Atualmente, as cepas de S. cerevisiae utilizadas na produção industrial de álcool combustível no Brasil são leveduras diplóides que metabolizam a sacarose através da sua hidrolise extracelular pela invertase periplasmática, seguida pelo transporte para o interior da célula das moléculas de glicose e frutose formadas, e posterior metabolização pela glicólise. Neste trabalho, utilizando técnicas de engenharia genética e posteriores cruzamentos, foram desenvolvidas cepas diplóides de S. cerevisiae capazes de consumir este açúcar por meio da sua captação direta pelo transportador codificado pelo gene AGT1, e hidrólise intracelular pela invertase citoplasmática codificada por uma versão modificada do gene SUC2. Estas modificações permitiram alcançar um rendimento 10% maior na produção de etanol, além de aumentar a velocidade de consumo da sacarose e diminuir a quantidade de açúcares residuais ao final do processo. Estes resultados abrem novos horizontes para tornar a produção de etanol no Brasil mais eficiente. / Currently, the S. cerevisiae strains used in industrial production of fuel alcohol in Brazil are diploid yeasts that of metabolize sucrose through its extracellular hydrolysis by the periplasmic invertase, followed by the transport of the formed glucose and fructose into the cells, and further metabolism through glycolysis. In this study, utilizing genetic engineering techniques and further crosses, diploid strains of S. cerevisiae were developed that are able to consume this sugar through its direct uptake by the transporter encoded by the AGT1 gene, and intracellular hydrolysis by the cytoplasmic invertase encoded by a modified version of the SUC2 gene. These modifications allowed achieving a 10% higher yield in the production of ethanol, besides increasing the sucrose consumption rate and decreasing the amount of residual sugars at the end of the process. These results open new horizons to turn ethanol production in Brazil more efficient.

Distal enhancer elements regulate the tissue-specific expression of their target genes via the establishment of physical interactions with the gene promoter. In mice, a cluster of five enhancers, jointly classified as a super-enhancer, specifically upregulate α-globin gene expression during erythroid differentiation. Aside from the Nprl3 gene, whose promoter is located inside this enhancer region, expression-levels of other genes within a short distance (&lt,50kb) of the enhancer region are not affected by the activation of the enhancer in erythroid cells, despite being located within the same sub-TAD in erythroid cells. The CCCTC-binding factor (CTCF) is implicated in the organisation of chromosome topology through the formation of interactions between its binding sites in an orientation-dependent manner. In this thesis, I demonstrate that CTCF functions in vivo as a boundary to maintain α-globin enhancer-promoter specificity in erythroid cells. The study of the local chromatin architecture by next-generation Capture-C reveals that α-globin enhancer and promoter interactions are constrained to a compartment of roughly 70kb. The unidirectional interaction profiles of the α-globin enhancers are delimited by the interactions between two genomic domains flanking the α-globin cluster. Further investigation shows that each of these domains contains several CTCF binding sites orientated in tandem, such that CTCF binding orientation between domains is convergent. Although CTCF binding across the α-globin locus is identical between mouse embryonic stem (ES) cells and erythroid cells, interaction between these domains occurs only in erythroid cells suggesting it is dependent on the formation of tissue-specific α-globin enhancer-promoter interactions. By generating a series of mouse models, deleting CTCF binding sites at the α-globin enhancers singly and in combination, I show that the deletion of two CTCF binding sites directly flanking the enhancer cluster results in a shift in interactions between flanking domains, away from the enhancer region. This leads to an expansion of enhancer interactions to include two genes directly upstream of the α-globin enhancers: Rhbdf1 and Mpg. Despite the Rhbdf1 gene being subject to polycomb group protein-mediated gene repression in erythroid cells, ablation of CTCF binding results in increased interactions between both the Rhbdf1 and Mpg gene promoters and the α-globin enhancers and concurrent strong transcriptional upregulation of both genes. The Rhbdf1 gene promoter acquires the active histone mark H3K4me3, but doesn't lose Polycomb Repressive Complex 2 (PRC2) mark H3K27me3 or binding of its catalytic component Ezh2. Despite the presence of this repressive mark, robust levels of Rhbdf1 expression are detected at levels higher than those in ES cells where this gene is actively expressed under the influence of its own enhancer. I conclude that regulation of the direction of enhancer interactions by CTCF is required for the promoter specificity of enhancers and the maintenance of transcriptional states of nearby genes.

Saccharomyces cerevisiae é o organismo de escolha para produção de etanol combustível, apresentando-se adaptadas ao ambiente hostil das dornas de fermentação. Adaptações como floculação, formação de espuma e de biofilme são características fenotípicas indesejáveis ao processo brasileiro. Por isso, a pronta identificação destas características faz-se necessária no intuito de minimizar perdas de rendimento e diminuir o custo da produção. Sabe-se que proteínas codificadas pela família de genes FLO estão relacionadas a estes fenótipos, os quais são regulados pela proteína Flo8p, a qual possui papel fundamental na apresentação destas características fenotípicas indesejáveis. Desta forma, o presente trabalho teve como objetivo analisar o fenótipo e o comportamento metabólico, relacionando-os com a regulação via Flo8p. Os resultados obtidos revelam o papel fundamental de Flo8p na regulação positiva destes fenótipos em linhagem isolada do ambiente industrial (FT02). Verificou-se uma forte tendência da participação do gene FLO11 na formação de espuma, via Flo8p. Além disso, através da deleção do FLO8 da linhagem FT02, observou-se a influência positiva de Flo8p na floculação e hidrofobicidade celular, filamentação e poder invasivo. / Saccharomyces cerevisiae is the chosen organism for fuel ethanol production, presenting adapted to the hostile environment of the fermentation vats. Adaptations like flocculation, d foam formation and biofilm are undesirable phenotypes for the Brazilian process. Hence, the early identification of these characteristics is necessary to minimize yield losses and lower the production cost. Proteins codified by FLO gene family are related with these phenotypes, which are positively regulated by Flo8p protein, showing fundamental role in the expression of the undesirable phenotypes. So, the present work aimed to analyze the phenotypes and the metabolic behavior of an industrial strain isolated directly from Brazilian ethanol production process (FT02), relating them with the Flo8p regulation. Our results showed that the Flo8p plays a fundamental role in the regulation of these characteristics in the industrial strain FT02. We also verified a strong tendency of the FLO11 gene participation in the foam formation, by Flo8p. Moreover, by FLO8 deletion in FT02 strain, we could identify the positive influences of the Flo8p in flocculation and cellular hydrophobicity, filamentation and invasive growth.

Gene regulatory networks are composed of interconnected regulatory nodes created by regulatory factors of multiple types. All organisms finely tune gene expression in order to adapt to and survive within their current niche. Obligate parasitic bacteria are under extreme pressure to quickly and appropriately adapt their gene regulatory programs in order to survive within their given host. Borrelia burgdorferi is one such organism and persists in nature by alternating between two hosts; Ixodes spp. ticks and small vertebrate animals. These two hosts represent drastically different environments; requiring a unique gene regulatory program to survive and transmit between them. Microbiologists have long sought to better understand exactly what stimuli pathogens sense and how that information is relayed in to physiologic adaptation. In this work I aimed to examine two parts of this interesting field. First, I sought to better understand the stimuli B. burgdorferi sense in order to adapt to their hosts by testing several hypotheses centered on the general notion that B. burgdorferi senses both internal and external metabolic cues as primary signals for adaptation. I demonstrated that a second messenger system immediately downstream of a critical metabolic pathway is important during vertebrate infection and that a key regulator of virulence is itself regulated by a factor involved in DNA replication. Second, I sought to better define the topology of gene regulatory networks, known and unknown, that are important for the ability of the bacteria to adapt. The work in this section focus on the idea that B. burgdorferi gene regulatory networks are extremely complex and are not currently well defined in the literature. My studies revealed that B. burgdorferi possesses a large number of previously undefined regulatory targets, including extended 5’ and 3’ UTRs of known genes, and encodes several hundred-putative small non-coding RNAs. Furthermore, I demonstrate that two essential regulatory factors share substantial, independent, overlap in their regulons highlighting the still undefined complexity of regulatory networks at play in B. burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, exists in a defined enzootic cycle involving Ixodes scapularis ticks and various vertebrates. Humans can serve as an accidental host, if a tick colonized with B. burgdorferi happens to feed on a human. B. burgdorferi are also accidental pathogens: they do not make toxins, or destroy host tissue by other mechanisms. They merely transmit between vector and host to survive. In order to do this, they must effectively sense their current environment, and appropriately alter cellular processes. Understanding the regulatory mechanisms of how B. burgdorferi manages to do this has been a focus of the Stevenson lab for many years. Previous work identified SpoVG as a DNA-binding protein. Although a homologue of this protein had been implicated to serve a regulatory role in other bacteria, the Stevenson lab was the first to demonstrate a function for the protein, both for B. burgdorferi and two other bacteria. Studies contained in this body of work aim to provide insight into regulation of SpoVG by B. burgdorferi as well the impact that it has on gene regulation. By using genetic mutants, we determined that SpoVG is regulated at the levels of transcription and translation in culture by growth rate, temperature, and other regulatory factors. Additionally, we provide evidence that SpoVG regulates its own expression. Numerous genes are under control of SpoVG. Biochemical analyses revealed that SpoVG specifically interacts with DNAs and RNAs associated with genes found to be under its regulatory control. Finally, we provide evidence for SpoVG acting in concert with other known regulatory factors such as other DNA-binding proteins and the cyclic di-nucleotide second messengers cyclic-di-GMP and cyclic-di-AMP. All together, these studies provide insight into how B. burgdorferi broadly regulates cellular processes during different stages of the enzootic cycle. We hypothesize that SpoVG does this through globally manipulating the three-dimensional structure of the bacterial chromosome, and that exactly how SpoVG acts at any given point will be dependent on the other regulatory factors that are also present in the cell.

Doctor of Philosophy (PhD) / Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.

Present in almost all living organisms, cytochrome P450s form one of the biggest enzyme superfamilies. They are versatile biocatalysts, capable of performing a range of biochemical reactions and are involved in a wide spectrum of biological functions. The vinegar fly, Drosophila melanogaster, has 85 P450s in its sequenced genome. Six of these have been found to catalyse the synthesis of the important insect molting hormone, 20-hydroxyecdysone and a handful have been implicated in insecticide resistance. The other P450s remained largely uncharacterised. / In the first half of this thesis, the expression patterns of P450s in the D. melanogaster genome were characterised by in situ hybridisation at the third instar larval stage. Most P450s have defined expression patterns at this stage of development. A majority of P450s are expressed in the midgut, Malpighian tubules and fat body, tissues that are involved in the metabolism of xenobiotics. Other P450s are expressed in specific tissues, such as the prothoracic glands, the salivary glands and the gonads, where they might have roles in development or reproduction. In particular, Cyp6g2 is expressed in the corpus allatum (CA), where it could play a role in juvenile hormone synthesis. An RNAi lethality screen using lines that were available from the Vienna Drosophila RNAi Centre identified a number of P450s which are essential for development and viability. / In the second half of the thesis, the transcriptional regulation of a P450 involved in insecticide resistance, Cyp6g1, was investigated. Cyp6g1 was regulated by two discrete cis-regulatory modules/enhancers, one controlling expression in the Malpighian tubules and one controlling expression in the midgut and fat body. Phenobarbital induction of Cyp6g1 is tissue-specific and is mediated by a fragment in the 5’ regulatory region that interacts with both enhancers. Characterisation of the long terminal repeat (LTR) of the Accord transposable element in the 5’ region of Cyp6g1, present in insecticide resistant populations, shows that the Accord LTR contains cis-regulatory elements which increase expression of Cyp6g1 in the fat body, midgut and Malpighian tubules, and contribute to insecticide resistance in these populations. / This study shows that the diverse tissue distribution of different P450s in D. melanogaster is related to the diverse biological functions of the enzymes encoded. This is exemplified by the detailed examination of the regulation of the insecticide resistance-conferring P450, Cyp6g1. Its expression pattern reflects its detoxification function in the fly. The role of transposable element insertions in changing gene expression patterns and contributing to selectable variation in genomes is also demonstrated through the Cyp6g1 study.

Previous work carried out in our laboratory has identified that phase variation of type III R-M systems found in Haemophilus influenzae, Neisseria meningitidis and N. gonorrhoeae is reversible, and occurs at high frequency, as seen both through mod::lacZ fusions, and by measuring changes in repeat tract length. In addition, phase variation of the methyltransferases results in coordinated switching of expression of a distinct group of genes in each of the strains studied so far. WE have termed this phenomenon the PHASEVARION, for phase variable regulon, to identify the set of genes whose expression is affected by moe phase variation. Many of the genes found to be regulated by mod phase variation are known virulence factors and even include some genes investigated as candidates for vaccine development (Srikhanta et al., 2005 and 2009. The aims of this project was to further the investigation of how these R-M systems regulated the expression of genes which hitherto had not been predicted to phase vary. The first step in the process of investigating how phase variable R-M systems influence expression of unrelated genes is to identify the DNA sequences methylated by the methyltransferases of interest. As discussed in Chapter 3, elucidation of the ModA1 methylation target site was in part facilitated by predictions that the phase variable methyltransferase found in H. influenzae strain Rd methylated the same sequence as did HinfIII, isolated from H. influenzae strain Rf. This hypothesis was confirmed by methylation dependent inhibition of digestion, revealing that ModA1 methylates the second A in its recognition sequence, 5’-CGAAT-3’. Once confirmed, the genes found to be regulated by modA1 phase variation in the initial phasevarion study could be investigated for the presence of ModA1 methylation sites within their promoters or upstream of their transcriptional regulators. Two such methylation target sites were located just upstream of the dnaK ORF. Transcriptional start site analysis of the dnaK gene revealed three transcripiotnal start sites, one of which is unduced by heat shock. Exactly 10 nucleotides upstream of this heat shock induced transcriptional start site lies one of these ModA1 methylation target sequences. Ongoing invetigations are looking into the importance of this ModA1 site located within the dnaK promoter, and whether this is the site responsible for ModA1 dependent variations in dnaK expression. Although numerous methods were investigated for their potential to identify all sites methylated by the different modA alleles, the only method which resulted in identification of any methylation target sites was methylation dependent inhibition of restriction. This method allowed us to confirm the ModA1 recongition sequence, and to discover the methylation sequence, and adenine targeted by the modA13 allele, which is found in many clinically relevant N. gonorrhoeae strains. As will be discussed in Chapter 5, ModA13 dependent inhibition of restriction was first observed when the Neisserial plasmid pCmGFP was extracted from modA13 ON and modA13::kan cells, and further investigated and confirmed using a Southern blot approach to determine whether ModA13 dependent inhibtion could be detected as differential methylation of the chromosome. It was found that ModA13 recognised the sequence 5’-AGAAA-3’, with methylation occurring on the second last A. This sequence was mapped not only to the genes found to be regulated by modA13 phase variation, but also to the entire FA1090 chromosome, and this information will be used in future studies to investigate the direct molecular mechanisms by which modA13 phase variation results in subpopulations with different phenotypes in relation to antimicrobial resistance and biofilm/cell invasion.

<p>Since plants can not escape their predators by walking, they use some other defense systems, like induction or repression of defense genes. A microarray experiment performed with barley attacked by the bird cherry-oat aphid (Rhopalosiphum padi), led to the hypothesis that contig 16360 (similar to ser/thr kinases) could be linked with the resistance of barley against R. padi, and contig 6519 (similar to WIR 1A) with the susceptibility. Time course experiments showed that contig16360 and AJ250283 (similar to BCI-4) are almost induced in the same way, each, by two different aphids (R. padi and Metopolophium dirhodum). Genomic PCR was used to test the hypothesis that when plants have the gene for contig 16360, they are more likely to be resistant against aphid attack, and when plants have the gene for contig 6519, they are more likely to be susceptible. This test was performed with 69 barley lines: wild, commercial or breeding lines. Results were that the presence of WIR 1A gene has no correlation with the susceptibility, while presence of ser/thr kinase seems to be correlated with resistance.</p>

<p>Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. The life cycle of this oncogenic DNA tumour virus is strictly associated with the differentiation program of the infected epithelial cells. Expression of the viral capsid genes L1 and L2 can only be detected in the terminally differentiated epithelial cells. The studies here focus on the regulation of HPV-16 late gene expression, which is under tight regulation. </p><p>Our experimental system consisted of almost the full length HPV-16 genome driven by a strong CMV promoter. This plasmid and mutants thereof could be transfected into HeLa cells and RNA levels monitored. Using this system, we identified an hnRNP A1-dependent splicing silencer between positions 178 and 226 of the L1 gene. This silencer inhibited the use of the 3' splice site, located immediately upstream of the L1 AUG. We speculate that this splicing silencer plays an essential role in preventing late gene expression at an early stage of the viral life cycle. We subsequently identified a splicing enhancer located in the first 17 nucleotides of L1 that may be needed to counteract the multiple hnRNP A1 dependent splicing silencers in the L1 coding region. A 55kDa protein specifically bound to this splicing enhancer. We also demonstrated that binding of the cellular factors to the splicing silencer in the L1 coding region had an inhibitory effect on expression from L1 cDNA expression plasmids.</p><p>The HPV-16 genome is divided into the early region and the late region, separated by the early poly(A) signal (pAE). pAE is used preferentially early in infection, thereby efficiently blocking late gene expression. We demonstrated that a 57 nucleotide U-rich region of the early 3’untranslated region (3’eUTR) acted as an enhancing upstream element on the usage of pAE. We demonstrated that this U-rich region specifically interacts with hFip1, CstF-64, hnRNP C1/C2 and PTB, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 pAE. </p><p>In conclusion, two regulatory RNA elements that both act to prevent late gene expression at an early stage in the viral life cycle and in proliferating cells were identified: a splicing silencer in the late region and an upstream u-rich element at the pAE.</p>