Measuring chromosome-end fusions in fission yeast

Dissertation presented to obtain the Ph.D degree in Molecular Biology

Studies on the wine spoilage yeast Brettanomyces bruxellensis: 4-ethylphenol production and improved detection

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Fermentation of fruit juices by the osmotolerant yeast Candida magnoliae

This study focuses on the assessment of the fermentation conditions required to modulate the metabolic flux in the osmotolerant yeast Candida magnoliae and evaluate its potential to produce low-alcoholic and low-caloric fermented beverages. For that purpose, two strains, PYCC 2903 and PYCC 3191, were used and fermentation conditions as oxygenation, sugar concentration and the ratio of glucose to fructose were studied using synthetic culture media. Candida magnoliae PYCC 2903 was subsequently used to ferment real industrial fructose-rich substrates such as fruit juices. Sugar consumption profiles for C.magnoliae PYCC 2903 incubated aerobically in the presence of high fructose and glucose concentrations (15%, 10% and 5%) showed a selective utilization of fructose, denoting a preference for this sugar over glucose. The lower ratio between ethanol and sugar alcohols yield was obtained for both strains incubated under oxygen limitation simulating industrial fructose-rich substrates, confirming the ability of this yeast to direct fermentation towards alternative products. Enzymatic assays for hexokinase activity in terms of capacity and affinity for glucose and fructose were performed, aiming to elucidate its contribution to the fructophilic behaviour of this yeast. Enzymatic assays for both strains showed that the Vmax is two to threefold higher for fructose than for glucose but Km is also 10-20-fold higher for this sugar than for glucose. Hence, hexokinase kinetic properties do not explain fructophily in C.magnoliae. This indicates that fructose transport is probably determining in this respect, as observed for other fructophilic yeasts. Fruit juice fermentations with C.magnoliae PYCC 2903 revealed a potential for the production of beverages with interesting sensorial properties. Pear and peach fermentations exhibited the best results with the lowest ratio between ethanol and sugar alcohols yield and the most pleasant organoleptic features.

Evaluating the potential of yeast strains to produce added value products for the food and/or pharmaceutical industries

This study focus in the valorization of the apple pomace with the main goal of obtaining added value products. For that, hot compressed water technology was used for the extraction of phenolic compounds and hydrolysis of polysaccharides presents in the lignocellulosic structure of apple pomace to obtain simple sugars. The sugars have been utilized as alternative carbon source for growth, lipid accumulation and carotenoids production by five different yeast Yarrowia lipolytica, Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodosporidium babjevae and Rhodosporidium toruloides. Hydrolysis experiments were carried out with constant pressure of 100 bar, flow rate of 2mL/min and temperatures between 50°C and 250°C. The amount of total sugars present in apple pomace hydrolysates showed maximum values for the hydrolysis temperatures of 110°C and 190°C. In fact, these temperatures revealed the best results regarding the monosaccharides quantities. The amount of 5-HMF and furfural in each hydrolysate varied through the different temperatures. Maximum values for 5-HMF were obtained with 170°C, while furfural showed to be maximum at 210°C. Extraction of phenolic compounds were performed in simultaneously with hydrolysis reactions. Total phenolic compounds (TPC) increased along the temperature, however with small variations between 170°C and 250°C. Hydrolysates were then used as alternative carbon source to yeast growth. R. mucilaginosa shows the highest optical density, with the hydrolysate obtained at 130°C. Carotenoids produced by these yeast scored a total of 7.02μg carotenoids/g cell dry weight, while for the control assay, the same yeast scored 9.31μg caratonoides/g cell dry weight. β-carotene was quantified by HPLC, were 33% of the carotenoid production by R. mucilaginosa with hydrolysate as carbon source, corresponded to β-caroteno.

In vitro antifungal activities of leaf extracts of Lippia alba (Verbenaceae) against clinically important yeast species

Introduction There are few studies reporting the antifungal activities of Lippia alba extracts. Methods A broth microdilution assay was used to evaluate the antifungal effects of Lippia alba extracts against seven yeast species of Candida and Cryptococcus. The butanol fraction was investigated by gas chromatography-mass spectrometry. Results The butanol fraction showed the highest activity against Candida glabrata. The fraction also acted synergistically with itraconazole and fluconazole against C. glabrata. The dominant compounds in the butanol fraction were 2,2,5-trimethyl-3,4-hexanedione, 3,5-dimethyl-4-octanone and hexadecane. Conclusions The butanol fraction may be a good candidate in the search for new drugs from natural products with antifungal activity.

Contribution to drug discovery and development for tauopathies using yeast as a model

This work aimed to contribute to drug discovery and development (DDD) for tauopathies, while expanding our knowledge on this group of neurodegenerative disorders, including Alzheimer’s disease (AD). Using yeast, a recognized model for neurodegeneration studies, useful models were produced for the study of tau interaction with beta-amyloid (Aβ), both AD hallmark proteins. The characterization of these models suggests that these proteins co-localize and that Aβ1-42, which is toxic to yeast, is involved in tau40 phosphorylation (Ser396/404) via the GSK-3β yeast orthologue, whereas tau seems to facilitate Aβ1-42 oligomerization. The mapping of tau’s interactome in yeast, achieved with a tau toxicity enhancer screen using the yeast deletion collection, provided a novel framework, composed of 31 genes, to identify new mechanisms associated with tau pathology, as well as to identify new drug targets or biomarkers. This genomic screen also allowed to select the yeast strain mir1Δ-tau40 for development of a new GPSD2TM drug discovery screening system. A library of unique 138 marine bacteria extracts, obtained from the Mid-Atlantic Ridge hydrothermal vents, was screened with mir1Δ-tau40. Three extracts were identified as suppressors of tau toxicity and constitute good starting points for DDD programs. mir1Δ strain was sensitive to tau toxicity, relating tau pathology with mitochondrial function. SLC25A3, the human homologue of MIR1, codes for the mitochondrial phosphate carrier protein (PiC). Resorting to iRNA, SLC25A3 expression was silenced in human neuroglioma cells, as a first step towards the engineering of a neural model for replicating the results obtained in yeast. This model is essential to understand the mechanisms of tau toxicity at the mitochondrial level and to validate PiC as a relevant drug target. The set of DDD tools here presented will foster the development of innovative and efficacious therapies, urgently needed to cope with tau-related disorders of high human and social-economic impact.

The role of Pep4p, the vacuolar yeast protease ortholog of human cathepsin D, in mitochondria-dependent apoptosis

Doctoral Thesis (PhD Programm on Molecular and Environmental Biology)

The brewing yeast

The concept of brewing science is very recent when compared with the history of beer. It began with the microscopic observations of Louis Pasteur and evolved through the last century with improvements in engineering, microbiology, and instrumental analysis. However, the most profound insight into brewing processes only emerged in the past decades through the advances in molecular biology and genetic engineering. These techniques allowed scientists to not only affirm their experiences and past findings, but also to clarify a vast number of links between cellular structures and their role within the metabolic pathways in yeast. This chapter is therefore dedicated to the behavior of the brewing yeast during fermentation. The discussion puts together the recent findings in the core carbon and nitrogen metabolism of the model yeast Saccharomyces cerevisiae and their fermentation performance.

Toxicity induced by a metal mixture (Cd, Pb and Zn) in the yeast Pichia kudriavzevii: The role of oxidative stress

The present work aims to contribute for the elucidation of the role of oxidative stress in the toxicity associated with the exposure of Pichia kudriavzevii to multi-metals (Cd, Pb and Zn). Cells of the non-conventional yeast P. kudriavzevii exposed for 6 h to the action of multi-metals accumulated intracellular reactive oxygen species (ROS), evaluated through the oxidation of the probe 2,7-dichlorodihydrofluorescein diacetate. A progressive loss of membrane integrity (monitored using propidium iodide) was observed in multi-metal-treated cells. The triggering of intracellular ROS accumulation preceded the loss of membrane integrity. These results suggest that the disruption of membrane integrity can be attributed to the oxidative stress. The exposure of yeast cells to single metal showed that, under the concentrations tested, Pb was the metal responsible for the induction of the oxidative stress. Yeast cells coexposed to an antioxidant (ascorbic acid) and multi-metals did not accumulate intracellular ROS, but loss proliferation capacity. Together, the data obtained indicated that intracellular ROS accumulation contributed to metal toxicity, namely for the disruption of membrane integrity of the yeast P. kudriavzevii. It was proposed that Pb toxicity (the metal responsible for the toxic symptoms under the conditions tested) result from the combination of an ionic mechanism and the intracellular ROS accumulation.

Spéciation of chromium in a chromium enriched yeast.

The sample under investigation in this project is an experimental chromium enriched yeast used as a possible additive in animal foodstuff, which was produced by growing yeast in the presence of chromium (III) chloride. Chromium on its own in not biologically active but chromium in the form of chromium enriched yeast is biologically active. The objective of this project was to show the complete absence of chromium(VI) from the sample. A literature survey describing previous work carried out on the speciation of Cr(VI) has been carried out. The principal methods of detection of Cr(VI) used in this project are Polarography, G.F.A.A. Spectroscopy, U.V. Spectroscopy and H.P.L.C. For each of the above methods a calibration curve was obtained and each method was applied to the yeast extract. The H.P.L.C. and U.V. spectroscopic method are specific for Cr(VI) but polarography and G.F.A.A. spectroscopy measure total chromium. Tris-NaOH buffer has been investigated for the extraction of chromium(VT). Problems associated with air oxidation of Cr(III) in alkaline solution have identified and procedures described for the suppression of air oxidation. Procedures are described for the application of the extraction procedure to the yeast extract and for the determination of Cr(VI) in the extract. Procedures are also described for the preconcentration of Cr(VI) on a HPLC column and for the application to the yeast extract. The rate of reduction of Cr(VI) by ascorbic acid is investigated and found to be first order with respect to ascorbic acid concentration. The reduction capacity of the yeast is also investigated and it was found that in acid solution the yeast will reduce Cr(VI) but in neutral or basic solution the reduction capacity is diminished. Conclusions regarding the objectives of the project are drawn and suggestions for further work are given.

Influence of quantities of brewer yeast on the performance of Anastrepha obliqua wild females (Diptera, Tephritidae)

Using artificial solid diets, experiments were performed with Anastrepha obliqua (Macquart, 1835) wild females in order to verify the influence of different quantities of brewer yeast on the performance and compensation behavior to unbalanced diets ingestion. The observed parameters were egg production, ingestion, diet efficiency and survival in the reproductive phase. Results indicated that there was no compensatory ingestion to different quantities of yeast and that the diet with 12.5g of yeast provided the best performance. The absence of compensatory ingestion is discussed based on the yeast phagostimulation and on the costs involved in solid diets ingestion. The relation between the analyzed parameters and the protein quantities in the diet were discussed.

Shaping fission yeast with microtubules.

For cell morphogenesis, the cell must establish distinct spatial domains at specified locations at the cell surface. Here, we review the molecular mechanisms of cell polarity in the fission yeast Schizosaccharomyces pombe. These are simple rod-shaped cells that form cortical domains at cell tips for cell growth and at the cell middle for cytokinesis. In both cases, microtubule-based systems help to shape the cell by breaking symmetry, providing endogenous spatial cues to position these sites. The plus ends of dynamic microtubules deliver polarity factors to the cell tips, leading to local activation of the GTPase cdc42p and the actin assembly machinery. Microtubule bundles contribute to positioning the division plane through the nucleus and the cytokinesis factor mid1p. Recent advances illustrate how the spatial and temporal regulation of cell polarization integrates many elements, including historical landmarks, positive and negative controls, and competition between pathways.

Translocation of the yeast dolichol-phosphate-mannose synthase into microsomal membranes.

Dolichol-phosphate-mannose synthase catalyzes the formation of Dolichol-phosphate-mannose from Dolichol-phosphate and GDP-mannose. Analysis of the primary amino acid sequence of the yeast enzyme predicts a luminal orientation of the enzyme in the endoplasmic reticulum. We analysed the translocation of the Dolichol-phosphate-mannose synthase into dog pancreatic microsomal membranes: resistance to proteolytic attack provides evidence of its luminal orientation and asks for a reevaluation of the topology of the reaction.

A medium for inducing conversion of Histoplasma capsulatum var. capsulatum into its yeast-like form

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that, under special conditions, converts from its more common mycelial form to a yeast-like form. Achieving this conversion, however, has been problematical for researchers. The present study tested conversion rates in ten Histoplasma capsulatum var. capsulatum strains using seven culture media, four of wich were conventional and three novel. One of our novel media, MLGema, induced complete conversion, of two strains within five days of incubation at 35 degrees centigrades, and of all strains that eventually converted by the time of the second subculturing transfer, under defined experimental conditions. MLGema is also inexpensive and easy to produce.

The dynamics of yeast telomeres and silencing proteins through the cell cycle.

Genes integrated near the telomeres of budding yeast have a variegated pattern of gene repression that is mediated by the silent information regulatory proteins Sir2p, Sir3p, and Sir4p. Immunolocalization and fluorescence in situ hybridization (FISH) reveal 6-10 perinuclear foci in which silencing proteins and subtelomeric sequences colocalize, suggesting that these are sites of Sir-mediated repression. Telomeres lacking subtelomeric repeat elements and the silent mating locus, HML, also localize to the periphery of the nucleus. Conditions that disrupt telomere proximal repression disrupt the focal staining pattern of Sir proteins, but not necessarily the localization of telomeric DNA. To monitor the telomere-associated pools of heterochromatin-binding proteins (Sir and Rap1 proteins) during mitotic cell division, we have performed immunofluorescence and telomeric FISH on populations of yeast cells synchronously traversing the cell cycle. We observe a partial release of Rap1p from telomeres in late G2/M, although telomeres appear to stay clustered during G2-phase and throughout mitosis. A partial release of Sir3p and Sir4p during mitosis also occurs. This is not observed upon HU arrest, although other types of DNA damage cause a dramatic relocalization of Sir and Rap1 proteins. The observed cell cycle dynamics were confirmed by direct epifluorescence of a GFP-Rap1p fusion. Using live GFP fluorescence we show that the diffuse mitotic distribution of GFP-Rap1p is restored to the interphase pattern of foci in early G1-phase.