With the remarkable advancement in the nanoparticles (NPs)-based drug delivery systems (DDS) over the past several decades, the pharmacological properties associated with conventional free drugs delivery are improved. In this thesis, we report potential candidates for the next-generation NP-based DDS. While natural DDS are promising as they possess exceptional delivery mechanisms and selective targeting, synthetic DDS are more favorable for their low immunogenicity. Our developed natural DDS called magnetotactic bacterial cages (MBC), which is based on magnetotactic bacteria (MTB) as a guidable delivery vehicle for DNA functionalized gold nanoparticles (AuNPs). Loading DNA functionalized AuNPs in MTB aided in increasing the maximum-tolerated dose of DNA functionalized AuNPs and tackled issues related to DNA functionalized AuNPs stability and systemic delivery. Natural DDS hold great advantages; however, it is difficult to make complete prediction about their immunogenicity and toxicity on the basis of preclinical trials. Thus, we assessed the efficacy of synthetic NP-based DDS. Using inorganic platforms, we were able to develop the first visual monitoring system of bacteria-NPs interaction. The system offers simultaneous sensing and inhibition of bacteria in infected cells. The system is comprised of Au nanoclusters @lysozyme (AuNC@lys) colloids MSN loaded with antibacterial agents. The applicability of the inorganic DDS in the biomedical field has been limited by the high bioaccumulation risks. Hybrid materials combine the advantages of organic, inorganic and natural carriers, offering opportunities for enhanced stability, manipulating release behavior and combine two or more functions in a single platform. To further enhance the properties our inorganic DDS, we incorporated light-responsive organic ligands to silicabased NPs. Plasmid DNA was loaded on the light-responsive bridged silsesquioxane nanocomposites (BS NPs). Light irradiation was performed to reverse the surface charge of NPs via a photoreaction of the organic fragments (silsesquioxane) within the NPs, that resulted in the release of plasmid DNA in HeLa cancer cells. Finally, we assessed a new class of organic-inorganic DDS composed of inorganic metal ions and organic linkers, zeolite imidazolate frameworks-8 (ZIF-8). These NPs showed exceptional ability to entrap large cargo due to their tunable porosity and structural flexibility.

The autophagy pathway is an essential component of the innate immune response, capable of rapidly targeting intracellular bacteria, which are subsequently degraded by lysosomal enzymes. Recent work has begun to elucidate the regulatory signalling for autophagy induction in response to pathogenic bacteria. However, the initial signalling regulating autophagy induction in response to the detection of pathogens remains largely unclear. Here we report that AMPK, an important upstream activator of the autophagy pathway, is rapidly stimulated upon detection of pathogenic bacteria, prior to bacterial invasion. Bacterial recognition is initially achieved through detection of outer membrane vesicles (OMVs). Additionally, we show that AMPK signalling relieves mTORC1-mediated repression of the autophagy pathway in response to Salmonella infection, positioning the cell for a rapid induction of autophagy. Surprisingly, we found that the activation of AMPK and inhibition of mTORC1 in response to extracellular Salmonella are not accompanied by an induction of bulk autophagy. However, upon Salmonella invasion AMPK signalling is required for efficient and selective targeting of bacteria-containing vesicles by the autophagy pathway through activation of pro-autophagic kinase complexes. Collectively, these results demonstrate a key role for AMPK signalling in coordinating the rapid autophagic response prior to invasion of pathogenic bacteria.

Orientador: Maria Regina Bentlin / Resumo: INTRODUÇÃO: As infecções primárias de corrente sanguínea (IPCS) são responsáveis por aumento da morbimortalidade em recém-nascidos. OBJETIVOS: Determinar a incidência e a mortalidade das IPCS com confirmação laboratorial, após 72 horas de vida, em função dos agentes etiológicos, avaliar a resistência antimicrobiana dos agentes mais frequentes e rever o esquema de terapia empírica da Unidade. MÉTODOS: Estudo de coorte, retrospectivo, realizado na UTI Neonatal do Hospital das Clínicas - Faculdade de Medicina de Botucatu (UNESP), entre 2014 - 2016, após aprovação do Comitê de Ética. Critérios de inclusão: recém-nascidos com IPCS e hemocultura positiva após 72 horas de vida. Não incluídos agentes contaminantes e o crescimento de estafilococos coagulase negativa (SCoN) em apenas uma hemocultura. A amostra foi constituída por 71 recém-nascidos com 72 hemoculturas positivas. Foram estudadas variáveis maternas, gestacionais e do parto, procedimentos e resistência antimicrobiana. Desfechos: choque séptico e óbito. Os agentes foram comparados em grupos: Gram-positivos e Gram-negativos. Analise estatística: descritiva e comparação entre grupos com testes paramétricos e não paramétricos, com significância estatística de 5%. RESULTADOS: A incidência de IPCS foi de 10,2%, sendo 57% por Gram-positivos, 40% por Gram-negativos e 3% por fungos. A mortalidade foi de 21% e o óbito diretamente relacionado à IPCS ocorreu em 10% dos Gram-positivos e 14% dos Gram-negativos. Ventilação mecânica, uso ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

PhD, Faculty of Health Sciences, University of the Witwatersrand / The South African gold-mining workforce has an unusually high incidence of Mycobacterium kansasii disease, yet little is known about the possible sources of M. kansasii infection, genetic diversity and the basis for this organism’s pathogenicity. The purpose of this study was to investigate these issues in a gold-mining environment. Five M. kansasii isolates and 10 other potentially pathogenic mycobacteria were cultured mainly from showerhead biofilms. PCR-restriction analysis (PRA) of the hsp65 gene on 191 clinical and on the 5 environmental M. kansasii isolates revealed 160 subtype I (157 clinical and 3 environmental), 8 subtype II (clinical) and 6 subtype IV (5 clinical and 1 environmental) strains. Twenty-two isolates (21 clinical and 1 environmental) did not show the typical M. kansasii PRA patterns. After confirmation by DNA sequencing as belonging to the M. kansasii species, the results suggested that these isolates were probably new subtypes of M. kansasii. In contrast to the clonal population structure found amongst the subtype I isolates from studies in other countries, DNA fingerprinting of 114 subtype I clinical and environmental isolates showed genetic diversity amongst the isolates. One of the 2 environmental isolates showed 100% identity with a clinical isolate, suggesting that water distribution systems are the possible sources of M. kansasii infection for the miners. An investigation into the genetic differences between clinical (subtype I) and environmental (III, IV and V) isolates, using Hybridisation Monitored Differential Analysis (HMDA), identified 45 open reading frames (ORFs) encoding predominantly membrane-associated proteins that include six potential virulence factors, two family members of transcription regulators for drug and xenobiotic metabolism, three family members of multidrug efflux systems, a number of proteins associated with lipid and carbohydrate metabolism and transport, and a number of hypothetical proteins with unknown function. Immunological analysis of M. kansasii isolates, using the Lymphocyte Transformation and Cytometric Bead Array assays, showed that M. kansasii modulates immune responses through suppression of lymphocyte blastogenesis and by altering the expression of Th1/Th2/Th17 cytokines by human lymphocytes in vivo for its own survival. This study demonstrated for the first time that water distribution systems in South Africa are possible sources of M. kansasii infection, and showed that subtype I strains of M. kansasii from the study region display genetic diversity and have unique or divergent genes not found in other subtypes. It also demonstrated that immunosuppression is one of the pathogenic mechanisms employed by M. kansasii.

The impact of AIDS and the dread of acquiring HIV infection from patients have led to the resurgence in infection-control practices among health care workers. Recent reports of blood-borne pathogen transmission in health care settings, including oral health, have caused considerable public health concern. Transmission has been reported from patient to patient, patient to health care workers, but rarely from health care worker to patient. The risks of dental clinicians acquiring serious infections have been well documented but the risk to dental assistants has received less attention, especially in South Africa. Aim: To assess infection-control practices of dental assistants and their level of adherence to universal precautions in public health care facilities in Limpopo Province. Objectives: To establish the prevalence and the type of occupational exposures among dental assistants working in public health care facilities in Limpopo Province. Methods: A cross-sectional survey was conducted among dental assistants in Limpopo Province in 2005. The study population comprised all 73 employees who performed the functions of a dental assistant in public dental facilities. A self-administered questionnaire was used to collect information regarding work experiences and training, infection-control practice and knowledge, and the nature, incidences and reporting of any occupational exposures they had experienced. A follow-up telephone call was made to these dental assistants, after they had received the questionnaire, to re-iterate the importance of the survey and to request them to complete and return the questionnaire in the prepaid envelope they had been given. The facilities were clustered according to the six districts in Limpopo Province. Ethical approval was given by the University of the Witwatersrand and the Department of Health and Welfare in Limpopo Province. Results: Fifty-nine dental assistants returned the completed questionnaire, giving a response rate of 80.8%. Epi Info Version 3.3.2 programme was used to analyze the data. The majority of respondents were female (95%), with a mean age of 40.2 years (age range 23-54). More than 90% of the respondents had no formal training for their occupation, half (49.1%) did not have any health training, 22% were auxiliary nurses, 18.6% were “correspondence-trained” assistants who had been trained via distance learning and had no practical clinical training and only 10.2% of the respondents had received training at a technikon or university . The majority of the dental facilities (57.6%) had one dental assistant working alone, followed by those with two or three assistants (39.5%). The number of respondents assisting more than two oral clinicians in a day was 93.3%. The mean number of clinicians assisted per day was 3.8. The total numbers of dental assistants who experienced occupational exposures while working at the various dental clinics were 26 (44.1%), with 11.5% experiencing multiple injuries within the preceding six months. Auxiliary nurses and trained assistants were significantly more likely than untrained assistants to be aware of universal precautions, their protective effects, needle stick protocols, and of the need for personal protective equipments to be worn for all procedures (p=0.001). Compliance with infection-control practices was low overall. More than twothirds of the assistants routinely wore gloves during procedures. The lowest compliance reported was the use of protective eye shields, whilst more than 62.7% were not vaccinated against hepatitis B virus. More than two-thirds of the assistants were injured in the process of removing and or cleaning instruments; 65.3% of the injuries were direct punctures. Twenty-three percent did not report the injury. The risk of injury for the untrained assistants was 9.9 times higher than that for auxiliary nurses, p=0.008. A small percentage (23.8%) of those with sharp injuries was placed on antiretroviral drugs. Surprisingly, a significant high percentage of respondents were given wound cleaning only as treatment of their occupational exposures (78.4%) and sharp injuries (83%). Conclusion and recommendation More than 90% of the respondents had no formal training for their occupation. Dental assistants were understaffed and had increased workload. The greatest incidence of injury was associated with the handling of sharp objects, and this included recapping used needles. Occupational exposures to infectious material were found to be relatively high whilst compliance to some basic infection-control guidelines was low among dental assistants. The training of dental assistants should be regulated. More suitably qualified dental assistants should be appointed and existing ones should be given inservice training on the importance of infection-control practices and compliance with universal precautions.

MSc (Med) Epidemiology and Biostatistics, Faculty of Health Sciences, University of the Witwatersrand, 2009 / Focus in public health research is shifting to the role of socio-economic factors in the promotion of health. Hence, an understanding of the roles socio-economic factors plays in improving health and health-seeking behaviour is important for public health policy. This study examined the relationship between socio-economic factors and HIV infection in rural Limpopo Province South Africa, an area characterized by poverty differentials and migration. Various possible social and economic risk factors (such as nationality, education status, marital status, employment status, migration status and socio-economic status) for HIV infection are analysed and discussed. This is secondary data analysis was carried out during the period June 2001 to March 2005 among 2345 14-35 year old residents in eight (8) villages in rural Limpopo. Married participants (OR 0.53 [95%CI 0.28 – 1.00]), those from poor (OR 0.49 [95%CI 0.28-0.85]) and less poor households (0.38 [95%CI 0.21-0.70]) are less likely to contract HIV infection. Noteworthy from these analyses also is the increased risk for HIV infection seen among female participants, those not currently schooling (OR 1.9 [95%CI 1.2 – 3.3]) and non-South African citizens (OR 5.18 [95%CI 1.04-25.8]). Conclusion: Women, out-of school youths and non South African citizens are shown to be high-risk population groups for HIV infection. HIV prevention programs that target identified vulnerable population groups and increased social support for the family may contribute to mitigating the spread of HIV in rural South Africa.

Introduction and Background: Central venous catheters (CVCs) are extensively used worldwide. Mechanical, infectious and thrombotic complications are well described with their use and may be associated with prolonged hospitalisation, increased medical costs and mortality. CVCs account for an estimated 90% of all catheter-related bloodstream infections (CRBSI) and a host of risk factors for CVC-related infections have been documented. These include, most importantly, the duration of catheterisation. The duration of use of CVCs remains controversial and the length of time such devices can safely be left in place has not been fully and objectively addressed in the critically ill patient. Over the past few years, antimicrobial impregnated catheters have been introduced in an attempt to limit catheter-related infection (CRI) and increase the time that CVCs can safely be left in situ. Recent meta-analyses concluded that antimicrobial-impregnated CVCs appear to be effective in reducing CRI. Materials and Methods: This was a prospective randomised double-blind study performed in the adult multidisciplinary Intensive Care Unit (ICU) at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) over a four year period. The study entailed a comparison of standard triple-lumen versus antimicrobial impregnated CVCs on the rate of CRI. The aim was to determine whether the duration of catheter insertion time could safely be increased from the standard practice of seven days at the CMJAH adult multidisciplinary ICU to 14 days, to assess the influence of the antimicrobial impregnated catheter on the incidence of CRI, and to elucidate the epidemiology and risks of CRI. Results: One hundred and eighteen critically ill patients were included in the study which spanned 34 951.5 catheter hours (3.99 catheter years). Sixty-two patients received a standard triple-lumen catheter and 56, a chlorhexidine-silver sulfadiazine (CSS) impregnated triple-lumen catheter. The mean duration of placement for the full sample of 118 CVCs was 12.3 days (range, 1-14). No statistically significant difference in CRI rates between the two types of catheters could be demonstrated. The most common source of primary CRBSI was skin, followed by hub and infusate. The site of CVC insertion (internal jugular versus subclavian vein) and the use of parenteral nutrition were not noted to be risk factors for catheter infection. There was no clinical evidence of catheter-related thrombosis in either of the study groups. Conclusion: This study was unable to demonstrate that antimicrobial catheters provided any significant benefit over standard catheters, which it is felt, can safely be left in place for up to 14 days with appropriate infection control measures. The most common source of CRI was the skin. The administration of parenteral nutrition and the site of catheter insertion (internal jugular vein versus subclavian vein) were not noted to be risk factors for CRI. There was no clinical evidence of thrombotic complications in either of the study groups. This study offers direction for the use of CVCs in critically ill patients and addresses many of the controversies that exist.

Nos hospedeiros mamíferos, os parasitas do gênero Leishmania vivem nos macrófagos se evadindo de mecanismos microbicidas dessas células, tais como a produção de óxido nítrico (NO). A produção de NO pela enzima óxido nítrico sintase induzida (iNOS) nos macrófagos requer L-arginina como substrato, o mesmo aminoácido utilizado pela arginase para produzir ornitina e uréia. Logo, a arginase pode atuar na sobrevivência de Leishmania no hospedeiro competindo com a iNOS, reduzindo a produção de NO, além de seu papel na via de poliaminas, essencial para a replicação dessas células. Com isso, o objetivo desse estudo é elucidar o papel da arginase de L. (L.) amazonensis durante o ciclo de vida do parasita, particularmente, sua função no estabelecimento e na manutenção da infecção da célula hospedeira, e como esse papel seria exercido. Nesse sentido, obtivemos soros policlonais anti-arginase, a partir da arginase recombinante de L. (L.) amazonensis purificada, e esses soros foram utilizados na imunomarcação da enzima em preparações com formas promastigotas e macrófagos infectados com amastigotas de L. (L.) amazonensis. Assim, determinamos a compartimentalização da arginase nos glicossomos tanto na forma promastigota do parasita como na forma amastigota, durante a infecção. Além disso, obtivemos diversos mutantes com a expressão de arginase modificada quanto à quantidade e localização que nos permitiram avaliar a importância da compartimentalização dessa enzima nos glicossomos. Entre esses mutantes temos: superexpressores de arginase, com e sem sinal de endereçamento para glicossomo; parasitas com um alelo de arginase nocauteado e o outro substituído pelo cassete contendo o segmento ddFKBP-ARG, que teriam a expressão de arginase regulada pelo domínio ddFKB sendo nocautes funcionais de arginase; e finalmente, também obtivemos parasitas nocaute nulo de arginase. A análise desses mutantes permitiu conclusões importantes para o conhecimento da fisiologia do parasita e sua relação com o macrófago, revelando que o papel da arginase de Leishmania parece ser muito mais complexo do que o inicialmente postulado, participando na regulação de outras vias metabólicas do próprio parasita e da célula hospedeira. Paralelamente, também determinamos que o sistema ddFKBP é funcional em L. (L.) amazonensis, e assim pode ser utilizado no estudo funcional de outras proteínas importantes para esses parasitas. / In the mammal host, Leishmania parasites live inside macrophages escaping from their microbicidal mechanisms, such as the nitric oxide (NO) production. The macrophage NO production by inducible nitric oxide synthase (iNOS) requires L-arginine as substrate, the same amino acid required by arginase to generate ornithine and urea. So, arginase may play a dual role in Leishmania survival reducing the NO by competing with iNOS, and participating in the polyamines pathway, which is essential for the cells replication. Considering this, the aim of this study is to elucidate the role of L. (L.) amazonensis arginase during the parasite life cycle, mainly its function for the establishment and maintenance of the host cell infection, besides to elucidate the way that this enzyme plays its role. With this in mind, we obtained polyclonal anti-arginase sera using purified recombinant L. (L.) amazonensis arginase, these sera were used in immunolabelling assays of L. (L.) amazonensis promastigotes and macrophages infected with L. (L.) amazonensis amastigotes. These experiments determined that arginase is compartmentalized in the glycosomes of both promastigotes and amastigotes, during infection. Besides, we obtained several mutants with altered arginase expression, modified in terms of quantity and location, which permitted us to evaluate the importance of glycosome arginase compartmentalization. Among these mutants are: overexpressors of arginase, with and without glycosomal addressing signal; parasites with one arginase allele knocked out and the other one replaced by a sequence containing the ddFKBP-ARG fusion that would allow us to regulate arginase expression, working like a functional arginase knockout; and finally, we also obtained arginase null knockouts parasites. The mutants analyses lead us to important conclusions for the knowledge of the parasite physiology and its relationship with the host macrophage, revealing that the Leishmania arginase role appears to be more complex than previously thought, playing an important role in the regulation of other metabolic pathways, of the own parasite and of the host cell. In the other hand, we also determined that the ddFKBP system is functional in L. (L.) amazonensis, and then can be used for functional studies of other important parasite´s proteins.

Co-infection with Hepatitis C Virus (HCV) is a common occurrence in Human Immunodeficiency Virus (HIV)-positive patients and an increasing cause of morbidity and mortality. Little is known however of the burden or the natural history of these infections or their interactions in most parts of sub-Saharan Africa, where both viruses are endemic. In this study a total of 1500 people aged 11 months to 76 years referred to the serology unit of Royal Victoria Teaching Hospital between the months of July to December 2003 were evaluated for anti-HIV, anti-HCV and CD4+ T-cell count and compared with the subjects' socio-demographic and risk factors. HIV and HIV/ HCV seropositive persons who consented to a follow-up study were age and sex matched with HIV and HCV seronegative control subjects and followed for 18 months with biannual monitoring of trends in CD4 count against a possible HIV or HCV seroconversion of the seronegative control subjects. The overall prevalence of antibodies to HIV and HCV was 6.7% (101/1500) (Cl, 5.6-8.2) and 2.1% (31/1500) (95 % CI, 1.4-2.9) respectively. HIV rates in asymptomatic adults were 3.6 %( 43/1189) (OR: 0.16; Cl: 0.13-0.28) and 1.0 %( 12/1189 (OR: 0.16; Cl: 0.08-0.34) for HCV. HIV/HCV co-infections rate was 0.6% among all the subjects sampled and 8.6% in HIV positive persons. The HIV rate in this study is twice the UNAIDS/WHO estimate for the country and twice the numbers of women than men were infected with HIV at a comparatively younger age, while males 55 years and over had higher HIV rates than those below 35. HCV and HIV/HCV coinfection was more commonly associated with males than females. This study showed that Hepatitis C serotype 2 is the most prevalent type in the country and was predominantly associated with HIV-1, and suggests that HCV serotype 2 spread earlier than serotypes 1 and 3. The mean CD4 count of apparently healthy males and females was 489/μl and 496/μl respectively, while the mean CD4 count at diagnosis (CD4dx) of HIV, and HIV/HCV persons was 310 cells/μl and 306 cells/μl respectively. Only about half of the apparently healthy population had CD4 counts of 500 cells and over (51 %), while 1.1 % (15/1377) had counts below 200 cells per microlitre for no explained reasons. HN/HCV co-infected person recorded a lower CD4 count at diagnosis than HIV alone infected persons and also a more significant decline in CD4+ than HIV infected alone persons. The study shows that high HIV rates were independent of the educational status of the individual, while history of sexually transmitted diseases, high income earning and involvements in polygamous marriages were all significant risk factors for HIV, HCV and HIV/HCV co-infection. Female circumcision, knowledge and use of condoms, blood oath, histories of blood transfusion and wife inheritance were not associated with HIV or HCV transmission. The study found an HIV incidence rate of 1.4% (4/288) during the 18 months follow-up period and identified Sexually Transmitted Diseases (STDs) as the associated risk factor. There is need for a new CD4+ staging in the country based on the population within the country and the initiation of a large scale longitudinal study to elucidate the risk factors associated with HCV in the country. The study has provided baseline data on CD4 and its trends in co-infected persons and also a baseline on the distribution and epidemiological pattern and associated risk factors of co-infection between HIV and HCV in the country. It has also determined the incidence of HIV and its associated risk factors in the country. The study has therefore contributed to our understanding of the natural history of these infections and provided an important frame work for possible intervention.

Bacteria are under constant threat by their viral parasites, the bacteriophages (phages) and have evolved a range of anti-phage systems to defend themselves. One of these systems is termed abortive infection (Abi) where, upon phage infection, an Abi system may be activated which initiate a bacteriostatic or bactericidal response. While the infected bacteria do not obviously benefit from the activation of these systems, the cessation of bacterial growth or premature cellular suicide prevents the release of phage progeny. Thus Abi can be viewed as an altruistic process as only the remaining clonal bacterial population benefits. The Type III toxin-antitoxin systems have previously been shown to be involved in Abi, however the mechanisms through which these systems are activated are still poorly understood. A common approach to reveal the phage product involved in triggering these systems is to first determine the mutations that a previously sensitive phage evolves to escape after exposure to an Abi system. Analysis of viral "escape" mutants has been used in this study to try to elucidate the activation mechanism(s) of two Type III systems (ToxIN$_P$$_a$ and TenpIN$_P$$_l$) of several environmental phages. Several new phage products were identified in escape mutants as candidate factors involved in circumventing Abi - and possible roles in phage metabolism predicted. Furthermore, the genomes of several phages that could not evolve escapes, or were insensitive to Abi, are sequenced and these data exposed interesting curiosities regarding Abi (as well as the discovery of several novel and rare phages). Previously, no coliphage was identified that was capable of escape of the ToxIN$_P$$_a$ or TenpIN$_P$$_l$ systems. However, this study defined and characterised the first ToxIN$_P$$_a$ and TenpIN$_P$$_l$ coliphage escapes as well as a new method for isolating host-dependent coliphage escapes. Finally, multiple phages that infect the insect pathogen $\textit{Photorhabdus luminescens}$ TT01 (the bacterial strain from which the TenpIN$_P$$_l$ system originated) were isolated, genomically sequenced and characterised in terms of host range. The results revealed a large superfamily of flagellum-dependent phages that exhibit remarkable host promiscuity, possibly defining the most promiscuous phages thus far identified.