Orientadora: Márcia Ferreira da Rosa Sobreira / Coorientador: Hélio José Montassier / Banca: Ketherson Rodrigues Silva / Banca: Ruben Pablo Schocken Iturrino / Banca: Marita Vedovelli Cardozo / Banca: Diane Meyre Rassi / Resumo: A cinomose canina é uma das enfermidades infectocontagiosas mais importantes que acomete os cães. É uma doença causada pelo vírus da Cinomose ("Canine Distemper Virus" - CDV), um Paramyxovirus, do gênero Morbillivirus, de ocorrência mundial, sem sazonalidade, sem predileção de sexo ou raça, apresenta maior incidência em animais jovens, podendo acometer todas as idades. No Brasil, pesquisas sobre o uso de técnicas que comparam os diferentes testes diagnósticos direto para o vírus da cinomose (CDV) associado a análise molecular e filogenética do mesmo ainda continuam escassas. Além disso, são poucos os estudos sobre epidemiologia molecular que relatam diferenças marcantes na análise filogenética do gene hemaglutinina (H) do CDV entre os isolados de campo e as cepas de referência do CDV, especialmente àquelas utilizadas na produção de vacinas. Dessa maneira, o presente trabalho tem como principal objetivo avaliar os principais métodos diagnósticos laboratoriais para identificação do CDV em cães com suspeita de cinomose associando à análise filogenética do gene H dos isolados de campo. O Ensaio imunocromatografico direto (IC), a RT-PCR e a Nested-PCR foram avaliados para detecção do CDV em diferentes amostras clínicas (urina, suabes retais e conjuntivais) de 62 animais suspeitos de cinomose, provenientes do atendimento ambulatorial do hospital veterinário da Unesp (Jaboticabal-SP) e de clínicas particulares da região. Após detecção viral as amostras foram submetidas a sequenciame... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Canine distemper is one of the most important infectious diseases that affects dogs. It is a Canine Distemper Virus (CDV), a Paramyxovirus, of the genus Morbillivirus, a global deforest, without seasonality, without predilection of sex or race, that is constituted by the majority of the animals, being able to affect all ages. In Brazil, research on the use of techniques that compare the different direct diagnostic tests for the distemper virus (CDV) associated with a molecular and phylogenetic analysis of the same are still scarce. In addition, there are some studies on molecular epidemiology that report differences in the phylogenetic analysis of the hemagglutinin (H) gene from CDV between the fields of action and as a reference for CDV, especially the practices used in the production of vaccines. Thus, the main objective of this study is to identify laboratory diagnoses for the identification of CDV in dogs with suspected pair association to the phylogenetic analysis of the H gene of the field isolates. The direct immunochromatographic (CI) assay, RT-PCR and nested-PCR were evaluated for the detection of CDV in different animal species (urine, swabs and conjunctival) of 62 suspected cases of distemper from the outpatient clinic of the Unesp (Jaboticabal-SP) and the private medical records of the region. After viral detection as sequential sequencing followed by phylogenetic analysis of the CDV H gene. A total of 42 animals were positive at least one of the experiment techni... (Complete abstract click electronic access below) / Doutor

Resumo:A disfunção mitocondrial está associada com a manifestação e com a origem de doenças e distúrbios metabólitos. Um novo paradigma complementa um dogma recente relacionado com esta função, onde moléculas presentes no meio extra ou intra-mitocondrial podem atuar como reguladores da resposta imunológica nata, decorrente de um estresse e/ou de uma infecção. O vírus da cinomose canina é responsável por um quadro de depleção quando se encontra em fase de produção viral, principalmente nas células imunocompetentes. Neste sentido, células mononucleares de sangue periférico canino (CMPC) foram coletadas de cães saudáveis, cultivadas e infectadas pela estirpe vacinal CDV (Onderstepoort). Após 24h post-infection (p.i.), as enzimas superóxido dismutase 1 (SOD1), proteína antioxidante 1 (AOP-1) e estresse térmico 70 (Hsp-70) foram detectadas pela reação de imunofluorescência. A expressão do mRNA dos respectivos genes foi realizada em CMPC CDV+ e CDV - após 24h de infecção com a reação em cadeia da ™ polimerase em tempo real. As sondas fluorescentes JC-1 e MitoTracker Green foram utilizadas para avaliar potencial de membrana e função mitocondrial, respectivamente. A estirpe vacinal induziu a perda da viabilidade em mais de 80% das células infectadas em comparação ao grupo controle (p = 0,001) após 24 h. A permeabilidade da membrana mitocondrial (Δψ) detectada pelo uso da sonda MitoTracker™Green e JC-1 revelou um aumento da Δψ e da atividade mitocondrial no grupo CDV + (p = 0,0012) em comparação com as CMPC não infectadas. Em contraste, a expressão do mRNA dos genes AOP-1 e SOD1 foram considerados superiores, enquanto o Hsp-70 não apresentou diferença na sua expressão nos grupos CDV + e CDV-. As CMPC infectadas pelo CDV aumentaram a transcrição dos genes AOP-1 e SOD1, uma defesa antioxidante celular, concomitante com a redução de viabilidade...(resumo completo, clicar acesso eletrônico abaixo) / Abstract:itochondrial dysfunction is associated with the manifestation and origin of diseases and disorders. The new paradigm complements the current mitochondrial dogma, whereby molecules present on or inside the mitochondria may act as immune regulators in response to stress or pathogens. Canine distemper virus infection (CDV) is responsible to immunosuppressive stage when the virus replicates among immune cells. For this purpose, canine peripheral blood mononuclear cells (PBMC) collected from healthy dogs were cultured and infected by CDV vaccine strain (Onderstepoort) and after 24 h post-infection (p.i.) superoxide dismutase (SOD1), antioxidant like protein 1 (AOP-1) and heat shock protein 70 (Hsp-70) enzymes were search in PBMC by immunofluorescence. The expression of mRNA of respective genes was performed in infected and uninfected canine PBMC at 24 h post-infection by real time polymerase chain reaction. Mitochondrial dysfunction was evaluated by the use of MitoTracker™Green and JC-1 probes at the same post-infection time. The vaccine strain induced loss of PBMC viability in more than 80% of infected cells in comparison to control group (p<0.001) at 24h post-infection. The mitochondrial membrane permeability (Δψ) searched by MitoTracker™ Green and JC-1 probes revealed an increase of Δψ in the CDV + group (p<0.0012) in comparison to uninfected PBMC. In contrast, the expression of mRNA of AOP-1 and SOD 1 were considered higher, whereas the Hsp-70 has no difference in its expression between CDV+ and CDV- groups. PBMC infected by CDV increased AOP-1 and SOD1 gene transcription, an antioxidant cell defense, concomitant to a reduce level of PBMC viability. The viral replication also seems to regulate mitochondrial function by modify the membrane potential. However, at this point, host cells have developed an defense producing mediators related to protect against oxidative insult. This is the first... / Orientador: Tereza Cristina Cardoso da Silva / Banca: Vera Cláudia Lorenzetti Magalhães Curci / Banca: Roberto Gameiro Carvalho / Mestre

Made available in DSpace on 2015-04-09T12:28:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-21Bitstream added on 2015-04-09T12:48:05Z : No. of bitstreams: 1 000814238.pdf: 438890 bytes, checksum: f7cc8219db858e87f195b5d08dbb8b04 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / itochondrial dysfunction is associated with the manifestation and origin of diseases and disorders. The new paradigm complements the current mitochondrial dogma, whereby molecules present on or inside the mitochondria may act as immune regulators in response to stress or pathogens. Canine distemper virus infection (CDV) is responsible to immunosuppressive stage when the virus replicates among immune cells. For this purpose, canine peripheral blood mononuclear cells (PBMC) collected from healthy dogs were cultured and infected by CDV vaccine strain (Onderstepoort) and after 24 h post-infection (p.i.) superoxide dismutase (SOD1), antioxidant like protein 1 (AOP-1) and heat shock protein 70 (Hsp-70) enzymes were search in PBMC by immunofluorescence. The expression of mRNA of respective genes was performed in infected and uninfected canine PBMC at 24 h post-infection by real time polymerase chain reaction. Mitochondrial dysfunction was evaluated by the use of MitoTracker™Green and JC-1 probes at the same post-infection time. The vaccine strain induced loss of PBMC viability in more than 80% of infected cells in comparison to control group (p<0.001) at 24h post-infection. The mitochondrial membrane permeability (Δψ) searched by MitoTracker™ Green and JC-1 probes revealed an increase of Δψ in the CDV + group (p<0.0012) in comparison to uninfected PBMC. In contrast, the expression of mRNA of AOP-1 and SOD 1 were considered higher, whereas the Hsp-70 has no difference in its expression between CDV+ and CDV- groups. PBMC infected by CDV increased AOP-1 and SOD1 gene transcription, an antioxidant cell defense, concomitant to a reduce level of PBMC viability. The viral replication also seems to regulate mitochondrial function by modify the membrane potential. However, at this point, host cells have developed an defense producing mediators related to protect against oxidative insult. This is the first... / FAPESP: 2012/06897-0

Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-12-11Bitstream added on 2014-06-13T20:16:24Z : No. of bitstreams: 1 roncatti_ftlb_me_araca.pdf: 389281 bytes, checksum: a97475a27c32aac594bae9073a20134e (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Morte celular programada (MCP) ou apoptose é um fenômeno correspondente a resposta do hospedeiro a uma infecção viral, envolvida na morte e/ou sobrevivência da célula infectada. O vírus da cinomose, canine distemper virus (CDV) é capaz de induzir a apoptose em células linfoides circulantes, tecidos linfóides e cerebelo de cães naturalmente infectados. O CDV também afeta as células Vero e linhagens de células de tumor cervical modulando várias etapas da MCP. Além disso, a fase de imunossupressão, estabelecida a partir da infecção pelo CDV, nem sempre está associada à detecção de partículas virais nas células infectadas. No presente estudo, células mononucleares de sangue periférico de cães (CMSP) foram coletadas, cultivadas e infectadas com a estirpe vacinal de CDV (Onderstepoort) e após 24 h da infecção os eventos iniciais da apoptose foram evidenciados pela marcação e quantificação das proteínas anexina v™ e ApoTrace™. A expressão do RNA mensageiro relacionado ao gene Bax, BCl-2, caspase-3, 8 e 9 e survivin foi realizada em culturas de CMPC infectadas e não infectadas após 24h pela reação de cadeia de polimerase em tempo real. A infecção viral foi responsável pela perda da viabilidade CMPC com expressão da anexina™ V FITC e Apo-Trace ™ FITC em mais de 80% das células infectadas em comparação ao grupo controle (p < 0,001) após 24 h. Os valores elevados da anexina™ V FITC e Apo-Trace ™ FITC encontrados estão em contraste com a maior expressão do mRNA do survivin e da BCl-2, considerados marcadores anti-apoptotic (p < 0,0002). Além disso, Bax, caspase 3, 8 e 9 foram expressos na mesma proporção nas CMPC infectado e não infectado, sem diferenças entre elas (p < 0,005). Em síntese, as células infectadas pelo CDV aumentaram a expressão de survivin e transcrição de gene BCL-2, um mediador de... / Programmed cell death or apoptosis is a central axis of the host response to virus infection involved in cell survival and death. In this respect, several responses are developed by host cells that may control virus replication and infection. On the other hand, viruses have developed strategies to counteract host responses. Canine distemper virus (CDV) has been described to induce apoptosis in lymphoid tissues, lymphoid cells and cerebellum of naturally infected dogs. It has been also described affecting Vero cells and cervical tumor derived cells line by modulating programmed cell death pathways. In addition, the immunosuppression stage established from CDV infection is not always associated to virus detection in damaged cells. Examining the host response at early stages of immunosuppression, especially the expression of activators and/or inhibitors of apoptosis processes can provide important understanding of how the immune responses to CDV are orchestrated. For this purpose, canine peripheral blood mononuclear cells (PBMC) collected from healthy dogs were cultured and infected by CDV vaccine strain (Onderstepoort) and after 24 h post-infection (p.i.) early events of apoptosis were detected by search for annexin V™ and ApoTrace™ markers. The expression of mRNA of Bax, BCl-2, caspase-3, 8 and 9 and also survivin genes was performed in infected and uninfected canine PBMC at 24 h post- infection by real time polymerase chain reaction. The vaccine strain induced loss of PBMC viability with expression of annexin™ V FITC and Apo-Trace™ FITC in more than 80% of infected cells in comparison to control group (p<0.001) at 24h post-infection. The initial of early apoptosis after 24h post- infection is in contrast with higher expression of mRNA of surviving and BCl-2, two major anti-apoptotic proteins (p < 0.0002...(Complete abstract click eletronic acces below)

Orientador: Tereza Cristina Cardoso da Silva / Banca: Vera Claudia Lorenzetti Magalhães Cursi / Banca: Cristina Maria Rodrigues Monteiro / Resumo: Morte celular programada (MCP) ou apoptose é um fenômeno correspondente a resposta do hospedeiro a uma infecção viral, envolvida na morte e/ou sobrevivência da célula infectada. O vírus da cinomose, canine distemper virus (CDV) é capaz de induzir a apoptose em células linfoides circulantes, tecidos linfóides e cerebelo de cães naturalmente infectados. O CDV também afeta as células Vero e linhagens de células de tumor cervical modulando várias etapas da MCP. Além disso, a fase de imunossupressão, estabelecida a partir da infecção pelo CDV, nem sempre está associada à detecção de partículas virais nas células infectadas. No presente estudo, células mononucleares de sangue periférico de cães (CMSP) foram coletadas, cultivadas e infectadas com a estirpe vacinal de CDV (Onderstepoort) e após 24 h da infecção os eventos iniciais da apoptose foram evidenciados pela marcação e quantificação das proteínas anexina v™ e ApoTrace™. A expressão do RNA mensageiro relacionado ao gene Bax, BCl-2, caspase-3, 8 e 9 e survivin foi realizada em culturas de CMPC infectadas e não infectadas após 24h pela reação de cadeia de polimerase em tempo real. A infecção viral foi responsável pela perda da viabilidade CMPC com expressão da anexina™ V FITC e Apo-Trace ™ FITC em mais de 80% das células infectadas em comparação ao grupo controle (p < 0,001) após 24 h. Os valores elevados da anexina™ V FITC e Apo-Trace ™ FITC encontrados estão em contraste com a maior expressão do mRNA do survivin e da BCl-2, considerados marcadores anti-apoptotic (p < 0,0002). Além disso, Bax, caspase 3, 8 e 9 foram expressos na mesma proporção nas CMPC infectado e não infectado, sem diferenças entre elas (p < 0,005). Em síntese, as células infectadas pelo CDV aumentaram a expressão de survivin e transcrição de gene BCL-2, um mediador de... (Resumo completo, clicar acesso eletronico abaixo) / Abstract:Programmed cell death or apoptosis is a central axis of the host response to virus infection involved in cell survival and death. In this respect, several responses are developed by host cells that may control virus replication and infection. On the other hand, viruses have developed strategies to counteract host responses. Canine distemper virus (CDV) has been described to induce apoptosis in lymphoid tissues, lymphoid cells and cerebellum of naturally infected dogs. It has been also described affecting Vero cells and cervical tumor derived cells line by modulating programmed cell death pathways. In addition, the immunosuppression stage established from CDV infection is not always associated to virus detection in damaged cells. Examining the host response at early stages of immunosuppression, especially the expression of activators and/or inhibitors of apoptosis processes can provide important understanding of how the immune responses to CDV are orchestrated. For this purpose, canine peripheral blood mononuclear cells (PBMC) collected from healthy dogs were cultured and infected by CDV vaccine strain (Onderstepoort) and after 24 h post-infection (p.i.) early events of apoptosis were detected by search for annexin V™ and ApoTrace™ markers. The expression of mRNA of Bax, BCl-2, caspase-3, 8 and 9 and also survivin genes was performed in infected and uninfected canine PBMC at 24 h post- infection by real time polymerase chain reaction. The vaccine strain induced loss of PBMC viability with expression of annexin™ V FITC and Apo-Trace™ FITC in more than 80% of infected cells in comparison to control group (p<0.001) at 24h post-infection. The initial of early apoptosis after 24h post- infection is in contrast with higher expression of mRNA of surviving and BCl-2, two major anti-apoptotic proteins (p < 0.0002...(Complete abstract click eletronic acces below) / Mestre

Orientador: Rogério Martins Amorim / Resumo: As células estromais mesenquimais multipotentes (MSCs) são células adultas que residem em diversos tecidos do corpo e possuem capacidade proliferativa, imunomoduladora, anti-inflamatória e neuroregenerativa representado pela secreção de citocinas e fatores neurotróficos. O tecido neural possui baixa capacidade regenerativa após lesões traumáticas, inflamatórias e degenerativas, sendo alvo de estudos com uso de MSCs devido ao seu potencial terapêutico. A cinomose é uma doença viral infectocontagiosa comum em cães que acomete o sistema nervoso central refletindo em sinais clínicos neurológicos causadas por lesões desmielinizantes, resultantes da replicação viral no interior dos oligodendrócitos e de células da microglia, pela regulação do complexo de histocompatibilidade de classe I e infiltração de células inflamatórias. Nesse sentido, com o intuito de reduzir os sinais clínicos neurológicos, o objetivo desse estudo foi avaliar a eficácia terapêutica das MSCs derivadas do tecido adiposo canino pela via intratecal (IT) em cães com encefalomielite viral por cinomose. Foram utilizados 10 cães, sem raça definida, apresentando sinais neurológicos de início agudo e progressivo com diagnóstico positivo pelo teste de Reação em Cadeia pela Polimerase quantitativo (qPCR). No momento 0 (M0), foi realizado exame neurológico, teste qPCR, hemograma e bioquímica sérica. No momento 1 (M1), os animais foram submetidos à anestesia para exame de ressonância magnética (RM), seguido da colheita do... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Multipotent mesenchymal stromal cells (MSCs) are adult cells that reside in various tissues of the body and have proliferative, immunomodulatory, anti-inflammatory and neurooregenerative capacity represented by secretion of cytokines and neurotrophic factors. Neural tissue has low regenerative capacity after traumatic lesions, inflammatory and degenerative, being the target of studies with the use of MSCs due to its therapeutic potential. Distemper is a common viral disease, infectious and contagious of dogs that affects the central nervous system reflecting on clinical neurological signs caused by demyelinating lesions, resulting from viral replication within the oligodendrocytes and microglial cells, by regulation of the class I histocompatibility complex and infiltration of inflammatory cells. Thus, in order to reduce neurological clinical signs, the objective of this study was to evaluate the therapeutic efficacy of MSCs derived from canine adipose tissue by the intrathecal route (IT) in dogs with viral encephalomyelitis due to distemper. Ten dogs were used, mixed breed, presenting neurological signs of acute and progressive onset with positive diagnosis by quantitative Polymerase Chain Reaction (qPCR). At time 0 (M0), neurological examination, qPCR test, blood count and serum biochemistry were performed. At the moment 1 (M1), the animals were submitted to anesthesia for magnetic resonance imaging (MRI), followed by cerebrospinal fluid (CSF) harvest, and then transplantat... (Complete abstract click electronic access below) / Doutor