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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
91

Prevalência, fatores de risco e alterações clínicas e laboratoriais na infecção pelos hemoplasmas canino e felino em cães no Hospital Veterinário da Universidade de Passo Fundo / Prevalence, risk factors, laboratorials findings on canine and feline hemoplasma infection in dogs from Veterinary Teaching Hospital at Universidade de Passo Fundo

Valle, Stella de Faria January 2011 (has links)
Os micoplasmas hemotróficos (hemoplasmas) são organismos pleomórficos, epicelulares, gram negativos que infectam a superfície dos eritrócitos de diversas espécies. Devido ao fato de ser incultivável em meios de cultura tradicionais, o diagnóstico é baseado em técnicas moleculares. Em cães, a infecção pelos hemoplasmas pode causar anemia hemolítica na fase aguda, enquanto na doença crônica os sinais são inaparentes, sendo que a imunodepressão e a esplenectomia podem desencadear a doença aguda. Os objetivos do presente estudo foram avaliar a prevalência dos hemoplasmas em cães submetidos a atendimento clínico e cirúrgico no Hospital Veterinário da Universidade de Passo Fundo (HV-UPF), identificar os fatores de risco para infecçao pelos hemoplasmas e as condições clínicas na infecção natural. Para isso, foram selecionadas amostras de sangue com EDTA submetidas com propósito diagnóstico ao Laboratório de Análises Clínicas do HV-UPF. Após a verificação do controle da extração (PCR para o gene GAPDH), as amostras foram encaminhadas ao Department of Comparative Pathobiology, Purdue University (IN, USA) para as análises moleculares. Foi realizada reação em cadeia da polimerase (PCR) para os hemoplasmas felinos (‘Candidatus Mycoplasma turicencis’, ‘Candidatus Mycoplasma haemominutum’) e caninos (Mycoplasma haemocanis e ‘Candidatus Mycoplasma haematoparvum’). Ao total, foram analisadas 347 amostras, sendo que 16 foram negativas para o controle GAPDH e excluídas do estudo. A prevalência para hemoplasma foi de 6,4% (21/331) sendo 5,1% (17/331) para Mycoplasma haemocanis e 1,8% (6/331) para um organismo geneticamente semelhante ao hemoplasma felino ‘Candidatus Mycoplasma haemominutum’. Não foram encontradas amostras positivas para ‘Candidatus Mycoplasma turicencis’ e ‘Candidatus Mycoplasma haematoparvum’. Os cães positivos para hemoplasmas tinham como fatores de risco a presença de ectoparasitas (carrapatos e/ou pulgas), idade avançada e habitavam em casa. Embora tenha sido identificada a presença de correlação entre as neoplasias e feridas causadas por brigas com outros cães e a infecção pelos hemoplasmas, tais resultados foram atribuídos a uma influência da idade e a presença de ectoparasitas. Não houve variações hematológicas e bioquímicas em decorrência da infecção pelo Mycoplasma haemocanis. Refere-se, no presente estudo, o primeiro estudo relacionando a freqüência da infecção pelos hemoplasmas em cães no Brasil. / The hemotropic mycoplasmas (haemoplasmas) are pleomorphic epicellular and gramnegative organisms that do not grow in conventional culture media. The organisms infect the red blood cell surface of several species and nowadays the diagnosis is based on molecular techniques. In dogs, the acute infection causes severe hemolytic anemia, while in the chronic disease the clinical signs are unapparent. In addition splenectomy or immunosuppression may result in the acute disease. The aims of the present study were to evaluate the prevalence of haemoplasmas in dogs that were submitted to clinical or chirurgical procedures at the Veterinary Teaching Hospital of the University of Passo Fundo (UPF-HV), to identify the risk factors for infection and the clinical conditions of haemoplasma natural infection. EDTA blood samples that were submitted for diagnosis purpose at the Laboratório de Análises Clínicas of the HV-UPF were randomly selected and clinical data were obtained from the hospital database. After DNA extraction and the internal control, the samples were sent to the Department of Comparative Pathobiology at the Purdue University (IN, USA), School of Veterinary Medicine, for molecular analysis. A polymerase chain reaction (PCR) for feline haemoplasmas ('Candidatus Mycoplasma turicencis' and 'Candidatus Mycoplasma haemominutum') and canine haemoplasmas (Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum') was performed. A total of 347 samples were analyzed, while 16 were negative for the control GAPDH and excluded from the study. The total prevalence was 6.4% (21/331), 5.1% (17/331) for Mycoplasma Haemocanis and 1.8% (6/331) for an organism genetically similar to feline 'Candidatus Mycoplasma haemominutum' and no samples were found positive to 'Candidatus Mycoplasma turicencis' and 'Candidatus Mycoplasma haematoparvum'. The haemoplasma positive dogs lived at private homes, are old and were in contact with ectoparasites (ticks and/or fleas). Although it was identified a positive correlation between neoplasic disease, wounds caused by dog fights and the haemoplasma infection, these results were attributed to the influence of age and the vector. Hematological and biochemical variations were not identified at Mycoplasma haemocanis positive samples. This is the first study frequency of haemoplasma infection in dogs in Brazil.
92

Prevalência, fatores de risco e alterações clínicas e laboratoriais na infecção pelos hemoplasmas canino e felino em cães no Hospital Veterinário da Universidade de Passo Fundo / Prevalence, risk factors, laboratorials findings on canine and feline hemoplasma infection in dogs from Veterinary Teaching Hospital at Universidade de Passo Fundo

Valle, Stella de Faria January 2011 (has links)
Os micoplasmas hemotróficos (hemoplasmas) são organismos pleomórficos, epicelulares, gram negativos que infectam a superfície dos eritrócitos de diversas espécies. Devido ao fato de ser incultivável em meios de cultura tradicionais, o diagnóstico é baseado em técnicas moleculares. Em cães, a infecção pelos hemoplasmas pode causar anemia hemolítica na fase aguda, enquanto na doença crônica os sinais são inaparentes, sendo que a imunodepressão e a esplenectomia podem desencadear a doença aguda. Os objetivos do presente estudo foram avaliar a prevalência dos hemoplasmas em cães submetidos a atendimento clínico e cirúrgico no Hospital Veterinário da Universidade de Passo Fundo (HV-UPF), identificar os fatores de risco para infecçao pelos hemoplasmas e as condições clínicas na infecção natural. Para isso, foram selecionadas amostras de sangue com EDTA submetidas com propósito diagnóstico ao Laboratório de Análises Clínicas do HV-UPF. Após a verificação do controle da extração (PCR para o gene GAPDH), as amostras foram encaminhadas ao Department of Comparative Pathobiology, Purdue University (IN, USA) para as análises moleculares. Foi realizada reação em cadeia da polimerase (PCR) para os hemoplasmas felinos (‘Candidatus Mycoplasma turicencis’, ‘Candidatus Mycoplasma haemominutum’) e caninos (Mycoplasma haemocanis e ‘Candidatus Mycoplasma haematoparvum’). Ao total, foram analisadas 347 amostras, sendo que 16 foram negativas para o controle GAPDH e excluídas do estudo. A prevalência para hemoplasma foi de 6,4% (21/331) sendo 5,1% (17/331) para Mycoplasma haemocanis e 1,8% (6/331) para um organismo geneticamente semelhante ao hemoplasma felino ‘Candidatus Mycoplasma haemominutum’. Não foram encontradas amostras positivas para ‘Candidatus Mycoplasma turicencis’ e ‘Candidatus Mycoplasma haematoparvum’. Os cães positivos para hemoplasmas tinham como fatores de risco a presença de ectoparasitas (carrapatos e/ou pulgas), idade avançada e habitavam em casa. Embora tenha sido identificada a presença de correlação entre as neoplasias e feridas causadas por brigas com outros cães e a infecção pelos hemoplasmas, tais resultados foram atribuídos a uma influência da idade e a presença de ectoparasitas. Não houve variações hematológicas e bioquímicas em decorrência da infecção pelo Mycoplasma haemocanis. Refere-se, no presente estudo, o primeiro estudo relacionando a freqüência da infecção pelos hemoplasmas em cães no Brasil. / The hemotropic mycoplasmas (haemoplasmas) are pleomorphic epicellular and gramnegative organisms that do not grow in conventional culture media. The organisms infect the red blood cell surface of several species and nowadays the diagnosis is based on molecular techniques. In dogs, the acute infection causes severe hemolytic anemia, while in the chronic disease the clinical signs are unapparent. In addition splenectomy or immunosuppression may result in the acute disease. The aims of the present study were to evaluate the prevalence of haemoplasmas in dogs that were submitted to clinical or chirurgical procedures at the Veterinary Teaching Hospital of the University of Passo Fundo (UPF-HV), to identify the risk factors for infection and the clinical conditions of haemoplasma natural infection. EDTA blood samples that were submitted for diagnosis purpose at the Laboratório de Análises Clínicas of the HV-UPF were randomly selected and clinical data were obtained from the hospital database. After DNA extraction and the internal control, the samples were sent to the Department of Comparative Pathobiology at the Purdue University (IN, USA), School of Veterinary Medicine, for molecular analysis. A polymerase chain reaction (PCR) for feline haemoplasmas ('Candidatus Mycoplasma turicencis' and 'Candidatus Mycoplasma haemominutum') and canine haemoplasmas (Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum') was performed. A total of 347 samples were analyzed, while 16 were negative for the control GAPDH and excluded from the study. The total prevalence was 6.4% (21/331), 5.1% (17/331) for Mycoplasma Haemocanis and 1.8% (6/331) for an organism genetically similar to feline 'Candidatus Mycoplasma haemominutum' and no samples were found positive to 'Candidatus Mycoplasma turicencis' and 'Candidatus Mycoplasma haematoparvum'. The haemoplasma positive dogs lived at private homes, are old and were in contact with ectoparasites (ticks and/or fleas). Although it was identified a positive correlation between neoplasic disease, wounds caused by dog fights and the haemoplasma infection, these results were attributed to the influence of age and the vector. Hematological and biochemical variations were not identified at Mycoplasma haemocanis positive samples. This is the first study frequency of haemoplasma infection in dogs in Brazil.
93

An epidemiological investigation of respiratory disease in racehorses

Wood, James Lionel Norman January 1999 (has links)
No description available.
94

Mycoplasma arginini increases activation, energetic deregulation, and tumor progression of VM-M3 metastatic macrophage cells

Flores, Roberto Ettore January 2014 (has links)
Thesis advisor: Thomas N. Seyfried / Mycoplasmas are the smallest, self-replicating free-living prokaryotes, and have been associated with carcinogenesis. Mycoplasmas can be detected in a high percentage of a wide variety of primary human cancers. Some mycoplasma species such as M. fermentans and M. hyorhinis can transform normal murine and human cell lines into tumorigenic cells. Mycoplasma infection can activate oncogenes as well as inactivate tumor suppressor genes. These observations suggest that mycoplasmas can be both carcinogenic and or onco-modulatory. I found that the metastatic macrophage VM-M3 cell line (referred to as M3+) was infected with mycoplasmas. Mycoplasmal16S rDNA sequencing showed M3+ cells were infected by the mycoplasma species M. arginini. Antibiotic was used to eradicate M. arginini from M3+ cells (referred to as M3- cells). The energetics of the infected M3+ cells and the non-infected M3- cells was studied by measuring respiration (oxygen consumption) and fermentation (lactate production). Respiration was enhanced and fermentation was reduced in the M3- cells compared to the M3+ cells. Glucose enhanced the fermentation and reduced the respiration of both the M3+ and the M3- cells. The M3+ cells produced higher quantities of metabolites indicative of immunological activation (itaconic acid, succinate, and citrulline) compared to M3- cells. In addition, in-vitro proliferation was higher in the M3+ cells than in the M3- cells at high cell densities. Primary subcutaneous tumor growth and metastasis was less in mice inoculated with the M3- cells than with the M3+ cells. The survival of a VM mouse was longer when inoculated with the M3- cells compared to the M3+ cells. Altogether these data indicates that M. arginini is an onco-modulator associated with activation, deregulated energetics and enhanced tumor progression of VM-M3 metastatic macrophage cells. / Thesis (MS) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
95

Isolamento de micoplasma de suínos com problemas respiratórios e tipificação dos isolados pela PFGE e seqüenciamento do gene 16S rRNA. / Isolation of swine origin mycoplasmas from specimens with respiratory disturbances and typification of isolates by PFGE and 16S rRNA sequencing gene.

Yamaguti, Mauricio 03 June 2009 (has links)
As doenças respiratórias são responsáveis, na suinocultura, por grandes perdas econômicas e entre os agentes destacam-se os micoplasmas. Foram coletadas 126 amostras de muco nasal/tonsilar, e fragmentos de 78 pulmões, 2 de traquéia e 2 de tonsila. No isolamento foi utilizado o meio FRIIS modificado, na identificação, a Multiplex-PCR e na tipificação, a PFGE e sequenciamento do gene 16S rRNA. Foram obtidos 59 isolados identificados como M. hyopneumoniae (1,70%), M. flocculare (3,40%) e M. hyorhinis (94,90%). A PFGE dos isolados de M. hyorhinis, resultou em 10 e 9 perfis com a enzima AvaI e XhoI, respectivamente. O sequenciamento do gene 16S rRNA dos isolados M. hyorhinis apresentaram baixo polimorfismo quando comparados entre si e com a cepa de referência. A sequência do gene 16S rRNA do isolado de M. hyopneumoniae, quando comparada a seqüência da cepa J e os isolados 7448 e 232, resultaram em polimorfismo. O M. hyopneumoniae continua sendo o mais difícil de isolar. Os dendrogramas obtidos da PFGE resultaram em grande heterogeneidade entre os isolados de M. hyorhinis. O seqüenciamento do gene 16S rRNA permitiu o estudo de variabilidade interespecífica e intraespecífica dos isolados de micoplasmas. / Economic losses in swine production are common due the respiratory diseases in these animals. M. hyopneumoniae and M. hyorhinis are the most frequent microbial agents. The aim of this study was recover this isolates in FRIIS medium, indentify them by Multiplex PCR and detect their genotypic variations by PFGE and sequencing the 16s rRNA gene. One hundred twenty six swabs from tonsil and nasal mucus of swine with respiratory disturbances were analyzed. It was included 78 lungs, two trachea and two tonsils. It was obtained 59 isolates; 1.70% of M. hyopneumoniae, 3.40% of M. flocculare and 94.90% of M. hyorhinis. The PFGE of M. hyorhinis, allowed obtain 10 profiles with enzyme AvaI and nine profiles with XhoI. A low polymorphism of gene 16sRNS was detected in M. hyorhinis isolates when compared with the type strain at the GenBank. The M. hyopneumoniae isolates resulted in polymorphisms when comparated with strain J, 7448 and 232. M. hyopneumoniae is still the most difficult to isolate. M. hyorhinis isolates of different herds showed a large heterogenicity with enzymes AvaI e XhoI. The sequencing of gene 16S rRNA allowed analyse the interespecífic and intraespecífic variations of mycoplasmas isolated.
96

Detecção do vírus da Influenza Aviária, Paramyxovirus tipo 1 (vírus da Doença de Newcastle), Mycoplasma gallisepticum e Mycoplasma synoviae em aves silvestres e domésticas próximas às granjas avícolas comerciais nas regiões de Mogi das Cruzes e Louveira do Estado de São Paulo / Detection of Influenzavirus, Paramyxovirus I, Mycoplasma gallisepticum and Mycoplasma synoviae in free-ranging birds and backyard chicken around poultry farms in Mogi das Cruzes and Louveira, São Paulo state

Guimarães, Marta Brito 19 December 2012 (has links)
Objetivou-se, neste trabalho, detectar o vírus da Influenza aviária, Paramyxovirus tipo 1 (doença de Newcastle), Mycoplasma gallisepticum e Mycoplasma synoviae, respectivamente pelas técnicas de RT-PCR e PCR, em aves domésticas e aves em vida livre próximas às granjas avícolas nas cidades de Mogi das Cruzes e Louveira do Estado de São Paulo. As aves silvestres foram capturadas, anilhadas, submetidas à avaliação de estado geral e à coleta de suabes de orofaringe e cloaca. As aves de subsistência ou fundo de quintal seguiram o mesmo protocolo com a exceção do anilhamento, e tiveram amostras de sangue coletadas para a pesquisa de anticorpos contra o vírus da Doença de Newcastle, Mycoplasma gallisepticum e Mycoplasma synoviae pela técnica de ELISA indireto. Foram considerados os aspectos da biodiversidade entre as espécies silvestres capturadas e a biossegurança nas granjas. As aves silvestres apresentaram resultados negativos nesta pesquisa, no entanto, Mycoplasma gallisepticum e Mycoplasma synoviae foram detectados pela técnica da PCR nas aves de subsistência, assim como apresentaram títulos de anticorpos para os agentes acima citados e para o Paramyxovirus tipo I. Duas granjas não possuíam medidas de biosseguridade adequadas permitindo o contato de animais de vida livre com as aves de fundo de quintal e com as aves de produção, o que pode facilitar a disseminação de patógenos de interesse para a saúde pública e para a avicultura comercial. / The aim of this study is to detect avian influenza virus, Newcastle disease virus (Paramyxovirus I), Mycoplasma gallisepticum and Mycoplasma synoviae in backyard chicken and wildlife birds around commercial poultry farms using RT-PCR and PCR. The birds were captured with mist nets, identified with alluminium leg rings, subjected to the assessment of clinical conditions and samples were collected by oral and cloacal swabs. The same was done with backyard chicken without the identification with leg rings. Blood samples were collected from backyard chicken and tested for antibodies against Mycoplasma gallisepticum, Mycoplasma synoviae and Paramyxovirus I by indirect ELISA test. This study was conducted in Mogi das Cruzes and Louveira, São Paulo state, where the commercial poultry is considered an activity of great importance. The results were negative to wild birds, but we could detect Mycoplasma gallisepticum and Mycoplasma synoviae by PCR and antibodies titles for Mycoplasma gallisepticum, Mycoplasma synoviae and Newcastle disease in backyard chickens.Two farms didn´t have appropriate biosecurity measures, allowing intense contact with free-living birds, backyard chicken and poultry facilitating spread of pathogens with concern to human health and poultry farms.
97

Detecção do vírus da Influenza Aviária, Paramyxovirus tipo 1 (vírus da Doença de Newcastle), Mycoplasma gallisepticum e Mycoplasma synoviae em aves silvestres e domésticas próximas às granjas avícolas comerciais nas regiões de Mogi das Cruzes e Louveira do Estado de São Paulo / Detection of Influenzavirus, Paramyxovirus I, Mycoplasma gallisepticum and Mycoplasma synoviae in free-ranging birds and backyard chicken around poultry farms in Mogi das Cruzes and Louveira, São Paulo state

Marta Brito Guimarães 19 December 2012 (has links)
Objetivou-se, neste trabalho, detectar o vírus da Influenza aviária, Paramyxovirus tipo 1 (doença de Newcastle), Mycoplasma gallisepticum e Mycoplasma synoviae, respectivamente pelas técnicas de RT-PCR e PCR, em aves domésticas e aves em vida livre próximas às granjas avícolas nas cidades de Mogi das Cruzes e Louveira do Estado de São Paulo. As aves silvestres foram capturadas, anilhadas, submetidas à avaliação de estado geral e à coleta de suabes de orofaringe e cloaca. As aves de subsistência ou fundo de quintal seguiram o mesmo protocolo com a exceção do anilhamento, e tiveram amostras de sangue coletadas para a pesquisa de anticorpos contra o vírus da Doença de Newcastle, Mycoplasma gallisepticum e Mycoplasma synoviae pela técnica de ELISA indireto. Foram considerados os aspectos da biodiversidade entre as espécies silvestres capturadas e a biossegurança nas granjas. As aves silvestres apresentaram resultados negativos nesta pesquisa, no entanto, Mycoplasma gallisepticum e Mycoplasma synoviae foram detectados pela técnica da PCR nas aves de subsistência, assim como apresentaram títulos de anticorpos para os agentes acima citados e para o Paramyxovirus tipo I. Duas granjas não possuíam medidas de biosseguridade adequadas permitindo o contato de animais de vida livre com as aves de fundo de quintal e com as aves de produção, o que pode facilitar a disseminação de patógenos de interesse para a saúde pública e para a avicultura comercial. / The aim of this study is to detect avian influenza virus, Newcastle disease virus (Paramyxovirus I), Mycoplasma gallisepticum and Mycoplasma synoviae in backyard chicken and wildlife birds around commercial poultry farms using RT-PCR and PCR. The birds were captured with mist nets, identified with alluminium leg rings, subjected to the assessment of clinical conditions and samples were collected by oral and cloacal swabs. The same was done with backyard chicken without the identification with leg rings. Blood samples were collected from backyard chicken and tested for antibodies against Mycoplasma gallisepticum, Mycoplasma synoviae and Paramyxovirus I by indirect ELISA test. This study was conducted in Mogi das Cruzes and Louveira, São Paulo state, where the commercial poultry is considered an activity of great importance. The results were negative to wild birds, but we could detect Mycoplasma gallisepticum and Mycoplasma synoviae by PCR and antibodies titles for Mycoplasma gallisepticum, Mycoplasma synoviae and Newcastle disease in backyard chickens.Two farms didn´t have appropriate biosecurity measures, allowing intense contact with free-living birds, backyard chicken and poultry facilitating spread of pathogens with concern to human health and poultry farms.
98

Isolamento de micoplasma de suínos com problemas respiratórios e tipificação dos isolados pela PFGE e seqüenciamento do gene 16S rRNA. / Isolation of swine origin mycoplasmas from specimens with respiratory disturbances and typification of isolates by PFGE and 16S rRNA sequencing gene.

Mauricio Yamaguti 03 June 2009 (has links)
As doenças respiratórias são responsáveis, na suinocultura, por grandes perdas econômicas e entre os agentes destacam-se os micoplasmas. Foram coletadas 126 amostras de muco nasal/tonsilar, e fragmentos de 78 pulmões, 2 de traquéia e 2 de tonsila. No isolamento foi utilizado o meio FRIIS modificado, na identificação, a Multiplex-PCR e na tipificação, a PFGE e sequenciamento do gene 16S rRNA. Foram obtidos 59 isolados identificados como M. hyopneumoniae (1,70%), M. flocculare (3,40%) e M. hyorhinis (94,90%). A PFGE dos isolados de M. hyorhinis, resultou em 10 e 9 perfis com a enzima AvaI e XhoI, respectivamente. O sequenciamento do gene 16S rRNA dos isolados M. hyorhinis apresentaram baixo polimorfismo quando comparados entre si e com a cepa de referência. A sequência do gene 16S rRNA do isolado de M. hyopneumoniae, quando comparada a seqüência da cepa J e os isolados 7448 e 232, resultaram em polimorfismo. O M. hyopneumoniae continua sendo o mais difícil de isolar. Os dendrogramas obtidos da PFGE resultaram em grande heterogeneidade entre os isolados de M. hyorhinis. O seqüenciamento do gene 16S rRNA permitiu o estudo de variabilidade interespecífica e intraespecífica dos isolados de micoplasmas. / Economic losses in swine production are common due the respiratory diseases in these animals. M. hyopneumoniae and M. hyorhinis are the most frequent microbial agents. The aim of this study was recover this isolates in FRIIS medium, indentify them by Multiplex PCR and detect their genotypic variations by PFGE and sequencing the 16s rRNA gene. One hundred twenty six swabs from tonsil and nasal mucus of swine with respiratory disturbances were analyzed. It was included 78 lungs, two trachea and two tonsils. It was obtained 59 isolates; 1.70% of M. hyopneumoniae, 3.40% of M. flocculare and 94.90% of M. hyorhinis. The PFGE of M. hyorhinis, allowed obtain 10 profiles with enzyme AvaI and nine profiles with XhoI. A low polymorphism of gene 16sRNS was detected in M. hyorhinis isolates when compared with the type strain at the GenBank. The M. hyopneumoniae isolates resulted in polymorphisms when comparated with strain J, 7448 and 232. M. hyopneumoniae is still the most difficult to isolate. M. hyorhinis isolates of different herds showed a large heterogenicity with enzymes AvaI e XhoI. The sequencing of gene 16S rRNA allowed analyse the interespecífic and intraespecífic variations of mycoplasmas isolated.
99

Microbiological studies, principally upon the mollicutes / by Ronald H. Leach.

Leach, Ronald H. (Ronald Hubert) January 1994 (has links)
Includes bibliographical references. / 1v. (various pagings) ; / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Comprises four sections: published research papers; abstracts of major conference presentations; mycoplasmological subject and book reviews; and, academic theses. The published research papers comprising the main basis of the submission predominantly report original findings of laboratory investigations within microbiology dealing with Micobacteria, microbial water requirements, and Mollicutes (mycoplasms). / Thesis (D.Sc.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995
100

Paper de les Proteïnes P110, P140, MG312 i MG217 en l'Estructura i Funció de l'Organela Terminal de Mycoplasma genitalium

Burgos Castellanos, Raül 18 November 2009 (has links)
La tesis que es presenta comprèn tres treballs d'investigació independents, connectats entre sí per un mateix fil conductor: l'estudi de l'estructura i funció de l'organela terminal de Mycoplasma genitalium.En el primer treball s'han obtingut i caracteritzat mutants deficients per les proteïnes P140 i P110. Aquests estudis han revelat que les proteïnes P140 i P110 estan estabilitzades de forma recíproca, són els principals determinants de citadherència i la seva presència és necessària per al desenvolupament de l'organela terminal. A més, l'absència d'aquestes dues proteïnes també promou la inestabilitat de la proteïna de motilitat MG386 i un increment en els nivells de la proteïna DnaK. Per altra banda, també s'han analitzat un conjunt de mutants espontanis en adhesió cel·lular que apareixen amb una elevada freqüència. Aquest anàlisi ha permès determinar que aquests mutants s'originen com a conseqüència de grans delecions afectant als gens codificants de les proteïnes P140 i P110. A més, s'ha demostrat que aquestes delecions són producte de recombinacions entre seqüències dels gens afectats i seqüències repetides que es troben distribuïdes al llarg del genoma. Aquest origen recombinatiu explica l'alta freqüència d'aparició d'aquests mutants i obre la possibilitat de l'existència d'un mecanisme general de variació de fase de l'expressió de les proteïnes P140 i P110. En el segon treball s'ha investigat el paper de la proteïna MG312 en l'arquitectura i funció de l'organela terminal. Aquest estudi ha permès determinar que aquesta proteïna és un component estructural bàsic de l'organela terminal, essencial per al manteniment de la integritat d'aquesta estructura i d'alguns dels seus components. Com a conseqüència, els mutants deficients per la proteïna MG312 no desenvolupen organeles terminals, són parcialment deficients en adhesió i completament immòbils. D'altra banda, també s'ha analitzat la implicació de diferents dominis en la funció de la proteïna MG312. En general, aquest estudi ha permès identificar dos dominis funcionals importants: un domini C-terminal que manté la integritat estructural de l'organela terminal i un domini N-terminal amb una implicació més específica en motilitat i/o divisió cel·lular. També s'ha analitzat la implicació específica dels motius EAGR i Walker A presents en el domini N-terminal. Per últim, la proteïna MG312 ha estat expressada de forma recombinant. Estudis de microsocòpia electrònica sobre la proteïna recombinant indiquen que aquesta proteïna mostra una notable semblança estructural amb les proteïnes SMC, implicades en el manteniment estructural dels cromosomes. En el tercer i últim treball s'ha abordat la identificació de gens involucrats en el control del moviment dels micoplasmes. Es descriu el desenvolupament d'un mètode, que ens ha permès aïllar un mutant deficient per la proteïna MG217 i que exhibeix alteracions en els patrons de moviment. Les dades obtingudes de la caracterització d'aquest mutant indiquen que la proteïna MG217 és un nou component de l'organela terminal implicat en promoure la inclinació d'aquesta estructura respecte el cos cel·lular. De manera interessant, aquestes alteracions en el grau d'inclinació de l'organela terminal es correlacionen amb els canvis observats en els patrons de moviment, suggerint que l'organela terminal actua com a un timó i determina la direcció del desplaçament dels micoplasmes. / This thesis is composed of three independent research works focused on the study of the structure and function of the terminal organelle in Mycoplasma genitalium.In the first work, we obtained and characterized P140 and P110 deficient mutants. These studies revealed a reciprocal posttranslational stabilization between P140 and P110 proteins, and that these proteins are required for cell adhesion and terminal organelle development. Loss of either P140 or P110 also correlates with decreased levels of the MG386 motility related protein and increased levels of the DnaK protein. In addition, several cytadherence-negative mutants that appear spontaneously at unexpectedly high rates were also examined. We found that these mutants were originated as a consequence of large deletions affecting the P140 and P110 coding genes. Furthermore, we demonstrated that these deletions are the product of recombination events involving sequences of the P140 and P110 coding genes and DNA repetitive elements that are distributed along the M. genitalium genome. The recombinative origin of these deletions provides a plausible explanation for the unexpectedly high rates of appearance of these mutants and opens the possibility of the existence of a phase variation mechanism to switch on and off the expression of the M. genitalium P140 and P110 cytadhesins. In the second work, we investigated the role of the MG312 protein in the function and architecture of the terminal organelle. We found that MG312 protein is a structural component essential to maintain the integrity of the terminal organelle and its components. As a consequence, MG312 deficient mutants loss the terminal organelle and exhibit an intermediate-cytadherence phenotype and a complete absence of gliding motility. On the other hand, we also examined the contribution of different domains in the MG312 protein function. This study allowed us to identify two separate and functional domains: a C-terminal domain implicated in the terminal organelle assembly functions and an N-terminal domain involved specifically in gliding motility and/or cell division. The specific contribution of the EAGR box and Walker A box found in the N-terminal region was also examined. Finally, MG312 was expressed in a recombinant way and its structure was analyzed by using electron microscopy. Images obtained showed that this protein exhibits a surprising structural similarity with SMC proteins, involved in the structural maintenance of chromsomes.In the third and last work we aimed to identify genes related to the regulation of the mycoplasma cell movement. We developed a straightforward procedure that allowed us to isolate a MG217 deficient mutant exhibiting an altered gliding behaviour pattern. Further characterization of this mutant revealed that the MG217 protein is a novel terminal organelle component involved in the curving of this structure in M. genitalium cells. Interestingly, changes in the tilt angle between the terminal organelle and the cell body correlates with the observed changes in the gliding behaviour, suggesting that the terminal organelle operates as a guide to steer the mycoplasma cell in specific directions.

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