This study examines immune cell infiltration into the olfactory bulb (OB) of mice following nasal administration of inflammatory agents, focusing on SARS-CoV-2 (CoV2) infection and its potential implications for Alzheimer’s disease (AD). Although the OB is typically protected by the blood-brain barrier, external contaminants from the olfactory epithelium (OE) have been shown to disrupt brain function, as observed in COVID-19 patients with neurological manifestations. Understanding immune cell dynamics in the OB is critical for determining how peripheral inflammation affects the brain, potentially contributing to the progression of neurodegenerative diseases. Immunostaining was performed on OB tissue from mice treated with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly-IC), or phosphate-buffered saline (PBS) as a control. Additionally, RNAscope analysis was performed on OB tissue from both mice genetically predisposed to AD and mice without this predisposition, treated with either CoV2 or PBS. Preliminary results show that LPS, Poly(I:C), SARS-CoV-2 nasal inoculation, and LPS injection induced infiltration of monocytes/macrophages, although these cells were rare. Inflammatory markers identified through RNAscope were also expressed by infiltrating monocytes. These findings suggest that monocyte infiltration occurs upon CoV2 infection in the OE. The OB itself does not exhibit CoV2 infection, but crosstalk between the OE and OB appears to trigger OB inflammation. Whether AD-prone mice exhibit altered levels of infiltration remains unclear and requires further investigation. Future work will further explore the role of CoV2 in triggering immune responses in the brain, aiming to better understand the neurological consequences of the virus in the context of AD.

Enterococcus faecalis is a bacterium that causes many different types of infections in humans, including endocarditis, catheter-associated urinary tract infections, and root canal infections. The extracellular protease GelE is a significant virulence factor for E. faecalis. Its primary function is to degrade extracellular peptides and proteins, such as gelatin and host proteins. While the function of GelE for virulence and biofilm formation has been well studied, there is much to be understood about the genes that contribute to its regulation and activity. Therefore, the primary aim of this project is to determine the genes responsible for gelatinase expression and activity in E. faecalis. By having a full picture of the genes required for GelE activity, we may identify new targets for therapeutics that reduce the virulence of antibiotic-resistant E. faecalis strains. I am screening an E. faecalis transposon mutant library to determine strains that do not degrade gelatin. So far, I have identified 44 mutants that are required for gelatinase activity. The mutated genes encode peptidases, response regulators, and cold shock proteins. Identifying the genetic factors that influence GelE expression and activity will give us a better understanding of E. faecalis virulence and provide potential pathways in treating enterococcal infections more effectively.

Short stature is a broad clinical term describing individuals whose height is significantly below age and sex-matched population norms, often due to underlying skeletal dysplasia. For example, achondroplasia, or short-limbed dwarfism, involves a mutation in FGFR3 that leads to persistent SOX9 expression in hypertrophic chondrocytes, altering endochondral differentiation in the growth plate. Moreover, haploinsufficiency of SOX9 causes the severe skeletal malformation known as Campolemic Dysplasia (CD). Thus, the regulation of SOX9 expression and gene dosage is crucial for normal development and growth of endochondral bone. However, the mechanisms of regulation of SOX9 protein remains mostly unresolved. Our laboratory generated a mouse model with a hypomorphic in-frame microdeletion in the SOX9 transactivation domain (TAM), hereby Referred to as Sox9del. To evaluate the role of the TAM domain in postnatal long bone growth, we used micro-computed tomography (µCT) and histological analyses on femurs and showed that Sox9del mutants have reduced femur length, cortical thickness, and surface area mineralization. Histological assessments revealed reduced growth plate thickness, increased hypertrophy of chondrocytes, and disrupted chondrocyte organization, supporting that disruption of the SOX9 TAM domain impaired endochondral ossification. Future work will explore how the expression of SOX9 and other regulators of chondrocyte differentiation in the growth plate are effected in Sox9del mutant animals. Together, these findings demonstrate that the SOX9 TAM domain is necessary for optimal SOX9 function in the growth plate and suggest that low-effect variants in Sox9 may contribute to milder, short stature phenotypes, including forms of skeletal dysplasia such as achondroplasia.

Cupriavidus necator is a particular strain of bacteria showing promise in industrial applications producing high value compounds or in environmental microbiology and bioremediation due to its metabolic versatility and high tolerance to environmental stressors. Development of an efficient and controllable CRISPR Cas9 toolkit is essential for efficient modification of this strain for a more streamlined work flow. To work towards this goal we will: (1) construct a CRISPR Cas9 system based on the rhamnose induction system and a self-splicing intron-based riboswitch system, (2) optimize the CRISPR Cas9 system through sgRNA design screening, Cas9 expression level optimization, DNA donor template screening, and parameters such as induction growth phase, transformation recovery, and selection stringency, and (3) knock out the endogenous plasmid within Cupriavidus necator based on the CRISPR Cas9 system for improved efficiency in C1 feedstock based bioproduction applications. These efforts aim to provide a foundation for an efficient and modular genome engineering platform for Cupriavidus necator for future synthetic biology applications.