La paroi de la levure Saccharomyces cerevisiae est une structure très dynamique composée de beta-glucanes, de mannanes et de chitine (polymère de N-acétylglucosamine). Elle peut s’adapter à l’état physiologique et aux changements morphologiques des cellules, ainsi qu’aux contraintes environnementales. Cette remarquable plasticité est assurée par l’intervention de différentes voies de régulation et de signalisation dont la voie CWI (Cell Wall Integrity) et la voie de la Calcineurine ou Protein Phosphatase 2B. La toxine killer K9 est une petite protéine sécrétée par la levure Hansenula mrakii. Cette toxine exerce son action létale sur les souches contrôles de S. cerevisiae mais pas sur des mutants du gène KNR4. Elle inhibe in vitro la beta-(1,3)-glucan syntase. Ce travail a dans un premier temps utilisé la Microscope à Force Atomique (AFM) et mis en évidence que la paroi de S. cerevisiae contrôle et mutant knr4 sont affectées de façon similaires par un traitement par la toxine K9. Dans un second temps, nous avons pu démontrer que la localisation cellulaire de Knr4 aux sites de croissance polarisée est nécéssaire pour l’action létale de la toxine K9 sur les cellules de S. cerevisiae.Knr4 fait partie d’une famille de protéines très conservées dans le domaine fongique, impliquées dans le contrôle de l’intégrité pariétale et la morphogenèse. Elle constitue un élément coordinateur pour la voie CWI et la voie de la Calcineurine. Notre travail a mis en évidence que la phosphorylation des résidus serine 200 et serine 203 de Knr4 joue un rôle dans ce mécanisme de coordination / The aim of my thesis was to study the fuction of Knr4 in the cell wall synthesis, morphogenesis, and related signaling pathways. The content of my thesis is mainly divided into three parts: The first part concerns our search to find out unknown partners of Knr4 and to investigate the cellular pathways required for localization of Knr4 protein. To that end, we decided to use a series of deletion mutants interrupted in genes related to morphogenesis and establishment of cellular polarity. We selected candidate genes from the Saccharomyces cerevisiae genome database (SGD, Stanford), using the keywords “Morphogenesis” and “Cell Polarity”. After selection and addition, 25 genes related to the morphogenesis and cell polarity were chosen for our Knr4 localization analysis. Through analysis of the results, we got 10 interesting mutants related to morphogenesis and polarity in which knr4 protein localization was affected: bem2Δ, pcl1Δ, pcl2Δ, rrd1Δ, spa2Δ, tpd3Δ, bem1Δ, bnI1Δ, yck1Δ and bud6Δ, and two additional mutants pph21Δ related to the tpd3Δ and cna1Δ involved in the calcinerin pathway. The second part deals with a mutational analysis of in vivo phosphorylated residues of Knr4 in the function and localization the protein, as well as in the modulation of calcineurin activity and CWI pathway. We found that S200S203 phosphorylation mutants cannot rescue viability of a double mutant bck1Δknr4Δ, while they can rescue slt2Δknr4Δ. In addition, S200S203 phosphorylation mutants behave as the absence of Knr4 towards suppression of lethality caused by an hyperactivated Mkk1 allele. Also we found that the knr4with KNR4S200AS203A mutant can results in hyperactivation of the Calcineurin pathway compared to control situation. So serin 200 and serin 203 may be involved in the cross-talking with the calcineurin pathway and CWI pathway. The third part is the study of K9 killer toxin’s strong cytocidal activity against sensitive yeast strains, including Saccharomyces cerevisiae. Treatment with this toxin results in the formation of pores at the surface of the cells, and more specifically at places where cell wall synthesis is the most active, namely at the tip of growing buds or mating projections. Yeast cells treated with K9 toxin then die by releasing cytoplasm and cellular materials from these pores. In the yeast S. cerevisiae, Knr4 protein localizes at the sites of polarized growth (bud tips, shmoo tips), which are also the sites where the toxin forms pores in the cell wall. Mutants defective in KNR4 gene are remarkably resistant to this toxin. In this study, we analyzed for the first time the biophysical effects of K9 on the yeast cell wall using Atomic Force Microscopy (AFM), a cutting edge technology that allows measuring the nanomechanical properties of living yeast cells, and their alterations by various drugs. To this end, we measured the effects of K9 toxin on the nanomechanical properties of the cell wall of S. cerevisiae wild-type cells and mutants deleted for KNR4 gene, at the short (2 h) and long term (20 h). Our results reveal an important cell wall remodeling occurring in wild-type cells already after 2 hours and only visible in knr4 mutant after 20 hours of treatment. Moreover, we investigated the role of Knr4 protein in the cells sensitivity towards the toxin. We were able to show that the presence of the N-terminal domain of Knr4 protein, which is required for its correct cellular localization at the bud tip during cell cycle, is essential for the toxin K9 wild-type sensitivity. In addition, a series of deletion mutants from the YKO collection in which the Knr4 cellular localization is also lost display a reduced sensitivity to the K9 toxin. Taken together, these results shed light on the importance of the proper localization of Knr4 protein at sites of intensive cell wall growth for the wild-type cells sensitivity to K9 killer toxin.

Baker´s yeast (Saccharomyces cerevisiae) were magnetically modified by three different methods, namely, surface modification by magnetic fluid, entrapment of cells into alginate and covalent immobilization on particles of magnetic chitosan. The ability of H2O2 decomposition was tested for all types of modification. It is apparent that the most amount of hydrogen peroxid was degraded by magnetic fluid - modified cells (84-95%), while the efficiency of cell which were modified by other methods was much lower (40-60%). Thanks to immobilization on particles of magnetic chitosan, we made completely new type of magnetic material, which was tested for adsorption of Crystal violet and Safranin O. It was founded that magnetic chitosan adsorbs no dyes, so all adsorption belongs to immobilized yeast. The maximum adsorption capacities were determined using Langmuire isotherm at 69,4 mg/g for Crystal violet and 99,0 mg/g for Safranin O.

Made available in DSpace on 2016-06-02T19:56:52Z (GMT). No. of bitstreams: 1 5322.pdf: 3668179 bytes, checksum: e3612c159bebd3f0a5be1640868316ae (MD5) Previous issue date: 2013-03-21 / Financiadora de Estudos e Projetos / The conversion of the hemicellulosic fraction in ethanol is a factor that impacts on the economic viability of the second generation ethanol production process from sugar cane bagasse. Hemicellulose from bagasse is a heteropolymer constituted by pentoses and glucose, being xylose the predominant sugar (~ 21 %). Among the available technological alternatives for ethanol production from xylose, SIF process (Simultaneous Isomerization and Fermentation), consisting of xylose conversion to xylulose by glucose isomerase (GI) enzyme and xylulose fermentation by the yeast S. cerevisiae, is considered a promising alternative. The main objectives of the present work were: i) evaluate the performance of different S. cerevisiae strains towards xylulose intake and ethanol productivity; ii) assess the influence of cultivation conditions (temperature, oxygen availability and initial xylose concentration) upon ethanol and xylitol production by the selected strains; iii) define the operation conditions for the continuous SIF process, using a system of fixed bed reactors associated in series. Preliminary experiments were conducted in 50 mL flasks, containing 4 g of pellets with a load of 20 % of immobilized GI, co-immobilized with yeast (load of 10 %) in alginate gel. For the screening of yeasts showing better performance on ethanol production from xylose, two commercial baker´s yeast strains (Itaiquara® e Fleischmann®), three industrial strains (BG-1, CAT-1 e PE-2) and one lab strain (CEN.PK113-7D) were evaluated. These experiments were performed at 35 oC, using a medium composed by xylose (60 g/L), urea (5 g/L), CaCl2 (1.9g/L) and several salts, at initial pH of 5.6. Additional SIF studies were carried out with the selected yeasts Itaiquara®, BG-1 or CEN.PK113-7D under different temperature conditions (40 oC), aeration (15 mL flasks) and initial xylose concentration (130 g/L) for comparison with the results obtained at the standard conditions. For SIF cultures, samples were withdrawal and the concentrations of reducing sugars were determined by DNS method while xylose, xylulose, ethanol and by-products (xylitol, glycerol etc) concentrations were assessed by liquid chromatography. Cell viability was also measured at the beginning and end of the experiment. When comparing the different yeasts, Itaiquara® strain presented the best performance, reaching ethanol concentrations of 22.4 g/L, with a productivity of 2.1 g/Lh. The conversion of xylose was similar for all studied industrial strains as well as among the baker s yeast and lab strains. Concerning the group of additional experiments, at 40 oC, a decrease of viability and ethanol selectivity was observed for Itaiquara®, whereas productivity and selectivity for CEN.PK113-7D. was improved. For the studies conducted under semianaerobic conditions, the yeast BG-1 showed an increase in selectivity and yield. However, the reaction time increased to app. 45 hours. On the other hand, the performance of strain Itaiquara® was not altered by the lower level of oxygen tested. In the experiment with 120 g/L of xylose, more than 40 g/L of ethanol was obtained in 24 hours of cultivation. Thus, we conclude that the SIF process proposed in the present work is a viable alternative for the production of ethanol from xylose or lignocellulosic residues. For the operation of the continuous system composed by fixed bed reactors associated in series, the recommended conditions include the Itaiquara® yeast with a temperature no higher than 35 oC, keeping the total residence time around 10 hours for a feeding supply containing 60 g/L of xylose. / A conversão da fração hemicelulósica da biomassa em etanol é um dos fatores que impactam a viabilidade econômica do processo de produção de etanol de segunda geração a partir do bagaço de cana-de-açúcar. A hemicelulose do bagaço é um heteropolímero constituído por pentoses e glicose, sendo a xilose o açúcar predominante (~ 21 %). Dentre as diversas alternativas tecnológicas para a produção de etanol a partir de xilose, o processo SIF (Simultânea Isomerização e Fermentação), consistindo na isomerização da xilose em xilulose pela enzima glicose-isomerase (GI) e na fermentação da xilulose pela levedura S. cerevisiae, é considerado uma alternativa promissora. Os principais objetivos do presente trabalho foram: i) avaliar o desempenho de diferentes cepas de S. cerevisiae em termos de assimilação de xilulose e produtividade em etanol; ii) estudar a influência das condições de cultivo (disponibilidade de oxigênio, temperatura e da concentração inicial de xilose) na produção de etanol e xilitol pelas cepas selecionadas; iii) definir as condições de operação para um processo SIF contínuo em sistema de reatores de leito fixo associados em série. Os experimentos preliminares foram conduzidos em frascos de 50 mL contendo 4 g de pelletes com carga de 20 % de glicose isomerase imobilizada, coimobilizada com levedura (carga de 10%) em gel de alginato. Para a seleção da levedura com melhor desempenho na produção de etanol a partir de xilose, foram avaliadas duas linhagens de levedura de panificação comercial (Itaiquara® e Fleischmann®), três cepas industriais (BG-1, CAT-1 e PE-2) e uma utilizada em laboratório (CEN.PK113-7D). Esses experimentos SIF foram conduzidos a 35ºC utilizando meio composto por xilose (60 g/L), ureia (5 g/L), CaCl2 (1,9 g/L) e sais diversos, em pH inicial 5,6. Experimentos SIF complementares foram realizados com as leveduras selecionadas Itaiquara®, BG-1 ou CEN.PK113-7D em diferentes condições de temperatura (40oC), aeração (frascos de 15 mL) e concentração inicial de xilose (130 g/L) para comparação com os resultados obtidos nas condições padrão. Em todos os experimentos SIF, amostras foram retiradas para determinação da concentração de açúcares redutores (método DNS) e de xilose, xilulose, etanol e subprodutos (xilitol, glicerol etc.) por cromatografia em fase líquida. Foi também acompanhada a viabilidade celular ao longo do cultivo. Na comparação entre as diferentes leveduras, destacou-se especialmente a levedura Itaiquara®, alcançando concentrações de etanol de 22,4 g/L, com produtividade em etanol de 2,1 g/Lh. A conversão de xilose foi semelhante entre as leveduras industriais e entre as leveduras de panificação e a de laboratório. Quanto ao conjunto de experimentos complementares, na temperatura de 40ºC houve diminuição de viabilidade e seletividade em etanol para a Itaiquara® e melhora na produtividade e seletividade para a CEN.PK113-7D. Nos experimentos realizados em condições semianaeróbias, a levedura BG-1 apresentou aumento de seletividade e rendimento em etanol, porém para um tempo de reação de 45 horas, aproximadamente. Já a levedura Itaiquara® não teve seu desempenho influenciado pela menor disponibilidade de oxigênio. No experimento realizado com 130 g/L de xilose, alcançou-se mais de 40 g/L de etanol em 24 horas de cultivo. Conclui-se, assim, que o processo SIF de xilose, proposto no presente trabalho, é uma alternativa viável para a produção de etanol a partir de xilose ou de resíduos lignocelulósicos. Para a operação em sistema contínuo composto por reatores de leito fixo associados em série recomenda-se a utilização de levedura Itaiquara® e de temperatura de, no máximo, 35ºC, mantendo-se o tempo de residência total em torno de 10 horas para uma alimentação contendo 60 g/L de xilose.

Among the various external signals perceived by yeast cells, nutrient availability is a condition to which these cells show a highly diverse biological response. Diploid cells in response to different nutritional stress conditions shows different developmental outcomes. On nitrogen starvation, cells undergo dimorphic transition whereby a unicellular yeast form transforms to a multicellular pseudohyphal form. While in the complete absence of a nitrogen source and a fermentable carbon source, yeast cells enter into a complex developmental program termed sporulation which culminates in haploid spores. The main objective of this work was to understand the role played by S. cerevisiaeSUB1 in starvation-induced meiotic program of diploid cells, decipher its target in sporulation specific gene expression cascade, study the domain architecture of Sub1 and examine its functional homology to mammalian PC4. Role of Sub1 in induction of sporulation and other stress responses in S. cerevisiae In a previous whole-genome screen for mutants with altered sporulation efficiency in the Saccharomyces cerevisiae S288c strain, SUB1 locus was identified as a negative regulator of sporulation (Deutschbaueret al., 2002). Moreover, genome-wide gene expression analysis in SK1 strain had shown that SUB1 transcript levels are repressed during sporulation (Chu et al., 1998). Many studies in different yeast strain backgrounds implicate more than 1,000 genesout of 6,200 genes in yeast genome as being differentially expressed during the sporulation process (Chu et al., 1998; Primiget al., 2000; Deutschbaueret al., 2002). Interestingly, these studies show the number of regulatory genes that negatively affect sporulation is far lower than those that are activators of sporulation and moreover their mechanism of action is poorly studied. S. cerevisiae.SUB1 is one among negative regulators of sporulation(Deutschbaueret al., 2002). Global transcriptome of diploid yeast cells undergoing sporulation showed SUB1 transcripts are greatly reduced with time progression (Chu et al., 1998). To understand the role of SUB1 in sporulation, we generated deletion of both SUB1 alleles in the diploid S288c strain background and compared the kinetics of asci formation in this strain with that of the wild-type. We observed that cells lacking SUB1 exhibit ~5-fold increase in tetrad asci. Based on Eosin Y and Calcoflour White staining assays, we find no change in spore morphology in the mutant. Thus the increase in sporulation efficiency in sub1/sub1diploids is not accompanied by formation of defective spores. We validated the reduction in SUB1 transcript levels during sporulation in wild-type SK1 strain background. We also examined the Sub1 protein levels by epitope-tagging of the chromosomal SUB1 open reading frame and determining protein levels in this strain. We find that consistent with the data on transcript levels, Sub1-TAP tagged protein levels too decreased gradually on shift to sporulation medium. We created sub1alleles in diploids in the SK1 strain background and using this strain background we investigated Sub1 target genes and chose IME2 (early), SMK1, SPS2 (middle), DIT1, DIT2 (mid-late) and SPS100 (late) genes as representative sporulation genes. We observed that sub1∆/sub1∆cells have a significantly elevated expression of middle genes (SPS2 and SMK1) that followed normal induction kinetics i.e., 5 hours post transfer to sporulation medium. However, the expression levels or timing for other class of sporulation genes did not change in sub1∆strain as compared with the wild-type. In order to confirm these observations, we also studied the effects of over-expression of SUB1 from the GAL1 promoter by transforming the high copy plasmid. This was done in wild-type SK1 cells and the expression of sporulation genes were analyzed. We observed that expression of SMK1 and SPS2middle sporulation genes was reduced on over-expression of SUB1.We used the Sub1-TAP protein to assess if Sub1 directly regulates these genes by Chromatin immunoprecipitation assays. For these studies, we examined the recruitment of Sub1 to these loci through the time course of sporulation. In wild-type SK1 cells, Sub1 was to bound to middle sporulation genes and this was striking in cells at 5th hour post-induction of sporulation. These data establish that Sub1 directly associates with chromatin at these loci co-incident with the time points where expression levels of these changes is altered in cells lacking Sub1. Furthermore, to assess the role of Sub1 in other stress responses, such as pseudohyphae formation in response to nitrogen starvation, pheromone-induced agar invasion and secretory stress, we employed a genetic approach. Genetic interaction studies of SUB1 with RPB4, a subunit of RNA polymerase with functions in stress response and HOS2, a subunit of Set3 complex and a close homolog of mammalian HDAC3, reported to be involved in sporulation and secretory stress, were performed. Based on sporulation frequency and pseudohyphal formation in the double mutants we conclude that SUB1 is downstream of both these genes. Moreover, our results from assays of schmoo formation and pheromone-induced agar invasion suggest that SUB1 functionally interacts with HOS2. Study of domain architecture of Sub1 and homology to human PC4 Comparison of the S. cerevisiae Sub1 protein with its higher eukaryotic homologs showed that the N-terminal region of yeast Sub1 (32-105 aa) is highly conserved (Knauset al., 1996; Henry et al., 1996) with the 106-292 C -terminal amino acids being yeast-specific. We employed deletion analysis to generate partial Sub1 proteins and used them to understand the roles played by these domains in different phenotypes associated with Sub1. Our analysis of the localization of various Sub1-GFP fusion proteins shows that 146-172 aa in the C-terminal domain of Sub1 confers nuclear localization. Sporulation frequency analysis of the different domains of Sub1 suggests that both the N and C terminal domains are necessary for sporulation function of Sub1. The N terminal domain of yeast Sub1 shares homology with human PC4 and not surprisingly possesses ssDNA binding ability first attributed to human PC4 (Kaiser et al., 1995). In order to investigate whether the effects of SUB1 on kinetics of sporulation require its ssDNA binding function, we generated the sub1(Y66A) ssDNA binding mutant (Sikorskiet al., 2011) and over-expressed it in the S288c genetic background. We assessed sporulation efficiency of sub1∆/sub1∆cells over-expressing sub1(Y66A) mutant allele as compared to cells over-expressing wild-type SUB1. Interestingly, cells with over-expression of sub1(Y66A) have reduced sporulation efficiency that is equivalent to the levels achieved on over-expression of wild type SUB1. This data suggests that the ssDNA-binding ability of Sub1 is not important for its role in sporulation. Furthermore, we examined the ability of human PC4 to contribute to yeast sporulation process by complementation analysis. We observed that over-expression of PC4 complemented the phenotypes of sub1∆strain, suggesting that the function of Sub1/PC4 family is evolutionarily conserved. Studies on biochemical interactions of Sub1 with histone proteins Human PC4 is a chromatin-associated protein, present on metaphase chromosomes (Das et al., 2006). The short C-terminal domain of PC4(62-87 aa) interacts with core histones H3 and H2B in vitro and in vivo and this interaction mediates chromatin condensation. The homology between S. cerevisiaeSub1 (32-105 aa) and human PC4 (62-127 aa)is in the domain required for their DNA binding properties and coactivator functions, suggesting possible conservation in their interactions. We tested the interactions of yeast Sub1 with histone proteins by adopting both in vitro and in vivo interaction assays. We find recombinant Sub1 had strong interactions with rat and yeast histone H3in vitro. Moreover,Sub1 was found to interact with histone H2B, but not with H2A, in vivo, a binding specificity also shown by human PC4.Thus, we demonstrate conservation in the interaction of Sub1 with histone proteins.

Synthetic biology has ushered in a new age of molecular biology with the aim towards practical developments in disciplines ranging from medicine, agriculture, and industry. Presently, it remains difficult to manipulate the genomes of many organisms due to lack of genetic tools. These problems can be circumvented by cloning large fragments of DNA into strains where many genetic tools are in place, such as Saccharomyces cerevisiae. However, this organism is unable to directly transfer cloned DNA to other organisms and is unable to stably maintain DNA with a G+C content >40%. Many organisms relevant in biotechnology often have G+C content DNA >60%, and therefore are difficult to engineer. Here, the soil bacteria Sinorhizobium meliloti was chosen as a host strain to clone and manipulate large fragments of high G+C content DNA. S. meliloti is a Gram-negativeα-proteobacteria that forms symbiotic relationships with legumes to fix nitrogen. It has a multi-partite genome with a G+C content of 62.7% that includes a chromosome (3.65 Mb), the pSymA (1.35 Mb), and pSymB (1.68 Mb) replicons. A restriction endonuclease hsdR mutant strain lacking pSymA and pSymB was created and used in this study. Multi-host shuttle (MHS) vectors were constructed that allow for direct transfer and maintenance of DNA in E. coli, S. cerevisiae, and P. tricornutum. Characterization of strains was conducted to determine transduction, conjugation, and transformation frequencies, as well as stability of MHS plasmids. Furthermore, a proof-of-concept experiment was conducted to clone large plasmids (70-205 kb) with G+C content >58% via site-specific recombination at a landing pad in the MHS vector, which was then verified using colony PCR. This work demonstrates the usefulness of S. meliloti containing a MHS vector for cloning of large fragments with high G+C content DNA, a technology that may be used for several applications in both applied and basic research. / Thesis / Master of Science (MSc) / Synthetic biology is an emerging field that incorporates principles of molecular biology and engineering for the design and construction of biological systems for application in medicine, agriculture, and industry. Presently, it remains difficult to modify genomes of several organisms due to lack of available techniques. Yeast is currently used for the modification of large DNA pieces, however it is unable to transfer and maintain modified DNA with high G+C content. Here, the bacteria Sinorhizobium meliloti was used as a host organism to conduct genetic engineering due to its ability to maintain large DNA pieces with a high G+C content. Characterization experiments were conducted to assess the efficiency of this organism for this task. Using this strain, a proof-of-concept experiment to demonstrate the uptake and maintenance of large, high G+C DNA pieces was completed. This technology may be useful in biotechnology applications for engineering of large DNA pieces from industrially relevant organisms.

Understanding the molecular structure of DNA was considered as greatest achievement in modern biology. It helped in understanding fundamental cellular processes such as replication of DNA, nature of the genetic code and transcription. It also led to technological advancements such as DNA sequencing, genetic engineering and gene cloning. The DNA molecule is highly polymorphic in nature and its structure is dependent on environment, base composition and sequence context. B-DNA, A-DNA, Z-DNA and curved or kinked DNA are some of the well characterized double helical polymorphs. B-DNA is the most prevalent structure in vivo and it can undergo small local variations and global variations. In this thesis we refer to distinct structural property of any particular DNA sequence as deviation from fibre model B-DNA structural parameters or random sequence DNA. Structural properties of DNA are an outcome of the linear arrangement of the 4 chemically different nucleotide bases and the characteristic features of the two grooves (minor and major) arising due to the asymmetric position of glycosidic bonds of base pairs. DNA structure and properties are expected to vary along its length. Several structural features have been defined for DNA duplex, while DNA stability, bendability and intrinsic curvature are well studied and found to be biologically relevant. These three sequence dependent properties differ in their nature and information content and can be studied both at local and global levels, depending on the length of DNA fragment being examined. Majority of the work in this thesis focuses on the analysis of these three DNA structural features in promoter regions of different eukaryotic systems and their relationship with gene expression. The thesis work is divided in to five sections briefly described below. The sections discuss prevalence of the three structural features, DNA stability, bendability and intrinsic curvature in the promoter regions of six eukaryotic systems namely S. cerevisiae, D. melanogaster, C. elegans, zebrafish, mouse and human. The relationship between DNA structural features of promoter regions of S. cerevisiae with gene expression variability is discussed, followed by application of the structure-based promoter prediction algorithm ‘PromPredict’ in annotating promoter regions of six different eukaryotes. Finally, an analysis of structural features of the flanking sequences of transcription factor binding sites (TFBSs) of six transcription factors and their relationship with the DNA binding affinity is discussed. Each of the projects described below will appear as a separate chapters in the thesis. An overview of the eukaryotic transcription machinery, promoter elements and different DNA structural properties are discussed in the introduction of the thesis (chapter 1). The structural properties of DNA in the promoter regions of eukaryotic genes (chapter 2)Earlier studies in the lab reported that, apart from sequence motifs, promoter re- gions have distinct structural properties, such as lower stability, lesser bendability and more curvature compared to other genomic regions. But those studies were on small datasets and few model systems. Advancement in high-throughput tech- niques has made availability of transcription start site information for many model systems. This work was initiated with the aim of investigating the structural fea- tures in different eukaryotic systems belonging to different domains of life. The quantitative analysis of three different structural features of promoter regions of six different model systems S. cerevisiae, C. elegans, D. melanogaster, zebrafish, mouse and human has been carried out. Further, the composition of different k-mers (k=3, 4 and 6) A-tracts and G-quadruplexes has been studied. The analysis allowed us to understand the similarities and differences in struc- tural features of promoter sequences in different model systems. The core promoter sequences of S. cerevisiae, C. elegans, D. melanogaster, zebra fish, mouse and hu- man have been observed to be less stable and have lower preference for nucleosome formation. S. cerevisiae, C. elegans and D. melanogaster promoter sequences have been shown to be less bendable whereas zebrafish, mouse and human promoter se- quences are flexible in terms of bendability towards major groove as predicted fDNase 1 sensitivity model. S. cerevisiae, C. elegans, D. melanogaster core promoter regions have AT rich oligomers, whereas mouse and human core promoter regions have GC rich oligomers and G-quadruplex motifs. DNA structural features of TATA-containing andTATA-less promoters (chapter 3)Eukaryotic genes can be broadly classified as TATA-containing and TATA-less based on the presence of TATA-box in their promoter sequences. Experiments on both classes of genes have reported that, they have differences in regulation of gene ex- pression and cellular functions. In this chapter, the differences in compositional and structural features of TATA-containing and TATA-less promoters in the above mentioned model systems are discussed. The results suggested that DNA structural features of TATA-containing and TATA-less promoters are distinctly different in all eukaryotes. The TATA-containing promoters are less stable, more flexible and more curved compared to TATA-less promoters in lower eukaryotes. In mouse and hu- man genes, DNA duplex stability and G-quadruplex motifs are very distinguishing features in the two classes of promoters. DNA structural properties of eukaryotic promoter regions and gene expression variability (chapter 4) Gene expression is regulated by various external (environment and evolution) and internal (genetic) factors. Presence of sequence motifs, such as TFBSs and TATA- box, as well as DNA methylation has been implicated in the regulation of expression of some genes in vertebrates, but a large number of genes lack these sequences. Ear- lier analyses (described in previous sections) in S. cerevisiae, have shown that their promoter sequences have special structural properties, such as low stability, less bendability and more curvature compared to other genomic regions. These strutural features may play a role in transcription initiation and regulation of gene expression. This project was carried out to understand 1. What is the relationship between DNA structural features and gene expres- sion? 2. What is the relationship between gene expression and bidirectionality of a pro- moter region? For this purpose, the information of seven different gene expression variability measures, stochastic noise, responsiveness, stress response, trans variability, mu- tational variance, interstrain variation and expression divergence have been com- pared with structural features in the promoter regions. It is observed that a few of the variability measures of gene expression are linked to DNA structural prop- erties, along with nucleosome occupancy, TATA-box presence and bidirectionality of promoter regions. Interestingly, gene responsiveness is shown to be most, inti- mately correlated with DNA structural features and promoter architecture. The study highlights the importance of sequence dependent structural features in gene regulation. Promoter prediction in eukaryotes using DNA duplex stability (chapter 5) Structural property-based algorithms can discriminate promoter sequences from non-promoter sequences and are far better than sequence motif-based predictors. Compared to other structural features, low stability is found to be the most preva- lent feature in promoter regions. “PromPredict” (in-house algorithm) uses the din- ucleotide free energy values obtained from differential melting stability of DNA du- plexes as a predictor of promoters and has been successfully used earlier to annotate promoter sequences in prokaryotes and rice. Comprehensive analysis of the perfor- mance of PromPredict in S. cerevisiae, D. melanogaster, C. elegans, zebrafish, mouse and human as well as TATA-containing and TATA-less promoter regions of S. cere visiae with TSS data and 48 eukaryotic systems with translation start site (TLS) data revealed that differential stability is a good criterion for promoter prediction. DNA structure in flanking sequences of consensus motifs modulate transcription factor binding (chapter 6) Sequence specific DNA-protein interactions are essential for specific expression pat- terns during the development. There are several factors contribute to DNA-binding specificities of transcription factors (TFs). They include structure and flexibility of TFs, cofactors, chromatin environment and DNA sequence. Along with actual tran- scription factor binding sites (TFBSs), their sequence context (flanking sequences) is also shown to play a major role in gene regulation. Most of the studies have ad- dressed the sequence context at global level but very little is understood about the role of sequences flanking TFBSs in binding of transcription factors. This project was initiated with the aim of understanding the effect of flanking sequences of TFBSs in transcription factor binding affinity. In vitro DNA binding information of six different transcription factors (with three types of DNA bind- ing domains, Zinc finger (GATA4), home domain (AbdA, AbdB and Ubx) and bZIP (fos-jun and Nfil3)) was provided by Aseem Ansari’s lab. The compositional and structural features (minor groove width, propeller twist, wedge and free energy) are compared with the DNA binding profiles of 12mers (or 8mers) of six different transcription factors. It has been observed that some of the DNA structural proper- ties of flanking sequences are strongly correlated with binding affinity. For GATA4 sequences, binding affinity is negatively correlated to GC content or minor groove width at their 5′ -flanking region, showing the significance of narrow minor groove at 5′ -region. On the other hand, the binding affinity of bZIP proteins is negatively correlated to wedge angles, whereas in case of homeodomain proteins, it is posi- tively correlated to propeller twist and GC content. Thus, this study highlights the differential preference for flanking sequences outside the core binding motifs of six different TFs, which interact with DNA through α-helix. ‘The relationship between transcription pre-initiation complexes and gene ex- pression variability in S. cerevisiae’ is briefly described in the appendix section of the thesis. General conclusion Overall, the results presented in this thesis indicate that DNA sequence based structural features are unique to promoter regions and play an important role in gene regulation. Local structural features of flanking sequences of transcription factor binding sites are also instrumental in determining the DNA binding affinity of transcription factors.

Made available in DSpace on 2016-06-02T19:55:39Z (GMT). No. of bitstreams: 1 5990.pdf: 3991723 bytes, checksum: 8f7428459353354f21c1db08bd391507 (MD5) Previous issue date: 2014-02-27 / Universidade Federal de Sao Carlos / The industrial production of fuel ethanol and sugar generates the main byproduct of sugarcane bagasse, which is burned in boilers for power generation. However, as a lignocellulosic material (consisting basically of three polymers: cellulose, hemicellulose and lignin), bagasse can be reused for the production of second generation bioethanol (2G), which is a renewable and environmentally friendly biofuel. For industrial 2G bioethanol production becomes economically feasible, the use of all fermentable fractions present in the bagasse is required: C6 fraction (cellulose) and C5 fraction (hemicellulose). These fractions are subjected to hydrolysis processes that generate as main sugars glucose and xylose respectively. It is important, therefore, that the microorganism employed for the production of ethanol 2G is able to utilize all the sugars generated during the hydrolysis process. In this work we chose the yeast Saccharomyces cerevisiae to be the main microorganism used in the industrial production of ethanol, although unfortunately, this yeast is unable to ferment xylose. However, while S. cerevisiae does not use xylose, can ferment xylulose obtained by isomerization of xylose by the enzyme glucose isomerase. The objective of this study was to develop and evaluate technological alternatives for the production of ethanol 2G from hexoses and pentoses using wild S. cerevisiae. In relation to the C6 fraction, in this work two important aspects have been addressed: i) study of the operation regime of a fed-batch reactor enzymatic hydrolysis of the C6 fraction of bagasse from sugarcane, yielding values of final glucose concentration of 200 g.L-1, higher than 45 g.L-1 achieved in batch reactor; ii) kinetic modeling of complex systems (enzymatic hydrolysis of lignocellulosic substrates), in which an interpolator was developed using fuzzy logic as an important tool to represent the processes of enzymatic hydrolysis of lignocellulosic materials for rugged and reliable manner. Now, in relation to the C5 fraction initially applied simple techniques of Evolutionary Engineering, leading to the selection of a different strain of S. cerevisiae, adapted to assimilate xylulose in minimal medium and characterized by reduced formation of xylitol, which demonstrated a selectivity of ~7 getanol.gxilitol -1, significantly higher than the selectivity achieved by the wild strain of ~2 getanol.gxilitol -1. The selected strain was studied in batch cultures conducted in bench scale reactor under different conditions of oxygen limitation. It was found that the production of ethanol is favored over the formation of xylitol, keeping the flow of consumed xylulose above 0,5 mmol.gMS -1.h-1 for flow of oxygen consumption of 0.1 mmol.gMS -1.h-1, reaching in this condition selectivities around 4 getanol.gxilitol -1. For zero flow of oxygen (anaerobic culture) or above 0,3 mmol.gMS -1.h-1, ethanol production is drastically reduced , regardless of the flow xylulose assimilated by the cells. / A produção industrial de etanol combustível e de açúcar gera como principal subproduto o bagaço de cana de açúcar, que é queimado nas caldeiras para geração de energia. Entretanto, por ser um material lignocelulósico (constituído basicamente por três polímeros: celulose, hemicelulose e lignina), o bagaço pode ser reaproveitado para a produção de bioetanol de segunda geração (2G), que é um biocombustível renovável e ambientalmente amigável. Para que a produção industrial de etanol 2G se torne economicamente viável, é necessário o aproveitamento de todas as frações fermentescíveis presentes no bagaço de cana: fração C6 (celulose) e fração C5 (hemicelulose). Estas frações são submetidas a processos de hidrólise que geram como principais açúcares glicose e xilose respetivamente. É importante, portanto, que o microrganismo empregado para a produção de etanol 2G seja capaz de utilizar todos os açúcares gerados no processo de hidrólise. Neste trabalho foi escolhida a levedura Saccharomyces cerevisiae por ser o principal microrganismo utilizado na produção industrial de álcool combustível, embora, infelizmente, esta levedura seja incapaz de fermentar xilose. No entanto, embora S. cerevisiae não utilize xilose, pode fermentar a xilulose obtida pela isomerização de xilose pela enzima xilose isomerase conhecida industrialmente como glicose isomerase. Assim, o objetivo do presente trabalho foi desenvolver e avaliar alternativas tecnológicas para a produção de etanol 2G a partir de hexoses e pentoses, utilizando S. cerevisiae selvagem. Em relação à Fração C6, neste trabalho foram abordados dois aspectos importantes: i) estudo da operação em regime de batelada alimentada de um reator de hidrólise enzimática da fração C6 do bagaço de cana de açúcar, obtendo-se valores de concentração final de glicose de cerca de 200 g.L-1, superiores aos 45 g.L-1 alcançados em reator operado em bateladas simples; ii) modelagem cinética de sistemas complexos (hidrólise enzimática de substratos lignocelulósicos), no qual foi desenvolvido um interpolador utilizando a lógica fuzzy como uma ferramenta importante para representar os processos de hidrólise enzimática de materiais lignocelulósicos de forma robusta e confiável. Já em relação à Fração C5, inicialmente aplicou-se técnicas simples de Engenharia Evolutiva, levando à seleção de uma linhagem diferenciada de S. cerevisiae, adaptada à assimilação de xilulose em meio mínimo e caracterizada por reduzida formação de xilitol, a qual apresentou uma seletividade de ~7 getanol.gxilitol -1, valor significativamente superior à seletividade alcançada pela linhagem selvagem, de ~2 getanol.gxilitol -1. A linhagem selecionada foi então estudada em cultivos em batelada conduzidos em biorreator de bancada, sob diferentes condições de limitação por oxigênio. Verificou-se que a produção de etanol é favorecida, em detrimento da formação de xilitol, mantendo-se o fluxo de xilulose consumida acima de 0,5 mmol.gMS -1.h-1, para fluxo de oxigênio consumido de 0,1 mmol.gMS -1.h-1, alcançando-se nessa condição seletividades em torno de 4 getanol.gxilitol -1. Para fluxos de oxigênio nulo (cultivo anaeróbio) ou acima de 0,3 mmol.gMS -1.h-1, a produção de etanol é drasticamente reduzida, independentemente do fluxo de xilulose assimilado pelas células.

Made available in DSpace on 2017-07-10T18:08:04Z (GMT). No. of bitstreams: 1 Grettya Maria Assuncao.pdf: 2495923 bytes, checksum: 815b6e9d74269efe9f6ba6585d9b8a4f (MD5) Previous issue date: 2014-02-28 / The whey is a dairy industry effluent. Due to its high nutritional content , is used in the production of whey protein concentrate by ultrafiltration. This process generates another residue, cheese whey permeate which also contains high nutritional content, it is rich in lactose, and some minerals (P, K, Mg, Na and Ca). Can be reused in fermentation processes as a source of nutrients. One of these processes is the production of cell biomass from the growth of yeasts for use in food for human and animal consumption. Yeasts are used to obtain the GRAS (generally regarded the safe), in other words, be considered safe for use in foods. Some are: S. cerevisiae, used as a standard micro-organism to have its well-defined characteristics; S. boulardii, used in medicine as probiotic and marketed as a medicine, there are no reports of its production from waste; K. marxianus with potential of metabolizing lactose present in the permeate. This work aims to study the growth of three yeasts, using culture medium as the residue of cheese whey permeate, for the production of biomass. Two experimental design were performed to enzymatic hydrolysis in order to obtain the best condition for this process, and an experimental design was carried out for the fermentations in order to assess which variables are significant in the production of yeast biomass process. The analyzes carried out during the process were: absorbance, reducing sugars, indirect and direct dry weight and pH. Fermentation was carried out with addition of oxygen, aiming an increase in the production of cell biomass, BOD and COD analysis and analysis of the final concentration of ethanol. The best condition for the hydrolysis was obtained using [enzyme] 0.125% , T 35.35 °C and pH 7,7. For the production of biomass without added oxygen, the best values achieved for all yeasts were from 150 g L-1 and permeate temperature of 30 °C, the results were: 14.22 g L-1 of S. cerevisiae at pH 4, 1 g L-1 [(NH4)2SO4], 0.5 g L-1 [MgSO4], and 4 g L-1 [KH2PO4 ]; 22.85 g L-1 of S. boulardii at pH 5,5, 9 g L-1 [(NH4)2SO4], 1.2 g L-1 [MgSO4] and 9 g L-1 [KH2PO4]; and 16.90 g L-1 of K. marxianus at pH 6,5, 2 g L-1 [(NH4)2SO4], 2.5 g L-1 [MgSO4] and 1 g L-1 [KH2PO4]. The results of cell biomass obtained in these process conditions and with the addition of oxygen to the fermentation medium were 16.54 g L-1 of S. cerevisiae, 30.58 g L-1 of S. boulardii and 21.12 g L-1 K. marxianus. The ethanol concentration at the end of the process for each yeast was 28.8 g L-1 of ethanol from S. cerevisiae; 27 g L-1 of ethanol from S. boulardii and 52.1 g L-1 of ethanol from K. marxianus. And the reduction in BOD and COD parameters was 69.31% in COD and 97.19% in BOD using S. cerevisiae; 97.67 % in COD and 99.53 % in BOD using S. boulardii; and 83.53 % in COD and 93.14 % in BOD using K. marxianus. Thus, the study demonstrated that permeate cheese whey is a residue with a potential application in the production of yeast biomass, which allow the end of the fermentation process reduced the initial organic load of the waste being one of the bound parameters to reduce the environmental impact as well as the decreasing cost of the wastewater treatment processes. / O soro de queijo é um efluente da indústria láctea. Devido ao seu alto teor nutricional, é utilizado na produção de concentrado proteico de soro por ultrafiltração. Esse processo gera outro resíduo, o permeado de soro de queijo que também contém elevado teor nutricional, pois é rico em lactose, além de alguns sais minerais (P, K, Mg, Na e Ca). Pode ser reutilizado em processos fermentativos como fonte de nutrientes. Um destes processos é a produção de biomassa celular proveniente do crescimento de leveduras, para uso em alimentos para consumo humano e animal. As leveduras utilizadas devem obter o GRAS (generally regarded as safe), ou seja, ser consideradas seguras para o uso em alimentos. Algumas são: S. cerevisiae, utilizada como micro-organismo padrão por ter suas características bem definidas; S. boulardii, utilizada na medicina como probiótico e comercializada como medicamento, não havendo relatos da sua produção a partir de resíduos; K. marxianus com potencial de metabolizar a lactose presente no permeado. Este trabalho tem por objetivo estudar o crescimento das três leveduras utilizando como meio de cultivo o resíduo permeado de soro de queijo, para a produção de biomassa. Dois planejamento experimentais foram realizados para a hidrólise enzimática visando obter a melhor condição para esse processo, e um planejamento experimental foi realizado para as fermentações com o intuito de avaliar quais as variáveis são significativas no processo de produção de biomassa de leveduras. As análises realizadas durante o processo foram: absorbância, açúcares redutores, massa seca indireta e direta e pH. Foi realizada uma fermentação com adição de oxigênio, objetivando um aumento na produção de biomassa celular, análises de DBO e DQO e análise da concentração final de etanol. A melhor condição obtida para a hidrólise foi utilizando [enzima] 0,125%, T 35,35ºC e pH 7,7. Para a produção de biomassa sem adição de oxigênio, os melhores valores alcançados para todas as leveduras foram a partir de 150 g L-1 de permeado e a temperatura de 30 ºC, cujos resultados foram respectivamente: 14,22 g L-1 de S. cerevisiae em pH 4, 1 g L-1 [(NH4)2SO4], 0,5 g L-1 [MgSO4] e 4 g L-1 [KH2PO4]; 22,85 g L-1 de S. boulardii em pH 5,5, 9 g L-1 [(NH4)2SO4], 1,2 g L-1 [MgSO4] e 9 g L-1 [KH2PO4]; 16,90 g L-1 de K. marxianus em pH 6,5, 2 g L-1 [(NH4)2SO4], 2,5 g L-1, [MgSO4] e 1 g L-1 [KH2PO4]. Os resultados de biomassa celular obtidos nessas condições de processo e com a adição de oxigênio ao meio fermentativo foram 16,54 g L-1 de S. cerevisiae, 30,58 g L-1 de S. boulardii e 21,12 g L-1 de K. marxianus. A concentração de etanol ao final do processo para cada levedura foi de 28,8 g L-1 de etanol a partir de S. cerevisiae; 27,0 g L-1 de etanol a partir de S. boulardii e 52,1 g L-1 de etanol a partir de K. marxianus. E a redução nos parâmetros DBO e DQO foi de 69,31% na DQO e 97,19% na DBO utilizando S. cerevisiae; 97,67% na DQO e 99,53% na DBO utilizando S. boulardii; e 83,53% na DQO e 93,14% na DBO utilizando K. marxianus. Desta forma, o estudo demonstrou que o permeado de soro de queijo é um resíduo com grande potencial de aplicação na produção de biomassa de leveduras, as quais possibilitam ao final do processo fermentativo uma redução da carga orgânica inicial do resíduo sendo esse um dos parâmetros vinculados à redução do impacto ambiental, bem como à diminuição dos custos de processos de tratamento do efluente.

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