Myelodysplastic Syndrome/Myeloproliferative Neoplasm Unclassifiable (MDS/MPN-U) is a group of blood cancers that exhibit features of both MDS and MPN. Myeloproliferative Neoplasms (MPN) are cancers characterized by the proliferation of abnormal blood cells in the bone marrow, while Myelodysplastic Syndromes (MDS) are a group of cancers where immature blood cells in the bone marrow do not mature into healthy blood cells. Due to its overlapping features and the evolving morphology not being able to provide an accurate diagnosis, the molecular and cytogenetic abnormalities aimed at understanding the structure and properties of abnormal chromosomes could provide crucial insights for diagnosing and comprehending MDS/MPN-U. We hypothesized that the patients with MDS/MPN exhibit specific, unexplored molecular features that were not found in previous studies. We studied fourteen patients diagnosed with MDS/MPN-U overlaps and performed genetic analyses using proximity ligation sequencing (Hi-C sequencing) on Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens. Hi-C sequencing reconstructed all aberrations and rearrangements of the MDS/MPN-U genome. Cytogenetics and molecular data were collected and analyzed. New biomarkers and recurrent traits were identified. We expected Hi-C Sequencing to identify additional cytogenetics aberrations that were not detected using routine clinical cytogenetics techniques. Moreover, new insights into MDS/MPN overlap syndromes will help fill the knowledge gap in blood cancer, contributing to enhanced diagnostic accuracy, the development of treatment strategies, and improved patient care.

The causative agent of malaria, Plasmodium spp., generated 608,000 deaths worldwide in 2022 according to the World Health Organization and disproportionately threatens endemic areas of Africa. Plasmodium sporozoites infect the host by entering the bloodstream through the skin following bites by female Anopheles mosquitoes. From there, sporozoites migrate to the liver and infect hepatocytes. A single sporozoite-infected hepatocyte is capable of producing thousands of merozoites, which go on to enter the bloodstream. Complete elimination of infected hepatocytes is necessary to achieve sterile protection. In order to observe adaptive and innate immune cell localization towards infected hepatocytes, we applied fluorescence microscopy on livers in a BALB/c rodent model of malaria. Naïve unvaccinated mice were infected with sporozoites of Plasmodium yoelii, a rodent malaria parasite. Two important cell populations are recruited to infected hepatocytes. The first are tissue resident memory CD8+ T cells (Trm), which are crucial in pre-erythrocytic protection. The second are Kupffer cells, which are specialized liver macrophages. To measure these adaptive and innate cell populations, respectively, we applied fluorescently-labeled antibodies to mark the parasite as well as Trms and Kupffer cells. After staining the collected liver tissue and imaging with a widefield fluorescent microscope, we visualized recruitment and measured immune cell proximity quantitatively within a region of interest of the area surrounding infected hepatocytes using microscopy imaging analysis software. This method will be used to test the hypothesis that Trms and Kupffer cells are induced following sporozoite challenge in the rodent malaria model.

Rhabdomyosarcoma (RMS) is a devastating soft tissue sarcoma most commonly affecting children. The components in the RAS-PI3K-MAPK signaling pathway are frequently mutated in RMS and are being studied as a critical target for treatment. A MEK1/2 inhibitor, trametinib, has shown clinical efficacy singly or in combination with other drugs in improving survival outcomes of patients with cancer types harboring mutations in the MAPK pathway. In addition, vincristine is a common chemotherapeutic drug used to treat RMS, but its efficacy has been limited due to RMS cells developing resistance. The study aims to determine whether combined treatment of vincristine and trametinib could more significantly inhibit RMS cancer growth compared to treatment with each drug alone. For this study, a clonal zebrafish line bearing RMS tumors labeled with a fluorescent protein was first treated with a range of trametinib concentrations to find the appropriate dosage for optimal effectivity yet minimal toxicity. Then, RMS-bearing zebrafish received two doses of trametinib, vincristine, or a combination of both through intraperitoneal injections (IP). Finally, we collected tumors from each treatment group to assess treatment-induced changes in gene expression by quantitative RT-PCR and changes in tumor cell proliferation and cell death by immunohistochemical stains. The changes in tumor volumes were determined by quantifying the intensity of tumor fluorescence in the ImageJ software. Preliminary findings showed that zebrafish tumors treated with the combination of vincristine and trametinib significantly inhibited tumor growth compared to the other with each agent alone. Thus, the results of this in vivo study on zebrafish may introduce a drug dosage combination with translational potential in creating a new treatment composed of lower doses of each drug while increasing potency against chemotherapy-resistant tumors and lessening side effects in patients. The end results will translate into improved survival outcomes and well-being of RMS patients.

Colorectal Cancer (CRC) incidence is rapidly rising in patients younger than fifty with no prior family history. Recent research has revealed that very low frequency somatic mutations accumulate in normal tissue with aging. Many of these mutations occur in common cancer genes and are positively selected, producing clonal expansions, that might be linked to cancer risk. I hypothesize that the normal colon of patients with early onset CRC might carry clonal expansions driven by mutations in CRC genes, which could be detected with ultra deep duplex sequencing. Duplex sequencing is an error correction method that improves sequencing accuracy through double stranded molecular tagging. I used duplex sequencing to perform ultra deep sequencing (~3000x) of the main driver genes of colorectal cancer (CRC) which includes: BRAF, APC, FBXW7, KRAS, PIK3CA, SMAD4, and TP53. First, I performed adjustments of gene probes by increasing or decreasing the proportion of each probe in each gene pool to achieve comparable depth of sequencing across genes. Then, I sequenced normal colon tissue of two patients with and without CRC. Preliminary data showed deleterious mutations in tumor suppressor genes: APC, FBXW7, and TP53, which occurred at higher frequency in the patients with cancer. The next steps of this research involve analyzing the normal colon from a larger cohort of patients with and without CRC. Our results have demonstrated that clonal expansions are visible in early onset CRC with these sequencing methods, and I anticipate that I will identify more clonal expansions in patients with early onset CRC than in those without CRC. In conclusion, duplex sequencing allows for the detection of cancer driver mutations in normal tissue, allowing the discovery of early somatic events in CRC and offering potential for early CRC detection, prevention, and prediction.

TAR DNA binding protein 43 (TDP-43) is an RNA/DNA binding protein that forms pathological aggregates in most amyotrophic lateral sclerosis (ALS) and half of frontotemporal lobar degeneration (FTLD) cases. Knockout of TDP-43 in animal models leads to neurodegeneration and motor deficits, but overexpression of wildtype TDP43 leads to the same events; therefore, TDP43 protein homeostasis is critical to prevent ALS/FTLD. To achieve this homeostasis, TDP-43 autoregulates its own mRNA splicing, resulting in multiple TDP-43 isoforms, some of which go through non-sense mediated decay to regulate overall TDP43 levels. However, other isoforms encode unique proteins with differing C-termini, leading to variable cellular localization. It is unknown if these alternative, protein-coding isoforms are predominantly associated with ALS/FTLD or if aging changes the frequency of these isoforms. To determine how TDP43 overexpression yields these different isoforms and interacts with aging and ALS-like symptoms, we created a novel approach to overexpress human TDP43 via Adeno-Associated Virus (AAV) delivered through retro-orbital injection, leading to ALS-like motor deficits. Surprisingly, when tested in older and younger mice, we found the older mice were paradoxically protected from severe motor deficits and mortality. To determine if tardbp alternative splicing is linked to ALS-like symptoms and aging, I designed and validated primers and protocols to measure the nine tardbp mRNA isoforms in mice via quantitative real-time polymerase chain reaction (qRT-PCR). I have started to determine if hTDP43 overexpression leads to differential splicing compared to mice injected with a sham AAV in these old and young mice. Once this is done, we will clone the most interesting differentially spliced isoform in an AAV and inject that AAV and a full-length TDP43 AAV into mice to see if the spliceform causes increased toxicity, manifesting in worsening motor deficits and mortality.

Mice missing NADH: Ubiquinone Oxidoreductase Fe-S Protein 4 (NDUFS4) are a model of Leigh Syndrome, a mitochondrial disease. NDUFS4 KO mice show reduced lifespan and high levels of acetylation of mitochondrial proteins. The anti-diabetic acarbose increases survival in NDUFS4 knockout mice and reduces mitochondrial protein acetylation, as deduced by reduced acetylation of SOD2 on Lysine 68. SIRT3 is a NAD+/- dependent deacetylase that deacetylates mitochondrial proteins. We sought to determine whether acarbose reduces acetylation in NDUFS4 mice via SIRT3. NDUFS4 and SIRT3 double KO mice were fed acarbose and dissected at 35 days of age. Brain samples were collected and analyzed via western blot. We determined the levels of acetyl K68 SOD2, total SOD2, and actin for each sample. Our results show that acarbose reduces acetylation in knockout NDUFS4 regardless of the presence of SIRT3. Acarbose reshapes the intestinal microbiome and the production of short-chain fatty acids in NDUFS4 KO mice. We are currently devising in vitro experiments to determine whether short-chain fatty acids can reduce mitochondrial protein acetylation in NDUFS4 knockout cells. This research investigates if short-chain fatty acids are responsible for acetylation of mitochondrial proteins. The results can be significant because the acetylation of mitochondrial proteins can prevent diseases from being as deadly by extending lifespan. The work completed during this research is hopeful to be applied to a human model, in which this model can be used to treat mitochondrial diseases.

Staphylococcus aureus is an important human pathogen that has increasingly developed resistance to antibiotics and antimicrobials. It is important to understand how genetic mutations influence antibiotic resistance to anticipate how the organism is able to evolve and combat emerging resistance. My project aims to fully characterize the impact of variants in the norA gene, which is associated with efflux-mediated resistance. The Salipante lab has developed a system for precision genome engineering in S. aureus by constructing a vector that is able to conduct recombineering while suppressing DNA repair, and a separate vector for killing unmodified bacteria using programmed CRISPR/Cas9. Using this system, I will construct a library of genetic mutations in the S. aureus genome that will encompass all possible single mutations in norA and to subsequently test the fitness costs of those mutations in the presence and absence of antibiotics.We will recombineer into S. aureus randomly mutagenized oligonucleotides that encode a silent mutation that renders transgenic bacteria immune to targeted CRISPR/Cas9 cutting. After killing off unmodified bacteria, the transformed population will be composed entirely of different mutants, so that with a large population size, we can statistically ensure that all possible random mutations are represented. We will analyze the gene sequence from the initial population, after the population has expanded in the absence of drugs, and after exposure to different concentrations of drugs, to compare the relative representation of each specific mutation. Changes that are detrimental under a condition will be underrepresented relative to the starting population, while those that are beneficial will be overrepresented. Mutations that are lethal will not be recovered in the first place. Our project has the potential to fully and comprehensively address clinically important questions regarding antibiotic resistance evolution in this gene.

Lipotoxicity in cells occurs when lipids in biological tissue accrete to a toxic level. This toxic buildup is associated with obesity and type 2 diabetes, two of the leading causes of death around the world. Various studies have induced obesity in mice through a diet consisting of high levels of saturated fats. Some of these studies also investigated the effects Adefovir Dipivoxil, an antiviral and inhibitor of mitochondrial DNA replication, had on these obese mice. My research investigates fibroblasts that have been grown in a high-lipid environment and how Adefovir Dipivoxil affects these cells. We hypothesized that administering Adefovir to these cells would halt apoptosis and prevent further progression of lipotoxicity by stabilizing levels of triacylglycerol synthesis. To test this, I induced lipotoxicity in the cells using palmitic acid, a saturated fatty acid. Next, I treated the cells with Adefovir Dipivoxil. To measure the efficacy of the drug, I used a staining kit to measure the ratio of live to dead cells before and after administering the drug. Furthermore, I used a dye-based assay to measure the intracellular triglyceride levels before and after treatment. In addition to treating fibroblasts with palmitic acid, I treated a new set of fibroblasts with oleic acid, an unsaturated fatty acid, to determine how Adefovir acts on these cells. Data from this research will contribute to further understanding the mechanism of lipotoxicity on various cells, as well as the role that mediators of mitochondrial function like Adefovir Dipivoxil could play in treating lipotoxicity. On a broader scale, we hope that this research will provide insight into future treatments for obesity and type 2 diabetes.

The antibiotic penicillin is highly effective at treating the STI syphilis, caused by the bacterium T. pallidum. However, the United States has seen increases in syphilis cases every year for the past 20 years; congenital syphilis cases have risen more than 219% from 2017 to 2021 and overall syphilis cases have risen 32% from 2020 to 2021. This situation demonstrates the need for an effective vaccine as current approaches are not working. The aim of this project is to utilize phage immunoprecipitation sequencing (PhIP-Seq) techniques to assist in the development of an effective vaccine in rabbits and eventually humans. To this end I have been using PhIP-Seq techniques to systematically profile the immune responses to vaccine candidates and T. pallidum infections in rabbits. When rabbits are immunized with a cocktail of three strains of the protein TprC we saw a protective immune response against treponemes (resulting in no viable treponemes) whereas an immunization with TprD saw reduced immune protection. I used PhIP-Seq methods - informed by next-generation sequencing (NGS) and differential expression analysis - to determine the epitope-specificity of antibodies in polyclonal serum samples from rabbits immunized with these vaccine candidates. Epitope-specificity comparisons between the resulting antibodies of the two immunogens can shed light on regions of these proteins critical for protection against treponemes. In the next few months I plan to integrate alanine scanning mutagenesis into the project to assess amino acid binding specificity and accurately identify crucial residues for antibody-binding. The fusion of scanning mutagenesis with PhIP-Seq will allow me and the other research scientists assisting with the project to refine of the effectiveness of our existing vaccine candidates.

Renal cell carcinoma (RCC) is the most common type of kidney cancer, and patients with advanced RCC have a 15% survival rate. Some RCC patients respond well to immunotherapy, but the antigen targets recognized by RCC-reactive T cells remain largely unknown. There is a critical need to develop a time-efficient and high-throughput genomic screening platform that can link T-cell receptors (TCRs) to their cognate antigens and identify essential genes in RCC cells responsible for escape from T-cell killing. I am using a CRISPR-Cas9-based whole genome screening platform, the Brunello Library, which uses 76,456 single guide RNAs to target 19,114 genes in the human genome. To identify essential genes, I transduced A498-mCherry Cas9 cells with the Brunello library, selected the CRISPR-Cas9 engineered cells, and co-cultured them with T cells expressing an RCC tumor reactive TCR targeting the KIAA0020 gene. Through the first large screen, I obtained numerous hits in at least four expected genes that are known to be involved in antigen processing and presentation: TAP1, TAP2, B2M, and HLA-A. These results successfully demonstrated the proof of principle of the screening approach and also defined a threshold baseline of representation of the sgRNAs in the target population that is required to provide enough cells to be identified after T-cell selection. I will implement additional rounds of screening designed to increase the overall representation of sgRNAs. Knowing the tumor peptide antigen targets is crucial in understanding T-cell responses and accelerating the development of TCR-engineered T-cell immunotherapy for RCC.

Meningiomas, the most common type of primary human brain tumor, arise from the thin fibrous membrane that covers the brain and spinal cord. Most grow slowly and are diagnosed when they disrupt brain function or lead to persistent headaches. While many meningiomas can be cured by surgery, ~20% of them cannot be fully resected or display increased growth, invasion and destruction of adjacent brain and skull. Effective control or eradication of these ‘High Grade II/III’ meningiomas is clinically challenging. To identify new agents and treatment measures, our project uses both computational and experimental approaches in concert to identify new and potentially better therapies. As part of this effort, we are using PISCES, a machine learning model, together with augmented drug and radiation combination datasets to predict potential new therapy synergies. The best predictions from PISCES will then be tested experimentally in our cell line model versus standard-of-care treatments.  My presentation summarizes work to characterize genomic, drug and ionizing radiation sensitivity data on IOMM-Lee, a Grade III human meningioma cell line disease model. We detail how A.I.-driven analyses of IOMM-Lee and related meningioma datasets led us to test new drug pairs and drug-radiation combinations predicted by PISCES to be more effective in killing IOMM-Lee tumor cells. This translational cellular disease model and project are part of a long-term effort to develop better ways to rapidly and efficiently identify and validate new treatment options for brain tumors and other human cancers that can be taken directly to clinical trial.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure around the world. Inherited mutations cause the left ventricle of the heart to enlarge, thinning the heart muscle wall and decreasing the overall function of the heart. In my research project, I will determine if disrupting fibroblast function by knocking out a key developmental signaling factor, p38, can improve, or even reverse, DCM disease characteristics. Specific Aim 1 will be to determine the effects of p38 knockout-induced fibroblast dysfunction on cardiomyocyte function and structural remodeling in late-stage DCM. The rationale is that myocytes in DCM have poor contraction and structurally remodel to longer, thinner morphologies, which occurs in our DCM mouse model around 4 months of age. I expect to see less of these characteristics with the p38 knockout. Specific Aim 2 will assess cardiac fibroblast proliferation and fibrosis in response to disabling cardiac fibroblast function late into the DCM disease process. The rationale is that studying and observing the dynamics of the fibroblast population is critical when understanding the effects of fibroblasts and the p38 knockout model on DCM. In previous studies, the Davis lab identified that cardiac fibroblasts maladaptively respond to inherited DCM mutations in cardiac myocytes, worsening the whole heart. I expect to see less fibroblast proliferation in the p38 model. P38 is essential for fibroblast signaling pathways and functionality, so by knocking it out I will be able to test if fibroblasts are a viable therapeutic target for patients with DCM.

To form Human Immunodeficiency Virus type 1 (HIV-1) virions, the HIV-1 Gag polyprotein multimerizes, traffics to the cell membrane, assembles into virions, and buds as viral particles. HIV uses the infected cell proteins to support viral assembly and export from the cell. Although some cellular proteins have been identified that participate in viral assembly and budding, the spatial and temporal "cellular proteome" is not known. Insights as to the order of proteins involved in facilitating assembly and budding provide information on viral infection and potential therapy targets. I am utilizing a split-APEX2-mediated proximity labeling method to identify which host factors interact with Gag during assembly. The basis of the method is splitting APEX2 into two segments encoded into separate Gag sequences so that the APEX2 segments rejoin for complete enzymatic function when Gag dimerizes and multimerizes. Once the APEX2 enzyme reconstitutes, we activate it to biotinylate cellular host proteins within ~20 nm of Gag during the processes of virion formation. I aim to deliver this system to primary human CD4+ T-cells and macrophages via two lentiviral vectors containing transgenes for either AP-Gag-P2A-EGFP or EX-Gag-P2A-mCherry. Cells express the dual fluorescence markers EGFP and mCherry if both Split-APEX2 domains are present, and we enrich these cells by cell sorting. APEX2 is restored and enzymatically active, allowing proximity-dependent biotinylation of Gag-host cell proteins for SILAC-based quantitative proteomic mapping during virion assembly. We are using this method to quantitatively map the Gag-host protein "interactome" in the infected cellular microenvironment in subtypes of human primary CD4+ T-cells and macrophages. Therefore, this research studies Gag interactions of potential HIV-1 host dependency factors in CD4+ T-cells and macrophages, the natural cell populations for HIV-1 infection. Previously, we conducted a similar study in HEK293Ts, and we anticipate elucidating a similar Gag-host cell protein analysis with this study.

Almost every form of cardiac disease is characterized by fibrosis, or the accumulation of collagen, an extracellular matrix (ECM) protein, secreted by the cardiac fibroblast. The buildup of fibrosis is a major clinical burden, as it contributes to diastolic dysfunction, or the heart’s inability to relax, and arrythmias, or an irregular heartbeat. In previous studies, the Davis lab has found that in chronic injury, the heart likely undergoes minor offenses along with periods of rest which accrue over a lifetime. Even when exposed to repeat injury stimuli, the heart is able to recover, and the cardiac fibroblasts can transcriptionally regress. Yet, what remains unclear is when the heart experiences repetitive stress, which is common with hypertension, will these once-activated cardiac fibroblasts have a more aggressive response? And if so, are the activation cues stored in the primed external environment, or are they intrinsic to the cell? To address this, we developed a fibroblast isolation and injection protocol that will ultimately allow us to isolate discrete populations of fibroblasts and study them in hearts void of injury. Our results found that fibroblasts from donor hearts that were subjected to a myocardial infarction injury were detectable at 4 and 14 days post cardiac injection but had little proliferation. However, there was an increase in host fibroblasts recruited to the graft site, many of which were proliferating, and fibrosis was found within these same regions. These results demonstrate that cardiac fibroblasts from the same strain can be isolated and adoptively transferred to other hearts, without exogenous ECM. We can apply this baseline protocol to further examine fibroblast memory in vivo in a model of intermittent hypertension.

High-grade serous carcinoma (HGSC), the most common subtype of ovarian cancer, originates in the fallopian tube epithelium from precursor lesions carrying somatic TP53 mutations. Individuals with germline mutations in DNA repair genes are at high risk of HGSC but the reason is unknown. We hypothesize that individuals at high risk of HGSC carry an excess of pathogenic TP53 mutations in fallopian tube epithelium, which predisposes them to cancer. Preliminary data suggests that individuals with germline mutations in BRCA1 and BRCA2 (lifetime risk of HGSC 45% and 21%, respectively) have more TP53 mutations in fallopian tube than individuals without germline mutations, supporting our hypothesis. However, TP53 mutations have not yet been characterized in individuals with germline mutations in RAD51C/RAD51D, BRIP1 and PALB (lifetime risks of HGSC 10%, 6% and 5%, respectively). We aimed to conduct an ultra-sensitive characterization of TP53 mutations in patients with germline mutations in RAD51C/RAD51D, BRIP1, and PALB2, and compare their mutational profile with those of individuals without germline mutations in HGSC risk genes and those with BRCA1 or BRCA2 germline mutations. Right and left fallopian tube biopsies were collected, frozen, and macrodissected using a 1mm biopsy punch. DNA was extracted and sequenced for TP53 using ultra-deep (15,000x) duplex sequencing. Data from 6 patients revealed varying degrees of pathogenic mutations in individuals with germline mutations. BRIP1 and PALB2 patients showed low and moderate levels of TP53 pathogenic mutations (11% and 41%, respectively), while RAD51C patients showed the highest percentage of pathogenic mutations (67%), matching their higher HGSC risk. We plan to sequence 6 additional patients to get more comprehensive data. By showing the differences in TP53 mutation patterns among these distinct populations, our research seeks to enhance our understanding of the underlying mechanisms of ovarian cancer predisposition and design better tools for early cancer detection, prediction, and risk assessment.

Alzheimer’s disease (AD) is the most common neurodegenerative disease, with over 6 million Americans suffering from the illness and prevalence increasing each year. My work was conducted as part of an NIH-funded multi-institutional network called TREAT-AD (TaRget Enablement to Accelerate Therapy Development for AD) that aims to find potential therapies for AD. The bioinformatics core of this network identified genetic targets of interest using RNA-sequencing and proteomic analysis of post-mortem tissue from participants with AD. We hypothesized that manipulating expression of these target genes in a relevant human model would influence levels of AD-related biomarkers. To manipulate genetic expression efficiently, I used shRNA technology in human induced pluripotent stem cell (hiPSC) derived neurons. I then analyzed these hiPSC-derived neurons for AD-relevant readouts, such as soluble amyloid beta secretion and intracellular phosphorylation of Tau protein, relevant to the two main neuropathological hallmarks of AD. I ran quantitative polymerase chain reactions (qPCR) to measure neuronal expression levels of each gene target, and compared amyloid beta and phosphorylated tau outputs to control samples using MSD ELISA assays. I found four gene targets that have substantial neuronal expression and found that each affected AD-related output levels when gene expression was knocked down with shRNA. My findings provide direct molecular genetic evidence that links these genes to AD pathways, suggesting that these genes could serve as promising targets for therapeutic development.

Down syndrome (DS, trisomy 21) is the most common chromosomal condition, affecting 1 in 700 live births. In addition to neurodevelopmental issues, people with DS also have an increased susceptibility to infections such as influenza and COVID. Altered immune function is one likely driver. Additionally, non-immune factors may also contribute. For example, upregulation of TMPRSS2, an enzyme that promotes cellular entry of SARS-COV2 encoded on chromosome 21, may predispose people with DS to severe SARS-CoV2 infection. This project builds on recent data from the Khor lab that suggests a link between DS and susceptibility to malarial infection. P. vivax is a parasite that causes malaria and is endemic across wide regions, from South America to South Asia. P. vivax enters cells through a protein called Duffy which is a chemokine receptor on the surface of red blood cells (RBCs). Recent profiling of whole blood from patients with DS showed dysregulation of many genes. Geneset enrichment analyses identified heme metabolism as the second most affected pathway. Within these heme metabolism genes, ACKR1, the gene encoding Duffy, was particularly and disproportionately upregulated. This suggests that people with DS may express higher levels of the Duffy protein on their RBCs, which in turn may predispose to P. vivax infection. This project seeks to better understand and validate how Duffy expression is dysregulated in people with DS. The goal of the project is to quantify the level of Duffy present in the RBC’s of patients with and without DS. If validated, the next step will be to test the susceptibility of these cells to malaria. This may point to a need for altered clinical management of malaria in people with DS and inform further studies looking at the mechanistic differences in RBC biology in people with DS.

Alzheimer's Disease (AD) is clinically characterized as a predominantly amnestic (memory impairment) syndrome at presentation that progresses to affect other cognitive domains. AD is pathologically defined by the presence of amyloid plaques and neurofibrillary tangles of hyperphosphorylated tau (pTau) in stereotypical brain regions. AD shows clinical and pathological diversity, including non-amnestic subtypes, severity of tau pathology across brain regions, and co-pathologies such as aggregates of hyper-phosphorylated transactive response DNA-binding protein 43 (pTDP-43). This study aims to examine the association between pTau and pTDP-43 using new highly quantitative approaches. By examining the combined pathology, we hope to identify patterns of pTau related to pTDP-43 across the different clinical and pathologic subtypes. The University of Washington Alzheimer's Disease Research Center clinical core autopsy cohort was characterized and subdivided into amnestic and non-amnestic syndrome subtypes. The subjects were analyzed to identify the prevalence of pTDP-43 and its correlation to the subject's cognitive data and patterns of progression. This analysis was used to select a subset of 29 cases with non-amnestic dementia and a matched subset with an amnestic subtype for more in-depth neuropathological and molecular profiling of several brain regions. Using the HALO platform, I generated quantitative measures of pTau in the frontal, temporal, and parietal cortex, as well as the hippocampus. The integration of these findings aims to understand how pTDP-43 pathology influences tau distribution based on clinical presentation These results will allow us to select a small set of cases for further work that will include using NanoString GeoMx Digital Spatial Profiling to identify potential pathways relevant to the association between pTDP-43 and pTau severity concerning mechanisms of clinical and pathologic heterogeneity in AD. These insights will allow for further research of these pathways to determine their biological relevance and ways to mitigate their effects.

The HIV-1 capsid is a conical lattice comprised of over 1500 monomeric capsid proteins (CA) that acts as a protective casing for the viral genome. Assembly of the capsid core is driven by the small negatively charged molecule, inositol phosphate (IP6), which interacts with positively charged CA residues. The ability to inhibit viral replication through the disruption of CA assembly has been demonstrated by the novel capsid binding antiretroviral, lenacapavir. Antiretroviral research is also being done on “clickable” compounds which should covalently interact with CA through a Sulfur (VI) Fluoride Exchange (SuFEx) reaction. Our lab has received lenacapavir analogues as well as compounds that potentially interact with CA via a SuFEx reaction (SuFEx compounds). My aim is to assess the impact of lenacapavir and the synthesized analogues on CA assembly, in addition to validating the ability of 15 promising SuFEx compounds to covalently bind CA. In preparation, I transformed E. coli cells with a plasmid containing the HIV-1 CA sequence and induced expression using IPTG. The cells were then pelleted, lysed and the protein purified utilizing gel filtration, anion- and cation-exchange chromatography. Assembly of the purified CA was induced in vitro by the addition of IP6 in both the absence and presence of lenacapavir and the analogues. Relative to lenacapavir, two analogues promoted assembly to a greater extent, while one performed on par and three enhanced assembly to a lesser extent. These analogues will be further studied to determine antiviral activity. Samples of CA in the presence of the SuFEx compounds have also been prepared and sent for mass spectrometry analysis. It will then be determined which of the SuFEx compounds bind CA covalently in vitro and may aid in the development of new capsid targeting antiretrovirals.
 

Recent findings suggest that decreased chromatin acetylation leads to neurodegenerative diseases including Alzheimer's Disease (AD). Histone acetylation alters chromatin structure which regulates gene expression. One of the key proteins involved in this genetic modification is KAT5 (TIP60) acetylase. The Young Lab recently demonstrated that increased chromatin acetylation leads to the expression of genes involved in cognition and neuronal maturation. KAT5 is known to interact with the intracellular region of the Amyloid Precursor Protein (APP), which is a main player in the development of Alzheimer’s Disease (AD). In wild-type cells, the KAT5 protein is believed to be released from the membrane and translocated to the nucleus where it leads to increased chromatin acetylation and gene expression. Recent hypothesis suggest that an amyloidogenic cleavage of APP, either due to mutations or in AD conditions, prevents KAT5 nuclear translocation. To address this question, we will perform the following three experiments using human brain tissue and human induced pluripotent stem cell-derived neurons (hiPSC-Ns): 1. Immunohistochemistry for KAT5 on control and AD brains to see if we can detect a reduction in nuclear KAT5 localization in AD. 2. Immunocytochemistry on hiPSC-derived neurons harboring a familial AD (fAD) mutation in APP (Swedish mutation-K670N/M671L) as well as in neurons derived from cells that are deficient in APP (APP KO). 3. RNA-sequencing to determine differential gene expression between cells with fAD mutations and those that are deficient in APP, with a focus on genes regulated by KAT5. We expect to see aberrant KAT5 localization and gene expression in cells and tissues with AD and fAD mutations. Since treatments targeting the deposition of beta-amyloid led to many unsuccessful medical trials, we anticipate this study will demonstrate the importance of the absence of KAT5 signaling during early development of AD and devise new strategies for treatments.

The HIV-1 gag polyprotein consists of the core structural proteins of the virus. During the late stages of viral replication, gag assembles beneath the plasma membrane into a curved immature hexagonal lattice. This process is primarily mediated by the capsid (CA) and spacer peptide 1 (SP1) domains which form a six-helix bundle with the assistance of the naturally occurring small molecule inositol hexakisphosphate (IP₆). Following budding of the immature virion, the HIV-1 protease cleaves the gag domains and CA protein assembles into a mature conical capsid, the protective shell of the virus. Disruption of CA assembly has been shown to inhibit viral replication and in turn has led to the development of the novel antiretroviral drug lenacapavir. While lenacapavir can bind mature capsid cores, the exact mode in which this drug impacts viral assembly and specifically interacts with immature CA remains unclear. My project aims to investigate the effect of lenacapavir and chemically and structurally related analogues of this drug, which have been designed and synthesized by a collaborator, on immature CA assembly in vitro. For this study I optimized the expression and purification of a gag construct that spans the CA to NC domains with an additional N-terminal Serine residue (s-CANC). I transformed E. coli cells with a plasmid containing the s-CANC construct and then overexpressed the protein. I optimized the purification protocol involving gel filtration, ion exchange and immobilized metal affinity chromatography. The purified s-CANC was then used to perform assembly assays using IP₆ in the absence and presence of lenacapavir and the analogues. The formation of immature capsid cores was confirmed via negative staining electron microscopy. Insights from these studies aim to provide a better understanding of how lenacapavir impacts the assembly of immature CA in addition to aiding the development of new capsid targeting antiretroviral drugs.

Alzheimer’s disease (AD) is the most common cause of dementia in the aging population, characterized pathologically by the presence of amyloid plaques and tau neurofibrillary tangles in the brain. However, AD often coexists with other pathologies contributing to dementia, such as hyperphosphorylated aggregates of the protein TDP-43. TDP-43 induces a dementia syndrome similar to AD and the combination of AD and TDP-43 is associated with accelerated cognitive decline, greater brain atrophy, and increased AD pathologic burden. AD and TDP-43 pathology are definitively diagnosed post-mortem upon neuropathologic examination but there is a great need to be able to identify these pathologies in living patients using biomarkers. Currently there are accepted biomarkers for AD, including measures of amyloid beta and hyperphosphorylated tau proteins in cerebrospinal fluid (CSF), but there are no biomarkers for TDP-43. Leveraging the reliability of CSF in detecting pathologic proteins, we hypothesize that measurable hallmarks of underlying TDP-43 pathology also exist in CSF. We tested four groups of brain donors (n=36 per group) defined by presence or absence of AD and TDP-43 pathology at autopsy: healthy controls, AD only (amyloid plaques and tau tangles), TDP-43 only, and AD+TDP-43. Post-mortem CSF samples are analyzed for TDP-43, hyperphosphorylated tau (pTau-181), and the brain injury marker glial fibrillary acidic protein (GFAP) using the Quanterix SR-XTM Biomarker Detection System. Because these assays are intended for ante-mortem samples, the first aim of the study was to determine optimal sample preparation for post-mortem samples, followed by the second aim to determine if there are concentration differences between proteins in CSF across groups. Successful identification of reliable TDP-43 biomarkers in living patients would improve neurodegenerative disease diagnostics, enabling accurate underlying pathology diagnosis and facilitating tracking disease progression and treatment response as therapies for AD, TDP-43, and other causes of dementia emerge.

Human-induced pluripotent stem cells can be differentiated into many neuronal cell types, including astrocytes. Astrocytes are a critical brain cell, generally called glia, that interact and communicate with neurons. Their dysfunction is apparent in Alzheimer’s disease (AD), but how these processes contribute to AD development is not well understood. I created and refined an astrocyte differentiation protocol from hiPSCs to understand astrocyte dysfunction in AD. The induction protocol starts at neural progenitor cells (NPCs) already differentiated from hiPSCs. I used astrocyte-specific cell surface markers, CD44 and CD184 to purify astrocyte progenitors from this population, and then matured the cells by culturing them for 28 more days in Astrocyte-specific media. To analyze the purity of the astrocyte cultures and success of the induction, I firstly ran a glutamate uptake assay via a colorimetric glutamate assay kit. Secondly, via RT-qPCR I tested for an upregulation of astrocyte genes GFAP, ALDH1L1, P1TX1, EEAT1 as cells differentiated from NPCs to mature astrocytes. At the same time I tested if there is downregulation of NPC genes Nestin, Sox2, and Pax6. I stained for astrocyte markers such as GLAST, S100b, and GFAP via immunocytochemistry (ICC) to confirm the expression of astrocyte relevant proteins. Following the validation of the astrocyte differentiation protocol, I tested activation of the astrocytes using conditioned media from familial AD (FAD) neuronal cultures, differentiated from hiPSCs that have the FAD Swedish mutation, and represent AD-mimicking conditions. Next, I tested astrocyte activation in comparison to 2 groups: a negative control with regular media treatment, and a positive control with a known inflammatory cytokine such as IL-1β or LPS. Activation of astrocytes were analyzed via a cytokine ELISA kit and through ICC. This project helps elucidate in what ways astrocytes interact with disease progression of FAD, and how their activity is differentially triggered in FAD.

Traumatic brain injury (TBI) is recognized as a risk factor for neurodegenerative diseases, but the underlying mechanisms remain unclear. This study aimed to investigate how localized brain injury alters pathologic protein aggregation associated with neurodegenerative disease, focusing on hyperphosphorylated tau (p-tau) and beta-amyloid deposition in brain parenchyma adjacent to chronic contusion. Using brain donors from the University of Washington Brain Repository, cases with a diagnosis of chronic contusion were identified. Beta-amyloid and p-tau deposition were assessed in sections adjacent to the contusion and in contralateral sections without contusion (internal control). Manual counting and HALO imaging software were utilized to quantify the highest density of neuritic plaques/neurofibrillary tangles and overall deposition of abnormal protein in grey matter, respectively. Statistical analyses were performed to compare deposition in contusion versus control sections. Preliminary data was collected in 9 cases, predominantly male with a median age of 79 years. Neurofibrillary tangles were significantly higher in sections with contusion compared to internal controls (p=0.0188), with a similar trend observed for neuritic plaques (p=0.0723). HALO software analysis confirmed increased deposition of both proteins in the contusion compared to control sections (p=0.0391 for both p-tau and beta-amyloid). These findings support that TBI may modulate neurodegeneration by increasing p-tau and beta-amyloid deposition and underscores the importance of further research into the relationship between TBI and neurodegenerative diseases. Next, I will expand our cohort with more cases from the last few years and plan to compare brain contusion in mid-life to contusion occurring after the age of 65. In addition to assessing abnormal protein deposition, I will also examine the neuroinflammatory response. I expect to find greater levels of neuroinflammation as well as increased tau and beta-amyloid aggregation in the older age group as the neuroinflammatory response becomes prolonged during aging.

Traumatic brain injury (TBI) is one of the most prominent environmental risk factors for neurodegenerative disease, including Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE). Both AD and CTE are characterized by the accumulation of abnormal phosphorylated tau (p-tau) protein in neurons in the brain in addition to other pathologies. In CTE in particular, the p-tau deposition occurs around the vasculature at the sulcal depths of the brain. Inhibitory neurons are the brakes of neuronal circuits in the brain and many previous studies using animal models have revealed a deficit or loss of inhibitory neurons after TBI. Some inhibitory neurons even have a special role in monitoring the vasculature of the brain, where the p-tau accumulates in CTE. However, no one has evaluated whether these inhibitory neurons are affected by p-tau pathology after human TBI. Here, I evaluate whether p-tau pathology accumulates in inhibitory neurons in ten cases of CTE and ten cases of brain contusion that have associated p-tau pathology compared to a positive control group (ten cases of AD without any history of head injury). Double-staining immunohistochemistry labeled with different colored chromagens is performed using three different inhibitory neuron markers (parvalbumin (PV), somatostatin (SOM) and TAC1R) combined with an antibody for the abnormal phosphorylated tau protein. Using the Halo image analysis system, the colocalization module is used to determine if p-tau accumulates in inhibitory neuron subtypes. These results will be confirmed with immunofluorescence and 3D confocal microscopy. I anticipate that inhibitory neurons, especially ones associated with vasculature (TAC1R+) will be affected by p-tau pathology in contusion and CTE cases, but not in positive controls. This project will give further insight into possible mechanisms of circuit and neuronal dysfunction that may occur after head injury and have therapeutic implications.

Naive B-cells in a primary follicle upon antigen exposure, start to proliferate and form a germinal center (GC) that also has other cells such as T-cell subsets, histiocytes, and follicular dendritic cells. The proliferating GC cells or centroblasts mainly occupy the 'dark zone' of the GC. Centroblasts further differentiate into centrocytes that are predominantly seen in the 'light zone' of the GC, and some of the selected 'centrocytes' further differentiate into memory B-cells or plasma cells. We set out to identify ideal markers to distinguish centroblasts and centrocytes and investigate them as biomarkers of disease classification, grading, and subtyping of GC B-cell lymphomas. We interrogated publicly available in-situ mRNA expression data from reactive lymph nodes using GeoMx Digital Spatial Profiler (PMID: 33083730), single-cell transcriptomic data from reactive lymph nodes and follicular lymphoma (FL) grade 1-2 and grade 3A (PMID: 35687817). Several markers were selected for single-antibody immunohistochemistry and multiplex-immunohistochemistry experiments. We focused on AID, CXCR4, Ki67, and PHH3 as markers for centroblasts and CD40 as a marker for centrocytes and tested them on tissue microarrays (TMA) with cores of 15 low-grade FL, eight high-grade FL, and 14 diffuse large B-cell lymphomas (DLBCL; 8 GCB type & 4 non-GCB). After analyzing the expression of the biomarkers on HALOlink with algorithms developed in the laboratory, we observed a significantly higher proportion of cells in high-grade FL expressing AID and Ki67 than in low-grade FL. The other markers showed no statistically significant difference in the percentage of cells expressed between high-grade FL and low-grade FL. Evaluating the proportion of cells expressing AID would help in objectively grading FL samples, but the impact of AID-assisted grading of FL on patient outcomes must be further evaluated.

According to the World Health Organization, Alzheimer’s Disease (AD) is the most common form of dementia – a major and growing cause of disability and dependency among older people globally. The Seattle AD Brain Cell Atlas (SEA-AD) project is a collaboration between the University of Washington (UW) and the Allen Institute for Brain Science (AIBS) aimed at discovering early vulnerable cell types in AD. In SEA-AD, we hope to further our understanding of the etiology and early progression of AD to ultimately identify targets for effective therapeutic intervention. Eighty-four participant brain donors with a postmortem interval less than 12 hours from the UW AD Research Center (12/84) and Kaiser Adult Changes in Thought (72/84) studies were included in the SEA-AD cohort. At the time of procurement, one hemisphere was frozen in super-cooled isopentane for transcriptomic analysis at AIBS; the contralateral hemisphere was fixed in 10% neutral buffered formalin for neuropathologic assessment at UW. The middle temporal gyrus, medial entorhinal cortex, and hippocampus were sampled, processed, embedded in paraffin, and sectioned for  immunohistochemical (IHC) studies. Seven antibodies, including duplexed stains, targeting amyloid b (6e10) and microglia (IBA1), pTau (AT8) and pTDP-43 (1D3), monoplexed a-synuclein (LB509), astrocytes (GFAP), neurons (NeuN), and triplexed histochemical stain: hematoxylin, eosin, and Luxol fast blue were deployed to assess the neuropathology associated with the presence and progression of AD and related neuropathologic changes. The data obtained from the quantitative assessment of the IHC staining is integrated with the transcriptomic data generated by the Allen Institute to enhance our understanding of the cellular vulnerabilities and associated molecular processes of AD. Public access to this neuropathological data through the SEA-AD resource potentiates research efforts to understand and identify the mechanisms of AD progression.