Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: We construct and study a simple model for an active gel of exible polymer filaments crosslinked by a molecular motor cluster that perform reversible work while translating along the filaments. The filament end points are crosslinked to an elastic background. In this sense we employ a simplified model for motor clusters that act as slipping links that exert force while moving along the strands. Using the framework of replica theory, quenched averages are taken over the disorder which originates from permanent random crosslinking of network end points to the background. We investigate how a small motor force contributes to the elastic properties of the network. We learn that in addition to the normal elastic response for the network there is an extra contribution to the network elasticity from the motor activity. This depends on the ratio of the entropic spring constant for the linked bio-polymerchain to the spring constant of the tether of the motor. / AFRIKAANSE OPSOMMING: Ons konstrueer en bestudeer 'n eenvoudige model vir 'n aktiewe netwerk van eksieble polimeerfilamente wat deur grosse van molekulere motors aan mekaar verbind word wat omkeerbare werk doen terwyl dit langs die filamente transleer. Die eindpunte van die filamente is aan 'n elastiese agtergrond verbind. In hierdie sin benut ons 'n eenvoudige model vir motorclusters wat as verskuifbare verbindings krag op die filamente tydens beweging kan uitoefen. Nie-termiese wanorde gemiddeldes word geneem oor die wanorde wat deur die lukrake permanente verbindings van netwerk eindpunte aan die agtergrond veroorsaak word. Ons ondersoek hoe 'n klein motorkrag tot die elastiese eienskappe van die netwerk bydra. Ons leer dat daar bo en behalwe die gewone elastiese respons vir die netwerk 'n elastiese bydrae as gevolg van die motors se aktiwiteit voorkom. Dit hang af van die verhouding van die entropiese veerkonstante van die biopolimerketting tot die veerkonstante van die anker van die motor.

Doctor of Philosophy / Department of Biology / Susan J. Brown / RNA interference (RNAi) is a natural gene-silencing phenomenon triggered by dsRNA (dsRNA). While RNAi is an endogenous process that plays essential roles in regulating gene expression it can also be harnessed as a tool for the study of gene function. Introducing dsRNA that is homologous to target mRNA into a cell triggers the RNAi response causing the destruction of the homologous mRNA and a loss of function phenotype. In some organisms, such as the nematode Caenorhabditis elegans, once dsRNA is introduced into the body cavity, the RNAi effect is seen throughout the organism because the dsRNA is taken up by individual cells and is then spread from cell to cell. This process has been termed the systemic RNAi response. For other organisms, such as the fruit fly Drosophila melanogaster, introduction of dsRNA into the body cavity does not result in a systemic RNAi response. This may be due to the cell’s inability to take up dsRNA or spread that dsRNA from cell to cell. For other organisms, including mammals, introduction of dsRNA into the body cavity does not result in a systemic RNAi response because the immune response causes dsRNA destruction before it can be utilized in the RNAi pathway. For organisms that do not exhibit a systemic RNAi response, complex genetic methods are needed to introduce dsRNA into cells to induce the RNAi response. Therefore, one of the challenges in utilizing RNAi as a genetic tool is introducing the dsRNA into individual cells. In recent years, systemic RNAi responses have been documented in both model and non-model organisms, making RNAi an accessible genetic tool. The red flour beetle, Tribolium castaneum is an emerging model organism that has a robust systemic RNAi response. However, the mechanism of systemic RNAi and the specific parameters required to obtain a strong systemic RNAi response in this organism have not been thoroughly investigated. The aim of this work is to provide data that can allow RNAi to be better utilized as a genetic tool in Tribolium and to use this information as a basis for the use of RNAi in other insects in which it can be performed. Specifically we provide data on the essential parameters necessary to achieve an effective systemic response in Tribolium, we describe differences in the systemic RNAi response between Drosophila and Tribolium, we analyze the conservation and function of RNAi machinery genes in Tribolium and we provide information on the genes critical for a systemic RNAi response in Tribolium.

Chez la plupart des eucaryotes, la présence d’ARN double brin induit la mise en place de mécanismes qui peuvent inhiber l’expression de gènes sur la base d’une complémentarité de séquence. L’exemple le mieux connu est le cas de l’interférence par l’ARN telle qu’elle a été décrite initialement chez C. elegans, où les ARN double brin génèrent une endonucléase spécifique de séquence qui dégrade tout ARN parfaitement complémentaire du petit ARN guide contenu dans le complexe RISC. En plus de cette activité post-transcriptionnelle, il a été observé chez de nombreux eucaryotes l’existence de mécanismes apparentés à l’interférence par l’ARN et qui inhibent la transcription en agissant au niveau de la chromatine. Si ces mécanismes ont été clairement mis en évidence chez les plantes et les champignons il n’existe que quelques exemples de ce type de régulation chez les mammifères. De manière inattendue, le fait de cibler le promoteur d’un gène avec de petits ARN double brin peut conduire à une augmentation de son expression. Cette réponse paradoxale n’a été observée jusqu’à présent que dans des cellules de mammifère, et si elle suscite un intérêt en particulier pour stimuler l’expression de gènes suppresseurs de tumeurs, son mécanisme est encore inconnu.Mes travaux ont porté sur l’étude de l’induction de l’expression par des petits ARN. Ils reposent tout d’abord sur le développement d’une approche expérimentale qui permet de suivre l’activité du promoteur du gène ciblé. Pour cela, j’ai utilisé des constructions indicatrices organisées autour d’un promoteur bidirectionnel qui contrôle l’expression de deux protéines fluorescentes. Lorsque l’on cible le messager de l’une de ces protéines, l’expression de l’autre est augmentée et j’ai pu montrer que ceci corrèle avec la quantité d’ARN messager et de polymérase II présente sur le promoteur bidirectionnel. Ainsi, l’utilisation d’un promoteur bidirectionnel permet effectivement de suivre le niveau de transcription du gène ciblé par le petit ARN.Cette induction de l’expression détectée de manière « controlatérale » n’est pas due à un effet hors cible des petits ARN car elle nécessite la présence de la séquence cible sur l’un des transcrits de la construction indicatrice. L’induction peut être observée avec de nombreux petits ARN différents, y compris s’ils interagissent comme des micro ARN. Les constructions indicatrices que j’ai développées sont donc biaisées en faveur d’une réponse de type induction transcriptionnelle enréponse à un silencing. L’utilisation d’un promoteur bidirectionnel est probablement à l’origine de ce biais à travers la possibilité d’induire une transcription convergente sur les plasmides lorsqu’ils sont circulaires. De fait, la linéarisation de la construction indicatrice supprime l’induction, du moins pour les constructions les plus simples.Si le coeur du complexe RISC, la protéine Ago2, est nécessaire au silencing et à l’induction, j’ai pu montrer que dans le deuxième cas c’était en fait pour guider le complexe RISC sur les transcrits et non pas pour les couper. En effet, le silencing des protéines TNRC6A et B diminue fortement l’induction sans toucher au silencing s’il procède en mode siRNA. De plus l’ancrage sur le transcrit EGFP induit une réponse de même type que le petit ARN (silencing et induction). Cette approche d’ancrage m’a permis d’identifier les domaines nécessaires au silencing et à l’induction et de montrer qu’ils sont distincts.Ce travail permet donc de mettre en évidence que l’induction transcriptionnelle observée sur nos constructions indicatrices est due à une activité des partenaires des protéines Argonaute, la famille GW182/TNRC6. Cette observation ouvre la voie à une caractérisation du mécanisme de cette induction en montrant qu’elle relève d’une activité spécifique du complexe RISC. / In the majority of the eucaryote, the presence of double-strands RNA induce the inhibition of gene expression base on the complementary of sequence. The best known example is the case of RNA interference in C. elegans which is the first model described, in which the double-strands RNA generate an specific endonuclease who degrade all RNA complementary perfectly to the small RNA guide included in the complex RISC. In addition to this post-transcriptional activity, it has been observed in many eukaryotes the existence of mechanisms related to RNA interference and it inhibit transcription by acting at the chromatin. If these mechanisms have been clearly demonstrated in plants, fungi, there are only several examples of this type of regulation in mammals. Unexpectedly, the targeting the promoter of a gene with small double-stranded RNA can lead to increased expression. This paradoxical response has not been observed so far in mammalian cells, but it raises interest particularly to stimulate the expression of tumor suppressor genes, unfortunely the mechanism is still unknown.My work has focused on studying the induction of expression by small RNAs. They are based first on the development of an experimental approach that allows to monitor the promoter activity of the targeted gene. To do this I used indicator constructions organized around a bidirectional promoter that controls the expression of two fluorescent proteins. When targeting the messenger of one of these proteins, the expression of the other is increased and I was able to show that thisincrease correlates with the amount of RNA messenger polymerase II presented on the bidirectional promoter. Thus, the use of a bidirectional promoter can effectively monitor the level of transcription of the gene targeted by the small RNA. This induction of expression detected in a "contralateral" is not due to an off-target effect of siRNA because it requires the presence of the target sequence on one of the transcripts of the construction indicator. The induction can be observed with many different small RNAs, including the interact as micro RNA. Thus the construction indicator that I developed are biased in an induction response transcriptionally in response to a silencing. The use of a bidirectional promoter is probably the origin of this bias through the possibility of inducing a convergent transcription when the plasmids are circular. In fact, the linearization of the construction indicator removes the induction, at least for the simplest constructions. If the heart of the complex RISC is the protein Ago2, is necessary for the silencing and the induction, I was able to show that in the second case Ago2 was in fact to guide the RISC complex on the transcripts but not to cut it. Indeed, the silencing of proteins TNRC6A and B reduces induction significantly without affecting the silencing if it processe in the siRNA model. Also anchoring the transcript EGFP induces a response similar to the small RNA (silencing and induction). This anchor approach allowed me to identify domaines necessary for silencing and induction and show that they are distinct. This work makes it possible to demonstrate that the transcriptional induction observed in our constructions indicator is due to a activity partner ofArgonaute proteins, the GW182/TNRC6 family. This observation open the way for characterization of the mechanism of this induction by showing that it belongs to a specific activity of the RISC complex.

O presente estudo foi baseado na ocorrência, análises morfométricas, craniométricas e conteúdo estomacal de cetáceos odontocetes encontrados mortos em praias do sul do Estado de São Paulo (Ilha Comprida e Marujá) e norte do Estado do Paraná (Deserta), no período de abril de 1986 à abril de 1988. Para a espécie mais abundante estimou-se também a idade dos indivíduos pelo número de anéis na camada de dentina. Encontrou-se 100 animais compreendidos em 7 gêneros e 7 espécies pertencentes a 3 famílias. Para a região são caracterizadas pela primeira vez, Delphinus delphis (n=13), Stenella frontalis (n=3) , Kogia breviceps (n=1) e Globicephala macrorhynchus (n=1). Destas, para o litoral e São Paulo são novas S. frontalis e G. macrorhynchus, sendo esta última também descrita pela primeira vez para águas ocidentais do Atlântico Sul. Para o litoral do Estado do Paraná são todas ocorrências novas, com exceção de Sotalia fluviatilis. Esta última foi a espécie mais abundante (n=58) , seguida por Pontoporia blainvillei (n=21). Também foi encontrada a espécie Tursiops gephyreus (n=2) além de 1 delfinídeo não identificado. A ocorrência total dessas espécies seguiu um padrão pouco definido ao longo do ano, ocorrendo em maior número nos meses de inverno e verão. Para a espécie mais abundante, S. fluviatilis alguns dos parâmetros de crescimento foram estimados baseando-se em equações que descrevem relações interespecíficas desses parâmetros. Animais com 6 ou mais anéis foram classificados como maduros fisicamente. Construiu-se uma curva de crescimento a mão livre para a espécie, baseando-se em alguns dos dados fornecidos pelas equações e dados obtidos dos próprios exemplares como número de anéis dos dentes e comprimentos dos corpos; o método de von Bertalanffy para ajuste da curva de crescimento não mostrou-se satisfatório para todas as fases do crescimento da espécie. Ainda para essa espécie analisou-se 5 componentes funcionais do crânio dos exemplares encontrados, obtendo-se 6 anéis para a estabilização do crescimento desses componentes, com excessão do aparato alimentar que atingiu o patamar com 8 anéis. A análise da dieta alimentar dessa espécie mostrou a ocorrência de espécies de peixes demersais pelágicas de superfície comuns na região costeira, sendo a família Sciaenidae a mais abundante. Bicos de lulas pertencentes a 2 ou 3 espécies também foram encontrados em 100% dos estômagos analisados. Encontrou-se em um Estômago camarões pertencentes à espécie Penaeus brasiliensis. Parasitas estomacais pertencentes à família Anisakidae foram encontrados em 28,6% dos estômagos, além de mesma frequência para isópodos parasitas de brânquias de peixes. A maioria dos animais encontrados. Possivelmente foram mortos em redes de pesca na região costeira adjacente às praias estudadas. / The occurrence, morphometric and craniometric data and stomach contents of odontoceti cetaceans found in the south coast of São Paulo State and north coast of Paraná State (Ilha Comprida, Marujá and Deserta beaches) from April, 1986 to April, 1988 were analysed in this study. For the most abundant species we estimated the individual age by reading growth layers in the dentine. We found 100 animaIs belonging to 7 genera, 7 species and 3 famiIies. The first records for this region were: Delphinus delphis (n=13), Stenella frontalis (n=3). Kogia breviceps (n=1), and Globicephala macrorhynchus (n=1). The first ones for the coast of São Paulo State were: S. frontalis and G. macrorhynchus. This Iast species was also first described for the southwestern Atlantic. AlI of the species were new records for the coasts of Paraná State except S. fluviatilis. This Iast one was the most abundant species (n=58), followed by Pontoporia blainvillei (n=21). We also encountered Tursiops gephyreus (n=2) and an unidentified delfinid. There was little seasonal pattern in the total occurrence of the animals with a small increase in their abundance during the winter and summer seasons. For the most abundant species, Sotalis fluviatilis, we estimated some growth parameters using published equations which describe the interspecific relationships of these parameters. Physical maturity was considered in animals with for more dental layers. A growth curve was drawn by eye, using data obtained the from some of the above equations and data obtained from individuaIs analysed in this study, such as dental layers and total length; a growth curve by von Bertalanffy\'s method did not fit well. The species plateau development of 5 skull functional components for this showed 6 dental layers for the attainment of adult plateau except for the feeding apparatus which stops growing at 8 dental layers. The diet of S. fluviatilis consisted mainly of demersal and superficial pelagic common fishes found in the Brazilian southeastern coast. The Sciaenidae family was the most abundant. Beaks of squids belonging to 2 or 3 especies were found in all stomach contents analysed; we also encountered shrimps in one stomach content. Stomach parasites belonging to the Anisakidae family were found in 28,6% of the stomachs with the same frequency of fish gill parasite isopods. Most of the cetaceans were probably captured accidentally with gillnets in adjacent waters of the studied beaches.

This article develops a novel methodological framework to investigate the exposure of eco- nomic systems to the risk of physical capital stranding. Combining Input-Output (IO) and network theory, we define measures to identify both the sectors likely to trigger relevant capital stranding cascades and those most exposed to capital stranding risk. We show how, in a sample of ten European countries, mining is among the sectors with the highest external asset strand- ing multipliers. The sectors most affected by capital stranding triggered by decarbonisation include electricity and gas; coke and refined petroleum products; basic metals; and transporta- tion. From these sectors, stranding would frequently cascade down to chemicals; metal products; motor vehicles water and waste services; wholesale and retail trade; and public administration. Finally, we provide an estimate for the lower-bound amount of assets at risk of transition-related stranding, which is in the range of 0.6-8.2% of the overall productive capital stock for our sample of countries, mainly concentrated in the electricity and gas sector, manufacturing, and mining. These results confirm the systemic relevance of transition-related risks on European societies. / Series: Ecological Economic Papers

O presente estudo foi baseado na ocorrência, análises morfométricas, craniométricas e conteúdo estomacal de cetáceos odontocetes encontrados mortos em praias do sul do Estado de São Paulo (Ilha Comprida e Marujá) e norte do Estado do Paraná (Deserta), no período de abril de 1986 à abril de 1988. Para a espécie mais abundante estimou-se também a idade dos indivíduos pelo número de anéis na camada de dentina. Encontrou-se 100 animais compreendidos em 7 gêneros e 7 espécies pertencentes a 3 famílias. Para a região são caracterizadas pela primeira vez, Delphinus delphis (n=13), Stenella frontalis (n=3) , Kogia breviceps (n=1) e Globicephala macrorhynchus (n=1). Destas, para o litoral e São Paulo são novas S. frontalis e G. macrorhynchus, sendo esta última também descrita pela primeira vez para águas ocidentais do Atlântico Sul. Para o litoral do Estado do Paraná são todas ocorrências novas, com exceção de Sotalia fluviatilis. Esta última foi a espécie mais abundante (n=58) , seguida por Pontoporia blainvillei (n=21). Também foi encontrada a espécie Tursiops gephyreus (n=2) além de 1 delfinídeo não identificado. A ocorrência total dessas espécies seguiu um padrão pouco definido ao longo do ano, ocorrendo em maior número nos meses de inverno e verão. Para a espécie mais abundante, S. fluviatilis alguns dos parâmetros de crescimento foram estimados baseando-se em equações que descrevem relações interespecíficas desses parâmetros. Animais com 6 ou mais anéis foram classificados como maduros fisicamente. Construiu-se uma curva de crescimento a mão livre para a espécie, baseando-se em alguns dos dados fornecidos pelas equações e dados obtidos dos próprios exemplares como número de anéis dos dentes e comprimentos dos corpos; o método de von Bertalanffy para ajuste da curva de crescimento não mostrou-se satisfatório para todas as fases do crescimento da espécie. Ainda para essa espécie analisou-se 5 componentes funcionais do crânio dos exemplares encontrados, obtendo-se 6 anéis para a estabilização do crescimento desses componentes, com excessão do aparato alimentar que atingiu o patamar com 8 anéis. A análise da dieta alimentar dessa espécie mostrou a ocorrência de espécies de peixes demersais pelágicas de superfície comuns na região costeira, sendo a família Sciaenidae a mais abundante. Bicos de lulas pertencentes a 2 ou 3 espécies também foram encontrados em 100% dos estômagos analisados. Encontrou-se em um Estômago camarões pertencentes à espécie Penaeus brasiliensis. Parasitas estomacais pertencentes à família Anisakidae foram encontrados em 28,6% dos estômagos, além de mesma frequência para isópodos parasitas de brânquias de peixes. A maioria dos animais encontrados. Possivelmente foram mortos em redes de pesca na região costeira adjacente às praias estudadas. / The occurrence, morphometric and craniometric data and stomach contents of odontoceti cetaceans found in the south coast of São Paulo State and north coast of Paraná State (Ilha Comprida, Marujá and Deserta beaches) from April, 1986 to April, 1988 were analysed in this study. For the most abundant species we estimated the individual age by reading growth layers in the dentine. We found 100 animaIs belonging to 7 genera, 7 species and 3 famiIies. The first records for this region were: Delphinus delphis (n=13), Stenella frontalis (n=3). Kogia breviceps (n=1), and Globicephala macrorhynchus (n=1). The first ones for the coast of São Paulo State were: S. frontalis and G. macrorhynchus. This Iast species was also first described for the southwestern Atlantic. AlI of the species were new records for the coasts of Paraná State except S. fluviatilis. This Iast one was the most abundant species (n=58), followed by Pontoporia blainvillei (n=21). We also encountered Tursiops gephyreus (n=2) and an unidentified delfinid. There was little seasonal pattern in the total occurrence of the animals with a small increase in their abundance during the winter and summer seasons. For the most abundant species, Sotalis fluviatilis, we estimated some growth parameters using published equations which describe the interspecific relationships of these parameters. Physical maturity was considered in animals with for more dental layers. A growth curve was drawn by eye, using data obtained the from some of the above equations and data obtained from individuaIs analysed in this study, such as dental layers and total length; a growth curve by von Bertalanffy\'s method did not fit well. The species plateau development of 5 skull functional components for this showed 6 dental layers for the attainment of adult plateau except for the feeding apparatus which stops growing at 8 dental layers. The diet of S. fluviatilis consisted mainly of demersal and superficial pelagic common fishes found in the Brazilian southeastern coast. The Sciaenidae family was the most abundant. Beaks of squids belonging to 2 or 3 especies were found in all stomach contents analysed; we also encountered shrimps in one stomach content. Stomach parasites belonging to the Anisakidae family were found in 28,6% of the stomachs with the same frequency of fish gill parasite isopods. Most of the cetaceans were probably captured accidentally with gillnets in adjacent waters of the studied beaches.

DNA double strand breaks (DSBs), which may occur during DNA replication or due to the action of genotoxic agents, are extremely dangerous DNA lesions as they can cause chromosomal rearrangements and cell death. Therefore, accurate DSB repair is vital for genome stability and cell survival. Two main mechanisms serve to repair DNA DSBs: non-homologous end joining, which re-ligates DNA ends together, and homologous recombination (HR), which restores broken DNA using homologous sequence as a template for repair. One-ended DSBs are a subject for the specialised HR-dependent repair pathway known as break-induced replication (BIR). At low frequency, DNA breaks can also be healed by telomerase, which normally extends telomeres at natural chromosome ends, but may also add de novo telomeres to DSBs due to their similarity to chromosome ends. De novo telomere addition is a deleterious event, which is effectively inhibited by the nuclear Pif1 (nPif1) helicase phosphorylated at the TLSSAES motif in response to DNA damage. In this study, it is reported that the same regulatory motif of nPif1 is also required for DSB repair via BIR. The requirement of the nPif1 TLSSAES sequence in BIR is dependent on the functional DNA damage response (DDR). Thus, nPif1 phosphorylation by the DDR machinery might mediate the role of nPif1 in BIR. In contrast, the nPif1 regulatory motif is not essential for BIR at telomeres in cells lacking telomerase. These observations indicate that the mechanism of nPif1 function in DSB repair via BIR and in BIR at telomeres might be different. In this work, a protocol for nPif1 pull-down was optimized to reveal the mechanism of the phosphorylation-dependent nPif1 functions in cells undergoing DNA repair, i. e. the mechanism of nPif1-mediated inhibition of de novo telomere addition and promoting DSB repair via BIR. In future, this protocol can be used to dissect the role of nPif1 in DNA repair through the identification of its potential interacting partners. The Srs2 helicase negatively regulates HR via dismantling Rad51 filaments. According to preliminary data from the laboratory of Sveta Makovets, Srs2 also promotes de novo telomere addition at DSBs in a Rad51-dependent manner. The work presented here establishes that Srs2 is dispensable for telomerase-mediated addition of TG1-3 repeats to DSBs. Instead, Srs2 is required for the reconstitution of the complementary DNA strand after telomerase action, thus ensuring the completion of de novo telomere addition. Overall, this study demonstrates that recombination-dependent DSB repair and de novo telomere addition share common regulatory components, i. e. the nPif1 helicase phosphorylated in response to DNA damage and the Srs2 helicase. Phosphorylated nPif1 promotes DSB repair via BIR in addition to its known role in inhibition of telomerase at DSBs, whereas Srs2 uses its well established ability to remove Rad51 from ssDNA to promote the restoration of dsDNA and thus to complete de novo telomere addition.

<p>This thesis is based on six papers, Papers I-VI, focusing on the interplay between the stabilizing elements of nucleic acids self-assembly; hydrogen bonding, stacking and solvent effects. In Paper I we investigate how the substitution of the O4' for CH₂ in the sugar moiety of adenosine (2'-deoxyaristeromycin) at the A⁶ position of the Dickerson-Drew dodecamer makes the two modified bases exist in a dynamic equilibrium between Hoogsteen and Watson-Crick base pairing in the NMR time scale. Paper II is a structural study of the incorporation of 1-(1',3'-<i>O</i>-anhydro-<i>β</i>-D-psicofuranosyl)thymine in the T⁷ position of the Dickerson-Drew dodecamer. NMR constrained molecular dynamics and hydration studies show the base-base distortions caused by the introduction of a North-type locked sugar in an otherwise B-type DNA•DNA duplex. Paper III shows that the stacking distortion caused by the 1-(1',3'-<i>O</i>-anhydro-<i>β</i>-D-psicofuranosyl)thymine building block perturbs the charge transfer similar to a DNA mismatch. Paper IV highlights how the sequence context affects the physico-chemical properties, monitored by the p<i>K</i><i>a</i> of guanine itself as well as how the charge perturbation is experienced by the neighboring bases, in ssDNA and ssRNA. Paper V focuses on the differences between the structural equilibria of single-stranded ssDNA and ssRNA. Directional differences in single-stranded stacking between ssDNA and ssRNA are identified and provide a basis to explain directional differences in p<i>K</i><i>a</i> modulation and dangling-end stabilization. In Paper VI the thermodynamic gains of dangling ends on DNA and RNA core duplexes are found to correlate with the X-ray geometries of dangling nucleobases relative to the hydrogen bonds of the closing base pairs.</p>

This thesis is based on six papers, Papers I-VI, focusing on the interplay between the stabilizing elements of nucleic acids self-assembly; hydrogen bonding, stacking and solvent effects. In Paper I we investigate how the substitution of the O4' for CH2 in the sugar moiety of adenosine (2'-deoxyaristeromycin) at the A6 position of the Dickerson-Drew dodecamer makes the two modified bases exist in a dynamic equilibrium between Hoogsteen and Watson-Crick base pairing in the NMR time scale. Paper II is a structural study of the incorporation of 1-(1',3'-O-anhydro-β-D-psicofuranosyl)thymine in the T7 position of the Dickerson-Drew dodecamer. NMR constrained molecular dynamics and hydration studies show the base-base distortions caused by the introduction of a North-type locked sugar in an otherwise B-type DNA•DNA duplex. Paper III shows that the stacking distortion caused by the 1-(1',3'-O-anhydro-β-D-psicofuranosyl)thymine building block perturbs the charge transfer similar to a DNA mismatch. Paper IV highlights how the sequence context affects the physico-chemical properties, monitored by the pKa of guanine itself as well as how the charge perturbation is experienced by the neighboring bases, in ssDNA and ssRNA. Paper V focuses on the differences between the structural equilibria of single-stranded ssDNA and ssRNA. Directional differences in single-stranded stacking between ssDNA and ssRNA are identified and provide a basis to explain directional differences in pKa modulation and dangling-end stabilization. In Paper VI the thermodynamic gains of dangling ends on DNA and RNA core duplexes are found to correlate with the X-ray geometries of dangling nucleobases relative to the hydrogen bonds of the closing base pairs.

Mesozooplankton are of critical importance to marine food webs by transferring energy from the microbial food web to higher trophic levels and depositing energy to the deeper ocean layers through fecal deposition. While decades of research have shown that viruses have significant impacts in the oceans, and infect a wide range of organisms from bacteria to whales, there is still little known about the impacts of viruses on the mesozooplankton community. As copepods are the most abundant mesozooplankton group, this study sought to characterize the viruses present in natural populations of the calanoid copepods Acartia tonsa and Labidocera aestiva in Tampa Bay, Florida. Viral metagenomics revealed two virus genomes, named Acartia tonsa copepod circovirus (AcCopCV) and Labidocera aestiva copepod circovirus (LaCopCV), which were discovered in their respective copepod species. Both viruses show amino-acid similarities to known circoviruses, and phylogenetic and genomic analyses suggest they may be divergent members of the Circoviridae family. LaCopCV was found to be extremely prevalent in the L. aestiva population, with up to 100% of individuals infected. High viral loads for LaCopCV were observed by quantitative PCR, with an average viral load of 1.3x105 copies per individual. In addition, transcription of the LaCopCV replication gene was detected in L. aestiva, demonstrating active viral replication. AcCopCV could be detected sporadically in A. tonsa populations throughout the year. The circoviruses were specific to their respective hosts, and were not detected in the other copepod species or surrounding seawater. Virus-like particles were observed in A. tonsa and L. aestiva under transmission electron microscopy, demonstrating that viruses were actively proliferating in copepod connective tissue, as opposed to gut tissue, parasites, or symbionts. Preliminary results from in-situ hybridization show that the AcCopCV genome can be detected in A. tonsa tissue, linking the discovered genomes to virus propagation in copepod tissue. This is the first study describing viruses in copepods, as well as the first discovery of circoviruses infecting marine organisms. These results suggest that viruses impact marine copepod populations, necessitating further studies to determine the ecological impacts of viruses on the mesozooplankton community.