UCSF

Pandemic Potential Pathogens remain an ever-present scourge upon the human species. Epidemics and pandemics have ranged from nuances to having the power to shut the entire world down. Molecular biology and epidemiology have the synergistic power to understand, prepare for, and combat this horseman of the apocalypse. In this thesis, I share my humble part in this perpetual struggle, examining the potential mutations of note and their phenotypic properties of Enteroviruses as well as modeling the spread and transmission of Coronavirus. Using the powers invested through the mathematical lens of physical biology, I endeavored to add to both basic virology and epidemiology against these dangerous pathogens.

Parkinson’s disease (PD), like other neurodegenerative diseases, lacks a disease-modifying therapy. While we have solutions to counteract the dopaminergic depletion that ensues at the level of the striatum following the progressive loss of substantia nigra neurons in the midbrain, and therefore, are capable of reversing the motor symptoms experienced by patients, our understanding of the molecular mechanisms that trigger degeneration is minimal. Even following the discovery of single mutations in several genes that can cause familial forms of PD, like PINK1 and PRKN, we are still deprived of a deep appreciation of the basic biological mechanisms that drive disease initiation and progression. In Chapter 1 of this thesis, I describe the remarkable research progress that was made in the last two centuries following the original description of the disease. I detail how the mitochondrial hypothesis of PD emerged from brilliant discoveries, the advent of new biochemical and genomic tools, and even a few accidents, and how it may underlie the selective vulnerability of midbrain dopamine neurons that die in PD. Finally, I introduce some key unanswered questions in the field of PD research that I begin to address in this thesis. In Chapter 2, I describe the novel approach we developed to study a pathway of mitochondrial turnover that is critical in PD, which allowed us to track individual mitochondria at every step of the degradation process in neurons. We gained unprecedented insight into the fate of these mitochondria once they reach the lysosomal compartment which led to us to identify new steps of PINK1/PRKN-mediated mitochondrial degradation. These findings may have important implications for both basic biology and disease pathophysiology. In Chapter 3, I describe a mouse model we developed to assess the impact that chronic changes in neural activity have on the function and survival of midbrain dopamine neurons. We find that the substantia nigra dopamine neurons are preferentially vulnerable to chronic increases in neural activity, while other dopamine neurons are more resilient, suggesting a potential role for chronic hyperactivity as a driver of disease. Lastly, in Chapter 4, I elaborate and speculate on disease implications of the findings described in Chapter 2. I contextualize our contribution within the broader effort to understand why PINK1 and PRKN are so crucial to neurons under certain high-stress conditions, but are dispensable at baseline.

GPR174 is abundantly expressed in B and T lymphocytes and has a role in restraining T cell responses, but the function of GPR174 in B cells is less clear. Here we report that upon in vitro culture B cells undergo a spontaneous GPR174-dependent activation process that is associated with marked changes in gene expression, including up-regulation of Cd86, Nr4a1, Ccr7 and phosphodiesterases. B cells lacking Gαs show a block in induction of the GPR174-dependent program. Spontaneous up-regulation of CD86 in cultured B cells is dependent on protein kinase A. Both GPR174- and Gαs-deficient B cells show enhanced survival in culture. In vivo, GPR174 contributes to NUR77 expression in follicular B cells and is needed for establishing a marginal zone compartment of normal size. Treatment of mice with lysophosphatidylserine (lysoPS), a GPR174 ligand, is sufficient to promote CD86 up-regulation by follicular B cells. These findings demonstrate that GPR174 can signal via Gαs to modulate B cell gene expression and show this can occur in vivo in response to lysoPS. Additionally, the findings illuminate a pathway that might be targeted to improve systems for the in vitro study of B cell responses.

This dissertation traces Black AIDS Activism in the Bay Area and how the layered crises of the crack cocaine epidemic, chronic joblessness, and poverty shaped HIV/AIDS prevention and education efforts. It also compares Black health activism with ways policymakers, local media outlets, and health professionals’ interventions and perspectives helped or hindered Black communities. Many public health officials and journalists purported that poor and working-class African Americans’ behavior—particularly that of drug users, sex workers, gay men and men who had sex with men (but did not identify as being gay)—placed them at greater risk of infection. This approach ignores ways chronic joblessness, police surveillance, inadequate access to health care, and other structural inequities, disregarded by the medical community before HIV/AIDS was identified, contributed to African Americans’ disease susceptibility. As a result, Black activists paved the way for new, alternative, relevant methods to HIV/AIDS prevention, awareness, and outreach throughout the 1980s by contesting the liberalism of the “San Francisco Model of Care.” Their community care methods remain helpful for public health departments failing to develop effective harm reduction tactics in low-income communities of color.

Context – Unrelieved pain and sleep disturbance are common symptoms in patients receiving chemotherapy. Increased stress may be an underlying cause for both symptoms.Objectives – Purposes were to identify subgroups of outpatients with distinct co-occurring pain AND sleep disturbance profiles and to evaluate for differences among these subgroups in demographic and clinical characteristics. In addition, differences in global stress, disease-specific stress, and cumulative life stress, as well as resilience and coping were evaluated. Methods – Patients (n=1343) completed measures of pain, sleep disturbance, stress, resilience and coping. Latent profile analysis was used to identify subgroups of patients with distinct pain AND sleep disturbance profiles. Differences among the subgroups were evaluated using parametric and non-parametric tests. Results – Three distinct profiles were identified (i.e., No Pain + Moderate Sleep Disturbance (SD) (27.6%), Moderate Pain + Moderate SD (38.6%), Severe Pain + High SD (33.8%)). Compared to the other two classes, Severe Pain + High SD class was younger, had fewer years of education, was more likely to be female, more likely to live alone, less likely to be employed, and had a higher level of comorbidity. This class had the highest stress scores and were more likely to report higher rates of adverse childhood experiences (ACEs; e..g, physical and sexual abuse). Conclusions – Given that over 70% of our sample reported clinically meaningful levels of both symptoms and 33.8% reported relatively high rates of ACEs, clinicians need to perform routine assessments; initiate appropriate referrals, and provide effective symptom management interventions including cognitive behavioral therapy.

Living systems operate at many scales, from biochemical reactions of individual atoms and molecules to complex behaviors of cells and organisms, and even evolutionary adaptation of entire ecosystems. Understanding the relationships between these processes, namely how changes at one scale propagate to other scales, is a fundamental pursuit of biology. One such complex propagation is called a genotype-phenotype map, defined here as how a protein mutation impacts its function in the context of its molecular interaction network to ultimately alter cellular fitness. Our generally poor understanding of this propagation limits our prediction of the effects of disease mutations and our ability to rationally engineer mutations for precisely tuning protein function in the dynamic cellular environment. In this dissertation, I present two studies of the small GTPase switch Gsp1, the S. cerevisiae homolog of human Ran, which uncover novel allosteric mechanisms governing how the effects of point mutations propagate from the molecular to the cellular scale.In Chapter 1, I outline the systems biology approach to studying molecular interaction networks, introduce the components of the network of Ran/Gsp1, and motivate the use of mutagenesis in the study of protein structure and cellular function. In Chapter 2, I describe the genetic and physical interaction profiling of point mutations in Gsp1 partner interfaces, which led to the discovery of novel allosteric sites coupled to the GTPase switch, as confirmed by enzyme kinetics and 31P nuclear magnetic resonance. Analysis of the genetic interaction profiles showed that distinct cellular processes were sensitive to changes in either the rates of GTPase hydrolysis or nucleotide exchange, prompting a model for a single GTPase selectively and independently controlling different downstream pathways by regulated tuning of its switching. In Chapter 3, I describe a mutational scanning study which quantitatively measured the fitness effect of all possible point mutations in Gsp1. The scan revealed an unexpected widespread toxic/gain-of- function response, in which mutations were more deleterious than loss of gene function by truncation of Gsp1 via internal STOP codon. Sites enriched for toxic/gain-of-function mutations included a novel allosteric cluster of residues which stabilize the GDP-bound state of Gsp1, confirmed by enzyme kinetics. The study defined a functional map of allosteric regulatory sites in Gsp1 which generalizes to other GTPases and confirmed that perturbation of the switch mechanism is the dominant factor in the effect Gsp1 mutations exert at the cellular level. Finally, in Chapter 4, I discuss the implications of these findings for future studies of molecular switches and their interaction networks, as well as for the use of high-throughput genome-wide measurements to guide the engineering of protein function.

Background: Discoveries made through genome-wide association studies have revolutionized the field of human genetics by uncovering disease mechanisms and enabling precision medicine. Although there has been tremendous effort to obtain cohort sizes on the order of hundreds of thousands of individuals, there is an immense underrepresentation of non-European ancestries, which has potential to contribute to health inequity. Here, we present works on (i) an in-depth simulation to identify optimal approaches for achieving equitable accuracy for polygenic risk scores (PRS) in diverse populations and (ii) an application of trans-ancestry discovery of germline associations in the complex phenotype of multiple primary tumors. Methods: (i) Through our simulation framework, we implement many strategies for building PRS, including a local-ancestry specific approach, and measure accuracy in admixed and African ancestry individuals. (ii) We also conducted a whole-exome sequencing study of two large, multi-ancestry populations consisting of 6,429 multiple cancer cases, 29,091 single cancer cases, and 165,853 cancer-free controls. We employed single-variant and gene-based tests to characterize the genetic susceptibility to multiple primary tumors in comparison to individuals with one and, separately, no cancers through the investigation of rare and common variation. Results and Conclusions: (i) Variants discovered in African ancestry populations have greater potential to achieve unbiased PRS prediction across populations. Studies should prioritize the inclusion of diverse participants in GWAS, and care must be taken with the interpretation of currently available risk scores. (ii) Our applied trans-ancestry analysis of multiple primary tumors identifies rare loss-of-function variants and gene-level associations with cross-cancer pleiotropy and potential for prioritizing cancer survivors at high risk for developing subsequent tumors.

Background – Pain and fatigue are common symptoms in oncology patients.Objective – Study purposes, in a sample of oncology outpatients receiving chemotherapy (n=1342), were to: identify subgroups of patients with distinct worst pain AND morning fatigue profiles and evaluate for differences among the subgroups in demographic and clinical characteristics; as well as the severity of common symptoms and quality of life (QOL) outcomes. Methods – Oncology outpatients receiving chemotherapy (n=1342) completed self-report questionnaires to assess pain and morning fatigue, a total of six times over two cycles of chemotherapy. Joint latent profile analysis was used to identify subgroups of patients with distinct pain and morning fatigue profiles. Differences among the classes were evaluated using parametric and non-parametric tests. Results – Five distinct profiles were identified (no pain and no morning fatigue (27.6%), moderate pain and no morning fatigue (28.2%), moderate pain and morning fatigue (28%), moderate pain and increasing and decreasing morning fatigue (6.9%), severe pain and very high morning fatigue (9.3%). Patients in the three highest profiles had clinically meaningful levels of depression and sleep disturbance and decrements in QOL. Conclusions – Over 44% of the sample had moderate to high levels of both pain and morning fatigue. Unrelieved pain may contribute to disturbed sleep which results in higher levels of morning fatigue. Implications for practice – Clinicians need to assess for pain and fatigue, as well as sleep disturbance during chemotherapy.

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients¬, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC’s multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.

Oncology patients receiving chemotherapy report on average 14 concurrent symptoms. The co-occurrence of these symptoms is associated with poorer functional status, decrements in quality of life (QOL), and increased mortality. Given that symptoms rarely occur in isolation, the concept of a symptom cluster emerged in the literature in 2001. An increased understanding of how symptoms cluster together and the biological mechanism(s) that underlie them has the potential to lead to the development of targeted interventions to decrease symptom burden. Therefore, the overall aims of this dissertation research were to: 1) review the conceptual basis for using variable-centered versus patient-centered analytic approaches in symptom cluster research; 2) systematically review studies published since 2016 that evaluated for symptom clusters in patients receiving primary or adjuvant chemotherapy; 3) evaluate the stability and consistency of symptom clusters across time and across three symptom dimensions (i.e., occurrence, severity, and distress); 4) identify common and distinct symptom clusters across various types of cancer; and 5) evaluate for associations between psychological and gastrointestinal symptom clusters and epigenetic regulation of inflammatory genes in a heterogeneous sample of oncology patients. In terms of aim 1, a theoretical paper described two conceptual approaches that are used to evaluate symptom clusters; namely: “clustering” symptoms (i.e., variable-centered analytic approach) and “clustering” patients (i.e., person-centered analytic approach). Findings suggest that while each approach has unique strengths and weaknesses, conceptual clarity is needed when a study is designed and the specific research question(s) should guide the selection of the appropriate analytic method. The application of newer analytic approaches (e.g., network analysis (NA), natural language processing (NLP)) to study symptom clusters were reviewed. This paper summarized the paucity of research on the evaluation of the underlying mechanism(s) for symptom clusters. In terms of aim 2, in a systematic review, 23 studies were identified that evaluated for symptom clusters in patients receiving chemotherapy. Across these studies, the Memorial Symptom Assessment Scale (MSAS) was the most common instrument and exploratory factor analysis (EFA) was the most common statistical method used to identify symptom clusters. While psychological, gastrointestinal, and nutritional clusters were the most common clusters identified across studies, only the psychological cluster remained relatively stable over time. A major conclusion from this review was that clear criteria are needed to evaluate the stability of symptom clusters across time and dimensions. In addition, only five studies evaluated for secondary outcomes (e.g., functional status, QOL). Additional research is needed to evaluate the biological mechanism(s) for symptom clusters. In terms of aim 3, prior to the start of their second or third cycle of chemotherapy, outpatients reported an average of 13.9 (±7.2) concurrent symptoms. Lack of energy was both the most common and severe symptom while “I don’t look like myself” was the most distressing. Psychological, gastrointestinal, weight gain, respiratory, and hormonal clusters were the common symptom clusters identified across the three symptom dimensions. Our findings suggest that psychological, gastrointestinal, and weight gain clusters are common across various types of cancer while respiratory and hormonal clusters are cancer specific. In terms of aim 4, across a cycle of chemotherapy, the number of symptoms remained relatively stable over time, with patients reporting 13.9 (±7.2) symptoms prior to, 14.0 (±7.0) at one week after, and 12.2 (±6.8) at two weeks after receipt of chemotherapy. While the psychological, weight gain, respiratory, and gastrointestinal clusters were stable over time and dimensions, only the psychological, weight gain, and respiratory clusters were consistent across time and dimensions. In terms of aim 5, given the paucity of studies on the underlying mechanism(s) for the two most common symptom clusters (i.e., psychological, gastrointestinal), exploratory analyses were done to evaluate for associations between these clusters and epigenetic variation of inflammatory genes. Findings from both studies provide preliminary support for the hypothesis that epigenetic dysregulation of inflammatory processes contributes to the occurrence of psychological and gastrointestinal symptom clusters in patients receiving chemotherapy. For the psychological symptom cluster, cluster of differentiation (CD) 40 was differentially methylated across two independent samples (false discovery rate (FDR) = .017). Six expression-associated CpGs (i.e., eCpG; cg22232207, cg06571407, cg17929951, cg21601405, cg01943874, cg11841529) located in the promoter region of this gene were hypomethylated across both samples. For the gastrointestinal symptom cluster, one trans eCpG locus (i.e., cg03171795) that was associated with expression of the lymphotoxin beta (LTB) gene was associated with the occurrence of the gastrointestinal symptom cluster (FDR = 0.168). These findings warrant validation. This dissertation concludes with implications for clinical practice and future research.