Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-18T11:28:47Z No. of bitstreams: 1 mirelleneryhenrique.pdf: 3757005 bytes, checksum: f81cbf74fa9d4f4e52254d53c930d9aa (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-01T18:23:14Z (GMT) No. of bitstreams: 1 mirelleneryhenrique.pdf: 3757005 bytes, checksum: f81cbf74fa9d4f4e52254d53c930d9aa (MD5) / Made available in DSpace on 2016-07-01T18:23:14Z (GMT). No. of bitstreams: 1 mirelleneryhenrique.pdf: 3757005 bytes, checksum: f81cbf74fa9d4f4e52254d53c930d9aa (MD5) Previous issue date: 2012-03-29 / Este estudo investigou a presença de cândida em manifestações bucais de pacientes com doença renal crônica em pré-diálise, em hemodiálise e em indivíduos saudáveis, utilizando-se de parâmetros clínicos e microbiológicos. Foram selecionados 34 pacientes portadores de doença renal crônica que foram divididos em dois grupos: GE1 – 20 em pré-diálise e; GE2 – 14 em hemodiálise. Para o terceiro grupo (GE3), foram selecionados aleatoriamente 14 indivíduos saudáveis que estavam aguardando atendimento odontológico em clínica particular. Todos os participantes selecionados passaram por uma anamnese e os mesmos responderam a alguns questionamentos sobre a saúde de um modo geral e sobre hábitos particulares de alimentação e hábitos nocivos. A verificação das manifestações bucais foi realizada de forma visual e devidamente fotografada e anotada. Durante o exame intrabucal, nas áreas que apresentavam diagnóstico sugestivo de candidíase, foi coletado material com swab para exame microbiológico, utilizando-se a técnica da Alça Calibrada, onde o meio foi o CHROMagarTM Cândida, para detectar o crescimento de três tipos de cândida: C. albicans, C. krusei e C. tropicalis. Para obtenção dos resultados foi utilizada a prova de Qui-Quadrado, ANOVA (com Post-Hoc LSD) e o teste t de Student, todos com nível de significância de p < 0,05. Das manifestações bucais diagnosticadas, aquela mais frequentemente encontrada foi a “Língua fissurada” com 78,6% no GE2; 60,0% no GE1 e 26,7% no GE3. A “Língua saburrosa” foi a segunda manifestação bucal mais observada, sendo 71,4% no GE2 e 45,0% no GE1. O GE2 foi o grupo que mais apresentou manifestações bucais. Com relação ao diagnóstico visual de candidíase nas manifestações bucais, o GE2 foi prevalente com 28,6%, seguido do GE1 com 25,0%. A C. albicans esteve presente com 17,6% nos pacientes com DRC, sendo 10,0% para o GE1 e 28,6% para o GE2. No GE3 não foi observada a presença de C. albicans. Desta forma, pode-se concluir que nas manifestações bucais observadas, o microrganismo C. albicans esteve presente nos pacientes com doença renal crônica em pré-diálise e hemodiálise, porém sem significância para afirmar que o mesmo seja oportunista para desencadear a candidíase. / This study investigated the presence of oral manifestations of candidiasis in patients with chronic kidney disease in predialysis, hemodialysis and healthy subjects, using the clinical and microbiological parameters. We selected 34 patients with chronic kidney disease who were divided into two groups: EG1 – 20 in pre-dialysis; EG2 – 14 on hemodialysis. For the third group (EG3) were randomly selected 14 healthy subjects who were waiting for dental care in private practice. All selected participants underwent an interview and they answered some questions about health in general and on particular habits of diet and harmful habits. Verification of oral manifestations was performed visually and duly photographed and recorded. During the examination intraoral areas with the diagnosis suggestive of candidiasis, swab material was collected for microbiological examination, using the technique of calibrated loop where the medium was CHROMagarTM Candida, to detect the growth of three types of bleach: C. albicans, C. krusei and C. tropicalis. To obtain the results we used the chi-square test, ANOVA (with post-hoc LSD) and Student's t test, all with a significance level of p < 0.05. Of oral lesions diagnosed, that was the most frequently found "Fissured tongue" with 78.6% in EG2, 60.0% and 26.7% in the EG1 and EG3. The "Tongue furred" was the second most frequent oral manifestation, being 71.4% and 45.0% in the EG2 and EG1. The EG2 was the group that presented oral manifestations. With respect to visual diagnosis of candidiasis in the oral manifestations, the EG2 was prevalent in 28.6%, followed by EG1 with 25.0%. The C. albicans was present in 17.6% in patients with chronic kidney disease, and to GE1 10.0% and 28.6% for EG2. EG3 was not observed in the presence of C. albicans. Thus, it can be concluded that oral manifestations observed in the microorganism C. albicans was present in patients with chronic kidney disease in predialysis and hemodialysis, but no significance to claim that it is opportunistic to trigger candidiasis.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-11-06T12:40:20Z No. of bitstreams: 1 rafaeldeoliveiracorrea.pdf: 2314461 bytes, checksum: f68d67f14fca4892ff6a3da834575d63 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-11-09T14:20:46Z (GMT) No. of bitstreams: 1 rafaeldeoliveiracorrea.pdf: 2314461 bytes, checksum: f68d67f14fca4892ff6a3da834575d63 (MD5) / Made available in DSpace on 2017-11-09T14:20:46Z (GMT). No. of bitstreams: 1 rafaeldeoliveiracorrea.pdf: 2314461 bytes, checksum: f68d67f14fca4892ff6a3da834575d63 (MD5) Previous issue date: 2017-08-28 / A candidíase acomete a cavidade oral e é uma doença ocasionada pela Candida albicans e por outras espécies relacionadas, tais como: C. glabrata, C. krusei, C. guilliermondii. As infecções por cândida consistem atualmente em um problema de Saúde Pública. Isto se deve ao aumento de hospedeiros imunodeprimidos, tratamento prolongado com antibacterianos dentre outros. O uso de agentes antifúngicos eficientes contra leveduras é um importante meio de controle destas infecções bucais. Vários agentes antimicrobianos de origem vegetal estão sendo investigados devido as suas possíveis propriedades farmacológicas. O uso de plantas medicinais é uma prática usual pela população atual sendo que muitas plantas apresentam atividade biológica benéfica ao ser humano. Os extratos vegetais podem contribuir para a produção de medicamentos eficazes. Espécies vegetais como Ficus insipida Willd (caxinguba ou cuaxinguba), Brosimum parinarioides Ducke (amapá), Byrsonima intermedia A. Juss (saratudo), Curatella americana (caimbé), Anacardium spp (cajuí), apresentam notáveis propriedades antibacterianas, antifúngicas, anti-inflamatórias e antineoplásicas e por isso, neste estudo foram avaliados seus extratos etanólicos (brutos). Os principais objetivos foram: determinar o potencial antifúngico destes cinco extratos secos dessas espécies vegetais, frente às principais linhagens causadoras de candidíase oral (Candida albicans, C. glabrata, C. krusei e C. guilliermondii) além de estabelecer a concentração inibitória mínima (CIM) e a concentração fungicida mínima (CFM); verificar as alterações morfológicas e estruturais nas linhagens de Candida por meio da microscopia eletrônica de varredura (MEV); avaliar a toxicidade desses extratos em modelos de células de fibroblastos de camundongos (L929) e queratinócitos (HaCaT) e modelo de Artemia salina. Os extratos vegetais demonstraram potencial antifúngico com CIM e CFM de 0,0097µg/mL e 5µg/mL. Por meio das eletromicrografias foi possível evidenciar alterações morfológicas nas estruturas fúngicas das quatro espécies submetidas à ação dos extratos secos selecionados, o que demonstra sua ação sobre os fungos avaliados. Os extratos revelaram baixa toxicidade frente as células L929 e HaCaT e tóxicos para Artemia salina (IC50 = 80,70µg/mL). Por meio dos resultados apresentados neste trabalho é possível atribuir aos extratos ação antifúngica frente aos principais fungos causadores da candidíase oral. O extrato de Byrsonima intermedia apresentou melhor perfil antifúngico, demonstrando ser o mais promissor deles / Candidiasis affects the oral cavity and is a disease caused by Candida albicans and other related species, such as C. glabrata, C. krusei, C. guilliermondii. Candida infections are currently a public health problem. This is due to the increase of immunosuppressed hosts, prolonged treatment with antibacterial among others. The use of effective antifungal agents against yeasts is an important means of controlling these oral infections. Several antimicrobial agents of vegetable origin are being investigated because of their possible pharmacological properties. The use of medicinal plants is a common practice by the current population and many plants have biological activity that is beneficial to humans. Plant extracts can contribute to the production of effective medicines. Plant species such as Ficus insipida Willd (Caxinguba or Cuaxinguba), Brosimum parinarioides Ducke (amapá), Byrsonima intermedia A. juss (saratudo), Curatella americana (caimbe), Anacardium spp (cajuí), present remarkable antibacterial, antifungal, antiinflammatory properties and antineoplastic and therefore, in this study were evaluated their (crude) ethanol extracts. The main objectives were: to determine the antifungal potential of these five dry extracts of these plant species, against the main oral Candida albicans (Candida albicans, C. glabrata, C. krusei and C. guilliermondii) lines, besides establishing the minimum inhibitory concentration ) and the minimum fungicidal concentration (CFM); to verify the morphological and structural alterations in Candida strains by scanning electron microscopy (SEM); to evaluate the toxicity of these extracts in mouse fibroblast cell (L929) and keratinocyte (HaCaT) models and Artemia saline model. Plant extracts showed antifungal potential with MIC and CFM of 0.0097 μg / mL and 5 μg / mL. By means of the electromicrographs it was possible to show morphological changes in the fungal structures of the four species submitted to the action of the selected dry extracts, which demonstrates their action on the evaluated fungi. The extracts showed low toxicity to L929 and HaCaT cells and toxic to Artemia saline (IC 50 = 80.70 μg / ml). Through the results presented in this work it is possible to attribute antifungal extracts to the main fungi causing oral candidiasis. The extract of Byrsonima intermedia presented better antifungal profile, proving to be the most promising of them

Orientador: Márcio Ajudarte Lopes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T15:56:03Z (GMT). No. of bitstreams: 1 Gonzalez-Arriagada_WilfredoAlejandro_D.pdf: 1774961 bytes, checksum: b24f3765bd65d083c27d09fc2ffbd39b (MD5) Previous issue date: 2014 / Resumo: A radioterapia causa alterações na composição salivar e as proteínas PLUNC participam na resposta imune inata da cavidade oral. O objetivo desse estudo foi verificar se a radioterapia é capaz de modificar a expressão de PLUNC salivar e se essas proteínas estão associadas com os efeitos colaterais. MATERIAIS E MÉTODOS: Foi coletada saliva não estimulada de 65 voluntários (45 pacientes com câncer e 20 controles). No grupo de estudo a coleta foi realizada uma semana antes do início da radioterapia, no meio do tratamento e uma semana após o término. A expressão de SPLUNC1 e SPLUNC2A foi detectada por western blot, e foi analisada com os dados clínico-patológicos e efeitos colaterais. RESULTADOS: Foi notada uma redução do fluxo salivar durante e após o término da radioterapia, sendo mais acentuada nos pacientes que foram submetidos a radioterapia envolvendo a região facial. O campo de radiação facial foi correlacionado com os efeitos colaterais, principalmente com a presença (p=0,0110) e intensidade (p=0,0143) de mucosite. SPLUNC1 e SPLUNC2A foram detectadas na saliva dos pacientes sem tratamento em concentrações variáveis. O grupo de estudo mostrou níveis de SPLUNC2A significantemente maiores que o grupo controle, enquanto SPLUNC1 não mostrou diferenças. A concentração de PLUNC foi modificada pela radioterapia, observando diminuição dos níveis de SPLUNC2A na sua forma glicosilada (p<,0001) e aumento dos níveis de SPLUNC1 (p=0,0081) na segunda e terceira coletas. A única associação entre efeitos colaterais da radioterapia e PLUNC foi a presença (p=0,0363) e intensidade (p=0,0500) da mucosite com maiores níveis SPLUNC1. CONCLUSÕES: O presente estudo reportou que os níveis de SPLUNC1 e SPLUNC2A glicosilada são afetados pela radioterapia, sugerindo que essas proteínas podem ter importância no microambiente oral dos pacientes irradiados na cabeça e pescoço / Abstract: Radiotherapy causes alteration in saliva composition and PLUNC proteins participate in innate immunity of the oral cavity. The aim of this study was to verify if radiotherapy is able to modify the salivary PLUNC expression and if they are associated with radiotherapy side-effects. MATERIALS AND METHODS: Unstimulated whole-mouth saliva of 65 voluntaries (45 cancer patients and 20 controls) was collected. In the study group the collection was performed one week before the beginning of radiotherapy, in the middle of the treatment and one week after finishing. SPLUNC1 and SPLUNC2A expression were detected by western blotting and was analyzed with clinicopathological data and radiotherapy side-effects. RESULTS: Reduction of salivary flow rates was observed during and after conclusion of radiotherapy, being more accentuated in patients who underwent radiotherapy involving the facial region. Facial radiation field was correlated with collateral effects, mainly with the presence (p=0.0110) and severity (p=0.0143) of mucositis. SPLUNC1 and SPLUNC2A were detected in saliva of patients without treatment in variable concentrations. The study group showed levels of SPLUNC2A significantly higher than the control group, while SPLUNC1 did not show differences. Concentration of PLUNC was modified by radiotherapy, observing decreasing of glycosilated form of SPLUNC2A levels (p<.0001) and increasing of SPLUNC1 levels (p=0.0081) in the second and third collections. The only association between collateral effects of radiotherapy and PLUNC was the presence of mucositis (p=0.0363) and its severity (p=0.0500) with higher levels of SPLUNC1. CONCLUSIONS: The present study reported that levels of SPLUNC1 and glycosilated SPLUNC2A are affected by the radiotherapy, suggesting that these proteins may have importance in the oral microenvironment of irradiated head and neck cancer patients / Doutorado / Estomatologia / Doutor em Estomatopatologia

Orientador: Paulo César Giraldo, Fernando Guimarães / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T17:52:48Z (GMT). No. of bitstreams: 1 Ferreira_JozianiBeghiniJunqueiradeCarvalho_M.pdf: 1296627 bytes, checksum: ecb2857b529cc3c0e322efcb11c882e7 (MD5) Previous issue date: 2011 / Resumo: Introdução: As vulvovaginites (VV) são consideradas como agravos importantes à saúde da mulher, pois afetam suas vidas no âmbito sexual, afetivo, social, e psíquico. Muitos aspectos da fisiopatogênese destas afecções ainda precisam ser esclarecidos, entre eles os mecanismos imunes relacionados à instalação e propagação da infecção. Objetivos: Identificar e quantificar por citometria de fluxo (CF) os leucócitos presentes no conteúdo vaginal de mulheres com flora normal e VV. Avaliar a expressão do CD16 nos neutrófilos do conteúdo vaginal destas mulheres. Materiais e Métodos: Estudo de corte transversal, no período de junho de 2009 a outubro de 2010. O estudo incluiu 152 mulheres no menacme diagnosticadas com: flora vaginal normal (n=51), vaginose bacteriana (VB) (n=34), candidíase vulvovaginal (CV) (n=43) e VB associada a CV (VB+CV) (n=14). As mulheres foram submetidas a exame especular para medida de pH vaginal, teste de Whiff, coleta de material para bacterioscopia e cultura para fungos. A VB foi detectada pelos critérios de Amsel e/ou escore de Nugent ?7. A CV, pela presença de hifas ou esporos no conteúdo vaginal. VB+CV foi diagnosticada quando estas duas VV estavam presentes na mesma mulher segundo critérios já descritos. Foram consideradas com flora vaginal normal, as mulheres que apresentaram flora do tipo 1 e ausência de patógenos nos exames laboratoriais. Ao final do exame ginecológico, foi realizado um lavado da cavidade vaginal, o qual foi enviado ao laboratório para processamento. Os leucócitos foram marcados com anticorpos monoclonais conjugados a fluoróforos (anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD15, anti-CD16, anti-CD19, anti-CD24 e anti-CD56) e analisados por CF. Resultados: Os granulócitos neutrófilos foram as células predominantes em todos os grupos estudados. A média da porcentagem de granulócitos neutrófilos foi significativamente maior (p<0,05) na CV comparado à flora normal e VB. A média da porcentagem de granulócitos neutrófilos foi significativamente menor (p<0,05) na VB comparado à flora normal e a CV. Macrófagos e linfócitos foram detectados em percentuais bem menores que os granulócitos neutrófilos. Houve significância estatística (p<0,05) para os linfócitos TCD4 que apresentaram média da porcentagem maior na CV e VB em comparação à flora normal. Considerando-se a intensidade da expressão do CD16 nos neutrófilos, houve maior expressão nas VV (VB, CV, VB+CV) quando comparado à flora normal (p<0,05). Entre as VV, esta ocorrência foi maior na VB comparado à CV (p<0,05). Conclusões: Os granulócitos neutrófilos foram as células predominantes nos lavados vaginais e suas quantidades foram decrescentes considerando-se CV, flora normal e VB. Estes dados sugerem que, como em outros tecidos, na vagina, os neutrófilos são as principais células efetoras da resposta imune apresentando-se em maiores ou menores concentrações conforme o estímulo imunológico causado pelo micro-organismo. A maior intensidade da expressão do CD16 nos neutrófilos das mulheres com VV indica um atraso na apoptose dos neutrófilos envolvidos nestas infecções, predominando na VB / Abstract: Introduction: Vulvovaginitis (VV) is a serious health problem in women. This disease affects their lives in all aspects, be it, sexual, affective, social, and psychological. Many aspects of the pathophysiology of these infections have yet to be elucidated, including the immune mechanisms related to proliferation of microorganisms and maintenance of the infectious process. Objectives: One aim of this study was to identify and to quantify the immune cells present in the vaginal lumen of women with normal flora and VV by flow cytometry (FC). Another aim was to evaluate the expression of CD16 on neutrophils from the vaginal lumen of these women. Materials and Methods: A Cross-sectional study was performed from June 2009 to October 2010. The study included 152 women of childbearing age diagnosed with: normal vaginal flora (n=51), bacterial vaginosis (BV) (n=34), vulvovaginal candidiasis (VC) (n=43) and BV associated with VC (BV+VC) (n=14). The women underwent speculum examination for performing vaginal pH, Whiff test, Gram stain and culture for fungi. BV was diagnosed by the Amsel criteria and/or Nugent score ?7. VC was diagnosed by the presence of yeast or hyphae in the vaginal discharge. A diagnosis of BV+VC was made when both criteria were present in the same woman. Normal vaginal microflora was defined by the presence of type 1 flora and absence of pathogens in laboratorial exams. During gynecological examination, a lavage of the vaginal cavity was performed and sent to the laboratory for processing. Immune cells were labeled with fluorochrome-conjugated monoclonal-antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD15, anti-CD16, anti-CD19, anti-CD24 e anti-CD56) and analyzed by FC. Results: The neutrophil granulocytes were the predominant cells in all groups. The mean of the percentage of neutrophil granulocytes was significantly higher (p <0.05) in VC compared to the normal flora and BV. On the other hand, the mean of the percentage of neutrophil granulocytes was significantly lower (p <0.05) in BV compared to the normal flora and VC. Macrophages and lymphocytes were detected in percentages far lower than the neutrophil granulocytes. The mean percentage of CD4 lymphocytes was significantly higher (p <0.05) in BV and VC compared to the normal flora. The expression of CD16 on neutrophils was higher in VV (BV, VC, BV+VC) compared to the normal flora (p <0.05). Among the VV, it was higher in BV compared to VC (p <0.05). Conclusions: Neutrophil granulocytes were the predominant cells in the vaginal lavages. Higher amount of neutrophil granulocytes was observed in VC, normal flora and BV respectively. These data suggest that neutrophils are presented in higher or lower concentrations as the immune stimulation caused by the pathogen. The highest intensity of CD16 expression on neutrophils in women with VV indicates a delay in neutrophil apoptosis in these infections, predominantly in BV / Mestrado / Fisiopatologia Ginecológica / Mestre em Ciências da Saúde

Philosophiae Doctor - PhD / The reproductive health of individuals is severely compromised by HIV infection, with candidiasis being the most prevalent oral complication in patients. Although not usually associated with severe morbidity, oropharyngeal candidiasis can be clinically significant, as it can interfere with the administration of medications and adequate nutritional intake, and may spread to the esophagus. Azole antifungal agents are commonly prescribed for the treatment and prophylaxis of candidal infections. However, the emergence of drug resistant strains and dose limiting toxic effects have complicated the treatment of candidiasis. Consequently, safe and effective and affordable medicine is required to combat this fungus. Commercial garlic (Allium sativum) has been used time since immemorial as a natural antibiotic, however very little is known about the antifungal properties of two indigenous South African species of garlic, namely Tulbaghia alliacea and Tulbaghia violacea, that are used as folk medicines for a variety of infections. This study compares the in vitro anti-candidal activity of Tulbaghia alliacea, Tulbaghia violacea and Allium sativum extracts. It was found that the greatest concentrations of inhibitory components were extracted by chloroform or water. The IC50 concentrations of Tulbaghia alliacea were between 0.007 - 0.038% (w/v). Assays using S. cerevisiae revealed that the T. alliacea extract was fungicidal, with a killing half-life of approximately 2 hours. This inhibitory effect of the T. alliacea extracts was observed via TLC, and may be due to an active compound called Marasmicin, that was identified using NMR. This investigation confirms that extracts of T.alliacea exhibit anti-infective activity against candida species in vitro. / South Africa

Magister Scientiae Dentium - MSc(Dent) / Diabetes is often associated with a number of medical complications as a result of the metabolic changes taking place systemically. There is considerable evidence it is associated with oral complications including among others, gingivitis, periodontal disease, xerostomia, oral candidiasis, dental caries, periapical abscesses, lichen planus,burning mouth syndrome and an altered taste sensation (Lamster et al. 2008; Skamagas et al. 2008; Vernillo, 2003). The aim of the present study was to compare the oral health status in diabetic and non-diabetic patients with regards to their oral health problems, oral pathology and self perceived quality of life.A comparative cross-sectional study to determine the common oral complications prevalent in diabetics and non-diabetics was carried out in KwaZulu-Natal, at Prince Mshiyeni Memorial, EThekwini District, Umlazi. The study sample consisted of 150 diabetic patients and 150 non-diabetic patients attending the hospital. The oral health status was assessed clinically for each patient and recorded prior to the interview. The DMFT, plaque index and appearance of marginal gingiva were used to assess oral health status. An intra-oral examination was carried out to identify oral pathology lesions and other oral problems. Patients were then interviewed on the self perceived quality of life and the impact that diabetes has had on their lives. Plaque Index and DMFT were significantly higher among the diabetic group as opposed to the non-diabetic group. Periodontal disease was observed in more than half of the diabetic group as opposed to only 14.7% of the non-diabetic group. Except for tooth decay, the diabetic patients had more oral health problems compared to the non-diabetic group. More than half of the diabetic group reported having xerostomia compared to only 7.3% of the non-diabetic group. Altered taste sensation was also more prevalent in the diabetic group. In general, the diabetic group demonstrated a higher prevalence of oral pathology lesions and medical diabetes complications compared to the non-diabetic group.The self perceived quality of life was said to have deteriorated in 92% of diabetic subjects due to concomitant diabetic complications and 75% of this group indicated that they were not satisfied with their current quality of life.Diabetic patients were significantly less likely to perceive their quality of life as very satisfied after having adjusted for age and gender variables (OR=0.0188; CI: 0.0059-0.0594). Furthermore, diabetic patients were almost 6 times more likely to perceive themselves as “not satisfied” with their quality of life (QOL) as compared to non-diabetic patients.Individuals with diabetes exhibited poorer oral health when compared to non-diabetics.They exhibited a higher DMFT and had a significantly higher average number of missing teeth compared to the non-diabetic group. Special care needs to be given to diabetic patients because of the associated complications to improve their quality of life. A more detailed and in-depth study that utilises a diabetes-specific quality of life instrument may provide a more accurate way of determining the quality of life as well as periodontal disease in patients.

Philosophiae Doctor - PhD / One of the most common HIV-associated opportunistic infections is candidiasis, caused by Candida albicans or other Candida species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of Candida species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant Candida species. The mechanisms of Candida drug resistance in the African continent have also not been described. In this study, 255 Candida species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. Candida cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in Candida species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of Candida infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant Candida strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of Candida colonization mechanisms in HIV-infected African patients.

The 3Rs of reduction, refinement and replacement are the guiding principles of animal research and embedded in national and international legislation regulating the use of animals in scientific procedures. Awake imaging by MRI of rodents can offer a reduction by increasing the quality of scientific data through longitudinal imaging using less animals by avoiding a serial sacrifice design and refinement through reducing the stressful effects animals are exposed to, in comparison to existing models. Before awake imaging can become an established biomarker it must be demonstrated that pathology is traceable with comparable or an improvement on results using existing biomarkers. To validate awake imaging of rodents three study types were conducted in two different rodent species: imaging of the progression of Aspergillus fumigatus infection in the mouse lung using anaesthetized animals; analysis of stress in rats during imaging and imaging restraint; imaging of the abdomen in awake rats and a prospective study into the utility of this method for imaging the progression of Candida albicans renal infection. The first study type used an established model of invasive pulmonary aspergillosis in mice to test the utility of MRI for tracking infection in the lung parenchyma. Images of collapsed parenchyma were obtained and shown to increase as the infection progressed. Further work is required to establish this as a clinically relevant biomarker. The study type used restraint, blood collection and imaging as stressors using corticosterone levels as a surrogate of stress. Analysis of levels in blood and faeces by RIA and ELISA allowed comparison of stress during anaethetised and awake imaging for the first time. There were no differences in rat corticosterone levels during anaethetised and awake imaging indicating that awake imaging was no more stressful than currently used procedures. The third study type employed restrainers and acclimatisation to MRI scanner noise to acclimatize rats for awake abdomen imaging. Both anaethetised and awake rats were imaged with FLASH and IntraGate™ sequences. These methods were utilized in an established model of disseminated candidiasis by imaging the kidney. Comparable image quality was obtained in awake animals, with the utility of the method validated by imaging differences in renal pathology between vehicle and low and high dose treated animals. In conclusion, the first steps have been taken towards establishing awake animal imaging by MRI. The imaging is no more stressful than using an anaesthetic and was a useful biomarker in the rat abdomen and capable of tracking disease development. Further work is required to make the technique fully quantitative and automated and hence become a useful tool for monitoring progression of fungal infection and other pathology.

A gomesina é um peptídeo antimicrobiano catiônico, purificado dos hemócitos da aranha caranguejeira Acanthoscurria gomesiana. Possui amplo espectro de atividade contra bactérias, fungos, protozoários e células tumorais. Candida albicans é uma levedura comensal que faz parte da microbiota humana. O tratamento desta micose geralmente é feito com fluconazol, contudo casos de resistência vêm sendo reportados, com isso vários peptídeos antimicrobianos vêm sendo estudados a fim de se tornarem tratamentos alternativos. Este trabalho teve como objetivo avaliar a eficácia do tratamento com a gomesina em um modelo de candidíase disseminada e vaginal. O tratamento de gomesina foi eficaz no controle do fungo. Verificou-se um efeito imunomodulatório, pois seu tratamento aumentou as concentrações de IL-6, TNF-<font face=\"symbol\">a e INF-<font face=\"symbol\">g dos rins dos animais com candidíase disseminada. A gomesina não apresentou nenhum efeito tóxico para os animais. Os dados apresentados neste estudo reforçam o potencial da gomesina para ser um agente. / The gomesin is a cationic antimicrobial peptide, purified from hemocytes of the spider Acanthoscurria gomesiana. It has a broad spectrum of activity against bacteria, fungi, protozoa and tumor cells. Candida albicans is commensal yeast that is part of the human microbiota. The treatment of this mycosis is usually done with fluconazole although cases of resistance have been reported. With the emergence of microorganisms resistance, several antimicrobial peptides have been studied in order to become alternative treatments. This study aimed to evaluate the effectiveness of treatment with gomesin in a model of disseminated and vaginal candidiasis. The treatment with gomesin showed to be effective in controlling the fungus. There was also found an immunomodulatory effect as its treatment increased concentrations of IL-6, TNF-<font face=\"symbol\">a and INF-<font face=\"symbol\">g in the kidneys of animals with disseminated candidiasis. The gomesin did not show any toxic effect to animals. The data presented in this study reinforce the potential of gomesin to be an antifungal agent.

O aumento na incidência de infecções fúngicas e a alta toxicidade ou elevado índice de resistência associado aos antimicóticos comerciais, criou um mercado carente de novas drogas. Neste contexto, os peptídeos antimicrobianos (AMPs) surgem como uma alternativa promissora ou fonte de conhecimento por desempenhar ação inibidora de crescimento e/ ou letal contra bactérias Gram-positivas e Gram-negativas, fungos, parasitas e/ ou vírus, além de atividade antitumoral e efeito imunomodulador. Como os mecanismos pelos quais eles o fazem são diferentes daqueles das drogas não peptídicas, os AMPs estão pouco associados ao desenvolvimento de resistência microbiana. A hemoglobina (Hb) é uma fonte de peptídeos com funções biológicas diversas. O fragmento 33-61 (Hb33-61) da cadeia &#945; da Hb bovina foi o primeiro AMP descrito a ser gerado in vivo no trato gastrointestinal do carrapato Boophilus microplus. Nossos estudos posteriores usando CD e H1-RMN revelaram que a amidação C-terminal deste fragmento o tornava ainda mais ativo que o primeiro e que em presença de micelas de SDS o Hb33-61a apresenta uma dobra &#946; na porção N-terminal (Lys40-Phe43) e outra (Ser49- Ser52) seguida de &#945;-hélice no C-terminal (Ala53-Ala60), bem como um segmento Pro44-Leu48 capaz de mover-se independentemente e agir como uma dobradiça. Nossas investigações usando análogos sintéticos truncados do Hb33-61a mostraram que o Hb40-61a poderia ser sua porção mínima ativa por apresentar comportamento conformacional idêntico. Nossos estudos subsequentes enfocando as suas propriedades evidenciaram a sua capacidade de causar morte rápida de cepas de Candida, incluindo C. albicans resistentes ao fluconazol e extravasamento de conteúdo e formação de poros em LUVs, revelando sua ação permeabilizante de membrana. Em continuidade ao estudo do Hb40-61a, investigamos no presente trabalho as suas propriedades e o seu mecanismo de ação contra C. albicans. Para isso, sintetizamos, purificamos e caracterizamos esta hemocidina, o seu análogo inteiro composto por D aminoácidos (ent-Hb40-61a) e o seu análogo marcado com 5 (6) carboxifluoresceína (FAM-Hb40-61a). Ensaios com eritrócitos humanos confirmaram a baixa atividade hemolítica desses AMPs em meio de alta e baixa força iônica. O análogo ent-Hb40-61a apresentou a mesma atividade antifúngica que o análogo L, evidenciando um mecanismo de ação não-estereoespecífico. Análises de células de Candida tratadas com FAM-Hb40-61a por microscopia confocal mostraram que em ½ MIC e MIC o peptídeo deposita-se na membrana plasmática e é internalizado, respectivamente. Citometria de fluxo demonstrou que na MIC cerca de 97% das células encontram-se marcadas pelo peptídeo, confirmou a influência negativa da alta força iônica em sua atividade, mostrou que a internalização celular na MIC é independente da temperatura e que a alteração no metabolismo energético da célula afeta de maneira negativa a internalização do peptídeo. Ensaios de permeabilidade celular com Syto 09 e iodeto de propídeo confirmaram danos progressivos à membrana plasmática de C. albicans com o aumento da concentração do Hb40-61a. Experimentos usando DiBAC4(5) e de DPH revelaram que o Hb40-61a altera o potencial de membrana e afeta sua fluidez, respectivamente. Imagens preliminares das células tratadas e não tratadas com Hb40-61a por microscopia de força atômica (AFM) sugeriram alterações nas células de C. albicans após tratamento com a hemocidina. Medidas preliminares do diâmetro médio das células de C. albicans revelaram que elas diminuem após o tratamento com o peptídeo, o que pode ser mais um indício de dano à membrana plasmática por formação de poros e extravasamento de conteúdo intracelular. Assim, obtivemos fortes indícios de que o alvo do Hb40-61a é, de fato, a membrana plasmática das células de Candida, de que ele apresenta potencial de uso tópico para tratamento de candidíase e pode servir como modelo para o desenho de novas drogas antimicrobianas, peptídicas ou não, com propriedades ainda mais valiosas e índices terapêuticos mais elevados. Testes preliminares mostraram que é possível a adsorção do Hb40-58a à nanopartículas de PSS e que, em relação ao peptídeo livre, este arranjo mantém a atividade antifúngica com MIC superior e apresenta menor atividade hemolítica. / The increased incidence of fungal infections and the high toxicity or high level of resistance associated with conventional antimycotics created a demand for new drugs. Antimicrobial peptides (AMPs) constitute a promising alternative and/or an important source of knowledge due to their growth inhibitory action and/or lethality against Gram-positive and Gram-negative bacteria, fungi, parasites and/or viruses. Besides, AMPs display antitumoral and immunomodulator effects. As their mechanisms of action are different from those of non-peptide drugs, AMPs are less associated with the development of antimicrobial resistance. Hemoglobin (Hb) is a source of peptides with diverse biological functions. The fragment 33-61 (Hb33-61) of bovine Hb &#945; chain was the first AMP reported to be generated in vivo in gastrointestinal tract of Boophilus microplus. Our studies of Hb33-61 using CD and H1-NMR showed that amidation of its C-terminal (Hb33-61a) increases its activity; in the presence of SDS micelles, Hb33-61a is characterized by a central hinge joining the C-terminal region (containing a &#946; turn followed by a helical element) to the N-terminal region (that presents only a &#946; turn). Our previous investigations using synthetic truncated analogues of Hb33-61a suggested that Hb40-61a could be its minimal active portion as it presented equal biological and structural properties. Our subsequent studies focusing on its properties showed its ability to quickly kill Candida albicans strains (including those resistant to fluconazole) and to cause leakage of the contents of LUVs and pore formation in GUVs, revealing its membrane permeabilizing action. We further investigated the properties of Hb40-61a and its possible mechanism of action against C. albicans. To do it, we synthesized, purified and chemically characterized it, its all-D analogue (ent-Hb40-61a) and its analogue labeled with 5 (6) carboxyfluorescein (FAM-Hb40-61a). Tests using human erythrocytes confirmed the low toxicity of these hemocidins at high or low ionic strength. The ent-analogue was as active as the all-L compound suggesting a non stereospecific mechanism of action. Confocal microscopy analysis of Candida cells treated with FAM-Hb40-61a showed that at ½ MIC and MIC, the peptide deposits on the plasma membrane and is internalized, respectively. Flow cytometry results showed that at the MIC about 97% of the cells are marked by the peptide, confirmed the negative influence of high ionic strength on its antifungal activity and showed that the cellular internalization at the MIC is partially dependent on ATP, but independent on the temperature. Cell permeabilization assays using Syto 09 and propidium iodide confirmed progressive damage of the membrane as a function of Hb40-61a concentration. Experiments employing DiBAC4 (5) and DPH revealed that the Hb40- 61a alters the membrane potential and affects its fluidity, respectively. Preliminary atomic force microscopy (AFM) images of C. albicans cells before and after treatment with Hb40-61a suggested morphological changes in the plasma membrane. Preliminary measurements of the average diameters of the fungal cells indicated size reduction after treatment with the Hb40-61a probably resulting from pore formation and leakage of cell contents. Thus, we obtained strong evidences that the target of this peptide is indeed the plasma membrane of Candida cells. Thus, this hemocidin have the potential to be used topically for treating candidiasis and/or serve as model for the design of new antimicrobial drugs, peptide or non-peptide, with even more valuable properties and improved therapeutic indexes. Preliminary tests confirmed the possibility of adsorbing Hb40-58a to nanoparticles of polystyrene sulfate (PSS) and that resulting assembly is still active and less hemolytic than the free peptide.