Selective Activation of Src Family Kinases by the HIV-1 Nef Protein

Nef is a critical HIV-1 accessory factor shown to promote viral pathogenesis by altering host cell signaling pathways. Nef has been shown to bind several members of the Src family of protein-tyrosine kinases, and these interactions have been implicated in the pathogenesis of HIV/AIDS. The studies summarized below investigated this key interaction between virus and host cell proteins. We explored the direct effect of Nef interaction on Src family kinases (SFKs) using Saccharomyces cerevisiae, a well-defined system in which c-Src expression arrests yeast cell growth in a kinase-dependent manner. The seven SFKs found in HIV target cells wre expressed in yeast; each was found to be active alone, but repressed by co-expression of the negative regulatory kinase Csk. We then co-expressed each SFK with both Csk and HIV-1 Nef and found that Nef selectively activated Hck, Lyn, and c-Src among SFKs.We then used our yeast-based system to identify small molecule inhibitors of the active Nef:Hck complex using the auto-dowregulated Hck-YEEI molecule. Yeast expressing the Nef:Hck-YEEI complex were used to screen a library of small heterocyclic compounds based on their ability to rescue growth inhibition. Two compounds identified in this screen potently blocked Nef-dependent HIV replication, indicating Nef:SFK complexes as valid targets for anti-HIV drug therapy. Finally, we used the yeast assay to identify novel mechanisms of Nef:SFK interactions. We screened a panel of primary Nef alleles containing the known SH3-binding elements and discovered four alleles whose proteins demonstrated altered activation of SFKs. Sequence examination revealed the existence of amino acid changes in regions not previously suspected to be involved in SH3-mediated interaction. Particularly intriguing are residues in a large unstructured loop that projects from the Nef core. These findings suggest that critical residues outside of the known SH3-binding motifs may affect SFK binding and activation. Together, the results presented here advance the field of HIV research by furthering our understanding of the interaction between the HIV-1 Nef virulence factor and the Src kinase family, as well as validating this virus:host cell interaction as a rational target for anti-HIV drug discovery

CRISPR/CASTE: Functional Genomic Studies of the Major Evolutionary Innovations of Ants

Ants are social organisms that live in groups and depend intimately on their nestmates for growth and survival. Ants have evolved a number of highly sophisticated, social phenotypes that allow them to form coherent colonies. This thesis explores two particularly derived features of ant biology: complex chemical communication and caste plasticity. To study these features, I had a particular focus on generating and characterizing germ-line mutants. I believe that the study of mutants, and applying molecular biology methods more generally, can lead to insights in ant biology that would not be possible with more traditional methods. I first describe my efforts to develop a CRISPR protocol to make the first germ-line mutant ant lines. I conducted this work using a unique, clonal ant species, Ooceraea biroi, that has many properties making it favorable for laboratory genetics studies. Establishing this protocol required a multi-year optimization process to account for many of the particular features of ant biology, such as egg production and incubation, growing and maintaining lines, and optimizing experimental treatments to produce high mutagenesis rates. I next describe the mutants I generated using these methods, null mutants of a highly conserved insect protein called orco. Orco, or olfactory receptor co-receptor, is required for the function of an important class of chemosensory proteins, the odorant receptors, in insects. I created orco mutant ants and found that they have striking deficiencies in their social behavior and fitness, including an inability to nest with other ants or follow chemical pheromone trails and severely reduced life span and fecundity. These results supported the growing consensus that odorant receptors are key chemosensory proteins for pheromone perception in ants, and provided a new window into ant social behavior and collective organization. Unexpectedly, and unlike orco mutants in other types of insects, I also found that orco mutant ants have severe neuro-anatomical deficiencies, including a loss of most antennal lobe glomeruli and sensory neurons in the antenna. This surprising result implies that orco may play an important role in antennal lobe morphology in ants, and could provide insights into the development and evolution of complex olfactory systems. The following chapter is a critical literature review of the development and evolution of morphological castes, such as workers and queens, in ants. I describe a stereotyped and previously overlooked pattern of morphological variation in ants, and illustrate how this pattern may provide some early insights into the molecular mechanisms of caste plasticity. This chapter provides a falsifiable, mechanistic framework for caste development and suggests a route to start looking for the actual molecules that regulate this interesting process. Finally, I start to realize this promise by characterizing a caste mutant in the laboratory. I discovered a spontaneous ‘winged mutant’ that belongs to one of the known clonal lineages of O. biroi and aberrantly expresses queen-like morphology and behavior at worker-like body sizes. These mutants bear a striking resemblance to one class of ant species with derived caste systems, the recurrently evolved workerless social parasites. They could thus provide a window into the mutations that give rise to these unique ants. Overall, this thesis represents the first characterization of mutant lines in ants, and I hope it demonstrates how this approach can be used to generate robust conclusions about ant biology

Emerging adulthood: Defining the life stage and its developmental tasks

Emerging adulthood, as first proposed by Jeffrey Arnett, is the developmental period spanning ages 18-29. Culturally, it is a time of institutionalized role moratorium, especially in post-industrial societies. Emerging adults share the five characteristics of self-focus, instability, identity explorations, feeling in-between, and a sense of possibilities. Emerging adulthood takes place across racial, cultural, and socioeconomic groups, although the experience of emerging adulthood varies among groups. The present paper provides an overview of the theory of emerging adulthood and its expressions in American society. An original program called “The Something Potluck” is outlined in the Appendix. The Something Potluck is designed to facilitate the developmental growth of emerging adult participants by providing psychoeducation and community support

An Examination of NFL’s “A Crucial Catch” Campaign: NFL Athletes as Disease Prevention Advocates

This study investigated the National Football League\u27s (NFL) A Crucial Catch campaign, identification with NFL athletes, exposure, NFL fanship, and intention to schedule a breast cancer screening and to talk about screenings with others. Participants (N = 119) were solicited through various social media outlets, and through an e-mail listserv from the School of Communication at Rochester Institute of Technology. The health belief model (Rosenstock, 1966) is used as the main theoretical framework. Hypotheses and research questions are investigated through an online survey mirroring Brown\u27s (2011) valid survey questions investigating identification with NFL athletes. Statistically significant relationships were found between identification with NFL athletes and exposure, intention to schedule a breast cancer screening, and NFL fanship. NFL fanship was also significantly related to exposure to the campaign

A commercial porcine circovirus(PCV)type 2a-based vaccine reduces PCV2d viremia and shedding and prevents PCV2d transmission to naïve pigs under experimental conditions.

Porcine circovirus type 2 (PCV2) vaccination has been effective in protecting pigs from clinical disease and today is used extensively. Recent studies in vaccinated populations indicate a major PCV2 genotype shift from the predominant PCV2 genotype 2b towards 2d. The aims of this study were to determine the ability of the commercial inactivated PCV2a vaccine Circovac® to protect pigs against experimental challenge with a 2013 PCV2d strain and prevent transmission. Thirty-eight pigs were randomly divided into four groups with 9–10 pigs per group: NEG (sham-vaccinated, sham-challenged), VAC (PCV2a-vaccinated, sham-challenged), VAC + CHAL (PCV2a-vaccinated and PCV2d-challenged), and CHAL (sham-vaccinated, PCV2d-challenged). Vaccination was done at 3 weeks of age using Circovac® according to label instructions. The CHAL and VAC + CHAL groups were challenged with PCV2d at 7 weeks of age and all pigs were necropsied 21 days post-challenge (dpc). The VAC-CHAL pigs seroconverted to PCV2 by 21 days post vaccination (dpv). At PCV2d challenge on 28 dpv, 3/9 VAC and 1/9 VAC + CHAL pigs were seropositive. NEG pigs remained seronegative for the duration of the study. Vaccination significantly reduced PCV2d viremia (VAC + CHAL) at dpc 14 and 21, PCV2d fecal shedding at dpc 14 and 21 and PCV2d nasal shedding at dpc 7, 14 and 21 compared to CHAL pigs. Vaccination significantly reduced mean PCV2 antigen load in lymph nodes in VAC + CHAL pigs compared to CHAL pigs. When pooled serum or feces collected from VAC + CHAL and CHAL pigs at dpc 21 were used to expose single-housed PCV2 naïve pigs, a pooled fecal sample from CHAL pigs contained infectious PCV2 whereas this was not the case for VAC + CHAL pigs suggesting reduction of PCV2d transmission by vaccination. Under the study conditions, the PCV2a-based vaccine was effective in reducing PCV2d viremia, tissue loads, shedding and transmission indicating that PCV2a vaccination should be effective in PCV2d-infected herds

Globally invasive populations of the clonal raider ant are derived from Bangladesh.

Identifying the native range of invasive species is useful to understand their evolution and natural history, as well as to develop new methods to control potentially harmful introduced organisms. The clonal raider ant, Ooceraea biroi, is an introduced species and an increasingly important social insect model organism, but its native range remains unknown. Here, we report a new series of O. biroi collections from Bangladesh, Singapore, Vietnam and China. We use a molecular phylogeny constructed with five gene fragments from 27 samples to determine that invasive lineages of O. biroi originated in Bangladesh. These lineages may have spread from Bangladesh via the historically significant Bay of Bengal shipping ports. Ooceraea biroi shares multiple features of its biology with other introduced ants, including parthenogenesis, retention of heterozygosity and presence of multiple egg-layers in the colony. Using laboratory rearing and microsatellite markers, we show that colonies collected from disturbed habitat in Bangladesh have these traits in common with colonies from the invasive range. Ancestral populations with sexual reproduction in primary habitats either remain to be discovered or have gone extinct. Our findings advance our understanding of the global spread of the clonal raider ant and highlight a suite of general traits that make certain ants prone to becoming invasive

Comparison of host immune responses to homologous and heterologous type II porcine reproductive and respiratory syndrome virus (PRRSV) challenge in vaccinated and unvaccinated pigs

Porcine reproductive and respiratory syndrome (PRRS) is a high-consequence animal disease with current vaccines providing limited protection from infection due to the high degree of genetic variation of field PRRS virus. Therefore, understanding host immune responses elicited by different PRRSV strains will facilitate the development of more effective vaccines. Using IngelVac modified live PRRSV vaccine (MLV), its parental strain VR-2332, and the heterologous KS-06-72109 strain (a Kansas isolate of PRRSV), we compared immune responses induced by vaccination and/or PRRSV infection. Our results showed that MLV can provide complete protection from homologous virus (VR-2332) and partial protection from heterologous (KS-06) challenge. The protection was associated with the levels of PRRSV neutralizing antibodies at the time of challenge, with vaccinated pigs having higher titers to VR-2332 compared to KS-06 strain. Challenge strain did not alter the cytokine expression profiles in the serum of vaccinated pigs or subpopulations of T cells. However, higher frequencies of IFN-γ-secreting PBMCs were generated from pigs challenged with heterologous PRRSV in a recall response when PBMCs were re-stimulated with PRRSV. Thus, this study indicates that serum neutralizing antibody titers are associated with PRRSV vaccination-induced protection against homologous and heterologous challenge