Drug resistance is a major obstacle to management and control of malaria and currently progressing at a rapid rate across Africa. This thesis has examined factors influencing evolution of resistant P. falciparum at two sites in Africa, including parasite migration, cross mating and fitness cost of resistance. In Asar village, eastern Sudan, the frequencies of drug sensitive and resistant parasites were monitored throughout the dry season in the absence of anti-malarial drug usage to examine whether persistence of resistant parasites is reduced in the absence of drug pressure. Two cohorts of P. falciparum infected patients were treated with chloroquine in the transmission season (Oct-Dec), and followed monthly in the dry season into the next transmission season. A large proportion of the cohort maintained sub-patent asymptomatic P. falciparum infections throughout the entire study period. Alleles of the chloroquine resistance transporter (Pfcrt) and multi-drug resistance protein (Pfmdr1) were examined. Mutant alleles of Pfcrt reached fixation following CQ treatment and remained high in the transmission season. However, at the start of the dry season, wild type alleles of both genes started to emerge and increased significantly in frequency as the season progressed. The mutant Pfcrt haplotype was invariably CVIET, indicating migration of CQ resistant parasites into an area; otherwise the CVMNK haplotype is normal. In addition, microsatellite haplotypes of dihydrofolate reductase (dhfr) gene and dihydropteroate synthase (dhps) genes, which control the parasite response to pyrimethamine and sulfadoxine respectively, were characterized. One major dhfr haplotype with double dhfr mutations and two major mutant dhps haplotypes were seen in eastern Sudan. These haplotypes are distinct from those prevailing in other African countries, suggesting the likely local origin of dhfr and dhps haplotypes conferring drug resistance. Transmission capacities of different P. falciparum clones within a single infection in The Gambia have a high ability to produce gametocytes and infect Anopheles mosquitoes even when they exist at levels not detectable by microscopy and PCR. These findings emphasize the crucial role of gametocyte complexity and infectivity in generating the remarkable diversity of P. falciparum genotypes seen in infected people. Parasites with different resistant dihydrofolate reductase (dhfr) haplotypes have the ability to infect Anopheles mosquitoes following drug treatment, and cross-mating between parasites with different dhfr haplotypes was detected. Our results showed that the major dhfr haplotype in the Gambia is similar to the common one seen in other African countries, suggesting that parasite migration plays a major role in spread of resistance. Indeed, the dominant resistant haplotype seen in infected patients was readily transmitted to infect mosquitoes.

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 2014-06-11T19:28:01Z (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

No description available.

HIV-1 persists to be a major health problem worldwide. A prophylactic or therapeutic vaccine offers the best hope to restrain the HIV-1 epidemic, however a consistent correlate of immune protection is yet to be found. HLA class I expression and their restricting HIV-1 specific CD8+ T cell responses have been shown to play a vital role in the control of HIV-1 infection. The interactions between HIV-1 and CD8⁺ T cell responses are complex and the mechanisms involved in the success or failure to control viraemia remain uncertain. Thus, the aim of these studies was to help define what CD8+ T cell responses a vaccine needs to induce to achieve durable immune control of HIV-1 infection. Focusing initially on HLA-B*35, an allele that has consistently been associated with rapid HIV-1 disease progression in the context of B clade infection, this study shows substantial differences in markers of HIV-1 disease outcome associated with different HLA-B*35 subtypes. Preliminary data suggest that effective targeting of a single epitope in Gag may be associated with HLA-B*35 mediated control of HIV-1 disease progression. Increased breadth of the Gag- specific CD8⁺ T cell responses is found to be associated with decreasing viral loads. These data therefore support the Gag hypothesis, and suggest that targeting of certain regions of the HIV-1 genome may have a positive effect in disease outcome, even for individuals carrying “detrimental” alleles. The extensive diversity of the HIV-1 genome and rapid viral adaptation are the main chal- lenges to vaccine design. Previous studies have suggested that effective CD8⁺ T cell responses drive selection of escape mutations that reduce viral replication capacity (VRC). There is also evidence that certain escape mutations can be transmitted from one host to another allow- ing for its accumulation in a population. The second study looked at the impact of HLA driven evolution of HIV-1 in VRC at a population level. This study compared two ART-naïve HIV-1 B clade infected cohorts, in Mexico and Barbados, in which protective HLA alleles (HLA- B*27/57/58:01/81:01) are expressed at 10% and 35% respectively, to analyze differences in VRC at a population level. Viral loads (VL) were found to be significantly higher in Mexico compared to Barbados and median CD4⁺ T cell counts significantly lower. Analysis of VRC in a subset of subjects in each cohort matched by CD4⁺ T cell counts between 300-500 cells/μl revealed that VL and VRC was significantly higher in the Mexican subset. This VRC difference was associated with accumulations in Barbados of eight previously described Gag escape mutation where fitness cost has previously been implicated. Accumulation remained significant in mismatched subjects. These data suggest that VLs and disease progression rates may differ between distinct populations as a result of the frequency of protective alleles in the respective populations, and that CD4⁺ T cell count-based guidelines to initiate antiretroviral therapy (ART) may need to be modified accordingly, to optimize the effectiveness of treatment-for-prevention strategies and reduce HIV-1 transmission rates to the absolute minimum. The final project aimed to improve the HIV-1 replication fitness assays currently used in the context of C clade infection. In order to achieve this, we attempted to design a clade C infectious molecular clone for the testing of gal-pol gene regions. However, the clones produced were not replication competent. Sequence analysis showed a large quantity of stop codons, most located within env which may explain the lack of infectivity. Chapter 5 describes the methodology used in the construction of the clade C isolate and suggests future work. Although we were unsuccessful in producing a replication competent virus, the construction of a C clade backbone which replicates efficiently remain an aim due to its importance for research directed to the analysis of genetic determinants of C clade virus. Data presented in this thesis suggest that vaccine-induced immune responses should aim to focus on vulnerable regions of the virus. These are conserved regions that can not escape without a high fitness cost and with a complex and difficult selection of compensatory mutations. Although much work remains to be done to achieve an effective CD8⁺ T cell based vaccine, hope remains that the induction of HIV control may be possible.