ItemEvaluating Improvements and Challenges in Affinity Chromatography for AAV Purification(Johns Hopkins University, 2023-07-31) Dachenko, Alexandra; Betenbaugh, Michael; Hadidi, MahsaPurification process development is crucial to the Research and Development (R&D) sector in the biotechnology industry by designing and optimizing downstream unit operations capable of manufacturing safe treatments for clinical trials. Purification is crucial in manufacturing therapeutics such as monoclonal antibodies (mAbs), mRNA, viral vectors, and non-viral vectors. Recently, viral vector gene therapy modalities using adeno-associated viruses (AAVs) have been shown to be an effective therapeutic agent for both rare and common diseases in humans. One of the key polishing steps in the downstream manufacturing process of AAVs is affinity chromatography, where a ligand with specific binding affinity to an AAV serotype is coupled to a base matrix that separates AAVs from host cell contaminants produced in the cell culture process. Column resin is the most common affinity chromatography matrix, but it possesses mass transfer limitations. This report evaluated a prototype affinity membrane device coupled with AVB ligand designed to overcome these mass transfer limitations and decrease process times, while yielding comparable AAV recovery and purity to column resin. The evaluations included dynamic binding capacity (DBC) studies, ligand lifetime studies, and buffer optimizations. The performance of the device was compared to control experiments using two common column resins with the same ligand as the device. The novel membrane device can bind at least 2e14 vg/mL_membrane and can undergo at least 3 cycles without a major binding capacity drop. The recovery of AAV is comparable to column resins; additional process development studies were required to increase recovery values because process parameters used for resin with the same ligand were not effective. A comparison of AAV product purity cannot be made because impurity profiles were not tested. Some recommendations for future work with the availability of more devices include more DBC studies, buffer optimization studies, and testing of the device performance with different AAV serotypes and load material. The prototype membrane device offers unique benefits over column resin for affinity chromatography in its fast flow rates, reusability, and potentially lower costs due to its “prepacked” design; however, the evaluations demonstrated the device does not provide significant enough process improvements that would favor its implementation in place of column resin for the tested material. ItemAssessing the Effectiveness of U.S. Financial Regulations: A Comparative Analysis with E.U. Responses(Johns Hopkins University, 2023-09-12) Fassas, Vassilios A.; Wolfson, Dorothea; Rosenthal, Alexander; Harris, DougThis paper assesses the effectiveness of US financial regulation in carrying out its intended purpose, namely, to adequately protect investors from industry abuse, insider advantages, and fraud. Reviewing recent financial crises, the role of the SEC, high profile Supreme Court cases, and legislation, such findings call into question the legitimacy of the financial system as a whole and is worrying due to Americans’ sheer reliance on banks and securities markets. Furthermore, this paper then compares the U.S. regulatory response with that of the E.U. as a result of the Global Financial Crisis and found that E.U. regulations are more clear, more potent, and more effective in handling and preventing financial crises. This paper uses statistical data, legislative analysis, and testimonial evidence to conclude that there are severe ways in which the US regulatory regime is lacking. Particularly, through vague laws that do not take proper measures to adequately protect against a future crisis, along with the evaluation of the capacity of the SEC to enforce the financial laws in question, US financial regulation does not effectively carry out its intended purpose. ItemPROJECTING LEADERSHIP: THE INTERPLAY OF PERSONALITY AND POLICY IN U.S. PRESIDENTIAL CAMPAIGNS(Johns Hopkins University, 2023-08-31) Broadus, Twais; Wolfson, Dorothea; Rosenthal, AlexandeThis study delves into the intricate nexus between personality traits in politics and their influence on electoral outcomes, specifically within the context of U.S. presidential elections from 2000 to 2020. The research utilizes a three-pronged approach: a content analysis of presidential debates to discern strategic trait-mentions, an examination of American National Election Study (ANES) data to capture voter evaluations of candidates based on specific traits, and an exploration of candidates' foreign policy emphasis, reflecting their projection of leadership. Key findings suggest Republicans consistently emphasize traits more than Democrats in debates, and there is noticeable variability in trait preference across partisan lines, particularly among Independents. A shift from domestic to foreign policy considerations was also observed over the years, indicating an electorate more cognizant of international influences on domestic policy. This change allows candidates to demonstrate their leadership skills through their stance on international matters. The culmination of these insights presents a comprehensive view, underscoring the dynamic interplay of candidate personality portrayals, voter preferences, and policy considerations in shaping electoral dynamics. The research emphasizes the imperative for political transparency and robust representation, aiming to demystify leadership roles and promote informed electoral choices for the greater societal benefit. ItemWHERE DO POLITICAL AND CORPORATE CAMPAIGNS CONVERGE AND DIVERGE?(Johns Hopkins University, 2023-08-16) Farrow, Monica R; Wolfson, Dorothea; Rosenthal, AlexanderAbstract This review identifies specific articles and literature that provide insights into the history of and correlations between current political and corporate campaign strategies. The findings suggest that corporations should and do adopt some of the more refined campaign techniques used by political entities and that scholarly attention should be expanded to include more extensive reviews of corporate requirements in this area. The overarching goal of this study is to: (1) convey the history of both corporate and political marketing strategies, (2) summarize where political and corporate marketing constructs diverge, and (3) demonstrate the increasing converging entanglement of corporate and political marketing campaign tactics. This examination particularly highlights the more advanced methodologies utilized by campaign teams to galvanize the passions of the masses. While those in the political arena have accumulated considerable experience with these methodologies, corporations have made less use of certain types, and have, thus, failed to reap the benefits of their advanced techniques. The strategic parallels these institutions have shared for promoting acceptance and growth deserve intense analysis and reflection to determine how the tactics peculiar to their structures can be optimized to promote the success of each individually and both collectively. From the British East India Company’s take on lobbying to the corporate marketing campaigns of Bud Light, Chick Fil A, Delta, and Target, corporations and political factions have assumed vital roles in the development and continuing existence of our institutions, both public and private. This review will consider how the evolution and impact of societal sways have both informed and molded campaign strategies in both sectors. As a result, this analysis will, hopefully, help fill the void of academic information that is available in this area. Additionally, this examination will identify the parallels that exist between how the government achieves support of the population on the one hand with how corporations reel in clients on the other, (e.g., lobbying, PAC fundraising, targeting, messaging, third-party activists, and storytelling). This study will then concurrently seek to address insufficient research as relates to the importance of polling a population to assess the impact of corporate campaign messaging. ItemUNDERSTANDING AND IMPROVING DIVERSITY IN CLINICAL RESEARCH FOR BLACK PATIENTS: A QUALITATIVE META-ANALYSIS STUDY(Johns Hopkins University, 2023-08-14) Yogi, Sara Aiko; Kantor, JeffreyBlack patients remain underrepresented in clinical research, representing 5% of patients enrolled United States clinical trials (Alegria et al., 2021). Lack of diversity in clinical research creates blind spots about the effectiveness of treatments in underrepresented populations, resulting in substandard downstream care. Through analysis of available literature on Black patient engagement in clinical trials, proposals can be made to increase trial enrollment and equity. A qualitative meta-analysis was conducted to interpret research completed to date on increasing clinical research diversity for Black patients. A PubMed literature search yielded 720 possible articles, 35 were used for meta-analysis. Each article underwent multiphasic review to identify research characteristics; generate enrollment promotors and barriers, identify themes; identify significant findings across multiple articles; identify under-investigation and contradictory findings; and review solutions applicable for future research. Enrollment promotors included thorough explanation and information provision on study design, possible downstream beneficiaries; strong/longitudinal researcher-participant relationships and researcher-participant racial concordance; higher income and socioeconomic status of participants; research institution accessibility; compensation and increased study funding; community outreach and personal contact; and personal connections to research for both participants and researchers. Enrollment barriers included low income/socioeconomic status, low education, unfeasible time commitments, lack of transportation; lack of trust in medicine and clinical research teams; inadequate information on clinical research; studies requiring medical record review, blood samples, novel pharmaceutical interventions, and invasive procedures; and studies with comorbidities as exclusion criteria. Themes in research conducted to date include cancer research, investigations into Black women, and historical, structural, and personal factors. Historical factors that affect enrollment for Black patients include mistrust of the medical establishment, research institutions, and researchers. Structural barriers include income, socioeconomic status, time commitment and lack of transportation as well as exclusionary study designs and lack of resources for better clinical research engagement. Personal factors showcase the need for nuanced engagement around personal connections to research subjects, and benefits and pitfalls of utilizing religious institutions as partners depending on the research study subject. Solutions from current literature included better research explanation, improving diversity of clinical research teams, increasing interactive instruction, and using unique methods of increasing community around clinical research. ItemPERCEIVED COMPETITION, JOB PERFORMANCE, AND SENSE OF BELONGING AMONG RESEARCH ADMINISTRATORS(Johns Hopkins University, 2023-08-22) Hammett, Lauren Nicole; Woods, Marianne R; Kantor, JeffreyResearch administrators are personnel in the highly collaborative and ever-changing research enterprise. The focus on competition as a means to promote productivity has the potential to work against a perception of belonging that can motivate non-task-specific job performance that benefits the organization. This study investigated the perspectives of research administrators regarding the level of competition amongst coworkers in their work environments in relation to their level of perceived insider status and contextual performance. A sample of 112 adult research administrators employed in the United States from Johns Hopkins University and an online platform for research administrators, NCURA’s Collaborate, was used for hypothesis testing. Data were collected through a self-report survey hosted in Qualtrics. The results of Pearson’s correlation analyses found a significant positive association between competition influenced by coworkers and contextual performance. The simple linear regression analysis of competition influenced by coworkers and contextual performance indicated a statistically significant positive relationship. ItemSURVIVAL AND GROWTH OF NONPROFIT ORGANIZATIONS: A MIXED METHODS STUDY ON SMALL, RESEARCH FOCUSED NONPROFITS IN WASHINGTON, DC.(Johns Hopkins University, 2023-08-11) Howard, Rebecca; Kantor, JeffreyNonprofit organizations (NPOs) are created to carry out a beneficent purpose. Unlike the private sector, nonprofit organizations are purpose-driven so that profit is not the primary goal. Nevertheless, nonprofits require financial resources to operate and carry out their mission. The more financial resources a nonprofit has at its disposal the more mission-oriented success it can achieve. Nonprofits fundraise from a variety of sources and methods, including individual donations, government contracts, corporate giving, and many other sources. Although an abundance of literature exists on nonprofit management and governance, there is little on fundraising management, revenue optimization, and the drivers of financial success for nonprofits. This is surprising given the wealth of research focused on growing revenue and income in the private sector. The goal of this study was to investigate small, research-focused nonprofits based in Washington D.C. that significantly increased revenue in a short time. Qualitative and quantitative data was utilized in combination with mixed methods. Financial data alone was not enough to understand how nonprofits with substantial increases in annual revenue were able to achieve such fundraising success. Semi-structured interviews with nonprofit leaders were conducted to learn about fundraising strategies and best practices for income growth. The mixed methods of this two-part study revealed several common strategies and best practices among small, research-focused nonprofits in Washington, D.C. However, it was also apparent that each nonprofit required an individualized approach to fundraising based on the unique needs and structure of the organization. ItemTHE ROLE OF PROJECT MANAGEMENT IN SPONSORED RESEARCH(Johns Hopkins University, 2023-08-09) Roig Vitali, Alexandra N; Qureshi, Saiqa AProject management provides guidance, direction, and tools for projects ranging in scope and complexity. The role of applying project management principles to sponsored research planning is a crucial factor in the success of most research, as well as an effective method to remain within the scope, schedule, and budget of the project. Project management techniques may be allusively applied to research as informal practice by the Principal Investigators (PI), rather than a Project Manager (PM). It is critical to understand that project management principles may not be applicable to all research ventures as the ambiguity and undefined goals surrounding some research are not conducive to the practices and procedures of project management. This study evaluates the role of project management in research through an in-depth literature review, as well as a survey questionnaire to the research community to evaluate to what extent is project management presently applied to research. The evidence gathered shows that project management is currently being applied by research professionals to a great extent for research projects leading to better results, and with better impact on timelines, and budgets. Consequently, researchers face a number of challenges such as administrative burdens, and lack of resources in staffing, capital, and technology. Two principal approaches to implementing a sound research management strategy are flexibility and an analysis of the requirements to identify the best approach. It is found that PIs are mostly taking on this decision, and ultimately driving the research planning, execution, and oversight, as well as conducting the research and evaluating results. PIs are overburdened, and applying project management principles may help alleviate project planning and execution. ItemTHE RELATIONSHIP BETWEEN NIH PANDEMIC FUNDING AND RESEARCH PRODUCTIVITY(Johns Hopkins University, 2023-08-03) Clark, Tyler Bradford; Kantor, JeffreyThe COVID-19 pandemic has had a lasting effect on all aspects of society and required a quick response to stem the spread of the virus and mitigate its effects. In its efforts to combat the pandemic, the United States federal government appropriated research funding to the National Institutes of Health (NIH) to research the disease, its effects, and possible treatments. The NIH allocated this funding to its constituent institutes according to its mission, goals, and responsibilities in relation to the pandemic. This thesis utilized data from the NIH RePORTER website to analyze the overall changes in NIH funding for fiscal years 2017-2022. Additionally, an analysis of publication data for the same years was conducted to find a measure of research productivity over the course of the pandemic. The two analyses were then used to calculate the cost per publication for the NIH and its 27 constituent institutes. This study found that while the pandemic funding did result in a shift in NIH appropriations to its institutes, the publication costs were only indicative of a trend in research productivity for the NIH overall. A trend in publication costs could only be identified for specific institutes based on the relation of their focus of study to the pandemic. ItemCHARACTERIZING THE STRUCTURE OF THE TYPE III-B CRISPR-CAS CMR COMPLEX USING CRYO-EM(Johns Hopkins University, 2023-08-25) Durecki, Brynn; Bailey, Scott; Kavran, JenniferCRISPR-Cas systems are adaptive immune systems found in prokaryotes. Since their discovery, many CRISPR-Cas systems have been characterized biochemically and structurally, and they are divided into two main classes based on their components. Class 1 CRISPR-Cas systems consist of multi-subunit effector complexes (Makarova et al., 2015). Within Class 1, Type III systems are among the most ancient and prevalent CRISPR-Cas complexes found in bacteria and archaea. Consisting of multiple protein subunits and a CRISPR RNA (crRNA), type III effectors specifically bind complementary target RNA, which activates both RNA and DNA cleavage activity to provide defense against invading nucleic acids (Hale et al., 2009; Samai et al., 2015). The subtype III-B complex structure has been solved in many organisms, but little is known about the structural and mechanistic details of type III-B CRISPR-Cas mediated immunity in the system Thermotoga maritima. Previous work in the lab has established the biochemical basis for RNA-activated DNA cleavage by a particular type III-B complex known as the Cas RAMP module (Cmr) in T. maritima, as well as the tolerance of mismatches between the crRNA and the target RNA to elicit this cleavage (Estrella et al., 2016; Johnson et al., 2019). The objective of this thesis was to characterize the structure of the Cmr complex using cryo-EM to validate this biochemical data. The complex was purified by affinity chromatography and analytical gel filtration, the samples were frozen at cryogenic temperature, the complex was imaged, and the resulting images were averaged and processed to obtain a 3D density map of the target unbound complex. We hoped to elucidate the precise catalytic mechanism of target RNA cleavage by the Cmr complex; however, due to low resolution, particle-limited maps and the formation of a sub-stoichiometric subcomplex, we were unable to see the molecular basis for the Cmr complex’s interference activity. Future experiments will focus on optimizing purification protocols and obtaining higher resolution target unbound and target bound structures to investigate specific interactions between the target RNA and key amino acid residues of the complex’s catalytic subunit, Cmr4. This work will not only provide the structural basis for CRISPR-Cas interference in an understudied organism, but it may also give insights that can inform the development of genetic engineering tools. ItemEXPLORATION OF CELL-BASED METHODS TO DISSECT LNCRNA-PROTEIN INTERACTIONS(Johns Hopkins University, 2023-08-23) Huang, Yuzhou; Hwang, Taeyoung; Leung, Anthony K. L.Long noncoding RNAs (lncRNAs) have been increasingly important in understanding regulations of gene expressions, thanks to the discovery of non-protein-coding genes and transcripts with the advent of high-throughput sequencing. It is known that most of the lncRNA functioning mechanisms involve interactions with RNA-binding proteins. Various in vitro experiments, such as in vitro transcribed RNA pulldown and electrophoretic mobility shift assay (EMSA), have been applied to study lncRNA-protein binding. However, the mechanisms of their interactions in the cell environment remain mysterious for most lncRNAs. Our goal is to explore cell-based methods to explore lncRNA-protein interactions. These include MPRNA-IP (Massively Parallel RNA Assay-Immunoprecipitation), a high throughput experiment developed by our lab, and CLIP-qPCR (Crosslinking Immunoprecipitation and qPCR) with RNA deletion mutants or RNase treatment. We focused on two lncRNA-protein interactions, cytosolic interaction NORAD-PUM2 and intranuclear interaction HOTAIR-EZH2. We detected a well-known PUM2-binding RNA sequence motif in NORAD, validating our methods for RNA-protein interactions. Regarding EZH2-HOTAIR interaction, our experiments showed that the EZH2 protein interacts with the 5’ end of lncRNA HOTAIR, as previous in vitro assays showed. But we unexpectedly identified that EZH2 also has a binding affinity with the middle region of HOTAIR while inside the cell nucleus. Taken together, our results present representative examples of applying multiple methods to study lncRNA-protein interactions inside the cells and provide new insights into the mechanism of lncRNA-protein interactions. ItemCharacterization of the ferric reductase gene family in Candida albicans(Johns Hopkins University, 2023-08-23) Zhao, Hongyu; Culotta, Valeria; Matunis, MichaelMetals are essential for life, serving as structural cofactors and catalysts in metabolic processes. Nutritional immunity is a host defense mechanism that capitalizes on the essentiality of metals to combat pathogens. Through co-evolution, fungal pathogens have developed strategies to endure metal-depleted conditions. The heme-containing transmembrane ferric reductase domain (FRD) plays a crucial role in maintaining metal homeostasis in pathogenic fungi by converting extracellular Fe+3 or Cu+2 to bioavailable Fe+2 or Cu+1 for cellular uptake. FRDs can also function as NADPH oxidases (NOX), producing reactive oxygen species (ROS) by reducing oxygen to superoxide. The opportunistic fungal pathogen Candida albicans possesses a family of 17 FRDs, of which only 4 have confirmed functions. The goal of this study was to characterize the role of the remaining FRDs and investigate their regulation. We predicted that reductases for Fe+3 and Cu+2 would be induced during metal starvation, and NOXs would be induced when abundant ROS is produced in the hyphal form of C. albicans. We grew wild-type C. albicans in the presence of iron or copper specific metal chelators and induced other cultures to make hyphae, and then analyzed expression of all 17 FRDs by qRT-PCR. We found that 8 FRD genes (FRE1, FRE2, FRE4, FRE5, FRE7, FRE9, FRE10, FRP1) were induced under Fe-starvation conditions, and 2 FRD genes (FRE7, FRE30) were induced under Cu-starvation conditions. 5 FRD genes (FRE2, FRE7, FRE8, FRE17, FRE30) were induced in hyphal cells. To further study our predictions on FRD genes, we conducted ferric reductase and cupric reductase assays on mutants in specific FRD genes and found that several of the genes we predicted to be cupric or ferric reductases showed either an increase or decrease in metalloreductase activity. Additionally, we found that Fe-responsive transcription factor SEF1 regulates the iron starvation induced expression of FRE1, FRE2, FRE4, FRE5, and FRP1. However, Fe-regulation iii of FRE7 and FRE9 occurs independent of SEF1. In addition to intracellular proteins like SEF1, certain unknown small molecule(s) secreted by C. albicans into the media were observed to influence FRD gene expression in response to Fe-starvation. Overall, our studies provide insight into the possible functions of several FRD genes and their regulatory factors. ItemZIC3 IS AN ESSENTIAL REGULATOR OF CONE PHOTORECEPTOR DEVELOPMENT(Johns Hopkins University, 2023-08-22) An, Roujin; Blackshaw, Seth; Selimyan, Roza13-line ground squirrel(13-LGS) is one of the rare diurnal rodents with cone-dominant retinas. A comprehensive large-scale single-cell comparative genomics study previously conducted in our lab revealed significant differences in cone development patterns between the 13-LGS and the rod-dominant mouse, characterized by an extended period of cone generation into the postnatal period in 13-LGS. A list of candidate genes was selected based on their differential expression patterns between 13-LGS and mice, among which Zic3 was our top candidate for further investigation. While Zic3 expression is strictly restricted to early-stage retinal progenitors (RPCs) in mice, it is also robustly expressed in late-stage RPCs and cone cells in 13-LGS, indicating a potential role for Zic3 in promoting cone photoreceptor specification and differentiation. To investigate this potential role of Zic3 in regulating cone photoreceptor development, Zic3 conditional knockout and overexpression models were generated, then analyzed with immunostaining and single-cell RNA sequencing. Selective disruption of Zic3 expression during early retinal development resulted in a reduction of cone cell density, while in vivo overexpression of Zic3 in late-stage RPCs that do not normally generate cones induced photoreceptor to translocate towards the apical retina, mirroring the normal location of cone photoreceptor nuclei. Co-electroporation of Zic3 and Pou2f1, which also promotes cone photoreceptor specification and shows expanded expression in developing 13-LGS retina, in ex vivo retinal explants resulted in a synergistic increase in generation of early-born retinal cell types, including cone photoreceptors, and suppression of rod-specific genes. Ex vivo overexpression of the transcription factor Onecut1, which also shows expanded expression in late-stage RPCs in 13-LGS, also strongly inhibited expression of rod-specific genes such as Nr2e3. These findings validated the role of transcription factor Zic3 in promoting cone specification during retinal development and potentially provided a new insight for the realization of directed induction of cone cell differentiation in stem cell therapy. ItemThe Effect of Poly(ADP-ribose) in Promoting Liquid-Liquid Phase Separation of C9ORF72 Arginine-Containing Dipeptide Repeats(Johns Hopkins University, 2023-08-04) Zhong, Hanyu; Leung, Anthony K.L.; Cai, DanfengArginine-rich dipeptide repeats (R-DPRs) translated from the C9ORF72 GGGGCC hexanucleotide repeat expansions (HRE) is one of the most important causes of disease progression of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). These R-DPRs exhibit toxicity to neuronal cells, leading to compromised mitochondria function, neuronal DNA damage, and stress granule formation. An RNA-like polymer poly(ADP-ribose) (PAR) can promote phase separation of R-DPRs and the formation of stress granules. However, how PAR facilitates the phase separation of R-DPRs remains unknown. To address this, we develop a liquid-liquid phase separation assay (LLPS) to assess the potency and chain-length dependency of PAR-induced droplet formation of R-DPR, specifically poly(GR). Additionally, we employ Electrophoretic Mobility Shift Assays (EMSAs) to investigate the length-dependent binding affinity of poly(GR) to PAR. Our findings reveal that the phase separation process of PAR with poly(GR) is both dependent on the stoichiometry ratio and poly(GR) length, consistent with the EMSA analyses. ItemEPIGENETIC CONTROL OF TOPOISOMERASE1 LESION REPAIR BY A MACRO-HISTONE(Johns Hopkins University, 2023-07-31) Wu, Tongyu; Oberdoerffer, Philipp; Bowman, GregorySingle-stranded DNA lesions (SSLs) are among the most common types of DNA damage. One predominant example for SSLs is Topoisomerase 1 cleavage complex (TOP1cc), which forms as a result of TOP1 activity in response to torsional DNA stress during DNA transactions such as transcription and DNA replication. TOP1ccs are frequently found at transcriptional start sites (TSSs) and can cause DNA damage and chromosome aberrations if not properly resolved. TOP1 inhibitors, such as camptothecin (CPT), are used as a pharmacological tool to trap TOP1cc on DNA, causing excessive DNA damage that kills cancer cells. What promotes efficient TOP1 function at DNA supercoil-forming regions while simultaneously preventing excessive TOP1cc accumulation remains unknown. Given that chromatin plays a central role in regulating the repair of double strand DNA breaks (DSBs), we propose that epigenetic control may similarly be a driver of TOP1cc repair. In previous work, we uncovered the macro-histone variant macroH2A1.1, but not its alternatively spliced macroH2A1.2 isoform, as an interactor of several TOP1cc repair factors. Here, we apply state-of-the-art genome-wide mapping approaches to investigate the relationship between macroH2A1.1 and TOP1 activity. We further relate macroH2A1.1 alternative splicing in cancer TOP1 inhibitor sensitivity across a panel of breast cancer cells with distinct macroH2A1.1 expression levels. Together, my findings point to a role for macroH2A1.1 as an epigenetic regulator of TOP1cc turnover at the TSS and a predictor of therapy responsiveness to TOP1 inhibition. ItemEPIGENETIC CONTROL OF TOPOISOMERASE1 LESION REPAIR BY A MACRO-HISTONE(Johns Hopkins University, 2023-07-31) Wu, Tongyu; Oberdoerffer, Philipp; Bowman, GregorySingle-stranded DNA lesions (SSLs) are among the most common types of DNA damage. One predominant example for SSLs is Topoisomerase 1 cleavage complex (TOP1cc), which forms as a result of TOP1 activity in response to torsional DNA stress during DNA transactions such as transcription and DNA replication. TOP1ccs are frequently found at transcriptional start sites (TSSs) and can cause DNA damage and chromosome aberrations if not properly resolved. TOP1 inhibitors, such as camptothecin (CPT), are used as a pharmacological tool to trap TOP1cc on DNA, causing excessive DNA damage that kills cancer cells. What promotes efficient TOP1 function at DNA supercoil-forming regions while simultaneously preventing excessive TOP1cc accumulation remains unknown. Given that chromatin plays a central role in regulating the repair of double strand DNA breaks (DSBs), we propose that epigenetic control may similarly be a driver of TOP1cc repair. In previous work, we uncovered the macro-histone variant macroH2A1.1, but not its alternatively spliced macroH2A1.2 isoform, as an interactor of several TOP1cc repair factors. Here, we apply state-of-the-art genome-wide mapping approaches to investigate the relationship between macroH2A1.1 and TOP1 activity. We further relate macroH2A1.1 alternative splicing in cancer TOP1 inhibitor sensitivity across a panel of breast cancer cells with distinct macroH2A1.1 expression levels. Together, my findings point to a role for macroH2A1.1 as an epigenetic regulator of TOP1cc turnover at the TSS and a predictor of therapy responsiveness to TOP1 inhibition. ItemEnhancing DataTrail Data Science Education: The BaltimoreTrails R Package and Shiny Web-App(Johns Hopkins University, 2023-08-08) Fuchs, Franklin Thomas Hans; Rosenblum, Michael; Colantuoni, ElizabethThis thesis presents BaltimoreTrails, a tool designed to enhance data science education within the DataTrail curriculum. By integrating Baltimore-specific datasets into an interactive Shiny Web-App, BaltimoreTrails provides unique data access and an interactive learning experience for students and educators. Additionally, the tool aims to foster a deeper understanding of data science concepts through interactive data exploration and visualization, thereby enhancing the overall learning experience. The development of this tool fills a knowledge gap by providing a software tool that enables the easy incorporation of many Baltimore-specific data sets into a graphical user interface to be used by students to support the teaching goals of the DataTrail program and adds directly to the curriculum by providing additional interactive tutorials and exercises on each DataTrail chapter. ItemCreation of a Next-Generation MANAFEST Assay: A Proof-of-Concept(Johns Hopkins University, 2023-06-30) Iocco, Domenica Kathryn; Smith, Kellie N.; Housseau, FranckT-cells recognize antigens by utilizing their T-Cell Receptors (TCRs), α/β heterodimers that bind to peptide:MHC complexes. The TCRβ chain is commonly used to track specific clonotypes because of its increased diversity due to the presence of the D gene, which is not present on the TCRα chain. Both the α and β chains are involved in antigen recognition and are necessary for the cloning of TCRs to be used in cell-based therapies, but the current processes for obtaining paired TCRs are time-consuming and expensive. MANAFEST, Mutation Associated NeoAntigen Functional Expansion of Specific T-cells, is an assay wherein peptide-stimulated T-cell culture is combined with bulk TCR sequencing to identify TCR Vβ CDR3 clonotypes. We aimed to optimize the current assay to create a next-generation MANAFEST that allows for antigen-specificity determination and the identification of paired TCRs in a single workflow. To develop this method, we utilized single cell sequencing coupled with the next-generation MANAFEST to both streamline the approach to obtaining paired TCRs and create a more-cost effective workflow. Our goal was to establish proof-of-concept for the development of a streamlined, high-throughput assay that is simultaneously able to identify antigen-specific T-cells and their corresponding TCRs, and we hypothesized that utilizing a single cell transcriptomic panel and TCR sequencing on peptide-stimulated cells would allow us to achieve this goal. We approached this hypothesis with a ten-day cell culture and peptide stimulation followed by single cell analysis and TCR sequencing on a high throughput platform that examined a panel of about 1,000 immune-related genes. We expected that we would be able to discover tumor specific TCRs by identifying the upregulation of genes associated with an activated T-cell signature. Indeed, our bioinformatics analysis revealed an upregulation of genes we anticipated to see, such as IFNγ, CCL3, and GZMB, and we were successfully able to identify the TCRs of the cells expressing these upregulated genes. In doing so, we addressed our central hypothesis—that utilizing a targeted, single cell transcriptomic panel coupled with TCR sequencing on peptide-stimulated T-cells can allow us to simultaneously identify both tumor reactive T-cells and their paired TCRs in a single workflow. ItemFINGERPRINTING OF WALKING USING DATA FROM WRIST AND ANKLE-WORN ACCELEROMETERS(Johns Hopkins University, 2023-06-16) Zhang, Yan; Crainiceanu, Ciprian; Zipunnikov, VadimIdentifying an individual from accelerometry data collected during walking without reliance on step-cycle detection has not been achieved with high accuracy. Therefore, we propose an open-source reproducible method to: (1) create a unique, person-specific “walking fingerprint” from a sample of un-landmarked high-resolution data collected by wrist and ankle-worn accelerometers; and (2) predict who an individual is from their walking fingerprint. Accelerometry data were collected during walking from 32 individuals (19 females) aged from 23 to 52 years old for at least 380 s each. For this study’s purpose, data are not landmarked, nor synchronized. Individual walking fingerprints were created by: (1) partitioning the accelerometer time series in adjacent, non-overlapping one-second intervals; (2) transforming all one-second interval data for a given individual into a three-dimensional (3D) image obtained by plotting each one-second interval time series by the lagged time series for a series of lags; (3) partitioning these resulting participant-specific 3D images into a grid of cells; and (4) identifying the combinations of grid cells (areas in the 3D image) that best predict the individual. For every participant, the first 200 s of data were used as training and the last 180 s as testing. This approach does not segment individual strides but instead walking, resulting in reduced dependence on complementary algorithms and increasing its generalizability. This method correctly identified 100% of the participants in testing data for left-wrist worn accelerometry  and highlighted unique features of walking that characterize the individuals. This is significant as predicting the identity of an individual from their walking patterns has immediate implications that can complement or replace those of actual fingerprinting, voice, and image recognition. Furthermore, as walking may change with age or disease burden, individual walking fingerprints may be used as biomarkers of change in health status with potential clinical and epidemiologic implications. ItemMethod Validation for Measurement of Hair Nicotine Levels in Adolescents with Congenital Heart Disease (CHD)(Johns Hopkins University, 2023-05-03) Fang, Xin Tong; Rule, Ana; Latshaw, Megan; Fox, MaryThe use of e-cigarettes (i.e., vaping) among adolescents in the United States has recently doubled, as reported by SAMHSA in 2020. Adolescents who have survived congenital heart disease (CHD) are at an elevated risk to the harmful effects of e-cigarettes. When CHD survivors reach 12 years of age, they begin the process of transitioning care from a pediatric to an adult cardiologist which entails them assuming more responsibility for their health care. In the US, 1/3 of those with CHD discontinue cardiology care during the transition period, which is the time where they are exposed by peer pressure to start using e-cigarettes (Jackson et al., 2018). Therefore, there is a need to assess exposure to tobacco for adolescents with CHD in order to develop and implement prevention strategies. Hair nicotine concentration has been increasingly used as a biomarker for tobacco use and second-hand exposure due to the ease of collection and storage. Among chromatographic methods, hair analyses have been commonly performed with liquid chromatography–tandem mass spectrometry (LC–MS/MS) or GC/MS, due to their sensitivity and ability to specifically detect nicotine (Kim et al., 2008). Few studies, however, have examined an isotope dilution method for measuring hair nicotine in GC/MS. The aim of this study was to validate the isotope dilution GC/MS method for measuring nicotine in hair samples obtained from adolescents who have survived congenital heart disease. There are several ways to assess an individual’s exposure to nicotine, including collection of saliva, urine, and hair samples. This study found that GC/MS-based isotope dilution method is a valid approach for determining nicotine in hair samples. The validation process involved assessing linearity, precision, limit of detection, and recovery. Using calibration standards, linearity was strong amongst all batches (R2 > 0.99). The average of the limit of detection was 0.144, which was comparable to previous studies. The precision and recovery values were also within the acceptable range. Additionally, the correlation between primary and duplicate hair sample analyses indicated good reproducibility and consistency. The results contribute to the growing body of evidence supporting the use of hair nicotine as a reliable and non-invasive biomarker for assessing nicotine exposure in research and clinical settings.