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Selective isolation protocols of selenium (Se) species integrated to Se specific atomic absorption spectroscopy (AAS) detection were developed and optimized for Se speciation in food supplements, including selenized yeasts. By ultrafiltration, 69.18% of Se in the extract was found as a low molecular weight soluble form, the remaining 30.82% was bound to high molecular weight components. After a cation-exchange chromatography of the ultrafiltrate, 3.77% of the Se in the extract was found in the aqueous washings of the column indicating the presence of free inorganic anions of Se; the 65.41% of Se retained on the column corresponded to the free organic Se cations. The limit of detection for the HPLC-THG-AAS system was 1.85 ng of Se. Se was shown to be widely distributed over all the proteins with one sharp peak corresponding to the free forms of Se. Four major peaks were found at MW $>$ 250 000 Da (15.97% of Se recovered), between 102 330 and 117 490 Da (7.06%), between 48 977 and 53 703 Da (12.71%) and close to the dye migration band (17.25%). / Selective isolation and HPLC-AAS protocols were also developed and optimized for the determination of free organic forms e.g. selenomethionine (SeMet), selenocystine (SeCystine) and inorganic forms of selenium in aqueous solutions, and in complex matrices such as nutritional supplements and mixtures of free amino acids. The selenoamino acid in alkaline solution was first derivatized with 1-fluoro-2,4-dinitrobenzene. After removal of excess of reagent by partitioning with diethyl ether, the N-dinitrophenyl (DNP)-derivatized selenoamino acid was acidified and extracted with diethyl ether. Inorganic Se(IV) was extracted from the acidic aqueous phases by complexation with 1,2-phenylenediamine, forming a piazselenol. Se derivatives were determined selectively by HPLC-THG-AAS. A selective chromatographic mechanism based on $ pi$-electron interactions was optimized using a silica stationary phase derivatized with p-nitrophenyl moieties. Co-injections of DNP-SeMet, DNP-SeCystine and piazselenol save retention times of 3.7, 4.0 and 4.9 min, respectively, using a methanolic mobile phase containing 1.5% triethylamine and 0.013M acetic acid. Primary analytical validation parameters including stability, linearity and limits of detection were obtained using purified DNP-SeMet, DNP-SeCystine and piazselenol standards which were characterized by $ sp1$H-, $ sp{13}$C- and $ sp{77}$Se-NMR analysis and/or fast atom bombardment MS techniques. The calibration graphs for sequential dilutions of these Se standards were linear and the limits of detection from the resultant calibration graphs were 17 ng, 0.21 ng and 18.53 ng of Se, respectively. The purified DNP-SeMet and DNP-SeCystine were found to be photosensitive. The recovery of SeMet, SeCystine and inorganic Se from the stock solutions and/or nutritional supplements was virtually quantitative. In the presence of a 500-fold excess of other amino acids, the recovery of SeMet and SeCystine (96.1 $ pm$ 3.9% and 98.08 $ pm$ 4.2%, respec

The goal of my M.Sc. thesis research was to investigate the link between divergent adaptation and reproductive isolation in speciation, the process by which one species becomes two over time. Previous studies followed the evolution of 12 replicate experimental populations of the yeast Saccharomyces cerevisiae in either of two non-optimal environments, high-salt or low-glucose, over 500 generations. In this research, I re-sequenced the whole genomes of six diploid strains of these populations, three from high salt and three from low glucose. Among the replicates, there was remarkable degree of parallelism in the underlying determinants of adaptation including multiple mutations in PMA1, encoding the main proton exporter, in high-salt and in several genes in the ras signalling pathway in both environments. A variety of positive and negative epistatic interactions were found among mutations from within and among populations, emphasizing the importance of epistasis in adaptation and potentially in speciation.

S. cerevisiae have developed the ability to forage for nutrients when presented with conditions of starvation. This dimorphic adaptation is particularly noticeable when yeast are subject to nitrogen depravation and has been termed filamentous growth, as cells form filament-like projections away from the center of the colony. The regulation of this response is under the control of the well-characterized MAPK and cAMP pathways. Previous work showed that Cdk8p phosphorylated a key transcriptional activator of the filamentous response, Ste12p, and subsequently targeted the factor for degradation under conditions of limiting nitrogen. Data presented in this thesis suggests that Cdk8p is regulated by another kinase, Tpk2p. In vitro kinase assays demonstrate that Tpk2p directly phosphorylates Cdk8p on residue Thr37, leading to the destabilization of Cdk8p after growth for 4 hours in SLAD media. Lack of phosphorylation on Thr37 yields a hypo-hypofilamentous phenotype, whereas a phospho-mimic mutant, T37E displays a filamentous hyper-filamentous phenotype.

Successfully inducing malolactic fermentation in the production of grape wines can be challenging, especially in wines after finishing alcoholic fermentation with limited energy sources, low pH values and high ethanol concentrations. In this thesis, the kinetics of several chemicals of enological relevance were studied in a white wine (Chardonnay) and a red wine (Cab Franc) vinified by traditional, consecutive alcoholic (AF) and malolactic fermentations (MLF), and simultaneous AF/MLF, where bacteria were co-inoculated with yeast. The Chardonnay must was adjusted to four pH values (3.20, 3.35, 3.50 or 3.65), the cab Franc was kept as original pH value (3.56) and the concentrations of sugars, organic acids as well as acetaldehyde were followed throughout the fermentations. For Chardonnay the degradation of glucose and fructose was slower at the lowest must pH value (3.20) and independent from the time of bacterial inoculation. In all cases, malolactic conversion was faster after yeast-bacterial co-inoculation and was completed in simultaneous treatments at pH values of 3.35-3.65, and consecutive treatments at pH 3.50 and 3.65. No statistically significant difference was observed among the final acetic acid concentration, in all inoculation and pH treatments. For Cab Franc, it confirmed that co-inoculation shortened the fermentation periods while having minor effects on other parameters. Overall, simultaneous AF/MLF allowed for greatly reduced fermentation times, while the must pH remained a strong factor for fermentation success and determined the final concentration of various wine components. The time of inoculation influenced formation and degradation kinetics of organic acids and acetaldehyde significantly.

The budding yeast, Saccharomyces cerevisiae, serves as an excellent model for identifying fundamental mechanisms of DNA repair. A Local Coherence Detection (LCD) algorithm that uses biclustering to assign genes to multiple functional sub-groups was applied on the chromosome E-MAP containing genetic interactions among genes involved in nuclear processes. Using this method, we found that Asf1 and Rtt109, genes that are together required for histone H3K56 acetylation, cluster together with Ctf4, Ctf18, Ctf8 and Dcc1, genes important for efficient sister chromatid cohesion. It is known that H3K56 acetylation is required for post-repair chromatin reassembly at sites of DNA double-strand breaks (DSBs). The cohesion genes were previously implicated in the repair of some DNA DSBs, but the nature of their involvement has not been reported. The experimental data in my thesis work suggest that Ctf4, Ctf8, Ctf18 and Dcc1 function in the post-repair chromatin reassembly pathway.

Organelles within the endomembrane system of all eukaryotic cells exchange membrane lipids and proteins using membrane-bound transport vesicles. This highly conserved vesicular transport process is essential for life and is highly regulated. Much of this regulation is provided by small monomeric GTP-binding proteins such as Arf and Arl that act as molecular switches, cycling between inactive GDP-bound and active GTP-bound states. This cycle of GTP binding and hydrolysis is controlled by guanine-nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. I have investigated regulatory interactions involving two ArfGAPs, Age1 and Gcs1, involved in post-Golgi vesicular transport in the budding yeast Saccharomyces cerevisiae. In yeast, the Age2 + Gcs1 ArfGAP pair is essential and facilitates post-Golgi transport. I found that overexpression of either the poorly characterized ArfGAP Age1 or the Sfh2 phosphatidylinositol-transfer protein can bypass the requirement for Age2 and Gcs1. Indeed, endogenous Age1 is required for efficient Sfh2-bypass. Moreover, the yeast phospholipase D protein, Spo14, which is activated by Sfh2 and regulates membrane lipid composition, is required for Age1 to effectively alleviate the deleterious effects of defective Age2 + Gcs1 function. My findings suggest that Age1 is regulated by membrane lipid composition and can provide ArfGAP function for post-Golgi transport. Gcs1 is involved in multiple vesicular transport stages, is a dual-specificity GAP for both Arf and Arl1 proteins and, as shown here, also has functions independent of its GAP activity. The absence of Gcs1 causes cold sensitivity for growth and endocytic transport. The cold sensitivity of cells lacking Gcs1 is alleviated by the elimination of either the Arl1 or Ypt6 vesicle-tethering pathway at the trans-Golgi, or by overexpression of Imh1, an effector of the Arl1 pathway. I found elimination of the Ypt6 pathway also prevents Arl1 activation and membrane localization, that Arl1 binding by Imh1 is necessary and sufficient for alleviation, and that the Gcs1 function required for growth and transport in the cold is independent of any GAP activity. My findings suggest that in the absence of this GAP-independent function of Gcs1 the resulting dysregulated Arl1 causes the gcs1? defects through the sequestration of a yet-to-be-determined cellular factor.

In Saccharomyces cerevisiae, DNA replication initiates at distinct origins termed Autonomously Replicating Sequences (ARSs). A key element in ARS is the ACS/B1 sequence, which binds the Origin Recognition Complex (ORC). During early G1 phase, the pre-replication complex is assembled by ORC. These ARSs are termed replicators. In yeast, ORC is also involved in gene silencing. These loci also contain an ACS/B1 element; however at these positions ORC recruits the silencing SIR complex. ARSs found here are termed silencers. Therefore, ARSs have a dual function. Research has also shown that origin ARSs can be substituted for silencing ARSs and vice versa. Since a mutation in ACS abolishes replication activity, studies have turned to the B1 element to account for the functional duality, but results have been mixed. I hypothesize that the B1 element plays a key role in the dual nature of ARS. To test this hypothesis, silencer and replicator ARSs were subjected to site directed mutagenesis around the WTW motif of the B1 element. Their efficiency was then tested using routine silencing and replication assays. Results reveal that the silencing ability of silencer ARSs is unaffected by these mutations; however mutations within and around the WTW motif reduce silencing efficiency of replicator ARSs. Sequence alignments have also shown that silencer ARSs have a broader WTW consensus than replicator ARSs. Preliminary replication assays are consistent with the above results and other research, contributing to a conclusion that the B1 element is not the sole determining factor in the dual nature of ARS. / NSERC

Saccharomyces cerevisiae is confronted with continually changing and diverse nutrient conditions. As such, it has adapted to utilize both a wide variety of nutrient sources and to preferentially use the most nutrient-rich source to obtain a competitive advantage. The transcription, intracellular trafficking and protein turnover of nutrient transporters, including the hexose transporter proteins (Hxts), are strictly regulated in response to nutrient conditions. The low affinity hexose transporter Hxt3p is highly expressed and localizes to the plasma membrane during growth in abundant glucose where it plays a major role in the transport of this sugar. However, following a shift to ethanol as a sole carbon source, Hxt3p is endocytosed and targeted to the vacuole for degradation while its expression is also repressed. In contrast, the high affinity hexose transporter Hxt7p is actively expressed and functional in the plasma membrane when glucose is limiting and nitrogen is abundant. Upon nitrogen starvation or rapamycin treatment, HXT7 transcription decreases and the protein is targeted for degradation. The mechanisms that govern these regulatory steps are poorly understood. The Vid and Gid proteins, several of which compose the Vid30 complex (Vid30c), facilitate the nutrient-dependent degradation of the gluconeogenic enzymes FBPase and Mdh2p when glucose-starved cells are replenished with glucose. Here we show that components of the Vid30c are needed for the ethanol-induced turnover of Hxt3p and the rapamycin or nitrogen starvation-induced degradation of Hxt7p. In addition, we demonstrate that the signals for the ethanol-induced turnover of Hxt3p and the rapamycin-induced turnover of Hxt7p converge on the Vid30c upstream of the Ras/cAMP/PKA pathway, ultimately controlling the degradation of both these hexose transporters. Finally, we provide evidence that the Vid30c controls the localization of the Ras GEF Cdc25p and may therefore directly regulate the activity of the Ras/cAMP/PKA pathway.

Pretreatment of recalcitrant lignocellulosic biomass to release sugars for bioconversion into ethanol produces fermentation inhibitors. Increasing yeast inhibitor tolerance should reduce production time and cost. UV mutagenesis followed by genome shuffling using cross mating was performed on Scheffersomyces stipitis strain GS301, a genome shuffled strain with increased tolerance to spent sulphite liquor (SSL). The main fermentation inhibitors in SSL are acetic acid, hydroxymethylfurfural (HMF), and various phenolics. UV mutagenesis resulted in acetic acid tolerant mutants, but they were phenotypically unstable. However, two rounds of UV mutagenesis followed by five rounds of genome shuffling resulted in strains EVB105, EVB205 and EVB505 with increased SSL tolerance and improved acetic acid and HMF tolerance. When fermenting undiluted SSL at pH 5.5, the three strains utilized sugars faster producing higher maximum ethanol than GS301. This study demonstrates that UV mutagenesis with genome shuffling can significantly improve inhibitor tolerance and fermentation performance of yeast. / NSERC Bioconversion Network