xix, 111 p. : ill. (some col.) / Since discovery of its biological effects in the late 1960's, cisplatin (cis-diamminedichloroplatinum( II)) has become one of the most broadly-prescribed cancer drugs in use today. A majority of efforts to understand the metallobiochemistry of this drug have focused on describing the interactions of cisplatin-derived Pt(II) complexes with DNA. Drug binding to this "high value" cellular target is believed to trigger the apoptotic pathways that underlie cisplatin's cytotoxic effects. Although RNA is chemically similar to DNA and responsible for accurately transferring, regulating, and transforming the same genetic information that is stored within the DNA genome, surprisingly little is known about platinum(II) drug binding to RNA. Accordingly, the first three chapters of this dissertation describe efforts to address questions regarding cisplatin coordination to RNA on the molecular scale. Chapter I reviews fundamental aspects of how metal complexes interact with nucleic acids, highlighting the bioinorganic chemistry of platinum(II) antitumor drugs. This chapter also introduces the idea that drug binding to RNA may form an important part of how these complexes work in the cell. Chapter II describes cisplatin crosslinking between RNA nucleobases located on opposite sides of the internal loop of an RNA subdomain derived from the catalytic core of the spliceosome. Chapter III describes how platinum adducts disrupt the activity of RNA processing enzymes similar to those that are necessary for maturation, maintenance and recycling of the transcriptome. Chapter III also describes the reversal of RNA platination using thiourea. The chemistry of platinum(II) is also characterized by preferential coordination to sulfur ligands, or thiophilicity. Incorporating this property into RNA chemistry, Chapters IV and V describe the reaction of platinum(II) complexes with phosphorothioate-substituted RNAs. Chapter IV describes engineering platinum(II) crosslinks in the Hammerhead ribozyme through the targeting of a platinum(II) complex to a specific phosphorothioate substitution installed in the active site of this catalytic RNA. Chapter V outlines efforts to characterize the cleavage and isomerization reactions promoted by platinum(II) coordination to phosphorothioate-substituted RNAs. Finally, Chapter VI summarizes the insights gained throughout the course of our studies and provides an outlook on the future of platinum-RNA chemistry. This dissertation includes co-authored material and previously published results. / Committee in charge: Michael M. Haley, Chair; Victoria J. DeRose, Advisor; David R. Tyler; Andrew J. Berglund; Eric A. Johnson

O tubarão-martelo, Sphyrna lewini é um dos mais valiosos recursos marinhos, e o preço pago por suas barbatanas no mercado Asiático pode atingir acima dos U$ 100,00/kg. A análise da composição de tamanhos e idades nas capturas, o estudo do crescimento desta espécie de grande porte e a evolução temporal dos desembarques, indicaram que este recurso se encontra sobreexplotado no sudeste e sul do Brasil, como reflexo de diferentes modalidades pesqueiras atuando ao longo de todo o seu ciclo de vida e à baixa resiliência desta espécie à pesca, por apresentar um crescimento lento (L&#8734 = 329,12 cm; K = 0,071 ano-1; to = -2,37 ano; sexos combinados), longevidade acima dos 40 anos e mortalidade natural baixa (M = 0,1 ano-1 na fase adulta), padrões estes típicos de uma espécie K-estrategista . A sobrepesca de recrutamento, ocorre nas áreas costeiras, principalmente pela pesca de arrasto e emalhe costeiro, não havendo a proteção das áreas de parto na primavera-verão. Neste caso há grandes capturas de neonatos e juvenis até 8 anos de idade. A tração adulta por sua vez é reduzida pela pesca de espinheI e de emalhe de superfície principalmente na zona de talude. Modelos de análise de covariância indicaram maiores capturas desta espécie na pesca de espinheI de monofilamento de superfície durante os meses de primavera-verão, na zona de talude (200 e 3000 m) e a existência de uma relação linear positiva entre a captura em peso e o esforço em número de anzóis. Medidas de manejo e conservação para esta espécie são sugeridas. / The scalloped hammerhead shark Sphyrna lewini is one of the most valuable marine resources, due to its high-priced fins in the Asian market, which can reach U$ 100,00/kg. The analysis of the length and age composition in the catches, growth studies, and the annual development of its landings in southern Brazil, showed signs of overexploitation for the species. This effect was mainly caused by different fishing gears exploiting all the phases of its life-cycle and its low resilience to fishing pressure due to its slow-growing strategy (L&#8734 = 329,12 cm; K = 0,071 yr-1; to = -2,37 ano; both sexes), longevity (> 40 yrs.) and low natural mortality (M = 0,1 yr-1, during adult phase), which means a K\'strategic typical pattern. Recruitment overfishing use to happen in coastal areas by trawls and anchored gillnets activities which destroy the nurseries and juvenile grounds for the species, mainly in spring-summer months when the parturition occurs. On the other hand, the adult fraction of the stock is reduced by surface longline and driftnets activities along the continental slope. For the surface monofilament longline fisheries, covariance models detected the highest catches of scalloped hammerhead sharks along the slope (between 200 - 3000 m depth), during spring-summer months. There was also a positive linear relationship between catch (in weight) and effort (hook number). Management and conservation measures are recommended for this species.

Reconfigurable antennas are attractive for many military and commercial applications where it is required to have a single antenna that can be dynamically reconfigured to transmit or receive on multiple frequency bands and patterns. RF-MEMS is a promising technology that has the potential to revolutionize RF and microwave system implementation for next generation telecommunication applications. Despite the efforts of top industrial and academic labs, commercialization of RFMEMS switches has lagged expectations. These problems are connected with switch design (high actuation voltage, low restoring force, low power handling), packaging (contamination layers) and actuation control (high impact force, wear, fatique). This Thesis focuses on the design and control of a novel ohmic RF-MEMS switch specified for reconfigurable antennas applications. This new switch design focuses on the failure mechanisms restriction, the simplicity in fabrication, the power handling and consumption, as well as controllability. Finally, significant attention has been paid in the switch’s electromagnetic characteristics. Efficient switch control implies increased reliability. Towards this target three novel control modes are presented. 1) Optimization of a tailored pulse under Taguchi’s statistical method, which produces promising results but is also sensitive to fabrication tolerances. 2) Quantification of resistive damping control mode, which produces better results only during the pull-down phase of the switch while it is possible to be implemented successfully in very stiff devices. 3) The “Hybrid” control mode, which includes both aforementioned techniques, offering outstanding switching control, as well as immunity to fabrication tolerances, allowing an ensemble of switches rendering an antenna reconfigurable, to be used. Another issue that has been addressed throughout this work is the design and optimization of a reconfigurable, in pattern and frequency, three element Yagi-Uda antenna. The optimization of the antenna’s dimensions has been accomplished through the implementation of a novel technique based on Taguchi’s method, capable of systematically searching wider areas, named as “Grid-Taguchi” method.

The RNA World Hypothesis posits that RNA enzymes (ribozymes) catalyzed biochemical reactions in primitive cells prior to the emergence of proteins. However, the evolutionary mechanisms that gave rise to functional RNA sequences on early Earth remains largely unclear. Using a bottom-up approach that combines in vitro selection and high-throughput sequencing, we demonstrate how a self-cleaving RNA enzyme, the Hammerhead Ribozyme (HHR), may have evolved from non-catalytic sequences in the RNA World. Multiple starting libraries were generated by progressively increasing the number of randomized positions in the ribozyme’s catalytic core. The HHR was selected from each of these libraries following several rounds of amplification and enrichment. Deep sequencing analysis was then used to track evolutionary trends that gave rise to the wild-type sequence during each selection. This novel approach revealed a wide range of functional HHR variants. Notably, we discovered active hammerhead variants with mutations to previously identified essential nucleotides, shedding new light on the sequence requirements of the full-length, cis-acting ribozyme. We also demonstrate that the evolutionary trajectory of each nucleotide in the catalytic core directly correlates with their functional importance, potentially giving researchers a novel method to assess the sequence requirements of functional nucleic acids. Altogether, the in vitro evolution of ribozymes shows how complex molecules might have emerged from non-catalytic polymers in the RNA world, contributing to our understanding of the origin of life on Earth. / Thesis / Master of Science (MSc) / The origin of life is complicated by the interdependence between deoxyribonucleic acid (DNA), which stores genetic information, and protein, which performs essential cellular functions. The RNA World Hypothesis attempts to solve this paradox by underpinning ribonucleic acid (RNA) as the foundation of cellular based-life, due to its unique ability to store genetic information as well as perform complex chemical reactions. However, the way that functional RNA molecules (ribozymes) emerged on early Earth in the first place remains largely unclear. We simulated molecular evolution in the laboratory using a process known as in vitro selection to demonstrate how a self-cleaving RNA enzyme, the Hammerhead Ribozyme (HHR), may have evolved in the RNA World. We also discovered different versions of the HHR, shedding new light on its structure and function. Altogether, the results from this work pave the way for a deeper understanding of ribozyme evolution and the origins of life on Earth.

Three hammerhead sharks (Sphyrna spp.) are currently managed as part of the large coastal shark complex in the United States. Including multiple species in an assessment ignores the different stock dynamics of each individual species within the complex due to different life histories. This study completed individual assessments of scalloped (S. lewini), great (S. mokarran), and smooth (S. zygaena) hammerhead sharks in the U.S. Atlantic Ocean and Gulf of Mexico. Combined data for all three species and unclassified hammerhead sharks were also used to produce a stock assessment of the hammerhead shark complex. Depletions of 83%, 96%, and 91% were estimated for scalloped, great, and smooth hammerhead sharks, respectively, between 1981 and 2005. When modeled as a single stock, the hammerhead shark complex experienced a 90% decline over the same time period. All three stocks, and the complex were overfished (below population size associated with maximum sustainable yield (MSY)), and overfishing (fishing level above that associated with MSY) occurred in 2005. We found that scalloped hammerhead shark population recovery is likely to occur within 10 years if catch remains at or below 2005 levels. Great and smooth hammerhead sharks will likely still be overfished in 30 years unless catches are reduced. It appears that the species composition could be changing in this hammerhead shark complex. The faster-growing scalloped hammerhead sharks are able to withstand fishing pressure better than great or smooth hammerhead sharks. However, it is difficult to target any single large coastal shark species while fishing; hence they are subject to similar fishing pressure. The result is a greater decline in great and smooth hammerhead sharks than experienced by scalloped hammerhead sharks. Therefore, the proportion of scalloped hammerhead sharks increased between 1981 and 2005. Species-specific stock assessments, such as those presented here, allow managers to more closely monitor populations of slower-growing species and reduce the risk of overexploitation of those species. / Master of Science

Dans les dernières années, une explosion de la recherche sur les ARN a eu lieue à cause de nombreuses découvertes démontrant l’importance de l’ARN dans plusieurs processus biologiques. Ainsi, de grandes quantités d’ARN sont devenues indispensables au bon déroulement de plusieurs études, notamment pour la biologie structurale et la caractérisation fonctionnelle. Cependant, il existe encore peu de méthodes de purification simples, efficaces, fiables et produisant un ARN sous forme native. Dans les dernières années, le laboratoire Legault a mis au point une méthode de purification par affinité utilisant une étiquette ARiBo pour la purification d’ARN transcrits in vitro par la polymérase à ARN du phage T7. Cette méthode de purification d’ARN a été spécifiquement développée pour maximiser la pureté et le rendement. De plus, elle est très rapide et fonctionne avec plusieurs types d’ARN. Cependant, comme plusieurs autres méthodes de purification, cette méthode produit des ARN avec des extrémités 5′ hétérogènes. Dans ce mémoire, des solutions sont proposées pour remédier au problème d’hétérogénéité en 5ʹ′ des ARN transcrits avec la polymérase à ARN du phage T7 et purifiés par la méthode ARiBo. La première solution consiste à choisir la séquence en 5′ parmi celles des 32 séquences testées qui ne présentent pas d’hétérogénéité en 5ʹ′. La seconde solution est d’utiliser une étiquette clivable en 5ʹ′ de l’ARN d’intérêt, tel que le ribozyme hammerhead, déjà utilisée pour ce genre d’application, ou le système CRISPR/Cse3 que nous proposons dans l’article présenté dans ce mémoire. De plus, nous avons adapté la méthode ARiBo pour rendre possible la purification d’un long ARN de 614 nt, le polycistron miR-106b-25. Nous avons également démontré la possibilité d’utiliser la méthode ARiBo pour l’isolation de protéines qui se lient à un ARN donné, le précurseur de miRNA pre-miR-153-2. En conclusion, ce mémoire démontre la possibilité d’adapter la méthode ARiBo à plusieurs applications. / In recent years, the field of RNA research has exploded due to several discoveries demonstrating the importance of RNA in many biological processes. Along with the increased interest in this field, large amounts of RNA have become essential to the success of several studies, in particular for structural biology and functional characterization. However, there are still very few native purification methods that are simple, efficient and reliable. In the past few years, the Legault laboratory has established an affinity purification method using an ARiBo tag to purify RNAs produced by in vitro transcription with the T7 RNA polymerase. This RNA purification method was specifically developed to maximise purity and yield. In addition, this method is fast and works with several types of RNAs. However, like several other purification methods, this method produces RNAs with 5' heterogeneity. This Master’s thesis propose solutions to overcome the problem of 5' heterogeneity for RNAs transcribed with the T7 RNA polymerase and purified with the ARiBo method. The first solution proposed is to choose a 5' sequence among those of the 32 sequences tested that do not present 5'- heterogeneity. The other possibility is the use of a cleavable tag at the 5'-end of the RNA of interest, such as the hammerhead ribozyme, already used for this purpose or the CRISPR/Cse3 system, which is presented here. Furthermore, we have adapted the ARiBo method to purify an RNA of 614 nt, the miRNAs cluster miR- 106b-25. We also demonstrate the possibility to use the ARiBo method to isolate proteins that bind a given RNA, the miRNA precursor pre-miR-153-2. In conclusion, this Master’s thesis demonstrates the possibility of adapting the ARiBo method for several applications.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / Le ribozyme "hammerhead" ou "en tête de marteau" appartient à une classe d'ARN catalytiques qui fut découverte pour la première fois chez certains viroïdes de plantes. Le ribozyme hammerhead peut spécifiquement reconnaître et cliver des ARN cibles par simple appariement des paires de bases entre le substrat et le ribozyme. Cela le rend un candidat attirant pour inactiver l'expression de gènes spécifiques in vivo. Cependant, dans le but d'utiliser ce ribozyme comme un agent antiviral et anticancer, il est important d'augmenter l'efficacité et la reproductibilité de son activité in vivo. Dans ce projet, nous utilisons Escherichia coli comme système modèle pour étudier l'effet de facteurs cellulaires sur l'activité du ribozyme in vivo. Il a été proposé que le couplage des processus de transcription et de traduction chez les bactéries pouvait limiter l'accès du ribozyme à sa séquence cible. Nous avons évalué l'inhibition du gène de la chloramphénicol acétyle transférase (CAT), codé sur un plasmide, par le ribozyme hammerhead chez E. coli dans des conditions où la transcription et la traduction ont été découplées soit par une augmentation de la vitesse de transcription, soit par une réduction de la vitesse de traduction. Dans une souche mutante de E. coli où la vitesse de traduction est réduite à 5.6 a.a./sec (la vitesse normale est de 15 a.a./sec), nous avons trouvé que l'activité de la CAT et le niveau d'ARNm de la CAT étaient réduits de près de 60%, alors que la présence d'un ribozyme n'avait aucun effet sur l'activité de la CAT dans une souche sauvage de E. coli. L'addition de streptomycine dans le milieu de culture, qui augmente la vitesse de traduction dans la souche mutante, rétablit la pleine activité de la CAT. Pour pousser plus loin l'idée que la souche à ribosomes lents facilite l'activité du ribozyme, nous avons incorporé le gène de la CAT dans le génome des souches sauvage et mutante. Dans ce cas, l'activité du ribozyme était détectable dans la souche sauvage et elle était grandement augmentée dans la souche à ribosomes lents. D'autre part, l'expression du gène de la CAT avec un promoteur de l'ARN polymérase T7, ce qui augmente la vitesse de transcription de ce gène, n'est pas affectée par le ribozyme hammerhead. Puisque la rapidité de l'ARN polymérase T7 devrait dissocier la transcription de la traduction, la dissociation de ces processus n'est donc pas une condition suffisante pour augmenter l'activité du ribozyme in vivo. Pour tester l'hypothèse que la vitesse de traduction pourrait affecter l'activité du ribozyme, nous avons testé trois ribozymes dirigés contre différentes régions de l'ARNm de la CAT. Nous avons trouvé que les trois possèdent des activités similaires. Il semble donc que l'accessibilité des régions 5' et 3' de l'ARNm de la CAT soit équivalente pour les ribozymes. Nous avons aussi utilisé d'autres stratégies pour ralentir la vitesse de traduction, soit l'insertion de codons à faible usage dans le gène de la CAT et la mutation de la séquence Shine-Dalgarno du gène de la CAT. Cependant, aucune de ces stratégies n'augmente l'activité du ribozyme. Ainsi, une vitesse inférieure de traduction ne peut expliquer l'amélioration de l'activité du ribozyme dans la souche à ribosomes lents. Nos résultats suggèrent qu'un facteur cellulaire inconnu affecte l'activité du ribozyme in vivo. À savoir si l'allèle mutant dans la souche à ribosomes lents, le gène S12, et son activité de chaperonne pourrait être impliqué est maintenant sujet de plus amples travaux. Ce système bactérien offre une fenêtre unique pour l'étude des facteurs cellulaires pouvant affecter l'activité des ribozymes in vivo. Nous avons aussi été impliqués dans l'étude d'ARN catalytiques dans la levure (Saccharomyces cerevisiae) dans l'espoir de comprendre comment l'environnement cellulaire de la levure affecte la fonction d'un ARN catalytique. Il a été démontré au Chapitre IV que l'activité de clivage d'un ribozyme peut être détectée par une nouvelle technique, le PCR dépendant de la ligation d'un ARN, quand le gène cible ADE1 est exprimé en cis du ribozyme, mais aucune activité en trans n'a pu être détectée dans ce même système. Ceci peut suggérer que l'association du ribozyme avec sa cible ainsi que l'activité catalytique du ribozyme sont plus lentes que le métabolisme des ARN dans la levure. Au Chapitre V, ceci a été démontré par l'arrêt du cycle cellulaire de la levure par trois différents traitements permettant une pause des cellules en phase G1, ce qui ralentit le métabolisme des ARNm. Dans ce cas, l'activité du ribozyme hammerhead dans la levure est détectée par l'évaluation directe de la concentration de l'ARNm de ADE1. Ces résultats indiquent que l'activité du ribozyme est influencée par l'environnement cellulaire.

Chapter 1 DNA barcoding based on the mitochondrial cytochrome c oxidase subunit I (COI) gene sequence is emerging as a useful tool for identifying unknown, whole or partial organisms to species level. However, the application of only a single mitochondrial marker for robust species identification has also come under some criticism due to the possibility of erroneous identifications resulting from species hybridizations and/or the potential presence of nuclear-mitochondrial psuedogenes. The addition of a complementary nuclear DNA barcode has therefore been widely recommended to overcome these potential COI gene limitations, especially in wildlife law enforcement applications where greater confidence in the identifications is essential. In this study, we examined the comparative nucleotide sequence divergence and utility of the mitochondrial COI gene (N=182 animals) and nuclear ribosomal internal transcribed spacer 2 (ITS2) locus (N=190 animals) in the 8 known and 1 proposed cryptic species of globally widespread, hammerhead sharks (family Sphyrnidae). Since hammerhead sharks are under intense fishing pressure for their valuable fins with some species potentially set to receive CITES listing, tools for monitoring their fishery landings and tracking trade in their body parts is necessary to achieve effective management and conservation outcomes. Our results demonstrate that both COI and ITS2 loci function robustly as stand-alone barcodes for hammerhead shark species identification. Phylogenetic analyses of both loci independently and together accurately place each hammerhead species together in reciprocally monophyletic groups with strong bootstrap support. The two barcodes differed notably in levels of intraspecific divergence, with average intraspecific K2P distance an order of magnitude lower in the ITS2 (0.297% for COI and 0.0967% for ITS2). The COI barcode also showed phylogeographic separation in Sphyrna zygaena, S. lewini and S. tiburo, potentially providing a useful option for assigning unknown specimens (e.g. market fins) to a broad geographic origin. We suggest that COI supplemented by ITS2 DNA barcoding can be used in an integrated and robust approach for species assignment of unknown hammerhead sharks and their body parts in fisheries and international trade. Chapter 2 The gray reef shark (Carcharhinus amblyrhynchos) is an Indo-Pacific, coral reef associated species that likely plays an important role as apex predator in maintaining the integrity of coral reef ecosystems. Populations of this shark have declined substantially in some parts of its range due to over-fishing, with recent estimates suggesting a 17% decline per year on the Great Barrier Reef (GBR). Currently, there is no information on the population structure or genetic status of gray reef sharks to aid in their management and conservation. We assessed the genetic population structure and genetic diversity of this species by using complete mitochondrial control region sequences and 15 nuclear microsatellite markers. Gray reef shark samples (n=305) were obtained from 10 locations across the species’ known longitudinal Indo-Pacific range: western Indian Ocean (Madagascar), eastern Indian Ocean (Cocos [Keeling] Islands, Andaman Sea, Indonesia, and western Australia), central Pacific (Hawaii, Palmyra Atoll, and Fanning Atoll), and southwestern Pacific (eastern Australia – Great Barrier Reef). The mitochondrial and nuclear marker data were concordant in most cases with population-based analysis showing significant overall structure (FST = 0.27906 (pST = 0.071 ± 0.02), and significant pairwise genetic differentiation between nearly all of the putative populations sampled (i.e., 9 of the 10 for mitochondrial and 8 of the 10 for nuclear markers). Individual-based analysis of microsatellite genotypes identified at least 5 populations. The concordant mitochondrial and nuclear marker results are consistent with a scenario of very low to no appreciable connectivity (gene flow) among most of the sampled locations, suggesting that natural repopulation of overfished regions by sharks from distant reefs is unlikely. The results also indicate that conservation of genetic diversity in gray reef sharks will require management measures on relatively local scales. Our findings of extensive genetic structuring suggests that a high level of genetic isolation is also likely to be the case in unsampled populations of this species.

Sharks are considered top predators in many marine ecosystems, and can play an important role in structuring those communities. As a result, it is necessary to understand the factors that influence their abundance and distribution. This is particularly important as fishery managers develop fishery management plans for sharks that identify areas that serve as essential fish habitat (EFH). This includes nursery habitat where sharks are born and juveniles spend the early part of their life. However, our understanding of shark habitat use in the northeast Florida waters is limited. The goal of this thesis was to characterize the abundance and distribution of sharks in northeast Florida estuaries, and to examine the effect of abiotic and biotic factors affecting shark habitat use. A bottom longline survey conducted from 2009 – 2011 indicated that 11 shark species use the estuarine waters of northeast Florida during summer months. Atlantic sharpnose (Rhizoprionodon terraenovae), blacktip (Carcharhinus limbatus), bonnethead (Sphyrna tiburo), and sandbar sharks (Carcharhinus plumbeus) were the most abundant species and made up 87.1% of the total catch. Month, bottom water temperature, and depth were the most important factors determining the presence and abundance of these species. This study also examined the role of prey abundance in determining the abundance of Atlantic sharpnose sharks. The probability of catching an Atlantic sharpnose shark, and the abundance of Atlantic sharpnose sharks, were most influenced by site. Neither potential prey abundance nor preferred prey abundance were not significant factors effecting Atlantic sharpnose abundance. This may be a result of prey sampling not providing an accurate measure of the true availability of prey resources. Other factors, such as predation risk, may better explain habitat use patterns of Atlantic sharpnose sharks. Continued sampling will give a better understanding of the factors influencing shark habitat use in this area.