This dissertation is concerned with the expression patterns, functionality and mechanisms of disease vector mosquito odorant receptors. The principal afrotropical vector for malaria, Anopheles gambiae, discriminates human host cues through the use of a highly divergent family of odorant receptors (AgORs). In vivo, ORs form a functional complex consisting of a conserved and broadly expressed non-conventional OR co-receptor (ORco) and a conventional odor-binding OR, which provides coding specificity to each complex. I have used next-generation RNA sequencing to discriminate OR expression patterns, and identified novel candidate genes involved in chemosensory signal transduction. In addition, I have functionally characterized and identified conserved ORs between multiple disease vector mosquitoes. In an effort to identify novel modulators of AgORs for insect control, we have performed high-throughput, cell-based calcium imaging screens, and as a result, I have identified the first ORco family agonist. Through the heterologous expression of AgORco, we demonstrated the novel activity of this agonist, its effects upon the signaling properties of ORco, and elucidated a molecular mechanism of OR signaling. In addition, single unit electrophysiology studies demonstrate the ability of this ligand to activate AgORco-expressing cells in vivo. As such, ligand-mediated modulation serves as a proof-of-concept demonstration that ORco is a viable target for the development of behaviorally disruptive olfactory compounds (BDOCs) that could foster malaria reduction programs. These studies have established a new model for OR-signaling and identified a new class of molecules with the future potential to reduce global burdens of malaria and other vector borne diseases.

Alu elements are a primate-specific non-autonomous retrotransposon of ~300 base pairs (bp). A Short INterspersed Element (SINE), Alu elements are found in high copy number in genomes across the primate radiation. Alu elements have a history of effective use as a phylogenetic marker due to the presence-absence nature of their retrotransposition, and have been implicated in the cause of a number of genetic diseases. Various subfamilies of Alu elements have gone through periods of increased activity during the history of the primate radiation, with the overwhelming majority of elements classified as genomic "fossils" which no longer propagate but can serve as useful markers in elucidating phylogenies. Lemurs (Infraorder: Lemuriformes) are a monophyletic radiation of strepsirrhine primates indigenous to the Island of Madagascar. The Lemuriformes are taxonomically subdivided into 5 families and >100 species, with numerous questions remaining to be answered about evolutionary relationships at the family, genus, and species levels. We computationally examined the grey mouse lemur (Microcebus murinus) genome for the presence of informative Alu elements and constructed a phylogenetic tree of 22 lemur species and 2 out-group species using the 138 loci retrieved. Alu element activity level in primate genomes varies widely across species examined. The genome of the Western lowland gorilla (Gorilla gorilla gorilla) was sequenced in 2012 and made available for study. We examined this genome for the presence of AluY elements. AluY is the youngest Alu subfamily and the one most active in the hominid lineage. Our study elucidated 1,075 western lowland gorilla-specific AluY elements classified into 10 subfamilies based on diagnostic mutations in the sequence of the element.

Faithful DNA replication during each cell division cycle is carried out by multiprotein complexes acting at each replication fork. In this complex, DNA unwinding and synthesis activities, driven by the Mcm2-7 helicase and DNA polymerases α, delta and ε, are coordinated by non-enzymatic replication factors. One such protein, Mcm10 (mini-chromosome maintenance protein 10) interacts with single- and double-stranded DNA, pol α, and Mcm2-7. Mcm10 loads onto chromatin at the onset of S phase and is required for subsequent recruitment of downstream proteins, and is thus critical for the replisome assembly during replication initiation. Mcm10 has been reported to form homodimeric, trimeric, and hexameric assemblies. Vertebrate Mcm10 protein consists of an N-terminal oligomerization domain (NTD), followed by internal (ID) and C-terminal domains (CTD). ID and CTD each binds DNA and pol α. The NTD, containing a coiled-coil motif, was shown by analytical ultracentrifugation (AUC) to form a dimer, and deletion of this domain from the full-length protein results in a monomeric Mcm10 form. This dissertation describes structure-function analyses of the coiled-coiled (CC) region. AUC showed that the CC exists as dimer and trimer in solution, with trimeric assemblies stabilized by low pH. Crystal structure at 2.4 Å resolution at acidic pH showed the coiled-coil region as a 3-helix bundle. Mutation of aliphatic residues at the coiled-coil interface disrupted oligomerization of the CC and NTD observed by AUC. The same mutants disrupted FL Mcm10 oligomerization in vivo detected by yeast-2-hybrid. These studies more clearly define the role of the NTD in Mcm10 oligomerization and provide insights into the biological function of Mcm10 in DNA replication.

Polyamines are a diverse class of aliphatic molecules that are highly polycationic at physiological intracellular pH. Polyamine levels are regulated by the coordinated actions of arginase (Arg), ornithine decarboxylase (Odc), and polyamine oxidase (Pao). Although little is known of their functions in fish, polyamines have been implicated in diverse physiological processes, including RNA transcription, cell growth, cell proliferation, and cell apoptosis. The main objective of this study was to describe the transcription and enzymatic activities of key enzymes for polyamine metabolism, to measure polyamine levels, and to assess putative roles of polyamines in the gills of the Gulf killifish (Fundulus grandis) during hypoosmotic challenge. In addition, the influence of irreversible inhibition of Odc by alpha-DL-difluoromethylornithine (DFMO) was assessed in the gills. Furthermore, the transcription and enzymatic activities of Arg, Odc and Pao was assessed in other tissues such as intestine and liver during hypoosmoitc challenges. Adult F. grandis were reared in 5 ppt and acutely transferred to 5, 2, 1, 0.5, and 0.1 ppt water, and gills were sampled at 6 h, 1 d, 3 d, and 7 d post-transfer. Results showed that arg II and odc mRNA levels were highly upregulated in the gills during the first few days post-transfer to hypoosmotic water. Hypoosmotic exposure also produced increases in gill Odc activity and in the concentrations of putrescine, spermidine, and spermine. DFMO application inhibited Odc activity and reduced polyamine levels after hypoosmotic exposure (0.1 ppt). Although gill putrescine levels remained elevated throughout the first 7 d post transfer to 0.1 ppt, the concentrations of spermidine and spermine decreased over time. The ratio of putrescine level over the sum levels of spermidine and spermine increased after 0.1 ppt exposure at 1 d and beyond. Concomitant with freshwater acclimation, an increase in Pao activity suggested that polyamine catabolism was upregulated in the gills. The phenotype of mitochondrion-rich cells (MRCs) in the gill epithelium shifted from a seawater type to a freshwater type following transfer to 0.1 ppt water in correlation with the increase in mRNA levels of arg II and odc in MRCs. In addition, the isolated opercular epithelium pretreated with spermidine had a lower active Cl- secretion rate and membrane conductance following symmetrical hypotonic exposure. Transcription and enzymatic activities of Arg II, Odc, and Pao were upregulated in the intestine and liver during hypoosmotic exposure, suggesting that polyamine levels are regulated in multiple tissues of the killifish. The putative roles of polyamines include inducing cell apoptosis by increasing caspase-3 activity, stimulating cell proliferation by increasing the levels of c-fos and c-myc mRNA levels, and inducing cell swelling via the modulation of Cl- secretion in the gills following hypoosmotic challenges. In summary, fish gill and intestine of killifish transferred to fresh water underwent dramatic physiological and morphological alterations, and had significantly altered polyamine metabolism. Highly cationic polyamines mediate early phase compensatory responses in the euryhaline killifish when faced with osmotic challenges. Although gill spermidine and spermine increased transiently in killifish with freshwater exposure, putrescine remained elevated over time, suggesting a possible role of this polyamine in acclimation of killifish to fresh water. The putative role of polyamines may be involved in gill remodeling by regulating cell proliferation and apoptosis.

Prokaryotic cyanobacteria have been studied as the simplest organisms that exhibit circadian rhythms. The central S.elongatus clock is comprised of at least three components, the proteins KaiA, KaiB and KaiC. The phosphorylation status of KaiC can sustain a ca. 24-hour period in vitro with the presence of the two other Kai proteins and ATP. Synchronization of different oscillators can be achieved by mixing two KaiABC in vitro samples that are in different phosphorylation phases, with prior results indicating that their rhythms continue, yet with a synchronized shared phase. I have focused on investigating the synchronization of oscillators with different periods in vivo and in vitro with the goal of elucidating the underlying mechanisms. Interdisciplinary approaches including molecular biology, biochemistry and computational simulation are combined to demonstrate that co-existence of two oscillators with different periods displays a robustly synchronized rhythm with an intermediate period both in vivo and in vitro. Moreover, the computationally simulated results agree with the in vitro experiment, and imply that monomer exchange may work together with KaiA sequestration to secure the molecular synchronization of KaiC population in the in vitro reaction.

One commonly cited mechanism for the success of invasive species is their superior competitive ability relative to that of native species. Although 88% (22 of 25) of the empirical studies support the prediction that the strength of competition for native species increases with latitude, no studies to date have compared the competitive ability of native and co-occurring invasive species across a broad latitudinal range. In a greenhouse, I investigated whether the competitive ability of North American native and European invasive haplotypes of Phragmites australis vary with latitude. Another widespread, non-indigenous haplotype, the Gulf Coast haplotype, was also included for assessment of competitive ability only. Competitive ability of each haplotype was evaluated against a standardized plant species, Spartina alterniflora, which is a common co-inhabitant of coastal marshes. The competitive ability (measured in terms of the proportional reduction in biomass of plants grown in the presence and absence of a potential competitor) of native haplotypes decreased with increasing latitude, whereas the competitive ability of invasive haplotypes showed no relationship with latitude. This study provided the first evidence that native and invasive species (or haplotypes) exhibit non-parallel gradients in competitive ability. Overall, the invasive haplotype was competitively superior to the native haplotype - biomass production of S. alterniflora was 19% lower when grown with the former than the latter haplotype. Moreover, in the presence of an interspecific competitor, the invasive haplotype produced 45% more aboveground biomass and 50% more belowground biomass than the native haplotype. Results also indicated that the Gulf Coast haplotype was not significantly different from either the native or invasive haplotypes in terms of competitive ability. Because the invasive haplotype appeared to have the greatest competitive advantage over the natives at northern latitudes, it may be more successful in this region of its invaded range. The results from this research indicate that novel and important findings are possible when the mechanisms concerning invasion success, such as biotic resistance and competition, are examined from a biogeographical perspective.

Evidence suggests that 15-lipoxygenase-2 (15-LOX-2) plays an important role in the progression of atherosclerosis. Due to the medical need to discover isoform specific inhibitors of 15-LOX-2, a novel human enzyme, our goal was to determine the 3-dimensional structure. Using bacterial expression, we were able to obtain a usable quantity of enzyme with a sufficient amount of the catalytic iron bound to the enzyme, however it was sensitive to cleavage by proteases. This led to a ratio of roughly 1:1 of cleaved and uncleaved protein after the final step of purification. Solving the crystal structure revealed the reason for the protease sensitivity. In the polycistin-1 lipoxygenase alpha-toxin (PLAT) domain of the protein there is a large loop composed of nonpolar amino acids, which is flanked by calcium ions. The next step was to determine the purpose of calcium binding. Analytical size exclusion chromatography (SEC) experiments revealed that when calcium binding sites were mutated, the enzyme could not bind the synthetic phospholipid bilayer mimic, nanodiscs, which indicated that calcium is necessary for membrane binding. In the crystal structure, the active site of 15-LOX-2 contained a detergent molecule, C8E4, which was necessary for crystallization. This detergent molecule was later identified as a competitive inhibitor and its size and flexibility make it comparable to the enzymes natural substrate arachidonic acid (AA). Comparison of the structure of 15-LOX-2 with the stable 5-LOX structure suggested that the 5-LOX active site is occluded by the broken α2-helix and must open for substrate acquisition. This structure will provide information relevant to the design of isoform specific inhibitors which will aid in determining the function of 15-LOX-2 and could result in the development of therapeutic agents to protect against atherosclerotic plaque development.

No description available.

This study was undertaken to test whether frequent quizzing would have different learning outcomes with the different populations within the classroom. Normalized mean learning gains were compared among classes that were quizzed or not quizzed. Allied Health Honors, Allied Health Academic and Academic classes in a high school biology classroom were given pre- and posttests in three units of study: protein synthesis, genetics, and classification. The same student population was also analyzed based on academic achievement levels: high, medium and low. In each unit, the experimental group was taught with traditional power point based lectures and guided notes, laboratories, activities, frequent questioning, and post-lecture quizzes. The control group was instructed with the same methods but did not complete the post-lecture quizzes but was allowed independent study time in the classroom to account for class time when the experimental group was quizzing. Pretest, posttest, learning gains, and effect size were calculated across each class type and achievement level. All quizzed populations had a higher normalized learning gain than their non-quizzed counterparts in every unit. Even though the differences were not always statistically different, the student populations were positively affected by frequent quizzing. Frequent quizzing is an effective tool to increase learning in student populations in general, despite any differences in motivation or achievement. Further study with more groups of students may lead to a better understanding of how frequent quizzing impacts the learning of different student populations.

The purpose of this study was to determine if students learn more from completing web based homework (WBHW) compared to completing homework by traditional means with pen and paper. Determining the efficacy of WBHW is important because many schools are being pressured to implement technology in the classroom. To determine the effectiveness of WBHW, student normalized learning gains were calculated from pre and post-test scores for 62 students. Learning gains were then compared when students completed WBHW, traditional homework, or did not complete homework at all, over four units in a high school chemistry class. No difference in learning gains were observed between the students completing traditional homework and those completing electronic homework. However, those students that completed either type of homework had significantly higher learning gains than those that did not complete their homework. Students were more likely to complete their homework when assigned on paper (86.7% of students) than on the computer (64.4% of students). Students also self-reported a preference for paper homework to WBHW. For example, 66% of students reported good or great learning gains as a result of traditional homework, compared to only 10% of students reporting the same for WBHW. The results of this study, demonstrate the importance of educators assigning meaningful homework in a method that students are likely to complete. High school chemistry students showed a preference toward traditional methods but, either method of homework completion was related to positive learning gains for students.