Orientadores: Francisco Hideo Aoki, Ângela Maria de Assis / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-28T00:12:52Z (GMT). No. of bitstreams: 1 Santos_CristinaRodrigues_M.pdf: 1833064 bytes, checksum: 4087a171a36bf726cff1abdcd0bd4cf8 (MD5) Previous issue date: 2015 / Resumo: O Pneumocystis jirovecii é um fungo ascomiceto que pode causar pneumonia (PPC) em indivíduos imunossuprimidos e eventualmente em imunocompetentes. Os métodos laboratoriais de baixo custo para detecção ainda é a coloração em lâmina. Os métodos moleculares são considerados mais eficientes e tem oferecido alternativas para o entendimento da biologia e da doença causada por esse microrganismo. O objetivo desse estudo foi detectar a presença de Pneumocystis jirovecii em pacientes com doença pulmonar. O método adotado foi a técnica de coloração de lâmina com azul de toluidina para detecção de cistos, PCR para detecção do fragmento mitocondrial pAZ102-H/E, Nested PCR com primers internos pAZ102-X/Y e sequenciamento pelo método de Sanger para detecção de alterações moleculares na região genômica estudada. Foram coletadas 139 amostras, sendo 102 de lavado broncoalveolar (LBA), 7 de escarro, 10 amostras de sangue periférico (fracionadas em hemácias/leucócitos e plasma) e 10 amostras de soro. Nos resultados da coloração pela técnica de azul de toluidina, 2 amostras de escarro e 1 de LBA foram positivas. Através dos métodos moleculares, 6 amostras foram positivas na primeira PCR e 91 amostras foram positivas pela Nested PCR. Os fragmentos pAZ102-X de 51 amostras foram sequenciadas, 18 amostras apresentaram mutação nos seguintes códon/posição 4406/13217, 4419/13257, 4431/13294, 4439/13316, 4440/13321, 4446/13338, 4452/13357, 4456/13367 e 4457/13371. Os pacientes que apresentaram essas alterações tinham um perfil com doenças pulmonares ou fatores de risco associados à infecção por P. jirovecii. Como a detecção de P. jirovecii, diagnóstico e prevenção de PPC são ligadas a história clínica do paciente, os avanços moleculares podem ser um caminho para diagnóstico de PPC, diferenciando essa pneumonia dos casos de colonização por esse fungo / Abstract: Pneumocystis jirovecii is an ascomycete fungus that can cause pneumonia (PCP) in immunocompromised individuals and possibly in immunocompetent. The low cost of laboratory methods for detection is with conventional staining methods. Molecular methods are considered more efficient and have offered alternatives to the understanding of biology and disease caused by this organism. The aim of this study was to detect the target DNA fragment (mtLSUrRNA) of P. jirovecii in patients with lung disease. We compared the staining technique with molecular methods using toluidine blue for microscopy and "in house" DNA extraction, PCR and Nested PCR for molecular detection and sequencing by the Sanger method to detect molecular alterations in the genomic region studied. It was analyzed 139 samples, such as, 102 bronchoalveolar lavage (BAL), 7 sputum, 10 samples of peripheral blood (fractionated in red blood cells / leukocytes and plasma) and 10 serum samples. The results of the toluidine blue staining technique, two sputum samples and 1 BAL were positive. Through molecular methods, 6 samples were positive in the first PCR and 91 samples were positive by Nested PCR. The pAZ102-X fragments of 51 samples were sequenced, 18 samples had mutations in codon/position 4406/13217, 4419/13257, 4431/13294, 4439/13316, 4440/13321, 4446/13338, 4452/13357, 4456 / 13367 and 4457/13371. Patients who presented these alterations had a profile with lung diseases or risk factors associated with infection by P. jirovecii. As the detection of P. jirovecii, diagnosis and prevention of PCP are linked to clinical history, molecular advances can be a way for diagnosis of PCP, differentiating this pneumonia cases of colonization by the fungus. OBSERVAÇÃOPor favor manter essa palavra em itálico: Pneumocystis jirovecii / Mestrado / Clinica Medica / Mestra em Ciências

Orientador: Luiz Carlos Zeferino / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-28T09:15:40Z (GMT). No. of bitstreams: 1 CamposTeixeira_CirbiaSilva_M.pdf: 1128371 bytes, checksum: f7ee3c8128bb7ec40c81ec62521ca6bf (MD5) Previous issue date: 2015 / Resumo: Introdução: O câncer cérvico-uterino é causado pelo HPV e a vacinação contra este vírus poderá alterar a prevalência destes na população. Objetivo: avaliar o impacto da vacinação contra HPV na detecção dos diferentes tipos de HPV no período de 12 anos pós-vacinação. Métodos: Em 2001, 91 mulheres do Centro de Campinas para estudos clínicos com a vacina contra HPV-16/18 da GSK, receberam três doses da `vacina¿ contra HPV ou de placebo (Al[OH]3) de forma randomizada e duplo-cega. Elas foram seguidas e realizaram testes de HPV (SPF-10 LiPA) em amostras cervicais coletadas semestralmente até 2010. Informações epidemiológicas, reprodutivas e comportamentais foram obtidas em 2001, 2005, 2010. Em 2012, este estudo local, as participantes retornaram, atualizaram suas informações e coletaram nova amostra, testada por CLART-HPV2 test. Os resultados disponíveis foram agrupados com total de 1492 testes de HPV. Foi analisada a proporção de mulheres com detecção de HPV, por agrupamento viral, a ocorrência de infecção persistente por seis meses (IP6m) por um mesmo HPV de alto risco (HR-HPV) e a relação com idade, novo parceiro sexual nos últimos 12 meses, uso de contraceptivo hormonal ou de preservativos, tabagismo, tipo de vacinação e o tempo decorrido. A análise estatística foi realizada por momento e evolutiva em 12 anos e comparados com a vacina recebida. A análise utilizou os testes x2, exato de Fisher, Mann-Whitney, GEE (equações de estimativa generalizada) e odds ratio com intervalo de confiança de 95% e p<0.5 para significância estatística. Resultados: Os grupos de mulheres `vacinadas¿ e `placebo¿ não apresentaram diferenças na idade e fatores de risco relacionados à aquisição de HPV. Não foi observada diferença na detecção de HPV por momento de coleta da amostra nos 12 anos, avaliados por vacina recebida (53% se vacina contra HPV vs. 47,4% se placebo, p=0,90). Também não houve diferenças significativas para os agrupamentos de HR-HPV, HR-HPV não-HPV16/18, HPV-16/18 e HPV de baixo risco (LR-HPV). Na análise longitudinal a detecção de DNA-HPV apresentou uma tendência de aumento com o tempo para HR-HPV não-HPV16/18 (p=0,03), e de menor detecção de HPV-16/18 (p=0,05) e LR-HPV (p=0,04). Apenas para os HPV-16/18 esta diminuição esteve associada com a vacinação prévia (p=0,05). O uso regular de contraceptivo hormonal esteve associado com 2,4 vezes mais de detecção de LR-HPV (p=0,03), sem relação com a vacinação. Houve 44 episódios de IP6m de HR-HPV, sendo duas vezes mais frequentes em mulheres tabagistas (p=0,03), mas sem relação com a vacinação. Foi observada uma redução, embora não significativa, de IP6m de HR-HPV nas mulheres vacinadas ao longo do tempo (OR=0,68; 95% CI: 0,36-1,28; p=0,23). Conclusões: Não houve diferença na proporção de mulheres com detecção de HPV de qualquer tipo, HR-HPV não-HPV16/18, HPV-16/18 e LR-HPV em relação à vacinação contra HPV-16/18 ou com placebo, em avaliações repetidas por 12 anos. Nas avaliações longitudinais houve uma tendência de menor detecção de HPV-16/18 e menos casos de IP6m por um mesmo HR-HPV detectados nas mulheres previamente vacinadas contra HPV-16/18 / Abstract: Introduction: The cervix cancer is caused by HPV and the vaccination in population base against this virus can change their prevalence. Objective: To assess the impact of HPV vaccination in the detection of different types of HPV in 12-years post-vaccination period. Methods: In 2001, 91 women from Campinas Centre started their participation in clinical trial with HPV-16/18 vaccine (GSK) and received three doses of the HPV vaccine or placebo (Al [OH] 3) in a randomized and double-blinded study. They were followed and performed HPV testing (SPF-10 LiPA) in cervical samples collected every six months, until 2010. Information epidemiologic, reproductive and behavioral was obtained in 2001, 2005 and 2010. In 2012, the participants were invited to return in a local study, when the information were updated and a new cervix sample was collected and tested by CLART-HPV2 test. The available results were gathered with a total of 1492 HPV tests. We analyzed the proportion of women with HPV detection by virus group, the occurrence of 6-month persistent infection (6MPI) by the same high-risk HPV (HR-HPV) and the relationship with age, new sexual partner in the last 12 months, use of hormonal contraception or condoms, smoking, type of vaccination and over 12 years. The statistical analysis was performed by moment of sample collection and longitudinally for 12-year period studied and compared by vaccination performed in 2001. The analysis used the tests chi-square, Fisher's exact, Mann-Whitney, GEE (generalized estimating equations) and odds ratios with 95% confidence interval and p<0.5 for statistical significance. Results: the women from groups 'vaccinated' and 'placebo' did not differ in age and risk factors related to the HPV acquisition. There was no difference in HPV detection by time of sample collection for over 12 years, according to the received vaccine (53% for HPV vaccine vs. 47.4% for placebo, p=0.90). There were also no significant differences for HPV groupments, HR-HPV, HR-HPV non-HPV16/18, HPV-16/18 and low risk HPV (LR-HPV). The longitudinal analysis of DNA-HPV detection showed an increasing trend over time for HR-HPV non-HPV16/18 detection (p=0.03), and a decreasing trend for detection of HPV-16/18 (p=0.05) and LR-HPV (p=0.04). Just for HPV-16/18 the decrease trend was associated with prior HPV vaccination (p=0.05). Regular use of hormonal contraceptive was associated with 2.4 times more LR-HPV detection (p=0.03), but unrelated to vaccination. There were 44 episodes of HR-HPV 6MPI, and their occurred twice more if the women smokes (p=0.03), but unrelated to vaccination. The HR-HPV 6MPI over the 12-year studied had a decreasing pattern in HPV vaccinated women, although not significant (odds ratio=0.68, 95% CI: 0.36 - 1.28; p=0.23). Conclusions: There was no difference in the proportion of women with detection of HPV (any type), HR-HPV non-HPV16/18, HPV-16/18 and LR-HPV in relation to vaccination against HPV-16/18 or placebo in repeated cervix samples for 12 years. In the longitudinal assessments there was a decreasing trend for detecting HPV-16/18 and less episodes of 6MPI of the same HR-HPV in women previously vaccinated against HPV-16/18 / Mestrado / Oncologia Ginecológica e Mamária / Mestra em Ciências da Saúde

O vírus respiratório sincicial humano (HRSV) é principal agente causador de infecções do trato respiratório inferior em crianças e lactentes. Um diagnóstico rápido e preciso evitaria o uso desnecessário de antibióticos, nos casos em que a infecção é viral. A reação em cadeia da polimerase após transcrição reversa (RT-PCR) e o ensaio de imunofluorescência indireta (IFI) são considerados ferramentas importantes na detecção do HRSV, pela alta sensibilidade e especificidade. Visando simplificar e minimizar os riscos de contaminação freqüentes, em duas etapas, foi padronizada uma reação em tubo único para detecção do HRSV em amostras clínicas. Aspirados de nasofaringe de 226 crianças de 0-5 anos de idade, com doença respiratória, atendidas no Hospital Universitário da Universidade de São Paulo (HU-USP), foram testados por imunofluorescência indireta, RT semi Nested PCR e RT-PCR em tubo único. Cento e duas amostras (45,1%) foram positivas em pelo menos uma das técnicas e 75 (33,2%) em todas. Três (1,3%) amostras foram positivas por IFI e RT semi Nested PCR, 1 (0,4%) foi positiva por IFI e RT-PCR em tubo único, 5 (2,2%) amostras foram positivas somente por IFI, 2 (0,9%) somente por RT semi Nested PCR e 16 (7,1%) amostras foram positivas pela RT semi Nested PCR e RT-PCR em tubo único. A RT-PCR em tubo único mostrou ser uma técnica rápida, sensível e específica, e o uso combinado de dois métodos aumenta a detecção do HRSV. / Respiratory Syncytial Virus is the main cause of acute lower respiratory tract infection (ALTRs) in infants, elderly and immunodepressed patients. Rapid diagnosis of Respiratory Syncytial Virus (RSV) infection is necessary to efficient treatment, avoiding the unnecessary use of antibiotics and determining patient isolation requirements. The reverse trancriptase polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay (IFA) methods have been referred as important tools for virus detection considering the high sensitivity and specificity, respectively of such methods. In order to maximize the simplicity and minimize the risk of sample cross-contamination by two steps RT-PCR, we developed a RT-PCR using a single-tube to detect HRSV in clinical samples. Nasopharyngeal aspirates (Nas) of 226 patients with acute respiratory illness, ranging 0-5 years old, were collected at the University of São Paulo Hospital (HU-USP) in São Paulo city. Samples were tested by indirect immunofluorescence assay, RT semi Nested PCR and single-tube RT-PCR. One hundred two (45,1%) of the 226 samples were positive at least by one of the three methods tested and 75 (33,2%) were positive by all methods. Three (1,3%) samples were positive only by IFI and RT semi Nested PCR, 1 (0,4%) sample were positive only by IFI and RT-PCR single-tube, 5 (2,2%) were positive only by IFI, 2 (0,9%) were positive only by RT semi Nested PCR and 16 (7,1) were positive only RT semi Nested PCR and RT-PCR single-tube. RT-PCR single-tube, showed to be fast, sensitive and specific for diagnosis of RSV and the combined use of both methods enhanced HRSV detection.

A busca por relações entre a infecção pelo Vírus da Imunodeficiência Humana (HIV) e a agressividade da doença periodontal (DP) tem motivado inúmeras pesquisas clínicas. Em acréscimo, a literatura atual também é divergente em relação ao exato processo de sucessão bacteriana que ocorre com a progressão da DP em pacientes HIV-positivos (HIV+). O objetivo deste trabalho foi comparar parâmetros clínicos periodontais e microbiológicos (prevalência de Porphyromonas gingivalis- Pg) em pacientes portadores do HIV. Foram selecionados setenta pacientes (35 HIV+, selecionados no Serviço Ambulatorial Especializado da cidade de Ji-Paraná/RO e 35 HIV-, selecionados da clínica de Periodontia da Faculdade São Lucas de Porto-Velho/RO). Os parâmetros clínicos periodontais avaliados foram: Índice de placa bacteriana (IP); Índice gengival (IG); Profundidade de sondagem (PS); Nível de inserção clínica (NIC) e Índice de sangramento gengival (ISG). A análise microbiológica para a determinação da prevalência de Porphyromonas gingivalis foi realizada através da PCR. Os resultados de todos os parâmetros periodontais analisados no grupo teste (HIV+) mostraram-se estatisticamente superiores quando comparados ao grupo controle (HIV-). Adicionalmente, a prevalência total de Pg também se mostrou significantemente maior no grupo teste em relação ao controle (77,14% e 52,95% para os grupos: teste e controle respectivamente). O modelo experimental realizado permitiu concluir que os indivíduos portadores do HIV apresentaram maior gravidade da doença periodontal acompanhada de um aumento na prevalência de Porphyromonas gingivalis em relação a indivíduos HIV. Estes achados sugerem que a terapia de suporte periodontal periódica pode se constituir como importante aliado na manutenção da saúde bucal deste grupo de pacientes. / The search for relations between infection by the Human Immunodeficiency Virus (HIV) and the aggressiveness of periodontal disease (PD) has motivated many clinical research. In addition, the current literature differs in relation to the exact process of bacterian succession that occurs with the progression of PD in HIVpositive patients (HIV +). The objective of this study was to compare clinical periodontal parameters and microbiological (prevalence of Porphyromonas gingivalis-Pg) in HIV patients . Were selected seventy patients (35 HIV +, selected in the Specialized Ambulatory Service - the town Ji-Paraná/RO and 35 HIV+, selected from the Clinic of Periodontics of the St. Lukes Academy of Porto-Velho/RO). The periodontal clinical parameters were evaluated: plaque Index (PI); gingival index (GI); depth survey (DS); level of clinical Insertion(LCI) and gingival bleeding Index (GBI). The microbiological analysis to determination the prevalence of Porphyromonas gingivalis was performed by PCR. The results of all parameters examined in the test group periodontal (HIV +) showed a higher statistically when compared to the control group (HIV-). Additionally, the overall prevalence of Pg also was significantly higher in group testing in relation to the control (77.14% and 52.95% for the groups: test and control respectively). The experimental model done conducted found that those HIV people bearers had greater severity of periodontal disease accompanied by an increase in the prevalence of Porphyromonas gingivalis regarding HIV people. These findings suggest that periodontal therapy regular support can be as important ally in oral maintaining health of this group of patients.

Neurons in the olfactory epithelium that are responsible for detecting the odors we smell are constantly dying. However, the olfactory system has the unique ability to regenerate new neurons in order for the sense of smell to be maintained. After a new sensory neuron is born in the olfactory epithelium, it must extend a new axon that will travel to the olfactory bulb and make specific synaptic contact so that the odor information from the epithelium can be coded and sent to the higher cortical areas of the brain. The olfactory system’s ability to recover is also even more complex in that it is capable of regeneration after an injury in which a portion or even the entire olfactory epithelium is removed. A well established model for this type of injury in the olfactory epithelium is by inducing a chemical ablation by injection of the drug methimazole. A specific interest in the regenerative process after injury is the mechanism by which axonal debris from the dead neurons is removed. After ablation of the olfactory epithelium, the cell bodies of the neurons detach but their axons remain intact. The axonal debris must not only be removed, but must also be done so in a way that minimizes inflammation in order for new axons to be able to extend to the olfactory bulb. Axonal debris removal has been characterized both in vitro and during development. However, the mechanism of debris removal has yet to be characterized after an injury. Our lab has studied different engulfment proteins in the olfactory bulb after injury using RT-qPCR and found specific temporal expression profiles at 3, 14 and 21 days post injury. Our initial investigations involved some known engulfment proteins such as Jedi1, GULP, and Megf10. However, we found that these proteins are downregulated after an injury. Further investigation has shown that the proteins Cd11b and TLR2 are upregulated after injury. These changes in expression can begin to shed light on the mechanism of axonal debris removal after an injury and can further be used to study how inflammation is suppressed in order to allow for axon extension and synaptic contact to be reestablished.

Introducción: La actual pandemia de COVID-19 es causada por el virus SARS-Cov-2, un betacoronavirus que puede provocar una gripe o desencadenar una tormenta de citoquinas, llegando a causar una neumonía severa o una falla multiorgánica. Desde el inicio de la pandemia se ha venido discutiendo sobre el valor diagnóstico de las pruebas de imagen para la COVID-19, resaltando el uso de la TC de tórax. Objetivo: Evaluar el uso de la TC de tórax como prueba diagnóstica en pacientes sospechosos de neumonía por COVID-19 en la clínica Anglo Americana de Lima, Perú en el año 2020. Material y métodos: Estudio de precisión diagnóstica retrospectivo, en el cual se incluirán mediante censo a 627 pacientes que hayan ingresado por Emergencia Respiratoria de la clínica y cuenten con resultado de RT-PCR para SARS-CoV-2 y TC de tórax, en esta última se clasificará como positiva para compromiso pulmonar por COVID-19 si cumple con los hallazgos de las categorías CO-RADS 4 y 5. Se evaluará la sensibilidad, especificidad, VPP y VPN de la TC de tórax en comparación con la RT-PCR para SARS-CoV-2 para el diagnóstico de COVID-19.

The aim of the thesis was the comparison of two DNA microextraction methods with the use of magnetic beads from food of plant origin. Samples had disparate and complex matrices and were either raw (broccoli) or processed (strawberry jam). The first method uses a magnetic separator for the manipulation of magnetic beads and was used as a standart for the comparison. The second method uses a paramagnetic needle, the advantage of which should be the possibility to isolate DNA of higher quality without a significant contamination by polyphenolic compounds or proteins. The former method was validated by statistic analysis of results obtained from both methods. DNA quality was judged by testing the amplificability of isolated DNA via PCR. The amplified products were visualised on an agarose gel with electrophoresis.

In the food industry, it is very important to take care of the quality, safety and organoleptic properties of the products supplied. For this reason, food must be checked. However, not all information can be found using conventional techniques such as immunoassays, chromatographic techniques, etc. DNA-based techniques can be used for these cases where traditional procedures are insufficient. Among them, the best known technique is PCR. The aim of the thesis was to isolate DNA from vegetable samples (broccoli, beetroot, carrot and pepper). DNA was isolated using the magnetic particle method and the traditional CTAB method. Both methods were able to isolate the DNA from the vegetable samples in quality and at a concentration suitable for PCR, where the 35S rDNA gene region was amplified (more precisely about 700 bp of the 18S-ITS1-5,8S region). After amplification, the PCR products were subjected to restriction reactions and the results compared to bioinformatic analysis. These steps have succeeded in finding suitable enzymes for diferentiation of PCR products from the tested vegetable species.

The effectiveness of magnetic microparticles in isolation of DNA from Lactobacillus rhamnosus CCM 1825T and DNA from chicken erythrocytes were studied in diploma thesis. Magnetic HEMA based microparticles coated by carboxylic groups and hyperbranched styrene-divinylbenzene particles (IMC AS ČR, Prague, Czech Republic) were used for DNA isolation. Magnetic microparticles Dynabeads® DNA DIRECT™ Universal (Dynal, Norway) based on polystyrene and MPG® Uncoated (PureBiotech, USA) based on magnetic glass were used as a control. The dependence of amount of eluted DNA on concentration of DNA in the base solution and the dependence of amount of eluted DNA on concentration of magnetic microparticles were studied. The affinity of magnetic microparticles to RNA for various concentrations of RNA solution was studied, too. The ability of tested particles to isolate DNA from real samples was validated using milk product Actimel. The quality of isolated DNA of Lactobacillus genus was proved using genus specific PCR.

Micromethod of DNA isolation using magnetic particles is one of the modern technological methods used in DNA isolation, and makes the process simpler, more effective and faster. The main aim of this study was to isolate the DNA from various plant (tomato) food products, using different types of magnetic particles. The results were compared and the quantity, purity and the possibility of amplication of the isolated DNA among samples were found to be different. The DNA isolation method using magnetic particles P(HEMA-co-GMA) or HPS B-M-NH2 was shown to be the most effective in achieving the above mentiond parametres. DNAs from the analysed samples of plant food products were isolated in sufficient quantity and quality to be used in the conventional PCR. Differences in the possibility of the amplification of the isolated DNA stored at -20 °C during more than a half year were not found.