Spatiotemporal mechanisms for actomyosin ring assembly and contraction in budding yeast cell division

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

Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes

Clin Sci (Lond). 2002 Nov;103(5):475-85

The Role of Actin-Capping Protein and Src signalling in tissue growth and apoptosis during Drosophila wing development

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

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.

IL-17A promotes intracellular growth of Mycobacterium by inhibiting apoptosis of infected macrophages

The fate of infected macrophages is a critical aspect of immunity to mycobacteria. By depriving the pathogen of its intracellular niche, apoptotic death of the infected macrophage has been shown to be an important mechanism to control bacterial growth. Here, we show that IL-17 inhibits apoptosis of Mycobacterium bovis BCG- or Mycobacterium tuberculosis-infected macrophages thus hampering their ability to control bacterial growth. Mechanistically, we show that IL-17 inhibits p53, and impacts on the intrinsic apoptotic pathway, by increasing the Bcl2 and decreasing Bax expression, decreasing cytochrome c release from the mitochondria, and inhibiting caspase-3 activation. The same effect of IL-17 was observed in infected macrophages upon blockade of p53 nuclear translocation. These results reveal a previously unappreciated role for the IL-17/p53 axis in the regulation of mycobacteria-induced apoptosis and can have important implications in a broad spectrum of diseases where apoptosis of the infected cell is an important host defense mechanism.

Fluorescence-based approaches towards the assessment of structural and functional alterations in lysosomes and vacuoles induced by lactoferrin and acetic acid

Dissertação de mestrado em Biofísica e Bionanossistemas

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-d pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

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.

Análisis de componentes del sistema inmune innato en la infección VIH/SIDA: Apoptosis y expresión de moléculas y receptores en Polimorfonucleares Neutrófilos

El Virus de la Inmunodeficiencia Humana (VIH) afecta principalmente a la respuesta inmune específica causando una pérdida progresiva de los linfocitos T CD4+. Este virus también puede afectar a células del sistema inmune innato, como los Polimorfonucleares Neutrófilos (PMN). Los objetivos propuestos para esta etapa del proyecto son: a) investigar el efecto de la infección por VIH sobre la apoptosis de PMN, b) analizar la expresión de moléculas y receptores de reconocimiento de patrones moleculares asociados a patógenos en estas células y c) evaluar el impacto de la terapia antirretroviral sobre la apoptosis y expresión de moléculas y receptores en PMN. Se incluirán individuos en distintos estadios de la infección con o sin tratamiento antirretroviral y se determinarán parámetros hematológicos, inmunológicos y virológicos a fin de correlacionar el nivel de apoptosis y expresión de moléculas y receptores con el nivel de linfocitos T CD4+ y carga viral. La importancia de los PMN en el control de la infección por el VIH es actualmente un área de mucho interés, ya pueden ejercer un efecto anti-VIH directo, y al mismo tiempo, ser blancos de la infección viral. Comprender los aspectos claves en la cascada de la apoptosis de estas células podría en un futuro aportar posibles blancos terapéuticos, que permitan restaurar la función de los PMN durante la infección VIH/SIDA.