Hepatitis C virus (HCV) causes severe illness in millions of people worldwide, but the epidemic strains responsible for most infections arose within the past hundred years and represent only a small part of total HCV diversity. In this thesis I combine laboratory and computational methods to study HCV in Africa. I aim to characterize its current genetic diversity and its historical transmission prior to the global HCV epidemic. In Chapter 2 I begin by screening samples from the Democratic Republic of the Congo (DRC) for HCV and the related human pegivirus. I find high HCV sequence diversity, including a putative new subtype, and find significantly higher HCV prevalence in those born before 1950. Chapter 3 continues this screening, and combines the sequences obtained with those from online databases. Using molcular clock methods I estimate that genotype 4 originated in central Africa around 1733, and that multiple lineages, including subtype 4a which dominates the HCV epidemic in Egypt, have moved to north Africa since ~1850. In Chapter 4 I analyse sequences sampled from an elderly population in Kinshasa to estimate HCV’s transmission history there during the 20th century. The results indicate a rapid increase in HCV transmission between 1950 and 1970 in multiple independent lineages. Possible causes of this increase are discussed. This study population also exhibits high HCV genetic diversity, including the second genotype 7 sample discovered to date. Finally, Chapter 5 uses a range of sequencing techniques, including RNAseq, to characterise two putative HCV recombinants from Cameroon. I confirm that both sequences are recombinants, and generate a full genome sequence for one. I also develop new tools to distinguish between dual infection and recombination in next-generation sequencing data, and discuss how recombination might affect HCV diversity and treatment.

Electron-ion recombination has been under study for many years, but comparisons between theory and experiment have been very difficult, especially for conditions where the ion under evaluation was a minority in concentration. This study describes a direct measurement of the recombination-rate coefficient for the recombination of minority as well as majority ions in the afterglow.

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are linked to breast as well as ovarian cancers. However, most cancer-causing mutations within the BRCA1 gene have been found in the N’ and C’ terminal regions of the BRCA1 protein, both believed to be important for DNA double-strand break (DSB) repair. The BRCA1 C’ terminal (BRCT) repeats have been implicated in phospho-serine protein binding whereas the N’ terminal RING domain interacts with the BARD1 protein to form a hetero-dimeric complex with E3 ubiquitin ligase activity. The BRCA1 BRCT domain binds CtIP, BACH1, and RAP80, all of which have been directly implicated in homologous recombination repair (HRR). Lysine 1702 (K1702) of BRCA1 resides within the phospho-serine binding pocket of the first BRCT repeat of BRCA1. To determine the effect of manipulating the ability of BRCA1 to bind CtIP and other phospho-proteins binding to the BRCA1 BRCT domain on DSB repair, and specifically HRR, we introduced a K1702M mutation into BRCA1 known to impair BRCT binding to a pSer-X-X-Phe peptide representing BACH1. Surprisingly, instead of impairing HRR, we found that BRCA1 K1702M resulted in hyper-recombination with > 3-fold higher levels of HRR compared to wild-type BRCA1 using an HRR assay based on GFP expression in BRCA1-defective HCC1937 cells. This hyper-recombinogenic phenotype coincided with cell-cycle arrest in S/G2 suggesting that the potential lack of binding of critical proteins to the BRCA1 BRCT domain results in abnormal HRR by priming cells to undergo more HRR which is enhanced during the S and G2 phases of the cell-cycle. In line with the increased HRR seen with the HRR/GFP assay, HCC1937 cells expressing BRCA1 K1702M showed increased levels of RAD51 foci and nuclear staining suggesting that HRR was highly elevated. Interestingly, the hyper-recombinogenic phenotype of BRCA1 K1702M could be reduced to normal levels with a second mutation (I26A) in BRCA1 that affects BRCA1 and CtIP ubiquitination. These results reveal a hierarchal regulation of HRR with ubiquitination having a dominate role in DSB repair by BRCA1 and suggests that targeted disruption of BRCT-CtIP binding increases HRR that is in turn controlled by ubiquitination. In addition, we provide evidence that BRCA1 serine 1387 phosphorylation within the SQ cluster region of BRCA1 is involved in the cell survival and DNA damage response to IR. The BRCA1 S1387A mutant only partially increased the radiosurvival of HCC1937 cells compared to cells expressing wild-type BRCA1 and immunocytochemical analysis revealed wild-type BRCA1 was located in the nucleus whereas the S1387A mutant was cytoplasmic in response to IR. We also show that BRCA1 SQ cluster serine phosphorylation in addition to serine 1387 is involved in HRR. Altogether, these findings reveal the importance of various regions of BRCA1 in DSB repair and may lead to multiple strategies of modulating BRCA1 function in response to DNA damage.

Chez Saccharomyces cerevisiae, la methylation de la lysine 4 de l'histone H3 (H3K4) est catalysée par le complexe à activité methyltransférase Set1, conservé au cours de l'évolution. Durant la méiose, l'absence de Set1 conduit à un retard de démarrage de la phase S, et à un défaut dans la formation des coupures double-brin de l'ADN (CDBs). Nous avons cherché à mieux caractériser ces deux conséquences phénotypiques liées à l'absence de Set1. Nous montrons que le retard de réplication est lié à la perte de méthylation de H3K4 mais qu'il ne résulte pas d'un défaut d'activité des kinases responsables de l'activation des origines de réplication ou de l'activation des voies canoniques de surveillance moléculaire liées aux dommages de l'ADN. L'importante diminution de fréquence de CDB sur la majorité des points chauds chez le mutant set1∆ a été corrélée à l'absence de la marque de H3K4 triméthylée. Nous avons confirmé le role de la méthylation de H3K4 sur la base de la diminution générale de la fréquence des CDBs observée en absence des différentes sous-unités du complexe associé à Set1 (COMPASS) ou chez un mutant exprimant une histone H3 non-méthylable (H3K4R). Pour tester la relation de causalité entre méthylation et CDBs, différentes sous-unités du COMPASS, telles que Set1 et Spp1, ont été fusionnées avec le domaine de fixation à l'ADN de Gal4 pour les cibler vers des régions non méthylées et dépourvues de CDB. Gal4BD-Spp1 stimule fortement la fréquence des CDBs à certains loci, y compris en contexte mutant H3K4R. Ainsi, le ciblage de Spp1 peut etre suffisant pour recruter et/ou activer la machinerie de CDB. / In Saccharomyces cerevisiae, the methylation of the lysine 4 of histone H3 (H3K4) is catalysed by the evolutionary conserved Set1 methyltransferase complex. During meiosis, the absence of Set1 leads to a delay of S-phase onset and to a defect in the formation of double-strand breaks (DSBs). Our work was intended to give some clues about these two phenotypic consequences of Set1 loss. We show that the replication delay is linked to the absence of H3K4 trimethylation but does not result from a defect of the kinases responsible for the activation of replication origins or the activation of the canonical DNA-damage checkpoints. The severe decrease of DSB levels at the majority of recombination hotspots in set1∆ has been correlated with the specific marking of DSB sites by H3K4 trimethylation at some loci. We have confirmed the role of H3K4 methylation by observing a general decrease in DSB frequency similar to that of set1∆ in mutants lacking various subunits of the Set1- associated complex (COMPASS) or expressing a nonmethylatable histone H3 (H3K4R). To test for a causal relationship between H3K4 methylation and DSB formation, we have fused different proteins of the COMPASS, such as Spp1 or Set1, with the DNA binding domain of Gal4, in order to target them to H3K4-unmethylated and DSB-cold regions. Remarkably, Gal4BD-Spp1 strongly stimulates DSB formation in naturally cold DSB regions, even in the H3K4R mutant context. Thus, the specific tethering of Spp1 to a chromosome site is sufficient to recruit and/or activate the DSB machinery.

Successful bacterial circular chromosome segregation requires that any dimeric chromosomes, which arise by crossing over during homologous recombination, are converted to monomers. Resolution of dimers to monomers requires the action of the XerCD site-specific recombinase at dif in the chromosome replication terminus region. This reaction requires the DNA translocase, FtsK(C), which activates dimer resolution by catalysing an ATP hydrolysis-dependent switch in the catalytic state of the nucleoprotein recombination complex. We show that a 62-amino-acid fragment of FtsK(C) interacts directly with the XerD C-terminus in order to stimulate the cleavage by XerD of BSN, a dif-DNA suicide substrate containing a nick in the 'bottom' strand. The resulting recombinase-DNA covalent complex can undergo strand exchange with intact duplex dif in the absence of ATP. FtsK(C)-mediated stimulation of BSN cleavage by XerD requires synaptic complex formation. Mutational impairment of the XerD-FtsK(C) interaction leads to reduction in the in vitro stimulation of BSN cleavage by XerD and a concomitant deficiency in the resolution of chromosomal dimers at dif in vivo, although other XerD functions are not affected.

How life originated from physical and chemical processes is one of the great questions still unanswered today. Studies towards this effort have transitioned from the notion of a single self-replicating entity to the idea that a network of interacting molecules made this initial biological leap. In order to understand the chemical kinetic and thermodynamic mechanisms that could engender pre-life type networks we present an empirical characterization of a network of RNA recombinase molecules. We begin with 1-, 2-, and 3-molecular ensembles and provide a game theoretic analysis to describe the frequency dependent dynamics of competing and cooperating RNA genotypes. This is then extended to 4- and 5-membered networks where varying topologies are compared and mechanisms that could lead to preferential growth and selection of genotypes are described. At the core of these network connections is ribozyme catalysis initiated through a 3-nucleotide base-pairing interface. With the development of a fluorescence anisotropy method, we are able to illustrate a correlation between these binding thermodynamics and network outcomes. Finally, we consider how the heterogeneity of the environment could impact network dynamics and develop a spectrum of spatial inducing methods in which our chemical populations can be probed. These experiments illustrate simple chemical dynamics of RNA interactions, yet these very processes are the foundation for building complexity and ultimately from where selection and evolvability derive.

Glutamic Acid Decarboxylase (GAD) is a rate-limiting enzyme responsible for synthesis of the inhibitory neurotransmitter GABA. Dopaminergic denervation in rodents by unilateral injections of 6-OHDA or MPTP causes an increase in Gad67 mRNA in the striatum, which is further exacerbated by administration of L-Dopa (Horvath et al., 2011; Katz et al., 2005 Bacci et al., 2002). Denervation of nigrostriatal neurons is the key pathological hallmark of Parkinson's disease, which results in hypokinetic movement and rigidity. Medium spiny projection neurons of the striatum comprise 95% of the neuronal population and utilize Gad67 (encoded by the Gad1 gene) for the synthesis of basal levels of GABA. The contribution of Gad67 to GABA signaling in medium spiny projection neurons in the striatum has not been thoroughly understood in normal or Parkinsonian states. Mice with a deletion in Gad67 in Gpr88 expressing neurons were generated by crossing mice with a floxed exon 2 of Gad1 with mice expressing Cre recombinase under the control of the Gpr88 promoter. The aim of this study was first, to characterize mice with a deletion in striatal Gad67 by immunohistochmical, electriophysiological and behavioral examination to determine whether Gad67 expression contributes to sensorimotor and learning tasks. And next, to investigate whether a downregulation in striatal Gad67 would decrease dyskinesia and affect the impaired motor symptoms following dopaminergic denervation with a unilateral 6-OHDA lesion and subsequent treatment with L-Dopa. In this study, neuronal Gpr88 expression was indicated by GFP reporter expression, which resulted from Cre-mediated excision of exon 2 of the Gad1 gene. Gpr88 expression was confirmed in the striatum, olfactory tubercle, cortex and brain stem. Furthermore, Gpr88 was confined to striatonigral and striatopallidal MSNs in the striatum. Additionally, Cre-mediated GFP reporter expression indicated that Gpr88 expression occurs throughout various brain regions, including the motor and visual areas of the cortex, amygdala, hippocampus and cerebellum during development. The developmental expression of Gpr88 seems to be a highly regulated process that occurs throughout the brain. In the conditional knockout mouse, deleting striatal Gad67 resulted in an upregualtion of Gad67 in the globus pallidus and downregulation in the substantia nigra. The changes in Gad67 expression indicate the effects of inactivating GABAergic signaling in striatonigral and striatopallidal MSNs in the direct and indirect pathways. Mice with a deletion in striatal Gad67 demonstrated compromised performance in spatial learning in the Morris water maze, suggesting that GABAergic striatal signaling in the direct and indirect pathways accounts for cue-based learning and spatial memory. However, inactivation of GABAergic signaling in striatonigral and striatopallidal MSNs does not account for motor deficits such as bradykinesia, akinesia or hypokinesia in intact mice; instead it perpetuates hyperkinetic motor activity. In the second experiment of this study, dopaminergic denervation by a unilateral 6-OHDA lesion induced bradykinesia and hypokinetic motor behavior, as demonstrated by impaired performance in the rota-rod and pole test. Additionally, L-Dopa administration to 6-OHDA lesioned mice evoked abnormal involuntary movements (AIMs) to the same degree in all dyskinetic mice. A deletion in striatal Gad67 did not decrease symptoms of dyskinesia, nor cause a lessening of motor impairment caused by dopaminergic denervation. Complete inactivation of the indirect pathway is believed to limit the inhibition of unwanted actions and may perpetuate dyskinesia, even when striatonigral MSNs of the direct pathway are inactive.

O eucalipto é amplamente cultivado em diversos países, dentre os quais, o Brasil se destaca pela sua alta produção. A cultura tem grande importância comercial e atende à uma ampla variedade de setores do mercado, entre eles o de celulose. Apesar disso, a cultura ainda está nas fases iniciais de domesticação devido, principalmente, ao seu longo ciclo reprodutivo e tempo de rotação, uma vez que os cortes são feitos entre 5 e 15 anos. A aplicação de tecnologias de marcadores moleculares é uma proposta promissora para acelerar o melhoramento do eucalipto. Com o desenvolvimento de metodologias modernas de sequenciamento tornou-se acessível a obtenção de grande quantidade de marcadores a baixo custo. Uma das aplicações de tais marcadores é a construção de mapas genéticos de ligação, os quais permitem a caracterização genética de caracteres quantitativos, além de estudos comparativos entre populações e o auxílio na montagem de genomas. No presente trabalho, objetivou-se a construção de um mapa genético integrado em uma progênie F1 segregante com 200 indivíduos, proveniente do cruzamento entre Eucalyptus grandis e Eucalyptus urophylla. Para identificação dos marcadores, foi realizado o ressequenciamento do genoma completo (WGS) dos genitores e a genotipagem por sequenciamento (GBS) da progênie. A metodologia de construção de mapa foi adaptada para o conjunto de dados obtido, que apresenta grande quantidade de marcadores do tipo SNP, pouco informativos e com maior probabilidade de erro comparado com os marcadores tradicionais mais utilizados nos últimos anos. Para isso foram propostas duas estratégias: i) utilização da posição dos marcadores no genoma de referência para auxílio na ordenação dos marcadores no mapa; ii) alteração do parâmetro de probabilidade de erro da abordagem implementada no software Onemap. O mapa obtido apresentou padrão de taxa de recombinação semelhante a outros mapas construídos para eucalipto. O mapa apresentou tamanho total de 1471.91 cM e 1512 marcadores, com distância média entre eles de 1.85 cM. Os marcadores formaram 11 grupos de ligação, que corresponderam aos cromossomos do genoma de referência. Em média, foram cobertos 96.8% dos cromossomos. Também foram agrupados, junto aos 11 grupos, 61 marcadores localizados em outros scaffolds no genoma de referência, os quais podem servir para elucidação na montagem destes. Utilizando as estratégias propostas, foi obtido um mapa integrado adequado para o experimento em questão, considerando o tamanho da população de mapeamento. / The eucalyptus is widely cultivated in several countries, among which Brazil is highlighted by its high production. This culture has great comercial importance and supplies a wide variety of markets, including cellulose. However, the culture is still in the early stages of domestication due, mainly, to its long reproductive cycle and rotation time, since cuts are made between 5 and 15 years. The application of molecular marker technologies is a promising proposal to accelerate the improvement of eucalyptus. By the development of modern sequencing methodologies it was possible to obtain a large quantity of markers at low cost. One of the applications of such markers is the construction of genetic linkage maps, which allow the genetic characterization of quantitative traits, as well as comparative studies between populations and the support in the assembly of genomes. In the present work, the aim was to construct an integrated genetic map in a segregating F1 progeny with 200 individuals, derived from the cross between Eucalyptus grandis and Eucalyptus urophylla. For the identification of the markers, it was performed a complete genome re-sequencing (WGS) of the parents and genotyping-by-sequencing (GBS) of the progeny. The mapping methodology was adapted to the obtained data set, which presents a large amount of SNP-type markers, with little information and with a greater probability of error compared to the most used traditional markers in the last years. For this, two strategies were proposed: i) use of the position of the markers in the reference genome to aid in the ordering of the markers on the map; ii) change of the error probability parameter in the approach implemented in the software Onemap. The obtained map showed recombination rate pattern similar to other maps constructed for eucalyptus. The map presented a total size of 1471.91 cM and 1512 markers, with a mean distance between them of 1.85 cM. The markers formed 11 linkage groups, which corresponded to chromosomes of the reference genome. On average, 96.8 % of chromosomes were covered. 61 markers located in other scaffolds in the reference genome were grouped with the 11 groups. They may serve to elucidate the assembly of these. Using the proposed strategies, a suitable integrated map was obtained for the present experiment, considering the size of the mapping population.

Neste trabalho, investigamos a influência da estrutura de energia das minibandas dos estados eletrônicos ocupados no tempo de recombinação em poços quânticos múltiplos (MQW) fracamente acoplados de GaAs / AlGaAs. Um dos melhores métodos para estudar o efeito da estrutura energética consiste em medir o tempo de recombinação eletrônica em função de parâmetros expostas à influência externa que afeta a estrutura energética, por isso, aplicamos um campo magnético externo. O espectro da emissão de fotoluminescência foi composta pelas contribuições das minibandas da banda de condução, Γ – Γ e Γ – XZ. Observou-se um aumento notável do tempo de recombinação quando o campo magnético causou a despopulação da minibanda de maior energia, Γ – XZ. O efeito observado é atribuído à variação induzida pelo campo magnético na densidade dos estados eletrônicos. / In the present work, we investigate the influence of the miniband energy structure of the populated electron states on the recombination time in GaAs/AlGaAs weakly coupled multiple quantum wells (MQW). The best method to study the effect of the energy structure is to measure the recombination time in the same sample subject to external influence which affects the energy structure, therefore, we apply an external magnetic field. The photoluminescence emission was composed of the contributions from the Γ – Γ and Γ – XZ conduction band minibands. Remarkable enhancement of the recombination time was observed when the magnetic field caused depopulation of the higher energy Γ – XZ miniband. The observed effect is attributed to the magnetic field induced variation of the electron density of states.

Os rotavírus, membros da família Reoviridae, são uma importante causa de diarreia em mamíferos e aves. São partículas icosaédricas não envelopadas e seu genoma é formado por 11 segmentos de RNA fita dupla que codificam seis proteínas estruturais e seis proteínas não estruturais. O objetivo deste estudo foi caracterizar os genes codificadores das proteínas não estruturais (NSP1-5) e estruturais (VP1-4, VP6-7) dos rotavírus do grupo A em aves de criações comerciais (corte, postura, matrizeiros e avozeiros) localizadas em 12 estados brasileiros, seguido de análises de recombinação e pressão de seleção das amostras definidas. Um total de 226 amostras fecais foram triadas através de reações de RT-PCR tendo como alvo a amplificação da VP6 e NSP5. A frequência de ocorrência, baseada em cada uma destas provas, variou de 9,7% a 18,14%, respectivamente. Em seguida, 10 das amostras positivas foram processadas com primers específicos visando a amplificação dos demais genes, seguido do sequenciamento nucleotídico e filogenia baseada no método de maximum likelihood, tendo como modelos de substituição GTR (NSP1-3, VP1-3, VP4, VP6, VP7) e HKY (NSP4, NSP5) e 1.000 repetições de bootstrap. Foram definidas sequências parciais para os genes codificadores da VP1-4, VP6-7 e NSP1-4 e sequências completas para NSP5. As respectivas árvores demonstraram que as dez amostras definidas se agruparam em clados aviários previamente descritos. Duas constelações genotípicas foram caracterizadas: G19-P[31]-I11-R6-C6-M7-A16-N6-T8-E10-H8 e G19-P[31]-I4-R4-C4-M4-A16-N4-T4-E4-H4. Estes genotipos são tipicamente encontrados em aves, mas quando analisados em conjunto, esta é a primeira descrição destas constelações. Eventos de recombinação foram observados nos genes NSP2, VP1, VP3 e VP7. Pelo menos um códon com pressão de seleção positiva foi encontrado nos genes codificadores das proteínas NSP1, VP2 e VP3. Este estudo propicia um melhor entendimento acerca da epidemiologia e diversidade viral circulante nas criações aviárias brasileiras, servindo de base para o estabelecimento de medidas profiláticas mais eficazes. / Rotaviruses are members of the Reoviridae family and they are a common cause of acute diarrhea in several mammalian and avian species. They are non-enveloped icosahedral particles and its genome comprises 11 segments of double-stranded RNA, which encodes six structural proteins (VP1-4, VP6-7) and six nonstructural proteins (NSP1-6). The objective of this study was to characterize the RVA nonstructural and structural proteins coding genes (NSP1-NSP5, VP1-VP4, VP6 and VP7) from fecal samples from avian farms (broiler breeders, poultry, laying hens, and grandparents) raised in Brazilian commercial farms from 12 states, followed by recombination and selection pressure analysis from samples defined here. A total of 226 fecal samples were screened using a RT-PCR technique targeting the amplification of the VP6 and NSP5. The frequency of occurrence, using these techniques, ranging from 9.7% to 18,14%, respectively; and from these, ten samples were further processed with specific primers to amplify the remaining genes, followed by respective nucleotide sequencing of the amplicons and phylogeny based on method maximum likelihood, as substitutions models GTR (NSP1-3, VP1-3, VP4, VP6, VP7) and HKY (NSP4, NSP5) and 1.000 bootstrap repetitions. Partial nucleotide sequences of VP1-4, VP6-7, and NSP1-4, and complete from NSP5, were obtained in this study. The phylogenetic trees depicted that the ten Brazilian rotavirus strains segregated with previous avian RVA described elsewhere. Two avian genotype constellations have been characterized here: G19-P[31]-I11-R6-C6-M7-A16-N6-T8-E10-H8, and G19-P[31]-I4-R4-C4-M4-A16-N4-T4-E4-H4. These genotypes are typically found in avian species, although when analyzed together, this is the first report of such constellations. Recombination events were observed in NSP2, VP1, VP3, and VP7 coding genes. At least on positive selected site was observed in NSP1, VP2, and VP3 genes. This study provides a better understanding of rotavirus epidemiology, by the definition of genetic variability of circulating strains.