Leung, Ho Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 136-146). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.II / ACKNOWLEDGEMENT --- p.III / ABSTRACT --- p.IV / CHINESE ABSTRACT --- p.VI / TABLE OF CONTENT --- p.VII / OVERVIEW OF THIS PROJECT --- p.1 / Chapter SECTION 1: --- Purification and Characterization of hemagglutinins from French bean and mottled kidney bean / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.2 / Chapter 1.2 --- Physiological functions of plant lectins --- p.6 / Chapter 1.3 --- Physiological functions of animal lectins --- p.9 / Chapter 1.4 --- Biological functions of lectins --- p.12 / Chapter 1.5 --- Clinical and research applications of lectins --- p.16 / Chapter 1.6 --- Legume lectins --- p.17 / Chapter 1.7 --- Isolation and purification of lectins --- p.19 / Chapter 1.8 --- Objectives of the present study --- p.21 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Chemicals --- p.22 / Chapter 2.2 --- Assay of hemagglutinating activity --- p.24 / Chapter 2.3 --- Purification protocol --- p.26 / Chapter 2.4 --- Assay of saccharide inhibition of hemagglutination --- p.28 / Chapter 2.5 --- Assay of pH stability --- p.28 / Chapter 2.6 --- Molecular mass determination and N-terminal sequence determination --- p.28 / Chapter 2.7 --- Assay of mitogenic activity --- p.29 / Chapter 2.8 --- Assay of antiproliferative activity --- p.30 / Chapter 2.9 --- Assay for antifungal activity --- p.30 / Chapter 2.10 --- Assay of HIV-1 reverse transcriptase inhibitory activity --- p.31 / Chapter 2.11 --- Assay of stability towards trypsin and chymotrypsin --- p.31 / Chapter 2.12 --- Assay of nitric oxide production --- p.32 / Chapter 2.13 --- Assay ofHIV-1 integrase --- p.32 / Chapter Chapter 3 --- EXPERIMENTAL RESULTS / Chapter 3.1 --- Purification scheme --- p.35 / Chapter 3.2 --- Size determination and N-terminal sequencing --- p.36 / Chapter 3.3 --- Temperature stability assay --- p.37 / Chapter 3.4 --- pH stability assay --- p.37 / Chapter 3.5 --- Saccharides inhibition of hemagglutination --- p.37 / Chapter 3.6 --- Stability towards Trypsin and Chymotrypsin --- p.38 / Chapter 3.7 --- Anti-proliferative activity --- p.38 / Chapter 3.8 --- HTV-1 reverse transcriptase inhibition --- p.39 / Chapter 3.9 --- Mitogenic activity --- p.39 / Chapter 3.10 --- Nitric oxide production --- p.39 / Chapter 3.11 --- HIV-1 integrase --- p.39 / Chapter 3.12 --- Defensin --- p.40 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Purification scheme --- p.68 / Chapter 4.2 --- Sequence comparison --- p.69 / Chapter 4.3 --- Physical Stability of the hemagglutinins --- p.70 / Chapter 4.4 --- Protease Stability --- p.71 / Chapter 4.5 --- Sugar Specificity Assay --- p.72 / Chapter 4.6 --- Anti-proliferative Aactivity toward Cancer Cells --- p.73 / Chapter 4.7 --- HTV-1 reverse trancriptase and H̐ơþV integrase inhibition --- p.74 / Chapter 4.8 --- Mitogenic activity --- p.75 / Chapter 4.9 --- Antifungal protein --- p.76 / Chapter Chapter 5 --- CONCLUSION --- p.78 / Chapter SECTION 2: --- Purification and Characterization of flammulolysin from mushroom Flαmmulinα velutipes / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.79 / Chapter 1.2 --- Mechanisms of hemolysis --- p.80 / Chapter 1.3 --- Biological role of hemolysins --- p.80 / Chapter 1.4 --- Mushroom hemolysin --- p.82 / Chapter 1.5 --- Applications of hemolysins --- p.83 / Chapter 1.6 --- Objectives of the present study --- p.83 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.84 / Chapter Chapter 3 --- EXPERIMENTAL RESULTS / Chapter 3.1 --- Purification and sequence determination --- p.90 / Chapter 3.2 --- Effect of sugars and salts on hemolysin --- p.90 / Chapter 3.3 --- Effect of Temperature and pH on hemolysin --- p.91 / Chapter 3.4 --- Effect of Proteases on hemolysin --- p.91 / Chapter 3.5 --- Effect of osmotic protection on hemolysin --- p.91 / Chapter 3.6 --- Effect of hemolysin on tumor cells --- p.91 / Chapter 3.7 --- Effect of hemolysin on spleen cells --- p.92 / Chapter 3.8 --- Effect of hemolysin on bacterial growth --- p.92 / Chapter 3.9 --- Effect of hemolysin on fungal growth --- p.92 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Purification and sequence comparison of hemolysin --- p.103 / Chapter 4.2 --- Sugar and Salts inhibition --- p.104 / Chapter 4.3 --- Temperature stability --- p.105 / Chapter 4.4 --- pH stability --- p.106 / Chapter 4.5 --- Protease stability --- p.106 / Chapter 4.6 --- Osmotic Protection --- p.106 / Chapter 4.7 --- Anti-tumour activity of the hemolysin --- p.107 / Chapter 4.8 --- Anti-fungal activity --- p.108 / Chapter Chapter 5 --- CONCLUSION --- p.109 / Chapter SECTION 3: --- Purification and Characterization of antifungal peptide from buckwheat seeds Fagopyrum esculentum / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- Plant antiftmgal proteins --- p.110 / Chapter 1.2 --- Classification of antifungal proteins --- p.110 / Chapter 1.3 --- Distribution of antifungal proteins in plants --- p.111 / Chapter 1.4 --- Mechanisms of antifungal activity --- p.111 / Chapter 1.5 --- Future Perspectives of Antifungal proteins --- p.112 / Chapter 1.6 --- Antifungal peptide from Buckwheat --- p.112 / Chapter 1 .7 --- Objectives of the present study --- p.113 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.114 / Chapter Chapter 3 --- EXPERIMENTAL RESULTS / Chapter 3.1 --- Purification and sequence determination --- p.118 / Chapter 3.2 --- Effect on anti-fungal activity --- p.118 / Chapter 3.3 --- Effect of temperature and pH on antifungal activity --- p.118 / Chapter 3.4 --- Effect of the antifungal peptide on tumor cells --- p.119 / Chapter 3.5 --- Effect of antifungal peptide on HIV-1 Reverse transcriptase Activity --- p.119 / Chapter 3.6 --- Effect of antifungal peptide on spleen cells and NO Production --- p.119 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Purification scheme and N-terminal sequence --- p.130 / Chapter 4.2 --- Antifungal Activity --- p.131 / Chapter 4.3 --- Physical stability --- p.131 / Chapter 4.4 --- Anti-proliferative activity toward cancer cells --- p.131 / Chapter 4.5 --- HTV-1 Reverse Transcriptase Inhibitory activity --- p.132 / Chapter 4.6 --- Mitogenic activity and nitric oxide production --- p.132 / Chapter Chapter 5 --- CONCLUSION --- p.133 / OVERALL CONCLUSION --- p.134 / REFERENCES --- p.136

Submitted by Aline Lins de Lima (alineodontoufpb@yahoo.com.br) on 2018-08-07T17:00:06Z No. of bitstreams: 1 Tese ALLima.pdf: 1544888 bytes, checksum: abad9299fa11bc8ebf7e2fcbbc8c6aff (MD5) / Approved for entry into archive by Silvana Alvarez null (silvana@ict.unesp.br) on 2018-08-16T13:18:18Z (GMT) No. of bitstreams: 1 lima_al_dr_sjc.pdf: 1544888 bytes, checksum: abad9299fa11bc8ebf7e2fcbbc8c6aff (MD5) / Made available in DSpace on 2018-08-16T13:18:19Z (GMT). No. of bitstreams: 1 lima_al_dr_sjc.pdf: 1544888 bytes, checksum: abad9299fa11bc8ebf7e2fcbbc8c6aff (MD5) Previous issue date: 2018-06-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A estomatite protética é uma das afecções mais comuns e recorrentes em pacientes portadores de próteses totais. O fungo do gênero Candida, promotor dessa patologia, além de resistente se torna ainda mais complexo de ser combatido devido à dificuldade da ação de fármacos tópicos que só conseguem permanecer por um curto período no local da infecção, em virtude da dinâmica da cavidade bucal. O processo de Solution Blow Spinning permite a obtenção de fibras ultrafinas que podem ser aplicadas em vastas áreas, inclusive na bioengenharia. Uma das aplicabilidades das fibras ultrafinas é sua utilização para liberação controlada de fármacos de forma eficiente e duradoura. Dessa forma, o intuito do presente trabalho foi incorporar Cinamaldeído (CA), composto que possui propriedades antimicrobianas, a mantas de Poli(ácido lático) e Poli(etileno glicol) (PLA/PEG) e avaliá-las quanto à produção e caracterização por, Microscopia eletrônica de varredura (MEV), Termogravimetria (TGA), Calorimetria Exploratória Diferencial (DSC), Espectroscopia de infravermelho por transformada de Fourier (FTIR), Espectroscopia no ultravioleta visível (UV/vis), ensaios mecânicos e ação antifúngica. Para realização dos experimentos, foram fiadas as seguintes mantas: PLA, PLA/PEG e PLA/PEG 23,8% CA. As micrografias obtidas por MEV mostraram, que os diâmetros das fibras que não continham CA, apresentaram diâmetros semelhantes entre si, PLA (354±160 nm)a e PLA/PEG (428±250nm)a, sendo esses diâmetro menores dos que encontrados nas fibras de PLA/PEG 23,8% CA (749±370 nm)b . O ângulo de contato e tensão superficial não puderam ser verificados em virtude da proporção de polímeros nas blendas que apresentaram alta afinidade pelos solventes utilizados no teste. No ensaio de TGA, a curva de PLA/PEG com acréscimo de 23,8% CA exibiu uma maior estabilidade térmica. No teste de DSC o ponto de transição vítrea das mantas contendo 23,8% CA foi o que apresentou menor valor. A liberação de CA foi satisfatória ocorrendo até o 12° dia. No teste de ensaios mecânicos, o acréscimo de CA às mantas aumentaram significativamente o Módulo elástico (24,94±4,45) e a Tensão máxima de ruptura (0,99±0,16 MPa) com relação às mantas puras de PLA/PEG (18,74±3.41 MPa) and (0,85±0.09 MPa), esse acréscimo ainda promoveu redução estatisticamente significante (p˂ 0,05%) em mais de 50% nos biofilmes monotípicos de C. albicans e C. krusei e no multiespécie de C. albicans, C. krusei e C. glabrata. Mediante os resultados encontrados pode-se depreender que é possível se obter mantas de fibras ultrafinas de PLA/PEG contendo 23,8% de CA com propriedades antifúngicas e capacidade de liberação do agente antimicrobiano por cerca de 12 dias. / Denture stomatitis is one of the most common and recurrent conditions in patients with total dentures. The fungal of the genus Candida, the causer of this pathology, besides being resistant, becomes even more complex to be combated due to the difficulty of the action of topical drugs that can only remain for a short time at the site of infection due to the dynamic of the oral cavity. The Solution Blow Spinning process allows the production of ultrafine fibers that can be applied in large areas, including bioengineering. One of the applications of ultrafine fibers is their use for controlled release of drugs in an efficient and long-lasting manner. Thus, the aim of the present work was to incorporate Cinnamaldehyde (CA), a compound that has antimicrobial properties, to Poly (lactic acid) and Poly (ethylene glycol) blankets (PLA / PEG) and to evaluate them for the production and characterization by Scanning Electron Microscopy (SEM), Thermogravimetric (TGA), Differential Scanning Calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), Ultraviolet–visible (UV/vis) spectroscopy, mechanical properties and antifungal action. Antifungal activity was verified against C. albicans, C. krusey and C. glabrata by broth microdilution test, disk diffusion and anti-biofilm activity, in both multi-species and mono-species biofilms. For the experiment, three types of meshes were spun: pure PLA, PLA/PEG and PLA / PEG 23.8% CA. The micrographs obtained by SEM showed that the fibers that did not contain CA had similar diameters to each other and smaller than the fibers containing PLA / PEG 23, 8% CA. The contact angle and surface tension could not be measured by virtue of the proportion of polymers in the blends which showed high affinity for the solvents used in the test. In the TGA assay, the PLA/PEG curve with 23.8% CA increase exhibited a higher thermal stability while in the DSC test the glass transition point of the meshes containing 23.8% CA it was the one with the lowest value. The release of CA was satisfactory occurring until the 6th day. PLA membranes with fibres of diameter exhibited the lowest fibre diameter (354 ±160 nm)a followed by PLA/PEG (428±250nm)a and PLA/PEG/CA (749±370 nm)b. Addition of CA resulted in an increase in mechanical properties of the membranes from (24.94±4.85 MPa) the elastic modulus and (0.99±0.16 MPa) tensile strength in comparison to PLA/PEG (18.74±3.41 MPa) and (0,85±0.09 MPa). CA incorporation increased improved the thermal stability, with release of CA of 0.10 µg/mL over a 12 days period. The PLA/PEG CA membranes presented antifungal activities, showing reductions in more than 50% of the biofilm biomass, being statistically significant (p<0.05%) to the control group. Fibrous membranes of PLA/PEG/CA ultrathin fibres were produced by SBS that exhibited antifungal properties and release over a 12-day period.

PAF26 is a synthetic hexapeptide that has been shown to be highly effective at killing filamentous fungi whilst showing low toxicity against human and bacterial cells. Unlike membrane permeabilising antimicrobials, PAF26 at low fungicidal concentrations is endocytically internalised by fungal cells and accumulates in the vacuole, which undergoes expansion. At a certain point, PAF26 is transported out of the vacuole into the cytoplasm and this is followed by cell death. Previously, cytosolic free Ca2+ ([Ca2+]cyt) has been shown to exhibit a dose-dependent increase in response to PAF26 but the significance of this was not understood. The main objective of my PhD research was to analyse what role(s) Ca2+ signalling has in the mode-of-action of PAF26 using Neurospora crassa as an experimental model. In the first part of my research, evidence for Ca2+ signalling having a significant role in the PAF26 mode-of-action was obtained by testing the PAF26 sensitivity of deletion mutants defective in different components of their Ca2+ signalling machinery. The Deltacch-1, Deltayvc-1 and Deltanca-2 strains were found to exhibit the greatest resistance against PAF26. Screening of a range of vacuolar fusion mutants provided evidence for PAF26-induced vacuolar expansion involving the fusion of small vacuoles. Evidence was also obtained for PAF26-induced programmed cell death occurring via the pathway involved in HET-C-mediated heterokaryon incompatibility. In the second part of my research, I used live-cell imaging with fluorescently labelled PAF26 to investigate the pattern and kinetics of peptide interaction, internalization and distribution within the cells of the Deltacch-1, Deltayvc-1 and Deltanca-2 PAF26-resistant strains compared with the parental wild type. From these studies I obtained evidence for: CCH-1 being required for endocytic internalization of PAF26; YVC-1 being required for PAF26-induced vacuolar expansion; NCA-2 being involved in PAF26 binding to the cell envelope and subsequent endocytic cell internalization and transport to vacuoles; and cell death not being simply induced by PAF26 making contact with the cytoplasm. In the third part of my research I used the genetically encoded bioluminescent Ca2+ reporter aequorin to investigate differences in the average [Ca2+]cyt responses to PAF26 in cell populations of the Deltacch-1, Deltayvc-1 and Deltanca-2 mutant strains compared to that of the wild type. The Ca2+ signatures of the dose-dependent [Ca2+]cyt increases of each mutant were significantly different to each other and to the wild type, and external Ca2+ was required to initiate the [Ca2+]cyt responses. Evidence was obtained for an as yet unknown mechanism of Ca2+ entry into the fungal cell that was independent of the Ca2+ channels, CCH-1 and YVC-1.In the fourth part of my research, I used the genetically encoded fluorescent Ca2+ reporter GCaMP6 to visualise the Ca2+ signatures within individual cells in response to a low fungicidal concentration (3.5 M) of PAF26. This peptide treatment resulted in [Ca2+]cyt spiking at irregular intervals within all cells but there was considerable heterogeneity in the Ca2+ signatures of individual cells of a germling population. Furthermore, subcellular regions within which transient [Ca2+]cyt increases occurred were spatially correlated with increased membrane fusion events between vacuolar-like structures. In the fifth part of my research, YVC-1 and CCH-1 were labelled with GFP. Whilst YVC-1 was consistently associated with the vacuolar membrane, CCH-1 was harder to visualise. CCH-1 appeared to localise to intracellular membranes in conidia and germlings and to the plasma membrane in mature hyphae. In the final part of my thesis I investigated the hypothesis, based on previous evidence, that PAF26 disrupts pH homeostasis by using live cell imaging with the fluorescent, ratiometric pH dye, carboxy SNARF-1. PAF26 treatment was found to cause acidification of the cytoplasm.

Invasive pulmonary aspergillosis is a life-threatening fungal infection commonly observed in immunocompromised patients and has a mortality rate approaching 100% once the disease is disseminated. Aspergillus fumigatus is the most common pathogen. Early diagnosis improves the prognosis but is very difficult since most signs and symptoms are nonspecific. Antifungal therapy, usually based on sterol biosynthesis inhibitors, is also of limited efficacy. In my attempts to discover a diagnostic sterol marker for aspergillosis, I observed that A. fumigatus incorporates large amounts of cholesterol from serum-containing medium. This observation suggested the hypothesis that exogenous cholesterol from the host can be imported by A. fumigatus and used as a substitute for ergosterol in the cell membrane. This proposed mechanism would reduce the efficacy of antifungal drugs that act as sterol biosynthesis inhibitors. Experiments to test this hypothesis were designed to determine the effects of serum-free and serum-containing medium on growth of A. fumigatus in the presence and absence of azole antifungal agents. The results showed a marked increase in growth in the presence of human serum. Cultures in media containing cholesterol but no serum also showed enhanced growth, a result indicating that a non-cholesterol component of serum is not primarily responsible for the increased growth. However, sterol analysis of A. fumigatus cultured in the absence of inhibitors showed little or no change in ergosterol levels. This result suggested that the imported cholesterol was not being used as membrane sterol. However, in parallel experiments using Itraconazole™, an antifungal agent that attenuates sterol biosynthesis by inhibiting the sterol 14a-demethylase (ERG11), ergosterol levels decreased with increasing doses of inhibitor. Moreover, serum-containing medium partially rescued A. fumigatus from the effects of Itraconazole™, and a similar rescue effect was observed with serum-free media containing cholesterol. From the preceding results, it can be concluded that human serum enhances A. fumigatus growth, that cholesterol import rescues Aspergillus from the effects of antifungal agents, that the potency of some azole antifungals is decreased by cholesterol, and that imported cholesterol may substitute for membrane ergosterol in the presence of sterol biosynthesis inhibitors.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The grape and wine ecosystem contains fungi, bacteria and yeasts whose interactions contribute to the final wine product. While the non-Saccharomyces yeasts are dominant in the early stage of alcoholic fermentation, the later stage is always dominated by Saccharomyces cerevisiae. Although their presence in wine fermentation is often short-lived, the non-Saccharomyces yeasts are known to produce an array of extracellular hydrolytic enzymes which facilitate the extraction and release of aroma compounds, but might also play a role in microbial interactions. The present study aimed to investigate the microbial diversity of grape juice and to evaluate the potential of non-Saccharomyces yeasts to produce hydrolytic enzymes and display anti-fungal properties. To capture the microbial diversity, culture-dependent (plating) and –independent (Automated Ribosomal Intergenic Spacer Analysis (ARISA)) techniques were used in parallel. The fungal and bacterial ARISA displayed a wider range of operational taxonomic units (OTUs) in comparison to cultivation-based technique, demonstrating that ARISA is a powerful culture-independent technique applicable to ecological studies in wine. Some of the uncommon yeast isolates derived from our cultivation-based study were subjected to an enzymatic screening process. Hydrolases, such as chitinases, β-1,4-cellulases, β-1,3-1,6-glucanases, β-glucosidases, pectinases and acid proteases were specifically sought. Most of the yeast isolates exhibited chitinase, β-1,4-cellulase as well as β-1,3-1,6-glucanase activities. Only Metschnikowia chrysoperlae exhibited β-glucosidase activity. We also retrieved the partial chitinase gene sequences from M. chrysoperlae, Pichia burtonii, Hyphopichia pseudoburtonii that exhibited chitinase activity. Among the isolates, Pseudozyma fusiformata exhibited a strong antagonistic activity against the wine spoilage yeasts B. bruxellensis AWRI 1499 and B. anomalus IWBT Y105. Furthermore, we showed that the killer phenotype of P. fusiformata cannot be attributed to a viral encoded dsRNA. Finally, two metagenomic approaches were employed in an attempt to explore the indigenous microbiome in a more holistic manner, where we adopted whole metagenome Roche GS-FLX 454-pyrosequencing and construction of a fosmid library. The whole metagenome sequencing revealed a wide range of hydrolytic enzymes that showed homology to enzymes from different fungal and non-Saccharomyces yeast species. Moreover, the metagenomic library screening resulted in the retrieval of 22 chitinase and 11 β-glucosidase positive fosmid clones originating from yeasts. Two clones of interest, BgluFos-G10 and ChiFos-C21, were subjected to next generation sequencing. BgluFos-G10 revealed 2 ORFs exhibiting homology to glycosyl hydrolase family 16 proteins whereas no ORFs encoding chitinase enzymes could be identified in the ChiFos-C21 clone. However, all the potential ORFs identified exhibited homology to a gene cluster from Clavispora lusitaniae ATCC 42720, suggesting that the cloned DNA fragments belonged to a yeast species closely related to C. lusitaniae or members of the family Metschnikowiaceae. Overall, our study identified a variety of novel hydrolytic enzymes. However, retrieving the full gene sequences of these identified enzymes would be the immediate follow-up of our study. Moreover, the hydrolytic and antifungal activities exhibited by the yeast isolate could be of major interest in evaluating their potential as biocontrol agents against grapevine fungal pathogens and subsequently the wine spoilage yeasts. It would be interesting to evaluate as well the potential impact of these enzymes under wine making condition and could be our next step of investigation. / AFRIKAANSE OPSOMMING: Die druif en wyn ekosisteme bevat swamme, bakterië en giste en die interaksies van hierdie organismes dra by tot die finale wyn produk. Die nie-Saccharomyces giste is dominant in die vroeë stadium van die alkoholiese fermentasie, maar die latere fase word altyd gedomineer deur Saccharomyces cerevisiae. Alhoewel hulle teenwoordigheid in wyngistings gewoonlik kortstondig is, is die nie-Saccharomyces giste bekend vir die produksie van ‘n verskeidenheid ekstrasellulêre hidrolitiese ensieme wat die ekstraksie en vrylating van aroma komponente fasiliteer, en ook moontlik ‘n rol kan speel in mikrobiese interaksie. Hierdie studie beoog om die mikrobiese diversiteit van druiwesap te bestudeer en die potensiaal van nie-Saccharomyces giste te evalueer ten opsigte van die produksie van hidrolitiese ensieme, asook die demonstrasie van anti-swam eienskappe. Kweking-afhanklike (uitplating), asook –onafhanklike (Automatiese Ribosomale Intergeniese Spasieerder Analise (ARISA)) tegnieke is in parallel gebruik om die mikrobiese diversiteit te bepaal. Die swam en bakteriële ARISA het ‘n groter verskeidenheid van operasionele taksinomiese eenhede (OTUe) vertoon in vergelyking met die kweking-gebasseerde tegniek en dit demonstreer dat ARISA ‘n kragtige kweking-onafhanklike tegniek is, wat toepasbaar is in ekologiese studies van wyn . Sommige van die skaarser gisisolate, uit ons kweking -gebasseerde studie was vir ensiemaktiwiteite geskandeer. Daar is spesifiek gesoek vir hidrolases soos chitinases,β-1,4-sellulases, β-1,3-1,6-glukunases, β-glukosidases, pektinases en suur proteases. Die meeste gisisolate het chitinase,β-1,4-sellulase asook β-1,3-1,6-glukunase aktiwiteit vertoon. Slegs Metschinikowia chrysoperlae het β-glukosidase aktiwiteit vertoon. Ons het verder die gedeeltelike chitinase geensekwensies van M. chrysoperlae, Pichia burtonii en Hyphopichia pseudoburtonii wat chitinase aktiwiteit vertoon het, bepaal. Een isolaat, Pseudozyma fusiformata, het ‘n sterk antagonistiese aktiwiteit teenoor die wyn bederfgiste, Bretanomyces bruxellensis AWRI 1499 en B. anomalus IWBT Y105 vertoon. Verder het ons gewys dat die killer fenotipe van P. fusiformata nie gekoppel kan word aan’n viraal gekodeerde dsRNA nie. Ten laaste is twee metagenomiese benaderings, naamlik die volledige metagenoom Roche GS-FLX 454-pirovolgordebepaling en konstruksie van ‘n fosmied biblioteek, gebruik om die inheemse mikrobioom op ‘n meer holistiese wyse te bestudeer. Die volgordebepaling van die volledige metagenoom het ‘n wye verskeidenheid hidrolitiese ensieme aan die lig gebring wat homologie met ensieme van verskillende swamme en nie-Saccharomyces gisspesies getoon het. Verder het die skandering van die metagenomiese biblioteek die isolasie van fosmiedklone van gisoorsprong wat positief is vir chitinase aktiwiteit (22 klone) en β-glukosidase aktiwiteit (11 klone) tot gevolg gehad. Twee van hierdie klone, BgluFos-G10 en ChiFos-C21, is met volgende generasie volgordebepaling ontleed. BgluFos-G10 het twee oopleesrame (OLRe) wat homologie met glikosiel hidrolase familie 16 proteïene het, vertoon maar geen OLRe wat chitinase ensieme enkodeer kon in die ChiFos-C21 kloon geïdentifiseer word nie. Al die potensiële OLRe wat geïdentifiseer is, het homologie aan ‘n genepoel van Clavispora lusitaniae ATCC 42720 vertoon, wat daarop dui dat die gekloneerde DNS fragmente aan ‘n gisspesie behoort wat naverwant aan C. lusitaniae of lede van die Metschinikowiaceae familie is. In geheel gesien het ons studie ‘n verskeidenheid van nuwe hidrolitiese ensieme geïdentifiseer. Die bepaling van die volledige geenvolgordes van hierdie geïdentifiseerde ensieme sal die onmiddelike opvolg aksie van hierdie studie wees. Verder is die hidrolitiese en anti-swam aktiwiteite wat deur die gisisolate gedemonstreer is, van hoof belang, asook die evaluering van hulle potensiaal as biokontrole agente teen wingerd swampatogene en wyn bederfgiste. Dit sal ook interessant wees om die potensiële impak van hierdie ensieme onder wynmaakkondisies te bepaal, en dit kan dus ons volgende ondersoek stap wees.

Les travaux présentés dans ce mémoire ont été motivés par l'importance économique de l'élevage de la crevette bleue du pacifique Litopenaeus stylirostris dont les fortes mortalités sont principalement dues au développement de maladies bactériennes et virales. Ils ont consisté en la caractérisation des deux premiers membres d'une famille originale de peptides multifonctionnels présents chez les crevettes pénéides, les stylicines. Ces peptides, nommés stylicines 1 et 2, sont des peptides anioniques (pI < 6.0), formés d'une région amino-terminale riche en résidus de type proline et d'une région carboxy-terminale riche de treize résidus cystéines. Ces molécules sont synthétisées et stockées dans de petits granules présents dans le cytoplasme des hémocytes. Pour mieux appréhender leurs rôles dans la réponse immunitaire des crevettes à une infection par des Vibrio, leurs formes recombinantes ont été produites dans E. coli BL21 (DE3) plysS, purifiées et caractérisées. Les deux rstylicines présentent des activités antiproliférative et anticoagulante. Seule la rstylicine1 présente des activités antimicrobiennes : antifongique sur Fusarium oxysporum (CMI<2.5 µM), et antibactérienne (bactériostatique) sur Vibrio sp (CMI<80 µM). Ce peptide est également capable de se lier aux LPS des bactéries à Gram (-) (Kd= 9.6x10-8 M) et d'agglutiner V. penaeicida "in vitro". Enfin, l'existence de gènes codant des formes modifiées de la stylicine1, chez certaines crevettes, pourrait être en relation avec une diminution de la résistante des individus aux infections. / The work reported here was motivated by the economical importance of the pacific blue shrimp Litopenaeus stylirostris farming where high mortality rates are due to bacterial and viral diseases. It consists in the characterisation of two original peptides, the first members of a new multifunctional family of peptides from peneide shrimps, the stylicines. Those two peptides, named stylicines 1 and 2, are negatively charged (pI < 6.0), and characterised by a proline-rich N-terminal region and a C-terminal region containing 13 cysteine residues. Stylicines are synthesized by heamocytes where they are stored within small cytoplasmic granules. To understand the role of these peptides in the immune response of shrimps to a vibrio infection, their recombinant forms were produced in E. coli BL21 (DE3) plysS, purified and characterised. The two rstylicines display biological anti-proliferative and blood clotting activities. Only rstylicine 1 displays antimicrobial activities: antifungal against Fusarium oxysporum (MIC<2.5µM) and bacteriostatic against Gram (−) bacteria, Vibrio sp. (MIC<80µM). Moreover this peptide displays an LPS-binding activity (dissociation constant (Kd) of 9.6×10−8 M) and agglutinate Vibrio. penaeicida "in vitro". Finally, the presence of sequences coding for modified forms of stylicine 1 in some shrimp's genome may be in relation with their lower ability to survive infections.

It is important to realize that guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. Infectious Diseases Society of America considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances. Coccidioidomycosis, also known as San Joaquin Valley fever, is a systemic infection endemic to parts of the southwestern United States and elsewhere in the Western Hemisphere. Residence in and recent travel to these areas are critical elements for the accurate recognition of patients who develop this infection. In this practice guideline, we have organized our recommendations to address actionable questions concerning the entire spectrum of clinical syndromes. These can range from initial pulmonary infection, which eventually resolves whether or not antifungal therapy is administered, to a variety of pulmonary and extrapulmonary complications. Additional recommendations address management of coccidioidomycosis occurring for special at-risk populations. Finally, preemptive management strategies are outlined in certain at-risk populations and after unintentional laboratory exposure.

It is important to realize that guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. Infectious Diseases Society of America considers adherence to these guidelines to be voluntary, with the ultimate determination regarding their application to be made by the physician in the light of each patient's individual circumstances. Coccidioidomycosis, also known as San Joaquin Valley fever, is a systemic infection endemic to parts of the southwestern United States and elsewhere in the Western Hemisphere. Residence in and recent travel to these areas are critical elements for the accurate recognition of patients who develop this infection. In this practice guideline, we have organized our recommendations to address actionable questions concerning the entire spectrum of clinical syndromes. These can range from initial pulmonary infection, which eventually resolves whether or not antifungal therapy is administered, to a variety of pulmonary and extrapulmonary complications. Additional recommendations address management of coccidioidomycosis occurring for special at-risk populations. Finally, preemptive management strategies are outlined in certain at-risk populations and after unintentional laboratory exposure.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Michael Kanost / Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC₅₀ of 12 μM, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta. Biophysical analysis showed that diapausin-1 binds to the β-1,3 glucan component of the S. cerevisiae cell wall. A second insect peptide investigated in this project was M.sexta stress-response peptide 1(SRP1), an immune-related peptide upregulated under different stress conditions including immune-challenge. Preliminary results for NMR structure determination are presented. Most of the amino acid residue spin systems were assigned, and we determined the connectivities of many amino residues as a first step to solve the NMR structure. The circular dichroism spectrum of SRP1 indicates that the peptide lacks alpha-helical structure and may contain beta strands and turns.

Orientador: James Venturini / Resumo: A paracoccidioidomicose (PCM) é micose sistêmica causada por fungos do gênero Paracoccidioides. As principais formas clínicas da doença são aguda/subaguda e crônica (FC), sendo que nessa última, a maioria dos pacientes desenvolvem fibrose pulmonar e enfisema. Estudos prévios demonstraram que pacientes FC, na forma ativa da doença, apresentam elevada produção de mediadores inflamatórios, incluindo a IL-1β. Essa citocina, diferente das demais, é produzidas por uma plataforma protéica intracelular denominada inflamassoma que pode ser ativadas por patógenos e sinais de dano do hospedeiro. Considerando-se que os mecanismos de ativação do inflamassoma em pacientes com PCM não são conhecidos, o presente estudo teve por objetivo determinar a expressão de genes envolvidos na ativação do inflamassoma e a produção de citocinas por monócitos e macrófagos alveolares de pacientes com PCM em diferentes momentos do tratamento antifúngico.Nossos resultados demonstram a ativação do NLRP3-inflamassoma, caracterizada pela elevada expressão de NLRP3, CASP1 e IL1B por monócitos. Esses achados corroboram a contribuição do NLRP3-inflamassoma na patogênese da PCM também em pacientes. / Abstract: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by species of the genus Paracoccidioides . The main clinical forms of the disease are acute/subacute (AF) and chronic (CF), and in the latter, most patients develop pulmonary fibrosis and emphysema. Previous studies showed that CF patients in active disease, exhibit elevated production of inflammatory mediators, including IL-1β. This cytokine, different from the others, are produced by an intracellular multiprotein platform called inflammasome that can be activated by pathogens and host signs of damage. Considering that the activation mechanisms of the inflammasome in patients with PCM are not know, this study aimed to determine the expression of genes involved in inflammasome activation and cytokine production by monocytes and alveolar macrophages from PCM patients at different times of antifungal treatment. Our results demonstrate the activation of the NLRP3 inflammasome-characterized by high expression of NLRP3, CASP1 and IL1B by monocytes. These findings corroborate the contribution of NRLP3-inflamassome in pathogenesis of PCM also in patients. / Mestre