UT Southwestern Graduate School of Biomedical Sciences

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Welcome to the UT Southwestern Graduate School of Biomedical Sciences’ electronic theses and dissertations (ETD) collection.

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Print theses and dissertations from 1943 to 2004 are located in the Library's Special Collections and Archives (Room E3.314) and are available by appointment. (Note: Former students may request a digitized copy of their work by email, but other users may submit an Interlibrary Loan request.) For more information, contact archives@utsouthwestern.edu.

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Branched Chain Amino Acid Metabolism in Development and Cancer
(2022-08) Patrick, McKenzie Taylor; Agathocleous, Michalis; DeBerardinis, Ralph J.; Chung, Stephen S.; Xu, Jian
Dysregulation of branched chain amino acids (BCAAs) and/or the enzymes catalyzing the first step in their catabolism, branched chain amino acid transaminase (BCAT) 1 or 2, are associated with a multitude of diseases. Despite their importance in physiology and pathobiology, in vivo studies examining the roles of BCAT1 and BCAT2 in normal development and cancer are critically lacking. We generated new BCAT1 and/or BCAT2 constitutive or conditional knockout (KO) mice, as well as BCAT1 constitutive or conditional knockin overexpression (OE) mice, and systematically examined the impacts of BCAT1 and/or BCAT2 KO or BCAT1 OE on normal development, hematopoiesis, and leukemia initiation and maintenance in vivo. First, we discovered that loss of BCAT2 but not BCAT1 leads to accumulation of BCAAs and BCKAs, causing morbidity and mortality that can be ameliorated by dietary BCAA restriction. Through proximity labeling proteomics, enzymatic and isotope tracing assays, we provide evidence for the formation of a mitochondrial BCAA metabolon involving BCAT2 and BCKDH, the second enzyme catalyzing the rate-limiting step of BCAA catabolism. Disabling the metabolon contributes to BCAT2-deficiency-induced phenotypes, which can be reversed by BCAT1-mediated BCKA reamination. These findings establish a role for metabolon formation in BCAA metabolism in vivo, and suggest a new strategy to modulate this pathway in diseases involving dysfunctional BCAA metabolism. Next, we determined that BCAT1 is dispensable for normal hematopoiesis and development, but BCAT1 OE increases hematopoietic stem cell frequency and function. BCAT1 expression is significantly increased in human primary AML samples containing FLT3 and AML1-ETO mutations but decreased in AML with MLL abnormalities, raising a fundamental question about how distinct oncogenic drivers create selective metabolic dependencies. Using retroviral and genetic murine leukemia models, we discovered that BCAT1 is required for FLT3-ITD- and AE9a-induced leukemia development and FLT3-ITD leukemia maintenance, whereas BCAT1 OE accelerated FLT3-ITD leukemia progression. In contrast, modulating BCAT1 expression in MLL-AF9 had no effect on leukemia development or maintenance. Our findings establish BCAT1 and BCAA as a metabolic dependency of AML-initiating cells in vivo and uncover new insights into onco-genotype-specific metabolic rewiring as a selective and targetable vulnerability.
Investigating Atypical Signaling of N-Methyl-D-Aspartate Receptors
(2022-08) Altamirano, Robert M.; Hibbs, Ryan E.; Monteggia, Lisa; Kavalali, Ege T.; Bezprozvanny, Ilya; Konopka, Genevieve
N-methyl-D-aspartate (NMDA) receptors are ionotropic ligand-gated channels that require ligand binding jointly with membrane depolarization to become active. This unique characteristic of the NMDA receptor coupled with previously published data suggests they may signal in a metabotropic fashion i.e., ligand binding triggers downstream conformational changes and signaling events independent of ion influx. It has been shown that long-term depression (LTD) can still be maintained in hippocampal slices treated with the open channel blocker MK-801 while the ligand binding site antagonist D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5) blocks LTD. However, conflicting data was published suggesting MK-801 does not block LTD warranting further study. My work aimed to answer the following question: can NMDA receptors signal in a metabotropic fashion? First, live cell imaging in primary hippocampal cultures infected with GCaMP6s or GCaMP6s fused to postsynaptic density protein 95 (PSD95-GCaMP6s) were used to measure NMDA receptor dependent calcium signaling events. Next, phosphorylation levels of proteins that had been implicated in a proposed metabotropic signaling cascade were measured to determine the plausibility of this model and subsequent metabotropic NMDA receptor signaling. Lastly, extracellular field recordings were conducted to replicate and advance previously published data that originally suggested NMDA receptors signal in a metabotropic fashion. D-AP5 and MK-801 reduced NMDA receptor dependent calcium signaling events similarly in both GCaMP6s and PSD95-GCaMP6s infected cultures indicating ionotropic NMDA receptor signaling. The biochemical screen was unable to substantiate the previously published model of metabotropic signaling but did further support my calcium imaging data whereby MK-801 can decrease postsynaptic calcium influx within 20 minutes. In our system, metabotropic NMDA receptor signaling is merely an artifact from the confounding effects of picrotoxin and a higher concertation of MK-801. By using a more physiologically relevant experimental design I showed MK-801 is a less effective blocker of LTD and requires pretreatment while D-AP5 can block this form of plasticity in as little as 15-minutes. The data from my project helps advance our understanding of downstream postsynaptic excitatory signaling and could assist with the development of therapeutics that target the NMDA receptor.
Systematic Design, Synthesis, and Evaluation of Ionizable Lipids for Lipid Nanoparticles Delivering mRNA
(2022-08) Lee, Sang Min; Tambar, Uttam; Corey, David R.; Qin, Tian; Siegwart, Daniel J.
Nucleic acids offer immense potential to act as targeted therapeutics and overcome limitations of traditional small molecule drugs. Diseases that are conventionally deemed untreatable and "untargetable" by small molecules are being addressed by alternative approaches, including nucleic acid therapeutics. However, to enable the feasibility of nucleic acid therapeutics, the challenge of delivery must be answered. To deliver RNA, both extracellular and intracellular barriers must be overcome. Due to the immune system's ability to safeguard against foreign materials and RNA's propensity for degradation, the delivery of RNA requires a carrier to protect it. Lipid nanoparticles (LNPs) have been developed to provide a modality enabling delivery of genetic cargos and represent the most promising vehicle to date for its reproducibility, safety, and efficacy. The works within this dissertation provide an insight into how chemical structures of ionizable amino lipids can control the functional mRNA delivery. Ionizable amino lipids were synthesized to study the fundamental structure-activity relationship (SAR) between the lipids and the in vitro and in vivo delivery of mRNA. Identification of potent ionizable amino lipids led to analysis of its respective physiochemical properties as it relates to in vivo LNP potency, revealing a range of physiochemical properties that expanded beyond the previously established ranges. Additionally, a systematic design, synthesis, and evaluation of ionizable amino lipids containing unsaturated motifs was conducted, inspired by the importance of unsaturated lipids in nature. These studies revealed that increased number of unsaturation does not necessarily result in improved mRNA-LNP potency. The inclusion of unsaturated motifs derived from natural products were most successful. Mechanistic studies highlighted how unsaturation can improve fusogenicity and affect cellular trafficking of LNPs. Combining unsaturated lipids with saturated lipids resulted in the best performing LNP formulations, suggesting there needs to be a fine balance of both components for successful mRNA delivery. These studies reiterate the importance of the design of chemical structure of the ionizable lipid used in LNPs, but also highlights the complexity of delivering mRNA.
Regulation of Regeneration and Clonal Fitness in Normal and Diseased Liver
(2022-08) Jia, Yuemeng; Mendell, Joshua T.; Buszczak, Michael; Hon, Gary C.; Zhu, Hao
The potent regenerative capabilities of planaria, newts, and zebrafish are largely absent in mammals. In humans, impaired regeneration contributes to poor outcomes after acute trauma, tissue damage, or organ transplant, as well as in chronic diseases such as liver cirrhosis, inflammatory bowel disease, and diabetes. A central goal of my thesis is to identify unexpected pathways in tissue repair by establishing and exploiting in vivo forward genetic screening approaches, and two key questions were tackled with these platforms. The first goal was to identify druggable chromatin regulators of regeneration. To facilitate the discovery process, direct in vivo CRISPR knockout and CRISPR activation screening platforms in mouse livers were developed. Among 164 epigenetic regulators, both gain- and loss-of-function screens identified imitation-SWI (ISWI) chromatin remodeling components BAZ2A and BAZ2B. In vivo sgRNA, siRNA, and knockout mouse experiments against either paralog confirmed increased liver regeneration. Two distinct BAZ2 bromodomain inhibitors GSK2801 and BAZ2-ICR also resulted in accelerated liver healing after diverse injuries. Mechanistically, BAZ2 inhibition resulted in an increase in ribosomal biogenesis and protein synthesis by directly regulating the expressions of ribosomal proteins, resulting in an expanded reservoir of ribosomes, which allowed a more rapid cell cycle entry. The second part of the thesis was to understand the functional impact of somatic mutations on tissue regeneration in the context of tissue damage. Through exome and ultra-deep sequencing of 82 human cirrhotic liver samples, functional mutations in PKD1, KMT2D, and ARID1A were identified. Heterozygous deletion of these genes in mice promoted regeneration after liver injuries. The key concept from this work is that chronic injury, and perhaps aging, can select for mutations that promote adaptive or regenerative phenotypes rather than carcinogenesis. Together, these discoveries have established new methods for gene discovery in mammalian tissues, identified new targets for regenerative medicine, and uncovered adaptive mechanisms in chronic tissue injury that do not necessarily drive cancer.
Molecular Basis of Coupling Calcium Sensing to Fast Membrane Fusion by Synaptotagmin-1 in Neurotransmitter Release
(2024-08) Jaczynska, Klaudia Marta; Parker, Matthew W.; Tomchick, Diana R.; Wang, Weiwei; Rizo-Rey, José
Neuronal communication depends on the rapid release of neurotransmitters through Ca2+-triggered synaptic vesicle exocytosis. Synaptotagmin-1 (Syt1) acts as the Ca2+ sensor for fast, synchronous neurotransmitter release. Despite decades of research, the precise molecular mechanism of Syt1 action and the coupling of Ca2+ sensing to membrane fusion remains elusive. Addressing these questions requires an understanding of the cooperation between Syt1 and the SNARE complex, which drives membrane fusion. The SNARE proteins anchored on opposing membranes bring them together by forming a tight four-helix bundle, a process referred to as zippering. A recent molecular model of SNARE-mediated membrane fusion has revealed that the zippering of the SNARE motifs into the juxta-membrane linkers, which catalyzes encounters of acyl chains from opposing bilayers, is the key event leading to membrane fusion. The work presented in this thesis provides a thorough analysis of the interactions between Syt1 and the SNARE complex in solution and on membranes. Leveraging NMR and fluorescence spectroscopy, the included studies systematize the often-contradictory data and present a comprehensive model illustrating how Ca2+ signaling is coupled to SNARE-mediated fusion. In our model, in the primed state of synaptic vesicles Syt1 binds the SNARE complex through a primary interface and the plasma membrane through a polybasic region. In the absence of Ca2+, Syt1 both enables the formation of a partially assembled SNARE complex yet strongly inhibits complete helical zippering and hence membrane fusion. Upon Ca2+ influx, Ca2+ binding by Syt1 induces its membrane insertion, leading to reorientation of the Syt1-SNARE complex on the plasma membrane. The reorientation generates a lever-like action of Syt1 that pulls the SNARE complex, facilitating the zippering of the linkers that induces fast membrane fusion and subsequent neurotransmitter release.
Investigating the Role of Complex I Loss in Thyroid Tumorigenesis
(2024-08) Li, Vicky; Tu, Benjamin; DeBerardinis, Ralph J.; Liszczak, Glen; Pan, Duojia
Changes in cellular metabolism are considered one of the hallmarks of cancer. In 1924, Otto Warburg initially posited that defects in cellular respiration drive tumorigenesis after observing that tumor cells often perform aerobic glycolysis in culture. Since then, multiple studies have demonstrated that most tumors actually require intact cellular respiration for proliferation. However, cancers of the colon, kidney, and thyroid are enriched in loss-of-function mutations predicted to negatively impact respiration. A recent study has shown that a cohort of tall cell variant-papillary thyroid cancer (TCV-PTC) tumors demonstrate widespread loss of Complex I of the electron transport chain. TCV-PTC is a clinically aggressive form of papillary thyroid cancer, driven universally by the BRAF V600E activating mutation. I have developed a murine model of Braf-driven thyroid cancer to investigate the role of Complex I loss in both tumor initiation and progression.
Cooperativity: Physiologic Cornerstone and Emerging Therapeutic Design Principle
(2022-08) Wilhelm, Jonathan Louis; Sumer, Baran; Gao, Jinming; Li, Bo; Lea, Jayanthi; Farrar, J. David
Precise spatiotemporal control of molecular transport is vital to functional physiological systems. Nature evolved to apply macromolecular cooperativity to achieve precision over systemic delivery of important molecules. In drug delivery, conventional nanocarriers employ inert materials and rely on passive accumulation for tissue targeting and diffusion for drug release. Early clinical studies show these nanodrugs have not delivered the anticipated impact on therapy. Inspired by nature, this dissertation explores a design principle that incorporates nanoscale cooperativity and phase transition to sense and amplify physiological signals to improve the therapeutic outcome. Using ultra-pH-sensitive (UPS) nanoparticles as an example, I will first demonstrate how all-or-nothing protonation cooperativity during micelle assembly/disassembly can be exploited to increase dose accumulation and achieve rapid drug release in acidic microenvironments. In ongoing development, I will then present three facets of investigation into a UPS-based nanovaccine platform using the polymer PC7A for cancer immunotherapy. The first line of inquiry involves a systematic investigation of physicochemical properties of the nanovaccine with a detailed characterization of the biophysical relationship between PC7A nanoparticles and peptide-based antigens. This work identifies dual-cooperative phase transition of the micelle and peptide, both of which are necessary for downstream vaccine efficacy. The second line of inquiry comprises the development of a biodegradable series of UPS nanoparticles. This work culminates with the investigation of a biodegradable nanovaccine and its improved safety profiles compared with the nondegradable predecessor. Finally, the third line of inquiry explores the roles of myeloid cells in governing the T cell responses following vaccination by PC7A and subsequent therapeutic resistance. This work identifies a population of macrophages which interacts with and suppresses T cell activity, leading to therapeutic resistance, and culminates with the development of combinatorial therapies to overcome resistance. Together, these bodies of work cooperate to enable optimized nanoparticle vaccination strategies through the improvement of biophysical stability, physiologic safety profiles, and long-term efficacy in late-stage tumors.
Development and Validation of a New Naming Task: The Southwestern Item Fluency Test (SWIFT)
(2022-08) Presley, Chase Reid; Cullum, C. Munro; Hart, John, Jr.; Lacritz, Laura H.; Rossetti, Heidi; Jeon-Slaughter, Haekyung
Tests of confrontation naming play an important role in the clinical assessment of language. Commonly used naming tests have a variety of limitations including ceiling effects, outdated stimuli, differential item functioning across groups, and often ignore speed of word retrieval even though this is a prominent component of word-finding difficulties (WFD). The aim of the current study was to develop and validate a brief, psychometrically sound naming task that is sensitive to WFD. One-hundred items were selected from the Bank of Standardized Stimuli (BOSS) (Brodeur et al., 2014), an open-source collection of high-definition photographs, to create the Southwestern Item Fluency Test (SWIFT). All items were normed on visual characteristics and selected items were required to (1) be easily recognizable, (2) represent a range of semantic categories, (3) be high in object-name agreement, and (4) span a range of difficulty. Phase I (Test Development) -Phase I of the study involved development and analysis of the SWIFT, where it was hypothesized that (1) confrontation naming item difficulty could be predicted by psycholinguistic characteristics, and (2) using the most predictive psycholinguistic characteristics to rank SWIFT items would result in four stimulus pages with similar visual and psycholinguistic characteristics. Psycholinguistic predictors of Age of Acquisition and Lexical Frequency were found to best predict item difficulty based on previously published difficulty indices on the Boston Naming Test (BNT). SWIFT items were ranked by predicted difficulty and divided into 4 groups of 25 items that contained an equal distribution of semantic categories and level of difficulty. ANOVAs comparing the SWIFT pages on visual and psycholinguistic characteristics found no statistical differences, showing strong initial psychometric support for the SWIFT development. As a result, four SWIFT stimulus pages were created, each containing 25 items presented in 5x5 rows. Administration of the SWIFT consists of a timed portion, where participants have 30 seconds per page to name as many objects as quickly as possible, and an untimed portion that ensures each item is administered. Phase II explored the utility of the SWIFT in a clinical sample (N=53). The SWIFT was hypothesized to demonstrate: (1) a single underlying "naming ability" factor, (2) adequate internal consistency (≥.70), (3) convergent validity with other tests of word retrieval, (4) acceptable discriminant classification of individuals with impaired word-finding ability, (5) greater sensitivity to Clinician-Rated WFD (CRWFD) compared to the Boston Naming Test (BNT). Statistical Analyses included (1) Principal Components Analysis (PCA), (2) Cronbach's alpha, (3) Spearman's rho Coefficient Correlations, and (4) Receiver Operating Characteristic (ROC) curve analysis with Area Under the Curve (AUC). Patients had a variety of neurological, general medical, and psychiatric diagnoses. The PCA demonstrated that a single component best explained the SWIFT variance with very high internal consistency (Cronbach's alpha = 0.976). Both SWIFT total scores demonstrated convergent validity with word-retrieval tests, specifically the BNT, and divergent validity with visuospatial tests. It was more closely associated with speeded tasks (WAIS-IV Coding) and tests of executive functions (TMT Part A and B) compared with the BNT. Both SWIFT total scores were successful in classifying individuals with objective impairments on tasks of word-retrieval (AUC = 0.834 - 0.835) and were categorically better than the BNT (AUC = 0.646) at classifying individuals with CRWFD (AUC = 0.707 - 0.724). The SWIFT appears to be a promising new naming tool with good sensitivity to WFD in clinical samples and may prove valuable to clinicians when speed of naming is of particular interest in the assessment of naming abilities.
Group Intervention for Resiliency and Posttraumatic Growth Following Sexual Trauma in Women Veterans
(2022-08) Mckenzie, Victoria Denise; Shivakumar, Geetha; Foxwell, Aleksandra; Jeon-Slaughter, Haekyung; Pai, Anushka; Anderson, Elizabeth Hallen
Women service members have higher rates of childhood sexual trauma in comparison to civilian women and almost 50 percent of women reported joining the military to escape stressful home environments. Women Veterans also experience a myriad of traumatic stressors while in the military, including high rates of Military Sexual Trauma (MST), combat exposure, deployments, and/or perceived personal danger which can increase the risk of developing Posttraumatic Stress Disorder (PTSD). Research with Veteran populations demonstrates that individuals with a history of trauma who experience psychological distress are more likely to be exposed to future trauma and that multiple traumatic events across the lifespan can have an aggregate negative impact on well-being, particularly in the post-deployment adjustment stage, often depleting them of important coping resources. Furthermore, women Veterans with combat exposure and/or MST experience PTSD differently than civilian women or military men, and therefore may require tailored and integrative treatments. The current evidenced-based treatments offered at the VA do not necessarily target aspects of resiliency. Studies have shown that interventions that are based in resiliency may reduce susceptibility to Posttraumatic symptoms, depression and suicide, and mediate the development of mental illness following trauma exposure. Interventions that focus on resiliency and posttraumatic growth (PTG) may help decrease symptom presentation, increase quality of life, and reduce the utilization and/or cost of care. This pilot project developed and implemented a resiliency group intervention manual and client workbook specifically tailored to women Veterans with histories of military sexual trauma in a clinical treatment setting. We evaluated feasibility and explored the impact of the psychoeducation group as it relates to overall knowledge and utility of resiliency skills. Outcome measures of enrollment rate, completion rate, drop-out rate and a knowledge/satisfaction questionnaire showed that the resiliency skill building psychoeducation group was feasible and acceptable. Data also showed that that the intervention positively increased scores related to resilience and posttraumatic growth, reduced clinical symptoms as assessed by the PCL-5 and PHQ-9, and improved quality of life in women Veterans that have experienced MST.
Examining Moral Injury in Legal Involved Veterans
(2022-08) Martin, William Blake; LePage, James; Holliday, Ryan Phillip; Hughes, Jennifer L.; Jeon-Slaughter, Haekyung; Holder, Nicholas Davis
BACKGROUND: Legal-involved Veterans (LIV) currently comprise approximately 8% of the incarcerated U.S. population, with over 180,000 veterans estimated to be in prisons or jails and a far greater number currently on parole or probation. Furthermore, LIV appear to experience heightened risk for negative sequelae such as homelessness, suicide, unemployment and psychiatric diagnoses. While various factors may contribute to these negative outcomes, the potential for exposure to potentially morally injurious experiences (PMIEs) during legal involvement and the resulting expression of moral injury as contributors have not yet been examined in this population. As a result, we adapted measures of moral injury to assess moral injury in the legal context. HYPOTHESES: 1. LIV will report exposure to PMIEs and moral injury during their legal involvement. 2. The adapted moral injury measures will demonstrate adequate psychometric strength. 3. Exposure to PMIEs and the expression of moral injury will be associated with suicidal ideation and suicide attempts. METHOD: Study participants (N=100) were recruited from the VA North Texas Health Care System. Participants were adults (age 18+) who are veterans of the U.S. armed forces and have at least one felony conviction resulting in incarceration. Patients were administered a battery of measures including the Moral Injury Events Scale (MIES) the Expressions of Moral Injury Scale (EMIS) as well as adapted versions of each measure to assess legal-related moral injury (Moral Injury Events Scale-Legal Involved Persons [MIES-LIP]; Expressions of Moral Injury Scale-Legal Involved Persons [EMIS-LIP]). Measures also included assessments of depressive symptoms (PHQ-9), PTSD symptoms (PCL-5) and suicidality (SITBI-R). We examined the psychometric properties of the adapted measures by evaluating their reliability, validity, and factor structure. We utilized multinomial logistic regression to evaluate the relationship between moral injury and suicide risk. RESULTS: The vast majority of our sample endorsed witnessing a potentially morally injurious event while in involved with the legal system and in the military. Furthermore, the majority of our sample endorsed engaging in morally injurious behavior in the legal context and in the military. Both adapted scales of moral injury (MIES-LIP & EMIS-LIP) demonstrated psychometric strength as evidence by good reliability, as well as convergent and discriminant validity with other study measures. Furthermore, the EMIS-LIP scale significantly differentiated those with a prior suicide attempt from those with no prior suicidal ideation. DISCUSSION: Legal-related moral injury appears to be a salient factor affecting many legal-involved veterans, and our adapted measures of moral injury demonstrated good reliability and validity. Furthermore, legal-related moral injury as measured by the EMIS-LIP was significantly associated with lifetime suicide attempt in LIV. Future studies should examine drivers of legal-related moral injury in LIV, which may inform future assessment and treatment.
Feasibility and Acceptability of Utilizing a Single Session Problem-Solving Intervention with Caregivers of Pediatric Patients Receiving Chronic Transfusion to Treat Sickle Cell Disease
(2022-08) Burchette, Dennis Delaine, Jr.; Oppenheim, Jenna N.; Germann, Julie; Robbins, Mona; Nero, Alecia; Nakonezny, Paul
INTRODUCTION: For patients and caregivers of patients receiving chronic red blood cell transfusions (CT) to treat sickle cell disease (SCD), few studies have investigated the use of single session interventions, and none to address problem-solving in a single session. Feasibility and acceptability of such an intervention with caregivers of pediatric patients who receive CT to treat SCD were investigated. Efficacy was also explored. METHOD: Participants were twenty caregivers (95% female; 95% Black/African American, Mage = 42.45 years) and patients (55% male; 95% Black/African American, Mage = 13.55 years) who were approached either during their CT appointment or via a HIPAA-approved telehealth platform while at home. Participants in the intervention group received a single session problem-solving intervention (SSPSI) during their second visit, and all participants completed self-report measures during each of the four research visits. RESULTS: Data indicate 54% of participants approached agreed to participate in the study and 80% of participants who enrolled were retained through the end of the study. Regarding acceptability, 100% of participant pairs in the intervention group found the intervention to be acceptable at each visit of the study. DISCUSSION: Caregivers and patients were able to utilize this patient and family-centered, culturally sensitive SSPSI to identify strategies to address problems they were experiencing. The study demonstrated feasibility and acceptability of the SSPSI. Further investigation is needed to further investigate efficacy of such an intervention with this population. Utilizing a SSPSI with caregivers and patients who receive CT to treat SCD appears to be feasible and acceptable, within the specific setting investigated, in addressing problem-solving with this patient population.
Exploring the Experiences of Caregivers of Black Autistic Children: Barriers and Underrepresentation in Care
(2022-08) Baker, Eva Catherine; Bellone, Katherine; Moore, Jessica; Selders, Michael; Galusha, Jeanine M.; Caze, Todd, II
Approximately 1 in 54 children are diagnosed with autism spectrum disorder (ASD). Research suggests Black children are more likely to be diagnosed later and with more severe ASD than white children and Black families report being more dissatisfied with their child's autism-related care even when adjusting for socioeconomic status. Having an autistic child increases parental stress, and while studies have found support groups to be a helpful for this population, these studies tend be composed of exclusively white participants. To our knowledge, only one other study has examined the experiences of Black families who have an autistic child. Understanding the general experiences of these caregivers, with a special emphasis on coping with the stigma in the Black community is an important area of focus to increase the utilization and satisfaction of their care. Study aims were to: 1) explore the experiences of caregivers of Black autistic children related to obtaining the diagnosis for their child, participating in care at the Center for Autism and Developmental Disabilities (CADD), and coping with the diagnosis to improve future services and 2) understand the barriers to accessing care at CADD to address the underrepresentation of Black children receiving psychological services in order to improve awareness of barriers and develop a plan to mitigate this by improving access to care. Participants included 12 caregivers of Black autistic children. Qualitative analyses were guided by constructivist grounded theory and utilized the constant comparative approach. Guided by study aims, qualitative analysis generated four primary sections: Section I: Pre-Diagnosis Experiences; Section II: Post-Diagnosis Experiences; Section III: Autism in the Black Community; and Section IV: Center for Autism and Developmental Disabilities. Findings describe the experience of caregivers and further explore how their child's race has impacted their experiences. Results suggest caregivers find support in sharing their experiences and problems with their immediate family, with friends, particularly those who also have children with disabilities, and by learning and sharing information about ASD with loved ones. Study results also revealed barriers to treatment, identified treatment facilitators, and include recommendations for program improvement, specifically to increase access and utilization of care.
Prevention of Alzheimer's Disease Through NHE6 Depletion
(2022-08) Wong, Connie Hua; Kitamura, Takashi; Bezprozvanny, Ilya; Douglas, Peter; Moe, Orson W.; Herz, Joachim
Currently, 1 in 9 people over the age of 65 are living with Alzheimer's disease (AD). The highest genetic risk factor for AD is Apolipoprotein E isoform E4 (ApoE4), which accounts for 40-65% of AD patients. AD is characterized by brain atrophy, synaptic dysfunction, and the accumulation of amyloid and tau proteins. These AD phenotypes are exacerbated in the presence of ApoE4, yet the molecular mechanism remains unknown. To develop therapies for AD, it is of the utmost importance to understand the basic molecular and cellular mechanism by which ApoE4 accelerates AD onset and progression. We have previously identified that ApoE4 halts early endosomal recycling, which subsequently causes synaptic dysfunction and impaired learning and memory. We propose a novel approach for rescuing the ApoE4-mediated impairment in endosomal recycling and synaptic dysfunction by lowering the pH of the early endosomes through inhibition or depletion of the early endosomal proton leak channel Na+/H+ Exchanger 6 (NHE6). The long-term goal of our research is to develop a treatment strategy for ApoE4 carriers during the 10- to 15-year period preceding the onset of clinical symptoms. During this period, patients with preclinical AD start accumulating Abeta plaques in the neocortex. The first aim of my thesis project was to investigate how NHE6 depletion affects Abeta pathology. The second aim was to evaluate any untoward side effects such as neuroinflammation or neuronal loss caused by the complete genetic removal of NHE6. For the last portion of my thesis project, I investigated a translational approach to target NHE6 as a therapeutic strategy by using anti-sense oligonucleotides against NHE6. Overall, the goal of my thesis is to determine if NHE6 is a rational target for delaying and/or preventing AD. The findings from my thesis have provided some underlying mechanisms of how NHE6 reduces Abeta plaque load but also have opened new doors on other avenues we need to explore before moving from preclinical to clinical trials.
Hybrid Enzyme-Loaded Silica Nanoparticles for Potential Therapeutic Applications
(2022-08) Heble, Annie Yang; Siegwart, Daniel J.; Ready, Joseph M.; Liszczak, Glen; Lux, Jacques
Enzymes are powerful biological catalysts that enable selective chemical reactions. Their ability to convert substrates to products at physiological pH and temperature have made them attractive as potential therapeutic agents. Since most enzymes are non-humanderived, their clinical use faces two main challenges: short functional half-life and immunogenicity. To address these limitations, there is a need for a versatile strategy that increases the functional half-life and safety profile of enzymes while preserving their efficacy. In this dissertation, I will report the design, syntheses, and applications of a Hybrid Enzyme-Loaded silica nanoParticle (HELP) platform used for therapeutic purposes. In this platform, enzymes are embedded inside nanoporous silica nanoparticles that prevent their interaction with large biomacromolecules but allow them to interact with small molecule substrates. This versatile encapsulation method has the potential to improve the stability of enzymes in circulation, eliminate immune responses, and prolong their functional half-life. First, I used catalase as a model enzyme because it has oxygen-generating properties that can be used for potential hypoxia relief and radiosensitization of tumors. Catalase was encapsulated in silica nanoparticles by modifying surface lysines to introduce a silica precursor, followed by silication in mild, aqueous phase conditions. These nanoparticles had high enzyme activity, optimal protection from proteases, and excellent stability over time. An in vivo pilot study demonstrated the ability of these particles to oxygenate tissues upon hydrogen peroxide infusion. Additionally, targeting moieties and radiotracers could be conjugated to the surface of these particles post-formulation. The HELP platform was also applied to asparaginase, a therapeutic enzyme used as first-line treatment to treat acute lymphoblastic leukemia (ALL), the most common childhood cancer. Unfortunately, it is bacterially derived and can elicit immune responses in pediatric patients. Asparaginase-loaded silica nanoparticles were formulated using a direct modification method and subsequent reverse emulsion conditions. These particles were monodisperse, had a mean diameter less than 50 nm, and demonstrated excellent protection from proteases and antibodies in vitro. Both immunogenicity and functional half-life of these particles are currently being investigated in vivo. Future studies will look at the therapeutic potential of these nanoparticles in an animal model of ALL.
Search for Insulin Secretion Modulators, Novel Functions of WNK Signaling and Intramolecular Signal Transduction in ERK2
(2022-08) Grzemska, Magdalena Grazyna; Albanesi, Joseph P.; Cobb, Melanie H.; Whitehurst, Angelique Wright; Cleaver, Ondine
In this dissertation, the regulation of insulin secretion from pancreatic beta cells, WNK1 pathway involvement in cell migration, angiogenesis, breast cancer, and other processes, as well as ERK2 intradomain communication are discussed. Each chapter is dedicated to a separate project, but the common denominators for these projects are protein kinases involved in the WNK or ERK pathway. Through an assay developed in the lab to detect compounds that have an extended action on insulin secretion, novel insulin secretion modulators were discovered. Using a bioinformatics platform, previously unknown downstream effectors of the WNK pathway were identified. Depletion of WNK1 in endothelial cells and breast cancers further defined WNK1-sensitive processes involved in cell migration and metastasis of breast cancer. Finally, ERK2 mutants were characterized to explore allosteric regulation between substrate binding sites and the ERK activation mechanism.
The Role of Acculturation in Parent-Child Relationships for Adolescent and Young Adult Patients' Adjustment to Cancer
(2022-08) Khan, Talha; Germann, Julie; Stewart, Sunita M.; Bordes Edgar, Veronica; Olufunke Awosogba; Marisa Abbe
Approximately 70,000 adolescents/young adults (AYAs; ages 15-39) are diagnosed with cancer annually in the United States (NCI, 2015). This psychosocially at-risk group is less studied and understood than other age cohorts, and experience disparities in access to developmentally informed treatment. A sub-demographic amongst this group that is even more-so less studied are immigrant AYAs. As cancer impacts family dynamics and roles, immigrant AYAs face a unique challenge of navigating the cancer experience alongside the acculturative experience. To our knowledge, little to no research exists on the role of acculturation in AYAs and parent-child relationships when coping with cancer. Research exists on conflicts in parent-child relationships due to differences in acculturation known as the acculturative gap. Understanding the role of the acculturative gap as well as overall acculturative attitudes when coping with cancer may be a promising area of focus when conceptualizing care for cancer patients from various backgrounds. Study aims were to: 1) describe ways acculturation relates to how AYA patients adjust to cancer and 2) describe how acculturation across parent-child relationships relate to AYAs adjusting to cancer. Participants included 15 AYA cancer patients who have been diagnosed with cancer at Children's Health (M = 18.1) and 13 caregivers. Demographic variables were collected from medical records and other measures. Qualitative analysis was guided by the principles of grounded theory (Glaser & Strauss, 1967) and utilized the constant comparative approach. Guided by study aims, qualitative analysis generated three primary sections: Section I: Participants and Aspects of Culture; Section II: Cancer and Acculturation; Section III: Parent-Child Relationships, Cultural Attitudes, and Experiencing Cancer. Results suggest that cancer has a gravitational effect, pulling many participants closer to their heritage culture through various ways culture is performed to cope with the cancer experience. Results also aided in participants ability to recognize ways their cultural identity and parent-child acculturation differences played a role in the cancer treatment through conflicts and related to autonomy, control, and increased sense of burden. Acculturation theory is a framework to guide clinicians, families, peers, friends, and others through future intervention and support strategies to improve coping for immigrant AYA cancer patients. Study results also include recommendations, directly from AYA patient.
Toward a Better Understanding of Racial and Ethnic Differences in Cognitive Function in MDD
(2022-08) Joseph, Jeethu Kochupaingottu; Greer, Tracy L.; McClintock, Shawn Michael; Bordes Edgar, Veronica; Carmody, Thomas; Czysz, Andrew H.
Major Depressive Disorder (MDD) is a highly pervasive and disabling disease that is characterized by persistent sadness and loss of interest. It creates a host of complications for the individual and affects various domains of functioning and quality of life. Although the prevalence for MDD is estimated to be higher in the white, non-Hispanic population, MDD still has a significant prevalence among other races and ethnicities. Due to the disparities in diagnosis and treatment in MDD, the burden of disability is increased in minority populations. To begin understanding what factors could possibly contribute to disparities in MDD, it is important to examine key players in MDD such as cognitive functioning. One increasingly recognized impact of depression that can be seen across all populations and ages is cognitive dysfunction, which plays a role in depression severity and recurrence. The examination of potential differences in cognitive function associated with race and ethnicity in depressive disorders has been underexplored. Apart from race/ethnicity itself, there are independent factors highly prevalent in minority populations including social determinants of health (SDOH) such as age and education, and medical or metabolic comorbidities, that independently impact cognitive functioning and depression. This broad investigation was broken into two parts. Part One focused on understanding the current knowledge and findings of racial and ethnic differences in cognitive performance in adults with MDD and possible contributors to those differences. This literature review found associations between race/ethnicity and cognitive functioning as well as other possible contributors to cognitive performance such as age, education, and presence of medical comorbidities such as HIV, metabolic syndrome, and obesity. Part Two of the investigation was a secondary analysis that compared group differences (between Black/non-Hispanic, White/non-Hispanic, and White/Hispanic participants) on measures of cognitive functioning using the National Institute of Health (NIH) Toolbox - Cognition Battery (NIHTB-CB). Significant mean group differences were observed across the Fluid and Total NIHTB-CB cognitive composite scores, with Black participants scoring lower than White, non-Hispanic/Latinx participants. Several differences remained significant when controlling for depressive symptomatology, metabolic function, and medical comorbidity burden. These results indicate that Black, non-Hispanic/Latinx race is a possible contributor to differences in cognitive performance in depression that is independent of depression, metabolic factors, and medical comorbidity burden, although the size of the noted differences may not be clinically meaningful. Future studies are needed to better understand contributors to such differences in cognitive performance and further expand our understanding of depression and development of precision medicine.
Multiphase Coalescence Mediates Hippo Pathway Activation
(2022-08) Wang, Li; Krämer, Helmut; Rosen, Michael K.; Buszczak, Michael; Pan, Duojia
The formation of biomolecular condensates facilitates spatiotemporal control of biochemical reactions inside cells, and this process is often reversed by the dissolution of condensates. Whether the function of condensates can be suppressed without condensate dissolution remains unknown. Here we show that upstream regulators of the Hippo signaling pathway form functionally antagonizing condensates, and the coalescence of these distinct condensates into a common phase provides a novel mode of counteracting the function of biomolecular condensates without condensate dissolution. Specifically, the negative regulator SLMAP forms Hippo-inactivating condensates to facilitate pathway inhibition by the STRIPAK phosphatase complex. In response to cell-cell contact or osmotic stress, the positive regulators AMOT and KIBRA form Hippo-activating condensates to facilitate pathway activation. The functionally antagonizing SLMAP and AMOT/KIBRA condensates further coalesce into a common phase to inactivate STRIPAK function by excluding SIKE, an essential STRIPAK subunit that is normally enriched in the Hippo-inactivating SLMAP condensates. These findings provide a new paradigm for restricting the activity of biomolecular condensates without condensate dissolution, shed light on the molecular principles of multiphase organization, and offer a new conceptual framework for understanding upstream regulation of the Hippo signaling pathway.
Engineering Considerations for Treatment of Prosthetic Joint Infections Using Alternating Magnetic Fields (AMF)
(2022-08) Sadaphal, Varun; Madhuranthakam, Ananth; Chopra, Rajiv; Greenberg, David; Welch, Tre; Copley, Lawson A. B.
Treatment of infected orthopedic implants presents a major medical challenge, involving prolonged antibiotic therapy and revision surgery. This contributes to >1 billion USD in annual healthcare costs in the US alone. Exposure of metallic implants to alternating magnetic fields (AMF) generates heat that can provide a non-invasive means to target biofilm adhered to the surface. In this thesis, first an AMF system with a solenoid coil was constructed for targeting a metal plate surgically implanted in a sheep model. The system produced magnetic field strengths up to 12 mT and achieved plate temperatures of 65-80°C within 10-14 seconds. A tissue-mimicking phantom of the sheep leg was developed to evaluate heating with the system and to compare these results with numerical simulations. Single and intermittent AMF exposures of a tissue-mimicking phantom agreed with numerical simulations within ± 5%. Similar agreement between experimental measurements and simulations was also observed in the live sheep metal implant model. The simulations also predicted 2-3 mm of tissue damage using a CEM43 thermal dose model for 1-h AMF exposures targeting 65°C for pulse delays of 2.5 and 5 mins. This study confirmed that AMF technology can be scaled up to treat implants in a large animal model with the same rates of heating and peak temperature as observed in prior in vitro studies. After this, the thesis focused on designing a working prototype to target a clinical knee implant with AMF. For initial optimizations numerical simulations approximate interaction between AMF and metal implants were used to design AMF coils windings for heating clinical knee implant on horseshoe surface. These winding patters were engineered into a prototype, capable of running a total current upwards of 400 A, producing a magnetic field of 4 - 5 mT at 2000 W of input power. This coil was further characterized and calibrated to optimal AMF parameters. The AMF system was able cycle through this power for 60 s at each instance, producing uniform surface temperature on complex geometry of knee implant. This heating rate was equivalent of producing 70 - 80 °C on implant surface inside a simulated homogeneous tissue model. Equivalent simple Helmholtz coil was also designed to show its inability of producing a narrow temperature distribution on complex knee implant surface. This work will be further enhanced for the final design for clinical trials based on the findings presented in this thesis.
Overcoming Vulnerabilities of Machine Learning in Neuroimaging Applications
(2022-08) Nguyen, Kevin Phan; Rajaram, Satwik; Montillo, Albert A.; Minassian, Berge; Rypma, Bart; Vinogradov, Elena
Neuroimaging has been widely used to non-invasively probe the structural and functional changes in many neurological and psychiatric diseases. Techniques such as functional magnetic resonance imaging (fMRI) generate a wealth of dense and high-dimensional measurements of the brain. Through machine learning, a powerful class of statistical methods, such measurements can be used to make personalized predictions that inform clinical care, such as diagnoses, prognoses, or treatment outcome trajectories. However, applications of machine learning must meet two critical requirements: 1) there must be sufficient training data and 2) data samples should be independent and identically distributed (iid). In this dissertation, I propose two novel methods to overcome these vulnerabilities in neuroimaging and other biomedical data, and I develop two promising applications of machine learning and neuroimaging to address critical needs in neurological and psychiatric disease. First, I demonstrate how standard machine learning approaches can be applied to predict future Parkinson's Disease severity from resting-state fMRI. By generating personalized prognoses for this debilitating neurodegenerative disease, better care decisions can be made. Next, I address the requirement for sufficient training data in machine learning, without which models tend to overfit and generalize poorly to new data. I propose a method to perform data augmentation, where additional data is simulated by transforming existing data, for 4D fMRI timeseries. Using this augmentation method, I develop predictors of antidepressant response from pre-treatment task-based fMRI. This targets a pressing need for individualized treatment selection tools in depression care, where it currently takes weeks to months to test various medications until adequate relief is found. Finally, I propose a method to construct neural networks, a potent class of machine learning models, that can specially handle non-iid data. This is of broad importance in biomedical data, which is frequently non-iid with samples clustered by experimental batch, study site, etc. Inadequate handling of cluster effects leads to confounding biases and poor generalization in conventional models. My method, tested across several biomedical applications, improves both performance and generalization while affording greater interpretability of cluster effects in the data.

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