UCSF

Background - This study sought to replicate the findings from our previous candidate gene analyses of pro- and anti-inflammatory cytokines in a sample of patients who were assessed prior to and for six months following breast cancer surgery. Specifically phenotypic differences between the Resilient (n=155) and Subsyndromal (n=180) depressive symptom classes were evaluated as well as variations in cytokine genes between the two latent classes.

Methods - Among 398 breast cancer patients following surgery, growth mixture modeling was used to identify latent classes based on Center for Epidemiological Studies Depression (CES-D) Scale scores. The CES-D was completed prior to surgery and monthly for a total of six months following breast cancer surgery. A total of 103 single nucleotide polymorphisms and 35 haplotypes among 15 candidate cytokine genes were included in the genetic association analyses.

Results - Patients in the Subsyndromal class were significantly younger, more likely to be married or partnered, and reported a significantly lower KPS score than patients in the Resilient class. Variation in three cytokine genes (i.e., tumor necrosis factor alpha (TNFA), interferon gamma receptor 1 (IFNGR1), interleukin 6 (IL6)), as well as age and functional status predicted membership in the Subsyndromal versus the Resilient class.

Conclusion - Growth mixture modeling identified two distinct groups of patients who differ in their experience with depressive symptoms. Variations in cytokine genes may influence the trajectory of depressive symptoms in high risk patients.

Nature challenges healthy mammals with constant risk of infection by a wide variety of pathogens and with degradation of healthy tissue and control systems. I have been interested in drug delivery and improved delivery of therapeutics primarily to attack pathogens but with incidental value in assessing vital functions of a healthy mammal. My recent work uses protein design to approach such problems. Delivering biomacromolecules remains important both for therapeutics and in discerning and shaping functions of cells.My first thesis project focused on designing a better glomerular filtration rate (GFR) marker to facilitate assessment of renal function and seeking a marker reflecting water distribution in the body, which is relevant to distribution of highly soluble drugs [1]. Tritiated polyethylene glycol (PEG) was known to clear the kidneys effectively and correlated well with a GFR standard assay. A better radioactive label for PEG would allow for easy detection, including imaging. At the time of the project, only one biological conjugation with PEG was reported. Attaching an iodinatable moiety to polyethylene glycol (PEG) polymers of different sizes enabled tracking the compound using radioactive iodine. I made a series of related compounds and studied the pharmacokinetics (PK) and pharmacodynamics (PD) of these in rodents and ultimately in a dog. Using relatively long PEG polymers of molecular weight (MW) 5,000 to 6,000 daltons, the PEG dominated the behavior of the compounds, clearing rapidly through the kidneys. With shorter PEG polymers, the chemistry of the iodinatable group was more significant and the compounds were more likely to clear through the bile, to a degree making them unsuitable for a GFR marker but possibly useful to study liver function. Chapter 1, the published manuscript from my first thesis project, is cited in 20 scientific publications and 51 issued US patents. Variations on the design principles of my project have been used widely in the pharmaceutical industry. During a break in my PhD studies, I improved the formulation of a human Phase-2-ready antifungal drug and designed and organized extensive testing in mice and dogs to show that a sustained-release formulation would overcome PK limitations and made the drug much more potent. This work is discussed briefly in Chapter 2. My second thesis project studied brilacidin activity against 40 fungal isolates from 20 different species, showing useful activity against several important human pathogens [2]. The human and many other innate immune systems includes a variety of peptides known as defensins that weaken or kill a variety of pathogens, including bacteria, fungi, and viruses. Brilacidin is a synthetic defensin-mimic, designed to exhibit the physicochemical properties of defensins as a class. Brilacidin is in human Phase 2 trials. Despite its potential, Brilacidin's efficacy against fungi had not been comprehensively explored until my studies, which showcased its viability as a therapeutic agent against challenging-to-treat fungal infections, thereby offering a beacon of hope for future clinical interventions. Chapter 3, the published manuscript based on this second thesis project, has recently been submitted for review, available online in Preprints.

Background: Approximately 3.8 billion people in low- and middle-income countries use unclean fuels as a source of primary cooking fuel as well as for heating. For pregnant women, the toxic chemicals produced by combustion of unclean fuels not only affect women’s health directly, but particulate matter and carbon monoxide are absorbed in maternal blood and cross the placental barrier potentially affecting the fetus. Methods: This dissertation presents three manuscripts. The first is a systematic review examining birth outcomes related to household air pollution (HAP) from type of cooking fuel in low-and middle-income countries. PRISMA 2009 guidelines were used for this systematic review. The inclusion criteria were quantitative, peer reviewed journal articles published within a date range of May 1, 2013-June 12, 2021. The quality of available evidence was evaluated using the Office of Health Assessment and Translation (OHAT) risk of bias rating tool. The reviewed studies presented evidence for an increased risk of low birth weight (LBW), preterm birth (PTB), small for gestational age (SGA), stillbirth, neonatal mortality and reduction in birthweight with unclean fuel use compared to cleaner fuels. The second study’s objective was to evaluate the effects of a liquefied petroleum gas (LPG) stove and fuel intervention during pregnancy on congenital anomalies, stillbirth and neonatal mortality using data from the Household Air Pollution Intervention Network (HAPIN) Trial. The HAPIN trial is a randomized controlled trial of LPG stoves and fuel distribution in nearly 3200 households conducted across India, Guatemala, Peru and Rwanda. Participants in the HAPIN Trial were monitored for adverse and serious adverse events. All analyses were performed according to intention-to-treat (ITT) analysis, and binary outcomes of congenital anomalies, stillbirth and neonatal mortality were compared between the two arms using log binomial models. The third study’s objective was to evaluate the effects of a LPG stove and fuel intervention during pregnancy on maternal outcomes of spontaneous abortion, postpartum hemorrhage, hypertensive disorders of pregnancy, and maternal mortality using data from the Household Air Pollution Intervention Network (HAPIN) Trial. All analyses were performed according to intention-to-treat (ITT) analysis, and binary outcomes of spontaneous abortion, postpartum hemorrhage, preeclampsia/eclampsia, and maternal mortality were compared between the two arms using log binomial models. Results: Systematically reviewing the evidence and risk of bias ratings illuminated several gaps in the current literature related to exposure assessment, outcome measurement and adequacy of adjustment for confounding. Results for the second study showed that adverse fetal and neonatal outcomes (congenital anomalies, stillbirth, neonatal mortality) did not differ based on stove type across four country research sites. Pregnant women assigned to the LPG intervention arm had a slightly lower risk of congenital anomaly (RR 0.96, 95% CI: 0.55, 1.70) and neonatal mortality (RR 0.97, 95% CI: 0.53, 1.77) compared to women in the control arm. In regards to stillbirth, pregnant women assigned to the LPG intervention arm had a slightly higher risk of stillbirth (RR 1.01, 95% CI: 0.60, 1.70) compared to women in the control arm. However these results were not statistically significant even after adjusting for maternal education, household food insecurity and bank account. Results for the third study demonstrated adverse maternal outcomes (spontaneous abortion, hypertensive disorders of pregnancy, postpartum hemorrhage and maternal mortality) did not differ based on stove type across the four country research sites. In intention-to-treat analyses we found women assigned to the LPG intervention arm had a higher risk of spontaneous abortion (RR 2.69, 95% CI: 0.71, 10.12) and maternal mortality (RR 1.51, 95% CI: 0.25, 9.03) compared to women in the control arm. Pregnant women assigned to the LPG intervention arm had a lower risk of postpartum hemorrhage (RR 0.84, 95% CI: 0.26, 2.74) and hypertensive disorders of pregnancy (RR 0.84, 95% CI: 0.26, 2.74) compared to women in the control arm. However all results were not statistically significant even after adjusting for maternal education, household food insecurity and bank account. Conclusion: The systematic review presented evidence for an increased risk of LBW, PTB, SGA, stillbirth, neonatal mortality and reduction in birthweight with solid fuel and kerosene use compared to cleaner fuels like gas and LPG. The HAPIN trial was the first multi-country RCT collecting data on household air pollution and health outcomes on pregnant women across four countries. While the LPG stove intervention did not significantly reduce the relative risk of adverse birth outcomes, women assigned to the LPG intervention arm had a slightly lower risk of congenital anomaly, neonatal mortality, hypertensive disorders of pregnancy and postpartum hemorrhage compared to women in the control arm. Access to sustainable and affordable energy should remain a priority for the global community.

Despite numerous advances, direct reprogramming of fibroblasts to induced cardiomyocytes remains an inefficient process with largely unknown underlying mechanisms. To understand the molecular events associated with direct cardiac reprogramming, we characterized genome-wide transcriptional changes in individual and bulk reprogramming cell populations, examined genomic changes in chromatin accessibility and DNA methylation levels during reprogramming, and mapped DNA binding by Gata4, Mef2c, and Tbx5 (GMT) that initiates the transition from fibroblast to induced cardiomyocyte. We discovered that the transition to cardiac-like gene expression occurs within 48 hours of initiating transdifferentiation. This change is accompanied by rapid epigenomic remodeling and includes dynamic chromatin regions enriched for transcription factor motifs in addition to GMT, with different motif families associated with specific patterns of chromatin and DNA methylation changes. We found evidence for binding of the reprogramming factors to dynamically-opening distal regulatory regions linked to cardiac function. In addition, we document the mechanism through which TGFβ and Wnt signaling pathway inhibition improves reprogramming efficiency by limiting aberrant reprogramming outcomes. These results provide insight into the mechanisms that drive direct cardiac reprogramming and provide a hypothesis-building framework for improving this process.

Being able to accurately respond to the environment is a trait crucial to cellular survival. However, signaling pathways that mediate the internal response to an external signal frequently share components. To ensure that specific pathways remain insulted from one another such that signaling fidelity is maintained, regulatory mechanisms must be in place to ensure signaling specificity. In Saccharomyces cerevisiae, a pair of mitogen-activated protein kinase (MAPK) pathways involved in the distinct developmental programs of mating pheromone response and filamentous growth shares many components but avoid pathway cross-talk. We have identified one of the mechanisms that inhibits erroneous activation of the filamentous growth pathway during activation of the mating response pathway. To ensure signaling specificity, the mating pheromone response pathway specific MAPK Fus3 targets the destruction of the filamentous growth-pathway specific transcription factor Tec1. We have further found that this proteolysis of Tec1 is dependent on the dual phosphorylation of Tec1 by Fus3 and the subsequent recognition of phospho-Tec1 by the SCFCdc4 ubiquitin ligase, the complex responsible for the ubiquitylation that signals for proteolysis. Specifically, Cdc4, the F-box protein component of SCFCdc4, binds directly to Tec1 via phosphorylations on both threonine 273 and threonine 276 in a mode of substrate recognition that differs significantly from the previously characterized Cdc4 substrate binding motif (Cdc4 phospho-degron) and requires residues on Cdc4 outside the core substrate binding pocket. This mechanism of Tec1 regulation by destruction explains the conundrum of the absence of filamentation gene expression despite the activation of a population of Kss1, the filamentation pathway specific MAPK, during pheromone signaling. It also presents an example of a novel mode of Cdc4 substrate recognition that, taken together with recently published data on Sic1, another Cdc4 substrate, could represent a more general means of high affinity Cdc4 substrate binding.

This work stems from our group's interest in identifying the mechanisms that govern placental function in normal human pregnancy and in pregnancy complications. Cytotrophoblasts (CTBs) carry out many of the placenta's most important functions. CTBs in contact with the uterus differentiate and subsequently invade the uterine stroma and blood vessels, a process that anchors the placenta to the uterus and redirects maternal blood toward the embryo/fetus. During differentiation/invasion, CTBs undergo phenotypic changes that result in vascular mimicry and display a marked arterial tropism. However, in preeclampsia (PE) CTB differentiation goes awry, and CTB invasion, particularly the arterial component, is shallow.

First, we theorized that molecules involved in arterial differentiation/function might play a role during vascular invasion. We found that expression of Notch family members, which play important roles during arterial differentiation, was extensively modulated during human CTB invasion. Inhibition reduced CTB invasion and expression of an arterial marker. Using mouse models, we confirmed that Notch activity was highest in artery-associated trophoblasts. Analysis of mouse trophoblast stem cell differentiation in vitro suggested that Notch2 played an important role in remodeling the uterine arterioles in this species. Conditional deletion of Notch2 in invasive trophoblast lineages highlighted its central importance in coordinating increases in utero-placental blood flow; trophoblast invasion of maternal arterioles failed, the blood canals that supply the placenta were smaller, and placental perfusion was decreased. Lastly, we asked whether defects in CTB expression of Notch family members were evident in PE. An absence of endovascular TB expression of the Notch ligand, JAG1, was frequently observed, suggesting that failures in Notch signaling are an important part of the pathogenesis of this condition.

Second, we theorized that dysregulated CTB differentiation underlies preeclampsia. To test this hypothesis, we compared gene expression in differentiating CTBs derived from normal and PE placentas. Molecules known to be dysregulated in PE as well as novel candidate regulators were differentially expressed. Of these targets, we performed an expanded functional analysis on SEMA3B. The results helped to explain alterations in CTB and endothelial cell (EC) phenotypes that are commonly observed in PE.

Last, we designed and tested high throughput methods to profile miRNA expression. These were applied in studies we initiated to probe the molecular mechanisms involved trophoblast differentiation and to identify novel serum biomarkers associated with PE. First, we performed experiments to optimize our experimental workflow, in which we measured the quantitative sensitivity of the Taqman miRNA expression assays in a variety of experimental conditions. Next, we identified miRNAs expressed by differentiating CTBs from normal and preeclamptic pregnancies. Lastly, we found many miRNAs that were differentially expressed in serum samples from women who experienced normal pregnancies or those complicated by PE. We believe that the results of the aforementioned experiments will enable many new research directions in the study of the maternal-fetal vascular interactions that occur during normal placentation and in pathological pregnancies.

This dissertation is about an impoverished, African American neighborhood in Oakland, California where the murders of Antoine and Danny occurred. I map the conditions that made their tragic deaths both possible and logical within a racialized vernacular of social, political and economic tidings that enunciate what is seen as a usual, American experience for such a place--a place that is often defined by its high levels of violence. I do this by writing about my own experiences living in this neighborhood, having bought a house here in 1999, as well as those of my neighbors and the families of the victims. Furthermore, I ethnographically engage its storied history, documenting those structures that frame its violence and consequently, molded these deaths. Therefore I look at migration, displacement, and the evocation of privilege and the provocation of poverty. I write about overt and covert racism, struggle, promise and deception. I document the people and policies that greedily promote the hoarding of prosperity and the generous sharing of its losses. I notice the effects of transportation on geography, community, subjectivity and suffering. I learn how wars, both large and small, near and far, seem somehow to always penetrate our lives. And finally, about the unnatural effects of natural disasters; and specifically, how such a seemingly random event as an earthquake, "an act from God," can wiggle its way into a history of racialized oppression in the United States and emerge as a trigger of vulnerability in a homicide epidemic.

This thesis details my work, both published and ongoing projects, on assembling proteins with modular DNA scaffolds in order to develop new strategies for engineering antibodies and probing cellular signaling complexes. The organization of cell surface receptors on a nanometer length scale can influence and regulate a cell’s functional response to signals. My goal was to systematically explore the effect of receptor complex composition, valency, and geometry on cell signaling. Towards this aim, I used modular DNA-based scaffolds to combinatorially control the position of attached proteins with low nanometer resolution. This strategy required a simple and modular technique for site-specifically conjugating synthetic oligonucleotides to proteins. My approach uses the aldehyde tag, which can be genetically incorporated into proteins at either protein terminus or in an internal loop, which provides the potential for orientational control of proteins on DNA scaffolds. We designed and optimized four different reactions that generate DNA−protein conjugates to provide flexibility in linker chemistry. The protein-DNA conjugates can be efficiently assembled into structures with greater valency and complexity. We are expanding our DNA scaffold libraries to generate collections of macromolecular assemblies varying in valency and architecture. These protein-bearing DNA-scaffolded multivalent probes may have novel activities as antibodies with unique specificities and biological activities, such as the ability to deliver protein-based effectors, increasing avidity, or modulating specificity. In one application, we are using this strategy to prepare and screen multivalent DNA-scaffolded biparatopic antibodies for inhibiting urokinase plasminogen activator receptor in triple negative breast cancer lines. Additionally, we are using these modular DNA scaffolds to investigate the effect of nanoscale organization on the signaling profiles of the Epidermal Growth Factor Receptor (EGFR) family by systematically guiding the assembly of signaling complexes on cells. By dimerizing EGFR in the absence of ligand using our DNA scaffolds, we discovered that although dimerization of EGFR is sufficient for autophosphorylation and recruitment of adaptor proteins Grb2 and SOS, it is not sufficient for Ras activation, activation of the AKT or MAPK signaling pathways, nor for the concentration of the receptor into clathrin coated pits and endocytosis.

Persistent pain following breast cancer surgery is a significant clinical problem. Both inherited and acquired inflammatory factors (e.g., cytokines) appear to play a role in the development and maintenance of persistent pain. However, less is known about the molecular mechanisms of inflammation associated with the development of persistent pain following breast cancer surgery. Growth mixture modeling was used to identify persistent breast pain phenotypes based on pain assessments obtained prior to and monthly for six months following breast cancer surgery. The purpose of this dissertation is to evaluate for differences in demographic and clinical characteristics as well as to evaluate the associations between single nucleotide polymorphisms contained within candidate cytokine genes and pain group membership. In addition, methylation of the promoter region of the genes that harbored gene variations associated with pain group membership were evaluated in the no pain and mild pain classes. Different subsets of phenotypic characteristics (i.e., age, strange sensations prior to surgery, reconstruction performed at the time of surgery, re-excision/mastectomy done within six months and worst postoperative pain intensity) and genes (i.e., interleukin (IL) 1 receptor 2, IL4, IL10, IL13 and IL6, tumor necrosis factor alpha (TNFA)) were associated with the distinct phenotypes (i.e., mild persistent pain, severe persistent pain) and suggest that different mechanisms of heritable susceptibility may exist. In addition, CpG methylation within the TNFA promoter may provide an additional mechanism through which TNFA may alter the risk for mild persistent breast pain after breast cancer surgery. Coupled with phenotypic variations, these genetic and epigenetic variations may help to identify individuals who are predisposed to the development of persistent breast pain following breast cancer surgery, differentiate biological mechanisms, and facilitate the development of novel therapies.

Among the common apolipoprotein (apo) E isoforms (apoE2, apoE3, and apoE4), apoE4 is the major known genetic risk factor for Alzheimer's disease (AD). Previous in vitro and in vivo experiments showed that apoE4 preferentially undergoes aberrant cleavage in neurons, yielding neurotoxic C-terminal-truncated fragments. To study the effect of these fragments on amyloid-beta (Abeta) clearance/deposition and their potential synergy with Abeta in eliciting neuronal and behavioral deficits, transgenic mice expressing human apoE3, apoE4, or apoE4(delta272-299) were cross-bred with mice expressing human amyloid precursor protein harboring familial AD mutations (hAPPFAD). At 6-8 months of age, hAPPFAD mice expressing human apoE3 or apoE4 had 94% and 89% less hippocampal Abeta(1-x), respectively, than hAPPFAD mice lacking apoE, while hAPPFAD mice expressing mouse apoE had the highest Abeta levels. Abeta deposition in hAPPFAD mice expressing human apoE3 or apoE4 was 89% and 87% less than hAPPFAD mice without apoE, respectively. Thus, human apoE stimulates Abeta clearance, but mouse apoE does not. Expression of apoE4(delta272-299) reduced total Abeta levels by only 63% and Abeta deposition by 46%, compared to hAPPFAD mice without apoE. Unlike apoE3 and apoE4, the C-terminal-truncated apoE4 bound Abeta peptides poorly, leading to decreased Abeta clearance and increased Abeta deposition. Despite their lower levels of Abeta and Abeta deposition, hAPPFAD/apoE4(delta272-299) mice accumulated potential pathogenic Abeta oligomers and displayed neuronal and behavioral deficits similar to or more severe than those in hAPPFAD mice lacking apoE. Thus, the C-terminal-truncated apoE4 fragment inefficiently clears Abeta peptides and has enhanced toxicity in the presence of low levels of Abeta to elicit neuronal and behavioral deficits in mice.

Origins of replication are activated throughout the S phase of the cell cycle such that some origins fire early and others fire late to ensure that each chromosome is completely replicated in a timely fashion. However, in response to DNA damage or replication fork stalling, eukaryotic cells block activation of unfired origins. Human cells derived from patients with ataxia telangiectasia are deficient in this process due to the lack of a functional ataxia telangiectasia mutated (ATM) kinase and elicit radioresistant DNA synthesis after irradiation . This effect is conserved in budding yeast, as yeast cells lacking the related kinase Mec1 (ATM and Rad3-related (ATR in humans)) also fail to inhibit DNA synthesis in the presence of DNA damage. This intra-S-phase checkpoint actively regulates DNA synthesis by inhibiting the firing of late replicating origins, and this inhibition requires both Mec1 and the downstream checkpoint kinase Rad53 (Chk2 in humans). However, the Rad53 substrate(s) whose phosphorylation is required to mediate this function has remained unknown. Here we show that the replication initiation protein Sld3 is phosphorylated by Rad53, and that this phosphorylation, along with phosphorylation of the Cdc7 kinase regulatory subunit Dbf4, blocks late origin firing in Saccharomyces cerevisiae. Upon exposure to DNA damaging agents, cells expressing non-phosphorylatable alleles of SLD3 and DBF4 (SLD3-m25 and dbf4-m25, respectively) proceed through the S phase faster than wild-type cells by inappropriately firing late origins of replication. SLD3-m25 dbf4-m25 cells grow poorly in the presence of the replication inhibitor hydroxyurea and accumulate multiple Rad52 foci. Moreover, SLD3-m25 dbf4-m25 cells are delayed in recovering from transient blocks to replication and subsequently arrest at the DNA damage checkpoint. These data indicate that the intra-S-phase checkpoint functions to block late origin firing in adverse conditions to prevent genomic instability and maximize cell survival.

Abstract

Background and Objective: If lasers could selectively ablate residual composite with minimal damage to the enamel surface, decrease the clinician’s time, and improve the surface properties of underlying enamel, problems with current debonding techniques could be overcome. The overall objective of this work was to selectively remove residual orthodontic composite from the enamel surface using a rapidly scanned carbon dioxide laser controlled by spectral feedback without perceptible changes to the enamel.

Materials and Methods: A carbon dioxide laser operating at a wavelength of 9.3 µm with a pulse duration of 10-15 µs and a pulse repetition rate of ~200 Hz was used to selectively remove composite from the polished buccal surfaces of 30 extracted human teeth. Laser parameters were tested to determine the ideal settings to remove composite with the least amount of enamel damage. GrenGlooTM composite was used to better visualize residual composite. The amount of enamel surface modulation/roughness was measured with a three dimensional digital microscope. A spectral feedback system utilizing a miniature spectrometer was used to control the laser scanning system.

Results: The amount of enamel lost with our final laser parameters averaged 2.59 ± 0.63 µm. The laser settings that produced the best results were the following: Spot size= 450 µm, Fluence= 4.0 J/cm2, Overlap= 50 µm. These enamel-conserving parameters were still able to remove composite at clinically relevant speeds.

Conclusions: Residual orthodontic composite can be quickly removed from the tooth surface using a rapidly scanned CO2 laser with spectral feedback with minimal damage to the underlying enamel surface.

Background: Evidence suggests that lower levels of morning energy are associated with higher levels of stress and lower levels of resilience in patients receiving chemotherapy. Purpose: Study purposes were to identify subgroups of patients with distinct morning energy profiles; evaluate for differences among the profiles in demographic and clinical characteristics, as well as measures of stress, resilience, and coping. Methods: A total of 1,343 outpatients receiving chemotherapy completed a demographic questionnaire and measures of global, cancer-related, and cumulative life stress, and resilience at study enrollment. Morning energy was assessed using the Lee Fatigue Scale at six time points over two cycles of chemotherapy. Results: Latent profile analysis was used to identify subgroups of patients with distinct morning energy profiles. Three morning energy profiles were identified (i.e., High (17.3%), Low (60.3%), Very Low (22.4%)). Compared to High class, the other two morning energy classes were less likely to be employed; had a lower functional status and a higher comorbidity burden; and were more likely to self-report depression and back pain. For all three types of stress, significant differences were found among the three classes with scores that demonstrated a dose response effect (i.e., High < Low < Very Low; as decrements in morning energy increased, stress scores increased). Compared to High class, Very Low class reported higher rates of physical and sexual abuse. The resilience scores exhibited a dose response effect as well (i.e., High > Low > Very Low). Patients with the two worst energy profiles reported a higher use of disengagement coping strategies. Conclusions/Implications for Practice: Findings highlight the complex relationships among decrements in morning energy, various types of stress, resilience, and coping in patients undergoing chemotherapy. Clinicians need to assess for stress and adverse childhood experiences to develop individualized management plans to increase patients’ energy levels.

Caries Management by Risk Assessment in Children

Paul Aaron Johnson

Abstract

Purpose: To investigate the efficacy of a modified caries management by risk assessment (CAMBRA) protocol on reducing the cariogenic bacterial load, improving oral hygiene care and dietary habits on children aged 5-9 years old.

Methods: Sixty-six children aged 5-9 years old participated in a single blind randomized controlled clinical trial and were assigned to either the modified CAMBRA or regular treatment group. Parents of intervention group were asked to give child 6-8g of xylitol mints per day (8-12 mints/day) for a total of six months. These individuals were seen every 3 months for fluoride varnish application, oral hygiene and diet evaluation with counseling. Additional diet and oral hygiene information was sent home to parents. dmfs/DMFS scores, caries risk assessment, saliva samples were taken at baseline and at 6 months and MS, LB and TVC of bacterial levels tabulated.

Results: MS, LB and TVC at baseline and 6 month showed slight decrease but there was no statistical significance between the modified CAMBRA and regular treatment groups p<0.05. There was consistent trend of decreasing caries risk factors (tooth brush and snacking frequency, plaque scores) from baseline to 6 months however these changes between the two groups were not statistically significant as well.

Conclusion: 6-8g of daily xylitol mints show a minimal, but not statistically significant decrease in cariogenic bacterial load in the oral cavity. Increased frequency of professional oral hygiene and diet counseling shows a positive trend in decreasing caries risk behavior at six months in this population.

Background and Significance: The subject of human rights discourses and humanitarian intervention, limb disability and amputation are among the leading causes of disability in the developing world. From a comparative perspective, over 80% of the world’s amputees, yet only 2-5% have access to appropriate prosthetic and rehabilitative services. Aims: 1.) To investigate discursive constructions of (im)mobility and the value of prostheses from the perspective of humanitarian mobility workers and 2.) to examine representational politics and the moral economy of prosthetic limb supplementation in the developing world. Research Questions: This dissertation asks, “What are ways in which humanitarian workers conceive of mobility, and why are they invested in mobilizing amputees in the developing world? How do they create meaning, purpose, and value for themselves and potential donors?” Sample: Four U.S.-based humanitarian mobility organizations served as discursive sites for my study. Methods: From January 2016-2018, I pursued three strands of qualitative research: 1.) Twenty hours of ethnographic fieldwork at five humanitarian fundraising events, 2.) twenty-five hours of primary semi-structured qualitative interviews with sixteen U.S. mobility workers and board members from the four organizations of study, two informal interviews, and analysis of transcripts from four secondary interviews, and 3.) discourse analysis of one monograph, 60 blogs and social media posts, 20 photographs, and over three hours of promotional and documentary video footage. I analyzed all data using situational analysis. Findings: Study participants characterized mobilities as manifold, drawing upon their own experiences of immobility to mobilize others. Moreover, they confronted their privilege and criticized the social, cultural, and economic forces that generate and perpetuate immobility in the developing world; their initial tourist expeditions evolving into activist undertakings. Finally, they leveraged distinct representational strategies to appeal to an imagined, able-bodied, American donor public and garner funds to mobilize their work. Conclusions: This research offers a nuanced portrait of (im)mobility in the lives of humanitarian mobility workers, shifting the gaze from amputees in the developing world to the movements and emotional attachments of mobility workers themselves.

Major depressive disorder is one of the most common and debilitating psychiatric disorders. Psychopharmacological agents are the most widely used form of treatment, although they are not universally effective and can produce significant side effects in some patients. The most common psychopharmacological agents used to treat major depression are the selective serotonin reuptake inhibitors, or SSRIs. Often, these drugs take several weeks to relieve depressive symptoms. Individualized therapy would have great clinical utility by identifying patients that are likely to respond positively to SSRI therapy a priori. The goal of this thesis is to investigate the use of genetic markers for guiding treatment with SSRIs.

We utilized several complementary pharmacogenetic approaches and two depressed populations treated with SSRIs. The first was a small (N=96) population given fluoxetine, and the second was a large (N=1,953) population taking citalopram. We used the fluoxetine population and a variant discovery approach to uncover novel variation and previously unknown tagging SNPs in the molecular target of SSRIs, the serotonin transporter, then employed a linkage disequilibrium mapping approach to investigate variants for association to response. Several variants in the promoter region of the gene were associated with fluoxetine outcome. No markers were associated with response when investigated in our citalopram population.

We also investigated relevant candidate genes for association with citalopram response and tolerance. Variants within the FEV gene, a master transcription factor in the serotonin pathway, were associated with a number of response phenotypes and mouse work implicates this gene in citalopram response. None of our other candidate genes demonstrated association with citalopram response.

Utilizing a panel of approximately 20,000 non-synonymous cSNPs for association with citalopram response, one SNP in the gene LRP2 was significantly associated with response in the African American population. We also performed a whole genome association study using over 500,000 SNPs from across the genome. Using a two-stage study design, none of the most highly associated markers in the discovery sample were also associated in the validation sample.

Staphylococcus aureus (S. aureus) is a gram-positive bacterium that can cause severe infections such as pneumonia, osteomyelitis, and endocarditis when it breaches the skin. This study aimed to enhance the chemoenzymatic radiosynthesis of 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK), a radiotracer potentially useful for imaging S. aureus infections. By optimizing the synthesis of 2-deoxy-2-[19F]-fluoro-sakebiose ([19F]FSK), we identified key factors—such as increased enzyme concentration and decreased precursor levels—that significantly improved the yield. Applying these optimized conditions to the synthesis of [18F]FSK resulted in a 30% increase in the radiochemical yield (RCY%) from the control experiment. In vitro evaluation showed that [18F]FSK was successfully incorporated into two strains of S. aureus, suggesting its potential utility for imaging bacterial infections in vivo. This work lays the groundwork for using [18F]FSK in PET/CT imaging to diagnose and monitor S. aureus infections.

This dissertation describes the research carried out within the scope of two projects that deal with novel biomedical technologies and their use for advancing medical knowledge through statistical integration of genomics assays. I have explored and characterized epigenetic intratumoral heterogeneity and brain cancer evolution by creating customized statistical analyses and novel methodology for understanding and integrating RNA- seq, methylation arrays, and exome-seq data. To explore functional effects of the Ilf2 RNA-binding protein (RBP) through embryonic stem cell differentiation in mice, I created statistical pipelines to remove data- generation artifacts, applied various testing methods, and integrated the information utilizing small RNA-seq, ribosome profiling, and RNA-seq technologies.

Congenital heart defects (CHD) occur in nearly one percent of live births each year and are the leading cause of defect-associated infant mortality. In spite of the growing size of disease cohorts, the molecular underpinnings of most cases remain unexplained. Given its high recurrence rate in families, we expect much of this contribution to be found within patient genomes, but extensive genetic heterogeneity limits our ability to statistically confirm risk loci. Previously-validated causal mutations occur in a wide range of genes that encode for proteins in signaling and migration, chromatin remodelers that induce lineage specification, and transcription factors regulating the expression of these genes. In order to identify cryptic risk loci, my thesis has focused on creating novel computational approaches to overcome statistical challenges and broaden our understanding of the mechanisms that can lead to CHD. By integrating protein-protein interaction networks of cardiac transcription factors with whole exome sequencing, I showed that interactors are enriched for rare and de novo mutations in CHD patients. I developed a variant prioritization scheme for de novo variants, which identified a GLYR1 mutation that destabilizes its interaction with cardiac transcription factor GATA4. I describe GCOD, a novel algorithm that uses probabilistic modeling to identify sets of genes predicted to interact in the etiology of CHD, including a novel genetic interaction between GATA6 and POR. Finally, in addition to coding mutations, I aimed to assess whether disruption to chromatin organization contributes to disease by characterizing three CHD patient variants that I predicted would alter the regulatory landscape of heart-relevant genes. My work has increased our repertoire of known and suspected disease loci in CHD and related developmental co-morbidities, and provided evidence of oligogenic combinations and disrupted genome folding as a mechanism in CHD.

Protein turnover through endolysosomal degradation and the ubiquitin proteasome system are critical for maintaining protein homeostasis. These pathways protect the cell from accumulation of misfolded proteins, coordinate critical signaling processes, and facilitate recycling pathways that are central to cellular health. An integral member of these systems is the E3 ubiquitin ligase CHIP, which facilitates the turnover of damaged or terminally misfolded proteins. Canonically, substrate recognition by CHIP is dependent on Hsp70 or Hsp90 chaperones, which serve as intermediaries between a misfolded client and CHIP. However, emerging evidence suggests that CHIP also has the capacity to recognize substrates independent of a chaperone binding partner. In this dissertation, we explore the biological relevance of such chaperone-independent substrates by exploring interactions predicted by a biophysical scoring function we previously developed, termed CHIPscore.

In the first chapter, I and others describe one such interaction between CHIP and the relatively uncharacterized, membrane-anchored protein CHIC2. We find that CHIC2 binding strongly attenuates CHIP activity, and that CHIC2 knockout phenocopies CHIP knockout in certain cell types, implying that chaperone-independent interactions can sometimes predominate CHIP’s biological functions. Furthermore, loss of the CHIP-CHIC2 interaction induces neurodegeneration and shortens lifespan in C. elegans, demonstrating that formation of this chaperone-independent complex is important in animals. We propose that CHIC2 attenuates CHIP activity at the membrane, offering a novel mechanism by which this ubiquitin ligase can be regulated.

In the second chapter, I explore additional chaperone-independent interactors beyond CHIC2 that are predicted by CHIPscore. In preliminary results, I demonstrate interactions between CHIP and three additional proteins. These proteins are completely uncharacterized and reside at different subcellular localizations, suggesting that CHIP may be regulated at various locations within the cell through mechanisms that are not yet understood. This work opens substantial new avenues upon which future studies should be based.

During the past century the field of dental care has evolved to be stable, mature, and highly oriented to a set of institutional logics, particularly entrepreneurship and autonomy, that support the traditional organizational archetype (ideal type model) of solo private-practice dentistry. Since the 1970s, dental hygienists have been working to realign the occupational structure in oral health by challenging both the professional dominance of dentistry and the traditional organization of dental care services.

The study combines insights from the sociology of the professions on the agency of emerging occupations seeking to advance a professional project with insights from organization theory on institutional entrepreneurship to explore a case study of the development of Registered Dental Hygienists in Alternative Practice (RDHAP) in California. This mixed-method case study was developed from qualitative data, including key informant interviews, archival data, media analyses, and two quantitative surveys examining the RDHAP work force. A theoretical model for understanding the institutional agency of emerging professions was developed that provides a link between the study of professions and the study of organizations in health care.

This study explores the movement for independent hygiene practice, which entailed a politicized battle between organized dentistry and dental hygiene that played out in their respective professional associations, the legislature, the courts, the business community, and the media. This conflict led to the development of a new category of hygienists who are allowed to work independently in underserved areas and to the introduction of an alternative practice archetype that now co-exists in the field and is underpinned by a unique interpretive scheme (values and beliefs), structures, and systems. The alternative archetype is built on providing mobile preventive dental hygiene care in a collaborative manner within underserved communities and institutions. This new organizational form has not yet been fully institutionalized as it does not yet have the resources and legitimacy of the traditional model. Expansion of the specific model is hampered by a regulatory structure that inhibits the diffusion of innovation across state lines; however, similar efforts are underway in other states, contributing to the wider adoption of this general practice model.

In order to win the battle to end the global tuberculosis (TB) epidemic, we need new and better tools. Approximately one-third of the world's population is infected with the TB bacterium, and in 2015 over 10 million people become sick with the disease and almost 2 million people died, meaning TB is now one of the top ten causes of disease worldwide, outranking HIV and malaria. In addition, over 40% of TB cases each year do not get diagnosed or notified. These missed or delayed diagnoses lead to increased morbidity and mortality for those suffering from the disease, and help drive the TB epidemic through continued transmission. The World Health Organization’s (WHO’s) new “End TB Strategy” envisions a 90% reduction in TB incidence by 2035, but in order to achieve this target there is an urgent need to develop, evaluate, and deploy strategies to improve TB case detection in high burden settings.

There are numerous barriers to TB case detection, operating at several interacting levels. Those at highest risk of falling ill with TB are disproportionately from poor, disadvantaged, and marginalized populations that are much more likely to face barriers to accessing health care when sick. Those who do fall ill with TB can face numerous economic, geographic, and social/cultural barriers before reaching health care. And once they do reach care, patients can still be hampered by significant provider and health system barriers.

To address some of these barriers to TB case detection, my dissertation research focuses on three perspectives on improving case detection for TB: policy, implementation, and social implications. My first paper describes a tool I helped develop for the WHO called ScreenTB that assists National TB Control Programmes (NTPs) to develop TB screening programs well suited to their local epidemic by modeling the potential yields and costs of a variety of approaches to TB screening. My second paper presents the findings from a year-long pilot implementation of a TB screening intervention, “STAMP out TB”, in primary care clinics in Jakarta, Indonesia. My third paper explores the potential social implications of TB case-finding in Dar es Salaam, Tanzania.

Together these papers contribute to the field of TB case detection by providing tools for, and information about, the development and implementation of TB case-finding strategies in high burden settings. These three papers also characterize the diversity of my work in this field, as they each represent a distinct skillset that I have acquired during my time at UCSF. Put together, these papers will demonstrate that I have mastered an array of different tools and approaches used in the field of global infectious disease epidemiology.

Introduction: The purpose of this study was to explore palliative care provider self-competence, ethical concerns and priorities for education and training in an inpatient hospice setting in Kenya. Momentum is building in sub-Saharan Africa to improve the provision of palliative care services. However, provider perspectives are poorly understood. We hypothesized that mean self-competence and ethical concerns scores would differ according to provider type. Methods: A descriptive, cross-sectional study design was piloted at Kimbilio Hospice, a 26-bed, rural, inpatient facility in Kenya. A quantitative survey instrument entitled, "Self assessment of clinical competency and concerns in end-of-life care," was adapted for this setting and administered to participants by two research interviewers. Survey responses were collected from clinical staff, caregivers and support staff. Self-competence scores were reported as means for each variable in the Clinical Care and Patient and Family Interactions categories. Ethical concern scores were grouped by clinical scenario and ethical concern type. Data was analyzed using Kruskal Wallis to compare between means according to provider type. Results: The sample included 24 providers, with five clinical staff, 11 caregivers and eight support staff. Kruskal Wallis testing revealed statistically-significant differences between mean scores in five self-competence variables: performing a basic pain assessment, use of oral opioid analgesics, assessment and management of nausea/vomiting and constipation, and discussing an end-of-life prognosis with a patient's family member (p<0.05). The highest mean self-competence score for clinical staff was 4 for the use of oral opioid analgesics. The lowest scores were 2.8 for discussing inpatient hospice referral, use of IV opioid analgesics and assessment of pain in pediatric patients. Palliative care staff had the highest mean ethical concern score that withdrawing ventilator support from a dementia patient at the request of a family member would violate ethical norms of the community (mean=3.29, SD=1.04). Sixteen providers (21%) selected pediatric palliative care as their top priority for future education. Discussion: Our results support the hypothesis that palliative care providers have varying levels of self-competence. Improving educational and training programs to build palliative care competencies in pediatric palliative care and ethics in sub-Saharan Africa are recommended based on these findings.

Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder that is difficult to study and treat despite decades of progress. This is due to disease heterogeneity, lack of precise phenotyping, and limited understanding of molecular mechanisms underlying clinical manifestations. Electronic medical records (EMR) are emerging as a real-world dataset with abundance of longitudinal human data across diagnoses, medications, and measurements with opportunity to derive insights without predefined selection criteria or limitations in scope. Recent developments of integrative heterogeneous graph databases that combine knowledge across omics relationships provide a means to further identify molecular hypotheses underlying complex clinical phenotypes. We performed deep phenotyping to characterize AD and sex differences in the EMR against a control cohort, and identified sex and AD associated comorbidities, medication use, and lab values. Extending this work to apply machine learning, we utilize clinical information to predict AD onset and identify prioritized genes via knowledge networks (e.g., APOE, ACTB, IL6) and genetic colocalization analysis (e.g., MS4A6A with osteoporosis). Our findings suggest that AD onset risk can be predicted based on clinical data and that there are sex-specific relationships in AD including musculoskeletal disorders among females with AD and neurological or sensory disorders among males with AD. Extensions to knowledge networks and molecular datasets further prioritize genes depending on an individual’s comorbid conditions. By leveraging clinical data to identify hypotheses for complex disease, we can further make steps towards better understanding molecular mechanisms and advance personalized treatment approaches in AD.