FC Receptor-Mediated Activities of Env-Specific Monoclonal Antibodies Generated from Human Volunteers Receiving a DNA Prime-Protein Boost HIV Vaccine: A Dissertation
Human immunodeficiency type 1 (HIV-1) is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years’ infection and as a result, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with antibody dependent cellular cytotoxicity (ADCC) activity. Previous studies in the Lu lab demonstrated that in an HIV-1 vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities along with multiple Fc mediated effector functions. I developed a protocol for the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from these DP6-001 vaccinees. By utilizing a labeled gp120 bait to isolate Env specific B cells, paired heavy and light chain immunoglobulin (Ig) genes were cloned and allowed for the production of monoclonal antibodies with specificity for gp120. By using this protocol, 13 isolated mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens, with mostly conformational epitopes and a few V3 and C5 specific mAbs. Equally cross-reactive Fc-mediated functional activities, including ADCC and antibody dependent cellular phagocytosis (ADCP), were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional antibodies by the DNA prime- protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV-1 vaccine design.
Exploiting DNA Repair and ER Stress Response Pathways to Induce Apoptosis in Glioblastoma Multiforme: A Dissertation
Glioblastoma multiforme (GBM) is a deadly grade IV brain tumor characterized by a heterogeneous population of cells that are drug resistant, aggressive, and infiltrative. The current standard of care, which has not changed in over a decade, only provides GBM patients with 12-14 months survival post diagnosis. We asked if the addition of a novel endoplasmic reticulum (ER) stress inducing agent, JLK1486, to the standard chemotherapy, temozolomide (TMZ), which induces DNA double strand breaks (DSBs), would enhance TMZ’s efficacy. Because GBMs rely on the ER to mitigate their hypoxic environment and DNA repair to fix TMZ induced DSBs, we reasoned that DSBs occurring during heightened ER stress would be deleterious.
Treatment of GBM cells with TMZ+JLK1486 decreased cell viability and increased cell death due to apoptosis. We found that TMZ+JLK1486 prolonged ER stress induction, as indicated by elevated ER stress marker BiP, ATF4, and CHOP, while sustaining activation of the DNA damage response pathway. This combination produced unresolved DNA DSBs due to RAD51 reduction, a key DNA repair factor. The combination of TMZ+JLK1486 is a potential novel therapeutic combination and suggests an inverse relationship between ER stress and DNA repair pathways.
The SMURF2-YY1-C-MYC Axis in the Germinal Center Reaction and Diffuse Large B Cell Lymphoma: A Dissertation
Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma. Patients who fail conventional therapy (~50%) have a poor prognosis and few treatment options. It is essential to understand the underlying biological processes, the progression of the disease, and utilize this information to develop new therapeutics.
DLBCL patients with high C-MYC expression have a poor prognosis and new therapeutics for these patients are needed. This thesis describes work testing the hypothesis that JQ1, which can indirectly inhibit C-MYC in some tumors, can be used as an effective treatment for DLBCL. Some tumors have an unknown mechanism causing high C-MYC expression, leading me to investigate the underlying mechanisms. YY1 is a transcriptional regulator of c- Myc and has been implicated in DLBCL and as a potential regulator of the germinal center (GC) reaction. DLBCL arises from GC cells or post-GC cells. I tested the hypothesis that YY1 regulates the GC reaction. SMURF2 is an E3-ubiquitin ligase for YY1 and a tumor suppressor for DLBCL. I was interested in examining the mechanism underlying the suppression of DLBCL by SMURF2 leading to the hypothesis that SMURF2 regulates the GC.
This thesis shows JQ1 leads to cell death and cellular senescence in human DLBCL cells. I conclude that BRD4 inhibition by JQ1 or derivatives could provide a new therapeutic avenue for DLBCL patients. I also show loss of YY1 perturbs the GC by decreasing the dark zone and increasing apoptosis. Finally I show modulation of SMURF2 does not affect the GC, suggesting SMURF2 utilizes a different mechanism to act as a tumor suppressor and may not modulate YY1 in the context of the GC.
Background: Traumatic injury remains a major cause of mortality in the US. Older Americans experience lower rates of injury and higher rates of death at lower injury severity than their younger counterparts. The objectives of this study were to explore pre-injury factors and injury patterns that are associated with post-discharge mortality among injured elderly surviving index hospitalization.
Methods: We queried a 5% random sample of Medicare beneficiaries (n=2,002,420) for any hospitalization with a primary ICD-9 diagnosis code for injury. Patients admitted without urgent/emergent admission were excluded, as well as patients presenting from inpatient hospitalization or rehabilitation. The primary endpoint was all-cause mortality. Patients were categorized into three mortality groups: death within 0-30 days, 31-90 days, or 91- 365 days post-discharge from the index hospitalization. These groups were compared with those who survived greater than one year post-discharge. Univariate tests of association and multivariable logistic regression models were utilized to identify factors associated with mortality during the 3 examined periods.
Results: 83,439 elderly patients (4.2%) were admitted with new injuries. 63,628 met inclusion criteria. 1,936 patients (3.0%) died during their index hospitalization, 2,410 (3.8%) died within 0-30 days, 3,084 (4.8%) died within 31-90 days, and 5,718 (9.0%) died within 91- 365 days after discharge. In multivariable adjusted models, advanced age, male sex, and higher Elixhauser score were associated with post-discharge mortality. The presence of critical injury had the greatest effect on mortality early after injury (0-30 days, OR 1.81, CI 1.64-2.00). Discharge to anywhere other than home without services was associated with an increased odds of dying.
Conclusions: Socio-demographic characteristics, disposition, and co-morbid factors were the strongest predictors of post-discharge mortality. Efforts to reduce injury-related mortality should focus on injury prevention and modification of co-morbidities.
Development and aging, two fundamental aspects of life, remain key biological processes that researchers try to understand. Drosophila melanogaster, thanks to its various merits, serves as an excellent model to study both of these processes. This thesis includes two parts. In the first part, I discuss our finding that the presynaptic neuron controls a retrograde signaling pathway by releasing essential components via exosomes. During synaptic development, postsynaptic cells send retrograde signals to adjust the activity and growth of presynaptic cells. It remains unclear what the mechanism is which triggers the release of retrograde signals; and how presynaptic cells are involved in this signaling event. The first part of this thesis demonstrates that a retrograde signal mediated by Synaptotagmin4 (Syt4) depends on the anterograde delivery of Syt4 protein from the presynaptic neuron to the muscle compartment likely through exosomes. This trans-synaptic transfer of Syt4 is required for the retrograde control of activity-dependent synaptic growth at the Drosophila larval neuromuscular junction.
In the second part of this thesis, I talk about our discovery that the disruption of nuclear envelope (NE) budding, a novel RNA export pathway, is linked to the loss of mitochondrial integrity and premature aging in Drosophila. We demonstrate that several transcripts, which are essential for mitochondrial integrity and function, use NE-budding for nuclear export. Transgenic Drosophila expressing a LamC mutation modeling progeroid syndrome (PS), a premature aging disorder in humans, displays accelerated aging-related phenotypes including progressive mitochondrial degeneration as well as decreased levels of a specific mitochondrial transcript which is normally enriched at NE-budding site. The PS-modeled LamC mutants exhibit abnormal lamina organization that likely disrupts the egress of these RNAs via NE-budding. These results connect defective RNA export through NE-budding to progressive loss of mitochondrial integrity and premature aging in Drosophila.
For nearly five decades, the simple eukaryote Saccharomyces cerevisiae has been used as a model for understanding the eukaryotic cell cycle. One vein of this research has focused on understanding how chromosome structure is regulated in relation to the cell cycle. This work characterizes a new mechanism that modulates the chromatin organizing condensin complex, in hopes of furthering the understanding of chromosome structure regulation in eukaryotes.
During mitosis, chromosomes are condensed to facilitate their segregation through a process mediated by the condensin complex. Upon interphase onset, condensation is reversed, allowing for efficient transcription and replication of chromosomes. This work demonstrates that Ycg1, the Cap-G subunit of budding yeast condensin, is cell-cycle regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. The cyclical expression of Ycg1 is unique amongst condensin subunits, and is established by a combination of cell cycle-regulated transcription and constitutive proteasomal degradation. Interestingly, when cyclical expression of Ycg1 is disrupted, condensin formation and chromosome association increases, and cells exhibit a delay in cell-cycle entry. These results demonstrate that Ycg1 levels limit condensin function, and suggest that regulating the expression of an individual condensin subunit helps to coordinate chromosome conformation with the cell cycle. These data, along with recent corroborating results in Drosophila melanogaster suggest that condensin regulation through limiting the expression of a single condensin subunit may be broadly conserved amongst eukaryotes.
T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRb deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3b sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.
Despite the development of effective antiretroviral treatments, there is still no cure for HIV-1. Major barriers to HIV-1 eradication include the diversity of intrapatient viral quasispecies and the establishment of reservoirs in tissue sanctuary sites. A better understanding of these populations is required for targeted treatments. While previous studies have examined the relationship between brain and blood or immune tissues, few have looked at and compared the properties of viruses from other tissue compartments. In this study, 75 full length HIV-1 envelopes were isolated from the frontal lobe, occipital lobe, parietal lobe, colon, lung, and lymph node of an HIV-1 infected subject. No envelopes could be amplified from the plasma or serum. Envelopes were subjected to genotypic and phenotypic characterization. Of the 75 envelopes, 53 were able to infect HeLa TZM-bl cells. The greatest proportion of non-functional envelopes was from the lung, a result of APOBEC-induced hypermutation. Lower frequencies of hypermutation were also observed in the occipital lobe and colon. Envelopes from regions of the brain were almost all macrophage tropic, while those from the body were predominantly non-macrophage tropic. All envelopes used CCR5 as a coreceptor. Phylogenetic analyses showed that sequences were compartmentalized inside the brain. These findings were also observed using PacBio next generation sequencing to examine 32,152 full length sequences. Envelopes from tissues of the body displayed greater variation in sequence length, charge, and number of potential N-linked glycosylation sites in comparison to envelopes from tissues of the brain. Increased variation was also observed in IC50s for inhibition and neutralization assays using sCD4, maraviroc, b12, PG16, 17b, and 447-52D. The increased variation observed in envelopes from tissues outside the brain suggests that different pressures may be influencing the evolution of these viruses and emphasizes the importance of further studies in these tissue sites.
The Role of Late Antigen in CD4 Memory T Cell Formation during Influena [i.e. Influenza] Infection: A Dissertation
While memory CD4 T cells are critical for effective immunity to pathogens, the mechanisms underlying their generation are poorly defined. Although extensive work has been done to examine the role of antigen (Ag) in shaping memory formation, most studies focus on the requirements during the first few days of the response known as the priming phase. Little is known about whether or not Ag re-encounter by effector T cells (late Ag) alters CD4 memory T cell formation. Since influenza infection produces a large, heterogeneous, protective CD4 memory T cell population, I used this model to examine the role of late Ag in promoting CD4 memory T cell formation.
In the experiments presented in this thesis, I demonstrate that late Ag is required to rescue responding CD4 T cells from default apoptosis and to program the transition to long-lived memory. Responding cells that failed to re-encounter Ag had decreased memory marker expression and failed to produce multiple cytokines upon re-stimulation. Ag recognition is required at a defined stage, as short-term Ag presentation provided 6 days after infection is able to restore canonical memory formation even in the absence of viral infection. Finally, I find that memory CD4 T cell formation following cold-adapted influenza vaccination is boosted when Ag is administered at this stage. These findings imply that persistence of viral Ag presentation into the effector phase is the key factor that determines the efficiency of memory generation. They also suggest that administering Ag during the effector stage may improve vaccine efficacy.
Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent issue in the modern world, predisposing patients to serious pathology such as cirrhosis and hepatocellular carcinoma. Mitochondrial dysfunction, and in particular, diminished hepatic oxidative phosphorylation (OXPHOS) capacity, have been observed in NAFLD livers, which may participate in NAFLD pathogenesis.
To examine the role of OXPHOS in NAFLD, we generated a model of enhanced hepatic OXPHOS using mice with liver-specific transgenic expression of LRPPRC, a protein which activates mitochondrial transcription and augments OXPHOS capacity. When challenged with high-fat feeding, mice with enhanced hepatic OXPHOS were protected from the development of liver steatosis and inflammation, critical components in the pathogenesis of NAFLD. This protection corresponded to increased liver and whole-body insulin sensitivity. Moreover, mice with enhanced hepatic OXPHOS have increased availability of oxidized NAD+, which promotes complete fatty acid oxidation in hepatocytes.
Interestingly, mice with enhanced hepatic OXPHOS were also protected from obesogenic effects of long-term high-fat feeding. Consistent with this, enhanced hepatic OXPHOS increased energy expenditure and adipose tissue oxidative gene expression, suggesting a communication between the liver and adipose tissue to promote thermogenesis. Examination of pro-thermogenic molecules revealed altered bile acid composition in livers and serum of LRPPRC transgenic mice. These mice had increased expression of bile acid synthetic enzymes, genes which are induced by NAD+ dependent deacetylase SIRT1 activation of the transcriptional co-regulator PGC-1a. These findings suggest that enhanced hepatic OXPHOS transcriptionally regulates bile acid synthesis and dictates whole-body energy expenditure, culminating in protection from obesity.
Viruses Implicated in the Initiation of Type 1 Diabetes Affect β Cell Function and Antiviral Innate Immune Responses: A Dissertation
The increasing healthcare burden of type 1 diabetes (T1D) makes finding preventive or therapeutic strategies a global priority. This chronic disease is characterized by the autoimmune destruction of the insulin-producing β cells. This destruction leads to poorly controlled blood glucose and accompanying life threatening acute and chronic complications. The role of viral infections as initiating factors for T1D is probable, but contentious. Therefore, my goal is to better characterize the effects of viral infection on human β cells in their function of producing insulin and to define innate immune gene responses in β cells upon viral infection. These aspects were evaluated in various platforms including mice engrafted with primary human islets, cultured primary human islets, β cells derived from human stem cells, and a human β cell line. Furthermore, the contributions of cell-type specific innate immune responses are evaluated in flow cytometry-sorted primary human islet cells. Taken together, the results from these studies provide insights into the mechanisms of the loss of insulin production in β cells during virus infection, and characterize the antiviral innate immune responses that may contribute to the autoimmune destruction of these cells in T1D.
Characterizing the Disorder in Tristetraprolin and its Contribution to Post-Transcriptional Gene Regulation: A Dissertation
RNA-binding proteins (RBPs) are important for a wide variety of biological processes involved in gene regulation. However, the structural and dynamic contributions to their biological activity are poorly understood. The tristetraprolin (TTP) family of RBPs, including TTP, TIS11b and TIS11d, regulate the stability of mRNA transcripts encoding for key cancer-related proteins, such as tumor necrosis factor- and vascular endothelial growth factor. Biophysical studies have shown that the RNA binding domain, consisting of two CCCH zinc fingers (ZFs), is folded in the absence of RNA in TIS11d and TIS11b. In TTP, however, only ZF1 adopts a stable fold, while RNA is required to completely fold the tandem zinc finger (TZF). The focus of this research was to understand the origin and biological significance of the structural differences observed for the TZF domains of TTP and TIS11d. Three residues were shown to control the affinity for the structural Zn2+ and determine the folding of ZF2 in the absence of RNA. The partially-folded TZF domain of TTP has greater selectivity for RNA sequences than the fully folded TZF domain of TIS11d. The mRNA destabilizing activity of TTP was increased when the partially disordered RBD of TTP was replaced with the fully structured TZF domain of TIS11d. Disruption of the structure and/or dynamics of the TZF domain observed in the disease-associated mutations of TIS11d, P190L and D219E, results in aberrant cytoplasmic localization. This work demonstrates that the extent of RBD folding in the TTP family is important for differential RNA recognition, mRNA turnover, and protein localization in vivo.
Systematic Experimental Determination of Functional Constraints on Proteins and Adaptive Potential of Mutations: A Dissertation
Sequence-function relationship is a fundamental question for many branches of modern biomedical research. It connects the primary sequence of proteins to the function of proteins and fitness of organisms, holding answers for critical questions such as functional consequences of mutations identified in whole genome sequencing and adaptive potential of fast evolving pathogenic viruses and microbes. Many different approaches have been developed to delineate the genotype-phenotype map for different proteins, but are generally limited by their throughput or precision. To systematically quantify the fitness of large numbers of mutations, I modified a novel high throughput mutational scanning approach (EMPIRIC) to investigate the fitness landscape of mutations in important regions of essential proteins from the yeast or RNA viruses. Using EMPIRIC, I analyzed the interplay of the expression level and sequence of Hsp90 on the yeast growth and revealed latent effect of mutations at reduced expression levels of Hsp90. I also examined the functional constraint on the receptor binding site of the Env of Human Immunodeficiency Virus (HIV) and uncovered enhanced receptor binding capacity as a common pathway for adaptation of HIV to laboratory conditions. Moreover, I explored the adaptive potential of neuraminidase (NA) of influenza A virus to a NA inhibitor, oseltamivir, and identified novel oseltamivir resistance mutations with distinct molecular mechanisms. In summary, I applied a high throughput functional genomics approach to map the sequence-function relationship in various systems and examined the evolutionary constraints and adaptive potential of essential proteins ranging from molecular chaperones to drug-targetable viral proteins.
A key process underlying synapse development and plasticity is stimulus-dependent translation of localized mRNAs. This process entails RNA packaging into translationally silent granules and exporting them over long distances from the nucleus to the synapse. Little is know about (a) where ribonucleoprotein (RNP) complexes are assembled, and if in the nucleus, how do they exit the nucleus; (b) how RNPs are transported to specific synaptic sites.
At the Drosophila neuromuscular junction (NMJ), we uncovered a novel RNA export pathway for large RNP (megaRNP) granules assembled in the nucleus, which exit the nucleus by budding through the nuclear envelope. In this process, megaRNPs are enveloped by the inner nuclear membrane (INM), travel through the perinuclear space as membrane-bound granules, and are deenveloped at the outer nuclear membrane. We identified Torsin (an AAA-ATPase that in humans is linked to dystonia), as mediator of INM scission. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the mRNAs fail to localize to postsynaptic sites leading to abnormal NMJ development. We also found that nuclear envelope budding is additionally used for RNP export during Drosophila oogenesis.
Our studies also suggested that the nuclear envelope-associated protein, Nesprin1, forms striated F-actin-based filaments or ‘‘railroad tracks,’’ that span from muscle nuclei to postsynaptic sites at the NMJ. Nesprin1 railroad tracks wrap aoround the postsynaptic regions of immature synaptic boutons, and serve to direct RNPs to sites of new synaptic bouton formation. These studies elucidate novel cell biological mechanisms for nuclear RNP export and trafficking during synapse development.
Understanding the Sequence-Specificity and RNA Target Recognition Properties of the Oocyte Maturation Factor, OMA-1, in Caenorhabditis elegans: A Dissertation
Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood.
OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes.
Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.
The Role of VTA Gabaergic Nicotinic Acetylcholine Receptors Containing the α4 Subunit in Nicotine Dependence: A Dissertation
Nicotine dependence is hypothesized to be due to neuroadaptations that ultimately drive compulsive nicotine use. The studies in this thesis aim to understand how the “upregulation” of nicotinic acetylcholine receptors (nAChRs) caused by chronic exposure to nicotine contributes to nicotine reward and nicotine withdrawal. Previous studies have shown that chronic nicotine induces upregulation of nAChRs containing the α4 subunit (α4* nAChR) within the Ventral Tegmental Area (VTA), a brain region critical for the rewarding properties of all illicit drugs. Curiously, α4* nAChR upregulation occurs specifically in the inhibitory GABAergic neuronal subpopulation of the VTA. To determine if increased expression and activation of α4* nAChRs in VTA GABAergic neurons contributes to nicotine dependence behaviors, I devised a viral-mediated, Creregulated gene expression system that selectively expressed α4 nAChR subunits containing a “gain-of-function” point mutation (a leucine mutated to a serine residue at the TM2 9´ position: Leu9´Ser) in VTA GABAergic neurons of adult mice. Sub-reward doses of nicotine were sufficient to activate VTA GABAergic neurons in mice expressing Leu9´Ser α4 nAChR subunits in VTA GABAergic neurons (Gad2VTA: Leu9´Ser mice) and exhibited acute hypolocomotion upon initial injection of low doses of nicotine that developed tolerance with subsequent nicotine exposures compared to control animals. In the conditioned place preference procedure, nicotine was sufficient to condition a significant place preference in Gad2VTA: Leu9´Ser mice at low nicotine doses that failed to condition control animals. I conclude from these data that upregulating α4* nAChRs on VTA GABAergic neurons increases sensitivity to nicotine reward. In a separate study testing the hypothesis that overexpression of Leu9´Ser α4* nAChRs in VTA GABAergic neurons disrupts baseline behavior and promotes anxiety-like behaviors, I found that overexpressing Leu9´Ser α4* nAChRs in VTA GABAergic neurons had a minimal effect on unconditioned anxiety-like behaviors. Drug naïve Gad2VTA: Leu9´Ser and control mice failed to exhibit any behavioral differences in the open-field, marble burying test and elevated plus maze compared to control.
Together, these data indicate that overexpression of the “gain-of-function” α4* nAChRs in VTA GABAergic neurons contributes to reward sensitivity without increasing susceptibility to nicotine withdrawal symptoms. My data indicates that nAChRs expressed in VTA GABAergic neurons may be a suitable target for the development of better smoking cessation aids.
Activation and Inhibition of Multiple Inflammasome Pathways by the Yersinia Pestis Type Three Secretion System: A Dissertation
Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. Precursors of these cytokines are expressed downstream of TLR signaling and are then enzymatically processed into mature bioactive forms, typically by caspase-1 which is activated through a process dependent on multi-molecular structures called inflammasomes. Y. pestis evades immune detection in part by using a Type three secretion system (T3SS) to inject effector proteins (Yops) into host cells and suppress IL-1β and IL-18 production. We investigated the cooperation between two effectors, YopM and YopJ, in regulating inflammasome activation, and found that Y. pestis lacking both YopM and YopJ triggers robust caspase-1 activation and IL-1Β/IL-18 production in vitro. Furthermore, this strain is attenuated in a manner dependent upon caspase-1, IL-1β and IL-18 in vivo, yet neither effector appears essential for full virulence. We then demonstrate that YopM fails to inhibit NLRP3/NLRC4 mediated caspase-1 activation and is not a general caspase-1 inhibitor. Instead, YopM specifically prevents the activation of a Pyrin-dependent inflammasome by the Rho-GTPase inhibiting effector YopE. Mutations rendering Pyrin hyperactive are implicated in the autoinflammatory disease Familial Mediterranean Fever (FMF) in humans, and we discuss the potential significance of this disease in relation to plague. Altogether, the Y. pestis T3SS activates and inhibits several inflammasome pathways, and the fact that so many T3SS components are involved in manipulating IL-1β/IL-18 underscores the importance of these mechanisms in plague.
Mechanisms Regulating Early Mesendodermal Differentiation of Human Embryonic Stem Cells: A Dissertation
Key regulatory events take place at very early stages of human embryonic stem cell (hESC) differentiation to accommodate their ability to differentiate into different lineages; this work examines two separate regulatory events.
To investigate precise mechanisms that link alterations in the cell cycle and early differentiation, we examined the initial stages of mesendodermal lineage commitment and observed a cell cycle pause that occurred concurrently with an increase in genes that regulate the G2/M transition, including WEE1. Inhibition of WEE1 prevented the G2 pause. Directed differentiation of hESCs revealed that cells paused during commitment to the endo- and mesodermal, but not ectodermal, lineages. Functionally, WEE1 inhibition during meso- and endodermal differentiation selectively decreased expression of definitive endodermal markers SOX17 and FOXA2. These findings reveal a novel G2 cell cycle pause required for endodermal differentiation.
A role for phenotypic transcription factors in very early differentiation is unknown. From a screen of candidate factors during early mesendodermal differentiation, we found that RUNX1 is selectively and transiently up-regulated. Transcriptome and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that RUNX1 knockdown impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion compromised TGFβ2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFβ2, but not TGFβ1. These findings identify novel roles for RUNX1-TGFβ2 signaling in mesendodermal lineage commitment.
Evaluating Acceptability, Feasibility and Efficacy of a Diabetes Care Support Program Facilitated by Cellular-Enabled Glucose Meters: A Dissertation
Background. Diabetes requires significant disease management, patient-provider communication, and interaction between patients, family members, caregivers, and care teams. Emerging patient-facing technologies, such as cellular-enabled glucose meters, can facilitate additional care support and improve diabetes self-management. This study evaluated patient acceptability, feasibility, and efficacy of a diabetes care support program facilitated by cellular-enabled glucose meters.
Methods. A two-phase study approach was taken. Get In Touch – Phase 1 (GIT-1) was a 1-month pilot involving patients with type 1 and type 2 diabetes. Get In Touch – Phase 2 (GIT-2) was a 12-month randomized controlled crossover trial involving patients with poorly-controlled type 2 diabetes. Results from GIT-1 and preliminary results from GIT-2 are presented.
Results. GIT-1 participants with type 1 (n=6) and type 2 (n=10) diabetes reported the intervention and cellular-enabled glucose meter were easy to use and useful while identifying potential areas of improvement. GIT-2 participants in both the intervention (n=60) and control (n=60) groups saw significant improvements in treatment satisfaction and A1c change, with intervention participants experiencing slightly greater improvements in each after 6 months (p=0.09 and p=0.16, respectively) compared to control participants.
Conclusions. Patients reported favorable acceptability of the intervention. Preliminary results from a randomized trial demonstrated potential of intervention to improve patient-reported and physiological health outcomes. Future studies should evaluate feasibility and efficacy over a longer period of time, with a greater number of participants, and targeting different populations of patients with diabetes. Provider perspectives and changes in provider behavior, clinical work flow, and caregiver burden should also be assessed.
The Role of MDM2 Phosphorylation in P53 Responses to DNA Damage and Tumor Suppression: A Dissertation
The p53 tumor suppressor protein is upregulated in response to DNA damage and other stress signals. The upregulation of p53 involves freeing it from negative regulation imposed by Mdm2 and MdmX (Mdm4). Accumulating evidence indicates that phosphorylation of Mdm proteins by different stress-activated kinases such as ATM or c-Abl significantly impacts p53 functions. We have previously shown that ATM phosphorylation of Mdm2 Ser394 is required for robust p53 stabilization and activation following DNA damage.
This dissertation describes in vivo examination of the mechanism by which Mdm2 Ser394 phosphorylation impacts p53 activities and its contribution to suppression of oncogene and DNA damage-induced tumors. We determine that phosphorylation of Mdm2 Ser394 regulates p53 activity by modulating Mdm2 stability and paradoxically delays Myc-driven lymphomagenesis while increasing lymphomagenesis in sub-lethally irradiated mice. c-Abl phosphorylates the residue neighboring Mdm2 Ser394, Mdm2 Tyr393.
This dissertation describes the generation of a novel Mdm2Y393F mutant mouse to determine if c-Abl phosphorylation of Mdm2 regulates p53-mediated DNA damage responses or tumor suppression in vivo. Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes.
The studies presented herein further our understanding of the mechanisms by which DNA damage-associated kinases stabilize and activate p53, and influence p53-dependent responses and tumor suppression. A better understanding of the in vivo effects of Mdm2 phosphorylation may facilitate the development of novel therapeutics capable of stimulating p53 anti-tumor activity or alleviating p53- dependent toxicities in non-malignant tissues.