The field of proteomics focuses on identifying proteins from a given sample. It is useful in clinical applications such as disease diagnosis. Single cell proteomics is a relatively new and rapidly advancing field. Its main goal is to create protein samples using a single cell. Preserving spatial concepts of the cell as well as cell interactions are other focuses. This is done using methods of sample purification, peptide separation, and bioinformatic analysis. Several advancements have also been made in this subfield, such as the concept of deep visual proteomics. These techniques include liquid chromatography and mass spectrometry. Obtaining enough protein sample from a single cell as well as identifying proteins from it poses several challenges. These challenges include the suppression of less abundant proteins in samples. The research question proposed involved increasing the amount of protein samples and reducing the suppression of less abundant proteins. This was done using samples gained from cells developed in seed plates. The cells were prepped into protein samples using methods of cell lysis and enzyme cleavage. Protein samples were then inserted into a mass spectrometer for an extended period of time. This was done for identification of proteins in each sample. Spectra were recorded of the sample from the scans of the mass spectrometer and was interpreted using several data programs. These trials were repeated, using specific counts of cells in each sample. These counts decreased in amount, with the goal being to create and analyze a sample from a single cell.

Podocytes are specialized cells vital to maintaining overall renal health. Podocytes form a crucial component of the filtration apparatus in the nephron working units of the kidney, which function to clean the blood. The Hippo pathway, a conserved signaling pathway within vertebrates involved in organ size control, tissue homeostasis, and regeneration, has emerged as a key regulator of podocyte function and integrity. Yes-associated protein (YAP) and the transcriptional coactivator with PDZ-binding motif (TAZ) are transcriptional coactivators within the Hippo pathway, both are significantly expressed in the podocyte nuclei. Previous studies demonstrated that silencing YAP leaves podocytes significantly more vulnerable to apoptosis, whilst TAZ deletion has been shown to affect the morphology of the podocytes and impede their function. Though research has demonstrated the importance of Hippo pathway components in maintaining podocyte function, the mechanisms of how this occurs are not yet fully understood. In this study, we investigated the roles of yap1 and taz(wwtr1) in zebrafish and podocyte development through their effects on the expression of podocyte markers, nephrin, wt1a, and wt1b. Interestingly, our data indicate a role for yap1 in early podocyte formation and warrant future studies to explore the emergence of podocyte progenitors. By expanding on the existing knowledge in the field we may better understand the potential of the Hippo Pathway as a therapeutic target for kidney diseases.

Human Papillomavirus (HPV) is responsible for cancer in different areas of the human body such as cervical, oropharyngeal, and anogenital cancers. HPV infects a host cell and sets on a mission to inhibit and evade many different genes responsible for cell death and triggering an immune response. Many different studies have implicated host-virus interactions important for inhibiting and avoiding the immune system. For example, cGAS and STING are host proteins that play a crucial role in controlling viral infections and triggering an immune response. However, HPV has found ways to counteract the functions of these antiviral proteins. Our lab uses in vitro methods to study the arms race between HPV and the host. I have been focused on developing new methods for the lab. Methods such as CRISPR-Cas-9 based fluorescent tagging of the endogenous genes will allow us to track cGAS and STING proteins in HPV infected cells and directly observe their movement and how HPV alters their role. Another method being recently used in many studies is the sequencing of viral mRNA using MinION technology. I am optimizing approaches to directly sequence HPV mRNA from diverse tissues and samples. These efforts will provide a better understanding of how HPV genomes and the host interact at different stages of the viral lifecycle. Through my results we will gain a better understanding of how this virus alters our own genes for survival.

The noncancerous enlargement of the prostate is known as Benign Prostatic Hyperplasia (BPH). This is a chronic disease characterized as the enlargement of the prostate and manifested as Lower Urinary Tract Symptoms (LUTS). LUTS include frequent urination, painful urination, nocturia and more. While these symptoms are not all attributable to obstruction or enlargement, they are both large factors. BPH affects 50% of men by age 50 and 90% by age 80. Therefore, BPH is widely researched and studied using many models in animal subjects. While there are numerous models that are extremely useful in studying BPH, an additional model – Toxoplasma gondii – is capable of inducing most symptoms and pathological features of the condition, and it is associated with BPH in humans. A notable pathological feature is the formation of prostatic nodules, which had only previously been successful in the hormonal model due to its hormonal manipulation and considerable increase of estrogens. Although estrogen receptors (ER) play a key role in BPH, it is not known how ER alpha and beta react to Toxoplasma gondii infection, how they interact with each other in the prostate, or once infected with Toxoplasma gondii. Using a co-culture of human prostatic epithelial and stromal cells to recreate a prostatic environment, my study will allow for meaningful addition of scientific knowledge to the topics of ER alpha and beta, Toxoplasma gondii, and prostatic epithelial and stromal interaction that can be used to advance treatment options for those affected by BPH and improve their quality of life.