This thesis describes the cloning and characterisation of two <I>Candida albicans</I> genes which were candidates for having a role in the phenotypic switching phenomenon of <I>C. albicans</I>. Phenotypic switching in <I>C. albicans</I> is a spontaneously occurring event whereby the surface morphology and several physiological processes of the <I>C. albicans</I> colony can change. These spontaneous switching events occur at high frequency and there are a range of up to fifteen different morphological forms that have been described. Switching is reversible and interconvertible (between the different phenotypes). The candidate genes chosen to investigate phenotypic switching were <I>RAD52</I>, a DNA double strand break repair gene and <I>H4</I>, a histone. <I>RAD52</I> was isolated following homologous probing of a <I>C. albicans</I> genomic library using a fragment of the gene sequence which was available on a public data base [http://alces.med.umn.edu/candida/html], as a probe. <I>H4</I> was isolated following PCR probing of a cosmid library. The switching repertoire of the <I>ura</I>- CAI4 strain of <I>C. albicans</I> was characterised. Attempts were also made to characterise switching frequencies. This strain was then used as the host for both knockout and overexpression studies of the candidate genes. The effect of overexpression of these genes on phenotypic switching was observed by recording growth rates, phenotypes and phenotypic switching frequencies. It was found that overexpression of <I>RAD52</I> affected the morphotype and growth of the yeast colonies compared the CAI4 parental strain. Overexpression of the H4 gene did not appear to affect growth, but a fourth morphological form named "root" appeared that had not arisen during characterisation of the CAI4 phenotypic switching repertoire. The degree to which the "root" phenotype was manifest appeared to correlate with the degree of overexpression of the <I>H4</I> gene. The effect of knocking out a single copy of <I>H4</I> was also observed. Growth was not affected. Observations of colony morphologies showed a preponderance of one particular morphology ("irregular wrinkle"). This data suggests that altering the wild type levels of expression of these genes can affect phenotypic switching in <I>C. albicans</I>.

Microtubules (MTs) are important components of the eukaryotic cytoskeleton, with critical functions in intracellular trafficking, establishing and maintaining cell morphology, and segregating chromosomes during mitosis. MTs are hollow, cylindrical polymers composed of αβ-tubulin heterodimers. The longitudinal assembly of αβ-tubulin subunits generates protofilaments, and multiple protofilaments (typically 13 in vivo) interact laterally to form the wall of the MT. In vitro, the polymerization of MTs proceeds in two steps: nucleation and elongation. During the nucleation phase, several αβ-tubulin subunits associate to form a seed, from which further MT elongation then occurs. However, at the relatively low αβ-tubulin concentrations found in vivo, the spontaneous assembly of MTs is not favoured, due largely to the slow kinetics of MT nucleation. The nucleation of MTs in vivo requires the γ-tubulin complex (γ-TuC), a ring-like complex composed of γ-tubulin and γ-tubulin complex proteins (GCPs). Two copies of γ- tubulin associate with one copy each of GCP2 and GCP3 to produce the γ-tubulin small complex (γ-TuSC). Multiple γ-TuSCs, along with the additional GCPs 4,5, and 6, assemble to form the larger γ-tubulin ring complex (γ-TuRC). The γ-TuRC contains a ring of 13 γ-tubulins, which acts as a template for the nucleation of MTs. Typically, the γ-TuC nucleates MTs only when localized to specific subcellular sites, referred to as microtubule organizing centres (MTOCs). However, the precise mechanism by which the γ-TuC is activated at MTOCs remains unknown. In fission yeast, the proteins Mto1 and Mto2 form a complex (Mto1/2) required for the nucleation and organization of cytoplasmic MTs. Mto1/2 determines sites of MT nucleation by recruiting the γ-TuC to several different MTOCs. Different sequences in the Mto1 C-terminus independently confer γ-TuC localization to spindle pole bodies, MTs, and the cell equator. Here, I show that the Mto1 N-terminus is necessary for localization to the nuclear envelope (NE). By simultaneously removing the N- and C-terminal localization domains, I generated the "Mto1-bonsai" mutant, which fails to localize to any conventional MTOCs. In mto1-bonsai cells, MTs are still nucleated in the cytoplasm in an Mto1- dependent manner, but nucleation is spatially random. This reveals that targeting of the γ- TuC to conventional MTOCs is not necessary for MT nucleation, and suggests that Mto1/2 has a direct role in activating MT nucleation by the γ-TuC. Live-cell confocal microscopy allows us to detect individual MT nucleation events, in which newly nucleated MTs are associated with single γ-TuCs as well as Mto1/2-bonsai complexes. Fluorescence quantification reveals that these nucleating complexes contain approximately 13 molecules of both Mto1-bonsai and Mto2, matching the 13 copies of γ-tubulin anticipated for a single γ-TuC. We propose that Mto1/2 may contribute to γ-TuC activation by promoting γ-TuSC assembly and/or inducing conformational changes in the γ-TuC upon binding. I also expressed and purified recombinant Mto1/2-bonsai complex, using a baculovirus/insect cell system. This recombinant Mto1/2-bonsai self-assembles into higher-order complexes, comparable in size to the complexes analyzed in vivo by fluorescence microscopy.

This thesis describes the isolation and characterisation of the C. albicans NRG1 gene, which encodes a repressor of filamentous growth in this pathogenic fiingus. A C. albicans SBP1 cDNA was previously isolated in a screen for transacting factors that bind to a STRE-like element (consensus sequence: CCCCT) (Leng, 1999). In S. cerevisiae, STRE is a stress-responsive element that is required for the regulation of many stress-responsive genes (Marchler et al., 1993). In C. albicans, this element had been identified in the promoters of two hypha-specific genes, ALS8 and HYR1. Since many conditions that induce yeast-hypha morphogenesis in C. albicans impose a stress, it was proposed that the STRE- binding protein (Sbpl) might influence yeast-hypha morphogenesis and/or stress responses in this human pathogen. The cDNA was then used to isolate the complete C. albicans SBP1 locus by colony hybridisation. Both the cDNA and gene were sequenced, revealing an ORF capable of encoding a protein of 310 amino acids containing a C2H2-zinc finger motifs near its C-terminus. The zinc finger region of this protein displayed the highest sequence similarity to S. cerevisiae NRG1 (67 % identity), and hence the gene was renamed CaNRGl. To examine the role of CaNrgl, a C. albicans nrgl/nrgl null mutant and a mutant over-expressing the NRG1 gene were created. Overexpression of NRG1 did not reveal any obvious phenotypes, but inactivation of NRG1 caused constitutive filamentous and invasive growth, as well as increased sensitivity to some stresses. Also, the expression of the hypha-specific genes, ALS8, ECE1, HWP1 and HYR1, was derepressed in the nrgl/nrgl mutants. Similar phenotypes were observed for a C. albicans tupl/tupl null mutant. These observations suggest that Nrgl represses filamentous growth in C. albicans, possibly by recruiting Tupl to specific promoters. Unlike the tupl/tupl mutant, nrgl/nrgl cells formed normal hyphae following pH and serum stimulation, they generated chlamydospores at normal rates, and they grew at 42 C. Transcript profiling of 2002 C. albicans genes revealed that Nrgl regulates a subset of Tupl-repressed genes, which includes known hypha- specific genes and some virulence factors. The data also showed that Tupl regulates other genes, which are not regulated by Nrgl, including glucose sensitive genes, amino acid and sterol biosynthesis genes, and genes encoding other virulence determinants. Taken together, this study demonstrates that Nrgl is a transcriptional repressor that regulates a set of functions required for yeast-hypha morphogenesis and virulence in C. albicans.

The ability to respond to stress is universal in all domains of life. Failure to properly execute the stress response compromises the fitness of the organism. Several key stress pathways are conserved from unicellular organisms to higher eukaryotes, so knowledge of how these pathways operate in model organisms is crucial for understanding stress-related diseases and aging in humans. The mechanisms of stress tolerance have been well-studied in the budding yeast Saccharomyces cerevisiae. Yeast respond to diverse stresses by initiating both general and stress-specific responses that generally protect the cells during and after the stress exposure. While previous work has revealed mechanistic insights on adaptation and survival under mild and long-term exposure to stress, how they cope with acute exposure to lethal stress is not well understood. Here, we combined transcriptional profiling, fitness profiling, and laboratory evolution to investigate how S. cerevisiae survive acute exposure to lethal ethanol stress. By using high throughput methods such as RNA-seq and barcode sequencing of the pooled yeast deletion library, we were able to discover and characterize both existing and novel pathways that yeast utilize to adapt to and survive ethanol stress. We found both ethanol-specific and as well general stress response mechanisms. We were also able to evolve a strain of ethanol under lethal ethanol stress to exhibit a survival of at least an order of magnitude greater than the parental wild-type strain. Additionally, this evolved strain exhibited cross protection to other stresses without compromising bulk growth rate. We found that this strain adapted its global expression levels to a post-stress state, making it more robust to various stresses even under optimal growth conditions.

Malgré son potentiel, l’hétérosis a rarement été étudié, et encore moins exploité, chez les levures, espèces d’intérêt biotechnologique majeur. Ce travail avait pour objectif d’explorer ce phénomène chez deux espèces de levure, Saccharomyces cerevisiae et S. uvarum, dans des conditions proches de celles de l’œnologie. Pour la première fois des hybrides interspécifiques ont été inclus dans un dispositif diallèle complet. Un autre aspect original de ce travail résidait dans l’approche intégrative choisie, qui combinait l’étude de phénotypes aux niveaux métabolique, cellulaire et populationnel. Un panel de 66 souches (55 hybrides et leurs 11 parents) a été analysé pour 35 caractères à deux températures et avec trois réplicats, soit au total 396 fermentations alcooliques. Ces données nombreuses et complexes nous ont conduits non seulement à utiliser, mais aussi à développer divers outils statistiques et de modélisation originaux pour l’interprétation des données. Après avoir vérifié que les interactions nucléo-cytoplasmiques n’influençaient pas la variation des caractères étudiés, nous avons tout d’abord montré que les sources de variation (effet souche, effet température et interactions souche*température) différaient selon les types de caractères. Nous avons ensuite comparé globalement les trois groupes d’hybrides : intraspécifiques S. cerevisiae*S. cerevisiae, intraspécifiques S. uvarum*S. uvarum et interspécifiques S. cerevisiae*S. uvarum, et avons observé que l’hybridation interspécifique pouvait engendrer des phénotypes présentant de meilleures aptitudes œnologiques et une homéostasie supérieure à celle des hybrides intraspécifiques. Ce dernier résultat pourrait expliquer que l’hybridation interspécifique soit si fréquente chez les levures naturelles et domestiquées. / Despite its biotechnological interest, heterosis has not commonly been studied or exploited in the yeast genus. This work aimed to explore this phenomenon within two yeast species well adapted to oenological conditions, Saccharomyces cerevisiae and S. uvarum. Eleven parental strains and their 55 intra- and inter-specific hybrids were phenotyped under enological conditions, at two temperatures in three replicates. A total of 396 alcoholic fermentations were characterized in depth through 35 phenotypic traits with original statistical and modeling tools. We first showed that, depending on the types of trait - kinetics parameters, life-history traits, enological parameters and aromas -, the sources of variation (strain, temperature and strain*temperature effects) differed in a large extent. Then we compared globally three groups of hybrids and their parents at two growth temperatures: intraspecific hybrids S. cerevisiae*S. cerevisiae, intraspecific hybrids S. uvarum*S. uvarum and interspecific hybrids S. cerevisiae*S. uvarum. We found that hybridization could generate multi-trait phenotypes with improved oenological performances and better homeostasis with respect to temperature. These results could explain why interspecific hybridization is so common in natural and domesticated yeast, and open the way to applications for wine-making.

Thesis advisor: Charles Hoffman / Cyclic adenosine monophosphate (cAMP) and the second messengers that modulate several biological processes are regulated by adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterases (PDEs). ACs and PDEs are comprised of superfamilies of enzymes that are viewed as druggable targets due to their many distinct biological roles and tissue-specific distribution. As such, small molecule regulators of ACs and PDEs are important as chemical probes to study the roles of individual ACs or PDEs and as potential therapeutics. In the past, our lab has expressed 15 mammalian PDE genes in S. pombe, replacing the endogenous Cgs2 PDE. High throughput screens for PDE inhibitors identified novel compounds that show relevant biological activity in mammalian cell culture assays. The aim of this thesis is to develop tools to understand the mechanism of interaction between key regulators of the cAMP pathway and small molecules. The current study is comprised of two parts. In the first part of this thesis, I developed a genetic screen that detected alleles whose proteins remain active in the presence of BC54 and was to confirm the effect of the PDE4BT407A mutation using cell-based assays and in vitro enzyme assays. In the second part of this thesis, I developed and carried out HTSs using a PKA-repressed GFP reporter that can identify compounds that reduce PKA activity, which would include PDE activators and AC or GNAS1 inhibitors. To date, I have identified three AC inhibitors that appear to act on several of the ten different mammalian ACs. To our knowledge, this is the first time a large HTS has identified AC inhibitors, where inhibition was assessed inside the cells. The findings in this thesis will be useful in the design of more effective PDE inhibitors and in the development of novel chemical probes for studying cAMP signaling in mammalian cells. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Charles Hoffman / Cyclic adenosine monophosphate (cAMP) and the second messengers that modulate several biological processes are regulated by adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterases (PDEs). ACs and PDEs are comprised of superfamilies of enzymes that are viewed as druggable targets due to their many distinct biological roles and tissue-specific distribution. As such, small molecule regulators of ACs and PDEs are important as chemical probes to study the roles of individual ACs or PDEs and as potential therapeutics. In the past, our lab has expressed 15 mammalian PDE genes in S. pombe, replacing the endogenous Cgs2 PDE. High throughput screens for PDE inhibitors identified novel compounds that show relevant biological activity in mammalian cell culture assays. The aim of this thesis is to develop tools to understand the mechanism of interaction between key regulators of the cAMP pathway and small molecules. The current study is comprised of two parts. In the first part of this thesis, I developed a genetic screen that detected alleles whose proteins remain active in the presence of BC54 and was to confirm the effect of the PDE4BT407A mutation using cell-based assays and in vitro enzyme assays. In the second part of this thesis, I developed and carried out HTSs using a PKA-repressed GFP reporter that can identify compounds that reduce PKA activity, which would include PDE activators and AC or GNAS1 inhibitors. To date, I have identified three AC inhibitors that appear to act on several of the ten different mammalian ACs. To our knowledge, this is the first time a large HTS has identified AC inhibitors, where inhibition was assessed inside the cells. The findings in this thesis will be useful in the design of more effective PDE inhibitors and in the development of novel chemical probes for studying cAMP signaling in mammalian cells. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Abhishek Chatterjee / Abstract Unnatural amino acid (UAA) incorporation is a powerful tool used by biochemists to discover the nature of protein structure and function. The evolution of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs enables site-specific incorporation of UAAs proteins inside of living cells. The goal of this study was to further expand the repertoire of genetically encoded unnatural amino acids in E. coli as well as eukaryotes. We first attempted to engineer an aaRS, previously evolved for p-borono-phenylalanine (pBoF), to specifically charge 3-acetyl-p-borono-phenylalanine (AcpBoF). A randomized library of the pBoF-specific synthetases was generated and it was subjected to established selection schemes in a bacterial host. This report also describes the development of a yeast-based selection system to alter the substrate specificity of bacterial leucyl-tRNA synthetase, for genetic code expansion in eukaryotes. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

No description available.

Current research efforts are focused on 'second generation biofuels', which includes biofuels produced from lignocellulosic material. Lignocellulosic material is primarily composed of cellulose, a glucose polymer, xylose rich hemicellulose and non-fermentable lignin. Saccharomyces cerevisiae is widely used on an industrial scale for the production of ethanol from glucose; however, native S. cerevisiae does not contain the genes required for fermentation of xylose into ethanol. Others have sequentially expressed trans-genes from xylose fermenting organisms to engineer strains of S. cerevisiae capable of fermenting this pentose. The goal of this thesis was to generate a single cassette of 9 genes which have been shown to ferment xylose and arabinose. The 17 kb DNA fragment harboring all the genes necessary was introduced into the yeast genome using one-step homologous recombination based transformation. Expression of this cassette was verified by demonstrating that the first and last genes on this cassette were transcribed. The modified strain exhibited xylose utilization under microaerobic fermentation conditions. Further genetic and process engineering methods may be employed to improve the yield. The experiments described here demonstrate that generating a functional cassette of pentose fermenting genes is still achievable.