The effects of DNA damage signaling on the DNA replication of the polyomavirus SV40 are examined in this dissertation. Infection of monkey cells with SV40 results in large amounts of cellular DNA damage signaling through the cellular kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR). Literature pertaining to DNA replication, DNA repair, DNA damage signaling, and polyomaviral replication is first discussed. This is followed by a number of experiments testing the molecular mechanisms that necessitate DNA damage signaling by ATM and ATR during SV40 infection. Furthermore, the contribution of ATM and ATR to SV40-induced cell cycle arrest and repair protein recruitment to viral DNA replication centers is explored. After an examination of how my experiments fit in with relevant manuscripts, I discuss the possible consequences of the aberrant products found upon inhibition of DNA damage signaling during SV40 DNA replication and how my current studies can be advanced. Additionally, the connection between DNA damage signaling and DNA repair and the implications of my dissertation to cellular DNA replication are discussed.

This research utilizes aphids to address some of the emerging themes in social evolution. To begin to address these themes, I identified traits to measure sociality in aphids. I found that for the ecological and behavioural traits tested, housekeeping and defense, there were quantifiable differences between social and nonsocial species. However, there was no clear threshold that differentiated social from nonsocial species, meaning that definitions of sociality in aphids depend in part on the traits that are measured. Next, I explored a major theme of social evolution, the ability of groups to protect the nest from predators, by characterizing adaptations for defense. I found that aphid soldier elicit an overexpression of the melanization immune response in victims causing toxicity which leads to death, and successful defense of the nest. Finally, cooperation is inherently vulnerable to exploitation by cheaters. Aphids present a rare opportunity to study the effect of cheaters on groups due to multiple unique life history traits. I characterized the consequences of cheaters for weakly and nonsocial species and, contrary to our expectations, the presence of cheaters has the strongest negative effect on the nonsocial species, compared to the social or weakly social species. The results suggest that there is a more complex relationship between competition and relatedness than previously realized. Taken together, these results offers a new perspective on the role of defense, social immunity and kin selection in the evolution of sociality in this disparate group.

Small noncoding RNAs including miRNAs and siRNAs play significant roles in gene regulation via the mechanism of RNA interference (RNAi) and have been exploited to conduct loss-of-function studies. The discovery that these small RNAs can regulate genes at the post-transcriptional level by interacting with mRNA targets has enabled researchers to understand their importance in normal cellular processes as well as disease processes. Although extensive findings have been illuminated at the post- transcriptional level, there are still some questions regarding roles of small RNAs at the level of transcription (DNA) using higher vertebrate models. Using zebrafish, we explored a novel transcriptional gene silencing method and found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable silencing in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes. Silencing was mediated by the RNAi enzyme, Dicer and ChIP analyses detected an enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame. Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

Though among the most controversial topics in systematic and evolutionary biology, species are a fundamental unit in biology, and are utilized by and critical to a wide variety of studies in the life sciences. Despite this importance, little work has focused on developing and examining objective methods for species delimitation until recently. Further, New Guinea and the surrounding regions are among the most diverse and geologically complex regions globally, yet the region remains poorly explored biologically, and little work has examined the evolutionary history of the fauna in the region. To investigate the influence of factors such as sampling intensity, species richness, and phylogenetic structure on discovery methods for species delimitation, I combine simulated and empirical data. In Chapter 1, I use simulated data to examine the accuracy of three discovery methods for species delimitation under a variety of different sampling strategies. I find that genetic clustering algorithms, such as Structurama, can be highly accurate in identifying even recent divergences with limited sampling of individuals and of loci, and that Gaussian clustering can be similarly accurate, though somewhat less sensitive to detecting recent divergences. However, my results show that nonparametric delimitation is highly sensitive to errors in gene genealogy estimation, and generally fails to delimit species accurately when true coalescent gene genealogies are unknown, as in empirical applications. In Chapters 3 and 4, I apply these methods empirically to examine the species boundaries, as well as the phylogeny and other aspects of the evolutionary history of, scincid lizards of the C. bicarinata and C. fusca groups, respectively. My results in Chapter 3 indicate that species delimitation analyses may be prone to underestimating the number of species by identifying only higher levels of clustering in systems with deep phylogenetic structure. I additionally find evidence for several cryptic species in the group, including deep, species-level divergence among the populations of C. storri from Australia, the Aru Islands, and New Guinea, despite their recent connectivity via Sahul Shelf emergence during Pleistocene glaciations. Through also examining niche evolution in the group, I find evidence for niche conservatism among most species in the group, but two species, C. bicarinata and C. sp. Amau from eastern Papua New Guinea, show evidence for environmental niche divergence. Analyses of the C. fusca group in Chapter 4 provide further evidence for a tendency of discovery methods for species delimitation to under-detect species in groups with high diversity or deep phylogenetic structure. Genetic clustering algorithms based on the complete dataset only identify a small number of clusters that correspond largely to deep phylogenetic clades, but when restricted to within these clades, this method identifies clusters that correspond well to finer, putative species-level structure. I also find evidence for extensive cryptic diversity in this group, identifying 28 distinct species among my sampling of 16 currently recognized species, as well as other incongruence with current taxonomy, including synonymous species and mis-assigned populations, supporting previous evidence of the need for extensive taxonomic revision in the C. fusca group. My biogeographic analyses also providence evidence that the C. fusca group likely evolved in Australia or Australia and New Guinea before diversifying in New Guinea, dispersing at least twice across Lydekkers line into Wallacea, and possibly also recolonizing Australia. Finally, in Chapter 5, I take a more comprehensive approach, and combine genomic and morphological data to test the validity of and examine the demographic history of two putative species of Tribolonotus from the islands of Buka and Bougainville in the northwestern Solomon Archipelogo. I use next-generation sequencing to collect a genomic dataset of several thousand loci, and apply species discovery (genetic clustering algorithms) and species validation (Bayes factor delimitations) to test for speciation between these populations. My results support this speciation event, despite the recent connectivity between these islands. I also collect a suite of morphological characters for this group and provide evidence for morphological divergence and diagnosibility. Demographic analyses applied using approximate Bayesian computation and diffusion analysis further provide evidence for a complex demographic scenario in which migration between these populations continued for some time following their initial divergence, but subsequently decreased in rate or ceased entirely. Combined, these results yield extensive insight into the utility of several methods for species delimitation, the taxonomy and systematics of Carlia and Tribolonotus in New Guinea and the surrounding regions, and the complex processes responsible for driving the generation and maintenance of the phenomenal diversity in the Sahul shelf region.

Species interactions play a prominent role in the establishment and spread of many invasive species. However, rarely are invasions studied in more than a direct pairwise species context, or with consideration to how species interactions can vary biogeographically. Using field surveys combined with common garden and greenhouse experiments, I investigated how multitrophic above and belowground interactions influence plant invasions at large spatial scales. I focused on comparisons between sympatric native and invasive lineages of Phragmites australis, a wetland grass distributed throughout North America. I conducted a field survey to examine support for the enemy release hypothesis in a tritrophic framework. In North America, the invasive lineage of P. australis escaped from introduced Lipara gall-flies, attributed to greater vertebrate predation on Lipara infesting the invasive than the native lineage. A complementary common garden experiment revealed that enemy release of the invasive P. australis lineage from Lipara was driven by local environmental conditions rather than genetic differences between the two lineages. Importantly, local enemy release was strongest at northern latitudes, generated by genetically based non-parallel latitudinal gradients in Lipara herbivory for the native and invasive lineages. This phenomenon could translate to biogeographic variation in invasion success and is worthy of investigation across a range of invaded systems and species interactions. I also conducted a greenhouse experiment to examine the interactive effects of rhizosphere soil biota, interspecific competition, and nutrient availability on performance of P. australis and native smooth cordgrass, Spartina alterniflora. All lineages of P. australis suffered negative impacts from soil biota, suggesting this interaction does not directly facilitate the success of invasive P. australis. However, the most interesting result from this experiment was that soil biota from the invasive P. australis lineage negatively impacted S. alterniflora, whereas soil biota from the native lineage had a positive impact. This indirect spillover of pathogens and mutualists interaction may have important implications for invasion success and restoration. In summary, my dissertation highlights the importance of examining biological invasions in a biogeographic and multitrophic context and has broad implications for the understanding and management of biological invasions.

The sedge family, Cyperaceae, is a large group with approximately 5,000 species distributed among ca. 100 genera. Sedges are economically and ethnobotanically important. They are conspicuous members of many floras around the world and provide vital food and cover for wildlife. The focus of this dissertation is on the genus Cyperus, which includes about 900 species. Due to its large size, advancements in knowledge of Cyperus are made in small bites. The molecular phylogenetics component of this research focused on New World Cyperus. Chapter 2 presents a phylogentic analysis employing sequences from the nuclear ITS region. Chapter 3 presents a more robust analysis using five genic loci, including sequences from nuclear ITS plus four plastid loci. This research was the first to estimate the phylogenetic position of the monotypic genus Karinia. Karina has been included in Cyperus in the past, and is here resolved as a member of the Ficinia clade which is consistently estimated as sister to the Cyperus clade in molecular studies. Karinia was embedded in a clade with Sciproides. Its morphology, including perennial habit, dense head-like inflorscences, and spirally-arranged floral scales, are consistent with that of Scirpoides. Another important result of this work is the resolution of the predominantly Central American Cyperus andinus and C. seslerioides as belonging to section Leucocephali. This study strongly supports section Leucocephali, whose members utilize C3 photosynthesis, as sister to C4 Cyperus. Members of section Leucocephali are adapted to open seasonally dry grasslands, which may be the ecological intermediary to the evolution of C4 photosynthesis. Sampling for molecular studies included in this research enabled assessment of two taxonomic sections: Luzuloidei and Diclidium. The New World section Luzuloidei, which is composed of C3 members, is strongly supported as monophyletic. A morphological synapomorphy in section Luzuloidei is the presence of two-keeled floral scales. Section Diclidium, which is diagnosed as having spikelets which break into one- to two-fruited segments upon maturity, is polyphyletic. Therefore, this unique mode of spikelet shattering arose independently at least twice. Results provided some insight into several taxonomic problems in the Umbellati group and in section Strigosi; however, a greater number of samples are needed to assess these problems. The suspected relationship of the North American prairie species Cyperus cephalanthus to the South American Cyperus rigens species group was confirmed by the molecular phylogenetic analyses, as was the monophyly of the C. rigens group. Cyperus cephalanthus strongly resembles the South American C. impolitus. It was hypothesized that these taxa were morpholically indistinguishable. Morphometric analysis showed that several characters are statistically different, includeing floral scale dimensions, achene width, and achene shape. However, results of Principal Components Analisis (PCA) suggest that Cyperus cephalanthus and C. impolitus are the same morphological species. Results of PCA of Cyperus rigens and several of its infraspecific taxa were inconclusive, highlighting the need for more work with this highly variable South American taxon. Coastal prairie is one of the most imperiled habitats in North America. It is in this habitat where Cyperus cephalanthus is found, with fewer than 20 known populations in North America (Louisiana and Texas). This research included a floristic survey of wet coastal prairie sites in southwestern Louisiana. This work was partly stimulated by the preference of Cyperus cephalanthus for this habitat, and further motivated by the discovery of several promising prairie remnants quadrupaling the aerial extent of known coastal prairie in Louisiana. The wet coastal prairie flora included 512 minimum-rank taxa, with 461 being native. A total of 255 were estimated to be ecological conservative and characteristic of coastal prairie, with the balance consisting of weedy elements taking advantage of disturbance and habitat alteration. The wet coastal prairie was rich in sedges, with 72 species among nine genera. Cyperus was the most species rich genus in the entire wet prairire flora with 20 species. Coefficients of conservatism (C-values) were assigned to each taxon in the wet prairie flora. These C-values will allow computation of various Floristic Quality Indices (FQI) for sites ranging from unplowed prairie remnants to de novo restorations.

Selected species in nature are able to tolerate prolonged periods of severe water stress. Survival of cellular desiccation in these anhydrobiotic organisms is conferred by a number of mechanisms including accumulation of stabilizing solutes, such as trehalose, and expression of Late Embryogenesis Abundant (LEA) proteins. An additional feature shared by some anhydrobiotic animals is the capacity to suppress mitochondrial oxidative phosphorylation in preparation for desiccation. These mechanisms improve the physiological robustness of cells to stress. The primary aim of this dissertation is to evaluate forms of metabolic preconditioning and to test the most propitious as a means to improve desiccation tolerance of mammalian cells. Several chemical agents, termed hypoxia mimetics, were evaluated in this study for utility in metabolic preconditioning. My results demonstrate that although each treatment increased Hypoxia Inducible Factor-1α (HIF-α) to varying degrees, none of them emulated every aspect of the hypoxia response in mammalian cells. A key aspect of the cellular hypoxia response is the phosphorylation and inhibition of pyruvate dehydrogenase (PDH). CoCl2 unexpectedly and strikingly decreased the phosphorylation of PDH. Neither desferrioxamine nor the prolyl-hydroxylase inhibitor FG-4592 caused an increase in PDH phosphorylation. Although dimethyloxalolglycine (DMOG) increased PDH phosphorylation, further examination of mitochondrial respiration showed that routine respiration was not reestablished 24 h after the treatment was removed. Based on these results hypoxia preconditioning was determined to be the most promising avenue for metabolic preconditioning. The effects of hypoxia preconditioning on desiccation tolerance were investigated with HepG2 cells that had been modified to accumulate trehalose and express AfrLEA2 from the brine shrimp Artemia franciscana. Spin-drying was used to quickly dehydrate the cells to a residual water content of 0.225 g H2O/g dry mass. Cells were then immediately rehydrated. The growth profiles of cells that received hypoxic preconditioning, with and without the nitric oxide donor MitoSNO, were compared to the growth of control cells without preconditioning. Results indicated that hypoxia preconditioning significantly increased cell proliferation compared to controls, and proliferation was further bolstered by the addition of MitoSNO. These findings support the concept that metabolic preconditioning can improve the biostability of mammalian cells after desiccation.

There is growing evidence for the regulatory roles of extracellular RNAs (exRNAs) in mediating cell-to-cell communication. To test whether exosomal RNA might also contribute to changes in gene expression in recipient cells, and to test whether mutant KRAS might regulate the composition of secreted RNAs, we comprehensively profiled small and long RNAs of cells and matched exosomes from isogenic colorectal cancer (CRC) cell lines differing only in KRAS status by RNA sequencing. We found that exosomal profiles are distinct from cellular profiles, and differentially enriched for specific small RNA, circular RNA (circRNA) and long RNA transcripts in exosomes dependent on KRAS status. Our small RNA analysis found that miR-10b was selectively increased in wild type KRAS exosomes while miR-100 was increased in mutant KRAS exosomes. In Transwell co-culture experiments, mutant KRAS donor cells conferred miRNA-mediated target repression in wild type KRAS recipient cells. In addition, we developed a bioinformatics pipeline to identify and evaluate circRNA candidates from RNA-Seq data. We found a significant down-regulation of circRNAs at a global level in mutant KRAS cells compared to wild type KRAS cells, indicating a widespread effect of mutant KRAS on circRNA abundance. Interestingly, circRNAs were more abundant in exosomes than cells, independent of KRAS status. Our long RNA analysis revealed that distinct RNAs species, such as pseudogene and antisense transcripts, are enriched in exosomes compared to cellular profiles. Additionally, specific mRNAs, such as Rab13, are upregulated in mutant KRAS exosomes. Here, we present comprehensive data to identify the broad and diverse classes of extracellular RNAs secreted in exosomes and we demonstrate that export of specific RNA can be altered by oncogenic KRAS signaling to potentially function beyond the cell of origin. Collectively, this will advance our understanding of exRNA biology in CRC and facilitate the development of potential exRNA biomarkers.

In recent years, there has been a rise in antimicrobial-resistant bacteria caused by overdependence on, and misuse of, antibiotics. This has led to an increase in research for identifying alternatives to combat pathogens. One promising means of combating pathogenic bacteria, particularly for those residing in the gastrointestinal tract (GIT), is the use of probiotics. This thesis focuses on a potential probiotic strain Lactobacillus fermentum 3872, the genome sequence of which was circularised during the study, identifying genes that may contribute to probiotic activity. Several genes involved in GIT survival, such as acid symporters were discovered, along with genes that encode adhesion proteins such as those involved in mucus, fibronectin and collagen binding. The genes mentioned above may contribute to L. fermentum 3872 survivability within the GIT and have an antagonistic effect on enteric pathogens via competitive exclusion. Other interesting genes identified in L. fermentum 3872 were potentially involved in bacterial aggregation, exopolysaccharide and vitamin synthesis, along with four prophage encoding regions. Genes that encode a class III bacteriocin was also identified. An additional gene encoding a collagen binding protein (CBP) of a newly discovered plasmid pLF3872, was recognised. The chromosomal sequence also had a partial CBP encoding gene. pLF3872 has a toxin-antitoxin gene pair that ensures stable maintenance of the plasmid, along with conjugation-related genes. Functional analysis of the recombinant CBP via ELISA experiments found that the protein had the ability to bind to collagen I, a protein present on the epithelial lining of cells of the GIT. ELISA experiments also demonstrated that a common gastrointestinal pathogen, Campylobacter jejuni, can bind to collagen I in a concentration-dependent manner. In addition, mass spectrometry analysis identified that C. jejuni strains 11168H and 81-176 may utilise flagellar components (FlaA and FlaB) for adhesion. Furthermore, C. jejuni 11168H and 81-176 binding to collagen I was inhibited in the presence of either L. fermentum 3872 or CBP, thus reducing C. jejuni adherence via competitive exclusion. Using an in vitro assay, it was also demonstrated that L. fermentum 3872 cell-free supernatant could inhibit the growth of C. jejuni, due to the acidic environment brought about by L. fermentum 3872. During the completion of the genome sequence of L. fermentum 3872, comparison of various sequence assembly techniques which focused on the quality of the genome assembly was conducted. The results showed that further extension of the genome sequence during sequence assembly may lead to assembly errors when over-relying on a commonly-used sequence quality indicator, referred to as read mapping. It is suggested that care must also be taken when using long read technology to complete the genome sequence of a bacteria, as this may result in nucleotide sequence redundancies.

Colorectal cancer (CRC) is one of the most commonly diagnosed types of cancer in the developed countries and the incidence is rising in the developing regions. Chronic inflammation, which is propagated by overexpression of cyclooxygenase-2 (COX-2) and its major product prostaglandin E2 (PGE2), plays a key role in the development of CRC. Culinary herbs and spices (CHS) are rich in polyphenols, have a high anti-oxidant capacity and possess anti-inflammatory activity. It has been shown that CHS inhibit the growth of CRC cells, however, their anti-carcinogenic mechanisms are mainly unknown. Hence, the aim of this study was to identify the CHS that were most potent inhibiting the growth of CRC cells, and subsequently to elucidate their anti-carcinogenic mechanisms, in particular, focusing on COX-2, the Wnt/β-catenin signalling pathway, and proteins involved in apoptosis. Another goal was to investigate whether combining the CHS would result in synergistic effects on the above. This study demonstrated that CHS extracted in water/or ethanol and their combinations inhibited CRC cell growth. This study also revealed that the most potent CHS extracted in ethanol (turmeric (TE), bay leaf (BLE) and ginger (GE)) and combinations downregulated the expression of COX-2 and suppressed COX-2 activity by reducing PGE2 release; their effect was comparable to that of the selective COX-2 inhibitor Celecoxib (50 μM). These CHS also induced apoptosis in CRC cells by targeting several key proteins: p53, caspase-3, and PAPR. However, the CHS did not have an effect on Wnt signalling pathway, which partially could be due to insufficient treatment time. In conclusion, this study demonstrated that CHS and their combinations inhibited CRC cell growth, inhibited COX-2 expression and activity, and modulated several key molecules involved in the development of CRC. Based on these findings, CHS have the potential to be utilized for CRC chemoprevention and possibly be used as a complimentary treatment. However, in vivo studies are needed to establish the true potential of these foods.