At the onset of cell division microtubules growing from spindle pole bodies (SPB) interact with each other to form the mitotic spindle enabling proper chromosome positioning and segregation. However, the exact mechanism of microtubule dynamics and microtubule associated proteins (MAPs) underlying spindle assembly is still not well understood. We developed an in vivo method to observe spindle assembly in the fission yeast Schizosaccharomyces pombe by inducing depolymerization of already formed and grown spindles by subjecting the cells to low temperatures, followed by subsequent repolymerization at a permissive temperature. We observed that microtubules pivot, i.e., perform angular movement around the SPB in a random manner, exploring the intranuclear space. Eventually microtubules extending from opposite SPBs come into contact and establish an antiparallel connection thus reassembling the spindle. Mutant approaches revealed that deletion of ase1 and klp5 did not prevent spindle reassembly, however introduced aberrations during the spindle formation. Amazingly, cut7p showed direct colocalization with microtubule overlap during spindle reassembly. Abrogation of cut7p led to inability to form a functional spindle. Thus, cut7p is the main regulator of spindle formation in fission yeast. None of the mutant strains affected microtubule pivoting, confirming that microtubule pivoting is a random movement unrelated to MAPs.

The operating principles of complex regulatory networks are more easily understood with mathematical modelling than by intuitive reasoning. In this thesis, I study the dynamics of the mitotic exit control system in budding yeast. I present a comprehensive mathematical model, which provides a system’s-level understanding of the mitotic exit process. This model captures the dynamics of classic experimental situations reported in the literature, and overcomes a number of limitations present in previous models. Analysis of the model led to a number of breakthroughs in the understanding of mitotic exit control. Firstly, numerical analysis of the model quantified the dependence of mitotic exit on the proteolytic and non-proteolytic functions of separase. It was shown that the requirement for the non-proteolytic function of separase depends on cyclin-dependent kinase activity. Secondly, APC/Cdc20 is a critical node that controls the phosphatase (Cdc14) branch and both cyclin (Clb2 and Clb5) branches of the cell cycle regulatory network. Thirdly, the model proved to be a useful tool for the systematic analysis of the recently discovered phenomenon of Cdc14 endocycles. Most proteins belonging to the cell cycle control network are regulated at the level of synthesis, degradation and activity. Presumably, these multiple layers of regulation facilitate robust cell cycle behaviour in the face of genetic and environmental perturbations. To falsify this hypothesis, I subjected the model to global parameter perturbations and tested viability against pre-defined criteria. According to these analyses, the regulated transcription and degradation of proteins make different contributions to cell cycle control. Regulated degradation confers cell cycle oscillations with robustness against perturbations, while regulated transcription plays a major role in controlling the period of these oscillations. Both regulated transcription and degradation are part of important feedback loops, that combined promote robust behaviour in the face of parametric variations.

The use of antibiotic growth promoters has been limited all around the world because of the concerns about antibiotic resistant bacteria and the presence of antibiotic residues in poultry products. Yeast-derived products are rich sources of ß1,3-1,6-glucan, mannan polysaccharides, and nucleotides and are considered as possible antibiotic alternatives due to their potential intestinal health benefits, growth promotion, and immune system stimulation. The objectives of the current research were: (1) to the evaluate effect of yeast products derived from yeast Saccharomyces cerevisiae on growth performance, gut histomorphology, and innate immune response of broiler chickens; (2) to investigate the effect of yeast products, including distillers dried grains with solubles (DDGS), on innate and antibody-mediated immune response following immunization with different antigens; and (3) to examine the effect of yeast-derived products and DDGS on growth performance, incidence of necrotic enteritis (NE), and local innate immunity in broiler chickens challenged with Clostridium perfringens. Overall, supplementation of diets with yeast products did not affect growth performance of broilers. However, the diets containing yeast cell walls (YCW) and nucleotides increased the villus height in the jejunum and enhanced the number of goblet cells in the ileum. Inclusion of diets with yeast products did not activate the innate immune response of birds under non-pathogen challenge conditions. However, the diet containing YCW activated Th2 cell-mediated immune response in birds immunized with sheep red blood cells and bovine serum albumin. Furthermore, supplementation of diets with YCW and DDGS in birds challenged with Escherichia coli lipopolysaccharide, activated the systemic innate immune response. Regarding antibody-mediated immune response, when compared to the control, serum antibody titer and specific antibody response against different antigens were not affected by dietary treatments. In the C. perfringens challenge study, growth performance, NE lesions and C. perfringens counts in the intestine were not affected by yeast-derived products. However, diets containing YCW and nucleotides stimulated the local innate immune response of birds by upregulation of cytokines and receptors involved in innate immunity. Such findings suggest that the immune-adjuvant like properties of YCW and nucleotides activate the innate immunity of broiler chickens following immunization or challenge with different antigens. / October 2015

In a minimal medium supplemented with glucose and yeast extract, the optimum pH for the growth of C. poinsettias was found to be 7.5. The organism requires thiamine, biotin, and pantothenic acid for growth. No absolute requirement was found for any amino acid, purine or pyrimidine although an amino acid mixture was stimulatory. Casamino acids could be substituted for the synthetic amino acid mixture. Yeast extract provided an additional factor(s) necessary for maximal growth. The results suggest that the unknown factor found in yeast extract might be purified by a combination of solvent extraction, and adsorption and elution from charcoal.

Animals, fungi, and amoebas require an actomyosin contractile ring at the division site to perform cytokinesis. The contractile ring initiates and guides the invagination of the plasma membrane as it forms new barriers between the nuclei at the cell equator. Defects in the contractile ring can result in misdirected, delayed, or premature cytokinesis, which leads to abnormal chromosome numbers. Aneuploidies resulting from failed cytokinesis sometimes lead to aggressive forms of cancer. This dissertation was motivated by the goal of better understanding the properties of the contractile ring and how it drives cytokinesis. Actomyosin is initially recruited to the cell equator through the coordination of scaffolding factors, actin-binding proteins, and signaling cascades. Subsequently, the sliding of actin filaments by myosin reshapes the resulting meshwork into a compact ring. Once fully assembled, the contractile ring establishes tension, which leads the plasma membrane inward. The primary motor proteins in the contractile ring of animal cells are class-II nonmuscle myosins, which typically function as bipolar filaments. Filament assembly is activated by phosphorylation and plays a central role in myosin function during cytokinesis. However, many underlying processes that regulate contractile ring function are poorly understood. Current models of cytokinesis have been based on mechanistic insights provided by two decades of work in the fission yeast system Schizosaccharomyces pombe. In fission yeast, the class-II myosin Myo2 provides the major source of motor activity in the contractile ring. Myo2 is two-headed and has a rod-like tail, which is consistent with other class-II myosins. Yet, it was unknown whether Myo2 assembles into filaments, or how phosphorylation affects its activity. To investigate these features, recombinant Myo2 was purified from the baculovirus/Sf9 insect cell expression system. Hydrodynamic measurements were used to examine whether Myo2 forms filaments. These sedimentation velocity data gave no indication that Myo2 self-assembles under the typical physiological salt concentrations, which suggests that Myo2 is unlike any class-II myosin known to date. Myo2 was also treated in vitro with its native kinase Pak1. Phosphorylation of Myo2 molecules had no effect on self-assembly, however it reduced actin-binding in motility assays and increased steady-state ATPase rates by two fold. Our results imply that the function and regulation of fission yeast Myo2 during cytokinesis depends on a specific scaffolding scheme at the plasma membrane, which has not been observed in other eukaryotes. Another interest of this dissertation was how the contractile ring is regulated during cytokinesis. We examined one cytokinesis protein, Cyk3, believed to mediate between the ring and extracellular processes. Genetics and live cell imaging analyses indicated that Cyk3 functions through a catalytically-inactive enzyme domain, which implicated Cyk3's involvement in one of the primary cytokinesis signaling pathways. This dissertation sheds new light on core aspects of how fission yeast undergo cytokinesis, especially with respect to the mechanism of Myo2 activity in the contractile ring. Characterizing the physical and enzymatic properties of an essential myosin in a simple organism should provide insights into cytokinesis in higher organisms.

Orientador: Aulus Cavalieri Carciofi / Resumo: Derivados da parede celular de levedura têm sido estudados por seu efeito prebiótico. Recentemente, preparações purificadas e mais solúveis foram desenvolvidas, com vista a aumentar seu efeito fisiológico. Este estudo teve por objetivo avaliar os efeitos da inclusão de dois diferentes extratos da parede celular de leveduras, um convencional (PCL) e outro com maior porcentagem de mananoligossacarídeos solúveis (PCLs) sobre a digestibilidade, produtos de fermentação nas fezes e alguns parâmetros imunológicos de cães adultos hígidos. Foi empregada uma única formulação, desdobrada em três tratamentos: CON – controle, sem adição de parede celular de levedura; PCL – adição de 0,3% de parede celular de levedura convencional; PCLs - adição de 0,3% de parede celular de levedura com elevada solubilidade de mananoligossacarídeos (MOS). Foram utilizados 24 cães beagle adultos, com oito repetições por ração, em delineamento em blocos casualizados. Cada bloco teve duração 30 dias, sendo avaliados no início e no final do período as concentrações séricas de fator de necrose tumoral alfa e interleucinas 6 e 10, produção ex vivo de intermediários reativos do oxigênio e nitrogênio por células mononucleares e polimorfonucleares de sangue periférico, capacidade fagocítica de monócitos e neutrófilos e concentração de IgA nas fezes. Adicionalmente, foi avaliada a digestiblidade aparente dos nutrientes, produção e qualidade das fezes, ácidos graxos de cadeia curta e ramificada, lactato, amônia, pH e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Derivates of yeast cell wall have been studied by its prebiotic effect. Recently, more purified and soluble preparations were developed, attempting to increase their biological actions. This study evaluated the inclusion of two yeast cell wall preparations, one conventional (YCW), and another with higher solubility of the mannan oligosaccharide fraction (YCWs), on their effects on nutrient digestibility, fermentation products on feces, and some immunological parameters of dogs. A single food formulation was used, unfolded on the following treatments: CON – control, without yeast cell wall addition; YCW – addition of 0.3% of a conventional yeas cell wall extract; YCWs – addition of 0.3% of a yeast cell wall extraction with elevated mannan oligosaccharides solubility. Twenty-four adult beagle dogs were used, eight dogs per food, distributed on a randomized block design. Blocks lasted 30 days, and tumor necrosis factor alpha and interleukins 6 and 10, ex vivo production of hydrogen peroxide and nitric oxide by peripheral neutrophils and monocytes, phagocytic index, and fecal IgA were evaluated at the beginning and ending of each period. Additionally, nutrient digestibility, feces production and quality, and fermentation products, pH, and biogenic amines were quantified on feces. Results were evaluated by variance analysis and compared by Tukey test (P<0.05). For the immunological parameters, the basal values were used as a covariate. The inclusion of YCWs reduced fat digestibili... (Complete abstract click electronic access below) / Mestre

>Magister Scientiae - MSc / Bioethanol is currently the most widely used biofuel and can be used as a direct replacement for current fossil fuel based transportation fuels. Consolidated bioprocessing (CBP), in which bioethanol is produced in a single reactor by a single microorganism, is a cost-effective way of producing bioethanol in a second generation process using lignocellulosic biomass as feedstock. The yeast Saccharomyces cerevisiae possesses industrially desirable traits for ethanol production and is able to produce heterologous cellulases, which are required for CBP. However, S. cerevisiae produces low titres of cellulases and one suspected reason for this is the stress caused by the heterologous proteins that induce the unfolded protein response (UPR). The UPR is a stress response pathway that will either lead to increased folding capacity within the ER or to degradation of these proteins and possibly apoptosis of the cell. It is thus beneficial to be able to determine when and to what extent the UPR is active during CBP organism development. Current methods of measuring the UPR include RNA and reverse transcriptase qPCR (r.t.qPCR) measurements, which can be cumbersome and expensive. The purpose of this study was to develop a vector based biosensor that will detect and quantify UPR activation. The vector consisted of either the T.r.xyn2 or eGFP reporter genes under the control of the S. cerevisiae HAC1p or KAR2p promoters. HAC1 and KAR2 are important regulators of UPR as their activation allows the UPR to achieve its function. The eGFP reporter under the transcriptional control of KAR2p was shown to be the superior combination due to the improved dynamic range when the UPR was induced in transformed S. cerevisiae strains by the stress inducer, tunicamycin. This UPR biosensor also proved to be more sensitive when measuring UPR induction in cellulase producing strains, depicting significant differences, compared to previous r.t.qPCR based tests which were unable to detect these differences. We thus developed a UPR biosensor that has greater sensitivity for changes in UPR induction compared to RNA based methods and the first KAR2p based UPR biosensor plasmid that allowed for more accurate detection and measurement of the UPR in cellulase secreting S. cerevisiae strains. The ability to quantify UPR induction will assist in identifying candidate cellulase genes that do not greatly induce the UPR, making them ideal to use in developing CBP yeasts.

Communication is a ubiquitous component of life. While complexity and sophistication vary, both unicellular and multicellular organisms constantly interact with their environment. Unicellular organisms, once thought to be asocial, have since been demonstrated to display a multitude of social interactions and hierarchies. For example, quorum sensing enables a bacterial population to modulate gene expression in response to cell-population density, initiating social behavior and the exchange of resources. In eukaryotes, unicellular ascomycete fungi use mating GPCRs to detect secreted peptide pheromones, initiating changes in gene expression required for mating. An overview of communication in unicellular organisms is presented in Chapter 1. In general, these communication systems are characterized by a high degree of fidelity, and as such have been harvested by synthetic biologists to organize communication in synthetic systems. Quorum sensing modules have been employed for pattern formation and to coordinate biosynthesis processes across a community. However, fungal mating remains underutilized as a source of synthetic biology tools. In this dissertation, we leverage fungal mating G-protein-coupled receptors (GPCRs) and their peptide ligands to build responsive yeast systems. We use genome-mining to identify additional fungal peptide-GPCR pairs, which are then characterized in the yeast Saccharomyces cerevisiae. In Chapter 2, we exploit the high specificity and sensitivity of fungal mating GPCRs to design a yeast whole-cell biosensor that produces a visible output in response to detection of peptide biomarkers. In Chapter 3, we genome-mine additional peptide-GPCR pairs and use them as orthogonal signaling channels to build synthetic yeast communities. Finally, in Chapter 4, we use these synthetic yeast communities to provide sense-and-respond capabilities to an Engineered Living Material (ELM).

Master of Science - Molecular and Cell Biology / The bacterial contamination profile of a typical commercial yeast factory was assessed by three replicate microbiological surveys. In order to detect low-level contamination in samples, this study made use of a preliminary incubation technique (24h at 37°C), which boosted bacterial counts for the identification of sources of contamination. Numbers of bacteria were quantified by standard pour- and spread-plate techniques and various selective media. Raw materials were negligible in contributing to the bacterial contamination of commercial yeast, with the exception of soda ash, used to control the pH of fermentations, which contained 2 log CFU/ ml Enterococcus and aerobic bacteria. It was found that the scale up of seed yeast biomass was the primary site for contamination with Enterococcus, which progressively increased in number as the product passed down the production line. Coliforms were present at low levels, with significant increases (P < 0.05) observed during the storage of yeast cream; extrusion of compressed yeast; and packaging of dry yeast. The environment surrounding the compressed yeast production line was identified as a potential source of airborne contamination. Although Salmonella spp. and S. aureus were not detected, L. monocytogenes was isolated from compressed and dry yeast products. In addition, Bacillus spp. commonly associated with the rope-spoilage of bread, were isolated from 67% of all dry yeast product samples. Shelf-life investigations, showed that cream and compressed yeast samples were spoiled with lengthened storage periods, and especially at higher temperatures (>10°C), whilst vacuum-packaged dry yeast remained bacteriologically stable. During shelf-life studies, isolates from spoiled cream and compressed yeast samples were predominantly Lactobacillus (up to 78%), while populations of Enterococcaceae predominated in vacuum-packaged dry yeast samples (up to 68%). The use of stainless steel surfaces, attached to processing equipment used in the manufacturing of Baker’s compressed yeast, in conjunction with SEM illustrated the accumulation of yeast and bacterial cells with early stages of biofilm formation, with time. Where populations of Gram-positive members of the lactic acid bacteria family, Lactobacillus and Enterococcaceae, were isolated in the highest proportion from processing equipment surfaces used in the manufacturing of Baker’s compressed yeast (81-100%).

As leveduras são de relevante importância dentro da agroindústria sucroalcooleira devido sua participação no processo fermentativo de produção de álcool. Deste modo, faz-se necessário o conhecimento deste agente fermentativo com destaque para Saccharomyces cerevisiae, principal gênero. O objetivo deste trabalho foi estudar a linhagem de levedura S. cerevisiae Y904, exposta a condições não proliferantes, após fermentação em meio que sofreu adição de óleo vegetal e sua possível correlação com manutenção da viabilidade celular. Foram realizadas análises para contagem de unidades formadoras de colônias, viabilidade celular, concentração celular, nitrogênio total na levedura e no meio, carboidratos totais, trealose e glicogênio. As leveduras submetidas a condições não proliferantes apresentaram menores teores de carboidratos totais, com destaque para trealose e glicogênio, em relação às leveduras comerciais. Saccharomyces cerevisiae sofreu queda de viabilidade acentuada após 24 h em solução fisiológica, em condições não proliferantes, sob agitação de 90 rpm e temperatura de 30 ± 1°C, seguida de uma acentuada autólise a partir de 120 h (5°dia), provavelmente, devido ao teor de carboidratos de reserva da célula que se encontravam em valores extremamente baixos, da ordem de 0,15 mg de trealose em 100 mg da matéria seca e 4 mg de glicogênio em 100 mg da matéria seca. A partir desse ponto entraram em total desorganização celular. / Yeast is highly important in sugar and alcohol agroindustry due to its role in the fermentative process of alcohol production. Thus, it is necessary to know this microorganism, most specially the Saccharomyces cerevisiae, the main species. The objective of this work was to study the strain Y904 of the yeast Saccharomyces cerevisiae under non-proliferating conditions after fermentation in a medium in which it was added vegetable oil and verify its possible correlation with the maintenance of the cellular viability. Analyses were performed in order to determine colony forming units, cellular viability, cellular concentration, total nitrogen in yeast and in medium, total carbohydrates and trehalose and glycogen contents. The yeast submitted to non-proliferating conditions presented a lower content of total carbohydrates, specially trehalose and glycogen, when compared to commercial yeasts. The viability of the yeast Saccharomyces cerevisiae Y904 markedly decreased after 24 hours in physiological solution under non-proliferating conditions in a shaker for 90 rpm at 30 ± 1°C. It was observed an accentuated autolysis from the 120 th hour (5 th day) on. This was probably because of the very low content of the carbohydrates of reserve in the cells, 0.15 mg of trehalose and 4.0 mg of glycogen in 100 mg of dry weight. From this point the cells began a total cellular disorganization.