The retina is a unique neuronal structure in the eye that facilitates vision. Many diseases cause the death of retinal cells and this can lead to blindness. Frogs and fish have retinal stem cells that can repair the retina after retinal cell death; these stem cells are concentrated in a region called the ciliary marginal zone (CMZ). It was thought that humans lack these cells; however, we have discovered a region of the retina that has some features of the CMZ. We call this the Late Proliferative Zone (LPZ). One of my research goals was to determine whether the LPZ in humans also contains retinal stem cells that could be harnessed to repair the injured retinae. To start, I measured the area of small cuttings of fetal retinal tissue grown in culture, called retinospheres (RSs), over time and identified a window from 250-325 days gestation in which the LPZ of the human retina continues to grow after the rest of the retina is quiescent. This result shows that the cells of the LPZ can make new retinal cells much later than we thought, supporting the idea that these are retinal stem cells. My second goal was to find factors that can stimulate the growth of these cells. I tested several factors known to be important for the stem cells in frogs and fish. I found the effects of these factors on the types of neurons made by the LPZ. In sum, investigating different ways to manipulate the LPZ provides the field with insight into what is needed to regenerate cell types lost in blinding diseases.

Coccidioidomycosis, also known as Valley Fever (VF) is caused by the fungus Coccidioides. Pigtail macaques (PTMs) bred at the Washington National Primate Research Center (WaNPRC) in Mesa, AZ are naturally infected with Coccidioides and are similar to humans in their physiology, symptoms, and immune responses. Populations with a weakened immune system, notably older individuals, are at risk for severe complications from infection. Additionally, there is evidence that males have a higher incidence of VF than females in endemic areas. I characterized the immune responses in a PTM model across age and sex to better understand how VF affects the immune response of these populations. Forty-two PTMs (2.25-19.24 years, 3.66-18.29 kg, 37 female, 5 male) at the WaNPRC were sampled for blood. The frequencies of immune cell subsets in whole blood were characterized by flow cytometry and compared for significant differences based on age and sex. I analyzed sex-based differences with Brown-Forsythe and Welch ANOVA t-tests and found no statistically significant differences. For age-based differences, we used a simple linear regression to analyze differences by age in immune cell subsets. We found that old PTMs (10.07-19.24 years) have higher activation of CD8+ T cells, myeloid dendritic cells, intermediate monocytes, and higher frequency of γΔ T cells and CD4+ γΔ T cells than young PTMs (2.25-9.69 years). Young PTMs have a higher frequency of CD45+ granulocytes, PD-1 High CD8+ T cells, plasmacytoid dendritic cells, and NK cells. By correlating older PTMs with higher immune cell activation, and younger PTMs with higher immune cell frequency, we have a better understanding of how a vaccine or treatment could be developed to support older individuals, who are at greater risk of severe infection.

People living with untreated HIV have a compromised immune system, which increases the risk for enhanced inflammation and disease severity in those co-infected with SARS-CoV-2. This emphasizes the importance of understanding the underlying mechanisms during immunosuppression that impact SARS-CoV-2 pathogenesis and disease outcomes. Since the microbiome plays an important role in immunity, microbial dysbiosis during HIV infection could contribute to prolonged SARS-CoV-2 pathogenesis. Microbial dysbiosis can be determined through the loss of diversity and changes to the composition of the microbiome. There is an established link between the increase in HIV disease progression and gastrointestinal microbial dysbiosis, however, the understanding of HIV-induced microbial dysbiosis during COVID-19 progression is unknown. In this project, we will investigate the gastrointestinal microbiome diversity and composition during SIV infection, to serve as a basis for understanding this undefined association. Utilizing the SIV macaque model for AIDS, we will test the hypothesis that the microbiome diversity and composition during SIV infection will be dissimilar between different gastrointestinal areas (stool, rectal swabs). Seven female rhesus macaques were intravaginally infected with SIVmac251. Rectal swabs and stool samples from the macaques were collected at baseline: 17~34 weeks post-SIV infection, and 7 days pre-SARS-CoV-2 infection. We extracted genomic DNA using a QIAgen PowerFecal Pro DNA kit and sequenced the ribosomal RNA after 16s amplification. We use the bioinformatics platform, QIIME2, to analyze the sequencing data. I am probing the data for relative microbial abundance, and diversity of microbial communities through metrics of richness, evenness, and specific indexes. Preliminary findings report that during SIV infection, the overall diversity of the gut microbiome is similar in stool and rectal swabs, and the microbiota composition is different between them. The results from these studies will then be used to understand the role of SIV-induced microbial dysbiosis on SARS-CoV-2 virological and disease outcomes.
 

Damage to the spinal cord causes one of the most debilitating injuries to the human body. The challenge of promoting the regeneration of this dense network of neurons and glia after spinal cord injury has been seen as insurmountable. However, new techniques emerging from the field of regenerative medicine have illustrated the possibility of encouraging the body to repair these injuries on its own. In the Wills Lab, we study the model organism Xenopus tropicalis, or the Western clawed frog, which has the ability to regenerate its spinal cord and associated tissue following amputation. My project focuses on how X. tropicalis uses the developmental morphogen Sonic Hedgehog (Shh) to re-establish the dorsal-ventral (DV) patterning of the spinal cord during regeneration. I have used cyclopamine, a Shh inhibitor, and SAG, an agonist, in order to perturb Shh signaling during regeneration. I then monitored the effect on DV patterning via immunohistochemical labeling of dorsal and ventral markers. Work so far has shown that Shh signaling is in fact necessary to the establishment of proper DV domains in the regenerate spinal cord. However, my research has also hinted that this specification is complex. Shh appears to have a more proliferative role early on, with patterning effects coming later. In addition, there appears to be an interaction between Shh and other signals that specify anterior-posterior polarity. Overall, my research so far has generated new evidence for how developmental signals are repurposed in the context of regeneration. 

Biofilm is a community of bacteria enclosed in an extracellular polymeric substance (EPS) attached to a surface. Inside the biofilm, bacteria can collaborate to increase their survival. The EPS also protects bacteria from drug penetration, leading to increased antibiotic resistance. Therefore, biofilm formation is often linked with chronic bacterial infections. Pseudomonas aeruginosa is an opportunistic pathogen that often causes chronic lung infections in cystic fibrosis patients. It is also a common model for studying biofilm formation. The initial step for biofilm formation is bacteria attaching to and sensing a surface. Upon surface contact, P. aeruginosa may produce cyclic adenosine monophosphate (cAMP), which is a universal second messenger that regulates cellular functions in both eukaryotes and prokaryotes. In P. aeruginosa, cAMP is synthesized by two adenylate cyclases, CyaA & CyaB, and degraded by a cAMP phosphodiesterase, CpdA. cAMP is a key regulator for P. aeruginosa virulence by upregulating the production of the type III secretion system, the type II secretion system, and the type IV pili. However, recent observations in our lab suggest that cAMP may also contribute to the homeostasis of the cell envelope. To investigate this phenomenon, I used microscopy to characterize the cell morphology of strains with different cAMP levels and found that increased cAMP levels lead to longer cells. I also found that high cAMP strains are more sensitive to ꞵ-lactam antibiotics specifically, while low cAMP strains become more resistant. Ongoing work includes characterizing the genetic factors that connect cAMP and ꞵ-lactam sensitivity, as well as using microscopy to determine changes in cell envelop induced by cAMP. Overall, this work reveals an important role of cAMP in bacterial physiology and provides insight into the complex relationship between virulence and antimicrobial resistance.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen of wounds, cornea, and the Cystic Fibrosis lung. P. aeruginosa is also a model organism for the study of bacterial biofilm formation. Biofilms are multicellular communities that form from bacterial growth concomitant with the production of extracellular polymeric substances (EPS). EPS includes polymers such as polysaccharides, DNA, and proteins; these polymers provide structure and protection to the biofilm cells. Proteomics experiments by the Parsek Lab and others have demonstrated that a notable component of the biofilm matrix are the secreted proteases. Secreted proteases have defined roles in virulence and nutrient acquisition, but their role in the biofilm matrix of P. aeruginosa has not been explored. I hypothesize that these secreted proteases recycle nutrients, remove cell waste, and protect cells from host immunity. To test my hypothesis, I generated a mutant strain of P. aeruginosa that lacks the six major secreted proteases. While we see that loss of the proteases does not impact planktonic growth, preliminary data suggests that loss of proteolytic activity results in moderately increased biofilm formation. Using a general proteolysis assay relying on casein hydrolysis, I have determined the relative contribution of each of the six proteases to the total proteolytic capacity of P. aeruginosa in planktonic growth. I will further test the impact of the proteases on biofilm growth in different growth environments, including under flow conditions and in artificial sputum medium. I will also assess which proteases contribute the most to proteolysis during biofilm growth. My work fits into a growing body of literature that suggests that the biofilm matrix is not an inert scaffold, but is instead a dynamic and active network.