DNA glycation is the DNA damage induced by reactive carbonyls (such as methylglyoxal and glyoxal) in plants and mammals. Glycation damage is quantitatively as important as oxidative damage. It is one of the major in vivo DNA damage sources associated with increased mutation frequency, DNA strand breaks, and cytotoxicity. In humans, glycation damage may contribute to Parkinson’s disease, cancer, and oxidative stress-induced diseases. Glycation damage in plant organelle (Chloroplasts and Mitochondria) DNA remains poorly understood. We recently showed that the demise of plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) during maize seedling development is associated with an increase in DNA damage resulting from oxidative stress caused by reactive oxygen species. As oxidative and glycation stress are closely linked in plants, we hypothesize that glycation might be one of the causes of ptDNA and mtDNA damage during development. Glycation damage can be prevented by the activity of the protein deglycase DJ-1, also known as Parkinson's Disease Protein 7 (PARK7). Our objective is to quantify glycation lesions in the organelles of maize plant tissues in the presence and absence of DJ-1. Our approach involves the quantification of glycation and deglycation in ptDNA and mtDNA using PCR analysis and the enzyme-linked immunosorbent assay (ELISA). This research should better understand glycation damage in plant organelles and might lead to insights concerning human pathologies and neurodegenerative disease caused by glycation.

The skin is a highly innervated sensory organ, providing our bodies with the vital ability to understand and respond to external stimuli, such as pain, temperature and touch. However, many injuries to the skin result in the severance of somatosensory axons, causing temporary or permanent loss of feeling. To reestablish innervation, skin and neuronal cells launch wound healing responses. Although these responses are known to occur after an injury, the exact biological pathways and cellular components involved remain poorly defined. This project aims to characterize the transcriptional responses to skin injury using zebrafish (Danio rerio) as a model organism. Unlike human skin where complete regeneration is not observed, zebrafish have almost perfect regenerative abilities. To address our question, we plucked fish scales to induce a rapid regenerative response in somatosensory neurons and skin. I previously conducted RNA-seq analysis on populations of neurons and skin cells over the course of an induced injury response and curated a list of differentially expressed genes. Using gene ontology annotations to inform on these transcriptional changes, I have begun to identify the enrichment of well defined pathways, processes, and cellular components that are up or down regulated during the injury response. Thus far, I have identified a number of upregulated biological processes associated with changes in the extracellular matrix of skin-resident cells. The identification of enriched biological processes will help guide future experimental designs that study the effects of manipulations in these processes. With a deeper understanding of genes and mechanisms involved in zebrafish skin repair, I hope to unlock regenerative secrets that could apply to the treatment of human tactile maladies. 

In the US over 100 million people live with diabetes or pre-diabetes. The economic burden of this is approximately $327 billion every year. This study seeks to establish an alternative mode of insulin production using a polyethylene glycol (PEG) transformation of Pleurotus ostreatus. P. ostreatus is a valuable target for genetic transformation due to its lack of endotoxins, rapid growth, and fully sequenced genome. In this study, I transformed P. ostreatus using PEG with a plasmid containing the human insulin gene, a green fluorescent protein (GFP) reporter gene, and a selectable resistance gene. Transformed cells were selected using hygromycin, extracted, and regenerated on growth media. Confocal microscopy confirmed the presence of the GFP and presumably the human insulin gene. An ELISA for insulin and proinsulin will be used in the upcoming months to test for genetic expression, and to determine the efficacy of protein folding in the transgenic fungal cells. This has the potential to not only expand the market for diabetic treatment options, but it initiates a valuable conversation about the importance of diversifying production methods and costs in the treatment of diabetes.

Alzheimer’s Disease (AD) is a neurodegenerative condition that affects more than 50 million individuals worldwide. The progression of AD is hallmarked by the buildup of beta-amyloid plaques and neurofibrillary tangles, leading to neuronal death on a large scale. In the early stages of AD, the hippocampus is disproportionately affected by this heavy neuronal loss. The genetic elements and pathways contributing to this AD pathology are still poorly understood. Excitatory synapse assembly (ESA) processes have been previously shown to be affected by the pathology of AD, leading us to investigate the expression patterns of genes involved in ESA in different brain regions. A gene ontology (GO) analysis was conducted to isolate individual genes involved in ESA and analyze their relation to beta-amyloid plaque and pTau neurofibrillary tangle severity in patients who presented signs of dementia due to AD. ESA gene expression was shown to be strongly correlated with increasing levels of pTau in all brain regions except for the hippocampus, where there was no correlation, in a linear regression analysis. This implies that the hippocampus has a unique response to AD pathology with regards to ESA gene expression among the different brain regions. The inability of hippocampal cells to express neuronal repair genes in the presence of severe neurofibrillary tangles requires further analysis and could eventually confer a novel target for AD therapeutics.

Our project is mainly focusing on hemichordates, they are marine invertebrates, related to echinoderms. While no chordate is able to regenerate all regions of its central nervous system after deathly injury. Ptychodera flava, a hemichordate, exhibits an exceptional ability to regenerate its entire central nervous system and anterior structures in as little as two weeks. We are studying this process to understand the underlying gene networks involved in neural regeneration. I am currently investigating the expression of transcription factors involved in neural development with homeodomain-containing genes Pax6, Six3, DLX, Frizzled, Chordin, Pou, and Msx-2, which have been shown to be upregulated during P. flava regeneration through transcriptome analyses. I have previously cloned the isolated PCR products into plasmids to make RNA probes for in situ hybridization. I am generating RNA probes, performing in situ hybridization. I will use RT-PCR to examine when there is a high RNA expression at various stages of regeneration, then I will perform in situ hybridization on P. flava tissue samples at these different stages. I will also be sectioning these samples after in situ hybridization and staining to image the cellular and tissue structures to aid in seeing where these genes are expressed in specific tissues during regeneration. My research goal is to ascertain the gene networks underlying regeneration and then to compare these to the expression of the same genes during embryonic development in P. flava. Finally, we can ask whether these genes exhibit similar gene networks that have been reported during development in direct-developing hemichordates. Further experiments will be examining if these gene networks are necessary and sufficient in regeneration by knocking-out or overexpressing these specific genes at different stages of regeneration. By using P. flava as a model to study transcriptional regulation during regeneration, we are aiming to identify the genetic and morphological mechanisms to achieve sufficient central nervous system and whole-body regeneration in a stem deuterostome, which might also give us the insight into potential key regulator factors during human regeneration.

Humans hear through the conversion of pressure waves vibrating in the inner ear into chemical signals released by activated hair cells. Loss of function of hair cells due to damage can result in permanent hearing loss. Zebrafish are one of the model organisms used to study hair cells, since zebrafish have hair cells similar to humans. Unlike humans, zebrafish can regenerate their hair cells after damage. Regeneration occurs when support cells differentiate into hair cells. Determining how zebrafish support cells differentiate into hair cells is important to understand if human support cells can be induced to differentiate in a similar way. This requires comparing the structural differences between support cells and hair cells. One important aspect of neuronal signaling is the release of calcium ions from the cell’s endoplasmic reticulum (ER). This experiment looked to answer whether the ER in zebrafish hair and support cells was quantifiably different. Since structure mediates function and support cells do not signal to neurons, their ER structure should not be equivalent to the hair cells’. To test this, I manually segmented and reconstructed serial block-face scanning electron microscope (SBF-SEM) images of both cell types’ ER into 3D. Using SBF-SEM as a reconstruction method allowed for more detailed visualization of both the cell volume and the ER, in contrast to other methods such as confocal microscopy. Support cells had an ER volume of 50.51 µm3, while hair cells had an ER volume of 29.09 µm3. Support cells had a higher ER to cell volume and ER surface area to volume ratio than hair cells. It can be concluded that ER structures of support and hair cells are quantifiably different. Quantifying ER differences between support and hair cells is an important step toward discovering solutions to deafness caused by damage to human hair cells.

We can use human pluripotent stem cells to derive cardiomyocytes (hPSC-CMs) in vitro, with the goal of transplanting them into the hearts of individuals who have suffered from heart attacks and restore contractile function. After transplantation into animal models, however, hPSC-CMs produce arrhythmias (irregular heartbeats), likely caused by the immature state of hPSC-CMs. This immature state is associated with low expression of cardiac genes regulating heart muscle contraction and electrical properties. We aim to mature hPSC-CMs in vitro by controlling the expression of these genes, so we can engineer them to behave more like adult cardiomyocytes. To do this, I am looking at DNA methylation, a modification occurring at cytosine nucleotides that is associated with transcriptional repression or gene silencing. My project goal is to determine if DNA methylation plays a role in regulating gene expression patterns of cardiac genes in hPSC-CMs. To investigate this, I have treated hPSC-CM genomic DNA with bisulfite reagent, which converts unmethylated cytosine nucleotides to thymine nucleotides. This treatment will allow me to differentiate between unmethylated versus methylated DNA, and determine whether cardiac maturation genes are methylated at their promoters (where gene expression is typically regulated) by running PCR. Additionally, I have cultured hPSC-CMs with the DNA methylation inhibiting drug 5-azacytidine. By blocking DNA methylation, I will be able to determine if methylation has a direct effect on the expression of cardiac genes by measuring gene expression via quantitative real-time PCR. I hypothesize that DNA methylation regulates cardiac gene expression, and inhibiting methylation will cause expression to increase. Thus, if DNA methylation represses cardiac gene expression, we can mature hPSC-CMs by inhibiting methylation. Ultimately, we hope to prevent arrhythmias that occur after hPSC-CM engraftment and develop cell therapies using mature hPSC-CMs to restore heart function after a heart attack.