UCSF

cGMP is a ubiquitous second messenger implicated in a multitude of neurobiological processes, including sensory transduction, learning and memory. FRET-based and GFP-based cGMP reporters have been developed to complement the genetic and biochemical tools used to probe the role of cGMP in these processes. While FRET-based cGMP sensors have been expressed in C. elegans to explore the spatiotemporal regulation of cGMP in sensory neurons in response to stimuli, their use requires a dual emission system, which limits their ability to be coexpressed with other fluorophores, such as red calcium sensors that can indirectly read out neural activity. This work demonstrates that WincG2, a GFP-based cGMP sensor codon-optimized for use in C. elegans, can report changes in cGMP levels in living, behaving C. elegans. We demonstrate that coexpression of WincG2 and light-activatable guanylyl cyclases in body wall muscle cells results in an increase in WincG2 fluorescence upon light exposure that corresponds with the rate of cGMP production. Furthermore, WincG2 fluorescence changes in the cell bodies of the gustatory neuron ASER and the phasmid neuron PHB in response to [NaCl] step changes and sodium dodecyl sulfate, respectively. This provides the first visual evidence that suggests GMP levels change in these neurons in response to stimuli. Intriguingly, preliminary data suggest that in ASER, cGMP levels decrease linearly in the cell body while increasing transiently in the cilia in response to a [NaCl] downstep, which could have implications for cGMP’s potential role in both sensation and memory in ASER. Finally, we demonstrate that cGMP could act as a neuromodulator in a nociceptive neural circuit. WincG2 fluorescence increases in the nociceptive neuron ASH - which is not known to express guanylyl cyclases - in the absence of food while remaining relatively constant in the presence of LB. These results suggest that cGMP could be flowing from other neurons into ASH to signal food status, resulting in the modulation of ASH activity. Taken together, this work demonstrates that WincG2 could be used the uncover cGMP’s role in diverse neurobiological processes in living, behaving C. elegans.

Giardia lamblia is a eukaryotic protozoan parasite and the causative agent of giardiasis, a debilitating enteric disease resulting in much morbidity and mortality worldwide. It is of interest not only as a target for the development of improved giardiasis therapies, but also as a model eukaryotic system. Giardia represents the earliest branching clade of eukaryotic cells. It is thus an ideal system for investigating the evolution of cell processes, organelle compartmentalization, and critical protein families. Analysis of the structure and function of the Giardia endomembrane system, cysteine proteases, and clathrin orthologues are the focus of this dissertation project.

The ER has been studied recently as a putative endocytic organelle. Examination of Giardia endocytosis using fluorophore-labeled proteins revealed that proteins were rapidly trafficked to a tubulovesicular network with ER-like properties. Using reporter constructs, cysteine proteases that are orthologues of lysosomal hydrolases were localized to the same tubulovesicular network. Functional protease assays helped define the role that cysteine proteases play in the degradation of exogenous proteins. Organelle-specific markers were used to describe the tubulovesicular compartment in which endocytosis and subsequent proteolysis takes place.

Cysteine proteases have been implicated in life cycle transitions (encystation and excystation) of Giardia. The completion of the Giardia genome indicated that there are twenty-seven cysteine protease genes in Giardia. Cysteine protease 2 (GlCP2) was identified as the major expressed cysteine protease gene in Giardia. Biochemical analysis of heterologously expressed GlCP2 suggested that this gene indeed plays an important role in both encystation and excystation.

Giardia clathrin is also key to the processes of endocytosis and encystation. Clathrin is associated with the peripheral vacuoles of vegetative Giardia and may facilitate the inital endocytic uptake of proteins. During encystation, clathrin localizes to encystation specific vesicles and may function in cyst formation. A dominant negative clathrin heavy chain disrupted cyst formation but did not affect endocytosis.

Computational approaches for binding affinity prediction are most frequently demonstrated through cross-validation within a series of molecules or through performance shown on a blinded test set. Here, we show how such a system performs in two realistic applications: 1. An iterative, temporal lead optimization exercise, and 2. A hybrid strategy that leverages diversified information as input. In the first evaluation, a series of gyrase inhibitors with known synthetic order formed the set of molecules that could be selected for "synthesis." Beginning with a small number of molecules, based only on structures and activities, a model was constructed using the newly developed Surflex-Quantitative Modeling (QMOD) approach. Compound selection was done computationally, each time making five selections based on confident predictions of high activity and five selections based on a quantitative measure of three-dimensional structural novelty. Compound selection was followed by model refinement using the new data. Iterative computational candidate selection produced rapid improvements in selected compound activity, and incorporation of explicitly novel compounds uncovered much more diverse active inhibitors than strategies lacking active novelty selection.

For the second evaluation we present a hybrid structure-guided strategy that combines molecular similarity, docking, and multiple-instance learning such that information from protein structures can be used to inform models of structure-activity relationships. The Surflex-QMOD approach has been shown to produce accurate predictions of binding affinity by constructing an interpretable physical model of a binding site with no experimental binding site structural information. Here we introduce a methodological enhancement to integrate protein structure information into the model induction process in order to construct more robust physical models. The structure-guided models accurately predict binding affinities over a broad range of compounds while producing more accurate representations of the protein pockets and ligand binding modes. Structure-guidance for the QMOD method yielded significant performance improvements, especially in cases where predictions were made on ligands very different from those used for model induction.

Potassium (K+) channels are membrane-embedded proteins that selectively pass K+ ions in or out of cells in response to a variety of signals, such as membrane potential changes or binding of ligands. In an excitable cell, such as a neuron or cardiac muscle cell, delayed rectifier voltage-gated K+ channels respond to changes in membrane potential to restore the cell membrane to its resting state after an action potential.

In vertebrates, voltage-gated K+ (Kv) channels are tetramers of similar or identical subunits arranged around a central conducting pore. While these channels are primarily gated by membrane potential, their biophysical properties are set by the type of subunits in each tetramer and by interactions with other effector molecules, such as membrane phospholipids, calcium-binding proteins, kinases, and scaffolding proteins. In some cases, discrete intracellular domains control the specific assembly of pore-forming and accessory proteins. However, the molecular mechanisms that direct specific assembly of this wide range of components into a functional K channel complex are incompletely understood.

Chapter 2 of this thesis establishes the atomic-resolution structure of one such assembly domain from a Kv7 family channel (Kv7.4). This study suggests the structural basis for specific assembly properties and binding of scaffolding proteins by other members of the Kv7 channel family. Additional studies in Chapter 3 explore implications of the Kv7.4 assembly domain structure for oligomerization in other subtypes. The biochemical and functional effects of Kv7 mutations designed to disrupt or enhance assembly domain oligomerization further support the critical role for this domain in specific assembly of these channels.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to cause significant morbidity and mortality worldwide. Although most infections are mild and a majority of patients recover, some experience severe and often fatal systemic inflammation, cytokine storm, and acute respiratory distress syndrome. The innate immune system of the human body is the first line of defense against SARS-CoV-2, sensing the virus through pattern recognition receptors and activating inflammatory cascades that promote viral clearance. Simultaneously, the virus has evolved numerous strategies to escape detection and surveillance of the immune system for successful replication. An improved understanding of innate immunity and viral evasion strategies will help identify targeted therapies to mitigate disease and improve patient outcome. Here, we report two cellular epigenetic proteins, BRD4 and SIRT5, as anti- and proviral binding partners of SARS-CoV-2 envelope (E) and non-structural protein Nsp14, respectively. We identify bromodomain and extra-terminal (BET) proteins as critical antiviral factors as genetic or chemical inactivation of BRD4 exacerbates viral infection in cells and enhanced mortality in mice. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the “histone mimetic” E protein, supporting a model where the E protein evolved to antagonize the innate immune system via BET protein inhibition. Conversely, genetic or chemical inactivation of SIRT5 reduces SARS-CoV-2 replication in cells. While SIRT5 interacts with Nsp14 through its catalytic domain, Nsp14 does not appear to be directly deacylated by SIRT5. Depletion of SIRT5 results in higher basal levels of innate immunity and a stronger antiviral response during infection, indicating SIRT5 is a proviral factor necessary for efficient viral replication. Lastly, we compared the humoral immune responses elicited by SARS-CoV-2 variants, WA1, Delta, and Omicron. We show that without vaccination, infection with Omicron induces a limited humoral immune response in mice and humans. In contrast to WA1 and Delta, Omicron replicates at low levels in the lungs and brains of infected mice, leading to mild disease with reduced expression of proinflammatory cytokines, diminished activation of lung-resident T cells, and limited cross-variant immunity against non-Omicron variants in unvaccinated individuals. Collectively, these findings advance our understanding on the various host-pathogen interactions that need to be considered in designing novel SARS-CoV-2 therapeutics.

Eukaryotic genes are littered with intervening sequences, or introns, that are transcribed, but must be precisely excised from a messenger RNA before it can be properly translated into protein. While introns were once regarded as "junk DNA," they are not inconsequential. However, we currently lack the knowledge to accurately predict the functions, if any, of individual introns in any organism. Here, I describe efforts to better understand the evolution and function of introns, with the vision that we will soon be able to identify important introns and predict their functions.

In Chapter 2, my colleagues and I describe an unexpected consequence of intron presence on chromatin structure, which suggests that introns have a broader influence on the biology of eukaryotes than previously appreciated. By analyzing published surveys of nucleosomes and 41 chromatin marks in humans, we show that 5' intronic and 3' exonic regions of active genes are differentially marked by characteristic chromatin marks, thus contributing substantially to the patterns of histone modifications within active genes. Intriguingly, these modification patterns were stable despite dramatic changes in the frequency of splice site usage at two alternative spliced genes. Thus, similar to promoter marks, which are relatively stable to differences in productive transcription, we propose that intronic and exonic chromatin marks reflect exon definition, rather than splicing per se.

In Chapter 3, I describe my work to better understand why certain introns persist in eukaryotic genomes. Using comparative genomics, I show that the ribosomal protein genes of Saccharomyces cerevisiae have greatly resisted intron loss. Mimicking the effect of intron loss with directed mutagenesis, I perform experimental tests that demonstrate that these introns do not promote gene expression, but rather, provide a means for regulation. Specifically, I show that the genes encoding ribosomal protein S9, both in S. cerevisiae and Drosophilia melanogaster, autoregulate in an intron-dependent manner. Lastly, I summarize published gene expression data from diverse animals, which suggest that multiple forms of alternative splicing have evolved to autoregulate S9 gene expression. Thus, I propose that the introns of eukaryotic genes persist, in part, due to their propensity to evolve regulatory function.

Older Chinese Americans often defer end-of-life (EOL) care discussions. Moreover,healthcare providers find facilitating EOL care discussions challenging, especially with patients whose ethnicities differ from their own. Currently, there is still little guidance on how to initiate and facilitate such discussions with older Chinese Americans and their families. In order to increase the engagement of older Chinese Americans, families and healthcare providers in EOL care discussions, this dissertation aims to (1) identify prevalence of EOL care discussions among Chinese and Chinese-American populations, (2) understand the facilitators and barriers related to EOL care discussions with older Chinese Americans and their families, (3) explore potential strategies to facilitate EOL care discussions with older Chinese Americans and their families.

To address the aims, an integrative literature review and a qualitative study that utilized focused ethnography were conducted. In the qualitative study, individual, semi-structured interviews were conducted with 14 community-dwelling older Chinese Americans, nine adult

children, and seven healthcare providers. The data were analyzed using thematic and constant comparative analysis.

The results of this dissertation suggest that Chinese-American populations experience similar facilitators and barriers to EOL care discussions as do Chinese populations who reside in other countries. Superstition, societal taboos related to death and dying and family objections were identified as the common barriers to EOL care discussions. However, older Chinese Americans and their adult children would discuss EOL care when the discussion was introduced at “optimal times,” which included after triggering events (e.g., death of loved ones, fall accidents), with changes in health status, or with advanced age. Older Chinese Americans, adult children and healthcare providers emphasized the importance of assessing readiness for EOL care discussions and they all recommended using indirect communication approaches.

In conclusion, assessing readiness to discuss EOL care should be an essential and necessary action to initiate end-of-life care

discussions with older Chinese Americans. Healthcare providers should proactively assess their patients’ readiness to discuss EOL care, and engage them in EOL care discussions during identified optimal times.

The ability to coordinate movements involving multiple body parts is fundamental to behavior. How the brain coordinates several body parts with different ranges of motion and reference frames is unknown. Here we investigated this problem by studying the neural circuits and computations underlying coordinated head and eye movements in mice. It is commonly thought that neural control of coordinated movements entails a hierarchical series of computations that progress from specifying higher- to lower-level movement parameters. Here we reveal a different computational logic implemented in the mouse gaze system. We found that single neurons in the mouse superior colliculus (SC) specify a mixture of eye movement endpoints and head movement displacements. They do so by innervating two separate hindbrain populations that transform identical excitatory input into eye movements with fixed endpoints and head movements with fixed displacements. Neural recordings showed that head displacement information is present in SC, whereas saccade information does not emerge until the hindbrain. Thus, displacements for the head and eyes are computed at different anatomical stages and the desired overall movement is nowhere represented. These results reveal a non-hierarchical computational logic for coordinated movements.

Innate effector lymphocytes, including invariant natural killer T-cells (iNKT), are conserved and integral components of the vertebrate immune system that orchestrate the early host response to infection, yet the mechanisms by which developing thymocytes acquire either a naïve or innate effector identity remain unclear. Here we report that histone deacetylase 7 (HDAC7), a highly conserved signal-dependent transcriptional corepressor abundantly expressed in thymocytes, is a crucial regulatory factor that licenses innate effector development in iNKT cells. In a gain-of-function setting where HDAC7 is constitutively nuclear localized, innate effector development is blocked and iNKT cells become diverted to extremely rare, naïve-like T-cells with limited cytokine production and propensity to recirculate. Conversely, in a loss-of-function setting where HDAC7 is removed via conditional genetic deletion, naïve T-cell development is impeded and more thymocytes acquire an innate effector identity, particularly in an Eomesodermin-expressing CD8 peripheral subset that resembles so-called “innate memory” T-cells. Regulation of this fate decision hinges on the ability of HDAC7 to antagonize the transcriptional activity of Promyelocytic Leukemia Zinc Finger (PLZF), a signature innate effector transcription factor of innate effector development, which we demonstrate occurs in part through upstream transcriptional repression and direct physical binding. Finally, we find that in mice with a gain-of-function HDAC7 transgene which spontaneously develop tissue-specific autoimmunity directed mainly against the hepatobiliary tissues and gastrointestinal mucosa, restoring iNKT cells in vivo can mitigate tissue destruction and reduce mortality rate. These studies identify HDAC7 as an important epigenetic licensing factor that controls naïve versus innate effector development in thymocytes and implicates a heretofore underappreciated role for innate effectors in protecting against autoimmune disease.

Detailed knowledge of folding intermediate and transition state (TS) structures is critical for understanding protein folding mechanisms. For kinetically-stable proteins such as α-lytic protease (αLP) and its family members, their large free energy barrier to unfolding is central to their biological function. Thus their TS structure plays a crucial role in protein function. However, structural information regarding this important state has been completely lacking, mainly because standard techniques to probe TS structure are not realistically applicable for αLP. Therefore, I used the information embedded in the sequence of homologous proteases to discern the physical mechanisms by which kinetic stability can be modulated. This required experimental validation using various biophysical and biochemical techniques, such as mutagenesis, x-ray crystallography, and detailed kinetic analyses.

From these studies, I have shown that the conserved distortion of a sidechain significantly contributes to the destabilization of the TS for a large sub-class of αLP homologs. The strain from this deformation actually provides a biological advantage in that lifetime is greatly extended. This study was the first that shows that sidechain distortion has been shown to be used for a functional purpose and uncovers an unanticipated challenge for structural biology to identify potentially relevant distortions from high resolution structural studies.

My structural and kinetic analysis of a acid resistant αLP homolog, Nocardiopsis alba Protease (NAPase), identified the physical basis for this proteins acid stability, thus providing crucial structural information about unfolding mechanisms and leading to a model for the TS structure for these proteases. This study provided insight into the evolutionary benefits of kinetic stability as a paradigm for generation of extremophilic behavior.

From a similar study of a thermophilic αLP homolog, Thermobifida fusca Protease A (TFPA), I identified a substructure of these proteases, termed the domain bridge, which is used to modulate the degree of kinetic stability. This study refined our model for the unfolding TS, in which the domain bridge undocks and unfolds allowing the two domains of the protease to separate, with the newly formed crevice filling with solvent. These studies represent the first physical understanding of the structural basis for kinetic stability.

Intervertebral disc degeneration is a contributing cause for many spine-related disabilities, and is a significant health concern in the United States. Currently available treatments aim to gain symptomatic relief, and do not treat the underlying biological problem. Cell therapy aims to target the cause of disc degeneration by repopulating the disc with cells capable of synthesizing appropriate matrix, restoring its biomechanical properties. The goal of this research was to investigate the use of mesenchymal stem cells (MSCs) for nucleus pulposus regeneration.

An in vivo injection model was used to characterize the regeneration potential of undifferentiated MSCs, juvenile chondrocytes, and fibrin carrier in rat coccygeal discs. Juvenile chondrocyte-injected discs displayed significantly improved MRI index over fibrin-injected discs, and histology indicated that injecting a more mature cell type resulted in maintenance of normal disc morphology.

To advance our understanding of the phenotypic changes that might arise in MSCs after transplantion to the disc environment, a novel bioreactor system was developed that mimics they hypoxia and hydrostatic pressure found in the intervertebral disc. Culturing MSCs in this system demonstrated that the physiochemical environment of the disc is sufficient to induce differentiation of MSCs, as evidenced by GAG production and increase in chondrogenic genes such as aggrecan, Sox9, and collagen II.

Pre-treatment of MSCs with TGF-β3 prior to bioreactor culture indicated that differentiated cells may function more effectively in disc-mimetic conditions, as evidenced by increased cell viability, GAG production, and persistence of TGF-β3-induced gene expression. More importantly, environment-dependent differences in gene expression highlighted the necessity of mimicking several aspects of the disc environment in concert when evaluating the fate of cells post-implantation. To better mimic the degenerated disc environment, inflammation was incorporated into the bioreactor. Expression levels of aggrecan, collagen II, and collagen X suggest that when MSCs are appropriately pre-differentiated and evaluated in a disc-mimetic environment, inflammation may not be as detrimental as seen in previous in vitro experiments.

This research highlights the importance of representing the salient features of the anticipated environment when evaluating cells for in situ disc regeneration, and demonstrates that MSCs may be optimized for nucleus pulposus regeneration.

Genomic variation does not only include nucleotide changes, it also comprises changes in DNA shape, structure, epigenetic marks, and expression, all of which can occur over generations, cellular differentiation, the span of a few hours or a few millennia. This doctoral thesis explores the implications and opportunities presented by these multiple forms of genomic variation for genome editing, cellular differentiation, genome regulation and comparative genomics, all towards improving our understanding of genome evolution and development and benefiting human health.

Cells have evolved proteins that detect foreign DNA, RNA, or proteins which then activate cellular pathways to combat bacterial or viral infection.1–4 Apolipoprotein B editing complex 3 (APOBEC3 or A3) is a host cytidine deaminase that deaminates cytidine to uridine in viral DNA in the cytoplasm causing hyper G→A mutation leading to destabilization and degradation of the viral genome.5–8 HIV viral infectivity factor (vif) has evolved to regulate A3 degradation of viral DNA.9 Vif hijacks host ubiquitination machinery to degrade A3s and prevent packaging of A3 into the viral particle.10,11 Vif hijacks the cotranscription factor core factor binding unit beta (CBF-beta), Elongin B (ELOB) and Elongin C (ELOC) to form the VCBC complex.12–15 VCBC binds A3s and Cullin 5 (Cul5) for ubiquitination and degradation of A3 thereby preventing packaging.12,16 Antigen binding fragments (Fabs) were generated against VCBC using a naïve B-cell Fab phage display library to isolate biological tools that are specific for the host-virus interaction.17 Two high affinity Fabs were found to bind at distinct epitopes on VCBC and produced distinct phenotypes when expressed in cells as single chain variable fragments (scFvs).17 The Fab 3C9 shields A3F from ubiquitination and restores packaging of A3F into the viral particle. The Fab 1D1 blocks binding of Cul5 and ubiquitination in vitro. The scFv 1D1 prevented ubiquitination of A3F but did not restore packaging. Affinity purification mass spectroscopy (AP-MS) in HEK293Ts with 3C9 scFv and 1D1 scFv showed different interactomes in the presence of vif. AP-MS with 1D1 scFv did not interact with CBF-beta, an important component of the VCBC complex. Spatial and temporal elucidation of proteins interacting with the complex will further determine events effecting viral infectivity.

TNF receptor family member HVEM is one of the most frequently mutated surface proteins in germinal center (GC) derived B cell lymphomas, yet the role of HVEM in normal GCs is unknown. We found that HVEM-deficiency intrinsically increased B cell competitiveness during pre-GC and GC responses. The Ig superfamily molecule BTLA was identified as the ligand regulating these responses, and B cell-intrinsic signaling via HVEM and BTLA was not required. Instead, BTLA signaling into the T cell through SHP1 reduced TCR signaling and the amount of preformed CD40L mobilized to the immunological synapse and thus diminished the help delivered to B cells. Moreover, T cell-deficiency in BTLA cooperated with B cell Bcl-2-overexpression in leading to GC B cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells in a manner that influences GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis.

Metformin is used first line for treatment of type 2 diabetes (T2D) and is one of the most frequently prescribed drugs worldwide. As the global incidence of T2D rapidly increases, the low cost of metformin makes this treatment option particularly attractive in developing nations. Understanding metformin’s efficacy in different patient populations with diverse genetic backgrounds will be critical in managing this deleterious metabolic disorder. The major goal of this dissertation research was to use novel, quantitative approaches to elucidate genetic and non-genetic components that predict metformin disposition and glycemic response. As a first goal, the role of transcription factor variants on metformin pharmacokinetics and pharmacodynamics was investigated. From this analysis, five variants in SP1 were significantly associated with changes in treatment HbA1c (p < 0.01) and metformin secretory clearance (p < 0.05). Genetic variants in transcription factors PPAR-alpha and HNF4-alpha were significantly associated with HbA1c change only, but were not significantly associated with pharmacokinetics. A plausible biological mechanism by which genetic variants affected the pharmacological variation of metformin was determined using gene expression levels linked to genetic variants (eQTLs). The focus was on transporter expression. From this study, we discovered that genomic regions proximal to metformin transporters were linked to expression levels of SLC47A1, SLC22A3, and SLC22A2, with a potential transcription factor-binding hypothesis for SP1. We also found variants in transcription factor HNF4-alpha were the most influential trans-eQTLs, accounting for expression level variation in both SLC47A1 and SLC22A1. Finally, we developed a mathematical model to quantify disease progression on metformin therapy using HbA1c data with the goal of explaining long-term HbA1c variability through the investigation of genetic, demographic, and clinical factors. From this analysis, we found two SNPs in CSMD1 (rs2617102, rs2954625) and one SNP in SLC22A2 (rs316009) as significantly influencing the long-term variance in HbA1c. Overall, this dissertation research enhances our current knowledge of the pharmacogenetic landscape by expanding the set of pharmacologically relevant genes and providing a pharmacokinetic and biological basis for some of these genes. Future research will continue to focus on replication and uncovering the mechanism driving the pharmacological genes highlighted here.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common known cause for Parkinson’s disease (PD) and are associated with several other diseases, including cancer and autoimmune disease. Excitingly, several strategies to modulate LRRK2 activity in the brain are currently being tested in clinical trials for PD. Yet, there remain many unanswered questions as to how LRRK2 mutations affect cellular homeostasis and ultimately cause disease. Increasing our understanding of the cellular biology surrounding LRRK2 function is certain to aid in the acceleration of drug discovery and development in PD. In Chapter 1, I broadly summarize the role of LRRK2 in PD and reported LRRK2 molecular and cellular functions. In particular, I describe the unanswered question of how PD-causing mutations in the kinase and GTPase domains of LRRK2 differentially affect observed LRRK2 kinase activity in cells. In Chapter 2, I present my published work detailing distinct cellular effects of PD-causing mutations in either the GTPase or kinase domain of LRRK2. Specifically, I found that GTPase-inactivating mutations strongly increase LRRK2 localization to endosomes upon inhibition of endosomal maturation. Under the same conditions, kinase-activating mutations only modestly affect LRRK2 localization and wild-type LRRK2 localization is minimally affected. I further demonstrate that the extent of LRRK2 endosomal localization is directly related to observed levels of LRRK2 substrate phosphorylation. Through this work, I provide a rationale for why PD-causing mutations across LRRK2 domains lead to differing levels of substrate phosphorylation in cells. Overall, my work highlights the importance of LRRK2’s GTPase domain for the protein’s cellular localization and activity and suggests that therapeutic strategies targeting LRRK2 GTPase function or localization may be beneficial for PD.

Effective and cost-efficient healthcare is at the forefront of public discussion; on both personal and policy levels, technologies that improve therapeutic efficacy without the use of painful hypodermic needle injections or the use of harsh chemicals would prove beneficial to patients. Nanostructured surfaces as structure-mediated permeability enhancers introduce a potentially revolutionary approach to the field of drug delivery. Parental administration routes have been the mainstay technologies for delivering biologics because these therapeutics are too large to permeate epithelial barriers. However, there is a significant patient dislike for hypodermic needles resulting in reduced patient compliance and poor therapeutic results. We present an alternative strategy to harness the body's naturally occurring biological processes and transport mechanisms to enhance the drug transport of biologics across the epithelium. Our strategy offers a paradigm shift from traditional biochemical drug delivery vehicles by using nanotopography to loosen the epithelial barrier.

Herein, we demonstrate that nanotopographical cues can be used to enable biologics > 66 kDa to be transported across epithelial monolayers by increasing paracellular transport. When placed in contact with epithelial cells, nanostructured films significantly increase the transport of albumin, IgG, and a model therapeutic, etanercept. Our work highlights the potential to use drug delivery systems which incorporate nanotopographical cues to increase the transport of biologics across epithelial tissue.

Furthermore, we describe current advancements in nano- and microfabrication for applications in anti-fibrosis and wound healing. Influencing cellular responses to biomaterials is crucial in the field of tissue engineering and regenerative medicine. Since cells are surrounded by extracellular matrix features that are on the nanoscale, identifying nanostructures for imparting desirable cellular function could greatly impact the field. Due to the rise in micro and nanofabrication techniques borrowed from the advances in the microelectronics industry, previously unattainable nanostructured surfaces on a variety of biomaterials can be generated. We investigated how nanostructured surfaces with varying nanofeature aspect ratios can influence fibrosis. Thus, nanostructured surfaces show substantial progress for therapeutic applications in drug delivery and wound healing.

Prostate-specific membrane antigen (PSMA) is a cell surface enzyme highly over expressed in prostate cancer cells that can be employed as a target for prostate cancer imaging and drug delivery. Boron Neutron Capture Therapy (BNCT) is an emerging noninvasive therapeutic modality for treating locally invasive malignant tumors by selective delivery of high boron content to the tumour and then subjecting the tumour to epithermal neutron beam radiation. In this study, we develop carborane encapsulated amphiphilic polymer nanoparticles by conjugating urea based PSMA inhibitors (ACUPA) and 89Zr chelating deferoxamine B (DFB) ligand and have investigated their efficacy to deliver enhanced boron payload to PSMA positive prostate cancer cells with simultaneous positron emission tomography (PET) imaging . Three different carborane encapsulated PLGA-b-PEG nanoparticles (NPs) were formulated with and without the PSMA targeting ligand, out of which two selected formulations; DFB(25)ACUPA(75) NPs and DFB(25) NPs radiolabelled with 89Zr were administered to mice bearing dual PSMA(+) PC3-Pip and PSMA(-) PC3-Flu xenografts. PET imaging and biodistribution studies were performed to demonstrate the in vivo uptake in mice. The NPs showed 2-fold higher uptake in PSMA(+) PC3-Pip tumors to that of PSMA(-) PC3-Flu tumors with a very high tumor/blood ratio of 20. However, no significant influence of the ACUPA ligands were observed. Additionally, the NPs demonstrated fast release of carborane with low delivery of boron to tumors in vivo. Although the in vivo afficacy of those NPs remain limited, a significant progress towards the synthesis, characterization and initial biological evaluation of the polymer nanoparticles is proposed in this report and the results presented could guide the future design of amphiphilic polymer NPs for theranostic applications.

The pancreas is a highly branched, compound gland whose development requires the diversification of many distinct cell lineages. Cells in exocrine compartment of the pancreas synthesize and secrete digestive enzymes required for food digestion, whereas cells in the endocrine compartment are responsible for maintaining glucose homeostasis through the production of various hormones. In this work, we sought to characterize the diversity of cell types present in the developing pancreas and chart their differentiation through multiple developmental stages. We start in Chapter 2 by identifying novel and known cell populations within the mesenchymal and epithelial compartments of the developing murine pancreas and mapping their developmental progress across multiple embryonic stages using single-cell RNA-sequencing. We uncover significant cellular diversity within the mesenchymal compartment and utilize single-cell transcriptomic data to reconstruct lineage relationships among the developing pancreatic mesothelium and several previously uncharacterized mesenchymal populations in mouse pancreatic development. In the epithelial compartment, we uncover a novel endocrine progenitor stage defined by high expression of a transcription factor named Fev. Through genetic lineage tracing, we demonstrate that this Fev+ progenitor population is derived from Ngn3+ endocrine progenitors and that all hormone-expressing endocrine lineages of the murine pancreas transit through a Fev-expressing cell stage. Through in silico reconstruction of endocrine lineages, we identify candidate regulators of alpha and beta lineage allocation expressed during this novel Fev+ progenitor stage. In Chapter 3, we apply our findings of endocrine lineage dynamics in murine pancreatic development to that of human and uncover similar FEV-expressing endocrine progenitor populations in human pancreatic development. We map the transcriptional dynamics of human endocrine cell differentiation and identify novel candidate lineage regulators of human alpha and beta lineage allocation. We subsequently identify major disparities between the developmental paths that human endocrine cells follow in vivo versus in vitro during directed differentiation of hESCs towards the beta lineage. Our analysis reveals a lineage that may be mis-differentiated as hESC-derived progenitors differentiate towards a beta cell fate. Blocking the generation of this mis-differentiated lineage during in vitro beta cell differentiation represents a powerful solution in making in vitro beta cell differentiation more robust. Finally, given that FEV+ progenitors constitute a major stage in human endocrine cell differentiation, we have generated novel tools to study both the function of FEV and FEV-expressing cells in in vitro beta cell differentiation. Our work forms a foundation on which improvements to in vitro beta cell differentiation can be made to more closely reflect proper endocrine cell development in vivo, thereby increasing beta cell yield at the end of this process and generating beta cells that are functional.

The opportunistic human pathogen Pseudomonas aeruginosa utilizes a type III secretion system to deliver virulence factors that disrupt host cell processes and host innate immunity. Four type III-secreted toxins have been identified in this bacterium: ExoS, ExoT, ExoU and ExoY. ExoT is a bifunctional enzyme whose N-terminus encodes a GTPase activating protein with activity towards Rho, Rac, and Cdc42 while the C-terminus encodes an ADP ribosyl transferase with activity towards Crk. ExoT inhibits bacterial internalization into host cells, disrupts the actin cytoskeleton, and blocks wound repair. We previously showed that ExoT is degraded in a proteasomal dependent manner and that in vivo resistance against this toxin is Cbl-b-dependent. Though Cbl-b is known to influence actin cytoskeleton and neutrophil infiltration into LPS treated host tissues, we show that Cbl-b is not necessary for phagocytosis of P. aeruginosa by macrophages or neutrophil infiltration into P. aeruginosa infected host tissues. In addition, we demostrate that ExoT lysine residues 49 and 74 control its proteasomal dependent degradation in the host cytoplasm. We further demonstrate by host cell rounding assays and an acute pneumonia mouse model that mutating these lysine residues to arginine increases the virulence of P. aeruginosa. Interestingly, mutating lysine 49 and 74 did not alter the virulence of P. aeruginosa in cbl-b-/- mice, which suggests that Cbl-b-mediated protection of the host against P. aeruginosa must depend on the targeting of these two lysine residues in the toxin. Although mutation of lysine 49 to arginine is able to increase toxin secretion rate into the medium, we found that mutations in both lysine 49 and 74 are necessary for increased levels of translocated toxin in the host cytoplasm. Collectively, these data demonstrate that ExoT lysine residues at positions 49 and 74 play a key role in toxin degradation by the host proteasome, which leads to an alteration of ExoT-mediated virulence of P. aeruginosa. Our findings also suggest that Cbl-b targets these residues through either ubiquitination or another yet unidentified molecular process. Experiments are underway to determine whether Cbl-b is able to polyubiquitinate the mutant toxin as it does on wild type ExoT.

Viruses are obligate parasites, unable to replicate outside of the host to which they are adapted. The adaptation of viruses to their accustomed host cell milieu is exquisite, contacting hundreds or thousands of host proteins in order to hijack host machinery and avoid antiviral defenses. Identifying the key functional interactions between virus and host is a critical step towards interfering with viral replication, as implicated host proteins are attractive and often highly-tractable therapeutic targets. This identification remains challenging, especially as it is best done directly in the primary cells or tissues in which the virus typically replicates. We have built on recent developments using CRISPR-Cas9 ribonucleoproteins that allowed perturbation of genomic sequences in primary human CD4+ T cells to functionally interrogate HIV-human interactions, identifying 86 that significantly alter HIV infection, including 44 not previously reported and 24 that harbor restrictive activity. We sequenced each knockout locus to illuminate the cell-type-specific DNA repair processes in T cells and built an algorithm for enhanced prediction of their CRISPR editing outcomes. We then adapted the CRISPR-Cas9 ribonucleoprotein editing for use in primary human myeloid cells, allowing for interrogation of host factors for many additional pathogens. Finally, faced with a viral pandemic, we identified questions we were well-positioned to answer, first assessing the performance of commercial SARS-CoV-2 antibody assays before returning to host-pathogen interaction mapping. We carried out comparative viral-human protein-protein interaction and viral protein localization analysis for all three pathogenic coronaviruses SARS-CoV-1, MERS-CoV and SARS-CoV-2. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 Orf9b, an interaction we structurally characterized using cryo-EM. Combining genetically-validated host factors with both COVID-19 patient genetic data and medical billing records identified important molecular mechanisms and potential drug treatments with effectiveness against COVID-19 that merit further molecular and clinical study.

Purpose: The aim of this mixed method study was to identify barriers for children with special health care needs (SHCN) to receiving routine preventive dental care following dental care with general anesthesia (GA).

Methods: Electronic health records were reviewed for inclusion criteria and demographic data. Caregivers of children with SHCN were contacted to participate in qualitative interviews. Interview topics explored child, family, and community level influences to accessing dental care. Qualitative analysis identified key themes of barriers and enablers to care.

Results: A total of 1,708 children received dental care with GA during the two-year study period, of which 498 (29.16%) had a diagnosis of a SHCN. The most common type of SHCN was neurodevelopmental disorders (n=142, 28.51%). The mean age at time of GA was 8.6 years. Fifty caregivers completed interviews. Identified barriers to obtaining routine dental care included child stress/anxiety, finding an accepting provider, dismissive providers, and proximity of provider/transportation to dental care. Enablers to obtaining care included effective behavior management, continuity of provider/care, positive provider attitude, and referral to an accepting provider.

Conclusion: Adequately trained and local providers with an accepting attitude are essential to enabling children with SHCN to obtain equitable access to routine preventive dental care.

At present, there are no means to counter functional loss in the aging brain underlying both normal cognitive decline and dementia-related neurodegenerative disease in the elderly. As such, defining key regulatory molecular signatures in the aging brain associated with age-related functional decline may help identify potential targets to restore cognition at old age. Given onset of cognitive impairments is not uniform across cognitive modalities, we sought to delineate the ages at which individual learning and memory processes exhibit dynamic deterioration. To define the temporal kinetics of age-related cognitive decline, we first assessed hippocampal-dependent learning and memory in the mouse species across the lifespan at young (3 months), mature (6 months), middle-aged (12 months), aged (18 months) and old (24 months) ages. We observed that the rate of cognitive decline was indeed different across distinct types of learning and memory, with recognition and associative memory impairments occurring at faster kinetics compared to spatial and working memory deficits. Young mice outperformed all age groups in recognition and associative memory, while mice that were middle-aged performed comparable to younger groups in spatial memory and working memory tasks. To define molecular changes associated with age-related cognitive decline, we performed single nucleus RNA sequencing (snRNA seq) and single nucleus chromatin accessibility sequencing (snATAC-seq) on hippocampi from young and aged mice and characterized differences in cell-type specific gene expression patterns and DNA regulatory elements during aging. DNA methylation has emerged as a critical modulator of neuronal plasticity and cognitive function. Notwithstanding, the role of enzymes that demethylate DNA remain to be fully explored. Here, we report that loss of ten-eleven translocation methylcytosine dioxygenase 2 (Tet2), which catalyzes oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), in adult neurons enhances cognitive function. In the adult mouse hippocampus, we detected an enrichment of Tet2 in neurons. Viral-mediated neuronal overexpression and RNA interference of Tet2 altered dendritic complexity and synaptic-plasticity-related gene expression in vitro. Overexpression of neuronal Tet2 in adult hippocampus, and loss of Tet2 in adult glutamatergic neurons, resulted in differential hydroxymethylation associated with genes involved in synaptic transmission. Functionally, overexpression of neuronal Tet2 impaired hippocampal-dependent memory, while loss of neuronal Tet2 enhanced memory. Ultimately, these data identify neuronal Tet2 as a molecular target to boost cognitive function.

DNA methylation and histone H3 lysine 9 methylation (H3K9me) are hallmarks of heterochromatin in plants and mammals and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes H3K9me2 via a chromodomain (CD) and bromo-adjacent homology domain (BAH), which flank the catalytic domain. Here, we show that the H3K9me mark allosterically stimulates ZMET2 activity and also substantially increases the specificity of ZMET2 for hemimethylated versus unmethylated DNA. Interestingly, dinucleosomes are the preferred ZMET2 substrate, with DNA methylation targeted primarily to linker DNA. Further, while the CD stabilizes ZMET2 binding, the BAH domain is critical for catalysis and serves as an allosteric regulator of the enzyme. Negative stain electron microscopy shows a single ZMET2 molecule bridging two nucleosomes within a dinucleosome, consistent with the biochemical analyses. We propose a model in which CD-mediated binding and allosteric activation via BAH/H3K9me interactions coupled with nucleosome bridging allows ZMET2 to correctly read the chromatin context and thereby faithfully maintain DNA methylation and reinforce heterochromatic elements across the genome.

Models are frequently and ubiquitously used in all pharmacokinetic investigations. The familiar inductive equation-based pharmacokinetic models formulate hypotheses about data; they alone cannot provide mechanistic insights. We need models that have extant, working mechanisms that generate emergent properties analogous to how phenomena emerge during wet-laboratory experiments.

In this dissertation, I report a new class of synthetic, agent-based, discrete events models and simulations, with the objective to provide mechanistic insights. Validated, biomimetic software components are plugged together to form in silico analogues of the referent experimental systems. Each synthetic analogue is a mechanistic hypothesis: execution produces an observable phenomenon.

The recirculating in silico livers (RISLs) are in silico analogues of isolated perfused rat livers during an experiment in which digoxin is administered, alone or in combination with either an uptake or efflux inhibitor. A RISL that comprised four time-variant mechanisms and new enzyme and transporter components achieved the most stringent similarity measure: simulated digoxin and metabolite perfusate levels were experimentally indistinguishable from the referent data. The mechanisms simulated unanticipated loss of hepatic viability during the original experiments: erosion of hepatic accessibility and of enzyme and transporter activities.

In silico experimental Caco-2 (cell monolayer) cultures (ISECC) are analogues of the confluent, asymmetric cell monolayer used in vectorial transport studies. To seek an explanation for the observed paradoxical saquinavir transport data, I followed an iterative refinement protocol that enabled discovery of plausible, new mechanistic details. The ISECC surviving the most stringent similarity challenge produced transport data statistically indistinguishable from referent observations. It required heterogeneous intracellular spaces; a biased distribution of metabolizing enzymes; and restrictions on intracellular drug movement.

Experimenting on synthetic analogues, such as RISLs and ISECCs, provides a formerly unavailable means of discovering and testing new mechanistic hypotheses. It is a powerful expansion of the scientific method: an independent, scientific means to challenge, explore, and improve any inductive mechanism. Validated, biomimetic analogues are concrete instances of hypothetical yet plausible mechanisms, and would replace vague, unverified concepts. The collection of mechanisms, rules, assemblies, and interactions of components can be subjected to testing and falsification, and in the absence of other competing theories, stands as the current best mechanistic hypothesis for the phenomena.

BACKGROUND:

Multiple studies have found that AAs are more likely to use crisis and acute care services and less likely to use outpatient services than Whites with severe mental illness (SMI). This difference might be related to less access to outpatient services by AAs.

PURPOSE:

The purpose of this study is to determine predictors of the number of crisis, inpatient, and residential services used in 12 months.

METHODS:

This study was a secondary analysis of the Clinical Trial for Wellness Training (NR05350-04), a randomized controlled trial. Data were extracted from interviews and mental health service utilization records. Data were analyzed using descriptive methods, logistic regression, and negative binomial and Poisson regression. The Behavioral Model for Vulnerable Populations, a model that proposes health service utilization is predicted by predisposing characteristics, enabling resources, and need, was used as the theoretical framework.

RESULTS:

On bivariate analysis, only the number of residential services used varied by race. In the regression analyses, which controlled for multiple factors, race was no longer an influence, despite AAs having higher rates of homelessness and greater likelihood of victimization. The number of crisis services used was predicted by drug use, receipt of social security benefits, and age. The number of inpatient services used was predicted by drug use and receipt of social security benefits. And the number of residential services used was predicted only by enrollment in an outpatient mental health program.

CONCLUSIONS:

Crisis and inpatient service use was roughly equal between AAs and Whites. Far fewer subjects used inpatient services than crisis indicating that access to this particular service is severely limited. Predisposing characteristics and enabling resources rather than need predicted service use. Residential service use was predicted only by enabling resources in that patients enrolled in outpatient programs were most likely to use this service. It is possible that professionals in outpatient mental health programs might refer AAs less to residential services. Another consideration regarding crisis and inpatient service utilization is that the extreme vulnerability of the subjects might have obscured racial differences in this study.

The semi-allogeneic fetus derives half of its genetic maternal from the mother. The other half, inherited from the father, leads to the expression of proteins that are foreign to the mother. In danger of potential immune recognition and rejection, the fetus is dependent on maternal immune regulation. Multiple mechanisms are in place to ensure the mother and fetus live in harmony during pregnancy, as outlined in Chapter one. This dissertation discusses what happens to some of these mechanisms during fetal intervention in mice.

Chapter two focuses on T cell specific mechanisms that prevent maternal T cell activation during pregnancy. These mechanisms include constraint in antigen presentation to maternal T cells, deletion of maternal T cells aware of fetal antigens and the lack of recruitment of maternal T cells to the uterine environment. Fetal intervention in mice results in enhanced antigen presentation with a reduction in apoptosis of activated cells and a more prominent presence of maternal T cells in the uterus. Maternal T cells also play a role in fetal demise (preterm labor) during fetal intervention.

Chapter three discusses changes in trafficking maternal cells during fetal intervention. Trafficking of maternal cells into fetal blood during normal pregnancy encourages generation of fetal Tregs that can suppress an anti-maternal T cell response. During fetal intervention, the presence of maternal T cells, maternal microchimerism (MMc), increases and may play a role in limiting engraftment of cells transplanted in utero. Because fetal intervention results in preterm labor, changes in maternal microchimerism during fetal intervention may also play a role in mediating fetal rejection during preterm labor. Using a mouse model of preterm labor, we saw enhanced maternal microchimerism in fetal blood. The contribution of maternal cells in fetal blood to the pathogenesis of preterm labor is an open field for further investigation.

Chapter four deals with the role of maternal Tregs in regulating maternal immune responses during fetal intervention. This chapter more specifically examines maternal antibody production against "conceptus-derived" antigens in the absence of Tregs during normal pregnancy and fetal intervention. Tregs prevent production of maternal antibodies during pregnancy. During fetal intervention, however, maternal antibody production does not increase, even in the absence of Tregs. The presence of "conceptus-derived" antigens in maternal circulation may play a role in preventing detection of maternal antibodies during fetal intervention. Because maternal antibodies do not have an effect on the success of pregnancy, alternative mechanisms may play a role in protecting pregnancy.

This dissertation discusses how the regulatory mechanisms protecting the semi-allogeneic fetus during pregnancy are disrupted during fetal intervention and may play a role in preterm labor. The results discussed in this dissertation provide support for a novel role of the maternal adaptive immune system in the rejection of the fetus during pregnancy complications. Finally, Chapter five discusses the future directions that have come about because of this thesis work.