantamento Rápido do Índice de Infestação por Aedes aegypti. O presente trabalho teve como objetivo conhecer da dengue no estado de Sergipe no primeiro semestre de 2016 relacionando-os com alguns parâmetros epidemiológicos. Os 437 pacientes suspeitos de dengue, de janeiro a junho do presente ano, foram cadastrados no LACEN/SE. Após critérios de exclusão, 382 amostras séricas destes pacientes com diagnóstico e resultado negativo para dengue através da metodologia ELISA NS1 Antígeno do kit Platelia foram analisadas pela técnica de RT-PCR em tempo real no sistema multiplex para a confirmação dos casos suspeitos de dengue. A distribuição dos pacientes de acordo com as variáveis epidemiológicas e demográficas revelou que houve predominância dos casos na região centro-sul do estado e da grande Aracaju (53,5%) podendo-se inferir a facilidade no acesso ao atendimento ambulatorial e laboratorial nessa região, o gênero mais acometido foi o feminino (62,8%) e a faixa etária de maior incidência foi 20 a 59 anos (59,3%). A maior frequência foi na zona urbana (84,9%) e os três primeiros dias dos sintomas (63,9%) com maior índice de coletas. A incidência de dengue nos casos que incialmente se apresentaram como suspeita foi de 22,5% (86) distribuídas entre os sorotipos DENV4 82,5% (71), DENV1 9,3% (8) e DENV3 8,1% (7). Em comparação aos dados oficias que informavam que não houve casos de dengue no referido período, este estudo revela a positividade para dengue com co-circulação de três sorotipos distintos sendo o DENV4 o predominante. Ademias, relata a primeira evidência, dos últimos dez anos, de circulação do sorotipo DENV3 no estado. A análise dos dados permitiu visualizar uma subnotificação dos casos triados e, assim, sugerir uma reavaliação dos métodos utilizados como triagem diagnóstica para que se possam tomar medidas de controle para riscos potenciais de formas graves da doença numa população. / Lagarto, SE

Orientador: Ricardo Della Coletta / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-11T04:25:53Z (GMT). No. of bitstreams: 1 Destro_MariaFernandadeSousaSetubal_M.pdf: 4049997 bytes, checksum: c65b11d1e2e17169065f897077fbfb75 (MD5) Previous issue date: 2008 / Resumo: Genes homeobox transcrevem fatores de transcrição com participação importante na organogênese através do controle da proliferação e diferenciação celular. Dentre estes genes destacam-se os membros da família HOX de genes homeobox, os quais estão envolvidos em processos celulares cruciais como controle do ciclo celular, diferenciação e apoptose. Os genes HOX estão relacionados com o surgimento de diferentes tipos de neoplasias, incluindo cânceres de mama, ovário, próstata, rins, pulmão, pele e leucemias. Na cavidade oral a participação destes genes é desconhecida. O objetivo deste estudo foi comparar os níveis de expressão dos membros da família HOX de genes homeobox dos loci B e C entre amostras orais de mucosa normal e carcinoma espinocelular (CEC). Para a realização deste trabalho, amostras de mucosa oral normal de pacientes não expostos aos principais fatores de risco para o câncer oral (hábito de fumar e consumir bebidas alcoólicas) e amostras orais de mucosa normal e CEC provenientes do mesmo paciente foram submetidos a ensaios semi-quantitativos de transcriptase reversa-reação em cadeia da polimerase (RT-PCR) ¿duplex¿, utilizando-se primers para o gene controle GAPDH e primers específicos para cada um dos membros dos loci B e C. Em adição, os níveis de expressão destes genes foram analisados em uma linhagem de queratinócito normal (HaCAT) e em 4 linhagens celulares derivadas de CEC oral. As linhagens celulares de CEC oral expressaram todos os membros do loci C, sendo que os genes HOXC4, HOXC5 e HOXC6 apresentaram maiores níveis de expressão quando comparado com a linhagem HaCAT. HOXB13 foi expresso por todas as linhagens celulares de CEC oral. Os genes HOXB1, HOXB3, HOXB5, HOXB8 e HOXC12 não foram expressos por nenhuma das amostras de mucosa oral normal, independente da origem, e de CEC oral. O padrão de expressão dos genes HOX foi muito similar entre os dois grupos de mucosa oral normal. As expressões dos membros HOXB7, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10 e HOXC11 foram significantemente maiores nas amostras de CEC oral comparado com amostras de mucosa oral normal. Os níveis de expressão dos membros HOXB2 e HOXC13 foram significantemente maiores nas amostras de CEC oral quando comparado com os níveis de expressão encontrados nas amostras de mucosa normal de pacientes livres de fatores de risco. Estes resultados sugerem que a expressão alterada de alguns membros da família HOX de genes homeobox pode estar associada com o desenvolvimento e/ou progressão do CEC oral / Abstract: Homeobox genes encode transcription factors with an important role during normal development by controlling cellular proliferation and differentiation. Among those genes, the HOX family is involved in crucial biological processes such as the control of the cell cycle, differentiation and apoptosis. HOX genes are related to many cancers, including those of the breast, ovary, prostate, kidney, lung, skin and leukemias. In the oral cavity, the role of those genes is unclear. The aim of this study was to compare the expression levels of HOX genes from loci B and C between normal oral mucosa and oral squamous cell carcinoma (SCC). Samples of normal oral mucosa and oral SCC obtained from the same patient, and samples of normal oral mucosa from patients without history of exposition to risk factors related to oral SCC (smoking habit and alcohol consumption) were analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) duplex method with specific primers for the control gene GAPDH and for each of the HOXB and HOXC members. Additionally, we analyzed the expression profile of those genes in a normal keratinocyte cell line (HaCAT) and 4 oral SCC cell lines. Oral SCC cell lines expressed all members of the locus C, and the expression of HOXC4, HOXC5 and HOXC6 was higher in those cell lines compared with HaCAT. Only HOXB13 was expressed for all oral SCC cell lines. None of the normal oral mucosa and oral SCC samples expressed HOXB1, HOXB3, HOXB5, HOXB8 and HOXC12. The HOX expression profile of the 2 groups of normal oral mucosa was quite similar. Regardless of the oral normal mucosa source, the expression of HOXB7, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10 and HOXC11 was statistically higher in oral SCC samples. HOXB2 and HOXC13 were significantly overexpressed in oral SCC when compared with normal oral mucosa from patients without risk factors related to oral SCC. These results suggest that a dysregulated expression of HOX genes from clusters B and C may be related to the tumorigenesis and/or tumor progression of oral SCCs / Mestrado / Patologia / Mestre em Estomatopatologia

Orientador: Maria Leticia Cintra / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T19:11:09Z (GMT). No. of bitstreams: 1 Scarpelli_EdsonMartins_M.pdf: 3928334 bytes, checksum: 3ef78d4f7a3f4ec5c95969a5e584a0e9 (MD5) Previous issue date: 2008 / Resumo: A cinomose canina (CC) é uma doença viral sistêmica de carnívoros, cujo diagnóstico é embasado em sinais neurológicos e extra-neurais. A desmielinização que ocorre nesta doença assemelha-se à das doenças desmielinizantes humanas, o que a torna um modelo animal para estudo de doenças neurodegenerativas humanas. Lesões iniciais do SNC caracterizam-se por desmielinização sem considerável inflamação e lesões tardias por um processo inflamatório crônico. Vários métodos são empregados para o diagnóstico da CC, destacando-se a RT-PCR como precisa e sensível. Estudar, prospectivamente, os achados clínicos e histopatológicos do cérebro de 49 cães com cinomose diagnosticada clinicamente, e confrontá-los com os resultados da RT-PCR foi o nosso objetivo. A presença de sinais neurológicos foi significante para o diagnóstico da doença, especialmente a mioclonia e ataxia. O comprometimento inflamatório era significante no cerebelo, hipocampo e diencéfalo sendo que a reação inflamatória era maior no diencéfalo. Quanto maior o peso cerebral, menor era a desmielinização. Nos animais na fase clínica aguda, a freqüência de desmielinização cerebelar foi maior. Foi significante a localização linização, mas, também, havia forte comprometimento cortical, embasando os comemorativos clínicos. A pesquisa do vírus da CC pela RT-PCR foi negativa em 34% dos animais doentes; 88% deles encontravam-se na fase crônica, onde o vírus esta praticamente ausente. Em todos os cães sadios, a RT-PCR resultou positiva, provavelmente por vacinação ou doença prévia. Nossos achados ressaltam a importância da avaliação clínica para o diagnóstico da CC e dos achados anatomopatológicos respaldando a clínica / Abstract: Introduction: canine distemper (CD) is a carnivores viral systemic disease whose clinical diagnosis is based on neurological and extra neural signs. The CD demyelination resembles those of human demyelinating diseases; therefore it becomes an animal model for human neurodegenerative diseases study. Central nervous system (CNS) initial lesions are characterized by demyelination with no considerable inflammation and late lesions by a chronic inflammatory process. Several methods are used for the diagnosis of CD; and RT-PCR stands out for being precise and sensitive. Purpose: to study, prospectively, clinical and histopathologic signs of 49 dogs' brains with distemper clinically diagnosed, and compare them to RT-PCR results. Results and Conclusions: the presence of neurological signs was significant for the diagnosis of the disease, especially myoclonus and ataxia. The inflammatory reaction was significant in the cerebellum, the hippocampus and the diencephalons; the inflammatory reaction was larger in the diencephalon. The higher the cerebral weight the lowest the demyelination. In animals on the acute clinical form, the demyelination frequency in the cerebellum was higher. Cerebellum and diencephalons localization in the demyelination was significant, but there was also strong cortical compromising, embasing clinical observations. RT-PCR for CD virus tested negative in 34% of the sick animals; 88% of them were in the chronic phase, in which the virus is practically non-existent. In all healthy dogs, RT-PCR resulted positive, probably due to vaccination or previous disease. Our findings stress the importance of clinical evaluation for CD diagnosis and histopathological examination supporting clinical findings / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Background: The infection caused by Mayaro virus (MAYV), which presents as an acute febrile illness, is considered a neglected tropical disease. The virus is an endemic and emerging pathogen in South America and the Caribbean, responsible for occasional and poorly characterized outbreaks. Currently there is limited information about its expansion and risk areas. Methods: A cross-sectional study was performed in 10 urban primary care health centers in the Cajamarca region of Peru from January to June 2017. A total of 359 patients with suspected febrile illness were assessed. RNA was extracted from serum samples, following which MAYV real-time reverse transcriptase PCR (RT-PCR) for the detection of the nsP1 gene was performed. Results: MAYV was detected in 11.1% (40/359) of samples after RT-PCR amplification and confirmatory DNA sequencing. Most infections were detected in the adult population aged 18–39 years (40%) and 40–59 years (32.5%). Headache was the most frequent symptom in patients with MAYV infection (77.5%), followed by fever (72.5%), myalgia (55.0%), and arthralgia (50.0%). During the study, most of the MAYV cases were seen in May (47.5%) and April (35.0%), corresponding to the dry season (months without rain). Conclusions: This study is novel in describing the presence of MAYV in Cajamarca, an Andean region of Peru. Symptoms are non-specific and can be confused with those of other arbovirus or bacterial infections. Molecular biology methods such as RT-PCR allow the timely and accurate detection of MAYV and could thus be considered as a tool for surveillance in endemic areas. / This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. 2015M3A9B6073666 ). This study was supported by CONCYTEC Peru , under the contract No 164-2016-FONDECYT, Lima, Peru. Incentive for Research of the Universidad Peruana de Ciencias Aplicadas (No. UPC-C-01-2019), Lima, Peru. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. / Revisión por pares

Morphological studies have shown pathology of neurons and glia in many brain disorders, including psychiatric disorders such as major depression. However, most biochemical characterizations of postmortem human brain tissue have not made a distinction between neurons and glia. Laser capture microdissection (LCM) to isolate specific cell types has the potential to advance our understanding of human brain pathologies. Here, RT-PCR was used to evaluate the utility of LCM in the capture of noradrenergic neurons, astrocytes and oligodendrocytes from the locus coeruleus (LC) of postmortem human brain. The 3 LC cell types were individually identified using modifications of established histological and morphological methods. LCM settings were optimized for each cell type and captured cell bodies were those having no nearby cell body of a different phenotype. LC neurons (200), astrocytes (500), and oligodendrocytes (500) were captured within the LC from 3 postmortem brains. RNA was isolated, reversed transcribed, and markers for neurons (tyrosine hydroxylase [TH], dopamine beta-hydroxylase [DBH]), astrocytes (glial fibrillary acidic protein [GFAP]), and oligodendrocytes (myelin oligodendrocyte glycoprotein [MOG]), along with 3 references (actin, GAPDH, ubiquitin C) were PCR amplified and quantified by standardized end-point PCR. RNA quality as assessed by RIN was not altered by LCM as compared to RNA isolated from homogenized tissue. TH gene expression was found only in neurons in 2 of the 3 brains. DBH gene expression was ~5-fold greater in neurons than in astrocytes and oligodendrocytes. GFAP gene expression in astrocytes was 7- and 5-fold greater than that in neurons and oligodendrocytes, respectively. MOG gene expression was only detected in oligodendrocytes. Different expression ratios of marker genes between neurons and glia suggest that simple cross contamination of mRNA is unlikely. Glial cells may contain DBH mRNA. Alternatively, DBH, but not TH, mRNA may occur in neuronal dendrites or axons in close association with glial cells that become captured with glia during LCM. GFAP may be expressed in low levels in neurons and oligodendrocytes, or alternatively, GFAP mRNA may be located in astrocytic processes in close association with neuronal and oligodendrocyte cell bodies. Use of a single marker to identify a cell type may be insufficient; other cell types for comparison or additional markers may be required. Multiple well-characterized markers can be used to evaluate clarity of cell capture for each sample. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of specific cell types in postmortem human brain.

Silene latifolia and Silene dioica are closely related dioecious plant species where sex is determined by the presence of sex chromosomes, X and Y. The sex chromosomes of S. latifolia and S. dioica are younger (10-20 million years) than in mammals (about 200 million years). These two species are thus important models for the study of early stages of sex chromosomes evolution. In this work a group of genes linked to sex chromosomes (Sl3, Sl7, DD44) was analyzed. These genes were de-novo isolated from S. dioica by screening of the BAC library. The expression of these genes in different tissues was compared using Real-Time PCR and HRM in S. latifolia and S. dioica. Using the low-copy FISH (fluorescence in situ hybridization) method, a physical map for individual genes was created. This map is the most detailed physical map created for closely related Silene species.

Real-time RT-PCR was used to assess the effects of interspecific microbial interactions on the expression of genes associated with lignin peroxidase, manganese peroxidase and alcohol oxidase production during the wood decay process. Expression levels of genes encoding the selected lignolytic enzymes were quantitated in one-, two- and multiple-organism interaction tests with the basidiomycetes Trametes elegans, Phanerochaete chrysosporium, Gloeophyllum sepiarium and Gloeophyllum trabeum. Compression strength loss was measured for each decay sample and correlated with gene expression data for each species. Soil microflora actively producing lignolytic enzymes during wood decay were also assessed and identified using degenerative PCR coupled with denaturing gradient gel electrophoresis, cloning and cycle sequencing. Differential expression was detected in three genes in the two-organism interaction tests: manganese peroxidase in T. elegans interactions, lignin peroxidase A in P. chrysosporium interactions and alcohol oxidase in G. sepiarium interactions. A positive linear correlation was observed between lignin peroxidase A expression and compression strength loss in P. chrysosporium interactions.

No description available.