The Southeastern United States has the most diverse and imperiled freshwater mussel (Unionidae) fauna in the world. The community structure and decline of these organisms is the result of complex interactions between biotic and abiotic factors, but the limited spatial scale of most community studies has failed to elucidate the underlying mechanisms shaping community structure. Basing community assessments solely on microhabitat variables alone has lead to stark contradictions in management recommendations and opposing definitions of habitat requirements for these organisms. However, with the introduction of GIS technology into aquatic management, it is now feasible to include variables from larger spatial scales and investigate previously undetectable mechanisms influencing unionid community structure. In my study, I tested the hypothesis that patterns of mussel species richness and abundance in the Tickfaw, Tangipahoa Bogue Chitto and West Pearl rivers in south-eastern Louisiana were related to a combination of local-scale habitat variables, riparian-scale land use and geology variables, and sub-segment scale land use and geology variables. ANOVA results indicated a significant difference between the upper three sub-segments and lower three sub-segments of these rivers. The Principal Component Factor Analysis (PCFA) results revealed that geology, land-use and fine sediment are components working together across spatial scales to produce a hydrologic variability mechanism, and a simple regression model based on the factor scores of each site was successful in predicting abundance and species richness. Through comparing the ANOVA results with the factor score results for each site, I conclude that hydrologic variability, defined by geology type and land-use as well as fine sediment, is influencing the pattern in freshwater mussel abundance and species richness found in the Florida Parishes of Louisiana.

Effects of hydrocarbons, salinity, tidal height, bioenergetics and competition on the oyster reef fauna were studied. Dried oyster shell was exposed to crude oil in the laboratory and plastic trays filled with control and oil-exposed shell were then deployed at two locations, in two seasons and at two tidal levels, with immersion periods of two and five weeks. Salinity exhibited a significant effect with sub tidal site having higher diversity. Hydrocarbon effects were less prominent. To determine effects of cleaners, Corexit 9580 was applied both alone and in combination with oil on cultch. At high concentrations, the cleaner ameliorated hydrocarbon effects. Hydrocarbon effects seemed to be less prominent than salinity and aerial exposure. In the next chapter, effects of salinity on the survival and bioenergetics of mud crabs Panopeus simpsoni and Eurypanopeus depressus were examined. Crabs were exposed to a range of salinities to determine the effects of salinity on tolerance. P. simpsoni exhibited a 28d LC50 of 6.97 PSU while E. depressus had a 28d LC50 of 0.19 PSU. Crabs were exposed to four salinities for bioenergetic measurements. Energy expenditure was highest at the lowest salinity and decreased as salinity increased. Scope for growth declined below 17.5 PSU. E. depressus was capable of surviving lower salinities than P. simpsoni. However, the physiological responses do not significantly differ between the two species. In the next chapter laboratory experiments were conducted to see if these two crab species differ in resource holding potential. Crabs were placed in aquaria at two salinities and refugia were checked daily for shelter occupancy. To determine if resource holding potential for refugia influenced predation risk, a second laboratory experiment was performed with a blue crab predator in each tank. The numbers of mud crabs of each species surviving were recorded. E. depressus was dominant over P. simpsoni in occupying the shelters at both salinity levels. E. depressus exhibited a higher survival in the presence of the predator. E. depressuss ability to tolerate lower salinities, and its dominance in resource holding potential, may lessen predation risk and allow colonization of more estuarine sites.

Analyses of ITS, ETS and matK sequences for genera of the subtribe Hinterhuberinae, including representatives of 17 subtribes from the tribe Astereae, rooted on Anthemideae, Calenduleae and Inuleae, provide no support for the monophyly of the Hinterhuberinae. Chiliotrichum and Celmisia groups, Olearia in part, Novenia, Oritrophium and Pteronia are in the basal group that also includes subtribe Felicinae. Madagaster, Remya, Olearia in part, Achnophora and Hinterhubera group lie in the Southern Hemisphere grade, with Asterinae, Bellidinae, Brachycominae, Grangeinae, Podocominae, Lagenophorinae and Baccharidinae. Monophyly of Asterinae, Grangeinae, Podocominae and Lagenophorinae is not supported. Instead South American representatives of Lagenophorinae, Grangeinae and Podocominae group with Baccharidinae and Hinterhubera group in the South American clade that is nested in the Southern Hemisphere grade. They are sister to the North American subtribes. Remya, Achnophora, Olearia in part and Australasian, European and African representatives of Asterinae, Brachycominae, Grangeinae, Podocominae and Lagenophorinae are grouped in a polytomy that is basal to the South American clade. Laennecia (Podocominae) is sister to Westoniella (Hinterhuberinae), and Myriactis panamensis (Lagenophorinae) and Plagiocheilus (Grangeinae) group with Archibaccharis (Baccharidinae). The Celmisia group is monophyletic. The radiate Diplostephium and Guynesomia do not group with other radiate genera from the Chiliotrichum group, but are in the Southern Hemisphere grade and close to the disciform Hinterhubera group. Transfer of Blakiella from Podocominae to Hinterhuberinae is supported by the three datasets. Madagaster is isolated from the other Hinterhuberinae in having basal position in the Southern Hemisphere grade. Evolution of dioecy in Baccharidinae and Aztecaster (Hinterhuberinae) evolved independently. Woody habit, epaleate receptacle, bisexual disk florets and ligulate peripheral florets are ancestral states in the tribe Astereae. Staminate disk florets and paleate receptacles are derived characters that evolved in several lineages independently. South America, southern Africa or New Zealand are proposed centers of origin for Astereae and for the Chiliotrichum and Celmisia groups. The Hinterhubera group evolved from the South American autochthonous elements, probably from the lower altitude plants during the uplift of the Andes and the development of páramo habitat.

Coevolution between amino acid residues and its context-dependence are important for exploring protein structure and function, and critical for understanding protein structural and functional evolution. Coevolution has long been ignored because of its complexity and the lack of computing power. In the research presented here, I developed an efficient coevolution analysis methodology based on likelihood comparisons of statistical models. Likelihood ratios and Bayes factors, calculated using the Markov chain Monte Carlo algorithm, were employed as the statistics. Two types of models, 2-state and 3-state, were developed to allow for the context-dependence of coevolution. Computer programs implementing this methodology were coded in C/C++ and were run on the Beowulf clusters of our laboratory and the super computers of LSU. Using these programs and custom Perl scripts, residue coevolution in cytochrome c oxidase and photolyases/cryptochromes protein superfamily was analyzed. I found that pairwise coevolution between residues is highly dependent on protein tertiary structures and functions. I detected extensive coevolving pairs in all our analyses, and these pairs were primary localized in regions of known structural and/or functional importance. I also found that coevolution is related to evolutionary rate and concentrated in moderately conserved sites. In supporting the importance of functional constraints, I detected a non-negligible coevolutionary signal between complex subunits and stronger coevolution in proteins of functional importance. I also found that the interaction between subunits can serve as a local coevolutionary constraint on one subunit rather than driving coevolution between two subunits. Based on coevolutionary patterns, I suggested that a domain without any previously supposed function actually operates as a folding core in the proteins of photolyase/cryptochrome superfamily. The coevolutionary patterns also provided clues regarding the functional evolution of electron transfer in this superfamily. I also found that coevolving sites with double substitutions along a branch tend to occur only at physically contacting sites, and that salt-bridge stabilization and secondary structure stabilization are important forces of residue coevolution. The methodology and programs developed in this research are powerful tools for coevolutionary analysis, which can provide valuable information for characterization of protein structural/functional domains and exploration of protein structural/functional evolution.

Recently, much evidence has been brought forth into the scientific community supporting the idea that RNA Polymerase III transcribed regions of DNA may serve as chromosomal landmarks for silencing. Transfer RNA genes are known to involve themselves in several extra-transcriptional functions within the chromosome, including the pausing of replication forks, Ty element integration, tRNA position effects (repression of neighboring genes), acting as a barrier to the spread of heterochromatin, and over-riding nuclosome positioning sequences. Our results suggest that many tRNA genes may serve these functions as well as exhibiting behavior similar to metazoan insulators. Also, ETC (Extra TFIIIC) sites within Saccharomyces cerevisiae, which bind only TFIIIC may also act as barriers or insulators. Our results support the idea that extra-transcriptional functions of RNA Polymerase III factors may be widespread and important contributors to genome biology.

Epigenetic modification is another mechanism involved in the cancer development besides classic mutations such as deletion. Aberrant promoter methylation and associated chromatin modification have been frequently reported in various tumors of different clinical stages. Hypermethylation has been frequently observed in tumor suppressor genes and causes reduced transcripts of these genes. Hypomethylation also has been reported to be involved in activation of oncogenes. Inactivation of the X chromosome and the imprinting of gamete DNA depend on the methylation patterns in the promoter region. Understanding methylation mechanisms could be helpful to diagnosis of early stage tumorgenesis or offer molecular markers for detecting cancers. Changes in methylation patterns based on human vulva pathological tissue type of four genes have been studied in the current project. RASSF1A and DAPK-1 are tumor suppressor genes. BRCA2 is considered highly associated with breast and ovarian cancer. And H19 is a maternal imprinted gene. DAPK-1 and BRCA2 have been found to be significantly hypermethylated in Lichen Sclerosis (LS) and Squamous Cell Carcinoma (SCC). And RASSF1A has displayed an interesting methylation pattern in the post transcription region, where the frequency of methylation significantly decreased from normal tissue to LS tissue, but then dramatically increased to SCC. H19 failed to show any changes in the methylation pattern with methods tested. This was most likely due to interference of primer dimers by SYBR-green in the real-time PCR analyses. Instead of using the same promoter sequence reported by previous papers, extended estimated promoter regions and partial transcription regions were obtained from Genome Browser and additional CpG island sites have been studied in current project. Methylation patterns have been detected that differ from the literature in these genes. These results may provide more information to find a more precise active promoter region and epigenetic involved sequences for future research.

LINE-1s (Long interspersed elements or L1s) and Alus are highly successful non-long terminal repeat retrotransposons with copy numbers of ~520,000 and >1 million within the human genome, respectively. They are associated with human genetic variation and genomic rearrangement. Although they are abundant throughout primate genomes, their propagation strategy remains poorly understood. The recently released human and chimpanzee draft genome sequences provide the opportunity to compare the human genome with the chimpanzee genome. Thus, we were able to assess how these elements expanded in primate genomes and how they create genomic instability during their integration into the host genome as well as subsequent post-insertion recombination between elements. To understand the expansion of Alu elements, we first analyzed the evolutionary history of the AluYb lineage which is one of most active Alu lineages in the human genome. We suggest that the evolutionary success of Alu elements is driven at least in part by stealth driver elements that maintain low retrotransposition activity over extended periods of time and occasionally produce short-lived hyperactive copies responsible for the formation and remarkable expansion of Alu elements within the genome. Second, we conducted a detailed characterization of chimpanzee-specific L1 subfamily diversity. Our results showed that L1 elements have experienced different evolutionary fates in humans and chimpanzees lineages. These differential evolutionary paths may be the result of random variation or the product of competition between L1 subfamily lineages. Third, we report 50 deletion events in human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of ~18 kb of human genomic sequence and ~15 kb of chimpanzee genomic sequence. This study provides the basis for developing models of the mechanisms for small and large L1 insertion-mediated deletions. Fourth, we analyzed the magnitude of Alu recombination-mediated deletions in the human lineage subsequent to the human-chimpanzee divergence. We identified 492 human-specific deletions (for a total of ~400 kb of sequence) attributable to this process. The majority of the deletions coincide with known or predicted genes, which implicates this process in creating a substantial portion of the genomic differences between humans and chimpanzees.

In this dissertation I describe a number of patterns and interesting aspects associated with the evolution of snake mitochondrial genomes (mtDNA). I also attempt to resolve the phylogeny of squamates, focusing on the relationship between the snakes and lizards. The results of this study indicate that snakes and worm lizards (amphisbaenians) appear to share an exclusive common ancestor, and snakes appear to have undergone strong selective pressure that shaped snake mtDNAs. Snake mtDNAs have several unique features, including a compact size, duplicated control regions, and an elevated evolutionary rate. Based on the correlation resulting from the asymmetric replication of mtDNA, the usage of control regions was inferred to be species specific. In snake mtDNAs, the magnitude of the rate acceleration varied considerably among genes and over time, and it appears that these changes at the nucleotide and protein level co-occurred with snake mtDNAs incurring a reduction in size and a duplication of the control region. In snake mtDNA, many unique amino acid substitutions were identified in all protein-coding genes. In the Cytochrome C Oxidase subunit I (COX1) protein, one of three proposed proton transfer channels was enhanced by several unique substitutions. Additionally, strong positive selection was detected on the COX1 gene of alethinophidian snakes. These may be causally related to the energetic demands imposed by the radical energy requirement in the early digestion period of alethinophidian snakes. Observations of change in COX1 gene suggest that, due to the relaxation of selective pressure or a population bottleneck, numerous deleterious substitutions accumulated on snake ancestral lineages. Then the impaired functions were recovered, or even enhanced by adaptation. During this period, the evolutionary rate of snakes was accelerated as well. In this research, the phylogenetic placement of snakes was inferred using the complete mtDNA of 65 vertebrates by maximum likelihood (ML) and partitioned-Bayesian inference. Snakes were placed as the sister taxon to worm lizards, and this branching pattern is strongly supported by Bayesian inference-derived posterior probability. The jackknife simulation also supports the sister relationship between snakes and worm lizards, cumulatively rejecting the hypothesis of marine origins of snakes.

SVA is a composite repetitive element named after its main components, SINE, VNTR and Alu. There are ~3000 SVA elements in the human genome. A genomic distribution analysis indicates that SVA elements are enriched in G+C-rich regions but have no preferences for inter- or intra-genic regions. A phylogenetic analysis of these elements resulted in the recovery of six subfamilies that were named SVA_A to SVA_F. The composition, age and genomic distribution of the different subfamilies have been examined. Subfamily age estimates indicate that the expansion of four SVA subfamilies (SVA_A, SVA_B, SVA_C and SVA_D) began before the divergence of human, chimpanzee and gorilla, while subfamilies SVA_E and SVA_F are restricted to the human lineage. Furthermore, I examined the amplification dynamics of SVA elements throughout the primate order and traced their origin back to the beginnings of hominid primate evolution, approximately 18 to 25 million years ago, which makes SVA elements the youngest family of retrotransposons in the primate order. Gene duplication is one of the most important mechanisms for creating new genes and generating genomic novelty. Retrotransposon-mediated sequence transduction (i.e. the process by which a retrotransposon carries flanking sequence during its own mobilization) has been proposed as a gene duplication mechanism. SVA elements are capable transducing 3 flanking sequence during retrotransposition. I examined all the full-length SVA elements in the human genome to assess the frequency and impact of SVA-mediated 3 sequence transduction. The results showed that ~53 kb of genomic sequence has been duplicated by 143 different SVA-mediated transduction events. In particular, I identified one group of SVA elements that has duplicated the entire AMAC gene three times in the human genome via SVA-mediated transduction events, which happened before the divergence of humans and African great apes. In addition to the original AMAC gene, the three transduced AMAC copies contain intact open reading frames (ORFs) in the human genome and at least two are actively transcribed in different human tissues. Thus, duplication of entire genes and creation of new gene families via retrotransposon-mediated sequence transduction represent an important mechanism by which mobile elements impact their host genomes.

Vibrio vulnificus is a Gram-negative organism that is found in estuarine environments. The bacterium is capable of causing gastroenteritis, wound infections, and septicemia, often resulting in death for susceptible individuals. Monoclonal antibodies (MAbs) to five lipopolysaccharide (LPS) O-serovars have previously been used as a serotyping system in epidemiological studies. Here is the first report of two additional MAbs capable of recognizing two new LPS serovars (O6 and O7), further increasing the number of V. vulnificus that can be typed. Analysis of V. vulnificus isolates from the United States, Europe, and Asia demonstrated that the O1/O5 serovar is dominate in clinical isolates, serovars O3 and O4 are dominate in the environmental isolates of Europe, and that most environmental isolates from United States are nontypeable. Additionally, MAb, RS1, was chosen for further analysis by having DNA coding for its light and heavy chains cloned into a phage-display system. The published primers, designed to be used with the pComb3X display system, produced primer dimers and were unsuccessful in generating the single chain variable fragment (scFv) to be cloned into the phage vector. Modification of the existing primers inhibited the formation of primer dimers and allowed for the successful generation of the scFv. The resulting phage-displayed antibody, PJ1, displayed similar binding characteristics as RS1 showing the usefulness of phage-display as an alternative method of maintaining pre-existing MAbs. Lastly, it was proposed that as V. vulnificus undergoes capsular switching it is possible that LPS switching occurs as well. Analysis of the data suggests that capsule and LPS switching are not directly related. Interestingly, LPS switching seems to occur in a unidirectional manner on two separate branches that converge on serovar O5 suggesting that O5 may be a group of sugars that are used as a building block for other serovars. This allowed for the formation of a speculative LPS model. The stability of serovars O6 and O7 in the LPS switching assay make them difficult to place in relationship to serovar O5 and further analysis will be needed to more accurately place them on the LPS model.