The lateral hypothalamus (LHA) is an important brain region for motivated behaviors including feeding. The LHA contains GABAergic (inhibitory), Glutamatergic (excitatory), and other neuropeptide neuron populations. Previous research demonstrated that optogenetic stimulation of LHA GABA neurons increases food consumption while stimulation of glutamate neurons decreases food consumption, but both populations increase in activity during consumption. The caveats to these previous research experiments are that they do not isolate consummatory behaviors from appetitive behaviors, and they only focus on the role of neuronal stimulation on caloric consumption, not on non-caloric rewards or aversive tastants. In our experiments, we use a multi-spout head-fixed mouse behavioral system to isolate consumption from other behavioral variables, and measure consumption over a range of different concentrations of either rewarding or aversive taste solutions. Using fiber photometry, I recorded calcium dynamics from both neuronal cell types of interest simultaneously, and we found that GABA neuron activity scales with increased lick rate regardless of the solution, while glutamate neuron activity scales with aversive but not rewarding solutions. When I stimulate LHA GABA neurons during consumption using the red-shifted excitatory opsin, Chrimson, we see an increase in licking regardless of solution. Stimulation of LHA vglut2 neurons reduced licking regardless of solution. We also ran inhibition experiments using the red-shifted inhibitory opsin, JAWS, of the two populations and saw that GABA inhibition reduces consumption, while glutamate inhibition increases consumption. Our research has shown how both populations work to drive consummatory behaviors and how their activity level influences consumption. This research is important because it uncovered the function of LHA GABA and Glut neurons in bidirectionally mediating consummatory behaviors for both rewarding and aversive solutions and will contribute to understanding of health issues related to consumption such as obesity.

To test questions on the evolutionary history of a species, it is important to consider the drivers of genetic diversification that lead to speciation. Species diversification is often driven by the formation of geographical boundaries, ecological diversity, sexual preference, or a combination of these factors. However, for the Western Banded Gecko (Coleonyx variegatus), a species native to the southwestern region of the United States, few of these factors exist. Despite the lack of clear barriers to gene flow, prior research identified several distinct populations of C. variegatus in this region, though there is some uncertainty with these distinctions as they relied solely on the signal from one mitochondrial DNA (mtDNA) locus. The aim of this project is to instead use genomic data to assess the validity of the C. variegatus populations defined by mtDNA, and to investigate how these populations are distributed across the geographic region they inhabit. Using genomic data allows us to more confidently define population boundaries and assess how they have evolved through time. To explore the aim of this project, we sequenced reduced representation genomic data for 224 individuals across the range of the species to determine how populations of C. variegatus are structured. We then built species trees to assess the relatedness of these populations with respect to each other and the overall evolutionary history of the species. Our findings show that there are consistencies between both the genomic and mitochondrial data population definitions, but distinct differences are also present, with mtDNA overestimating the number of populations. Thus, relying on mtDNA data alone may be insufficient for confidently ascribing population boundaries for C. variegatus. Accurately defining populations can have great implications for the conservation of native biodiversity, but as shown by our study, relying on a single mtDNA locus may mislead this crucial process.

Traditional vaccines use inactivated or live attenuated pathogens to elicit an effective adaptive immune response, but these vaccines can lack safety for immunocompromised individuals. Subunit vaccines–which display characteristic components of pathogens–are safe, stable, and readily engineered, but struggle to elicit a strong immune response. These next-generation vaccines require adjuvants (substances that stimulate the immune system) to increase efficacy. However, many currently used adjuvants lack well-understood mechanisms or wide applicability across vaccines. There is a need for new adjuvant platforms, and protein-based adjuvants are appealing because they are stable, readily engineered, and can be co-delivered with antigens on subunit vaccines. Toll-like Receptor (TLR) proteins are promising adjuvant targets that bind pathogen-associated molecules to activate the innate immune system. Of this family, TLR3 binds viral double-stranded RNA (dsRNA) and TLR5 binds the bacterial protein flagellin. Neither native agonist is a suitable adjuvant candidate; dsRNA is unstable and nonspecific and flagellin is degradation and aggregation-prone. Therefore, this project aims to design, test, and characterize novel protein-based adjuvants that can bind TLRs 3 and 5 and activate the immune system. Here, I test and characterize de novo mini-proteins that I have computationally designed to bind mouse TLR3 (mTLR3) and mouse TLR5 (mTLR5). I use yeast surface display, biolayer interferometry, and cell-surface binding assays to identify and characterize successful binders. Preliminary results show de novo mini-proteins specifically bind mTLR3 and mTLR5. Ultimately, this work hopes to provide a mouse model for these novel protein-based vaccine adjuvants with clinical aims. This project has wide-reaching public health implications, as vaccines offer the potential to improve the health and lives of countless individuals.

Group B Streptococci (GBS) are gram-positive bacteria that asymptomatically colonizes the vaginal tract of approximately 18% of women worldwide. However, during pregnancy GBS in the lower genital tract can ascend into the uterus and infect the placenta and baby resulting in preterm birth, stillbirth, and neonatal infection. We have used a nonhuman primate (NHP; pigtail macaque, Macaca nemestrina) model to determine differences between GBS strains that confer different levels of invasiveness. The objective of the study was to determine if there were differences in gene expression among animals infected with a “progressive” versus a “localized/resolved” infection. We hypothesized that a greater inflammatory response would be associated with a “progressive” GBS infection compared to the “localized/resolved”. Twenty one NHP received either a choriodecidual inoculation of: 1) 1-3 X 10^8 colony forming units (CFU) of hypervirulent GBSΔcovR (n=15) or, 2) saline (n=6). Cesarean section was performed at preterm labor or 1-3 days after GBS infection or 7 days after saline inoculation. Placental chorioamniotic membranes were sampled near the inoculation site. GBS infections were categorized as “progressive infections”, “localized/resolved infections”, or “resolved” infections at the time of preterm labor or 3 days after GBS inoculation. Next, we prepared mRNA libraries from placental chorioamniotic membranes near the GBS inoculation site, which were sequenced using the NextSeq 550 platform. Data were normalized and then analyzed by Single Gene Analysis, Gene Set Analysis, and Ingenuity Pathway Analysis. The analysis is currently ongoing and will be ready to summarize during the “Revision Window”. Prevention of GBS infection in pregnancy is complex and is likely influenced by multiple factors, including pathogenicity, host factors, and the vaginal microbiome. Understanding mechanisms influencing the invasiveness of GBS infections during pregnancy will facilitate the development of novel therapeutics and vaccines.