Orientadores: Ljubica Tasic, Priscyla Daniely Marcato Gaspari / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-25T19:45:44Z (GMT). No. of bitstreams: 1 Ballottin_DanielaPottMarinho_D.pdf: 4687735 bytes, checksum: 78a932e6ae81a2c4a51ae94b6bf0c65f (MD5) Previous issue date: 2014 / Resumo: Esta tese compreende a produção biogênica e extracelular de nanopartículas de prata (AgNP) utilizando o fungo Fusarium oxysporum, a caracterização físico-química e bioquímica de AgNP e de proteínas, respectivamente. As partículas obtidas foram caracterizadas por técnicas microscópicas, espectroscópicas e de espalhamento dinâmico de luz, as quais forneceram informações em relação a morfologia, tamanho, composição elementar, carga superficial e cristalinidade das partículas. A presença de proteínas estabilizadoras ao redor das partículas foi evidenciada por UV-Vis e TEM. Estas macromoléculas também foram estudas e caracterizadas por diferentes técnicas, tais como, fluorescência, dicroísmo circular, FTIR e Raman. Além disso, foram realizadas análises de eletroforese em gel sendo possível estimar a massa molar das proteínas que estabilizam as AgNP. Algumas destas proteínas foram identificadas por espectrometria de massas, a qual permitiu a obtenção de resultados promissores e inéditos, uma vez que não há na literatura nenhum relato sobre a identificação de proteínas envolvidas na síntese e/ou na estabilização das AgNP. Adicionalmente, foi estudada a atividade antimicrobiana das AgNP frente a diversos micro-organismos patogênicos, como duas espécies de Candida sp. (C. albicans e C. parapsilosis), as quais causam infecções hospitalares, e Xanthomonas axonopodis pv. citri (Xac), uma bactéria patogênica causadora do cancro cítrico, doença com sérias consequências para citricultura brasileira. Nestes estudos foi verificada a alta atividade antimicrobiana das partículas com a atividade inibitória mínima (MIC) da ordem de ?g mL-1. Cito- e genotoxicidade em diferentes organismos e células também foram investigadas, demonstrando que em concentrações utilizadas neste trabalho, as AgNP não apresentam efeito cito- ou genotóxico. São mostrados também resultados da impregnação das AgNP em tecido de algodão e a atividade antimicrobiana deste material frente a C. albicans, C. parapsilosis e Xac / Abstract: This thesis comprises the biogenic and extracellular production os silver nanoparticles (AgNP) using the Fusarium oxysporum fungus, the physic-chemical and biochemical characterization of AgNP and proteins, respectively. The obtained particles were characterized by microscopic, spectroscopic and dynamic light scattering techniques, which provided information regarding the morphology, size, elemental composition, particle surface charge and cristallinity. The presence of surrounding proteins, which stabilize particles was evidenced by UV-Vis and TEM. These macromolecules have also been studied and characterized by different techniques, such as, fluorescence, circular dichroism, Raman and FTIR. Moreover, del electrophoresis analysis were performed ans it was possible to estimate the molar weight of proteins. Some of these were identified by mass spectrometry, which allowed to obtain novel and promising results since there is no reports on the identification of proteins involved in synthesis and/or stabilization of AgNP. Additionally, it was studied the antimicrobial activity of AgNP against several pathogenic micro-organisms, such as, Candida sp. (C. albicans and C. parapsilosis), which cause nosocomial infections, and Xanthomonas axonopodis pv. citri (Xac), a pathogenic bacterium that causes citrus canker, a disease with serious consequences for the Brazilian citrus industry. In these studies it was investigated the high antimicrobial activity of particles through the minimum inhibitory concentration (MIC) values (order of ?g mL-1). Cyto- and genotoxicity in different organisms and cells have also been investigated, showing thet at determined concentrations, AgNP have no cyto- and genotoxic effects. Results of impregnation of AgNP in cotton fabrics and its antimicrobial activity are also shown against C. albicans, C. parapsilosis and Xac / Doutorado / Quimica Organica / Doutora em Ciências

Orientadores: Gonçalo Amarante Guimarães Pereira, Sandra Martha Gomes Dias / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T22:03:27Z (GMT). No. of bitstreams: 1 Barsottini_MarioRamosdeOliveira_M.pdf: 16356564 bytes, checksum: 9cf8e738f13f95639e35471c9a7da160 (MD5) Previous issue date: 2013 / Resumo: O fungo Moniliophthora perniciosa desperta grande interesse agroeconômico, pois é o agente etiológico da Vassoura-de-Bruxa do cacau. A cultura do cacaueiro é de grande importância no cenário nacional e na América Latina, sendo o entendimento dos mecanismos moleculares desta doença de grande valia. Durante a interação entre patógeno e hospedeiro, o primeiro produz moléculas para evadir ou alterar as respostas normais de defesa do segundo. O sequenciamento do genoma de M. perniciosa levou à identificação de proteínas-chave potencialmente envolvidas no processo patogênico do fungo, dentre as quais, estão: proteínas pertencentes à família das Cerato-plataninas (MpCPs), bem como proteínas pertencentes à família das Necrosis- and Ethylene-inducing Proteins (MpNEPs). As CPs são amplamente associadas à interação fungo-hospedeiro, agindo como toxinas, indutoras de resposta de defesa ou alergenos. As NEPs induzem morte celular e necrose em plantas dicotiledônes através da permeabilização da membrana celular. O objetivo desse projeto foi caracterizar funcionalmente as MpCPs e as MpNEPs, determinando assim sua relevância durante a Vassoura-de-Bruxa. A partir do transcriptoma de M. perniciosa e da análise filogenética das doze MpCPs encontradas, foi revelada uma correlação entre grupos de MpCPs e sua expressão diferencial ao longo da doença. Quatro MpCPs foram clonadas, expressas em sistema heterólogo e tiveram sua estrutura cristalográfica resolvida. Ensaios bioquímicos e biofísicos confirmaram que as MpCPs presentes em diferentes grupos filogenéticos apresentam capacidades distintas no tocante à interação com o açúcar N-acetilglicosamina e de formar agregados ordenados. Estudos funcionais indicaram que estas características estão potencialmente relacionadas ao bloqueio de resposta de defesa da planta e ao crescimento do fungo, respectivamente. Quanto às MpNEPs, somente a isoforma MpNEP2 foi detectada durante a Vassoura-de-Bruxa. A partir da estrutura cristalográfica dessa proteína e ensaios de mutação sítio-dirigida, foi identificado um hairpin hidrofóbico exposto ao solvente, possivelemte associado à ancoragem da MpNEP2 na membrana celular, o qual é tão importante quanto o sítio ativo da proteína para a atividade biológica da mesma / Abstract: Moniliophthora perniciosa is the causal agent of Witches' Broom Disease of cocoa and a major agroeconomic concern in Brazil and Latin America. In order to efficiently control this disease, it is crucial to understand the molecular basis underlying its progression. During the attack to the plant, a pathogen releases molecules to suppress or alter the regular defense response of the host. Results obtained from the genome sequencing of M. perniciosa lead to the identification putative virulence factors belonging to the Cerato-platanin protein family (MpCPs), and to the Necrosis- and Ethylene-inducing Proteins (MpNEPs). CPs are important to fungus-host interaction process, acting as toxins, defense response-inducing molecules or allergens. NEPs are toxin-like pore-forming proteins, which affect only dicot plants. This project aimed at the functional characterization of the MpCPs and MpNEPs, as well as understanding their importance for the Witches' Broom Disease progression. Twelve MpCP-coding genes were identified, and comprehensive transcriptome and phylogenetic analyses showed a correlation between MpCPs evolutionary clusters and their expression patterns throughout the disease. Four representative MpCPs had their crystal structure resolved. Biophysical and biochemical characterizations showed a correlation between the MpCP clusters, regarding sugar (N-acetylglucosamine) binding and protein self-assembling, which are possibly related to plant defense response suppression and hyphal growth, respectively. As for the MpNEPs, only the isoform MpNEP2 was shown to be expressed during the Whitches' Broom Disease. Its crystalloghaphic structure, along with site-directed mutagenesis and functional assays revealed that, besides the protein's active site, an hydrophobic hairpin exposed to te solvent is important to the necrosis-promoting activity, probably mediating the contact of MpNEP2 with the cell membrane / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Among fungal pathogens responsible for opportunistic infections, species of the genus Candida have a major role (Mitchell, 1998). Various Candida species cause superficial infections which can be cured by the currently available antifungal arsenal (Noble and Johnson, 2007). However, species of the genus Candida are also responsible for life-threatening systemic infections, particularly in immunocompromised patients with weakened immune system. Among Candida species, C. albicans, which can also be a commensal of the skin and the gastrointestinal and genitourinary tracts, is responsible for the majority of Candida bloodstream infections. However, there is an increasing incidence of infections caused by C. glabrata because it is less susceptible to azoles. Other medically important Candida species include C. parapsilosis, C. tropicalis and C. dubliniensis. The problem has been further worsened by the emergence of many drug resistant isolates which pose a major hurdle during a given treatment regimen. Therefore, there is a dire need to identify novel drug targets and the current study focuses on one such protein found in C. albicans and related Candida species. CaGAL102 does not encode a functional galactose epimerase CaGAL102 was previously identified in the lab as a paralog of CaGAL10. CaGAL10 endoes a functional UDP-galactose 4-epimerase and it can complement a Scgal10 null strain. Further, work on the Gal10 protein in the encapsulated yeast Cryptococcus neoformans identified two Gal10 paralogs in the genome, Uge1 and Uge2 with distinct functions (Moyrand et al., 2008). A similar scenario is found in S. pombe in which two Gal10 sequence homologs have been annotated. In the light of these observations, we wanted to test if CaGAL102 also encodes a functional ScGAL10 homolog. We found that CaGAL102 could not complement Scgal10 null strain though there was a strong conservation in the cofactor and the catalytic motif in both the proteins. We found after a careful literature review that Gal10 belongs to a family of proteins called the short chain dehydratase/reductase family (SDR) (Jornvall et al., 1995), members of which are characterised by the presence of glycine rich cofactor binding motif at the N-terminus and an YXXXK catalytic motif. Proteins belonging to the SDR family have a residue level identity of 15-30% indicating early duplication and divergence. Based on our literature survey we carried out a BLAST search in the NCBI protein database using CaGal102 as the bait protein. We found that CaGal102 is 32% identical at the protein level to dTDP-glucose 4,6 dehydratase (RmlB), another member of the SDR family. RmlB is the second enzyme of the rhamnose biosynthetic pathway which gives rise to dTDP-rhamnose. This pathway is involved in cell wall biosynthesis in bacteria and it has been shown that rmlB is essential for growth of Mycobacterium smegmatis (Li et al, 2006). Interestingly rhamnose is not present in the cell wall of C. albicans. Biochemical characterisation of CaCaGal102 A plant homolog of RmlB is found in A. thaliana which uses UDP-glucose as the substrate (Oka et al., 2007). Based on our alignment data we identified many critical residues in CaGal102. Most importantly we identified that lysine at position 159 lies in the YXXXK motif and could be important for activity. We therefore, mutated the lysine at position 159 to alanine. In order to find out the biochemical function of CaGal102 in vitro, we cloned expressed and purified recombinant wild type and catalytic mutant proteins from E. coli and used the purified proteins for our assays. We found that CaGal102 uses UDP-glucose as the preferred substrate. To further substantiate our data, we reintegrated the wild type or the mutant alleles in the native locus of CaGAL102 and checked for the rescue of morphology defects like filamentation and sensitivity to cell wall damaging agents. We also found that the Cagal102∆/∆ strain is avirulent in a mouse model of systemic infection. We have also carried out infection studies with the null mutant and the wild type and the catalytic mutant reintegrant strains. Our observation suggests that reintegrating one copy of the wild type allele rescues the virulence defect. Interestingly the strain harbouring one copy of the mutant allele behaves like the null mutant in a mouse model of systemic infection. We have also identified sequence homologs of CaGal102 in related Candida species. It is plausible to think that the homologs in related species also have similar effects and hence targeting this protein by a small molecule could help in treating candidiasis caused by related species. CaGAL102 is involved in cell wall architecture in C. albicans To elucidate the role of CaGal102 in C. albicans we generated a knockout out strain and studied various mutant phenotypes. The most striking observation was that the cells of the null mutant were filamentous as compared to the wild type control when grown in normal rich media. Further the cells were sensitive to various cell wall damaging agents and also to hygromycin B. We reasoned that lack of CaGal102 causes perturbation in the cell wall architecture rendering the cells sensitive to various cell wall damaging agents. To further strengthen this hypothesis, we decided to study the genetic interaction of CaGAL102 with genes known to be involved in cell wall biosynthesis in C. albicans. One of the candidate genes we chose for our study was GAL10, deletion of which in C. albicans renders the cells sensitive to various cell wall damaging agents. Loss of function of UGE1 in C. neoformans impaired biosynthesis of a cell wall component, galactoxylomannan. We found that cells lacking both Gal102 and Gal10 adhered to nylon membranes poorly as compared to single mutants or the wild type control. The second gene we chose was a P-type ATPase, PMR1 deletion of which causes increased sensitivity to cell wall damaging agents and hyper-activation of the cell wall integrity pathway similar to Cagal102∆/∆ strain. We found that cells lacking both Pmr1 and Gal102 were more sensitive to hygromycin B as compared to the single mutants. This confirmed our idea that CaGal102 is a novel gene involved in cell wall biogenesis in C. albicans. REFERENCES: Mitchell, A.P. (1998) Dimorphism and virulence in Candida albicans. Curr Opin Microbiol, 1, 687-692. Noble, S.M. and Johnson, A.D. (2007) Genetics of Candida albicans, a diploid human fungal pathogen. Annu Rev Genet, 41, 193-211. Moyrand, F., Lafontaine, I., Fontaine, T. and Janbon, G. (2008) UGE1 and UGE2 regulate the UDP-glucose/UDP-galactose equilibrium in Cryptococcus neoformans. Eukaryot Cell, 7: 2069-2077. Jornvall Hans, Persson Bengt, Krook Maria,‟ Atrian Silvia, Gonzalez-Duarte Roser, Jeffery Jonathan, and Ghosh Debashis (1995). Short-Chain Dehydrogenases Reductases (SDR). Biochemistry, 34: 6004-13. Li, W., Xin, Y., McNeil, M.R. and Ma, Y. (2006) rmlB and rmlC genes are essential for growth of mycobacteria. Biochem Biophys Res Commun, 342: 170-178. Oka, T., Nemoto, T. and Jigami, Y. (2007) Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion. J Biol Chem, 282: 5389-5403.