Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-08-01T19:55:39Z No. of bitstreams: 3 Dissertação- Reginaldo Teixeira Nunes - 2014.pdf: 2255035 bytes, checksum: 70c145eb933df8fd6b89f116a91cade4 (MD5) Ata da Defesa de Mestrado. Reginaldo Texeira Nunes.jpg: 2607204 bytes, checksum: fb38ba2b64baafd566c2d99c81304371 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-02T12:26:59Z (GMT) No. of bitstreams: 3 Dissertação- Reginaldo Teixeira Nunes - 2014.pdf: 2255035 bytes, checksum: 70c145eb933df8fd6b89f116a91cade4 (MD5) Ata da Defesa de Mestrado. Reginaldo Texeira Nunes.jpg: 2607204 bytes, checksum: fb38ba2b64baafd566c2d99c81304371 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-02T12:26:59Z (GMT). No. of bitstreams: 3 Dissertação- Reginaldo Teixeira Nunes - 2014.pdf: 2255035 bytes, checksum: 70c145eb933df8fd6b89f116a91cade4 (MD5) Ata da Defesa de Mestrado. Reginaldo Texeira Nunes.jpg: 2607204 bytes, checksum: fb38ba2b64baafd566c2d99c81304371 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-07-04 / The basidiomycete Cryptococcus neoformans is a pathogenic yeast responsible for causing meningoencephalitis in patients with a compromised immune system, leading to high mortality rates worldwide. Presently available antifungal drugs for treating this disease are not always efficient and they may lead to serious side effects and sometimes to fungal resistance. This situation has caused an increasing search for natural products derived from plants with antifungal action. Cymbopogon winterianus Jowitt (Poaceae), popularly known as citronella and Thymus vulgaris L. (Labiatae), known as thyme, are two plants commonly used in folk medicine. This study aimed at evaluating the biological activity of both C. winterianus and T. vulgaris essential oils (EOs) on C. neoformans clinical isolates. We evaluated the in vitro susceptibility of C. neoformans to each EO by the broth microdilution method, the in vitro interaction between EOs and fluconazole by the checkerboard method, their toxicity on human erythrocytes and their effects on the mitochondrial metabolism of fungal cells. Both EOs showed to possess antifungal activity against the tested isolates, with minimum inhibitory concentrations (MICs) ranging from 32 to 256 μg/mL for C. winterianus and from 32 to 128 μg/mL for T. vulgaris. No synergism between the EOs and fluconazole was observed. C. winterianus and T. vulgaris EOs did not cause hemolysis on human erythrocytes at concentrations corresponding to MIC values, indicating, therefore, low toxicity. The MTT assays showed no alterations on mitochondrial metabolism of fungal cells by C. winterianus at concentrations ≤ 128μg/mL, as well as no alterations at any concentrations corresponding to MIC values by T. vulgaris, suggesting that the main mechanism of action of both EOs is not by interfering with mitochondrial activity. Results show that C. winterianus and T. vulgaris EOs may be promising on the production of new antifungal drugs. / O basidiomiceto Cryptococcus neoformans é uma levedura patogênica causadora de meningoencefalite em pacientes imunocomprometidos, levando a altas taxas de mortalidade em todo o mundo. Os antifúngicos atualmente disponíveis para o tratamento dessa enfermidade nem sempre são eficazes, podendo causar efeitos colaterais graves e levar a casos de resistência. Diante dessa situação, a busca por produtos naturais, com ação antifúngica, oriundos de plantas tem crescido consideravelmente. Cymbopogon winterianus Jowitt (Poaceae), conhecida popularmente como citronela e Thymus vulgaris L. (Labiatae), conhecida como tomilho-branco, são duas plantas comumente utilizadas na medicina popular. Este trabalho avaliou a atividade biológica dos óleos essenciais (OEs) de ambas, em isolados clínicos de C. neoformans. Foram avaliados a suscetibilidade in vitro destes isolados ao OE de cada planta pelo método de microdiluição em caldo, a interação in vitro entre os OEs e fluconazol pelo método de checkerboard, sua toxicidade em eritrócitos humanos e seus efeitos no metabolismo mitocondrial de células fúngicas. Ambos os OEs apresentaram atividade antifúngica, com concentrações inibitórias mínimas (CIMs) variando de 32 a 256μg/mL, para C. winterianus, e de 32 a 128μg/mL para T. vulgaris. Não houve sinergismo entre os OEs e fluconazol. Os OEs de C. winterianus e T. vulgaris não causaram hemólise em eritrócitos humanos nas concentrações correspondentes às CIMs, mostrando-se de baixa toxicidade. Nos ensaios com MTT, observou-se que não houve alterações no metabolismo mitocondrial das células fúngicas por C. winterianus em concentrações iguais ou inferiores a 128μg/mL, e em nenhum dos valores correspondentes às CIMs por T. vulgaris, sugerindo que o principal mecanismo de ação antifúngica desses OEs nas CIMs não ocorre por interferência na atividade mitocondrial. Os resultados obtidos demonstram que OEs de C. winterianus e T. vulgaris podem ser promissores para a produção de novos antifúngicos.

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Ra-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anticryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines preexisting anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons.

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: INTRODUCTION Mycobacterium tuberculosis (MTB) and Cryptococcus neoformans are the most common causes of chronic meningitis in South Africa. Conventional microbiology has limited utility in diagnosing these pathogens due to the paucibacillary nature of cerebrospinal fluid (CSF) and the diagnostic delay associated with culturing methods. This study aimed to evaluate the utility of an in-house polymerase chain reaction (PCR) method for the detection of the etiological agent of chronic meningitis. METHODS CSF samples (where volume exceeded 5ml) were submitted to the Medical Microbiology diagnostic laboratory of the Tygerberg Hospital from patients with suspected tuberculosis meningitis (TBM). Following routine bacteriology, the sample was used to inoculate two mycobacterial growth indicator tubes (MGIT A and B) and subsequently incubated in the BACTEC 960 automated system. MGIT A followed standard operating procedures and the time to culture positivity was noted. Weekly aliquots (up to 6 weeks) were removed from MGIT B. These samples were boiled to inactivate the bacteria and then the DNA was extracted using the Promega Wizard SV Genomic DNA kit. The DNA was then speciated by PCR and high-resolution melting analysis (HRM) by using primers specific to either the RD9 region of MTB complex or primers specific to the partial internal transcribed spacer 1 (ITS1), 5.8S rRNA gene and partial ITS2 sequence of C. neoformans. RESULTS Routine CSF microscopy indicated that 14 of the 78 patients (17.9%) had typical CSF findings of TBM (lymphocytes predominant, increased protein levels and decreased glucose levels). IV Ziehl-Neelsen (ZN) stains were positive for 12 (15.4%) samples, and MTB was cultured from 19 samples (24.4%). Our optimized PCR and HRM method was able to detect M. tuberculosis in 17 of the 19 culture positive specimens with a sensitivity of 89.5% and a specificity of 62.7%. The sensitivity of this method was higher than that of direct microscopy. In all of the PCR positive samples, the time to detection, compared to culture, could be shortened by 1 to 2 weeks. Only one sample was positive for Cryptococcus culture and another sample was positive with a Cryptococcus latex test. PCR for Cryptococcus was positive in 2 cases (n=78), sensitivities and specificities could not be reported due to the low number of positive cases. CONCLUSION We demonstrated that a short culture period and the use of commercial DNA extraction kit on CSF samples increases the sensitivity of molecular tests to diagnose tuberculosis. Furthermore, the molecular techniques could significantly reduce the time to positivity of results, when compared to culture. Due to the low occurrence of Cryptococcus in the samples included in our study, we could not comment on the diagnostic utility of PCR in the diagnosis of Cryptococcal meningitis, when compared to the conventional methods. / AFRIKAANSE OPSOMMING: INLEIDING Mycobacterium tuberculosis (MTB) en Cryptococcus neoformans is die mees algemeenste oorsake van kroniese meningitis in Suid-Afrika. Routine mikroskopie dra beperkte waarde in die diagnose van hierdie patogene as gevolg van die klein hoeveelhede organismes wat in die SSV (serobrospinale vog) voorkom en die lang tyd wat dit benodig om hierdie organisms te kweek. Hierdie studie beoog om die diagnostiese waarde van ‘n polymerase ketting reaksie (PKR) metode wat intern ontwerp is te evalueer vir die identifikasie van patogene verantwoordelik vir kroniese meningitis. METODES SSV monsters (waarvan die volume 5ml oorskry) en waar daar ‘n kliniese vermoede van tuberkulose meningitis (TBM) was, is na die diagnostiese Mediese Mikrobiologie laboratorium van Tygerberg hospitaal gestuur vir roetine bakteriologiese ontleding. Die oorblywende monsters is gebruik om twee mikobakteriële groei-indikasiebuise (MGIT A en B) te innokuleer en hulle is geïnkubeer in ‘n BACTEC 960 geautomatiseerde sisteem. MGIT A is volgens roetine diagnostiese metodes geanaliseer en die tyd tot ‘n positiewe resultaat is aangeteken Weeklikse monsters (tot en met week 6) is uit MGIT B verwyder en die monsters is gekook om sodoende die bakterië te inaktiveer. Die Promega Wizard SV Genomiese DNS ekstraksiemetode is gebruik om die DNS te versuiwer. Spesiëring van die DNS is deur middel van ‘n intern ontwerpte PKR en hoëresolusiesmeltingsmetode (HRS) gedoen met inleiers wat spesifiek is tot die RD9 gedeelte van die MTB kompleks en inleiers spesifiek tot die gedeeltelike interne getranskribeerde spasieerder 1 (ITS1), 5.8S rRNS geen en die gedeeltelike ITS2 DNS volgorde van C. neoformans. VI RESULTATE Roetine SSV mikroskopie het aangedui dat 14 uit 78 (17.9%) pasiënte tipiese SSV bevindings van TBM (oorwegend limfosiete, verhoogde proteïene en verlaagde glukose) gehad het. Ziehl- Neelsen (ZN) kleurings was positief vir 12 (15.4%) monsters, en MTB is gekweek in 19 (24.4%) van hierdie monsters. Ons geoptimaliseerde PKR en HRS metode het daarin geslaag om M. tuberculosis in 17 van die 19 kultuurpositiewe monsters aan te toon met ‘n sensitiviteit van 89.5% en ‘n spesifisitiet van 62.7%. Die sensitiwiteit van die direkte PKR was hoër in vergelyking met mikroskopie. In al die PKR positiewe monsters was die tyd tot aantoning, in vergelyking met kultuur, verkort met 1 tot 2 weke. Slegs een monster het C. neoformans gekweek en ‘n ander monster was positief met die kriptokokkale latekstoets. PKR vir C. neoformans was positief in 2 gevalle (n=78). Die sensitiwiteit en spesifisiteit van die C. neoformans PKR kon nie bepaal word nie weens te min gevalle. GEVOLGTREKKINGS Ons het aangetoon dat ‘n verkorte inkubasieperiode en die gebruik van ‘n kommersiële DNS ekstraksiemetode op SSV monsters die sensitiwiteit van die molekulêre tegniek vir die diagnose van tuberkulose verhoog en dat hierdie metode die tyd na positiwiteit aansienlik verkort in vergelyking met kultuur. Weens die lae getalle van kriptokokkale meningitis in ons studie kon ons nie kommentaar lewer op die akkuraatheid van PKR in die diagnose van kriptokokkale meningitis, in vergelyking met meer konvensionele metodes, nie.

La cryptococcose chez les patients atteints du VIH-1 est principalement causée par Cryptococcus neoformans var. grubii tandis que Cryptococcus gattii infecte surtout les personnes immunocompétentes. Afin d’élucider les mécanismes causant la susceptibilité différentielle à l’égard de ces deux espèces de Cryptococcus dans le contexte de l’infection au VIH-1, nous avons utilisé un modèle novateur de la cryptococcose chez la souris transgénique CD4C/HIVMutA, qui exprime les gènes nef, env et rev du VIH-1. L’expression du transgène VIH-1 a augmenté le recrutement pulmonaire des macrophages alvéolaires mais a diminué celui des lymphocytes T CD4+ et CD8+ en réponse à l’infection par le C. neoformans ou le C. gattii. La production pulmonaire des chimiokines MCP-1 (CCL2) et RANTES (CCL5) était également réduite chez les souris transgéniques infectées par l’une ou l’autre de ces espèces de Cryptococcus. La production pulmonaire de MIP-1α, MIP-1β, TNF-α, TGF-β, IL-2, IL-4 et IL-13 était augmentée chez la souris infectée au C. neoformans comparativement à C. gattii. In vitro, les macrophages alvéolaires prélevés chez la souris Tg et stimulés par des agonistes ont produit davantage de MIP-1β, alors que les chimiokines MCP-1 et RANTES n’ont pas été détectées. / The most common cause of cryptococcosis in HIV-1-infected patients is Cryptococcus neoformans var. grubii, while Cryptococcus gattii usually infects immunocompetent people. We used a novel inhalation model of cryptococcosis in CD4C/HIVMutA transgenic mice expressing nef, env, and rev of HIV-1 to examine the mechanisms that cause differential susceptibility to these species of Cryptococcus in the context of HIV-1 infection. HIV-1 transgene expression increased alveolar macrophage but decreased pulmonary CD4+ and CD8+ T lymphocyte recruitment. Pulmonary production of the CC chemokines MCP-1 (CCL2) and RANTES (CCL5) was reduced in transgenic mice infected with C. neoformans or C. gattii, and concentrations were lower after infection with C. gattii compared to C. neoformans. Production of MIP-1α, MIP-1β, TNF-α, TGF-β, IL-2, IL-4 and IL-13 was increased in mice infected with C. neoformans compared to C. gattii. Production of MIP-1β by alveolar macrophages harvested from Tg mice was enhanced after agonist exposure in vitro, but production of the chemokines MCP-1 and RANTES was undetectable.

Die Detektion von Umweltsignalen und die gezielte zelluläre Reaktion ist eine zentrale und für das Überleben aller Lebewesen essentielle Fähigkeit. Candida albicans, als dominierender humanpathogener Pilz, ist hochgradig verschiedenen biochemischen und physikalischen Umweltbedingungen ausgesetzt, welche sowohl die Zellmorphologie als auch die Virulenz dieses Erregers beeinflussen. In der vorliegenden Arbeit wurde der Einfluss von Kohlendioxid, als ubiquitär vorkommendes Gasmolekül, auf die Zellmorphologie und Virulenz untersucht. Erhöhte Konzentrationen von Kohlendioxid stellen ein äußerst robustes Umweltsignal dar, welches die morphologische Transition vom Hefewachstum zum hyphalen Wachstum, einem Hauptvirulenzfaktor, in Candida albicans stimuliert. In diesem Zusammenhang wurde die Rolle der putativen Carboanhydrase Nce103 durch die Generation von knock – out Mutanten untersucht. Die Disruption von NCE103 in C. albicans führt zu einem Kohlendioxid – abhängigen Phänotyp, welcher Wachstum unter aeroben Bedingungen (ca. 0,033% CO2) nicht zulässt, jedoch unter Bedingungen mit einem erhöhten CO2 Gehalt von ca. 5% ermöglicht. NCE103 ist also für das Wachstum von C. albicans in Wirtsnischen mit aeroben Bedingungen essentiell. Durch Untersuchungen zur Enzymkinetik mittels Stopped – flow wurde in dieser Arbeit gezeigt, dass Nce103 die Funktion einer Carboanhydrase erfüllt. Die biochemische Funktion dieser Carboanhydrase besteht in der Fixation von CO2 bzw. HCO3ˉ in der Zelle zur Unterhaltung der wesentlichen metabolischen Reaktionen. Weiterhin konnte gezeigt werden, dass die Induktion hyphalen Wachstums durch CO2 in C. albicans nicht durch den Transport von CO2 mittels des Aquaporins Aqy1 beeinflusst wird. CO2 bzw. HCO3ˉ aktiviert in der Zelle direkt eine Adenylylcyclase (Cdc35), welche sich grundlegend von den bisher gut charakterisierten G-Protein gekoppelten Adenylylcylasen unterscheidet. Die Generation von cAMP beeinflusst in der Folge direkt die Transkription hyphenspezifischer Gene und nachfolgend die morphologische Transition vom Hefewachstum zum elongierten, hyphalen Wachstum. Dieser Mechanismus konnte sowohl in Candida albicans als auch in Cryptococcus neoformans nachgewiesen werden, was auf einen panfungal konservierten Signaltransduktionsmechanismus schliessen lässt. Die Inhibition dieser spezifischen Kaskade eröffnet neue Ansätze zur Entwicklung spezifischer antimykotischer Wirkstoffe. / Detection of environmental signals and subsequently directed reaction is essential for the survival of all living organisms. Candida albicans, as the predominant human fungal pathogen is exposed to severely different physical and chemical conditions, which influence cell morphology as well as virulence in human. In the present work, the influence of carbon dioxide as ubiquitous gaseous molecule on virulence and cell morphology was analysed. Elevated concentrations of carbon dioxide are a robust signal to induce the morphological transition from yeast growth to an elongated hyphal growth form, which is believed to be one of the main virulence factors in Candida albicans. The role of the putative carbonic anhydrase Nce103p in carbon dioxide signalling is reviewed by generating knockout mutant strains, which exhibited a carbon dioxide dependent phenotype. Growth under aerobic conditions (0,033 % carbon dioxide) is inhibited but feasible in 5% carbon dioxide. Therefore, Nce103p is essential for growth in host niches with aerobic conditions. Analysis of the biochemical properties of Nce103p by stopped – flow kinetics revealed carbonic anhydrase activity. It is hypothesised, that Nce103p is essential for fixation of carbon dioxide and bicarbonate within the cell in order to sustain basic metabolic reactions. Furthermore, the induction of hyphal growth was independent of aquaporine-mediated transport of carbon dioxide. Bicarbonate rather carbon dioxide activates directly the adenylyl cyclase Cdc35p generating cyclic AMP as second messenger and influencing the transcription of hyphal specific genes in Candida albicans thus promoting the morphological transition from yeast growth to elongated hyphal growth. This signal transduction cascade is present in Candida albicans as well as Cryptococcus neoformans and it is believed to be a pan fungal signal transduction cascade. The specific inhibition of carbon dioxide mediated polymorphism may serve as a new target for antifungal therapeutic agents.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-03-19T21:12:44Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_NeurocriptococosePediatricaEstado.pdf: 41426488 bytes, checksum: e03544c0c40fd8cd2c95fbcae35a23b6 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-03-20T15:31:35Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_NeurocriptococosePediatricaEstado.pdf: 41426488 bytes, checksum: e03544c0c40fd8cd2c95fbcae35a23b6 (MD5) / Made available in DSpace on 2013-03-20T15:31:35Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_NeurocriptococosePediatricaEstado.pdf: 41426488 bytes, checksum: e03544c0c40fd8cd2c95fbcae35a23b6 (MD5) Previous issue date: 2001 / Este estudo mostra o espectro de lesões cerebrais, através de tomografia computadorizada, na neurocriptococose da infância, por Cryptococcus neoformans var. gattii, no Estado do Pará. Analisamos os achados tomográficos de onze crianças (menores de 13 anos de idade), com infecção comprovada do sistema nervoso central por Cryptococcus neoformans var. gattii, internados no Hospital Universitário João de Barros Barreto, Belém PA, entre janeiro de 1992 a dezembro de 2000. A neurocriptococose foi definida pela identificação de leveduras encapsuladas ao exame microscópico, isolamento de Cryptococcus neoformans do líquor e identificação positiva da variedade gattii, através do meio composto de canavanina, glicina e azul-de-bromotimol com, pelo menos, o estudo tomográfico no momento do diagnóstico. A idade das crianças estudadas variou entre 6 a 12 anos, com média de 8,8 anos. Cincos eram meninos e seis meninas. Os principais achados clínicos foram cefaléia, febre e rigidez de nuca (n=11), náuseas e vômitos (n=10). O tempo médio entre o início dos sintomas e o diagnóstico foi de 4,2 semanas (variando de 2 a 8 semanas). Todas as tomografias de crânio foram anormais. Em todos os pacientes foram observados nódulos hipodensos. As demais anormalidades tomográficas observadas foram: 6 pacientes com hidrocefalia, 9 com atrofia difusa e 5 com associação de hidrocefalia e atrofia difusa. Descreve-se, pela primeira vez, achados tomográficos em série de casos de neurocriptococose por Cryptococcus neoformans var. gattii em crianças imunocompetentes. Este estudo mostra que esta infecção desenvolve múltiplos nódulos hipodensos, especialmente na região dos gânglios da base e na substância branca cerebral. As lesões, aparentemente progridem para importante atrofia da substância branca, com dilatação ventricular e proeminência de sulcos cerebrais, provavelmente, conseqüência de hidrocefalia compensatória. Em geral, estes pacientes apresentaram alterações leves do córtex cerebral. / This study shows the spectrum of computed tomography (CT) findings in children's neurocryptococcosis due to Cryptococcus neoformans var. gatfii, in the State of Para. We analysed the cranial CT scan appearance of eleven children (younger than 13 years of age) with proven central nervous Cryptococcus neoformans var. gattii infection, between January 1992 and December 2000, who were horizontalized were referred to Hospital universitário João de Barros Barreto, Belém, PA, Brazil. Intracranial cryptococcosis was defined by identification of encapsulated yeast by microscopic examination and isolation of Cryptococcus neoforman from cerebrospinal fluid and positive identification of the var. gattii with use of canavanine-glycine-bromothymol blue agar media, with at least a cranial CT study obtained at the time of the diagnosis. The age range was 6 years to 12 years, with a mean age of 8,8 years. Six were girls and five were boys. The most common clinical findings were headache, fever and nuchal rigidity (n=11 ); nausea and vomiting (n=10). The mean time from onset of symptoms to diagnosis was 4.2 weeks (range, 2 to 8 weeks). No normal cranial CT was detected in any patient. In all patients were observed hypodense nodules. The remaning scan abnomalities were as follows: six had hydrocephalus, nine had diffuse atrophy, and five had hydrocephalus coexistant with diffuse atrophy. We described by the first time cranial CT scan findings in series of case of neurocryptococcosis due Cryptococcus neoformans var. gaftii in immunocompetent children. This study shows that this infection most commonly develops multiple hypodense nodules, mainly in the basal ganglia region and in the cerebral white matter. These lesions apparently progress to important atrophy of the cerebral white matter, with ventricular dilatation and prominence of cerebral sulci, consequent of a presumable compensatory hydrocephalus. In general, these patients present mild changes in cerebral cortex.

Made available in DSpace on 2016-12-23T13:56:02Z (GMT). No. of bitstreams: 1 Ludmila de Matos Baltazar _ dissertacao.pdf: 1344528 bytes, checksum: 3adf6afe644493dc3f572d95071acc58 (MD5) Previous issue date: 2009-03-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The cryptococcosis is a systemic mycosis, which the meningoencephalitis is the most severe and most common clinical manifestation. The main species involved in your etiology are Cryptococcus neoformans and Cryptococcus gattii. The ecology of these species is not well, known, C. neoformans has the excrement of birds, mainly pigeons, as its principal habitat and C. gattii is commonly associate to the trees. The presence of C. gattii was investigated in different regions of Espirito Santo State and it was found in only two (0,7%) of the 209 samples of trees analyzed whereas C. neoformans was isolated in 9 (17%) of the 54 pigeon excrements analyzed. The occurrence of C. neoformans (63 isolates) at both clinical and environmental samples was higher than C. gattii (4 isolates). In the total, 67 isolates, clinical and environmental, of C. neoformans and C. gattii, were analyzed and all of then were sensitive to the voriconazole (VCZ) and amphotericin B (AMB). For the drug itraconazole (ITZ), 82% were sensitive (S), 18% sensitive-dose-dependent (SDD) and for fluconazole (FCZ), 75% S, 24% SDD and 1% resistant (R). Considering the origin of the isolates, the susceptibility profile was similar between the clinical and environmental origin. Among the azoles, the VCZ was the drug that showed higher activity and FCZ which showed less activity in vitro. There was no crossresistance between FCZ and VCZ. Regard the production of the phospholipase enzyme, 84% of the isolates had high production and only 3% showed no production of the enzyme. All the 67 isolates showed activity of phenoloxidase enzyme, being 71% low and none of then with negative activity. Lower production of phospholipase and higher phenoloxidase activity were observed in strains isolated from HIV-pos. All the clinical and environmental strains of C. neoformans belonged to genotype VNI and all C. gattii to VGII genotype. These results showed similarity in the genetic pattern, susceptibility profile of the drugs and production of extracellular enzymes, phospholipase and phenoloxidase between environmental and clinical isolates and confirm that the infection can be from environmental source. / A criptococose é uma micose sistêmica e a meningoencefalite é a sua manifestação clínica mais grave e mais comum. As principais espécies envolvidas na etiologia são Cryptococcus neoformans e Cryptococcus gattii. A ecologia destas espécies não é ainda bem conhecida, C. neformans tem como habitat, fezes de aves, principalmente pombos e C. gattii é comumente associada a árvores. A presença de C. gattii foi investigada em diferentes regiões do Estado do Espírito Santo e foi encontrada em apenas duas (0,7%) das 209 amostras de árvores analisadas enquanto C. neoformans foi isolado em 9 (17%) das 54 amostras de excrementos de aves analisadas. A ocorrência de Cryptococcus neoformans (63 isolados) tanto em amostras ambientais como clínicas foi maior que a de Cryptococcus gattii (4 isolados). No total, 67 isolados, clínicos e ambientais, de C. neoformans e C. gattii, foram analisados e todos foram sensíveis as drogas voriconazol (VCZ) e anfotericina B (AMB). Para a droga itraconazol (ITZ), 82% foram sensíveis (S), 18% sensível-dosedependente (SDD) e para fluconazol (FCZ), 75% S, 24% SDD e 1% resistente (R). Considerando a origem dos isolados, o perfil de suscetibilidade foi semelhante entre os de origem clínica e os ambientais. Entre os azólicos, o voriconazol foi o fármaco que apresentou maior atividade e o fluconazol o que apresentou menor atividade in vitro. Não foi observada resistência cruzada entre voriconazol e fluconazol. Quanto à produção da enzima fosfolipase, 84% dos isolados apresentaram produção alta e apenas 3% não produziram a enzima. Todos os 67 isolados mostraram atividade da enzima fenoloxidase, sendo 71% com atividade baixa e nenhum com atividade negativa. Produção mais baixa da fosfolipase e níveis mais altos de atividade da fenoloxidase foram observados em cepas isoladas de pacientes HIV-positivos. Todas as cepas clínicas e ambientais de C. neoformans pertenceram ao genótipo VNI e todas C. gattii ao genótipo VGII. Esses resultados revelaram semelhança no padrão genético, perfil de suscetibilidade a drogas e produção das enzimas extracelulares, fosfolipase e fenoloxidase, entre isolados ambientais e clínicos e confirmam que a infecção pode ocorrer a partir de fontes ambientais.

Adjacent cells communicate through gap junctions (GJs). These GJs are formed by head to head docking of two hemichannels (HCs) from two adjacent cells. HCs are connexin hexamer proteins. Connexin mutation is the most frequent cause of childhood hearing loss. This hearing impairment affects 2 in every 2000 children. Inhibition of the HCs might be the key factor to treat such disorders. A library of amphiphilic kanamycins was synthesized to be tested as HC inhibitors. These compounds showed excellent inhibition activity in comparison with the parent compound (kanamycin A) with less toxicity. A library of monosaccharide esters with varying carbon chain lengths (acetyl (C2) to hexadecyl (C16)) were synthesized, characterized, and tested for bioactivity. Carbohydrate esters showed low toxicity while remaining active against bacteria and fungi. The compound 6-O-tetradecanoyl-D-mannopyranose (MAN014), a mannose ester with a fourteen-carbon chain, showed the greatest antibacterial and antifungal properties. A mode of action study was tested against Staphylococcus aureus (bacteria) and Fusarium graminearum (fungus) and found the compound perturbed the cell membrum.

Thesis (Ph.D. (Botany)) -- University of Limpopo, 2023 / The use of medicinal plants as a source of treatment for various ailments including fungal infections is still practised in South Africa and across the globe. Fungal infections especially of Cryptococcus, Candida and Aspergillus species are the main cause of morbidity and mortality worldwide, particularly in developing countries. Traditional medicine is used as a source of remedies worldwide and has contributed extensively towards the development of modern medicine. Twelve selected medicinal plants (Kleinia longiflora DC. Berchemia discolor (Klotzsch) Hemsl., Persea americana Mill., Sansevieria hyacinthoides (L.) Druce, Dichrostachys cinerea (L.) Wright &Arn, Withania somnifera Dunal (Ashgandh), Momordica balsamina L., Lonchocarpus capassa, Pappea capensis, Rhus lancea L. fil, Peltophorum africanum, Maytenus heterophylla (Eckl. & Zeyh.) Robson) were analysed qualitatively for antifungal activities against Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. The plant materials were extracted with solvents of various polarities such as acetone, dichloromethane, methanol, hexane, and water. Methanol extracted the highest amount of crude extracts from all the plant species as compared to other organic solvents. Chemical components of the extracts were analysed using aluminum backed Thin Layer Chromatography (TLC) plates and developed using three different eluent systems: Ethyl acetate: methanol: water [EMW], Chloroform: ethyl acetate: formic acid [CEF] and Benzene: ethanol: ammonia hydroxide [BEA]. CEF was the best eluent solvent system since it separated more compounds from plant extracts. This indicates that the active compounds were relatively non-polar. More chemical compounds were observed in TLC chromatograms separated with CEF, followed by BEA and EMW. All plant extracts had shown different chemical components when separated from the three solvent systems. The bioautography and serial dilution assays were used to determine the biological activity of plant extracts against the tested microorganisms, respectively. All the tested plant extracts revealed some varying degrees of fungal inhibition, with minimum inhibitory concentrations (MIC) values ranging between 0.02 mg/ml and 2.5 mg/ml. The aqueous extracts had shown some activity against the tested microorganisms. Noteworthy, antifungal activity was observed in acetone, DCM, hexane, and methanol root extracts of D. cinerea against the three tested microorganisms with MIC values ranging between 0.02 mg/ml and 0.04 mg/ml. Furthermore, acetone extracts of D. cinerea and P. africanum had excellent activity against three fungal pathogens with MIC values of 0.02 mg/ml and 0.08 mg/ml. Active compounds were observed in dichloromethane extracts of W. somnifera with Rf values of 0.40 and 0.64. In TLC chromatograms separated with BEA, active compounds were observed in acetone, hexane, and methanol leaf extract of P. americana, this indicates that the fungal compounds were relatively non-polar. No active compounds were observed in plant extracts of K. longiflora. Active compounds were visible in all extracts of P. capensis in TLC chromatograms developed in CEF and EMW. The antioxidant present in plants prevents the free radicals from causing various diseases in humans by inhibiting the oxidation of free radicals at the cellular level. The qualitative and quantitative 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods were used to determine the antioxidant activities of plant extracts. The presence of antioxidant compounds was indicated by yellow bands against the purple background on the TLC plates. More antioxidant compounds were observed in acetone and dichloromethane extracts of S. hyacinthoides developed in BEA compared to other plant species tested. Methanol, hexane, and water extracts of L. capassa revealed good antioxidant activity against DPPH by having a high percentage of inhibition compared to other solvents. Noticeably, extracts of P. africanum possess strong antioxidant activity as compared to other plant species. Solvent-solvent fractionation using column chromatography of the acetone extract led to the isolation of six compounds. The biological activity of the isolated compounds of L. capassa was investigated against the tested pathogenic fungi. The isolated compounds revealed some varying degrees of inhibition to the fungal pathogens. The largest quantity was isolated from compound 1 (80 mg), compound 4 (39 mg), compound 3 (27 mg), compounds 2 and 5 (14 mg) and the least was compound 6 (4.8 mg). However only three compounds were successfully identified as Lupeol (compound 1), Friedelin (compound 3) and 6-(γ,γ-Dimethylallyl)-3’,4’-dimethoxy-6”,6”- dimethylpyrano-[2”,3”:7,8]-flavanone (compound 4). Compounds 2, 5 and were not identified due to some impurities. More importantly, the isolated compounds exhibited good antioxidant activity in qualitative and quantitative scavenging assays, which indicates that isolated compounds of L. capassa can scavenge the free radicals causing fungal infections in humans. The results support the traditional use of the selected plants to combat fungal infections and related ailments by the local people and traditional health practitioners in Vhembe District, Limpopo Province. The (3-(4,5-dimethylthiazol) -2,5-diphenyltetrazoliumbromide) (MTT) assay was used to determine the toxic effects of the plant crude extract and isolated compounds. Lupeol and 6-(γ,γ-Dimethylallyl)-3’,4’-dimethoxy-6”,6”-dimethylpyrano-[2”,3”:7,8]- flavanone revealed the same degree of cytotoxicity against the Vero monkey kidney cells. All the compounds were not toxic with an LC50 value of ˃ 0.2 mg/ml. / University of Limpopo and National Research Foundation

Cryptococcus neoformans (C. neoformans), um fungo que se encontra disseminado em várias partes do mundo, inclusive no Brasil, é o responsável pela criptococose infecção oportunista mais comum em pacientes com a síndrome da imunodeficiência adquirida (SIDA). O caráter sistêmico da criptococose pode levar esses pacientes a óbito. A finalidade de se obter um diagnóstico laboratorial rápido e acurado de C. neoformans, principalmente para o seguimento dos pacientes HIV nos levou a investigar \"seqüências iniciadoras\" (Si) A, B e C das regiões 18SrDNA, 5,8SrDNA e ITS do Cryptococcus spp. Pela Nested PCR com estas seqüências, sugerimos a melhor delas para o desenvolvimento de um diagnóstico molecular em relação aos métodos usuais. Para tal, foram avaliadas amostras de soro e líquor de 39 pacientes, que já haviam recebido tratamento clínico. Todos os casos foram selecionados em grupos, como segue:7 com criptococose (GIII), 14 HIV positivos (GIV), 18 HIV positivos associados com a criptococose (GV) em relação a 10 controles - indivíduos sadios (GI) e amostras de culturas referência (GII). Os resultados obtidos pela Nested PCR com as \"Sis\" A, B e C foram comparados àqueles obtidos pelos métodos de diagnóstico convencionais. As análises desse estudo mostraram que as \"Sis\" A, B e C detectam C. neoformans com especificidade variada, tanto no soro (SiA 91,66%, SiB-100%, SiC 75%), como no líquor (SiA 83,33%, SiB 100% e SiC 75%) mas não apresentaram falso positivo, quando esses resultados foram comparados aos obtidos das culturas heterólogas (GVI). A SiB, em líquor, apresentou sensibilidade, acurácia, valores preditivo positivo e negativo, e especificidade de 100% para a detecção de C. neoformans da mesma forma que no soro, porém neste o valor preditivo negativo foi 89%, acurácia 94% e a sensibilidade 88%. Em amostras de soro e líquor, os testes Tinta da China e Látex, mostraram resultados falso positivos para o GIV e falso negativos nos grupos GIII e GV. As análises comparativas entre as técnicas mostraram que a ordem de eficiência da sensibilidade para detecção de C. neoformans no soro foi SiB>SiA=Látex>SiC e no líquor foi SiB> SiA>tinta da China>Látex=SiC. No soro, a especificidade entre as técnicas foi SiB>SiA=Látex>SiC e no líquor foi SiB=tinta da China>Látex>SiA>SiC. De acordo com nossos dados, concluímos que, independente da doença associada (HIV) ou se o paciente for tratado, a SiB foi a melhor seqüência para a detecção de C. neoformans, tanto em amostras diretamente de soro, como de líquor para todos os grupos estudados. Tendo em vista os fatos, acreditamos que, a aplicação da técnica da Nested PCR com a \"SiB\", em amostras de líquor e soro, é um método viável e acurado para realizar o diagnóstico molecular do C. neoformans em pacientes HIV. O uso de amostras de soro, para o segmento dos pacientes com SIDA, durante o tratamento, pode ser a forma menos invasiva em relação ao líquor para a detecção do C. neoformans, com vantagens sobre os métodos utilizados / Cryptococcus neoformans (C. neoformans), a fungus that is widespread in many parts of the world, including Brazil, is responsible for cryptococcosis the most common opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). The systemic character of cryptococcosis may be fatal. In order to obtain a rapid and accurate laboratory diagnosis to follow - up of HIV-cryptococcosis patients led us to investigate the sensibility and especificity of three primers (A, B and C) of 18SrDNA, 5,8SrDNA and ITS regions of Cryptococcus spp. Using Nested PCR with those primers we suggest the best among them to be used as a method of molecular diagnosis in relation to the usual techniques. For this purpose, the serum and cerebrospinal fluid (CSF) of 39 patients, who had received medical treatment, were evaluated. All cases were separated in groups, as follows: 7 with cryptococcosis (group III), 14 HIV positive (group IV), 18 HIV positive associated with cryptococcosis (group V) were compared to, 10 healthy subjects (group I) controls, as well as to reference cultures (group II) samples. The results obtained by nested PCR with primers A and B and C were compared to those obtained by conventional diagnostic methods. The analyses of primers A, B and C detected C. neoformans both in serum (SiA 91,6%, SiB-100%, SiC 75%), and in CSF (SiA 83,3%, SiB 100% e SiC 75%). Besides, they were specific for the identification of C. neoformans and showed that there were no false positives when compared with heterologous cultures (group VI) samples. The primer B in CSF showed 100% sensitivity, 100% accuracy and 100% predictive values (positive and negative), and 100% specificity for the detection of C. neoformans, the same that occurs in serum, but in this case, with 88% in sensitivity, 89% predictive value negative and 94% accuracy. In serum and CSF samples the China Ink and the Latex tests showed false positive results for group IV and false negative for III and V groups. The comparative analysis among the techniques (Nested PCR, China Ink, Latex) indicated that the efficiency order of sensitivity for the detection of C. neoformans were PrB> PrA = Latex> PrC in serum and PrB > PrA > China Ink> Latex = PrC in CSF. For the serum and CSF specificities the same techniques were used with the following results: PrB>PrA=Latex>PrC and PrB=China Ink>Latex>PrA>PrC. According to our data we conclude that, whether the patients had been under treatment or not, the Nested PCR by PrB was the best way to detect C. neoformans both in serum and in CSF for all groups. The following up of AIDS patients, throughout the course of therapy was found to be feasible, accurate and less invasive to detect C. neoformans by using serum samples (directly)