Rhodopsin, a visual pigment protein found in retinal photoreceptors, mediates vision at low-light levels. Recent studies focusing primarily in human and mouse have challenged the assumption of neutral evolution of synonymous substitutions in mammals. Using recently developed likelihood-based codon models accounting for mutational bias and selection, we find significant evidence for selective constraint on synonymous substitutions in mammalian rhodopsins, and a preference for cytosine at 3rd codon positions. A second project investigated adaptive evolution in rhodopsin, in view of theories of nocturnality in early mammals. We detected a significant acceleration of non-synonymous substitution rates at the origins of therian mammals, and a tendency of synonymous substitutions towards C-ending codons prior to that. These findings suggest an evolutionary scenario in which synonymous substitutions that increase mRNA stability and/or translation efficiency may have preceded adaptive non-synonymous evolution in early mammalian rhodopsins. These findings have important implications for theories of early mammalian nocturnality.

La génétique inverse est devenue une méthode clé pour la production de virus à ARN génétiquement modifiés et pour comprendre les propriétés cellulaires et biologiques des virus. Cependant les méthodes les plus fréquemment utilisées, basées sur le clonage de génomes viraux complets dans des plasmides, sont laborieuses et imprévisibles. La première partie de cette thèse présente des études sur la mise au point d'un nouveau système de génétique inverse, appelé méthode ISA (Amplicons-Sous génomique-Infectieux), qui permet la génération, en quelques jours, de virus infectieux sauvages et génétiquement modifiés appartenant à trois familles différentes de virus à ARN simple brin de polarité positive, avec une grande maîtrise des séquences virales. Dans la deuxième partie de cette thèse, nous avons appliqué pour la première fois à un arbovirus (CHIKV), le ré-encodage des codons - une méthode développée récemment et très excitante pour le développement de vaccins vivants atténués. En utilisant une approche aléatoire de ré-encodage des codons qui attribue au hasard des codons sur la base de la séquence en acides aminés correspondante, nous avons mis en évidence des pertes importantes de fitness réplicatif sur des cellules de primates et d'arthropodes. La diminution du fitness réplicatif est en corrélation avec le degré de ré-encodage, une observation qui peut aider à la modulation de l'atténuation virale. En utilisant l'expérience acquise avec le CHIKV, nous avons transposé avec succès ce mécanisme d'atténuation au JEV et amélioré notre maîtrise du processus d'atténuation en utilisant une combinaison de la synthèse de novo et de la méthode ISA. / Reverse genetics has become a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but the most commonly used methods, based on the preparation of plasmid-based complete viral genomes, are laborious and unpredictable. The first part of this thesis presents studies relating to the development of a new reverse genetics system, designated the ISA (Infectious-Subgenomic-Amplicons) method, which enabled the generation of both wild-type and genetically modified infectious viruses belonging to three different families of positive, single stranded RNA viruses within days with great control of the viral sequences. In the second part of this thesis, we applied for the first time to an arbovirus (CHIKV), codon re-encoding - a recently developed and very exciting method for the development of live attenuated vaccines. Using a random codon re-encoding approach which randomly attributed nucleotide codons based on their corresponding amino acid sequence, we identified major fitness losses of CHIKV in both primate and arthropod cells. The decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. Detailed analysis of these observed replicative fitness losses indicated that they are the consequence of several independent re-encoding induced events. Using the experience acquired on the CHIKV, we successfully transposed this attenuation mechanism to JEV and improved our control of the attenuation process by using a combination of de novo synthesis and the ISA method.

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Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five regardless of hygiene or water quality. The currently licensed vaccines succeeded in reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the vaccines in developing countries. One of the main reasons for this can be due to the difference in strains, since the strains used to develop the currently licensed vaccines (RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2, G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A second shortcoming of the currently licensed vaccines is the cost of these vaccines. The vaccines are very expensive and most developing countries cannot afford the vaccines as well as the fact that the manufacturing companies cannot produce enough vaccines for all the countries. An attractive alternative to the currently licensed rotavirus vaccines is the non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer and efficacious alternative or complement the currently licensed vaccines. Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/ ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously obtained from a stool sample and the nucleotide consensus sequence was determined for both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised coding regions or open reading frames were used in this study. The open reading frames (ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria. The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa). Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon optimised genome segments into the expression vector. Only the expression of the VP6 protein in bacteria was observed with Coomassie stained SDS-PAGE. The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild type GR10924 G9P[6] strain were cloned into the wide range yeast expression system vector, pKM173, which allows for the simultaneous expression of more than one gene. Several yeast strains were used in this study namely Kluyveromyces marxianus, Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for western blot analysis to evaluate successful integration into the yeast genomes. Only a few of the colonies contained either both of the genome segments or only one of the two genome segments of interest. To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924 G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

Rotavirus infection causes severe gastroenteritis, affecting all children under the age of five regardless of hygiene or water quality. The currently licensed vaccines succeeded in reducing diarrhoea worldwide, but they still have shortcomings, especially the efficacy of the vaccines in developing countries. One of the main reasons for this can be due to the difference in strains, since the strains used to develop the currently licensed vaccines (RotaTeq and Rotarix) were selected from strains circulating in the developed world (G1, G2, G3 and G4), while the main strains present in Africa (G8, G9 and G12) were not included. A second shortcoming of the currently licensed vaccines is the cost of these vaccines. The vaccines are very expensive and most developing countries cannot afford the vaccines as well as the fact that the manufacturing companies cannot produce enough vaccines for all the countries. An attractive alternative to the currently licensed rotavirus vaccines is the non-live vaccine candidate, virus-like particles, which can provide a possible cheaper, safer and efficacious alternative or complement the currently licensed vaccines. Therefore, in this study a South African G9P[6] rotavirus strain, RVA/Humanwt/ ZAF/GR10924/1999/G9P[6], was used to determine whether or not co-expression of the structural proteins VP2 (genome segment 2) and VP6 (genome segment 6) was possible in bacteria and yeast. The South African GR10924 G9P[6] neonatal strain was previously obtained from a stool sample and the nucleotide consensus sequence was determined for both genome segment 2 (VP2) and genome segment 6 (VP6). Bacterial codon optimised coding regions or open reading frames were used in this study. The open reading frames (ORFs) of the genome segments encoding, VP2 and VP6, were cloned into the expression vector pETDuet-1, which allows for the simultaneous expression of two genes in bacteria. The ORF of genome segment 6 was purchased from GeneScript and the ORF of genome segment 2 was obtained from Dr AC Potgieter (Deltamune (Pty) Ltd R&D, South Africa). Compatible restriction enzyme sites were used to sub-clone the ORF of the bacterial codon optimised genome segments into the expression vector. Only the expression of the VP6 protein in bacteria was observed with Coomassie stained SDS-PAGE. The ORFs encoding VP2 (genome segment 2) and VP6 (genome segment 6) of the wild type GR10924 G9P[6] strain were cloned into the wide range yeast expression system vector, pKM173, which allows for the simultaneous expression of more than one gene. Several yeast strains were used in this study namely Kluyveromyces marxianus, Kluyveromyces lactis, Candida deformans, Saccharomyces cerevisiae, Yarrowia lipolytica, Arxula adeninivorans, Hansenula polymorpha and Debaryomyces hansenii. Expression of both proteins was not detected in the several yeast strains, as seen with western blot analysis. DNA extractions were done on two colonies of each yeast strain that were used for western blot analysis to evaluate successful integration into the yeast genomes. Only a few of the colonies contained either both of the genome segments or only one of the two genome segments of interest. To summarise, the simultaneous expression of VP2 and VP6 from rotavirus GR10924 G9P[6] was not successful in bacteria or yeast, but it was possible to soluble express the bacterial codon optimised GR10924 G9P[6] VP6 in bacteria using the pETDuet-1 as expression vector. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

A fragmentação e a invasão de florestas tropicais e a crescente concentração de assentamentos humanos aumentaram exponencialmente as chances de exposição a vírus emergentes e emergentes. Dado o grande potencial de espalhamento de patógenos em população humanas, a identificação e caracterização de agentes patogênicos circulantes podem melhorar a atenção primária e as capacidades de diagnóstico para um agente emergente futuro. As abordagens moleculares e metagenômicas que utilizam as tecnologias de sequenciação da próxima geração levaram a descoberta e caracterização de muitos vírus emergentes na última década. Além disso, as abordagens in silico também podem ajudar a identificar vírus emergentes usando apenas dados de sequenciamento publicamente disponíveis. Além disso, estimar a ascendência filogenética e até mesmo analisar as mudanças no uso de codons são ferramentas adicionais que podem melhorar a nossa compreensão de vírus emergentes ou reemergentes. Este projeto visou aplicar essas ferramentas em ambos os vírus que poderiam estar circulando no Brasil: Parvovírus B19 e vírus da Febre Amarela. Também exploramos as aplicações de modelos ocultos de Markov e índice de adaptação de codons usando dados publicamente disponíveis. Esperamos que este trabalho forneça uma prova de conceito para futuros projetos metagenômicos e demonstre a utilidade das várias técnicas moleculares e bioinformáticas no estudo de vírus emergentes. / Fragmentation and encroachment of tropical rainforests and the growing concentration of human settlements have exponentially increased chances of exposure to re-emerging and emerging viruses. Given the large potential for pathogens to spillover and spread in a population, identifying and characterizing circulating human pathogens could improve the readiness and diagnostic capabilities for a future emergence. Molecular and metagenomic approaches using next-generation sequencing technologies have led to the discovery and characterization of many emerging viruses over the last decade. In complement, in silico approaches can also help identify emerging viruses using only publicly available sequencing data. Moreover, estimating the phylogenetic ancestry and even analyzing changes in codon usage are additional tools that can improve our understanding of an emerging or re-emerging virus. This project aimed to apply these tools to two viruses that could be circulating in Brazil: Parvovirus B19 and Yellow Fever virus. We also explored the applications of Hidden Markov models and codon adaptation index using publicly available data. We expect this work to provide a proof-of-concept for future metagenomic projects, and demonstrate the utility for several molecular and bioinformatics techniques in the study of emerging viruses.

Nos últimos anos, inúmeros estudos epidemiológicos evidenciaram a forte associação entre o carcinoma do colo uterino e a infecção por papilomavírus humano (HPV). Esta associação deriva do reconhecimento de que estes vírus codificam oncoproteínas, dentre as quais E6 e E7, que apresentam propriedades transformantes. O produto do gene E7 se liga ao produto do gene retinoblastoma que perde a sua função de regular negativamente o ciclo celular. O produto do gene E6 se liga ao produto do gene supressor de tumor p53 levando a sua degradação pela via de proteólise dependente de ubiquitina. O gene p53 é um supressor tumoral com função de regulação do ciclo celular que apresenta vários polimorfismos distintos em diversos grupos étnicos e tem sido amplamente estudado tanto em tecidos normais quanto em tecidos tumorais. O polimorfismo do códon 72 do gene i>p53 é o mais estudado e pode apresentar três alelos diferentes na população. Um alelo codifica arginina (Arg), um codifica prolina (Pro) e outro, raramente encontrado, codifica cisteína (Cys). Em 1993 foi iniciado um estudo epidemiológico da história natural da infecção por HPV e neoplasia da cérvice uterina em uma população feminina de baixa renda em São Paulo (Brasil), uma das áreas de maior risco em todo o mundo. O estudo focaliza a infecção persistente por tipos oncogênicos de HPV como evento precursor que leva à carcinogênese do colo do útero e visa entender os atributos da história natural da infecção viral e das doenças associadas ao colo uterino. Um dos objetivos deste estudo é avaliar se o polimorfismo do códon 72 do gene p53 pode, ou não, ser utilizado como marcador de predisposição ao câncer do colo do útero uma vez que um estudo inicial relatou que pacientes portando o genótipo p53Arg homozigoto seriam 7 vezes mais susceptíveis ao desenvolvimento de neoplasia da cérvice uterina que pacientes contendo o genótipo p53Pro e heterozigoto p53Pro/ Arg. Contudo, vários estudos posteriores contradizem e corroboram esses achados. O presente projeto teve como objetivos, portanto, verificar se o polimorfismo do códon 72 do gene p53 poderia estar associado a infecções persistentes por HPV e ao risco de neoplasia do colo do útero, além de comparar metodologias de detecção utilizadas por outros estudos, visando esclarecer se os motivos que levam à discordância dos resultados podem ser atribuídos a ocorrência de erros classificatórios metodológicos. Ao todo, sete metodologias de detecção foram comparadas. Apenas uma delas, PCR alelo-específica, apresentou resultado discordante das demais utilizadas. Coincidentemente, essa metodologia foi amplamente utilizada por muitos estudos que encontraram associações tanto positivas quanto negativas. Isso poderia nos dar indícios de que os erros classificatórios dependentes de metodologia poderiam influenciar os resultados de correlação entre o polimorfismo do códon 72 e o risco de neoplasia do colo do útero. As correlações observadas por este trabalho entre este polimorfismo do códon 72 e o risco de neoplasia do colo uterino não mostraram associação deste polimorfismo com o risco de infecções persistentes por HPV e as lesões precursoras do carcinoma do colo uterino. / In recent years, a number of epidemiological studies have pointed toward a strong association between cervical cancer and infection by Human Papillomavirus (HPV). This association derives from the discovery that these viruses code for oncoproteins, among them E6 and E7 that have transforming properties. The E7 gene product associates with the retinoblastoma gene product, causing the latter to lose its function as a negative regulator of the cell cycle. The E6 gene product interacts with the tumor suppressor p53 gene product, resulting in its degradation via ubiquitin dependent proteolysis. The p53 gene is a tumor suppressor that funcions in the regulation of the cell cycle. It presents a number of distinct polymorfisms in diverse ethnic groups, and has been widely studied, both in normal and tumor tissues. The polymorfism of codon 72 is the most studied, and may present three different alleles in the population. One allele codes for arginine (Arg), another codes for proline (Pro), and a third, rarely found, codes for cystein (Cys). In 1993 an epidemilogical study of the natural history of infection by HPV and its possible association with cervical neoplasia was initiated in a population of low income females in São Paulo, Brazil, one of the areas of greatest risk in the world. The study focuses on persistent infection by oncogenic types of HPV as a precursor to carcinogenesis of the cervix, and seeks to understand the attributes of the natural history of viral infection and of illnesses associated with the cervix. One of the objectives of the study is to evaluate if the polymorfisms of codon 72 of p53 can or not be used as a marker of predisposition to cervical cancer, given the finding in the initial study that patients who were homozygous for the p53Arg genotype were 7 times more susceptible to developing cervical neoplasias than those patients who were homozygous for p53Pro, or heterozygous p53 Pro/ Arg. Previous studies have been realized both supporting and disputing these findings. The current study had two main objectives: to verify if the polymorfism of p53 codon 72 could be associated with persistent infections of HPV and the risk of cervical neoplasia, as well as to compare methods of detection used by other studies, in an attempt to clarify if the discording results of past studies could be due to methodological classification errors. Seven detection methods were compared. Only one of these, allele specific PCR, presented discording results from the rest. Coincidentally, this method was widely used in a number of studies which found both positive and negative associations. This might indicate that the method-dependent classification errors could influence the results of correlation between codon 72 polymorphism and the risk of cervical neoplasia. The correlations observed by this study did not demonstrate an association between codon 72 polymorphism and the risk of persistent HPV infection and precursor lesions of cervical cancer.

Unnatural amino acids (uAA) expand the structural and functional possibilities of proteins. Numerous previous studies have demonstrated uAA as a powerful tool for protein engineering, but challenges also remain. Three notable such challenges include: (1) the fitness of uAA-incorporated proteins are difficult to predict and time-consuming to screen with conventional methods, (2) uAA incorporation in difficult-to-express proteins (e.g. membrane proteins such as G-protein coupled receptors) remain challenging, and (3) the incorporation of multiple types of uAA are still limited. In response, we pose cell-free protein synthesis (CFPS), a rapid and versatile in vitro expression system, as a platform to explore solutions to these challenges. The "cell-free" nature of CFPS enables it to accelerate protein expression and tolerate extensive modifications to its translational environment. In this work, these advantages were utilized to address the aforementioned challenges by: (1) rapidly expressing and screening uAA-containing proteins, (2) incorporating uAA in functional G-protein coupled receptor in the presence of membrane-mimicking lipid additives, and (3) engineer the translational environment extensively towards multiple uAA incorporation.