Now showing items 1-20 of 168

    • The murine absolute visual threshold: behavior & retinal pathways

      LaMagna, Sam (2024-05-17)
      Connexin 36 (Cx36) gap junctions are important for governing the sensitivity of the dark-adapted retina. Despite its importance for physiological sensitivity, the degree to which retinal Cx36 governs the psychophysical absolute threshold is not known. The purpose of this work is to study to what extent inner- and outer-retinal Cx36 governs the absolute visual threshold. In Chapter 2 we developed a one-alternative forced choice (1AFC) task for measuring murine absolute visual thresholds to full-field flash stimuli. We found that our 1AFC task, in conjunction with the theory of signal detection, gave response bias-independent absolute visual threshold estimated. Using this assay, we found that decision criteria are related to response times. In Chapter 3 we used the 1AFC task and the power of transgenic mice to assess the relative contributions of inner and outer retinal Cx36 to the absolute visual threshold. We concluded that inner, not outer, retinal Cx36 is most responsible for governing the absolute visual threshold. In parallel, by testing mice with disrupted rod vision, we determined that rod OFF pathways, and not cones, set the absolute visual threshold in the absence of Cx36. Finally, we studied the impact of Cx36 on temporal summation at absolute threshold, by obtaining thresholds for a range of flash durations. Threshold-vs-duration data was then fit with a model of temporal summation that allowed us to determine whether Cx36 influences the temporal filtering properties of scotopic vision. Our model fits suggest that photoreceptor Cx36 may play a role in temporal processing at absolute visual threshold. Overall, this work sheds new light on the behavioral dynamics and neural underpinnings of rod mediated vision.
    • Battle of arms: human cytomegalovirus manipulates monocyte survival for viral dissemination.

      Geiler, Brittany (2024-05-16)
      Human cytomegalovirus (HCMV) is a highly prevalent pathogen with seropositivity rates reaching upwards of 90% in the United States. Most primary infections are asymptomatic in immunocompetent individuals, but HCMV poses a significant risk in immunocompromised and immunonaїve individuals including transplant patients and developing fetuses in utero. The key to systemic dissemination of HCMV relies upon the infection of monocytes, which function as non-permissive vehicles to deliver virus to end organ tissues. These primary infected monocytes also travel to the bone marrow, infecting CD34+ stem cells, leading to the establishment of a lifelong HCMV infection. HCMV can reactivate at any point throughout the host's lifetime, leading to HCMV-infected stem cells exiting the bone marrow as monocytes, disseminating to end-organ tissue, and perpetuating HCMV disease. In circulation, monocytes have a short life span of 48 hours that can be accelerated by the cellular death pathway, apoptosis, as a cellular defense mechanism against viral infection. Our lab has shown during primary infection, HCMV circumvents intrinsic apoptotic pathways, however, the mechanism by which HCMV blocks extrinsic apoptosis is unclear. The studies in this thesis reveal that HCMV induces cFLIP expression, inhibiting extrinsic apoptosis effector caspase 8. This effective inhibition of intrinsic and extrinsic apoptosis prompts trap-door death pathway necroptosis. However, the mechanism in which this pathway is activated and how HCMV modulates this pathway to promote cell survival is unknown. In these works, we identified TLR3 as the death receptor responsible for inducing necroptosis. To circumvent this activation, HCMV upregulates autophagy, a ubiquitous cellular recycling process. We saw the inhibition of autophagy altered nucleocytoplasmic shuttling and activation of executioner kinase, MLKL, culminating in necrotic cell death. This work highlights the delicate balance between pro-survival and pro-death elements in HCMV infected monocytes. However, investigating how HCMV modulates cellular death pathways in a primary infection monocyte model does not fully encapsulate the role of monocytes in HCMV dissemination. Once HCMV latency is established in CD34+ stem cells, this allows HCMV the ability to persist in the host for their entire life span as monocytes derived from latently infected stem cells that can re-seed HCMV to peripheral organs to establish a chronic lytic infection. To investigate this understudied secondary population of HCMV-infected monocytes, we developed a model in which primary HCMV-infected monocytes and infected monocytes derived from latently infected stem cells are on the same genetic background by differentiating a CD34+ myeloblastic cell line. Though preliminary, we believe that this model, combined with investigations of mechanisms in which HCMV promotes survival in primary infected monocytes, will allow for the development of novel therapies that specifically target HCMV-infected monocytes, thus preventing viral dissemination and the establishment of disease.
    • Novel signaling pathways driving experience-dependent maturation in dentate gyrus granule cells: a deep-sequencing approach

      Thompson, Jacqueline (2024-04-15)
      The granule cells in the dentate gyrus of the hippocampus are a cell type that is critical for learning and memory ability. Dentate gyrus granule cells exhibit the unique capacity to differentiate and mature throughout an individual's lifetime. Decades of dedicated research has revealed many transcription factors that facilitate the differentiation of granule cells from neural progenitors. The goal of this research project is to identify key molecular and transcriptional pathways that contribute to the maturation of dentate gyrus granule cells. We performed single-cell RNA sequencing and multiomic single-nuclei RNA and ATAC sequencing in an activity-dependent mouse reporter model to examine the influence of neuronal activity on the transcriptome and chromatin accessibility within individual granule cells. These experiments were performed between postnatal day 14 and postnatal day 24 due to the increased abundance of developing granule cells. We implemented an environmental enrichment paradigm where mice were reared in complex, dynamic, and socially-enriched housing. This paradigm allowed us to examine the impact of chronically increased circuit activity on granule cell maturation. This study reveals novel heterogeneity in the maturing granule cell population that is associated with previous neuronal activity and synaptic function. A follow-up investigation identifies new transcription factor candidates that appear to orchestrate the transition between maturity stages. We propose a new model where granule cell identity is established through an activity-independent transcriptional network. The subsequent experience of neuronal activity appears to drive the emergence of a distinct transcriptional network that is poised to facilitate long-lasting granule cell maturity.
    • Therapeutic hydrogel for wound healing applications

      Yang, Xiguang (2024-04-11)
      Managing chronic wounds is a complex challenge, often aggravated by the presence of inflammation and infection. Hydrogel materials stand out for their remarkable properties such as biocompatibility and drug-loading capabilities, making them ideal for applications in wound dressing and tissue regeneration. A compelling treatment strategy involves not only resolving persistent hyperinflammation but also effectively eliminating infection. The demand for an "all-in-one" hydrogel platform with dual functions, targeting both inflammation and infection in chronic wounds, is huge in clinical settings. This thesis presents two promising dual-functional hydrogel platforms specifically crafted for potent topical application in the treatment of wounds. First platform was named "pull and push" hydrogel, which is essentially micro-sized hydrogel functionalized with innovative and versatile telodendrimers (TDs). By rational design and synthesis, these conjugated TDs can strongly grab or trap inflammatory factors such as cytokines and endotoxins, as well as hold drug payload such as antibiotics and release out in a desired manner. We demonstrated the TD hydrogel's capability to scavenge proinflammatory cytokines using in vitro assays, and shown the TD hydrogel's effect in scavenging cytokines in a mouse model with skin inflammation. We also demonstrated TD hydrogel's structure dependent capabilities in encapsulating different antibiotics, and sustainable release profile. The drug maintained its efficacy with prolonged therapeutic window and reduced toxicity, as evidenced by in vitro assays and in vivo animal skin and soft tissue infection model. Second hydrogel platform was termed "kill and block", which is essentially a bulky photo-crosslinked hydrogel incorporated with telodendrimer nano-formulated antibiotics. Nanodrug released from topically applied bulky photogel can further dissociate into drug and nanocarrier telodendrimer. The released drug functions as antimicrobial to "kill" bacteria. Dead bacteria still potentially stimulate inflammation. The payload-free nanocarrier can simultaneously modulate local immune response by "blocking" inflammatory pathway. In addition, this platform address issues of wound morphology adaptiveness. We demonstrated the tunable drug release profiles, reserved drug efficacy after TD encapsulation and phtogel incorporation, and superior anti-inflammation efficacy in vitro and in vivo. Collectively, we organically combined the advantages of nanotechnology and hydrogel platform, providing promising solutions for the chronic wound treatment from immune modulation and drug delivery.
    • Exploring the roles of the connecting cilium in photoreceptor health

      Liu, Yu (2024-03-26)
      Defects in proteins functioning at the photoreceptor connecting cilium/transition zone (CC/TZ) have been linked to retinal degenerative disorders such as retinitis pigmentosa (RP) and cone-rod dystrophy (CRD). Mutations in eyes shut homolog (EYS, RP25), a secreted ciliary protein with laminin globular (LG) domains, have been linked to RP and CRD. Previously, some LG domains have been shown to interact with O-mannosyl glycans of α-dystroglycan (α-DG). Additionally, mutations in pomgnt1, an enzyme that plays a critical role in the synthesis of these glycans, have also been linked to RP (RP76). At the CC/TZ, the tectonic protein complex functions to maintain the unique biochemical environments of the inner segments (IS) and outer segments (OS) of photoreceptors. Mutations in tectonic complex proteins have been linked to ciliopathies that often include ocular abnormalities. The pathogenic mechanism underlying these mutations are poorly understood; thus, we hypothesized that EYS is an extracellular ciliary protein that interacts with α-DG and the tectonic complex. This project investigated the role of EYS, TMEM216, a member of the tectonic complex, and O-mannosyl glycans of α-DG in photoreceptor health. We determined that the C-terminal LG domains of EYS interacted with the O-mannosyl glycan epitope of α-DG. In pomgnt1 zebrafish mutants, EYS-glycan binding was reduced, and the secretion of EYS to the CC/TZ was significantly disrupted. Furthermore, in the pomgnt1 mutant retina, a substantial accumulation of EYS protein was observed in the soma of photoreceptors. Interestingly, deletion of pomgnt1 resulted in a pattern of photoreceptor degeneration similar to that previously observed in eys zebrafish mutants. By contrast, deletion of TMEM216 did not disrupt localization of EYS or of other tectonic complex proteins, yet photoreceptor degeneration was still observed in these animals. Our study has identified a previously unknown interaction between the LG domain-containing EYS and O-mannosyl glycans. These findings provide novel insight into the functional role of EYS around the CC/TZ and suggest the importance of O-mannosyl glycosylation in the regulation of protein secretion. Furthermore, our results suggest a mechanistic link between the disruption of glycosylation and photoreceptor degeneration, providing a new perspective on the underlying mechanisms behind RP25 and RP76.
    • IPSC-derived neurons as a model for studying the role of RELN in autism

      Mohktari, Ryan (2024-03-11)
      RELN is strongly associated with Autism Spectrum Disorder (ASD). Homozygous loss of the encoded protein REELIN is associated with severe neurodevelopmental phenotypes characterized by lissencephaly and cerebellar hypoplasia, yet the ASD linked variants are typically heterozygous and appear to require additional genetic risk to cause ASD. To functionally characterize a RELN variant in a patient with ASD, we used induced pluripotent stem cells (iPSCs) from a family of non-autistic parents and their son who had ASD (the proband). The proband has a maternally-inherited missense variant (R2457C) in the RXR motif of the REELIN protein. We differentiated the iPSCs into two types of neurons, inhibitory neurons which model the inhibitory forebrain neurons that secrete REELIN, and excitatory neurons which model the cortical pyramidal neurons that respond to REELIN. Immunoblotting revealed that the proband inhibitory neurons had a lower ratio of extracellular/intracellular REELIN compared to that of the parental neurons, suggesting a decreased REELIN secretion. Sholl analysis on the proband excitatory neurons showed reduced dendritic complexity and reduced total length compared to the parental neurons. REELIN treatment increased the dendritic length and complexity in proband neurons up to the level of parental neurons. CRISPR/Cas9-mediated RELN KO did not change the dendritic phenotype in the excitatory neurons, ruling out a cell autonomous role for REELIN in these neurons. The proband excitatory neurons also had lower mRNA expression of WNT target genes in response to WNT3a, suggesting an underactive WNT signaling, as well as higher total GSK3β protein and lower phosphorylation at the inhibitory S9 site, indicating an overactive GSK3β signaling. Inhibition of GSK3β improved the proband neurons dendritic complexity in the proximal parts of the dendritic arbor. However, inhibition of mTOR signaling, which has shown to regulate REELIN signaling, did not change the dendritic morphology. In conclusion, the pathophysiology of ASD in the proband likely consists of a reduced REELIN secretion from the inhibitory neurons and an additional vulnerability in the REELIN-responding excitatory neurons, the latter likely being an overactive GSK3β and an underactive WNT signaling, all of which result in reduced dendritic complexity.
    • Monomeric DENV-reactive IgA contributes protective and non-pathologic functions during DENV infection

      Wegman, Adam (2024-01)
      Dengue, caused by the 4 serotypes of dengue viruses (DENVs), is a tropical and subtropical vector-borne febrile illness which causes a significant global disease burden. A particular immunological feature contributing to severe disease is antibody-dependent enhancement (ADE), in which IgG isotype antibodies raised during a primary DENV infection opsonize and enhance the infectivity of DENVs during a secondary heterotypic infection. We and colleagues have described a monomeric serum IgA response during dengue infection. Here, we report on the functional characteristics of monomeric IgA in DENV infection. We show that isotype conversion of IgG to IgA preserves neutralization capacity while abrogating enhancing capacity. We show that DENV-specific IgA competitively antagonizes both IgG-mediated infection and downstream secretion of pro-inflammatory cytokines. This effect is largely attributable to the lower avidity of IgA-DENV immune complexes for permissive cells compared to IgG-DENV complexes. These findings have implications for serodiagnosis, therapeutics, and assessing risk of severe disease.
    • Finding Diamonds in the Rough: Uncovering Genetic Variants, Transcripts, and Biological Processes Associated with Resilience to Alzheimer's Disease

      Hou, Jiahui (2024-01-30)
      Late-onset Alzheimer's disease (LOAD) is a multifactorial disease with a strong genetic component. The growing understanding of the genetic basis and molecular mechanisms underlying LOAD risk presents an opportunity to uncover the factors that counter the risk and protect individuals from developing LOAD. The phenomenon wherein individuals demonstrate adaptability to the burden of disease risk can be referred to as "resilience". In this dissertation, I presented three studies that focused on the resilience to LOAD. Because resilience depends on and interacts with risk, we employed a risk-informed strategy to uncover resilience factors. This approach leveraged the current best-estimated LOAD risk to identify resilient individuals who, despite facing the highest LOAD risk, exhibit no dementia symptoms in old age. In Chapter 1, we demonstrated that a large number of risk-independent common genetic variants could reduce the penetrance of heightened genetic risk burden in LOAD. This study provided insights into the genetic architecture of resilience to LOAD, addressing a significant knowledge gap that requires attention. In addition, this study yielded a polygenic resilience score, enabling the assessment of the relative genetic resilience levels among individuals. In Chapters 2 and 3, we explored resilience to LOAD at the transcriptomic level. The study in Chapter 2 meta-analyzed all publicly available blood and brain transcriptomic studies of AD. This study laid the groundwork for investigating the resilience-conferring genes and pathways by establishing the best-estimated transcriptomic risk features in LOAD. In Chapter 3, we capitalized on the transcriptomic risk defined in Chapter 2 and examined the risk-residual genes that might confer resilience to increased transcriptomic risk of LOAD. This study implicated a couple of interesting pathways in resilience to LOAD and suggested that resilience and risk may operate in the same biological pathways. Taken together, our findings corroborated the idea that resilience in LOAD has a polygenic basis and highlighted the need to gain a deeper understanding of the genetic components, biological mechanisms, and phenotypic characteristics of resilience to LOAD risk. The dissertation contextualized these findings with the existing literature and suggested potential future directions to help further address the gaps in understanding resilience in LOAD.
    • Effort-based decision making and psychopathology in children and adults

      Nguyen, Nicholas (2022-05)
      The Research Domain Criteria (RDoC) initiative was introduced by the National Institute of Mental Health as a new research framework aimed at addressing longstanding pitfalls within the field of psychiatric research. This framework focuses on the study of fundamental units of human psychological behavior across multiple levels of information to inform our understanding of psychopathology. These units are categorized into larger domains of similar function, such as the Positive Valence Systems domain, which encompasses aspects of human reward-related behavior. This dissertation centers on the 'effort' component of human reward behavior, defined as the moderating effect of the perceived costs of physical or cognitive requirements on the valuation of a reinforcer. Three studies are presented from a program of research spanning five years on the study of 'effort' utilizing the RDoC research framework from both a behavior and genetics perspective. The first study (Chapter 2) is an exploratory study with the first application of RDoC to the study of 'effort' in relation to psychopathology in children and adults. It finds that behavioral measures of 'effort' in children and adults were associated with specific types of psychopathologies and with differing profiles between sexes. The second study (Chapter 3) assesses the cross-generational stability, divergent validity, and replicability of 'effort' and its associations with psychopathology in children and adults. It finds that 'effort' has divergent validity from other RDoC constructs of reward behavior, and that the specific associations with psychopathology initially observed in the first study were replicated in a larger population. It also finds that 'effort' does not display cross-generational stability between children and their parents. The third study (Chapter 4) examined the genetic contributions to 'effort', and the moderating effect of 'effort genes' on psychopathology. It found that genetic loci on three different chromosomes had genome-wide significant associations with quantitative measures of 'effort', and that polygenic risk scores generated from these measures were significant predictors of parent-reported levels of psychopathology in children. Together, this program of research provides the first comprehensive application of RDoC to the study of effort-based behavior and psychopathology in children and adults and has important implications for the advancement of the RDoC framework and future research in this area.
    • Neurotransmitter-mediated calcium signaling in apical dendrite initiation of cortical projection neurons and proposal for the role of Cajal-Retzius neurons

      Enck, Joshua (2023-12)
      This thesis investigates the developmental processes in the mouse cortex during embryonic days 13 to 15, corresponding to human gestational weeks 8 to 11. The primary focus is on the calcium signaling pathways in neurons, particularly as they transition from the migratory phase to the dendrite initiation and growth phase. The study places special emphasis on Cajal-Retzius neurons (CRNs), a transient population of excitatory neurons in the developing cortex. These neurons are known for their secretion of Reelin, a glycoprotein that has a role in regulating the position of glutamatergic neurons of the cortex. While the role of Reelin and its downstream effects on cortical organization have been well-documented, this work investigates a previously unexplored transient circuit between CRNs and cortical projection neurons (CPNs). The central questions addressed in this thesis include: What is the calcium signal as neurons transition from the migratory phase to dendrite initiation and growth? Is the signal important for CPN maturation and dendrite growth? How does the activity and neurotransmitter release by CRNs serve as a mechanistic substrate that informs and coordinates CPN development during the foundational phases of cortical development? What neurotransmitter systems are involved and how are they functioning? Our research employs a multidisciplinary approach, leveraging whole embryonic hemisphere explants and multiphoton microscopy to study the calcium signaling profiles of CRNs and CPNs. The findings reveal that CRNs not only secrete Reelin but also exhibit spontaneous activity and the potential for neurotransmitter release, specifically glutamate. This activity significantly influences intracellular calcium levels in CPNs, thereby affecting their dendritic growth and migration patterns. This work opens up a new avenue for understanding early cortical development by offering a novel framework that extends beyond the established role of CRNs and Reelin secretion. It provides compelling evidence that CRNs play a more multifaceted role in cortical development than previously thought, serving in a transient circuit that informs and coordinates CPN development. This thesis, therefore, not only fills a gap in our understanding of early cortical development but also sets the stage for future research into the pathophysiology of neurodevelopmental disorders.
    • Bulged G-quadruplexes in the human genome: identification and characterization of a novel type of non-canonical G-quadruplex

      Papp, Csaba (2023-11)
      G-quadruplexes (G4s) are secondary nucleic acid structures that are abundant in the human genome and have been linked to numerous physiological and pathological conditions. The underlying DNA sequence is a major determinant of the topology and stability of folded G4s. G4s containing bulges are a subpopulation of G4-like structures, whose genome-wide localization and functions are still unknown. Our study focuses on addressing this gap. We utilized a data-driven approach to establish models for the computational genome-wide prediction of potential sites of bulge containing G4 formation (pG4-BS). We also showed that the majority of human protein coding genes contain these pG4-BS, especially in their promoters and exon-intron junctions, suggesting roles in transcription and splicing processes. Furthermore, we validated our computational models using both thermodynamic assays and datasets derived from high-throughput technologies. We also showed that bulged G4s and R-loops, another type of secondary nucleic acid structure, show strong association with each other, hinting at functional interplay between these structures.
    • A specialized HSP90 co-chaperone network regulates steroid hormone receptor response to ligand

      Backe, Sarah (2022-04)
      Heat shock protein-90 (Hsp90) is an essential molecular chaperone responsible for the stability and activation of over 300 client proteins, many of which have been implicated in tumorigenesis. The chaperone function of Hsp90 is dependent on its ability to hydrolyze ATP. The ATP hydrolysis cycle, and therefore chaperone activity, is tightly regulated by a group of proteins known as co-chaperones and post-translational modifications. Folliculin-interacting proteins 1 and 2 (FNIP1 and FNIP2) are recently recognized Hsp90 co-chaperones with 74% amino acid sequence homology and largely overlapping reported functions. Another recently identified co-chaperone with similar functionality is the tumor suppressor Tsc1. FNIPs and Tsc1 co-chaperone activities have been characterized, however there remains a gap in understanding the functional differences between FNIP1, FNIP2 and Tsc1. Here, we dissected the impact of these newly identified co-chaperones towards Hsp90 client activity. Our data show that Tsc1 and FNIP2 form mutually exclusive complexes with FNIP1 and that unlike Tsc1, FNIP1/2 interact with the catalytic residue of Hsp90. We further demonstrate that FNIP1 and Tsc1 interact with a large set of overlapping proteins, whereas FNIP2 interactors are predominantly unique. Functionally, these co-chaperone complexes increase the affinity of the steroid hormone receptors glucocorticoid receptor and estrogen receptor to their ligands in vivo. We provide a model for the responsiveness of the steroid hormone receptor activation upon ligand binding as a consequence of their association with specific Hsp90:co-chaperone subpopulations. The results presented here demonstrate physiological and mechanistic differences between the co-chaperones FNIP1, FNIP2 and Tsc1.
    • Investigating the mechanism of interaction of R-loops and the Fragile X protein, FMRP: an entanglement of disordered tails and multivalency

      Li, Jing (2023-08)
      Fragile X syndrome (FXS) is one of the most prevalent forms of inherited intellectual disability and is the leading monogenetic cause of autism spectral disorder. FXS is caused by lack of expression or mutations of the FMR1 gene which encodes fragile X messenger ribonucleoprotein, FMRP. Recently, FMRP has been shown to undergo liquid-liquid phase separation (LLPS) in vitro and to localize different isoforms in distinct membrane-less organelles in cellulo. Despite three decades of research, the molecular mechanisms by which FMRP functions are still not fully understood. FMRP is best known as a cytoplasmic mRNA-binding translational regulator. Although the presence of a small fraction of FMRP in the nucleus has long been realized, it was only recently that studies are beginning to uncover its role in influencing genomic function and stability [1]. The Feng lab recently discovered a novel genome protective role for FMRP. FXS patient-derived cells undergo higher level of DNA double-strand breaks (DSBs) than normal cells, especially during DNA replication stress. These DSBs occur at sequences prone to forming R-loops, which are co-transcriptional RNA:DNA hybrids associated with multiple functions including genome instability. Exogenously expressed WT FMRP, but not an I304N disease-causing mutant abates R-loop-induced DSBs. This unexpected finding suggests that FMRP promotes genome integrity by preventing R-loop accumulation and chromosome breakage. However, the mechanism by which FMRP performs this critical function, and how disease-causing mutations affect this process is not fully understood. Here, we set out to elucidate the mechanism underlying FMRP's role in maintaining genome stability. First, we demonstrate that FMRP directly binds R-loops primarily through its C-terminal Intrinsically Disordered Region (C-IDR). In FMR1 CRISPR knock-out HEK293T cells, we observed dynamic condensates in WT FMRP but not in I304N mutant, suggesting that this mutation, located in the central RNA binding KH2 domain, disrupted the ability of I304N to assemble into higher order condensates. Furthermore, unlike the I304N FMRP, WT FMRP show increase in nuclear condensates that overlap with R-loops under replication stress. While we found that WT and I304N mutant can co-phase separate with R-loops in vitro, WT FMRP tends to form hollow droplets with R-loop substrates localized at the periphery, but the vast majority of I304N droplets are filled with dispersed R-loops substrates. Taken together, these data support the hypothesis that the ability of FMRP to form higher order assemblies with R-loops is critical to maintaining genome stability. Our study sets the stage to test the proposed phase separation-function paradigm in other FXS disease mutants.
    • Skewed Distribution Models: Data Analysis, Identification, and Applications in Biomolecular Systems and R-loop Biology of Cancer

      Grageda, Andre (2023-09)
      Modeling and computational analysis can be used to crystallize, integrate, and extract knowledge from large datasets generated by biology, medicine, and next-generation sequencing. The use of probability models, multifactor hypothesis testing, and computational analyses is crucial to studies in systems biology. These studies provide insights into understanding large and diverse molecular biology data sets. It is no longer enough to study individual molecules, their properties, and their interactions with other molecules in cells and organisms. In addition to generating numerous case studies with unique data, such studies provide a limited understanding of the underlying complexity and dynamics of the leading mechanisms determining the states and behaviors of a whole biological system. Sequencing and multi-omics experiments generate big data needed to model processes, organization and behavior of biological systems in a more comprehensive, less biased manner. Analysis of such enormously heterogeneous and complex information requires mathematical models and computational algorithms. It is the motivation and challenge of current systems biology and medicine. Applied to cancer systems biology, we will consider basic probabilistic aspects of big data. We study skewed frequency distributions commonly observed in diverse omics experiments. We focus on modeling and developing computational algorithms to quantify big data's statistical characteristics, aiming for accurate and unbiased characterization of the systems variation. In several applications, we focus on the identification of the skewed distributions for quantification and differentiation of the of R-loop formation patterns in non-cancer, pre-malignant states and cancer genomes. Current studies involving R-loops rely on the S9.6 antibody which generates noisy signals. We show that using R-loop forming sequences for filtering specific S9.6 signals selects biologically meaningful signals. R-loops have been shown to play a role in tumorigenesis. Using our R-loop forming sequence enrichment method, we investigate the roles of R-loops in tumorigenesis across different detection modalities primarily in breast cancer.
    • Loss of Nicotinamide Nucleotide Transhydrogenase Potentiates Autoimmunity in the C57BL/6J Mouse Strain

      Wyman, Brandon (2023-06)
      In Chapter I, we will discuss recent studies showing that mTOR pathway activation plays a critical role in the pathogenesis of autoimmune diseases. The mTOR pathway is a central regulator of growth and survival signals, integrating environmental cues to control cell proliferation and differentiation. Activation of mTOR underlies inflammatory lineage specification, and mTOR blockade-based therapies show promising efficacy in several autoimmune diseases. In Chapter II, we will discuss nicotinamide nucleotide transhydrogenase, NNT, an enzyme localized to the inner mitochondrial membrane which contributes to mitochondrial NADPH production. In C57BL/6J mice, the spontaneous loss of NNT creates a natural model for researching oxidative stress and its ability to potentiate autoimmune disease via the mTOR/AKT pathway. We identify the loss of NNT as a driver of autoimmune pathogenesis, including in multiple sclerosis and ulcerative colitis models. In sum, we highlight the link between upstream pathways of mTOR activation, particularly oxidative stress, and the downstream pathological shift in autoimmune disease due to mTOR activation. We show the novel finding that loss of NNT in the C57BL/6J mouse potentiates autoimmune pathogenesis, and that restoration of wild-type NNT reduces disease burden in select autoimmune models via restoration of redox balance.
    • The neuroinflammatory basis of schizophrenia and bipolar disorder: spotlight on brain macrophages, cytokines, and the blood-brain barrier

      Weickert, Cyndi Shannon; Zhu, Yunting (2023-08)
      Schizophrenia is a severe mental disorder that has been associated with dysregulation of the immune system. Using inflammatory cytokine transcript levels, we found approximately 40% of individuals with schizophrenia are classified as having elevated levels of inflammation with worse neuropathology. The aim of this study was to investigate the extent to which neuroinflammation is associated with schizophrenia in the dorsolateral prefrontal cortex (DLPFC) and midbrain. We used postmortem human brain tissue to investigate multiple inflammation-related molecular mechanisms. First, in the DLPFC, we found macrophages and astrocytes, rather than microglia, contributed more to neuroinflammation in schizophrenia, by showing unchanged or decreased microglial markers and elevated macrophage markers. Macrophage marker expression was more related to pro-inflammatory marker and macrophage recruitment chemokine expression. In addition, we classified "high" and "low" inflammatory (HI and LI) subgroups using inflammatory cytokine and macrophage marker protein levels as discriminators in the DLPFC for the first time. 30% of controls (CTRL) and 56% schizophrenia (SCZ) cases were classified as high inflammation individuals. We found higher CD163+ macrophage density in the DLPFC of SCZ-HI subgroup mainly around small blood vessels of both grey and white matter. In the midbrain, we characterized a substantial proportion of individuals in schizophrenia (46%) and bipolar disorder (29%) expressing elevated inflammatory mRNA (IL6, IL1, TNF and SERPINA3), which were termed the "high" inflammatory (HI) subgroups, and we confirmed increases in IL6 and IL1 at the protein level in these subgroups. Furthermore, we showed elevated macrophage and chemokine marker expression in schizophrenia and bipolar disorder HI subgroups which were associated with changed blood-brain barrier markers, indicating potential macrophage transmigration, supported by altered mRNA and protein levels in the adhesion molecules, tight junction proteins, basement membrane proteins, and angiogenic factors related to blood vessel regulation. Importantly, we observed a novel but unknown neuropathology of more frequent claudin-5 "cell bursts" between blood vessel fragments in schizophrenia and bipolar high inflammatory subgroups. These findings have implications for new immune-related treatments, therapy development, and potential targets for measuring disease progression or early detection of schizophrenia and bipolar disorder.
    • Analysis of phosphatidylinositol 3-phosphate binding to the erlin complex

      Wojcikiewicz, Richard; Hua, Fanghui (2023-08)
      The Endoplasmic reticulum (ER) membrane lipid raft-associated proteins erlin 1 (E1) and erlin2 (E2) are ~40 kDa proteins, and they are members of a superfamily of stomatin/prohibitin/ flotillin/HflK/C (SPFH) domain-containing proteins. E1 and E2 form a massive (~2 MDa) hetero-oligomeric complex that is an essential mediator of inositol 1,4,5-trisphosphate receptor (IP3R) ubiquitination and degradation. Mutations of E1 and E2 are involved in many pathological processes in neurological disorders, such as Hereditary Spastic Paraplegia (HSP), with unknown molecular mechanisms. The Wojcikiewicz laboratory has previously provided evidence that the erlin complex, immunopurified from mammalian cells binds to phosphatidylinositol 3-phosphate (PI(3)P), a key player in membrane dynamics and trafficking regulation in endocytosis and autophagy. In addition, the erlin complex may be involved in different cellular processes beyond IP3Rs degradation, but the exact nature of these roles has remained elusive. My research described in this thesis has uncovered intriguing new insights into the erlin complex and its role in previously unknown aspects of cellular biology. Through the application of diverse biochemical and molecular biology assays, I successfully identified specific regions on E2 that are essential for its binding to PI(3)P. Additionally, my research revealed that the erlin complex plays an important role in regulating cellular PI(3)P levels through its interaction and stabilization with this lipid. This binding and stabilization of PI(3)P are crucial for the regulation of autophagy and lysosome function. These findings contribute to our understanding of the erlin complex's importance in cellular biology and provide valuable knowledge about related processes that have implications for human health and disease.
    • Distinct Interaction Modes for the Eukaryotic RNA Polymerase Alpha-like Subunits and Implications for Disease Modeling

      Knutson, Bruce; Belkevich, Alana (2023-08)
      Eukaryotic DNA-dependent RNA Polymerases (Pols I-III) encode two distinct ⍺- like heterodimers where one is shared between Pols I and III, and the other is unique to Pol II. Human alpha-like subunit mutations are associated with several diseases including Treacher Collins Syndrome (TCS), 4H Leukodystrophy, and Primary Ovarian Sufficiency. Yeast is commonly used to model human disease mutations, yet it remains unclear whether the alpha-like subunit interactions are functionally similar between yeast and human homologs. To examine this, we mutated several regions of the yeast and human small alpha-like subunits and used biochemical and genetic assays to establish the regions and residues required for heterodimerization with their corresponding large alpha-like subunits. Here we show that different regions of the small alpha-like subunits serve differential roles in heterodimerization, in a polymerase- and species-specific manner. We found that the small human alpha-like subunits are more sensitive to mutations, including a "humanized" yeast that we used to characterize the molecular consequence of the TCS-causing POLR1D G52E mutation. These findings help explain why some alpha subunit associated disease mutations have little to no effect when made in their yeast orthologs and offer a better yeast model to assess the molecular basis of POLR1D associated disease mutations.
    • Disorder in the Loop: Identification of a Role for Intrinsic Disorder and Liquid-Liquid Phase Separation in R-Loop Biology

      Bah, Alaji; Garcia Dettori, Leonardo (2023-08)
      R-loops are non-canonical nucleic acid structures composed of a DNA:RNA hybrid, a displaced single-stranded (ss)DNA, and a trailing ssRNA overhang. R-loops perform critical biological functions under normal and disease conditions. To elucidate their cellular functions, we need to understand the mechanisms underlying R-loop formation, recognition, signaling, and resolution. Previous high-throughput screens identified multiple proteins that bind R-loops, including Enzymes and non-enzymatic Readers. However, the precise mechanisms by which these proteins modulate R-loop functions are not fully known. The Fragile X Protein (FMRP) has been recently shown to prevent R-loop-mediated DNA double strand breaks (DSBs), but the mechanism was unknown. FMRP has been previously shown to undergo Liquid-Liquid Phase Separation (LLPS) by itself and with another non-canonical nucleic acid structure, RNA G-quadruplexes, via its C-terminal intrinsically disordered region (C-IDR). Here, we identified the same C-IDR as the predominant R-loop binding site. This unexpected discovery prompted us to explore the hypothesis that disordered regions of other R-loop binding proteins are also utilized to recognize R-loops. Our analysis of the R-loop interactome revealed that low-complexity IDRs are prevalent in this interactome, and that Readers and Enzyme IDRs are distinct (Gly, Ser, Arg, and Pro-rich vs. Glu, Lys, Arg, and Ser-rich, respectively). Furthermore, like FMRP, both R-loop Readers and Enzymes are not only modular, (i.e., contain folded domain(s) interspersed with IDRs), but are also predicted to undergo LLPS. Next, we demonstrated that the IDRs from the R-loop binding protein RBM3 and the R-loop helicase DDX21 also bind to R-loops, providing additional examples of IDR-mediated R-loop binding from an R-loop Reader and Enzyme, respectively. Finally, we demonstrated that FMRP C-IDR and DDX21 N-IDR can undergo co-LLPS with R-loops suggesting that IDR-based R-loop binding and co-LLPS is a universal mechanism shared by all members of the interactome. Therefore, we propose that IDRs can provide a functional link between R-loop recognition and downstream signaling through the assembly of LLPS-mediated membrane-less R-loop foci, where the activities of the folded domains are coordinated to regulate the biological functions of R-loops. Mutations or dysregulation of the function of IDR-enriched R-loop interactors can potentially lead to severe genomic defects, such as the R-loop-mediated DSBs observed in Fragile X patient-derived cells.
    • Effects of the hepatic glucocorticoid receptor in the setting of sepsis, infection, and inflammation

      Lu, Hong; Winkler, Rebecca (2023-08)
      Each year hundreds of thousands of people develop life-threatening sepsis, defined by a combination of infection and organ dysfunction. Although many affected biological pathways are typically regulated by the glucocorticoid receptor (GR), during sepsis this is deficient and supplementation with exogenous glucocorticoids is often ineffective in reducing mortality. The GR has different effects in different organs. In liver many effects are beneficial, whereas in immune or muscular systems many effects are deleterious. A sampling of this literature is reviewed in chapter 1. With the hypothesis that liver-specific glucocorticoid therapy will be more clinically beneficial than systemic therapy, we studied liver-specific GR effects in infection, inflammation, or sepsis. Chapter 2 describes in-vitro chemokine alterations from GR activation in primary human hepatocytes, with inflammation or infection modeled by TNFα and/or lipopolysaccharide. Comparisons were made in primary human hepatocytes, the human hepatoma cell line HEPG2, and the non-liver cell line HEK293t. Chapter 3 outlines outcomes of liver-specific GR deficiency using mice with inducible liver-specific GR knockout, modeling sepsis with cecal ligation and puncture. Results of these two models show mRNA and/or protein changes induced by GR in chemokines; transcription factors; genes related to protein degradation, metabolism, metal management, inflammation, liver regeneration, and hemodynamic stability. Results of these 2 models demonstrate a significant role of the hepatic GR in many pathways dysregulated during sepsis. Therefore, targeting GR therapy to the liver instead of systemic treatment may prove more clinically beneficial to reducing the morbidity and mortality of sepsis.