Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 132-154).

The superinfection exclusion gene (sieB) of Salmonella phage P22 was mapped using phage deletion mutants. The DNA sequence in the region was re-examined in order to find an open reading frame consistent with the deletion mapping. Several discrepancies from the previously published sequence were discovered. The revised sequence revealed a single open reading frame of 242 codons with six likely translation initiation codons. On the basis of deletion and amber mutant phenotypes the second of these six sites was inferred to be the translation initiation site of the sieB gene. The sieB gene encodes a polypeptide with 192 amino acid residues with a calculated molecular weight of 22,442, which is in reasonable agreement with that estimated from polyacrylamide gels. The transcription start-site of sieB was identified by the use of an RNAase protection assay. The sieB promoter thus identified was inactivated by a two-base substitution in its -10 hexamer. The sieB gene of coliphage λ was also identified. The promoter for λ sieB was identified by homology to that of P22 sieB. sieB aborts the lytic development of some phages. P22 itself is insensitive to the lethal effect of SieB because it harbours a determinant called esc. It was found that the sieB gene encodes two polypeptides-SieB, which is the exclusion protein, and Esc, which is a truncated version of SieB that inhibits its action. Superinfecting P22 synthesizes an antisense RNA, sas, that inhibits synthesis of SieB but allows continued synthesis of Esc, thus allowing P22 to by-pass SieB-mediated exclusion. This translational switch induced by sas RNA is essential to vegetatively developing P22; a mutation that prevents this switch causes P22 to commit SieB-mediated suicide. It was also found that P22's Esc allows it to circumvent the SieB-mediated exclusion system of bacteriophage λ.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-06-21Bitstream added on 2014-06-13T19:14:39Z : No. of bitstreams: 1 pereira_dfa_me_sjc.pdf: 933077 bytes, checksum: 1cf55e305807271fd95bf2e3e95495ba (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A espondilite anquilosante (EA) é uma doença inflamatória crônica de etiologia desconhecida, caracterizada pelo acometimento predominante do esqueleto axial. A terapia convencional inclui o uso de antiinflamatórios não hormonais e drogas anti-reumáticas modificadoras da evolução da doença (DMARDs). O uso de agentes imunobiológicos, como o anti-TNF, tem sido considerada uma excelente opção terapêutica em casos mais graves e refratários, porém estudos prévios demonstraram maior risco de infecções após o tratamento. Reservatórios bucais de microrganismos oportunistas podem causar infecções sistêmicas, uma vez que a cavidade bucal representa uma porta de entrada para patógenos, especialmente em pacientes imunocomprometidos. O objetivo do presente estudo foi avaliar a presença e a sensibilidade aos antimicrobianos de Candida spp., Staphylococcus spp., Enterobacteriaceae e Pseudomonas spp. na cavidade bucal de pacientes com EA em uso de anti-TNF comparando-os com indivíduos controle. Foram incluídos no estudo: grupo anti-TNF (35 indivíduos, diagnosticados portadores de EA com idade entre 17 a 63 anos e sob terapia anti-TNF); grupo Convencional (35 pacientes portadores de EA, com idade entre 21 e 74 anos sob tratamento convencional não imunobiológico; e respectivos grupos controle composto por indivíduos saudáveis pareados na idade, gênero e condições bucais aos grupos com EA. Foram realizados exame clínico, anamnese, bem como coleta de enxágüe bucal de cada indivíduo a qual foi semeada em meios de cultura específicos para cada microrganismo e posterior obtenção do número de unidades formadoras de colônia por militros (UFC/mL). Os isolados foram identificados pelo Sistema API, bem como fora realizados testes de sensibilidade aos antifúngicos dos isolados de leveduras, e, antibióticos dos isolados bacterianos... / The Ankylosing Spondylitis (AS) is a chronic inflammatory disease of unknown etiology, particularly characterized by a compromise of the axial skeleton. The conventional therapy includes the use of non-hormonal anti-inflammatory and Disease-modifying antirheumatic drugs (DMARDs). The use of immunobiological agents, such as anti-TNF, has been considered an excellent therapeutical option in more serious and refractory cases. However, previous studies demonstrated an increased risk of infections after the treatment is completed. Oral reservoirs of opportunist microorganisms can cause systemic infections, once that the oral cavity represents a door of entrance for pathogens, especially in immunocompromised patients. The aim of the present study was to evaluate the presence and antimicrobial susceptibility of Candida spp., Staphylococcus spp., Enterobacteriaceae and Pseudomonas spp. in the oral cavity of patients with AS treated with anti-TNF in comparison to healthy individuals. The following groups were included in the study: anti-TNF group (35 AS patients, aged between 17 and 63 years and under anti-TNF therapy); Conventional group (35 AS patients aged between 21 and 74 years under non-immunobiological conventional treatment); and respective Control groups (composed by healthy individuals paired in age, gender and oral conditions with AS groups). After clinical examination and anamnesis, oral rinses of each individual was collected. The rinses were plated in specific culture media for each microorganism. The number of colony-forming units per milliters (CFU/mL) was obtained. Isolates were identified by API system. Next, sensitivity tests to antifungicals agents were done for yeasts isolates and antibiotics for bacterial isolates. For Staphylococcus spp., the CFU counts for the anti-TNF group and Conventional group was statistically higher than the respective control groups... (Complete abstract click electronic access below)

Resumo: A espondilite anquilosante (EA) é uma doença inflamatória crônica de etiologia desconhecida, caracterizada pelo acometimento predominante do esqueleto axial. A terapia convencional inclui o uso de antiinflamatórios não hormonais e drogas anti-reumáticas modificadoras da evolução da doença (DMARDs). O uso de agentes imunobiológicos, como o anti-TNF, tem sido considerada uma excelente opção terapêutica em casos mais graves e refratários, porém estudos prévios demonstraram maior risco de infecções após o tratamento. Reservatórios bucais de microrganismos oportunistas podem causar infecções sistêmicas, uma vez que a cavidade bucal representa uma porta de entrada para patógenos, especialmente em pacientes imunocomprometidos. O objetivo do presente estudo foi avaliar a presença e a sensibilidade aos antimicrobianos de Candida spp., Staphylococcus spp., Enterobacteriaceae e Pseudomonas spp. na cavidade bucal de pacientes com EA em uso de anti-TNF comparando-os com indivíduos controle. Foram incluídos no estudo: grupo anti-TNF (35 indivíduos, diagnosticados portadores de EA com idade entre 17 a 63 anos e sob terapia anti-TNF); grupo Convencional (35 pacientes portadores de EA, com idade entre 21 e 74 anos sob tratamento convencional não imunobiológico; e respectivos grupos controle composto por indivíduos saudáveis pareados na idade, gênero e condições bucais aos grupos com EA. Foram realizados exame clínico, anamnese, bem como coleta de enxágüe bucal de cada indivíduo a qual foi semeada em meios de cultura específicos para cada microrganismo e posterior obtenção do número de unidades formadoras de colônia por militros (UFC/mL). Os isolados foram identificados pelo Sistema API, bem como fora realizados testes de sensibilidade aos antifúngicos dos isolados de leveduras, e, antibióticos dos isolados bacterianos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Ankylosing Spondylitis (AS) is a chronic inflammatory disease of unknown etiology, particularly characterized by a compromise of the axial skeleton. The conventional therapy includes the use of non-hormonal anti-inflammatory and Disease-modifying antirheumatic drugs (DMARDs). The use of immunobiological agents, such as anti-TNF, has been considered an excellent therapeutical option in more serious and refractory cases. However, previous studies demonstrated an increased risk of infections after the treatment is completed. Oral reservoirs of opportunist microorganisms can cause systemic infections, once that the oral cavity represents a door of entrance for pathogens, especially in immunocompromised patients. The aim of the present study was to evaluate the presence and antimicrobial susceptibility of Candida spp., Staphylococcus spp., Enterobacteriaceae and Pseudomonas spp. in the oral cavity of patients with AS treated with anti-TNF in comparison to healthy individuals. The following groups were included in the study: anti-TNF group (35 AS patients, aged between 17 and 63 years and under anti-TNF therapy); Conventional group (35 AS patients aged between 21 and 74 years under non-immunobiological conventional treatment); and respective Control groups (composed by healthy individuals paired in age, gender and oral conditions with AS groups). After clinical examination and anamnesis, oral rinses of each individual was collected. The rinses were plated in specific culture media for each microorganism. The number of colony-forming units per milliters (CFU/mL) was obtained. Isolates were identified by API system. Next, sensitivity tests to antifungicals agents were done for yeasts isolates and antibiotics for bacterial isolates. For Staphylococcus spp., the CFU counts for the anti-TNF group and Conventional group was statistically higher than the respective control groups... (Complete abstract click electronic access below) / Orientador: Cristiane Yumi Koga Ito / Coorientador: Marcelo de Medeiros Pinheiro / Banca: Fernanda Lourenção Brighenti / Banca: Emilia Inoue Sato / Mestre

Secondary bacterial superinfections have been a significant source of debilitating morbidity and mortality outcomes during both seasonal influenza outbreaks and historical pandemics. As many as 40% of those infected with influenza that develop a secondary bacterial infection such as S. aureus or S. pneumoniae will succumb to the infection. The complex relationships between the immune system, the pathogens, and host response makes this facet of biomedical research a topic of continual discoveries. During superinfection, there is a novel hypothesis that the failure of resolution causes a cascade of uncontrolled inflammatory responses leading to excessive morbidity or death. Here, we investigate a specific metabolite receptor, PPARα, and how its induction during superinfection affects the host immune response and the ability to resolve the infection. During superinfections, previous studies from our lab has found that the CYP450 metabolites are produced in abundance compared to their respective levels during singular viral or bacterial infections. These metabolites furthermore induced PPARα, which has been found to enhance necroptosis during superinfection conditions. Understanding programming of cell death provides insights on the resulting inflammation during an active infection. We further examined PPARα properties by inducing it without the presence of pathogens and found PPAR’s mechanisms to be context dependent. When PPARα is stimulated solely with a chemical agonist WY14643, the induction drives macrophages to apoptosis. When we started examining PPARα induction while inducing programmed cell death profiles, we found that the effect of PPARα to be uninfluential to apoptosis, but highly influential in necroptotic cell death. Latest studies associate very long chain fatty acid accumulation in cells undergoing necroptosis, and fatty acid isolation and analysis was completed. Fatty acid isolation showed an accumulation of ceramides and a significant decrease of both vital eicosanoid precursors and cell membrane associated phospholipids. In these studies, we next examined how induction of PPARα affects macrophage immune response and ultimately hinders resolution of infection. In vivo animal studies showed that macrophages were polarizing toward anti-inflammatory M2 phenotypes during the superinfection. When flow cytometry studies were performed to examine if metabolites stimulating PPARα were responsible for this change, we found that macrophages were polarizing to the M2b phenotype. This finding has been highly intriguing to our studies, as M2b macrophages are most abundantly present when resolution occurs in influenza infection and mice have successfully produced anti-influenza antibodies. Further examination was done to see how macrophage immune response was affected. Using Nanostring and examining PPARα activated macrophages via microscopy, several cytokines and chemokines for immune response were dampened. Microscopy specifically showed the nuclear localization of NFκB is effectively diminished during PPAR activation, demonstrating that the immune response is impaired. Finally, macrophage function was considered and analyzed by CFU assays and live cell microscopy. Both indicated that phagocytosis was impaired in macrophages, but microscopy elucidated that there was a lack of bacterial killing due to PPAR activation. Understanding the mechanisms of superinfection and how to effectively ameliorate them has potential to not only reduce the amount of morbidity and mortality around influenza each year, but advance the understandings of how these different systems come together in other immune contexts. The understanding of programmed cell-death, inflammatory gene networks, immune response and function all have changed by lipid profiling and how these metabolites can influence traditionally well studied systems. Having a greater appreciation for how metabolites induce immune, transcriptional, or cellular changes, as well as how metabolites and lipid profiling can be altered would be groundbreaking for inflammation research of several diseases and conditions that are not fully understood. / Biomedical Sciences

Au sein des populations naturelles d'êtres vivants circulent une diversité de parasites, qu'il s'agisse de plusieurs espèces, souches ou plus généralement types. Si certains modèles d'épidémiologie évolutive intègrent déjà le polymorphisme des parasites, rares sont ceux pour lesquels la dynamique épidémiologique dépend de la croissance intra-hôte et des interactions que les parasites entretiennent lorsqu’ils infectent le même hôte. Les complexité combinatoire et dynamique explique pourquoi il n'y a pour l'heure pas de prédiction générale de l'évolution de la virulence dans de tels contextes d'infections multiples. À la recherche d'une tendance générale d'évolution de la virulence, nous modélisons chaque niveau de dynamique sur lequel l'évolution des parasites repose. En particulier, nous étudions explicitement les interactions et l'issue de la compétition au sein des hôtes, les dynamiques épidémiologique et enfin adaptative. Sur l'exemple des infections chroniques causées par des micro-parasites transmis horizontalement, nous employons les approches propres aux systèmes dynamiques et aux probabilités pour emboîter cette suite de dynamiques afin d'en explorer les conséquences évolutives. Nous définissions notamment le concept de patron d'infection, à savoir l'ensemble des issues intra-hôte associées à chaque configuration d'inoculation et décrivons cinq patrons jusqu'ici non décrits, lesquels échappent à la dichotomie classique entre super- et coinfection. Cette typologie nous permet par la suite d'envisager l'évolution de la virulence dans un cadre général. Nous observons en particulier une inéluctable mais bornée croissance évolutive de la virulence. / Natural populations of living beings are exposed to a diversity of parasites, be they several species, strains or more generally types. While some evolutionary epidemiology models already incorporate parasite polymorphism, few make the connection between between-host dynamics and within-host parasite growth. As parasite polymorphism can even occur within the same host, distinct parasite types can interact in various ways and thus interfere with their transmission and therefore their evolution. The combinatorial and dynamical complexity explains why we still lack general predictions regarding the evolution of virulence in such multiple infection contexts. Seeking for a general trend in virulence evolution, we model each dynamical level on which parasite evolution relies. In particular, we explicitly investigate the within-host interactions and competition outcomes, the epidemiological and adaptive dynamics. Focusing on chronic infections caused by horizontally-transmitted microparasites, we apply both dynamical systems and probabilistic approaches to this nested sequence of dynamics to explore the evolutionary outcomes. We notably define the concept of infection pattern, that is the set of within-host outcomes of all inoculation challenges and identify five yet undescribed patterns that escape from the classical super/coinfection dichotomy. This typology then allows us to address virulence evolution under a general framework. We in particular observe an unavoidable but bounded evolutionary increase in virulence.

Made available in DSpace on 2014-06-11T19:30:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-24Bitstream added on 2014-06-13T20:21:34Z : No. of bitstreams: 1 querido_smr_dr_sjc.pdf: 723832 bytes, checksum: 435196e1fab0a833edc2844b87c88a74 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Superinfecção resulta no desenvolvimento de patógenos residentes prejudiciais ou oportunistas em conseqüência de alteração da microbiota benéfica e pode representar uma complicação na terapia antimicrobiana. O presente estudo avaliou a presença de leveduras, estafilococos, enterobactérias e Pseudomonas na cavidade bucal e sulco gengival de indivíduos submetidos à terapia antibiótica para tratamento de tuberculose pulmonar e indivíduos controle. Foram incluídos cinqüenta indivíduos diagnosticados com tuberculose pulmonar e que estivessem sob antibioticoterapia por um período mínimo de 45 dias e máximo de seis meses. Para controle foram incluídos indivíduos sistemicamente saudáveis. A coleta de material da cavidade bucal foi realizada pela técnica de enxágüe bucal e do sulco gengival utilizando-se cones de papel absorvente esterilizados. As amostras coletadas foram semeadas em duplicata em placas de Petri contendo ágar Sabouraud dextrose com cloranfenicol, ágar Manitol e ágar MacConkey. As amostras de Candida spp. e de estafilococos coagulase-positivas foram identificadas por provas bioquímicas específicas e as amostras de estafilococos coagulase-negativas, enterobactérias e Pseudomonas foram identificadas utilizando-se o sistema API. Os resultados foram analisados pelo teste estatístico de Mann-Whitney (p<0,05). Candida spp. foram detectadas em número significativamente maior na cavidade bucal de indivíduos com tuberculose pulmonar em relação aos controles, sendo C. albicans mais prevalente em ambos os grupos. Não foram observadas diferenças estatisticamente significativas entre os grupos para Staphylococcus spp., enterocatérias e Pseudomonas. Concluiu-se que os indivíduos sob antibioticoterapia para tratamento de tuberculose pulmonar apresentaram maior prevalência de Candida spp. na cavidade bucal em relação aos controles. / Superinfection results an overgrowth of potentially harmful resident or other opportunistic pathogens and can further represent a complication of an antimicrobial therapy that alters a beneficial microbiota. The present study evaluated the presence of yeasts, staphylococci, enteric rods and pseudomonads in the oral cavity and gingival sulcus of individuals with pulmonary tuberculosis undergoing antibiotic treatment and controls individuals. There were included 50 individuals diagnosed with pulmonary tuberculosis that were submitted to antibiotic therapy during a minimal period of 45 days and maximum 6 months. Systemically health individuals were included in the control group. Samples from oral cavity were collected through oral rinses and subgingival samples were taken using sterile paper points. Samples were cultured in duplicate onto Sabouraud dextrose agar with chloramphenicol, manitol agar and MacConkey agar. The isolates of Candida spp. and coagulase-positive staphylococci were identified by phenothypic characterization and the isolates of coagulase-negative staphylococci, enteric rods and pseudomonads were identified by API system. Data were analyzed statistically by Mann-Whitney test (p<0.05). Candida spp. were detected in the oral cavity of pulmonary tuberculosis individuals in a significant higher number when compared to controls, and C. albicans was the most prevalent specie found in both groups. No significant statistical differences were observed between the groups related to Staphylococcus spp., enteric rods and pseudomonads. It was concluded that individuals with pulmonary tuberculosis showed significant increase in the quantity of Candida spp. concerning controls.

More than 25 years have passed since the first cases of HIV/AIDS were reported and a decade since highly active antiretroviral therapy (HAART) was introduced as part of the continuum of care for people living with HIV/AIDS (PLWHA). The introduction of HAART as a continuum of care for PLWHA positively changed the characteristics of HIV/AIDS epidemic, contributing for significant declines of HIV/AIDS-related morbidity and mortality rates. Thus, a new era began with HAART, often referred as the “HAART era”. However, HAART also brought with it new challenges and issues for researchers working in the field of HIV/AIDS. This thesis comprises several studies that were designed to gain understanding of some issues on prevention and management of HIV/AIDS that emerged in the current HAART era. The review of the literature points out for many emergent issues in HAART era, but only some issues were researched and presented in this thesis.

Orientador: Antonio Olavo Cardoso Jorge / Banca: Mariella Vieira Pereira Leão / Banca: Elizabete Brasil dos Santos / Banca: Juliana Campos Junqueira / Banca: Cristiane Yumi Koga-Ito / Resumo: Superinfecção resulta no desenvolvimento de patógenos residentes prejudiciais ou oportunistas em conseqüência de alteração da microbiota benéfica e pode representar uma complicação na terapia antimicrobiana. O presente estudo avaliou a presença de leveduras, estafilococos, enterobactérias e Pseudomonas na cavidade bucal e sulco gengival de indivíduos submetidos à terapia antibiótica para tratamento de tuberculose pulmonar e indivíduos controle. Foram incluídos cinqüenta indivíduos diagnosticados com tuberculose pulmonar e que estivessem sob antibioticoterapia por um período mínimo de 45 dias e máximo de seis meses. Para controle foram incluídos indivíduos sistemicamente saudáveis. A coleta de material da cavidade bucal foi realizada pela técnica de enxágüe bucal e do sulco gengival utilizando-se cones de papel absorvente esterilizados. As amostras coletadas foram semeadas em duplicata em placas de Petri contendo ágar Sabouraud dextrose com cloranfenicol, ágar Manitol e ágar MacConkey. As amostras de Candida spp. e de estafilococos coagulase-positivas foram identificadas por provas bioquímicas específicas e as amostras de estafilococos coagulase-negativas, enterobactérias e Pseudomonas foram identificadas utilizando-se o sistema API. Os resultados foram analisados pelo teste estatístico de Mann-Whitney (p<0,05). Candida spp. foram detectadas em número significativamente maior na cavidade bucal de indivíduos com tuberculose pulmonar em relação aos controles, sendo C. albicans mais prevalente em ambos os grupos. Não foram observadas diferenças estatisticamente significativas entre os grupos para Staphylococcus spp., enterocatérias e Pseudomonas. Concluiu-se que os indivíduos sob antibioticoterapia para tratamento de tuberculose pulmonar apresentaram maior prevalência de Candida spp. na cavidade bucal em relação aos controles. / Abstract: Superinfection results an overgrowth of potentially harmful resident or other opportunistic pathogens and can further represent a complication of an antimicrobial therapy that alters a beneficial microbiota. The present study evaluated the presence of yeasts, staphylococci, enteric rods and pseudomonads in the oral cavity and gingival sulcus of individuals with pulmonary tuberculosis undergoing antibiotic treatment and controls individuals. There were included 50 individuals diagnosed with pulmonary tuberculosis that were submitted to antibiotic therapy during a minimal period of 45 days and maximum 6 months. Systemically health individuals were included in the control group. Samples from oral cavity were collected through oral rinses and subgingival samples were taken using sterile paper points. Samples were cultured in duplicate onto Sabouraud dextrose agar with chloramphenicol, manitol agar and MacConkey agar. The isolates of Candida spp. and coagulase-positive staphylococci were identified by phenothypic characterization and the isolates of coagulase-negative staphylococci, enteric rods and pseudomonads were identified by API system. Data were analyzed statistically by Mann-Whitney test (p<0.05). Candida spp. were detected in the oral cavity of pulmonary tuberculosis individuals in a significant higher number when compared to controls, and C. albicans was the most prevalent specie found in both groups. No significant statistical differences were observed between the groups related to Staphylococcus spp., enteric rods and pseudomonads. It was concluded that individuals with pulmonary tuberculosis showed significant increase in the quantity of Candida spp. concerning controls. / Doutor

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