DNA is often correctly referred to as one of the building blocks of life. Understanding the combination of adenine, thymine, guanine, and cytosine that ultimately defines our characteristics is crucial to many genomic applications ranging from synthetic biology applications to our understanding of health and diseases. Modeling and understanding the causes and consequences of these base pair level interactions can impact the development of DNA origami and our understanding of genome stability in different health/disease states. DNA is widely known for its complex structures and uncovering, modeling, and predicting the structure and formation of DNA can provide valuable insights into various aspects of genomics including visualization and probe design. Modeling DNA’s inter-strand and intra-strand interactions has been the core focus of genomics for some time. Specifically, understanding intra-strand secondary structure formation across genomes has largely been unexplored due to computational limits. To further our understanding of DNA, I present the use of thermodynamic partition functions to model and predict intra-strand behavior of secondary structure formation. I have efficiently calculated secondary structure formation across hg38 at three relevant thermodynamic conditions and their corresponding length scales. With this information, I examine the distribution of the entire genome while also cross examining this information against specific regions with known biological features such as genic vs non-genic regions. Additionally, I apply the same predictive model to fluorescent in situ hybridization (FISH) probe design and provide additional quantitative metrics and insights in the development of optimal probe design. This research has the potential to further our understanding of the causes and consequences of non-random genes. Having additional information about the distribution of secondary structure formation of certain regions can be vital information about learning more about the selection of base pair sequences and its macro effects.

5-hydroxymethylcytosine (5hmC) is a DNA modification studied in mammalian cells and tissues that has been implicated in embryonic stem cell differentiation, neuronal development, and formation of tumors. However, much is still unknown about the regulatory function of 5hmC and its genomic localization in diverse cell types. Recently, a novel, enzyme-based method was developed for 5hmC identification, APOBEC-Coupled Epigenetic Sequencing (ACE-seq), that is highly accurate, requires low input, and does not degrade DNA as commonly used bisulfite treatment methods do. ACE-seq is especially relevant to cellular environments that are epigenetically dynamic, including brain tissue and embryonic cell cultures. My project uses ACE-seq to generate high fidelity 5hmC characterizations in the Elf1 cell line across culture conditions that mimic early human embryonic development in which dramatic changes in DNA methylation occur. ACE-seq will be used on three human embryonic stem cell (hESC) conditions that mimic the transition from before embryonic implantation, naïve hESCs (two conditions), to near the time of embryonic implantation, primed hESCs. We hope to gain insight into 5hmC localization and regulatory function during this physiologically significant developmental event occurring during early embryogenesis. A more robust understanding of 5hmC regional abundance during this transition will help us elucidate the regulatory circuitry underlying early development. Knowledge gained in this project is especially relevant to the field of personalized medicine, as thorough understanding of pluripotency transitions will be significant to future applications of stem cell based therapies and precision healthcare. 

 According to the Centers for Disease Control and Prevention, birth defects affect 1 in every 33 babies born in the United States, and severity within each condition varies widely. However, the genetic basis of isolated birth defects (iBD), that is having a single major birth defect, remains largely unknown. The research question for this study is, “What are the underlining genetic causes of different iBDs?” To answer this question, I visually screened patient data obtained from the University of Washington Center for Mendelian Genomics. I looked at the DNA sequences in families to confirm the presence of a genetic variant in the affected child and identify patterns of inheritance for children affected by glaucoma or omphalocele. Glaucoma is a condition affecting vision and omphalocele is a defect of the abdominal wall that causes organs to lay outside of the stomach. I looked at these birth defects because they are common causes of infant mortality in the first year of life. Early findings show that in our cohort, 7 out of 19 individuals with congenital glaucoma have overlapping genotypes with other disorders affecting vision. In the omphalocele cohort, there were no overlaps in their genotype with other similar disorders. These results suggest that within glaucoma, there are other possible candidate genes of interest for targeted sequencing, and that these individuals may be affected by other medical conditions relating to vision. Given these findings, I will continue to analyze data for other defects to expand our understanding of the phenotypes of iBDs. The next major defect I will study is tricuspid atresia which is a defect in the heart. This research will contribute to knowledge of the genetic basis for iBDs by narrowing down the candidate genes which can be used to identify targeted sequences for DNA sequencing.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is caused by the death of dopamine-secreting neurons in the midbrain. The onset of symptoms, including progressively worsening tremors, movement difficulty, and dementia are thought to be caused by protein aggregates called Lewy bodies, mitochondrial defects, and neuroinflammation. Mutations in the GBA gene, which codes for the enzyme glucocerebrosidase which breaks down the lipid glucosylceramide, accounts for 5-10% of all PD cases. Our lab has a GBA mutant fly model that features symptoms similar to human PD – neurodegeneration, shortened lifespan, motor deficits, and increased protein aggregation. We hypothesize that GBA mutants fail to break down glucosylceramide, a lipid that is common in membranes of many pathogens. This glucosylceramide accumulation is responsible for triggering the innate immune response system, in turn causing inflammation leading to neural death. I explore innate immune system activation in the Toll, lmd, and Jak/Stat pathways and determine if a GBA mutation causes glucosylceramide accumulation to trigger the activation causing neuron loss, and the associated phenotypes. To do this, I will use reporters to test expression of these transgenic lines in a chemical assay, observe gene activation and protein expression following manipulation of glucosylceramide production, and use RNA interference to understand the effect of specific genetic perturbations in mutants. It is extremely valuable to understand the pathway of neuroinflammation to neurodegeneration, the mechanisms behind this, and the cascading influences they may have. My novel research on the impact of innate immune response pathways streamlines our comprehension of the mechanistic influences of PD and many other neurodegenerative diseases, leading to treatment and prevention development.

Biological tubes are the foundation of most animal organs, and thus, tube formation is an important developmental process. We study tube formation in Drosophila melanogaster egg chambers by analyzing the formation and elongation of dorsal appendages (DAs), eggshell structures that facilitate gas exchange in the fully developed egg. For these tubes to form, a subset of follicle cells called the dorsal appendage patch (DA patch) must change shape and migrate towards the anterior end of the egg chamber. Polarity proteins play an important role in mediating these follicle cell behaviors, but the mechanisms through which they do so are not fully understood. My research aims to enhance our understanding of these mechanisms by establishing how the polarity protein Crumbs (Crb) is involved in the formation of the DAs. Preliminary data from another lab member suggests that crb transcripts are expressed at a higher level in the DA patches than in other follicle cells. Based on these data, I hypothesize that wild-type cells should have increased levels of Crb in the DA patches relative to other follicle cells, and may show unusual lateral Crb distribution. Additionally, polarity proteins that normally associate with Crb should be mislocalized in DA patch cells without Crb, causing irregular cell morphology. To test these hypotheses, I am observing Crb localization in the DA patch cells and comparing it to Crb localization in other follicle cells using antibody staining. I am also looking at the effects of losing Crb in the DA patch cells by studying DA patch cell morphology, DA shape, and the localization of other polarity proteins in crb-null clones. This research will give insight into how Crb functions in DA formation and better our understanding of the function of polarity proteins in tube formation across many model organisms and in humans.

MicroRNAs (miRNAs) are short 21-23 nucleotide (nt) nucleic acid species that have a seed-sequence complimentary to target RNAs which can then be negatively regulated via the RNA silencing pathway. miR-122 is the most highly expressed miRNA in the liver, accounting for 70% of the miRNA reads and is involved in hepatocyte differentiation and cholesterol/fatty-acid synthesis. Previous studies have indicated that total abrogation of miR-122 or competition of miR-122 by short hairpin RNAs (shRNAs) in mice caused an increased expression of the Dlk-Dio3 locus, which is highly expressed in development but downregulated in adults and ultimately leads to hepatocellular carcinoma (HCC). Furthermore, it is known that activation of the Dlk-Dio3 locus alone has been shown to lead to the development of HCC but the specifics of miR-122’s regulatory role have not been elucidated. To determine the effects of miR-122 on the Dlk-Dio3 locus, I am using human HCC cells that contain a 3-bp deletion down-stream from the miR-122 transcript. We predict that the 3-bp deletion prevents proper miR-122 transcription. By transfecting the cells with a miR-122 mimic transcript to introduce its expression, I can compare the levels of RNA expression in liver cells containing miR-122 to those without by using qPCR and small-RNA sequencing. Preliminary data indicates that cells transfected with the miR-122 mimic have decreased levels of expression of RNAs located in the Dlk-Dio3 locus such as Rtl1 and positive control targets such as Aldoa. To further confirm this relationship, I am planning on using CRISPR-mediated homology-directed repair to replace the 3-bp deletion in my cell line and endogenously re-express miR-122 in order to study its regulatory role at the Dlk-Dio3 locus.