The immune response to an infection is determined by a number of factors, which also affect the generation of memory T cells afterwards. The immune response can also affect the stability of the pre-existing memory populations. The memory developed after an infection can influence the response to subsequent infections with unrelated pathogens. This heterologous immunity may deviate the course of disease and alter the disease outcome. The generation and stability of memory CD8 T cells and the influence of the history of infections on subsequent heterologous infections are studied in this thesis using different viral infection sequences. Previous studies using mice lacking individual immunoproteasome catalytic subunits showed only modest alterations in the CD8 T cell response to lymphocytic choriomeningitis virus (LCMV). In this study, I found that the CD8 T cell response to LCMV was severely impaired in mice lacking all three catalytic subunits of the immunoproteasome, altering the immunodominance hierarchy of the CD8 T cell response and CD8 T cell memory. Adoptive transfer experiments suggested that both inefficient antigen presentation and altered T cell repertoire contribute to the reduction of the CD8 T cell response in the immunoproteasome knockout mice. Immune responses generated during infections can reduce pre-existing memory T cell populations. Memory CD8 T cells have been shown to be reduced by subsequent heterologous infections. In this study, I re-examined the phenomenon using immune mice infected with LCMV, murine cytomegalovirus (MCMV) and vaccinia virus (VACV) in different infection sequences. I confirmed that memory CD8 T cells were reduced by heterologous infections, and showed that LCMV-specific memory CD4 T cells were also reduced by heterologous infections. Reduction of the memory CD8 T cells is thought to be the result of apoptosis of memory CD8 T cells associated with the peak of type I interferon early during infection. I showed that memory CD4 T cells were similarly driven to apoptosis early during infection; however, Foxp3+ CD4+ regulatory T cells were relatively resistant to virus infection-induced apoptosis, and were stably maintained during LCMV infection. The stability of Treg cells during viral infections may explain the relatively low incidence of autoimmunity associated with infections. The history of infections can deviate the course of disease and affect the disease outcome, but this heterologous immunity is not necessarily reciprocal. Previous studies have shown the effects of heterologous immunity during acute infections. In this thesis, I showed that the history of LCMV infection led to higher viral titers during persistent MCMV infection, caused more severe immunopathology at the beginning of infection, and reduced the number of MCMV-specific inflationary memory CD8 T cells after the period of memory inflation. In a different context of infection, the history of LCMV infection can be beneficial. LCMV-immune mice have been shown to have lower viral titers after VACV infection, but VACV-immune mice are not protected during LCMV infection. I found that memory CD8 T cells generated from LCMV and VACV infections were phenotypically different, but the differences could not explain the nonreciprocity of heterologous immunoprotection. By increasing the number of crossreactive VACV A11R198-205-specific memory CD8 T cells, however, I showed that some VACV-immune mice displayed reduced viral titers upon LCMV challenge, suggesting that the low number of potentially cross-reactive CD8 T cells in VACV-immune mice may be part of the reasons for the non-reciprocity of immunoprotection between LCMV and VACV. Further analysis deduced that both number of potentially cross-reactive memory CD8 T cells and the private specificity of memory CD8 T cell repertoire played a part in determining the outcome of heterologous infections.

The innate immune system provides the first line of defense against infection. Pathogens are detected though a variety of Pattern Recognition Receptors (PRRs), which activate downstream signaling cascades. Effector molecules such as cytokines and chemokines are released upon activation and aid in cell recruitment, control of pathogen replication, and coordination of the adaptive immune response. Nucleic acids that are released into the cytosol during viral and bacterial infection are recognized through a special class of PRRs, coined cytosolic nucleic acid sensors. Upon recognition, these receptors induce the production of type I interferons and other cytokines to aid in pathogen clearance. Although many cytosolic nucleic acid sensors have been discovered, it is unclear how they work in concert to mediate these responses. The Interferon Gamma Inducible protein (IFI)16 and its proposed mouse orthologue IFI204 are cytosolic DNA sensors that have been linked to the detection of cytosolic DNA during infection with Herpes Simplex Virus (HSV-1). IFI16 binds dsDNA that has been released into the cytosol during viral infection and engages the adaptor molecule Stimulator of Interferon Genes (STING) leading to TANK binding kinase-1 (TBK1) dependent phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of type I interferons and interferon stimulated genes. In addition to its role as a sensor, in chapter two of this thesis we describe a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in anti-viral immunity. In an effort to better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as other inducers of IFN such as cyclic-dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to cytosolic DNA ligands and DNA viruses. In contrast, expression of the NF-κB regulated cytokines such as IL-6 and IL-1β were unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of expression of IFN-α and IFN stimulated genes such as RIG-I in response to cyclic GMP-AMP (cGAMP), a second messenger produced in response to cGAS, as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-α promoter in IFI16 knockdown cells suggesting that transcription of ISGs is dependent on IFI16. Since IFI16 knockdown compromised not only DNA virus driven pathways, we propose additional regulatory roles outside of DNA sensing. Collectively, these results indicate that IFI16 plays a role in the regulation of type I IFN gene transcription and production in response to both RNA and DNA viruses. The role of IFI16/IFI204 has been studied extensively in vitro, however the role of the receptors in vivo has yet to be determined. In chapter three of this thesis, we developed a mouse deficient in IFI204 to explore the role of IFI204 in in vivo immune responses to viruses. We investigated the ability of IFI204 deficient cells to induce type I interferons and other cytokines in response to a panel of DNA and RNA ligands in vitro. IFI204 deficient BMDMs displayed a partial defect in type I interferon induction in response to both DNA and RNA ligands and viruses as compared to WT mice. We also observed that this phenotype is time dependent, since there was no change in type I interferon induction after 12 hours post infection as compared to earlier time points. In contrast to these results, expression of the NF-κB regulated cytokines IL-6 and IL-1β were unaffected in IFI16 knockdown cells. These results suggest that IFI204 plays a partial role in the induction of type I interferons in response to both DNA and RNA ligands. Additionally, IFI204 may work in tandem with other receptors in a sequential manner to amplify the type I interferon response. We also studied the involvement of IFI204 in an in vivo model of HSV-1 infection. IFI204 knockout mice produce less brain and serum IFN-β, IL-6, and IL-1β 72 hours post intraperitoneal infection with HSV-1. Furthermore, IFI204 -/- mice are more susceptible to HSV-1 infection as compared to WT mice. These data indicate that IFI204 mediates the response to HSV-1 in vivo by inducing the production of cytokines that are necessary for the control of viral infection.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons, generally leading to paralysis and death within 3-5 years of onset. Over 50 different mutations in the gene encoding FUS/TLS (or FUS) will result in ALS, accounting for ~4% of all inherited cases. FUS is a multifunctional protein with important functions in DNA/RNA processing and stress response. How these mutations affect the structure or function of FUS protein and ultimately cause ALS is not known. The fact that mutations cause the protein to mislocalize from the nucleus to the cytoplasm of cells suggests that ALS pathogenesis may occur through a loss of nuclear function, gain of toxic cytoplasmic function, or both. Several FUS knockout animal models have been utilized for investigating a loss of function hypothesis and show phenotypes such as early lethality, reduced lifespan, and locomotor defects. To uncover cellular pathways affected by loss of FUS function, I have characterized the knockdown of FUS in a motor neuron-like cell line, NSC-34. In NSC-34 cells, the depletion of FUS severely impacts cellular proliferation and potentially causes increased levels of DNA damage. A quantitative proteomics analysis performed on cells undergoing various degrees of FUS knockdown revealed protein expression changes for known RNA targets of FUS, consistent with a loss of FUS function with respect to RNA processing. Proteins that changed in expression as a function of FUS knockdown were associated with vii multiple processes, some of which influence cell proliferation including cell-cycle regulation, cytoskeletal organization, oxidative stress and energy homeostasis. Importantly, cellular proliferation could be rescued by the re-expression of FUS and by treatment with the small-molecule, rolipram, indicative of potential therapeutic approaches. Collectively, the work presented in this dissertation demonstrates the importance of FUS for cell health and homeostasis, is suggestive of a role for FUS in DNA damage repair and identifies additional cellular pathways influenced by FUS depletion. Overall, this work provides mechanistic insight into ALS pathogenesis through loss of FUS/TLS function.

Cellular senescence is thought to play an essential role in many biological functions including tumor suppression and organismal aging. Senescent cells, which are permanently removed from the cell cycle, can be found both in vivo in many different tissue types and in vitro within cultures of non-immortalized cells. Despite their inability to proliferate, these cells persist and remain metabolically active for indefinite periods of time. This physiologic process occurs in response to a variety of cellular insults including oxidative stress, shortened telomeres, constitutive oncogene expression, and DNA damage, and can be initiated by upregulation of one of the two known senescent pathways, involving p16/Rb or p53/p21. The senescent cell phenotype is also characterized by changes to cell and nuclear morphology and to the secretory profile of the cell. Related to changes in nuclear morphology, epigenetic modifications to the packaging of DNA are thought to be key to the initiation and maintenance of the senescence program. While a large number of earlier studies focused on the findings that senescent cells gain regions of condensed heterochromatin, often in the form of Senescent Associated Heterochromatin Foci (SAHF), this thesis work shows that there is a marked loss of heterochromatin in the peri/centromeric regions of the genome. In fact, both α-satellite and satellite II sequences across the genome distend in a striking and unanticipated fashion; this can be readily visualized by fluorescence in situ hybridization (FISH) as their structure changes from a condensed spot to highly elongated and fine thread-like signals. We have termed this exceptional decondensation of constitutive heterochromatin Senescence Associated Distension of Satellites (SADS). Importantly, a series of experiments shows that SADS is both a consistent and an early event in the cell senescence process, which occurs as a result of every senescence induction method examined. We also observed that this distension was characteristic of both human and murine cells and in vivo in human benign Prostatic Intraepithelial Neoplasia (PIN) tissue. Furthermore, unlike SAHF formation, SADS can occur due to the activation of either of the two senescence pathways, p16/Rb or p53/p21. Additionally, the cytological dimensions of the thread-like satellite signals indicates that SADS represents “unraveling” of DNA on an unprecedented scale. Thus, it was surprising that this event was not facilitated by changes to several canonical histone modifications associated with condensed heterochromatin, namely H3K9Me3, H3K27Me3, or H3K4Me3, nor is it caused by loss of DNA methylation. Consequently, we believe that this marked distension of satellite DNA is due to changes in higher-order folding of the chromatin fiber. This is important for understanding fundamental events in the cell senescence process, but also provides a unique system for study of chromatin packaging that may provide new insights into the organization of DNA well beyond nucleosome packaging and the ten nanometer fiber. In fact, initial super resolution images of SADS suggest that the satellite sequences may be organized into domains or “globules”. Hence, we suggest that the changes to satellite sequence packaging may be facilitated by changes to higher-order nuclear structural proteins, such as LaminB1, which is reduced in senescent cells. Finally, this work provides analysis of the literature and preliminary experiments to consider the possibility that there are increased levels of cell senescence in Down syndrome (trisomy 21) cells. As individuals with Down syndrome (DS) experience many manifestations of premature aging (including early-onset Alzheimer’s Disease), have a resistance to solid tumor formation, are more susceptible to oxidative stress, and are trisomic for several genes implicated in causing senescence, our analysis provides plausibility for the hypothesis that accelerated rates of senescence may play a significant role in DS physiology. We also provide results of preliminary studies and outline the next steps for experimentation, using DS fibroblasts and a unique genetically engineered DS iPS cell system. As a final note, the quantification of cell senescence in trisomic versus disomic cells for these experiments relies substantially on the new single-cell marker of senescence discovered and established by this theses work, the Senescence-Associated Distension of Satellites.

Innate immunity is the first line of defense against invading pathogens. It provides immediate protection by initiating both cellular and humoral immune reactions in response to a wide range of infections. It is also important to the development of long-lasting and pathogen-specific adaptive immunity. Thus, studying of the innate immunity, especially the pathogen recognition and signaling modulation, is crucial for understanding the intrinsic mechanisms underlying the host defense, as well as contributing the development of the fight against infectious diseases. Drosophila is an ideal model organism for study of innate immunity. Comparing to mammals, Drosophila immunity is relative conserved and less redundant. A variety of molecular and genetic tools available add further convenience to the research in this system. My work is focused on the signaling modulation by post-translational modification after activation. In these studies I demonstrated in the center of Imd pathway, the Imd protein undergoes proteolytic cleavage, K63-polyubiquitination, phosphorylation, K63-deubiquitination and K48-polyubiquitination/degradation in a stimulation-dependent manner. These modifications of Imd play a crucial role in regulating signaling in response to infection. The characterization of ubiquitin-editing event provides a new insight into the molecular mechanisms underlying the activation and termination of insect immune signaling pathway.

The following work demonstrates that chromatin regulators play far more pronounced roles in dynamic gene expression than they do in steady-state. Histone modifications have been associated with transcription activity. However, previous analyses of gene expression in mutants affecting histone modifications show limited alteration. I systematically dissected the effects of 83 histone mutants and 119 gene deletion mutants on gene induction/repression in response to diamide stress in yeast. Importantly, I observed far more changes in gene induction/repression than changes in steady-state gene expression. The extensive dynamic gene expression profile of histone mutants and gene deletion mutants also allowed me to identify specific interactions between histone modifications and chromatin modifiers. Furthermore, by combining these functional results with genome-wide mapping of several histone modifications in the same time course, I was able to investigate the correspondence between histone modification occurrence and function. One such observation was the role of Set1-dependent H3K4 methylation in the repression of ribosomal protein genes (RPGs) during multiple stresses. I found that proper repression of RPGs in stress required the presence, but not the specific sequence, of an intron, an element which is almost unique to this gene class in Saccharomyces cerevisiae. This repression may be related to Set1’s role in antisense RNA-mediated gene silencing. Finally, I found a potential role for Set1 in producing or maintaining uncapped mRNAs in cells through a mechanism that does not involved nuclear exoribonucleases. Thus, deletion of Set1 in xrn1Δ suppresses the accumulation of uncapped transcripts observed in xrn1Δ. These findings reveal that Set1, along with other chromatin regulators, plays important roles in dynamic gene expression through diverse mechanisms and thus provides a coherent means of responding to environmental cues.

Neurological disorders – disorders of the brain, spine and associated nerves – are a leading contributor to global disease burden with a sizable economic cost. Adeno-associated viral (AAV) vectors have emerged as an effective platform for CNS gene therapy and have shown early promise in clinical trials. These trials involve direct infusion into brain parenchyma, an approach that may be suboptimal for treatment of neurodegenerative disorders, which often involve more than a single structure in the CNS. However, overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. We have developed novel capsids AAV-AS and AAV-B1 that lead to widespread gene delivery throughout the brain and spinal cord, particularly to neuronal populations. Both transduce the adult mouse brain >10-fold more efficiently than the clinical gold standard AAV9 upon intravascular infusion, with gene transfer to multiple neuronal sub-populations. These vectors are also capable of neuronal transduction in a normal cat. We have demonstrated the efficacy of AAV-AS in the context of Huntington's disease by knocking down huntingtin mRNA 33-50% after a single intravenous injection, which is better than what can be achieved by AAV9 at the particular dose. AAVB1 additionally transduces muscle, beta cells, pulmonary alveoli and retinal vasculature at high efficiency, and has reduced sensitivity to neutralizing antibodies in human sera. Generation of this vector toolbox represents a major step towards gaining genetic access to the entire CNS, and provides a platform to develop new gene therapies for neurodegenerative disorders.

Recently, the medial habenula-interpeduncular (MHb-IPN) axis has been hypothesized to modulate anxiety although neuronal populations and molecular mechanisms regulating affective behaviors in this circuit are unknown. Here we show that MHb cholinergic neuron activity directly regulates anxiety-like behavior. Optogenetic silencing of MHb cholinergic IPN inputs reduced anxiety-like behavior in mice. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their role as autoreceptors in these neurons has not been described. nAChRs are ligand-gated cation channels that are activated by the excitatory neurotransmitter, acetylcholine (ACh), as well as nicotine, the addictive component of tobacco smoke. We expressed novel nAChR subunits that render nAChRs hypersensitive to ACh, ACh detectors, selectively in MHb cholinergic neurons of adult mice. Mice expressing these ACh detectors exhibited increased baseline anxiety-like behavior that was alleviated by blocking the mutant receptors. Under stressful conditions, such as during nicotine withdrawal, nAChRs were functionally upregulated in MHb cholinergic neurons mediating an increase in anxiety-like behavior. Together, these data indicate that MHb cholinergic neurons regulate anxiety via signaling through nicotinic autoreceptors and point toward nAChRs in MHb as molecular targets for novel anxiolytic therapeutics.

In recent years, the transplant community has explored and adopted tools for quantifying clinical insight into illness severity and frailty. This dissertation work explores the interplay between objective and subjective assessments of physical health status and the implications for liver transplant candidate and recipient outcomes. The first aim characterizes national epidemiologic trends and the impact of Centers for Medicare and Medicaid quality improvement policies on likelihood of waitlist removal based on the patient being too frail to benefit from liver transplant (“too sick to transplant”). This aim includes more than a decade (2002–2012) of comprehensive national transplant waitlist data (Scientific Registry of Transplant Recipients (SRTR)). The second aim will assess and define objective parameters of liver transplant patient frailty by measuring decline in lean core muscle mass (“sarcopenia”) using abdominal CT scans collected retrospectively at a single U.S. transplant center between 2006 and 2015. The relationship between these objective sarcopenia measures and subjective functional status assessed using the Karnofsky Functional Performance (KPS) scale are described and quantified. The third aim quantifies the extent to which poor functional status (KPS) pre-transplant is associated with worse post-transplant survival and includes national data on liver transplantations conducted between 2005 and 2014 (SRTR). The results of this dissertation will help providers in the assessment of frailty and subsequent risk of adverse outcomes and has implications for strategic clinical management in anticipation of surgery. This research will also to serve to inform national policy on the design of transplant center performance measures.

Background: Racial and ethnic minorities are under-represented participants in health-related research. Comprehension and understanding of the research process are a barrier to research participation. A potential approach to engaging underserved populations in research is through improving research literacy, which we define as “the capacity to obtain, process and understand basic information needed to make informed decisions about research participation.” Methods: Through primary data collection and mixed-methods approaches, this doctoral thesis seeks to: 1) define and conceptualize the domains, determinants, and impacts of research literacy through the development of a multi-component comprehensive framework, 2) operationalize research literacy by developing and psychometrically testing the Research Literacy Scale, and 3) quantify differences in research literacy, measured by the Research Literacy Scale, by race/ethnicity, race-related factors, and other socio-demographic factors. Results: We created a framework outlining eight domains of research literacy and multi-faceted influences of societal, community, researcher, and participant factors that may influence an individual’s level of research literacy. The Research Literacy Scale created is comprised of 16 items, with a KR-20 estimate of 0.81 and test-retest reliability of 0.84. We found differences in mean scale scores by race/ethnicity, age, education, income, and health literacy (all p < 0.01). African-Americans and Latinos have lower research literacy scores, as compared to non-Latino Whites. Race-consciousness was associated with research literacy score. Conclusions: This study is the first to define, assess, and quantify factors associated with research literacy in a diverse community sample and may provide insights on approaches to enhance minority engagement in health-related research.