The aim of this dissertation is to investigate stabilization of lipid bilayers during water stress by protectants found in embryos of Artemia franciscana. Two LEA proteins were used: AfrLEA2 and AfrLEA3m. AfrLEA3m was experimentally demonstrated to reside in the matrix. Two detergents were used to differentially solubilize the outer and inner membranes of mitochondria isolated from A. franciscana. Release of AfrLEA3m occurred simultaneously with the release of fumarase, a matrix-resident marker. As a second independent method to corroborate the above findings, I demonstrated that recombinant AfrLEA3m can be imported into mitochondria isolated from rat liver. Molecular modeling of AfrLEA2 and AfrLEA3m revealed structural features that are consistent with amphipathic proteins able to interact with and stabilize cell membranes. The abilities of trehalose and LEA proteins to protect liposomes of various compositions from desiccation-induced damage were evaluated by carboxyfluorescein leakage. AfrLEA2 (cytoplasmic) and AfrLEA3m (mitochondrial) were able to offset damage during drying of liposomes that mimicked the lipid compositions of the inner mitochondrial membrane, outer mitochondrial membrane, and the inner leaflet of the plasma membrane. LEA proteins were more effective than trehalose at preventing desiccation-induced damage when these protectants were confined to one side of the lipid bilayer. When LEA proteins were used in conjunction with trehalose, additive protection was measured in some cases. Little to no additional damage occurred to liposomes dried for one week compared to liposomes dried overnight. The capacity of trehalose and LEA proteins to protect liposomes from freeze-thaw damage was also assessed. Damage to liposomes was less severe after freezing than desiccation. Trehalose provided liposomes with greater protection than LEA proteins from freeze-thaw damage. The greatest stabilization during freezing occurred when trehalose was present on both sides of liposome membranes. Only liposomes mimicking the outer mitochondrial membrane were significantly protected from freeze-thaw damage by LEA proteins. Based on bioenergetic properties assessed by respirometry, the outer membrane of isolated mitochondria (rat liver) remained intact after freezing in 300 mM trehalose solution. Respiratory control ratios were depressed by approximately 30% compared to non-frozen mitochondria, which indicated a limited retention of at least some inner-membrane-dependent properties.

The serotonergic raphe nuclei of the midbrain are principal centers from which serotonin neurons project to innervate cortical and sub-cortical structures. The dorsal raphe nuclei receive light input from the circadian visual system and indirect input from the biological clock nuclei. Dysregulation of serotonin neurotransmission is implicated in neurobehavioral and neurodevelopmental disorders, such as depression, anxiety and autism, and alterations in the serotonergic phenotype of raphe neurons has dramatic effects on affective behaviors in rodents. Here, I demonstrate that day length (photoperiod) during development induces enduring changes in mouse dorsal raphe serotonin neurons - programming their spontaneous neural activity, their responsiveness to noradrenergic stimulation, their intrinsic electrical properties, serotonin and norepinephrine content in the midbrain, gene expression of key serotonergic genes, the ratio of TPH2 positive cells to total cells in the dorsal raphe, as well as depression/anxiety related behavior in a melatonin receptor 1 (MT1) dependent manner. Our results establish mechanisms by which seasonal photoperiods may dramatically and persistently alter the function of serotonin neurons.

Nucleolar stress results when ribosome biogenesis is disrupted. An excellent example is the human Treacher Collins syndrome in which the loss of the nucleolar chaperone, Treacle, leads to p53-dependent apoptosis in embryonic neural crest cells, and ultimately to craniofacial birth defects. We show that depletion of the related nucleolar and Cajal body phospho-protein, Nopp140, in Drosophila melanogaster led to nucleolar stress and eventual lethality when multiple tissues were depleted of Nopp140 by RNAi. We used TEM, immuno-blot analysis, and metabolic protein labeling to show the loss of ribosomes. Targeted loss of Nopp140 only in larval wing discs caused apoptosis, which eventually led to defects in the adult wings. These defects were not rescued by a p53 gene deletion, as the craniofacial defects were in the murine model of TCS, thus suggesting that apoptosis caused by nucleolar stress in Drosophila is induced by a p53-independent mechanism. Loss of Nopp140 in larval polyploid midgut cells induced premature autophagy as marked by the accumulation of mCherry-ATG8a into autophagic vesicles. We also found elevated phenoloxidase A3 levels in whole larval lysates and within the hemolymph of Nopp140-depleted larvae, coincident with the appearance of melanotic tumors. The occurrence of apoptosis, autophagy, and phenoloxidase A3 release to the hemolymph upon nucleolar stress correlated well with the activation of JNK in Nopp140-depleted larvae. Nopp140 is considered a ribosome assembly factor, but its precise functions remain unknown. To approach this problem, we deleted the Nopp140 gene in Drosophila using FLP-FRT recombination. Genomic PCR, RT-PCR, and immunofluorescence microscopy confirmed the loss of Nopp140, its mRNA, and protein products. Nopp140-/- larvae arrested in the second instar stage and died within 8 days. While nucleoli appeared intact in Nopp140-/- cells, the C/D snoRNP methyl-transferase, fibrillarin, redistributed to the nucleoplasm in variable amounts; RT-PCRs showed that 2-O-methylation of rRNA in Nopp140-/- cells was reduced. Ultra structural analysis showed that Nopp140-/- cells were deficient in cytoplasmic ribosomes, but instead contained abnormal electron dense cytoplasmic granules. Furthermore, metabolic labeling showed a significant drop in protein translation. We believe the phenotypes described here define novel intracellular ribosomopathies.

In the sixth mass extinction, amphibians are being lost in greater numbers than almost any other taxon. The deadly skin disease, chytridiomycosis, has taken a great toll on amphibian populations around the globe. Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis, appears to have evolved as an obligate pathogen and thus must evade destruction by the amphibian defenses in order to reproduce. Several defenses along the skin mucosa likely inhibit the initial colonization by B. dendrobatidis zoospores, but B. dendrobatidis persists longer in the epithelium where it must survive any onslaught by innate and adaptive immune responses. Batrachochytrium dendrobatidis does this by evading phagocytes inside keratinocytes and impairing lymphocytes that might coordinate a robust immune response. Soluble molecules produced by B. dendrobatidis inhibited B and T cell proliferation and induced lymphocyte apoptosis. These molecules were only produced by cell-walled life-cycle stages that are present in host tissue. Inhibiting cell-wall biosynthesis with a chitin-synthase inhibitor greatly reduced its capacity to inhibit lymphocytes, suggesting that inhibitory factors are located in the cell wall. Treatments of inhibitory factors showed that the soluble molecules are not likely to be proteins or common structural components found in fungal cell walls. Although the B. dendrobatidis cell wall has not been characterized, fungal cell walls are largely composed of carbohydrate suggesting that inhibitory factors may be small molecules and/or carbohydrates. These data suggest that B. dendrobatidis has evolved a survival strategy targeting adaptive immune defenses in the skin of amphibian hosts.

Vesicular transport of proteins is a process essential for cell health and viability. Integral membrane proteins, called phospholipid flippases, play important roles in the formation of transport vesicles at sites of membrane budding. Flippases also establish and maintain membrane asymmetry. This is achieved by using the energy of ATP hydrolysis to unidirectionally translocate specific phospholipid molecules from the lumenal to the cytosolic side of the membrane bilayer. It is hypothesized that the resulting imbalance in phospholipid number between the bilayers causes membrane bending, captured by coat and accessory proteins. This leads to the formation of mature cargo-containing transport vesicles. Drs2p, the founding member of the flippase (P4-ATPase) family, has been implicated in the formation of clathrin-coated vesicles at the trans-Golgi network. The influence of Drs2p flippase activity on membrane curvature is explored in this thesis. When late Golgi membranes were purified, and Drs2p on these membranes were activated by ATP addition, tubulation, elongation, and membrane fusion phenotypes were observed. Furthermore, when purified Drs2p was reconstituted into giant unilamellar vesicles (GUVs), and activated by ATP addition, preliminary data indicates the formation of spindle-like tubes. Due to the presence of purified Drs2p (protein found most abundantly in this system) and dependence of this reaction to ATP addition, it is likely this membrane tubulation is Drs2p-dependent. Thus, these data implicate Drs2p in membrane curvature formation due to the ability to translocate phospholipids across the membrane bilayer. Work is also presented that looks at the role of the C-terminal tail of Drs2p on regulation of flippase activity. The C-terminal tail auto-regulates Drs2p function and loss of auto-inhibition by the tail either by proteolysis or interaction with Drs2p effectors stimulates the flippase activity of Drs2p. Furthermore, phosphatidylinositol 4-phosphate is shown to have a direct influence in binding the C-tail to stimulate Drs2p activity. This is the first evidence, in a P4-ATPase, that the C-tail functions as a domain responsible for regulating flippase activity, and this regulation can be relieved by interaction with a phosphoinositide.

Ku is central to the non-homologous end-joining pathway of DNA double strand break repair, first discovered in eukaryotes and more recently in prokaryotes and archaea. This study concerns the importance of two unique sequence features of Ku protein from Mycobacterium smegmatis that include a lysine-rich extension at the C-terminus and a zinc-binding motif in the DNA-binding bridge-region. The unique C-terminal tail of M. smegmatis Ku contains several lysine-rich low-complexity PAKKA repeats that are absent from homologs encoded by obligate parasitic mycobacteria, but present in other mycobacterial proteins such as histone-like proteins. Removal of the lysine-rich extension from Ku decreased thermal stability and abolished DNA end-joining. The tail contacts the core DNA binding domain of Ku and hinders DNA-protein interaction as evidenced by an increase in DNA binding affinity upon removal of the lysine-rich extension. In contrast to Ku lacking the C-terminus, full-length Ku can directly bind DNA without free ends and form multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer, suggesting that these properties are conferred by its C-terminus. My study suggests that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins and that extensions beyond the shared core domain may have independently evolved to expand Ku function. An in vitro metal binding assay showed zinc binding to a predicted zinc-binding motif in the bridge-region of M. smegmatis Ku, an event that stabilizes the protein and prevents cysteine oxidation, but has little effect on DNA binding. In vivo, zinc induced significant upregulation of the gene encoding Ku as well as a divergently oriented gene encoding a predicted zinc-dependent MarR family transcription factor. In addition, overexpression of Ku conferred zinc tolerance on E. coli. I speculate that zinc binding sites in Ku proteins from M. smegmatis and other mycobacterial species have been evolutionarily retained to provide protection against zinc toxicity. In all, my study identifies novel properties conferred by unique sequences present in M. smegmatis Ku protein, which suggests that the retention and evolution of unique sequences within a protein provides an adaptive advantage to microorganisms against environmental stress.

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.