In regions endemic for malaria, natural exposure results in an acquired immunity which protects individuals from severe disease. However, no vaccine against the blood-stage of malaria, against which naturally-acquired immunity is targeted, currently exists that is capable of emulating, or out-performing, natural protection. To rationally direct the next generation of blood-stage malaria vaccine development, a greater understanding of the immunological mechanisms involved in clinical protection is required. To date, the assessment of naturally-acquired and vaccine-induced immunity to the blood-stage of malaria has suffered from a paucity of in vitro immunological assays that are both robust and reproducible, whilst allowing for assessment of anti-parasitic activity induced by antibodies, either alone, or in conjunction with immune cells. Thus this Thesis describes the development of the antibody-dependent respiratory burst (ADRB) assay, for assessment of blood-stage immunity against Plasmodium falciparum, as well as the Duffy antigen receptor for chemokines (DARC) – Duffy binding protein (DBP) binding inhibition assay, for assessing antibody mediated immunity to P. vivax. A reproducible and standardised assay of ADRB activity was developed here and applied to studies of immunity in both mice and humans. ADRB activity, which assesses antibodies' ability to activate oxidative burst in neutrophils via Fc receptor (FcR)-dependent pathways, was shown to associate with clinical protection in a cohort from Mali where FcR-independent immunological assays, such as the assay of growth inhibition activity, did not. This work thus elucidates the importance of FcR-dependent immunity to P. falciparum malaria and establishes the ADRB assay as a useful tool for future vaccine development. In addition, the DARC-DBP binding inhibition assay was established and utilised to assess inhibitory activity of antibodies induced in the first Phase I clinical trial of this antigen. Results identify the need for significant improvements in vaccine design, and show the utility of the assay as a tool for assessing future blood-stage vaccine development efforts against this neglected parasite.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Phillip E. Klebba / Gram-positive bacteria are characterized by a single lipid bilayer with a thick peptidoglycan layer. This group of organisms contains bacteria commonly associated with human infection, including: Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis and Streptococcus pneumoniae among others. These bacteria have a common system for importing iron in the form of heme, which is acquired by proteins containing heme-binding NEAT (NEAr iron Transporter) domains. The heme acquisition system in S. aureus is termed the Iron Surface Determinant (Isd) system and in L. monocytogenes is termed Heme Binding Protein (Hbp) and Heme/Hemoglobin Uptake Protein (Hup). These proteins work together to obtain heme from hemoglobin and then transport the heme into the cytoplasm via well characterized ABC-transporters. Although there have been clinical trials with antibodies directed against Isd proteins, there are currently no antibiotics targeting iron uptake systems in bacteria in general. Building upon fluorescent approaches for detection of iron uptake in Gram negative organisms, this work develops fluorescent heme acquisition detection in Gram positive organisms. The spectrofluorimetric methodology facilitates the understanding of heme acquisition protein interactions and mechanisms in bacteria. This work could subsequently be used to identify inhibitors of Gram positive bacterial iron uptake systems, and develop a new target for antibiotic action.

Staphylococcus aureus (S. aureus) is a bacterium that commonly colonises the skin and nares of around one third of otherwise healthy individuals. While colonisation is benign, S. aureus can cross skin and mucosal barriers to cause infections that manifest as clinical disease. Clinical outcomes are diverse and range from mild, non-complicated and often self-limiting skin and soft tissue infections (including boils, abscesses and cellulitis) to more severe and life-threatening conditions including pneumonia, toxic shock syndrome and bacteraemia. Medication isn’t always needed for mild S. aureus infections as often they resolve with time but, for severe or persistent cases, antimicrobial treatment is generally required. Following decades of widespread and intensive usage of topical, enteral and parenteral antimicrobials to treat S. aureus infections; AMR has become an established and ubiquitous problem in the treatment of infections caused by this microorganism, especially when in the methicillin resistant form (i.e. MRSA). The aim of this thesis was to examine aspects of S. aureus epidemiology (including MRSA and methicillin-sensitive S. aureus (MSSA)) in Scotland using statistical methods and data from several large public health databases. More specifically this involved: descriptions of spatial and temporal trends of morbidity and mortality; comparisons of epidemiological and molecular attributes (including antimicrobial resistance) of (1) MSSA and MRSA, and (2) the dominant clones of MRSA (i.e. EMRSA-15 and EMRSA-16); descriptions of spatial and temporal trends of antimicrobial prescribing in primary and secondary care and any associations between prescribing rates and MRSA antimicrobial resistance; and carrying out a hospital-level risk factor analysis of MRSA, testing hypotheses that hospital size, hospital connectivity (through shared transfer patients) and hospital category have an effect on hospital-level incidences of MRSA in mainland Scottish hospitals. Results showed that total S .aureus bacteraemia and MRSA bacteraemia in Scotland statistically declined over time (p < 0.0001), but MSSA bacteraemia did not (p > 0.05). While combined mortality rates (i.e. all MSSA deaths (both primary and secondary cause), or all MRSA deaths (both primary and secondary cause)) mirror these findings; case-fatality ratios (CFR) show no declines over time for either MRSA or MSSA. Results also show that several epidemiological factors point towards a predominant community source for MSSA isolates (i.e. outside healthcare) and hospital source for MRSA. Evidence for this included: (1) the lack of resistance genes in the MSSA population, (2) MRSA was more associated with long-term care and high-risk patients in the specialties care of the elderly, high dependency units/intensive care units (HDU/ICU), and surgery and conversely MSSA with specialties that commonly served outpatients, and (3) the abundance of non-EMRSA-15/non- EMRSA-16 ‘other’ clones in the MSSA population as compared with the hospital-associated CC22 (EMRSA-15) and CC30 (EMRSA-16) clones. EMRSA-15 was by far the most dominant MRSA clone in Scotland with EMRSA-16 declining significantly and non-EMRSA-15/non-EMRSA-16 clones causing an increasing number of infections (over the time period 2003-2013). EMRSA-16 was resistant to a larger number of antimicrobials than EMRSA-15, typically 9 versus 5, and while resistance varied for EMRSA-16 over the study period, resistance remained stable for EMRSA-15. There was little difference between clinical and screening MRSA isolates. Analyses of antimicrobial prescribing showed that prescribing rates of several drugs increased over time (2003-2013). Prescribing was far higher in primary care settings than in secondary care, although this differed between antimicrobials. Significant positive associations between prescribing and resistance rates were found for gentamicin (pr - p<0.0001, se - p<0.0001) and trimethoprim (pr - p<0.01, se - p<0.0001) in both primary (pr) and secondary (se) care, and clindamycin (p<0.0001) in primary care only. Finally, in Scotland there is a threshold of connectivity above which the majority of hospitals, regardless of size, are positive for MRSA. Higher levels of MRSA are associated with the large, highly connected teaching hospitals with high ratios of patients to domestic staff. While there were a number of data limitations, this body of work provides a better understanding of the epidemiology of S. aureus including MRSA in Scotland.

Made available in DSpace on 2014-06-11T19:27:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-06-22Bitstream added on 2014-06-13T19:14:32Z : No. of bitstreams: 1 massa_r_me_jabo.pdf: 283971 bytes, checksum: 0b4319b9b239dc5b274d6820f39d809b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente estudo teve como objetivo avaliar a influência da brucelose, leptospirose, rinotraqueíte infecciosa bovina (IBR), diarreia viral bovina (BVD) e neosporose no indicador de reprodução mais comumente utilizado por pecuaristas brasileiros, a taxa de concepção (ou taxa de prenhez) após a estação de monta. Dois estudos epidemiológicos analíticos foram feitos: um estudo caso-controle, utilizando uma amostra das matrizes do rebanho que foram submetidas à estação de monta durante o período chuvoso, e um estudo de coortes retrospectivo, utilizando todas as novilhas submetidas à estação de monta durante a estação de “inverno”. A determinação do estado gestacional foi realizada por palpação retal entre 60 e 90 dias após o término da estação de monta. A pesquisa de anticorpos foi realizada pelos seguintes métodos: antígeno acidificado tamponado (AAT) e reação de fixação de complemento (RFC) para brucelose; soroaglutinação microscópica (SAM) para leptospirose; virusneutralização (VN) para IBR e BVD; e reação de imunofluorescência indireta (RIFI) para neosporose. A significância estatística da associação entre a doença e o fracasso na concepção foi avaliada pelos métodos de qui-quadrado ou teste exato de Fisher (p < 0,05) e pelo intervalo de confiança a 95% para o risco relativo (RR) ou a odds ratio (OR). Associações com relação causal possível foram encontradas entre falha na concepção e: infecção considerando qualquer sorovariedade de Leptospira spp. (Título ≥ 400) em primíparas (OR = 7,3636, IC 95%: 1,3373-40,5479; significativo), todas as quais foram infecções causadas por L. Wollfi; e infecção por L. Autmumnalis (título de ≥ 200), considerando todos os animais da estação de monta de “verão” (OR = 8,4058; IC95%: 1,0377 - 68,0882; significativo). Brucelose, neosporose IBR, BVD... / The present study aims to evaluate the influence of brucellosis, leptospirosis, infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD) and neosporosis in the reproductive indicator most commonly used among Brazilian ranchers, the conception rate (or pregnancy rate) after breeding season. Two analytical epidemiological studies were made: a case control study using a sample of matrices of the herd that were submitted to the breeding season during the rainy season, and a retrospective cohort study using all heifers submitted to the breeding season during the dry season. The determination of pregnancy status was performed by rectal palpation between 60 and 90 days after the end of the breeding season. The antibody search was performed by the following methods: rose bengal test and complement fixation test for brucellosis; microscopic agglutination test for leptospirosis, virus neutralization for IBR and BVD, and indirect immunofluorescence assay for neosporosis. The statistical significance of association between the disease and failure in the conception was held by the methods of chi-square or Fisher's exact test (p < 0,05) and by the 95% confidence interval for the relative risk (RR) or odds ratio (OR). Associations with possible causal relationship were found between conception failure and: Infection considering any serovar of Leptospira spp. (titer ≥ 400) in primiparous (OR = 7.3636, 95% CI: 1.3373 to 40.5479; significant), all of which were infections caused by L. Wollfi; and infection by L. Autmumnalis (titer of ≥ 200) considering all the animals of the rainy breeding season (OR = 8.4058; IC95%: 1.0377 – 68.0882; significative). Brucellosis, IBR, BVD, neosporosis and leptospirosis caused by other serovars did not influence the rate of conception in the breeding season... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:28:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2001Bitstream added on 2014-06-13T20:36:48Z : No. of bitstreams: 1 prodocimocalore_sa_me_botfm.pdf: 684440 bytes, checksum: 021d73a7a05b5ac24f7cf8c02d507263 (MD5) / A audição tem papel fundamental na vida do ser humano, possibilitando uma das nobres funções superiores do homem que é a comunicação. As doenças infecciosas podem alterar a integridade anatomofisiológica do sistema auditivo e também causar prejuízos no desenvolvimento global da criança. O presente estudo teve como objetivo delinear o perfil demográfico de 661 crianças matriculadas no Centro de Distúrbios da Audição, Linguagem e Visão (CEDALVI) do HRAC/USP, em Bauru-SP, reunindo os achados audiológicos das prováveis doenças infecciosas que causaram a deficiência auditiva nessa população. Os resultados desse estudo mostraram concentração maior de crianças na faixa etária de 2 a 3 anos de idade, procedentes da Região Sudeste do Brasil, do sexo masculino e nível sócio-econômico baixo. Quanto às características audiológicas, houve predomínio de deficiência auditiva do tipo sensorioneural, bilateral e graus grave a profundo. Concluiu-se que nas doenças infecciosas de origens congênita e adquirida, a rubéola e a meningite foram respectivamente as prováveis causas determinantes da deficiência auditiva na população estudada, devendo-se salientar a importância dos programas de vacinação e do diagnóstico precoce para que medidas profiláticas e terapêuticas possam ser elaboradas. / Hearing plays a fundamental role in human life, allowing one of the noble superior functions of mankind, which is the communication. The infectious diseases can alter the anatomicophysiological integrity of the auditory system and also bring about damages to the child’s global development. The present study aimed at delineating the demographic profile of 661 children enrolled at the Center for Attention to Hearing, Language and Vision Disturbances (CEDALVI) of the HRAC-USP, Bauru, Brazil, gathering the auditory findings of the probable infectious diseases causing the auditory deficiency in this population. The outcomes of this study showed a larger concentration of children aging 2 to 3 years old, from Southeastern Brazil, of the male gender and low socioeconomic level. Regarding the auditory characteristics, there was predominance of auditory deficiency of the sensorineural type, mainly bilateral of a severe to deep degree. It could be concluded that, among the congenital and acquired infectious disease, measles and the meningitis were respectively the probable determinant causes of the auditory deficiency in the studied population, thus highlighting the importance of the immunization programs and early diagnosis, so that prophylactic and therapeutic measures can be elaborated.

Made available in DSpace on 2014-10-09T12:53:08Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:58:06Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:54:15Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:43Z (GMT). No. of bitstreams: 1 12711.pdf: 2036091 bytes, checksum: a4517356221ed0ff2c40f070f9f8233a (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo, Sao Paulo

Made available in DSpace on 2014-10-09T12:53:08Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:58:06Z (GMT). No. of bitstreams: 0 / Atualmente, milhões de pessoas, por todo o globo, estão sob risco de serem infectados por um protozoário transmitido vetorialmente por pequenos insetos flebotomíneos. Este parasita é a Leishmania spp., causadora de uma patologia de amplo espectro, que varia desde a moléstia cutânea (tegumentar) até a visceral (kala-azar). A leishmaniose cutânea é a manifestação clínica de maior ocorrência (mais de 90% dos casos). A radiação ionizante, gerada em fonte de 60Co, tem sido utilizada com sucesso para promover alterações físico-químicas em diferentes protozoários, incluindo a Leishmania spp. Em trabalhos anteriores determinou-se que formas promastigotas de Leishmania amazonensis, irradiadas com diferentes doses de radiação gama, perdem sua viabilidade mantendo, porém, sua imunogenicidade. No presente trabalho, estudouse a utilização da radiação ionizante como ferramenta na seleção de formas metacíclicas do parasita em cultura axênica para a possível produção de imunógenos irradiados mais eficientes. Os resultados demonstram que culturas irradiadas com 400 Gy de radiação gama, possuem uma concentração de aproximadamente 75% de parasitas metacíclicos, capazes de produzir, in vitro, uma infecção que mimetiza a ocorrida naturalmente. Estes parasitas irradiados têm sua estrutura celular interna modificada mantendo, entretanto, seu arcabouço externo intacto. Camundongos de uma linhagem suscetível imunizados com leishmanias irradiadas com diferentes doses tiveram sua produção de imunoglobulinas aumentada, e mantiveram os títulos elevados após o desafio com parasitas não irradiados. Em outras linhagens pesquisadas este padrão se manteve, porém em títulos menores, sendo que camundongos imunodeficientes não responderam à imunização nem ao desafio. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:54:15Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:43Z (GMT). No. of bitstreams: 1 12711.pdf: 2036091 bytes, checksum: a4517356221ed0ff2c40f070f9f8233a (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo, Sao Paulo

Orientadores: José Dirceu Ribeiro, Renato Tetelbom Stein / Tese (doutorado) ¿ Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T01:47:34Z (GMT). No. of bitstreams: 1 Silva_EmersonRodriguesda_D.pdf: 2935794 bytes, checksum: a3a21e8b226c670f1ac8d027e5ed10a1 (MD5) Previous issue date: 2013 / Resumo: Introdução: A infecção do trato respiratório inferior (ITRI) é uma das principais causas de morbimortalidade em crianças, principalmente em países em desenvolvimento e em crianças menores de 3 anos de idade. Os vírus estão entre os principais agentes etiológicos das ITRI em crianças. No entanto, poucos estudos até o momento investigaram o impacto dos vírus respiratórios em populações de crianças de países em desenvolvimento e de que maneira as codetecções de dois ou mais vírus modificam a gravidade das infecções. Objetivo: identificar quais vírus respiratórios causam ITRI em lactentes e crianças até 3 anos de idade hospitalizadas e qual o impacto das codetecções virais sobre a gravidade destes episódios. Métodos: crianças de até 3 anos de idade internados em um hospital terciário no Brasil durante os meses de alta prevalência de vírus respiratórios tiveram amostras coletadas de aspiração nasofaríngea. Estas amostras foram testadas para 13 diferentes vírus respiratórios através de PCR em tempo real (RT-PCR). Os pacientes foram acompanhados durante a internação, e dados clínicos e das características da população foram registradas durante esse período e na alta para avaliar marcadores de gravidade, como tempo de internação e uso de oxigênio. Foi usada análise univariada para identificar potenciais fatores de risco e a regressão logística multivariada para determinar o impacto de detecções virais específicas sobre os desfechos, bem como para avaliar o efeito das codetecções sobre estes mesmos desfechos. Resultados: Foram analisados 260 episódios de ITRI com uma taxa de detecção viral de 85% (n = 222). Codetecção foi observada em 65% de todos os episódios de vírus-positivos. O vírus foi mais prevalente Vírus Sincicial Respiratório (RSV) (54%), seguido por Metapneumovirus humano (hMPV) (32%) e Rinovírus humano (HRV) (21%). Nos modelos multivariados, lactentes com codetecção de HRV + RSV permaneceram 4,5 dias a mais no hospital (p = 0,004), quando comparados ao grupo sem a codetecção. A mesma tendência foi observada para o número de dias de uso de oxigênio suplementar. Conclusões: Embora RSV permaneça como a principal causa da ITRI em crianças, mostrou-se um aumento no tempo de internação e uso de oxigênio em crianças com RSV e HRV codetectados por RT-PCR em comparação com aqueles com RSV mas sem HRV em codetecção. Além disso, nosso estudo identificou um número significativo de crianças infectadas por vírus recentemente identificados, tais como hMPV e bocavirus Humano (HBoV), e este é um achado relevante para comunidades pobres de países em desenvolvimento / Abstract: Introduction: lower respiratory tract infection (LRTI) is a major cause of morbidity and mortality in infants and children, especially in developing countries and in children under 3 years old. Viruses are among the major etiologic agents of LRTI in children. However, few studies to date have investigated the impact of respiratory viruses in populations of children in developing countries and how the co-detections of two or more viruses modify the severity of infections. Objective: To identify respiratory viruses in infants and children up to 3 years of age hospitalized due to LRTI and verify the impact of viral co-detections on the severity of these episodes. Methods: Children less than 3 years of age admitted to a tertiary hospital in Brazil during the months of high prevalence of respiratory viruses had collected samples of nasopharyngeal aspirate. These samples were tested for 13 different respiratory viruses by real-time PCR (RT-PCR). Patients were followed during hospitalization, and clinical and population characteristics were recorded during this period and at discharge to assess markers of severity, such as length of stay and use of oxygen. Univariate analysis was used to identify potential risk factors and multivariate logistic regression was used to determine the impact of specific viral detections on outcomes and to evaluate the effect of co-detections on these same outcomes. ix Results: We analyzed 260 episodes of LRTI with an overall viral detection rate of 85% (n = 222). Co-detection was observed in 65% of all virus-positive episodes. The most prevalent virus was Respiratory Syncytial Virus (RSV) (54%), followed by human metapneumovirus (hMPV) (32%) and human rhinovirus (HRV) (21%). In multivariate models, infants with co-detection of HRV + RSV stayed 4.5 days longer in the hospital (p = 0.004), when compared to the group without this co-detection. The same trend was observed for the number of days using supplemental oxygen. Conclusions: Although RSV remains the leading cause of LRTI in children, our study has shown an increase in the length of stay and use of oxygen in children with RSV and HRV co-detected by RT-PCR in comparison with those with RSV alone. Furthermore, our study identified a significant number of children infected by viruses recently identified, such as hMPV and Human bocavirus (HBoV), and this is an important finding for poor communities in developing countries / Doutorado / Pediatria / Doutor em Saude da Criança e do Adolescente