<p> In the practice of resource management conservation, it is common to introduce new members into small inbred populations in order to increase genetic diversity and reduce the negative effects of inbreeding. Although fitness often increases in the F1 generation immediately following intrapopulation hybridization, few studies have focused on the long-term fitness of such populations. The model organisms <i>Drosophila melanogaster</i> and <i> D. simulans</i> were used to investigate if heterosis, an increase in the fitness in the hybrid population, or outbreeding depression, a decline in the fitness in the hybrid population, occurs over an eight generation period. At each generation, a series of fitness related assays were performed to assess relative fitness. Results of this study were mixed. At best, only some hybridized populations showed slightly higher, but often nonsignificant increases in fitness, never in more than one assay. The lack of consistent strong persistent heterosis suggests that conservation efforts such as habitat preservation maybe be a better use of effort and money for assisting endangered species. </p>

<p> Impairments in various aspects of language, including the manipulation and comprehension of verbal and written language, are common in pediatric populations. Some disorders of language are secondary to other clinical presentations, while others, such as dyslexia (or reading disability [RD]), language impairment (LI), speech sound disorder (SSD), and autism spectrum disorders (ASD), have primary deficits in language skills. Each of these is a distinct disorder with unique clinical presentations and deficits. For instance, children with RD have deficits in reading and the use of written language, while those with LI have deficits in the manipulation and comprehension of verbal language. Additionally, children with SSD have difficulties in the production of speech sounds, while children with ASD may have delays or regressions in language and an inability to use complex, proper syntax and pragmatics. However, there is substantial comorbidity of these disorders, as children affected with one of these disorders are more likely to have or develop another disorder than their typically developing peers. These 'disorders&mdash;RD, LI, SSD, and ASD&mdash;are complex traits, with significant environmental and genetic components contributing to each. Similar to their phenotypic relationships, there is limited evidence that these disorders may share genetic contributors. In fact, these shared genetic components may explain the common phenotypic comorbidities of these disorders. Therefore, the overall goal of this project is to determine whether and to what extent RD, LI, SSD, and ASD share genetic associations with the hypothesis that these disorders have common genetic contributors. To accomplish this goal, I assess whether genetic associations were shared among these disorders or specific to individual disorders. First, I expand the association of the RD environmental risk factor, prenatal exposure to nicotine, to include LI and show the association of dopamine-related genes <i> ANKK1</i> and <i>DRD2</i> to LI. Second, two RD risk genes, <i> DCDC2</i> and <i>KIAA0319</i>, located within the DYX2 locus on chromosome 6p22, show associations with both LI and SSD. Third, I identify <i> ZNF385D</i> as a novel risk gene for subjects affected with comorbid RD and LI. I also assess the neuroimaging implications of DYX2 genes and <i> ZNF385D</i>, specifically in regards to cortical thickness, fiber tract volume, and fractional anisotropy. Finally, two LI risk genes, <i>ATP2C2 </i> and <i>CMIP</i> located within the SLI1 locus on chromosome 16, are associated with language skills of subjects with ASD. Taken together, these results characterize the relationship of previously identified risk genes to other related language disorders and identify novel risk genes that specifically contribute to language comorbidity. Shared genetic associations among these language disorders appear to be commonplace as opposed to the exception. However, the question remains of how these genetic variants interact with each other and other genes/exposures to ultimately lead to one or more of these language deficits seen clinically.</p>

<p> Evolutionary forces such as recombination, demography, and selection can shape patterns of genetic diversity within populations and contribute to phenotypic variation. While theoretical models exist for each of these forces independently, mathematically modeling their joint impact on patterns of genetic diversity remains very challenging. Fortunately, it is possible to perform forward-in-time computer simulations of DNA sequences that incorporate all of these forces simultaneously. Here, I show that there are trade-offs between computational efficiency and accuracy for simulations of a widely investigated model of recurrent positive selection. I develop a theoretical model to explain this trade-off, and a simple algorithm that obtains the best possible computational performance for a given error tolerance. I then pivot to develop a framework for simulations of human DNA sequences and genetically complex phenotypes, incorporating recently inferred demographic models of human continental groups and selection on genes and non-coding elements. I use these simulations to investigate the power of rare variant association tests in the context of rampant selection and non-equilibrium demography. I show that the power of rare variant association tests is in some cases quite sensitive to underlying assumptions about the relationship between selection and effect sizes. This work highlights both the challenge and the promise of applying forward simulations in genetic studies that seek to infer the parameters of evolutionary models and detect statistical associations.</p>

Mutations in the PHEX gene are responsible for X-linked hypophosphatemia, the most prevalent form of inherited rickets in humans. PHEX is an endopeptidase that is expressed in bone and parathyroid gland and shown to cleave parathyroid hormone [PTH]. To determine whether PHEX is regulated by PTH we used two models: (1) 5/6 nephrectomized [5/6 Nx] rats which exhibit significant hyperparathyroidism and (2) 1,25-dihydroxyvitamin D3 [1,25(OH)2D]-treated rats, which are characterized by marked hypoparathyroidism. We demonstrate that 5/6 Nx elicits an increase in PHEX mRNA and protein abundance in rat tibia whereas 1,25(OH)2D causes a decrease in both tibial parameters. In addition, administration of 1,25(OH)2D to 5/6 Nx rats blunts the increase in PHEX gene expression in bone. We also observe a significant correlation between serum PTH concentration and tibial PHEX expression. Our data thus provides evidence for a role of PTH in the regulation of PHEX expression.

Nascent polypeptide-associated complex And Coactivator alpha (alphaNAC) is a coactivator that potentiates AP-1-dependent transcription. alphaNAC has been shown to interact with the homodimeric transcription factor c-Jun, and more specifically in the N-terminal activation domain of the protein. This interaction leads to a greater stabilization of c-Jun on the AP-1 consensus site and consequently, an increased rate of transcription. alphaNAC is a substrate of the Integrin-Linked Kinase (ILK), which is activated through integrin-extracellular matrix interactions and stimulates c-Jun-mediated gene transcription. / By means of protein pulldown assays, we have demonstrated that phosphorylation of alphaNAC by ILK results in an enhanced interaction of phospho-alphaNAC with the phosphorylated form of c-Jun. Additionally, the c-Jun interaction domain on alphaNAC was identified. Affinity chromatography involving alphaNAC deletion mutants revealed that the c-Jun-binding site is located in the middle part of the protein, between amino acids 89 and 129. These results are consistent with earlier data from our laboratory demonstrating an interaction between alphaNAC and c-Jun as well as increased c-Jun-mediated transcription by the coactivating function of alphaNAC.

Human cytomegalovirus (HCMV) infection can cause life-threatening disease in immunodeficient hosts. Experimental infection in mice has revealed that natural resistance to murine cytomegalovirus (MCMV) is genetically determined and mediated by binding of the natural killer (NK) cell activating receptor Ly49H to the pathogen-encoded glycoprotein m157. The study of genetically diverse wild-derived strains revealed natural resistance in the PWK/Pas mouse. However, intracellular staining of NK cells indicated that PWK/Pas mice do not express Ly49H. Moreover, PWK/Pas were also found to be resistant to mutant virus lacking m157. Analysis of backcrosses to a susceptible strain demonstrated that MCMV-resistance is controlled by a major dominant gene effect linked to the Ly49 gene cluster and a modest influence of the major histocompatibility complex (H2). Ly49 cDNAs encoding four novel activating receptors were identified as candidate genes for the resistance phenotype. Our results provide genetic evidence for the existence of alternative NK cell receptors that mediate resistance to viral infection in mice by mechanisms other than recognition of m157. Functionally homologous innate resistance genes may be found in human populations.

Growing evidence suggests a partial genetic determination of suicide predisposition, with most studies focusing on serotonergic genes. Inconsistent results have characterized the serotonin transporter (5-HTT) gene, whereas few studies have investigated the recently discovered brain specific tryptophan hydroxylase 2 (TPH2) gene. At the same time, most of these studies have not controlled for associated psychopathology, an important confounder. Therefore, association studies were conducted to investigate the role of the 5-HTT and TPH2 genes in suicide susceptibility in the context of major depression and their effect on impulsive-aggressive behaviors (IABs), a possible intermediate phenotype. Finally, suicide predictors were investigated while controlling for genetic, clinical and behavioral risk factors. Certain 5-HTT and TPH2 genetic variants were significantly implicated in the vulnerability to suicide, however, no effect on levels of IABs was observed. Genetic and clinical suicide predictors were identified while controlling for other risk factors. Further studies in larger samples are necessary to replicate these findings and detect possible small genetic effects.

The beta-amyloid precursor protein (APP) is an extensively processed membrane protein with a single membrane spanning domain. One of the natural proteolytic products of APP is a peptide of 39 to 43 amino acids in length, beta-amyloid, that has been identified as a major constituent of senile plaques, a neuropathological hallmark of Alzheimer disease (AD). In Down syndrome (DS) (trisomy 21), overexpression of the APP gene, located on chromosome 21, is thought to be responsible for the early formation of senile plaques in the brain of DS patients. We investigated the transcriptional regulation of the APP gene in order to identify the cis-elements located in its promoter and the trans-acting factors recognizing these elements. Identification of the factors regulating APP gene expression, could help define conditions leading to the development of AD. The APP promoter possesses multiple transcriptional start sites, has a high percentage of GC and lacks a consensus TATA box. We have characterized, using electrophoretic mobility shift analysis (EMSA), DNase I footprinting, site-directed mutagenesis and transient transfection studies, three elements located in close proximity of the major transcriptional start sites of APP. We present evidence that two of the elements are recognized by the ubiquitous transcription factors Sp1 and USF respectively. Both the Sp1 and USF elements are essential for full activity of the APP promoter, regulate the APP promoter in an additive manner and synergize with the factor(s) recognizing a GC-rich element located upstream in transient transfection assays in NG108-15 and HepG2 cell lines. We also show that treatment by cyclic AMP of NG108-15 cells which had been transiently transfected with reporter constructs driven by different portions of the APP promoter caused an increase in reporter gene activity mediated by sequences located between --2991 to --488 and --303 to --204, even though the upstream sequences of APP do not contain a canoni

Hox genes encode homeodomain-containing transcription factors involved in the specification of structures along the anterior-posterior and secondary axes of the vertebrate embryo. Many pieces of evidence suggest that retinoic acid (RA) regulates Hox gene function. The present work involved the characterization of the transcription unit of a murine Hox gene, Hoxd4, its response to RA and its genetic interactions with Retinoic Acid Receptors (RARs) beta and gamma. / We have identified two Hoxd4 transcription start sites, P1 and P2. Multiple transcripts were identified by Northern blot, originating from both promoters and multiple polyadenylations signals. Expression of P1 transcripts in the neural tube showed an anterior border at the rhombomere 6/7 boundary, while P2 transcripts are detected more posteriorly. Somitic expression was strong up to somite 6 and weak in somite 5. After RA exposure in utero, only P1 showed a direct response to exogenous RA in the hindbrain, 4 or 24 hours after RA treatment on day 8.5 of gestation. In embryos collected 24 hours after RA treatment, wild-type mice showed a full rhombomere anteziorization of Hoxd4 expression. In contrast, Rarb null mice displayed only a partial anteriorization under the same conditions. The same holds true for the Hoxb4 gene. These results indicate that RARbeta is one of the mediators of the Hoxd4 and Hoxb4 RA-response. / Rarg null mutant mice display homeotic transformations of the cervical vertebrae that are similar to a Hoxd4 null mutant phenotype. In order to examine whether Hoxd4 and Rarg he in a common genetic pathway for patterning of the cervical region, we generated mice doubly mutant for these genes and analyzed their skeletons. For two malformations present in single mutants, namely an ectopic anterior arch in cervical vertebra 2 (C2) and the fusion of the basioccipital bone to C1, the penetrance and expressivity in double null mutants was significantly increased. Thus, we conclude that Hoxd4 and Rarg interact synergistically in the specification of these cervical vertebrae. In contrast to Rarg mutants, retinoic acid treatment of Hoxd4; Rarg double mutants on day 10.5 does not rescue normal development of C2. This result suggests that Hoxd4 mediates the retinoic acid-induced correction of this phenotype in Rarg mutants.

Double-strand breaks (DSBs) are one of the most lethal threats to genomic integrity. In the yeast Saccharomyces cerevisiae, double-strand breaks are primarily repaired via the homologous recombination repair pathway. While the mechanisms underlying this pathway have been generally characterized, the early steps leading to commitment of DSBs to homologous recombination repair are as yet unclear. In particular, it has been shown that the exonuclease processing of DSBs is a critical step prior to strand invasion of a homologous template. / Endo-exonucleases represent a class of nucleases that have been linked to recombinational repair processes in various organisms. Early on, a degradative role was ascribed to the bacterial recBCD endo-exonuclease in processing DSBs in a 5' to 3' directionality prior to recA-mediated homologous recombination in E. coli. However, the nuclease responsible for this nucleolytic processing has yet to be identified in yeast. In this dissertation, I demonstrate that RNC1 is a candidate gene that encodes a nuclease with characteristics similar to previously identified endo-exonucleases, and that RNC1 is implicated in homologous recombination repair of DSBs. / While the rnc1 null mutant is 10-fold more sensitive to persistent DSB induction than wild-type, a highly lethal rad52 null mutation is suppressed in an rnc1 knockout, indicating that RNC1 acts at an earlier step in the homologous recombination repair pathway than RAD52. Furthermore, in the absence of RNC1, the non-homologous end-joining pathway, mediated by the KU80 gene product, appears to play a more dominant role in repairing DSBs. Moreover, a ku80 rnc1 double mutant is hypersensitized to DSB induction. / Biochemical analysis of the purified RNC1 protein revealed that it has endonuclease activity on single-strand DNA as well as processive exonuclease activity on double-strand DNA with a 5' to 3' directionality. In vivo, this exonuclease activity was implicated in the processing of DSBs prior to repair. Our findings show that RNC1 possesses nucleolytic activities characteristic of previously identified endo-exonucleases, and confirm its role in the homologous recombination repair pathway. Intriguingly, RNC1 was also shown to exhibit methyltransferase activity which directs the modification of a specific base, m5U 54, in elongator tRNA. While the biological significance of this activity is as yet unclear, it is attributable to a domain in the RNC1 protein separate from that engaging in DNA repair processes.