Bibliography: leaves 132-155. / viii, 155 leaves : ill., plates, charts (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A putative callose synthase gene, designated HvGSL1 cDNA is siolated from barley and its involvement in callose biosynthesis is investigated. A near-full length HvGSL1 cDNA encoded a protein showing approximately 30% identitly with that of yeast FKS genes at the amino acid level. The function of this geen was investigated by heterologous expression, protein purification, immunochemistry and mass spectrometric analysis. Results provide strong evidence that the gene encodes a protein which is associated with callose synthase activity, and is likely to encode the catalytic subunit of the synthase complex. / Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Plant and Pest Science, 2003

Bibliography: leaves 132-155. / viii, 155 leaves : ill., plates, charts (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A putative callose synthase gene, designated HvGSL1 cDNA is siolated from barley and its involvement in callose biosynthesis is investigated. A near-full length HvGSL1 cDNA encoded a protein showing approximately 30% identitly with that of yeast FKS genes at the amino acid level. The function of this geen was investigated by heterologous expression, protein purification, immunochemistry and mass spectrometric analysis. Results provide strong evidence that the gene encodes a protein which is associated with callose synthase activity, and is likely to encode the catalytic subunit of the synthase complex. / Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Plant and Pest Science, 2003

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 119-147). Available also in a digital version from Dissertation Abstracts.

The opportunistic fungus Aspergillus fumigatus has emerged as one of the most common fungal human pathogens, causing severe and usually fatal systemic infections that account for more than 200,000 cases annually with mortality rates usually exceeding 50%. During infection, the virulence of A. fumigatus highly depends on its capacity to rapidly respond to external stress encounters in the human niche, such as the host immunological response and the activity of antifungal drugs. The echinocandin, caspofungin, is one of most commonly used antifungal drugs to treat intolerant or refractive patients suffering from invasive aspergillosis. Caspofungin disrupts the catalytic subunit of the β-1,3-glucan synthase complex, Fks1, resulting in the reduced production of the main cell wall component of A. fumigatus, the polysaccharide β-1,3-glucan. Despite its clinical relevance in patients with aspergillosis, caspofungin displays attenuated activity at high concentrations, a phenomenon known as ‘the paradoxical effect’. Little is known about the paradoxical growth of A. fumigatus during caspofungin treatment. Therefore, in this thesis, I investigated the key cellular and molecular responses of A. fumigatus upon caspofungin treatment, particularly during paradoxical growth by live-cell imaging. High-resolution confocal live-cell microscopy revealed that treatment with either low (0.5 µg/ml) or high (4 µg/ml) concentrations of caspofungin for 36 h caused similar abnormalities in A. fumigatus, including wider, hyperbranched hyphae, increased septation and repeated hyphal tip lysis. Regenerative intrahyphal growth occurred as a rapid adaptation to the lytic effects of caspofungin on hyphal tips and the dynamic relocation of Fks1 to vacuoles was a key feature observed in response to caspofungin treatment. The reduced amount of β-1,3-glucan resulting from caspofungin treatment was compensated by increased α-1,3-glucan and chitin content in mature hyphal tips. Interestingly, all lysed cells recovered by regenerative intrahyphal growth. However, after 48 h treatment, only cells exposed to high caspofungin concentrations developed paradoxical growth in leading hyphae. This response was associated with a relocalization of Fks1 at hyphal tips. Consistently, cells undergoing paradoxical growth showed normal morphology and ceased to undergo cell lysis, as well as having a normal content of β-1,3-glucan and α-1,3-glucan but not chitin, which remained high. Notably, the localization of the regulatory subunit of the β-1,3-glucan synthase complex, Rho1, was unaffected by caspofungin, but it was required for the development of paradoxical growth. Interestingly, the gene expression of the β-1,3-glucan synthase complex was downregulated by caspofungin treatment. In addition, caspofungin activity induced the nuclear translocation of the Ca+2 regulated transcription factor CrzA to nuclei and only hyphal tip cells in which this translocation occurred underwent cell lysis. Finally, similarly high concentrations of caspofungin also induced paradoxical growth of Aspergillus fumigatus during human A549 alveolar cell invasion. This thesis outlines several critical adaptations that occur at the cellular, subcellular and molecular levels at different times during exposure to high and low concentration of caspofungin.

Morphological transitions in Wangiella dermatitidis, a causative agent of human phaeohyphomycosis, influence virulence processes in this polymorphic fungus. My project first involved the cloning and characterizion of the β(1→3) glucan synthase gene WdFKS1, which encodes the enzyme's catalytic subunit, followed by cloning and characterizing the WdRHO1 gene, which encodes its regulatory subunit. To better understand the Rho-type GTPase-mediated regulation of cell polarity and its role in fungal morphological transitions, a homologue of WdRAC1 from a W. dermatitidis was subsequently identified by degenerate PCR and gene walking. Gene deletions of WdFKS1 and WdRHO1 in haploid W. dermatitidis were lethal, whereas the deletion of WdRAC1 was not. RNA interference on WdFKS1 mRNA expression resulted in incomplete septa and damaged cell wall integrity, as well as slow growth rate in W. dermatitidis. Overexpression studies, after site-specific integrations of WdRHO1 and WdRAC1 alleles under control of the glaA promoter into the nonessential WdPKS1 locus, showed the different alleles had different effects on the cell morphological development. For example, whereas overexpression of the wdrho1⁺ allele did not affect the growth rate of W. dermatitidis, the overexpression of wdrho1[superscript G14V], a constitutively active mutation, slowed growth and repressed true filamentous hyphal growth by promoting pseudohyphal growth. While the deletion of WdRAC1 did not affect growth, its loss retarded polarized hyphal growth in a hyphal-inducing minimal medium. Moreover, three new phenotypes of a previously derived WdCDC42 deletion mutant were discovered during this study: in the first, the wdcdc42[Delta] mutant displayed cell lysis when incubated in YPMaltose medium at 37°C; in the second, a dark budding neck abnormality was found after Calcoflour staining; and in the third, the wdcdc42[Delta] mutant displayed no branching during true hyphal growth. Interestingly, the overexpression of wdrac1[superscript G16V] complemented the second and the third phenotypes caused by the WdCDC42 deletion. In addition, the wdcdc42[Delta]/wdrac1[superscript G16V] double mutant unexpectedly displayed an interrupted carotenogenesis pathway. These results support that in W. dermatitidis, Rho-type GTPases play essential roles in growth rate determination and cellular morphogenesis, especially while producing polarized hyphal growth during its many morphological transitions. / text

Systemic fungal infection is a life-threatening problem. Anti-fungal drugs are the most effective clinical strategy to cure such infections. However, most current anti-fungal drugs either have high toxicity or have a narrow spectrum of effect. Meanwhile, anti-fungal drugs are losing their clinical efficacy due to emerging drug resistance. To protect us from these deadly pathogenic fungi, scientists need to study new drug targets and to solve problems related to drug resistance. The cell wall is essential for fungal cell survival and is absent from animal cells, so it is a promising reservoir for screening safe and effective drug targets. Alpha-1,3-glucan is one of the major cell wall carbohydrates and is important for the virulence of several pathogenic fungi. In this thesis, molecular biology and microscopy techniques were used to investigate the function and the synthesis process of α-1,3-glucan in the model fungus A. nidulans. My results showed that α-1,3-glucan comprises about 15% of A. nidulans cell wall dry weight, but also that α-1,3-glucan does not have an important role in cell wall formation and cell morphology. Deletion of α-1,3-glucan only affects conidial adhesion and cell sensitivity to calcofluor white. In contast, elevated α-1,3-glucan content can cause severe phenotypic defects. To study the α-1,3-glucan synthesis process, I systematically characterized four proteins, including two α-1,3-glucan synthases (AgsA and AgsB) and two amylase-like proteins (AmyD and AmyG). Results showed AgsA and AgsB are both functional synthases. AgsB is the major synthase due to its constant expression. AgsA mainly functions in conidiation stages. AmyG is a cytoplasmic protein that is critical for α-1,3-glucan synthesis, likely being required for an earlier step in the synthesis process. In contrast to the other three proteins, AmyD has a repressive effect on α-1,3-glucan accumulation. These results shed light on therapeutic strategies that might be developed against α-1,3-glucan. I also developed a strategy to investigate drug resistance mutations. The tractability of A. nidulans and the power of next generation sequencing enabled an easy approach to isolate single mutation strains and to identify the causal mutations from a genome scale efficiently. I suggest this strategy has applications to study the drug resistance mechanisms of current anti-fungal drugs and even possibly future ones.

The species of Brucella were isolated and characterized almost 120 years ago and their genomes sequenced for almost 4 years. Compared to other bacterial pathogens relatively, little is known about the factors contributing to their persistence in hosts and multiplication within phagocytic cells. Also, many aspects of the interactions between Brucella and its host remain unclear. Molecular characterization of intracellular survival processes of Brucella will provide guidance for additional prevention and control measures. One of the features that distinguishes Brucella is that they do not express classic virulence factors. Thus identification of virulence factors has been elusive and some of the identified virulence genes are putative. Disruption of putative virulence genes and studying the consequent effect on attenuation in cell lines or mouse models is a widely used method. However, in most cases it is not apparent whether the mutated genes encode virulence factors or merely affect normal metabolic or biological functions. Some mutations in Brucella can be compensated by redundancy or backup mechanisms. One method for identifying putative virulence genes involved in pathogenesis is to express these genes in a nonpathogenic host and isolate recombinants with increased virulence or survival ability either in cell culture or animal model. We hypothesize that over-expression of Brucella putative virulence genes in the non-pathogenic and close phylogenic relative Ochrobactrum anthropi should enhance its survival in infection models in vivo. O. anthropi is one of the closest Brucella relatives based on DNA, rRNA, and protein analyses but it is unable to establish chronic infection and considered as opportunistic pathogen that, under certain circumstances, may produce disease in immunocompromised humans. Therefore, we established enhanced expression system in Brucella and Ochrobactrum to identify B. suis virulence genes. We created an enhanced expression system that can be used for cloning and expression of heterologous genes in Brucella and Ochrobactrum. We studied the transcriptional activity of several promoters and created some tools to enhance the expression, detection and purification of Brucella recombinant protein in Ochrobactrum. The presumable importance of alkyl hydroperoxide reductases encoded by ahpC and ahpD genes and their contribution to intracellular survival of Brucella were studied by over-expressing them. The recombinant O. anthropi expressing B. suis ahpC and ahpD genes were able to resist in vitro killing by H2O2 and or cumene hydroperoxide and survived longer in the macrophage J774 A.1 cell line. The control O. anthropi was cleared from BALB/c mice in five days while the recombinants were recovered from spleens, livers and lungs of infected mice up to eight days post-infection. We tested the contribution of B. suis cyclic glucan synthetase gene (cgs) to virulence by over-expressing it in O. anthropi. We studied the ability of the recombinant O. anthropi to resist killing in vitro and in vivo. We generated evidence that B. suis cgs when over-expressed in O. anthropi increased the amount of cyclic glucans synthesized and accumulated in the periplasmic space. This accumulation changed the virulence of the microorganism from a soil bacterium that cleared from mice in less than five days into a pathogenic organism that could survive up to 9 days and at higher doses killed the mice. In summary, several vectors have been constructed for gene expression and protein purification in Brucella and Ochrobactrum. Novel useful tools for enhancement of heterologous gene expression were created and demonstrated to work in Brucella and Ochrobactrum. Brucella putative virulence genes were studied in Ochrobactrum using the newly constructed vectors and tools. Ochrobactrum as a gain of function model for studying putative virulence genes of intracellular pathogens in general and for Brucella in particular proved to be a very useful model. / Ph. D.

Orientadores: Marcia Regina Braga, Marcia Maria Camargo de Morais / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T04:21:10Z (GMT). No. of bitstreams: 1 Galvao_DaianeFelbergAntunes_D.pdf: 15313845 bytes, checksum: aa218f685858df886eb7062bfe4337dc (MD5) Previous issue date: 2014 / Resumo: Fruto-oligossacarídeos (FOS) são frutanos de baixo peso molecular produzidos por microorganismos. O interesse em FOS vem aumentando uma vez que eles são considerados ingredientes funcionais benéficos à saúde humana. Com o objetivo de analisar como a produção de FOS e a composição da parede celular de fungos filamentosos é afetada pela fonte de carbono, os fungos Fusarium solani (URM 3338) e Neocosmospora vasinfecta (URM 3329) foram cultivados em meios contendo cinco fontes de carbono diferentes (sacarose, inulina, glucose, frutose ou glucose mais frutose, todos a 1%) e coletas foram realizadas aos 5, 10 e 15 dias de crescimento. A partir do meio de cultivo filtrado foram analisados o pH, teores de açúcar total, açúcares redutores e proteínas, a presença de FOS e atividades enzimáticas invertásica e inulinásica. A partir do micélio, a biomassa foi quantificada e a parede celular foi isolada e sua composição em açúcares neutros, ácidos urônicos e quitina analisada. Foi avaliada também a expressão relativa de genes de síntese de parede celular b-1,3-glucano sintase e quitina sintases. Os dois fungos utilizaram todas as fontes de carbono crescendo nas diferentes condições. Atividade de hidrólise foi detectada no meio contendo sacarose ou inulina para o fungo F. solani, gerando glucose, frutose e fruto-oligossacarideos como produtos havendo utilização dos monossacarídeos. O micélio deste fungo apresentou alterações visíveis no crescimento em meio sólido apenas no meio com frutose, mas foi observada igual quantidade de quitina da parede celular deste fungo quando crescido por cinco dias em sacarose e inulina, mas em menor quantidade com relação aos demais meios. As análises de expressão relativa de genes mostraram indução do gene da b-1,3-glucano sintase e repressão do gene quitina sintase 5 em sacarose e inulina com relação a condição frutose. Estes dados sugerem que a alteração na composição da parede celular do F. solani pode ter relação com a secreção de enzimas nos meios sacarose e inulina. Para N. vasinfecta, quando crescido em sacarose foi observada atividade de transfrutosilação, com a liberação de glucose e síntese de 1-cestose (FOS) no meio. Transfrutosilação também foi observada no meio que teve inulina como fonte de carbono. O micélio deste fungo apresentou alterações visíveis em meio sólido nas condições frutose e inulina, sendo mais hialino do que nas demais condições. A quantidade de quitina na parede celular deste fungo crescido por cinco dias foi maior nas condições frutose e inulina com relação às demais. As análises de expressão relativa de genes mostraram indução dos genes de quitina sintase 4 e 5 nestas duas condições em relação à sacarose. A partir dos resultados, pode-se concluir que as fontes de carbono oferecidas foram utilizadas pelos fungos, que as mesmas afetaram a composição de açúcares da parede celular e a expressão de genes de síntese de componentes da parede e que estes fungos são promissores para a produção de FOS, pois possuem enzimas que hidrolisam a inulina, além de enzimas que sintetizam oligossacarídeos a partir de sacarose por transfrutosilação / Abstract: Fructooligosaccharides (FOS) are low molecular weight fructans produced by microbes and plants. Interest in FOS has been increasing since they are considered as functional food ingredients with benefical effects in human nutrition. With the aim of examining how the production of FOS and the composition of the cell wall of filamentous fungi are affected by the carbon source, Fusarium solani (URM 3338) and Neocosmospora vasinfecta (URM 3329) were cultured in media containing five different carbon sources (sucrose, inulin, glucose, fructose or glucose plus fructose) and samples were taken at 5, 10 and 15 days of growth. From the filtered culture medium, pH, total carbohydrates, reducing sugars and proteins, the presence of FOS and inulinase and invertase activities were analyzed. Mycelium biomass was measured and the cell wall was isolated and its composition in neutral sugars, uronic acids and chitin analyzed. The expression of b-1,3-glucan synthase and chitin synthase genes was also evaluated. Both fungi utilized all the carbon sources for growing. In sucrose- and inulin-containing media, hydrolytic activity was detected in F. solani generating glucose, fructose and FOS as products. When grown on solid culture media, visible changes were observed in mycelium of this fungus only in fructose, but the amount of chitin in the cell wall was higher in the sucrose and inulin-containing media when compared to other carbon sources. The expression b-1,3-glucan synthase gene was induced and chitin synthase 5 gene repressed on sucrose and inulin media. N. vasinfecta showed transfructosilation activity when was grown in sucrose, with release of glucose and synthesis of 1-kestose (FOS) in the culture medium. Transfructosilation was also observed in the inulin-containing medium. The mycelium showed visible changes when the fungus was cultured in solid medium with fructose or inulin as carbon sources. The amount of chitin in the cell wall of this fungus when grown for five days in inulin or fructose was higher in comparison to other carbon sources. The analysis of gene expression showed induction of chitin synthase 4 and 5 genes in these two conditions in relation to sucrose. From the results it can be concluded that the carbon sources affected growth, enzymic activity, composition of the cell wall and gene expression in F. solani and N. vasinfecta, and that these fungi are promising organisms for FOS production since they secrete enzymes that hydrolyze inulin or synthesize oligosaccharides from sucrose by transfructosylation / Doutorado / Biologia Celular / Doutora em Biologia Celular e Estrutural