Albert Einstein College of Medicine: Doctoral Dissertations

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    Transcriptional Dynamics During Lens Fiber Cell Differentiation and Novel Insights Into Their Denucleation Process
    (ProQuest Dissertations & Theses Global, 2018) Limi, Saima; Cvekl, Ales
    The temporal and spatial regulation of gene transcription is a central process governing mammalian development and differentiation. Cellular differentiation is executed through coordinated expression of multiple genes, including families of genes encoding proteins with highly specialized functions. Nascent transcription is not a constitutive event as genes are transcribed in sporadic pulses of activity termed transcriptional bursts. There are two transcriptional bursting parameters that regulate output of gene products, burst fraction and burst size/intensity. Transcriptional burst fraction refers to the number of active phases of transcription. Transcriptional burst size refers to the number of RNA molecules produced within a single burst event. It is not known how these parameters change during cell differentiation. Many protein-coding and non-coding genes can be located in gene-rich regions and their nascent transcription can be influenced by promoter location and orientation. Although transcription-coupled splicing generates mRNAs that leave the nuclei, recent studies indicate that specific spliced MRNAs can temporally accumulate in the nuclei. Lens specific transcription of α-, β-, and γ-crystallin genes is a unique system for studies of nascent transcription as the spatial organization of lens fiber cells within the lens correlates precisely with their differentiation status. In addition, lens transparency requires degradation of nuclei in mature lens fibers and this process remains poorly understood. Depletion of ATP dependent chromatin remodeling enzyme Snf2h in lens results in nuclear retention; nevertheless, how Snf2h influences nascent transcription and denucleation cascade remains unknown. My hypothesis predicts that a) Cellular differentiation impacts both transcriptional burst fraction and burst intensity and that individual crystallin genes employ these parameters differently, b) During lens fiber cell differentiation, condensation of chromatin and nuclear compaction prior the denucleation causes "premature" termination of nascent transcription, c) Disruption of lens differentiation by lensspecific depletion of Snf2h impairs transcription of differentiation-regulated genes as the nuclear degradation process, a key part of the differentiation cascade, is impaired, and d), Bi-directional nascent transcription from two crystallin promoters is a rare event due to the local competition for the RNA polymerase II convoys. Specific Aims: 1. To determine transcriptional burst parameters and their changes during lens fiber cell differentiation. 2. To examine the cellular and molecular mechanisms of lens fiber cell denucleation, including cessation of transcription in condensed nuclei and the role of ATPdependent chromatin remodeling enzyme Snf2h (Smarca5) in this process. 3. To investigate transcription from bi-directional promoters and from crystallin generich region of mouse chromosome 5. To study how transcriptional burst fraction and intensity of crystallin genes change during lens development and differentiation (Aim I) we performed RNA fluorescent in situ hybridization (RNA FISH) of αA-, βB1-,βB3-, βA4-, and γA-crystallins and β-actin in the lenses of embryonic day E12.5, E14.5, and E16.5 mouse embryos and newborns. RNA FISH detects nascent transcription sites to visualize active sites of transcription, and, thus, is used to quantify bursting parameters. Lens fiber cells were divided into four different areas, a, b, c, d from the periphery to the center of the lens, respectively, to represent successive differentiation stages. Burst fraction was quantified as percent alleles of a specific crystallin or the β-actin gene transcribed within a given region of the lens. We found that burst fraction changes dramatically during cellular differentiation throughout the lens fiber cell compartment. During early embryonic stages of lens development there was a general increase in burst fraction as cell differentiation progressed. Although burst intensity of crystallin genes also change during lens differentiation and denucleation, this change is moderate compared to the burst fraction parameter. We found unexpectedly that nascent transcription of βB I- and γA-crystallins and β-actin is retained in nuclei just prior their physical disintegration (Aim 2). Nuclear degradation is first marked by changes of the nuclear shape (from ovoid to round), reduction in its size and chromatin condensation. We found that these condensed nuclei transfer both histone and non-histone proteins, including Snf2h, from the nuclei into the cytoplasm. Lens-specific depletion of Snf2h disrupts the denucleation process in the lens; however, the persisting nuclei were still able to transcribe many crystallin genes. Disruption of this chromatin remodeler changed both burst intensity and fraction depending on the crystallin gene being studied (Aim 2). Our data show that nascent transcription from βA4- and βB 1-crystallin promoters can occur frequently from the same allele and that ~β3-crystallin spliced mRNAs are temporally retained within the nuclei of early lens fiber cells (Aim 3).
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    The Role of Heparan Sulfate Proteoglycans as Co-Receptors of Wnt-signaling during Neural Development.
    (ProQuest Dissertations & Theses Global, 2018) Saied-Santiago, Kristian; Buelow, Hannes E
    Cell migration is a hallmark of many biological processes, including development, immunity, and malignancies. In particular, structures part of the nervous system, including synapses and circuitries, are formed and maintained through the process of cell migration. The extracellular space plays a significant role in this process, as it facilitates exposure between the traveling cell and molecules within the extracellular environment. However, neurodevelopmental and physiological diseases, as well as metastatic cancer, can be caused by aberrant migrations. My thesis project aims to understand the role of heparan sulfate proteoglycans (HSPGs), extracellular glycoproteins present in the majority of cells, in cell migration and neural development. The polysaccharide chains attached to HSPGs contain a vast number of complex modification patterns in the form of acetylation, sulfation, and epimerization. Moreover, HSPGs have been shown to act as essential co-factors on multiple signaling pathways. To determine the significance of HSPGs in cellular migration, I created or obtained fluorescently labeled reporters that allowed me to visualize the positioning of single-cell(s) in live Caenorhabditis elegans. Using these reporters, I analyzed the migration and final positioning of five different cells in the worm. I found that membrane-bound HSPGs sdn-1/Syndecan and lon-2/Glypican are required for all the migration events tested; in contrast, some HSPGs are only needed in particular migrations. I also determined that a given HSPG is necessary to promote migration, while others are primarily required to establish the exact position of each moving cell(s). In sum, my genetic analyses suggest a unique combination of HSPGs modulate each migration event. While revising the literature, I found that a similar genetic study was conducted by surveying the role of different Wnt ligands and Frizzled receptors genes in cell migration. Previous research performed on loss-of-function mutants in Wnt-signaling genes demonstrated strikingly similar phenotypes to the ones observed in HSPG mutant animals. Based on these observations, I hypothesized HSPGs act together with Wnt ligands and frizzled receptors to control cellular migrations and positioning. Indeed, I found that the Wnt ligand EGL-20 requires the presence of SDN-1/Syndecan to promote cell migration. A second Wnt ligand, lin-44, functionally interacts with sdn-1 to promote axon guidance of a separate neuron. These findings suggest SDN-1 is capable of interacting with different Wnts in a context-dependent manner. To further determine the role of HSPGs with Wnt-signaling, I performed coimmunoprecipitation experiments between SDN-1/Syndecan, EGL-20/Wnt, and MIG1/Frizzled. These experiments suggested that SDN-1/Syndecan, EGL-20/Wnt, and MIG1/Frizzled form a biochemical complex when they are transfected in a heterologous system. These findings suggested SDN-1 is a co-receptor of the Wnt-signaling pathway. Next, I established the function of HS glycans during cellular and axonal migrations and demonstrated that SDN-1/Syndecan contains unique HS epitopes that may confer selectivity to the Wnt-Frizzled complexes acting in each migration event. Taken together, my thesis findings suggest HSPGs genetically and biochemically interact with Wnt-signaling genes to coordinate cell and axonal migrations, as well as positioning. To my knowledge, this is the first instance HSPGs and Wnt-signaling have been implicated in conjunction with neural development. I expect the research conducted during my thesis work will provide knowledge necessary to comprehend the process of cell migration in normal and diseased states.
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    How Genetic Variation influences Molecular Genomic Phenotypes
    (ProQuest Dissertations & Theses Global, 2018) Johnston, Andrew D.; Greally, John
    Variation of DNA sequence in the human genome has relatively predictable effects in protein-coding sequences, but is not well understood in the remaining 98% of the genome. A current focus of research is on the sites that regulate transcription, where DNA sequence variation can have effects to increase or decrease the expression of nearby genes. These functional variants are believed to exert their effects by altering the binding of transcription factors to the genome. While this is a widely-held assumption, and supported by many observations, the model still lacks formal proof. In order to test this model, I performed a comprehensive analysis of a 17-member family that has been intensively studied as part of the 1000 Genomes project as well as developed a method to efficiently edit lymphoblastoid cell lines (LCLs) using CRISPR/Cas9. First in Chapter 1, 1 review the genetic polymorphisms found within human populations and discuss the difficulty of ascribing disease risk to variants. l then introduce the various components of transcription regulation and how they are influenced by genotype. I further explore the use of lymphoblastoid cell lines as a model system and how this confers the manifestation of their molecular phenotypes. I then detail the application of CRISPR in both genome editing and epigenetic editing. In Chapter 2, 1 describe our rigorous approach to extensively characterize the molecule phenotypes of the LCLs derived from the 17-member family. The data revealed novel miRNAs, expression and chromatin accessibility quantitative trait loci (eQTLs, caQTLs) and allelic patterns of molecular organization of transcriptional regulatory processes. Through the combination of data generated in previous studies and by us, we prioritized an intronic enhancer, demonstrating allele specific occupancy of NF&kgr;B, at the TBC1 D4 locus for further studies. To assess the impact of the variant on regulatory phenotypes, I performed genome editing and validated molecular changes. Using a CRISPR/Cas9 system tethered to NF&kgr;B subunit p65, I retargeted NF&kgr;B to the locus to confirm the hypothesis that it is disruption of TF binding that mediates these altered molecular phenotypes. In Chapter 3, I discuss the development of a strategy to perform high efficiency genome editing in LCLs, a difficult cell-type to transfect and clone. I remark on its potential in future applications. Finally, in my Conclusions I discuss the limitations of and gained perspective from the studies. The technical difficulties of utilizing lymphoblastoid cell lines in CRISPR assays hindered further investigation of the mechanisms mediating genetic variant effects as well as increased sample sizes. The study serves as a foundation upon which questions regarding distal cis-regulatory sites could be explored. While we establish that the retargeting of a TF to a genetically-"silenced" cis-regulatory site can partially reconstitute the expression of the associated gene, the downstream mechanisms of the observed reactivation require follow-up investigation, particularly the roles of chromatin modulators. I also review CRISPR/Cas9 principles corroborated through the development of our high efficiency editing method in LCLs. The well-documented and thorough molecular characterization of the 17-member kindred studied in this thesis provides a wealth of data to act as a springboard for future studies.
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    The Role of Homeobox Transcription Factors Six3 and Six6 in Retinal Development
    (ProQuest Dissertations & Theses Global, 2018) Diacou, Raven Gwendolyn; Liu, Wei
    The potential of stem cell-based therapeutics has reignited interest and attention to the development of organs and cell fate specification. In patients, irreversible vision loss occurs due to neurodegenerative eye disease. A major goal in the field is identifying the mechanisms that are required to direct groups of stem cells towards the heterogenous cell types that comprise the retina. The retina serves as an easily accessible model of neural development as diverse neuronal cells are generated from a pool of multipotent progenitors. The genetic mechanisms regulating multipotent retinal progenitors and their differentiation are partially understood. Six3 and Six6 are homeobox transcription factors expressed in multipotent retinal progenitor cells. Although Six3 is required for eye specification, the role of Six3 and Six6 in retinal progenitor cells is largely undetermined as Six3-null mice fail to initiate eye development and until now Six3 was not studied in the context of retinal development after eye specification. The Six3;Six6 compound mutant retinas displayed novel retinal phenotypes (reduced proliferation, aberrant differentiation, and perturbed expression of several important transcription factors), which were not observed in either Six3 or Six6 null retinas. Although Six3 and Six6 regulate several known genes, the full gene regulatory network controlled by Six3 and Six6 has not been determined. Therefore, I investigated the genetic network regulated by Six3 and Six6 in retinal progenitor cells by 1) transcriptome profiling and 2) functional assessments of two molecular mechanisms downstream of Six3 and Six6 joint functions. In chapter 1, I review the process of eye development, followed by characterization of the genes required to maintain multipotent retinal progenitor cells and describe the histological and molecular phenotype observed. I describe two major retina phenotypes of the Six3;Six6 compound null retinal phenotype; expansion of ciliary margin fate and reduction of retinogenic genes in retinal progenitors resulting in reduced eye size. In chapter 2, I describe the mouse mating strategy used to generate Six3;Six6 compound null retinas and force expression of Sox2 in Six3;Six6 compound null retinas, detail methods to profile embryonic mouse retinas by RNA-sequencing and a method to culture embryonic mouse eyecups in soluble recombinant proteins. In chapter 3, I use RNA-sequencing to identify the genes dysregulated in the Six3;Six6 compound null retinas. From this analysis I identified 800 differentially expressed genes and confirmed the upregulation of ciliary margin fate and Wnt/Bcatenin pathway in the Six3;Six6 compound null retinas. Furthermore, Wnt3a, Wnt16, and receptor Fzd1 were identified from the RNAseq as a putative Wnt components regulating the CM expansion phenotype observed in Six3;Six6 compound null retinas. In chapter 4, I tested the effects of exogenous Wnt signaling on retinas and demonstrated Wnt3a was sufficient to promote ciliary margin fate. In chapter 5, I tested a candidate mediator of Six3 and Six6, transcription factor Sox2, by driving expression of Sox2 in Six3;Six6 compound null retinas through a genetic mouse mating strategy which partially restored aspects of the retinal phenotype observed in Six3;Six6 compound null retinas. This thesis provides a groundwork for identifying genes directly regulated by Six3 and Six6 during retinal development and may aid in the identification of novel biomarkers for patients with developmental eye disease and diseases of the anterior segment and inform genetic instruction of eye development in stem cell regeneration efforts. I propose Six3 and Six6 maintain neuroretinal progenitors by regulating several retinogenic programs, while antagonizing Wnt/B-catenin to suppress ciliary margin fate.
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    Runx1 Promotes Murine Erythroid Progenitor Proliferation and Inhibits Differentiation by Preventing Pu.1 downregulation
    (ProQuest Dissertations & Theses Global, 2018) Willcockson, Michael Alton; Skoultchi, Arthur I.
    Pu.1 is an ETS family transcription factor (TF) that plays critical roles in erythroid progenitors by promoting proliferation and blocking terminal differentiation. However, the mechanisms controlling expression and downregulation of Pu.1 during early erythropoiesis have not been defined. In this study, we identify the actions of Runx1 and Pu.1 itself at the Pu.1 gene Upstream Regulatory Element (URE) as major regulators of Pu.1 expression in Burst-Forming Unit erythrocytes (BFUe). During early erythropoiesis, Runx1 and Pu.1 levels decline and chromatin accessibility at the URE is lost. Ectopic expression of Runx1 or Pu.1, both of which bind the URE, prevents Pu.1 downregulation and blocks terminal erythroid differentiation, resulting in extensive ex vivo proliferation and immortalization of erythroid progenitors. dCas9 mediated occlusion of Runx1 binding sites within the URE leads to decreased Pu.1 expression and ectopic expression of Runx1 in BFUe lacking a URE fails to block terminal erythroid differentiation. Thus, Runx1, acting at the URE, and Pu.1 itself, directly regulate Pu.1 levels in erythroid cells and loss of both factors is critical for Pu.1 downregulation during terminal differentiation. The molecular mechanism of URE inactivation in erythroid cells through loss of TF binding represents a distinct pattern of Pu.1 regulation from those described in other hematopoietic cell types such as T-cells which downregulate Pu.1 through active repression. The importance of downregulation of Runx1 and Pu.1 in erythropoiesis is further supported by genome-wide analyses showing that their DNA-binding motifs are highly over-represented in regions that lose chromatin accessibility during early erythroid development.
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    The Molecular Basis For Receptor-Ligand Recognition In the Immunoglobulin Superfamily
    (ProQuest Dissertations & Theses Global, 2018) Gil, Nelson Homero, Jr.; Fiser, Andras
    The immunoglobulin superfamily (IgSF) is one of the largest in the human proteome, comprising nearly 500 cell-surface and secreted members critical to processes ranging from the modulation of the immune response to the formation of neural synapses. Molecular-level knowledge of IgSF binding interfaces remains scarce: only ~5% of IgSF proteins currently have crystallographic structures in complexed form. The primary motivation of this thesis has been to systematically elucidate the molecular-level details of IgSF binding interfaces in order to aid identification of potential binding partners. To this end, we have developed broadly-applicable computational approaches that extract functional information from protein sequence and structure data. Chapter I provides an expanded introduction to the IgSF and reviews the historical development of protein function prediction using sequence-based and structure-based methods. In addition, the principles of pharmacophore-based protein binding partner prediction are reviewed. Chapter II details the development of Selection of Alignment by Maximal Mutual Information (SAMMI), an algorithm to identify an optimal multiple sequence alignment (MSA) for functional residue detection by conservation analysis. This approach hypothesizes that the mutual information among aligned sequence positions will be maximal in those MSAs that include the most diverse set possible of structurally and functionally homogeneous sequence homologs. In Chapter III the performance of SAMMI is examined in the context of state-of-the-art functional residue prediction methods. We demonstrate that simple conservation analysis of SAMMI-selected MSAs improves upon modern methods, which mostly include sequence conservation as one of several input features for machine learning. We further show that a simple combinatorial MSA sampling algorithm will generally produce an MSA including an optimal set of homologs whose conservation analysis doubles state-of-the-art performance, at which point the primary source of error is due to binding site definition. In Chapter IV, we describe the development of a structure-based binding interface prediction algorithm in which binding sites of structurally homologous proteins are mapped onto a query protein of interest. We demonstrate that IgSFs tend to share the structural locations of their binding sites, and that the reliability of these predictions can be estimated based on their agreement with sequence-based conservation analysis. This allows us to provide binding interface predictions and associated reliability scores for IgSF proteins with unknown binding interfaces. In Chapter V, we provide a new quality indicator for the Protein Ligand Interface Design (ProtLID) algorithm, which predicts binding partners given a protein binding interface. ProtLID creates a residue-specific-(rs)-pharmacophore – a map of residue binding preferences that potential binding partners should satisfy. We implemented a novel statistical analysis of the rs-pharmacophore spatial matching distribution to provide a confidence measurement for any specific binding partner prediction. In summary, this thesis provides novel insights in the study of the IgSF and in the field of bioinformatics. SAMMI is the first objective approach to the selection of representative sequences for subsequent analysis. Our work also highlights the largely neglected importance of optimally composing MSAs for conservation analysis. Furthermore, we provide a systematic examination of IgSF binding interfaces and formalize the notion that IgSF proteins tend to bind at similar geometric locations on their structure. Finally, we provide a quality measure that can complement the ProtLID approach and prioritize experimental study of computational predictions.
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    Elucidating the Enhancer Code in 9p21 Locus Underlying Age-Related Disease Risks
    (ProQuest Dissertations & Theses Global, 2018) Zhu, Yizhou; Suh, Yousin; Morrow, Bernice
    The chromosomal region 9p21.3 is one of the first identified and most replicated risk loci for coronary artery disease (CAD) by genome-wide association studies (GWAS). This ~150 kb long non-coding region was later found to be associated with multiple disease phenotypes, most of which are age-related, such as cancers, glaucoma, and type II diabetes (T2D). Despite the strong evidence of disease associations from population studies, the mechanisms underlying the 9p21.3 locus remained largely elusive, similar to many other non-coding GWAS loci. Recent epigenetic annotation studies suggested an enrichment of enhancers in 9p21.3 and, at the genome-wide level, a general enrichment of GWAS variants around enhancer regions. I thus hypothesized that functional disease variants in the 9p21.3 locus cause alterations to enhancer functions and transcriptional regulatory networks, thus contributing to the risk of age-related diseases. In Chapter 2, I performed a comprehensive functional study of enhancers in the 9p21.3 locus. Using a human embryonic stem cell (hESC) differentiation panel, I confirmed CDKN2B expression was reduced by CAD causal variants specifically in vascular cells. A large fragment deletion study using CRISPR identified a causal enhancer region inside the CAD GWAS hotspot, together with other cell type specific enhancer elements. Together with the results from Chapter 3, I showed that these enhancers formed a synergistic interaction network that was significantly dampened by the risk genotype. In order to characterize the enhancer interaction network in 9p21.3, I designed a novel chromatin conformation capture method with substantially improved resolution. As described in Chapter 3, this new method, named Tri-4C, was able to map distal contacts to a hundred basepair resolution, leading to a much improved accuracy in the capture of enhancer loop interactions than existing methods. I demonstrated that Tri-4C was capable of revealing quantitative enhancer loop alterations under dynamic gene controls, as well as differentiating allele-specific loop interactions in heterozygote loci. In Chapter 4, I investigated the function of variants associated the diseases other than CAD. Using an in-silico prediction approach, I identified rs6475604, a glaucoma-associated enhancer variant showing reduced binding of the transcription factor YY1. Dampened YY1 activity in this locus may cause upregulation of its known repressive target, p16, leading to cell cycle arrest and premature senescence. Finally, in Chapter 5, to study whether 9p21.3 associates with extreme longevity, I performed a sequencing study of the locus in an Ashkenazi longevity cohort. I discovered an interesting mild depletion of combined age-related disease variants of this locus in centenarians. This finding supports the idea that reaching longevity is associated with evasion from risks of multiple, if not all, common age-related diseases. Taken together, these works established how causal variants in 9p21.3 were involved in the regulatory process of their target genes. The study resolved a key aspect of enhancer mechanisms underlying the disease risk associations of the 9p21.3 locus, which has remained largely a mystery in the past decade since its discovery. The effort to elucidate the enhancer regulatory networks in this locus used state-of-the-art model systems and techniques, which provide insights to the general question of how to address challenges in the functional interpretation of non-coding GWAS associations.
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    Myelodysplastic Syndrome Progression to Acute Myeloid Leukemia at the Stem Cell Level
    (ProQuest Dissertations & Theses Global, 2018) Chen, Jiahao; Steidl, Ulrich G.
    Myelodysplastic syndromes (MDS) are malignant hematopoietic stem cell-derived disease with poor clinical outcome and overall survival. Overall ~30% of patients with MDS progress to secondary acute myeloid leukemia (sAML), which has an exacerbated median survival of less than six months. Delaying the progression to sAML represents one of the key challenges in the clinical management of patients with MDS. Thus, cellular and molecular insights into the progression of MDS to sAML are needed. Previous seminal studies have investigated the clonal evolution during MDS to sAML progression by sequencing the longitudinal samples of bulk tumor cells from MDS and matched sAML of the same patient. However, the clonal origin of MDS and AML has been demonstrated to lie within the phenotypically and functionally defined stem cell compartment, representing a small subset of total bone marrow which cannot be effectively interrogated by bulk sequencing. Clonal evolution at the stem cell level has not yet been directly examined. To prospectively analyze subclonal composition at the stem cell level in MDS and study their clonal evolution during progression to sAML, we utilized longitudinal, paired samples from 7 patients with MDS who had later progressed to sAML. For both MDS and paired sAML samples, we utilized multi-parameter fluorescence-activated cell sorting (FACS) to isolate phenotypically defined stem cells and blasts from both stages. Specifically, we isolated hematopoietic stem and progenitor cells (HSPC, Lineage -CD34+CD38 -) expressing at least one of the leukemic stem cell (LSC) markers (CD45RA, CD123, or ILIRAP), to enrich for malignant stem cells (MDS-SC, AML-SC). At the same time, we isolated HSPCs that were triple-negative for CD45RA, CD123, and ILIRAP to enrich for pre-malignant stem cells (preMDS-SC, preAML-SC). We observed that phenotypically defined malignant stem cells were significantly expanded in the sAML compared to the matched MDS stage. Moreover, compared to preMDS-SC and preAML-SC, sorted MDS-SCs and AML-SCs showed impaired differentiation, with significantly myeloid-biased outputs in vivo and ex vivo, demonstrating that CD45RA/CD123/ILIRAP expressing HSPCs are indeed enriched for malignant stem cells in MDS and AML. To investigate the patterns of somatic mutations in stem cells and blasts, we performed targeted deep sequencing covering both coding and non-coding regulatory regions of around 600 MDS and AML relevant genes. Interestingly, we found that stem cells at both MDS and sAML stages have a significantly higher number of subclonal mutations compared to the blast population. Mutation signature analysis revealed that mutation patterns in all the cell populations were mainly associated with age-related signatures. Interestingly, in stem cell compartments, there are specific mutations associated with DNA repair pathways. We next directly compared the subclonal diversity of stem cells versus blasts by clonality analysis, which also revealed higher subclonal complexity at the stem cell populations compared to the blasts. In addition, in all the 7 patients studied, we observed shared dominant clones across all stem cell and blast populations, with key mutations such as TET2, TP53, or U2AF1. Remarkably, these shared mutations were also detectable in the T cells isolated from the same patients, suggesting that these mutations in TET2, TP53, and U2AF1 were acquired at the early stage during the initiation of MDS and AML. We next performed single-cell targeted re-sequencing of sorted stem and blast cells, to experimentally examine the clonal architecture and evolution during MDS to sAML progression. Our results revealed a pattern of non-linear, parallel clonal evolution, with distinct subclones within the MDS stem cells contributing to the generation of MDS blasts or the progression to sAML, respectively. The progression to sAML can be associated with rare subclones in MDS stem cells harboring mutations on RUNX1, NRAS, or NTRK3 and DUSP22 in 3 out of 7 patients, or a larger subclone with ERG and ATRX mutations in one patient. These MDS stem cell subclones were not detectable in MDS blasts, but became dominant upon sAML progression, indicating the early branching evolution at the MDS stem cells during the blast generation and disease progression to sAML. Additionally, in 3 other patients, we observed slightly later branching evolution at MDS stem cells, with dominant clones shared across MDS and sAML stages. In summary, using sorted stern cell populations from patients with MDS, we were able to capture of even small subclones at MDS stem cells that are crucial for eventual progression to sAML. Our data provide a comprehensive characterization of subclonal diversity, with important insights into the subclonal evolution of stem cells in MDS pathogenesis and transformation, as well as have implications for current bulk cell-focused precision oncology approaches.
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    Route Specific Effects of Lipoprotein Agonists of TLR2 on CD4 T Cell Priming
    (ProQuest Dissertations & Theses Global, 2018) Johndrow, Christopher Taylor; Porcelli, Steven A.
    Vaccines are currently the most successful prophylactic intervention against many infectious diseases. Subunit vaccines are a promising strategy for the development of novel effective vaccines, however, protein subunits are poorly immunogenic alone. Successful subunit vaccines require formulation with adjuvant compounds in order to stimulate cells of the innate immune system and generate protective adaptive immune responses. The development of vaccine adjuvants is critical for subunit vaccine development, but requires improved high throughput screening methods that reliably predict in vivo immune responses. Pattern recognition receptor (PRR) agonists are a growing class of potential vaccine adjuvants that are able to shape both the scale and character of the immune response to subunit vaccines through the direction of CD4 T cell polarization. We have applied a high-throughput in vitro assay to assess the CD4 T cell polarization potential of a panel of PRR agonists. Using this system, we identified lipopeptide TLR2 agonists as exceptional Th1 polarizing adjuvants. In vivo, we demonstrated that i.v. administration of TLR2 agonists replicates the findings from in vitro screening by promoting strong Th1 polarization. In contrast, these TLR2 agonists were found to inhibit priming of Th1 responses when administered intradermally in mice. We found that this route-specific suppression was mediated by infiltrating CCR2+ cells in the skin draining lymph nodes and was independent of skin-derived dendritic cells. We further demonstrated that CD4 T cell priming after immunization against Mycobacterium tuberculosis with BCG, a lipoprotein-rich bacteria recognized by TLR2, was dependent on immunization route, and Th1-like responses generated by intradermal administration of BCG were significantly improved when administered intravenously. We performed further studies to characterize the CD4 T cell priming environment in the skin draining lymph node, particularly dendritic cell and other innate myeloid cell trafficking into secondary lymphoid organs and expression of cytokines and co-inhibitory signals after treatment with TLR2 and TLR9 agonists. We assessed the expression of pro-inflammatory and anti-inflammatory cytokines by lymph node resident cells, expression of co-inhibitory molecules by dendritic cells, antigen trafficking and myeloid cell infiltration, and the effects of IL-10 and TGFβ neutralization on Th1 suppression. We also explored the effects of mycobacterial lipoproteins, either enriched directly from BCG or purified from recombinant expression on TLR2 activation and describe aspects of in vivo responses which suggest mycobacterial lipoproteins function similarly in the skin as synthetic TLR2 ligands and suppress optimal Th1 priming. Complete understanding of route-dependent TLR2 responses are critical for informed design of novel subunit vaccines and improvement of BCG and other vaccines based on live-attenuated organisms that express TLR2 ligands. It is the goal of this work to advance understanding of downstream effects of TLR2 signaling in various contexts and to contribute to future vaccine design strategies.
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    Iron Homeostasis-Regulatory Pathways mediate Hematopoietic Stem Cell Fate
    (ProQuest Dissertations & Theses Global, 2018) Kao, Yun-Ruei Christine; Will, Britta
    Iron homeostasis-regulatory pathways mediate hematopoietic stem cell fate Hematopoiesis is a highly regulated, step-wise process in which hematopoietic stem cells (HSCs) residing at the top of the hematopoietic hierarchy are capable of self-renewing to maintain the stem cell pool, and differentiating to give rise to blood cells of all lineages. Inefficient hematopoiesis is a frequent and critical clinical problem in aplastic anemia, myelodysplastic syndromes (MDS), immune thrombocytopenia, as well as chemotherapy-induced pancytopenia. Eltrombopag (EP), a small molecule initially designed as a thrombopoietin receptor (TPO-R) agonist, has emerged as a potent platelet-stimulating agent and has also shown remarkable efficacy in stimulating sustained multilineage hematopoiesis, suggesting an effect at the level of primitive HSCs. Apart from stimulating TPO signaling, EP has been reported to trigger TPO-R independent pathways involving iron chelation. Nevertheless, it remains to be determined whether EP exerts its effect at the HSC level, and whether the iron-chelating property is functionally relevant to the HSC stimulation by EP. We found that EP significantly enhanced not only multilineage differentiation, but also serial replating capacity of purified human HSCs. In addition, comparative analysis of stem cells in the bone marrow of patients receiving EP showed a marked increase in the number of functional stem cells compared to patients treated with romiplostim, another TPO-R agonist lacking iron-chelating ability. Microarray analysis of human HSCs also confirmed iron-associated molecular changes in EP-treated HSCs that were absent in TPO-treated HSCs. This cellular and molecular evidence strongly suggests a role of iron-mediated pathways in regulating HSC function that is distinct from TPO stimulation. Therefore, we utilized separation-of-function mouse models, including wild type and TPO receptor (TPOR) knockout models, to examine TPO-R independent effects of EP on HSC function ex vivo and in vivo. In both mouse models, we observed a significant increase of HSC self-renewal upon EP treatment, which was also consistently observed with two other clinically available iron chelators, Deferoxamine (DFO) and Deferasirox (DFX). Importantly, the increase of HSC self-renewal upon iron chelation was abrogated by preloading with ferric ammonium citrate (FAC), demonstrating the causative role of intracellular iron levels in the modulation of HSC self-renewal. Gene expression profiling of mouse HSCs treated ex vivo with DFO or EP revealed alterations in molecular pathways that are consistent with reduction of intracellular labile iron pools (LIP), including the activation of transferrin receptor (TfrciCD71) and Nuclear receptor coactivator 4 (encoded by Ncoa4). Intriguingly, simultaneous inhibition of CD71 and NOCA4 abrogated the increase of HSC self-renewal by iron chelators, suggesting the activation of iron-regulatory pathways following iron reduction mediated the HSC stimulatory effects. Further gene expression and metabolite profiling of cells exposed to iron chelators also revealed alterations in metabolic pathways associated with fatty acid oxidation (FAO), which was validated by Seahorse, an assay that directly measures the extracellular fluxes of oxygen consumption. Furthermore, iron chelation-mediated increase in HSC number was rescued by pharmacologic inhibition of CPT-1, a mitochondrial enzyme involved in the conjugation of fatty acids to carnitine for subsequent transfer inside mitochondria. Together, our data demonstrates the integral role of FAO in governing HSC fate transitions following reduction of LIP. Further molecular interrogation revealed an increase in free arachidonic acid (AA) following iron chelator treatment ex vivo, which we hypothesized could be partially regulated by NCOA4-mediated ferritinophagy. We designed short hairpin RNA (shRNA) constructs to knockdown Acsl4 , a member of the long-chain acyl-CoA synthetases that preferentially utilizes AA as substrates, to selectively inhibit the increase in FAO contributed by AA. We found that Acsl4 knockdown abrogated the increase in FAO rate stimulated by DFO, indicating that iron chelation increases the rate of FAO through the mobilization of intracellular AA stores. It has been previously described that upon nutrient deprivation, fatty acids packaged in lipid droplets mobilize to mitochondria and induce β-oxidation of the fatty acids. Inhibition of lipolysis by diethylumbelliferyl phosphate (DEUP) abrogated FAO stimulation upon iron chelation, suggesting the contribution of lipid droplets in fueling mitochondrial oxidation. Interestingly, simultaneous inactivation of AA and inhibition of lipolysis did not further decrease FAO. These findings indicate that AA fuels FAO by a mechanism that is predominantly dependent on lipid droplet mobilization and lipolysis. In conclusion, our data has provided proof-of-concept that experimental reduction of the intracellular labile iron pool, the most readily chelatable form of iron within cells, leads to an array of metabolic reprogramming and an increase in HSC numbers. In-depth investigation on the molecular underpinnings demonstrate that the intracellular labile iron pool reinforces stem cell-maintaining metabolic programs and acts as a rheostat in dividing HSCs. *Please refer to dissertation for diagrams.
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    Effect of βV-Tubulin on Cellular Function in Malignancy
    (ProQuest Dissertations & Theses Global, 2018) Mathew, Deepti; Horwitz, Susan Band
    This thesis work focuses on the Class V beta tubulin isotype (βV-tubulin) exploring the premise that cellular function or dysfunction may be mediated by the isotype composition of its microtubules. It expands on a previously published study that showed βV-tubulin to be highly expressed in tissue with secretory function. We hypothesize that altered βV-tubulin expression may be associated with the pathogenesis of ovarian cancer that is known to arise from secretory epithelial cells. Specifically, we test the association of βV-tubulin with chemosensitivity, invasion and secretion. Firstly, to evaluate the association of βV-tubulin expression with ovarian cancer pathogenesis, we performed immunohistochemistry (INC) using a highly specific antibody developed in our laboratory against human βV-tubulin to evaluate expression in the fallopian tube epithelium (FTE) of patients who underwent salpingectomy; dichotomizing analysis for high-risk (BRCA mutant) versus low-risk cohorts (n= 82 in total). These studies were complemented by evaluating βV-tubulin expression in serous ovarian neoplasms (n = 13). Observations were recorded by conventional pathological scoring metrics. We reported that in the FTE, βV-tubulin is expressed solely in the non-ciliated secretory cell type. While βV-tubulin expressing cells were rare in pathologically "normal" FTE, their frequency varied by reason for salpingectomy, and rose dramatically in FTE of patients with BRCA mutations. In tumor tissue, βV- tubulin expression was correlated with differentiation status and was elevated in high-grade serous ovarian carcinoma, relative to borderline serous tumors. Thus, these IHC-based analyses support the hypothesis that deregulated expression of 13V-tubulin is a pathologic feature of FTE from BRCAmutant women at high risk of developing ovarian carcinoma. A second component of these studies focused on genetically modulating βV-tubulin expression in cancer cell lines with high endogenous expression of the tubulin isotype. Cell models were selected to represent diseases of interest, namely ovarian and breast cancer; and cellular functions of interest, namely secretion and metastasis. The βV-tubulin encoding TUBB6 gene was targeted for knockout using CRISPR gene editing in two metastatic ovarian cancer and one triple negative breast cancer (TNBC) line. Knockout of βV-tubulin was confirmed in select clones by immunoblotting and immunofluorescence. Subsequent characterization evaluated the impact of βV-tubulin modulation on proliferation, chemosensitivity, invasion, mitochondrial density and activity, and secretory output and constituents. Complete knockout of βV-tubulin in cancer cell lines had no significant effect on proliferation rate or gross morphology. Nuanced though reproducible, changes were observed in cancer cell properties, including increased invasion and an increased ratio of polarized mitochondria in the absence of βV- tubulin. Moreover, βV-tubulin expression correlated with differential sensitivity to certain clinically relevant chemotherapeutics including Taxol, specifically conferring increased drug resistance. Finally, semi-quantitative proteomic analysis identified cohorts of differentially secreted proteins following modulation of βV-tubulin, and subsequent gene ontology analysis implicated modulation of functional pathways governing mRNA slicing and reproductive hormone regulation. Ongoing studies will interrogate the potential association of these functional pathways and candidate proteins with βV- tubulin. Overall, we conclude that expression of βV-Tubulin in secretory cells of the fallopian tube epithelium correlates with cellular atypia and potentially increased risk of BRCA-mutant ovarian cancer; though additional studies are required to corroborate these pilot data. Our findings add to the current understanding of the early pathogenesis of ovarian cancer, and highlight βV-tubulin as worthy of consideration as an IHC-based biomarker for precursor lesions in FTE. Moreover, βV-tubulin has modest but reproducible effects on defined cancer cell functions. The finding that βV-tubulin expression can potentially modulate the secretory output of cancer cells supports a specialized function for this isotype and lays the groundwork for additional studies. Thus, this research furthers understanding of the role of this understudied Class V isotype of β-tubulin in supporting the malignant phenotype of ovarian and breast cancer.
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    Exploring the PI3Kβ-Rab5 Interface
    (ProQuest Dissertations & Theses Global, 2018) Heitz, Samantha; Backer, Johnathan M.; Bresnick, Anne R.
    Class I Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that regulate cell motility, growth, and survival. Class IA PI3Ks are obligate heterodimers that contain a regulatory subunit (p85α/β, p55α/γ, or p50α) and a catalytic (p110α, β, or δ) subunit. The full-length regulatory p85 subunit consists of a N-terminal SH3 domain, two proline-rich domains (nPRD and cPRD) that flank a BCR homology domain (BCR), and two SH2 domains (nSH2 and cSH2) linked by the inter-SH2 (iSH2) domain. The p110 catalytic subunit contains an N-terminal adaptor binding domain (ABD), a Ras-binding domain (RBD), a C2 domain, a helical domain, and a C-terminal kinase domain. The Class IA PI3Kβ can be activated by both receptor tyrosine kinase (RTK) binding to the p85 SH2 domains and by G-protein coupled receptors (GPCRs), which stimulate G3γ binding to the C2-helical linker region of p110β (the G3y binding loop). In contrast to the other Class IA PI3Ks, which bind Ras, p110β binds the small GTPases Rac1 and Cdc42 via the RBD domain. RabS was first identified as a p110β binding partner in a screen for RabS effectors. RabS localizes to early endosomes and other vesicular structures, and P13Kβ has been implicated in endocytic trafficking of cell surface receptors. The Rab5-PI3Kβ interaction was found to be critical for macroautophagy induced by growth factor limitation. Our lab initially identified a region of p110β that mediates binding to Rab5 using a conservation-based mutagenesis approach that evaluated the C-terminal end of p110β (C2, helical, and kinase domains), which is sufficient to bind Rab5. From this screen, two residues in the helical domain were identified (Q596C and I597S) whose mutation disrupted binding to Rab5. Beyond these two residues, the extent of Rab5 binding to p110β is not known. The aim of my project was to define the Rab5 binding interface within PI3Kβ. To better define the Rab5-P13Kβ interface, we performed scanning alanine mutagenesis on the catalytic subunit p110β and analyzed binding using an in vitro pull down assay with active GST-Rab5. While it has been previously reported that the regulatory subunit p85 can bind to Rab5 and act as a GTPase activating protein (GAP), we failed to see any direct binding of p85 to GST-Rab5 using an in vitro pull down assay. Focusing on the catalytic subunit, we assessed binding of 35 helical domain mutants in p110β to Rab5. Of the helical domain mutants tested, only 12 were found to reduce binding to GST-Rab5 by more than 33% as compared to wildtype p110β. Based on research from another lab, we also tested p110β mutants from the RBD domain that were reported to affect Rab5 binding. In our hands, all the RBD domain mutants were able to bind to Rab5. All of the p110β mutants that were found to disrupt Rab5 binding were assessed for protein functionally. We used a GST-Rac1 in vitro pull down assay to confirm that the Rab5-uncoupled mutants could bind a known interactor of p110β. Using in vitro kinase assays, we found that mutations affecting Rab5 binding did not impact the basal or Gβγ-stimulated p110β kinase activity. Compiling the data, we found the Rab5 binding interface within p110β is restricted to two perpendicular α-helices in the helical domain that are adjacent to the initially identified Q596 and 1597 residues. Analysis of the Rab5-P13Kβ interactions by deuterium exchange-mass spectrometry identified p110β peptides that overlapped with these helices; no interactions were detected between Rab5 and other regions of p110β or p85α. Overall, we identified a discrete binding site for Rab5 in the helical domain of p110β. Using the Rab5-p110β interface data gathered, a collaborator selected a list of 31 small molecule compounds from a library of thousands, based on in silico modeling, that were predicted to disrupt the Rab5 binding interface. We adapted the GST-Rab5 in vitro pull down assay to assess the small molecule compounds for inhibitory action. Currently, we have not found any compounds to affect the interaction between p110β and Rab5. Despite this, we believe our data, which defines a single discrete Rab5 binding site in the p110β helical domain, will be useful for generating mutants and inhibitors to better define the physiological role of Rab5-PI3Kβ coupling in vivo.
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    Challenging Current Paradigms: Increasing the Efficacy of Radiation Therapy with Novel Radiation Schemes
    (ProQuest Dissertations & Theses Global, 2018) Savage, Talicia; Guha, Chandan
    Cancer is the second leading cause of death in the United States. Currently over 50% of all patients are treated with radiation therapy (RT). Although RT is frequently used for local control of solid tumors, recent reports have shown both immune activating and immunosuppressing roles of RT. We have studied the immunomodulatory effects of RT and have designed different regimens of RT with varying roles in immunomodulation and tumor control. We propose to use radiation as an immunomodulatory drug and classify three regimens of RT: low-dose tumor microenvironment modulatory RT (TMEM-RT), sub-lethal immunomodulatory RT (IM-RT), and high-dose immunoablative RT (IA-RT). Historically, RT fractionation has been divided into daily fractions of equal doses of radiation to reduce toxicity on the basis of time required for DNA repair in normal tissues. Although single high-dose IA-RT would release tumor associated antigens (TAA) and danger-associated molecular pattern (DAMP) signals for immune activation, it is also associated with reduced perfusion and an induction of regulatory T cells (Tregs) in the irradiated tumor microenvironment (TME). Low doses of RT have been shown to reprogram immunosuppressive macrophages and increase infiltration of immune effector cells. We, therefore, hypothesized that after a single high-dose IA-RT, post-ablation modulation (PAM) with low-dose TMEM-RT fractions would reprogram the THE by increasing inflammatory Ml macrophages, reducing the levels of Tregs and enabling the infiltration of immune effector cells, thereby increasing efficacy of tumor control. In this work, we challenge the current paradigm of equal fraction size in RT and propose a regimen of unequal radiation fractionation sizes by combining a single high-dose IA-RT fraction, followed by four 0.5 Gy fractions of TMEM-RT as PAM, applied either to increase local control or to suppress systemic metastases. Using image guided radiation, we found that we could significantly delay local tumor growth and increase survival in 3LL tumor bearing mice with a single 22Gy dose, followed by four 0.5Gy PAM doses to the primary tumor, as compared to a single 24Gy IA-RT dose alone. PAM-mediated increase in local control of the primary tumor was associated with an increase in infiltration of leukocytes and a decrease in immunosuppressive immune cell phenotypes in treated areas and secondary lymphoid organs. In a separate tumor model, we administered whole lung TMEM-RT of four fractions of 0.5 Gy, twelve days after a course of three IA-RT fractions of 20 Gy to the primary tumor and observed an increased survival with suppression of pulmonary metastases in mice with highly metastatic 4T1 breast cancer. Systemic PAM remodels the metastatic niche by reducing Treg and increasing the infiltration of immune effector cells, thereby, converting the "soil" of the metastases-prone organ, resistant to tumor growth. Thus, low dose TMEM-RT can be used as PAM for two applications: i) Local control with PAM administered directly after immune-ablative radiation to the primary tumor, and ii) Systemic control with PAM administered to potential metastatic prone organs, such as, whole lungs for targeted systemic control of tumors that can be locally controlled by IA-RT. By optimizing the immunomodulatory properties of radiation fractionation regimens, we laid the foundation for combining immunotherapeutics, such as tumor vaccines, checkpoint blockade and immune activating agents, for increasing local and systemic control of solid tumors.
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    Monosynaptic Cerebellar Modulation of the Substantia Nigra
    (ProQuest Dissertations & Theses Global, 2018) Wahburn, Samantha Gabrielle; Khodakhah, Kamran
    The survival of an organism depends on its ability to find and consume energy and reproduce in a constantly changing environment. Doing so necessitates a range of movements, but exactly how the brain plans, selects, and executes precise motor sequences remains unclear. Two subcortical structures, the cerebellum and basal ganglia, communicate with the cerebral cortex and thalamus to generate motor output. It has long been thought that the basal ganglia and cerebellum serve separate but complementary roles in motor control, and that information flows between the cortex and the basal ganglia or cerebellum through separate, parallel "loops." Thus integration of basal ganglia and cerebellar outputs occurs only at the level of the cortex. A growing body of evidence, however, suggests that these circuits are not as separate as originally thought. It has been shown across several species that there are a number of pathways in the brain capable of mediating communication directly from the cerebellum to the basal ganglia, and vice versa. In contrast to the long, multisynaptic loops through cortex, these pathways enable the cerebellum and basal ganglia to coordinate their outputs on faster timescales. In this thesis, I examine and characterize a pathway from the cerebellum to the substantia nigra and examine how the cerebellum might be involved in a movement disorder often attributed to basal ganglia dysfunction. The substantia nigra is a structure within the basal ganglia that consists of the pars compacta (SNc), a nucleus made of modulatory, dopaminergic neurons that project to and modulate the input structure of the basal ganglia, and the pars reticulata (SNr), which is a major output nucleus of the basal ganglia. There is anatomical evidence dating almost fifty years of a projection from the cerebellar nuclei to the dopaminergic neurons of the SNc, but whether neurons in the cerebellum can drive activity of these dopamine neurons on a fast timescale is unknown. In contrast, cerebellar projections to SNr have not previously been reported. Here, we test the hypothesis that the cerebellum can modulate the SNc and SNr through a direct, monosynaptic connection, using a combination of optogenetics and in vivo and in vitro electrophysiology. We found, in vivo, that neurons in SNc and SNr rapidly increased the number of spikes fired immediately following optogenetic stimulation of cerebellar fibers. Experiments in vitro determined that stimulation of cerebellar fibers evoked excitatory currents in both structures and confirmed that this modulation occurred through a monosynaptic pathway. Taken together, these data provide support for a cortex-independent pathway capable of relaying information directly from the cerebellum to the SNc and SNr and have important implications for understanding how the cerebellum and basal ganglia interact to generate movements under normal and pathological conditions. Previous work from our laboratory has shown that cerebellar-basal ganglia interactions are relevant in a number of movement disorders, including dystonia. The second part of this thesis examines how the cerebellum is implicated in a movement disorder typically associated with basal ganglia dysfunction. Myoclonus dystonia (DYT11) is an inherited movement disorder caused by loss-of-function mutations in SGCE and characterized by involuntary jerking of the upper body (myoclonus) and sustained contraction of agonist and antagonist muscles that result in painful, twisted postures (dystonia). A striking feature of this disorder is that patients frequently report improvement of motor symptoms after consumption of alcohol. Unfortunately, the neural basis of DYT11 is unclear, although the basal ganglia and cerebellum have been implicated. Here, we test the hypothesis that the cerebellum contributes to motor symptoms in DYT11. To that end, we generated a mouse model of DYT11 using short hairpin RNA (shRNA) to knock down sgce in the adult mouse. We found that knockdown of sgce in the cerebellum, but not the basal ganglia, produced dystonia and repetitive jerk-like movements in mice and showed that these symptoms improved after administration of ethanol, consistent with what is seen in patients. Further, we performed extracellular recordings from dystonic mice and found that cerebellar neurons fire aberrantly in dystonic mice. The work in this thesis demonstrates that the cerebellum and basal ganglia are more interconnected than previously thought. The cerebellum is capable of affecting the activity of substantia nigra pars compacta, a modulatory nucleus within the basal ganglia, and the pars reticulata, a major output nucleus of the basal ganglia. Further, this work illustrates how interactions between the basal ganglia and cerebellum may have significant implications for movement disorders, as disorders like dystonia frequently associated with the basal ganglia may also involve the cerebellum.
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    Generation of a Drosophila Model of Intellectual Disability to Investigate the Transcriptional, Behavioral and Neuronal Defects Caused by Mutations in Histone Demethylase KDMS.
    (ProQuest Dissertations & Theses Global, 2018) Zamurrad, Sumaira
    Mutations in lysine demethylase 5 (KDM5) family histone demethylases cause intellectual disability (ID) in humans. However, the mechanism underlying the cognitive impairment is unknown. KDM5 proteins are multi-domain transcriptional regulators, most well-known for their Jumonji C (JmjC) domain-encoded demethylase activity that specifically removes trimethylated lysine 4 of histone H3 (H3K4me3), a mark associated with active promoters. Current models of KDM5-induced ID suggest that it is the loss of demethylase activity that disrupts cognition. This is based primarily on a limited number of in vitro demethylase activity studies in which some, but not all, KDM5 missense mutations show reduced enzymatic activity of up to 2-fold. However, the in vivo effects of these mutations remain uncharacterized. In addition, the enzymatic defects, if any, of a majority of ID-associated KDM5 mutations proteins have also not been assessed in vitro or in vivo. It is also notable that no reported patient-associated mutations lie in catalytically critical residues within the Jumonji C domain. Indeed, most missense mutations lie outside of this domain. Thus, the causal link between KDMS's demethylase activity in neuronal function remains unclear. The role of KDM5 in neuronal function in general is evolutionarily conserved, with loss of KDM5 disrupting neurodevelopmental processes in model organisms including mice, worms and zebrafish. However, the in vivo defects of the specific reported KDM5 mutations on transcription, cognitive behaviors and neuronal development are not known. In addition, it is not currently clear whether missense mutations in distinct regions of KDM5 affect common downstream effectors through a demethylase-dependent mechanism or if each mutation has individual downstream consequences. Studies answering these questions are crucial to define the molecular mechanisms linking KDM5regulated transcription and cognition. Here, we establish Drosophila as a model system to understand this connection by generating seven fly strains harboring evolutionarily conserved missense mutations in KDM5 associated with ID. These mutations do not affect protein levels and produce viable, phenotypically normal flies. Significantly, only two of the seven mutations reduce histone demethylase activity levels in vivo, suggesting that reduced enzymatic activity may not be a necessary prerequisite for KDM5 mutations to cause ID. We carried out detailed studies including transcriptome analyses, cognitive assays and neuronal morphology on one ID-associated KDM5 mutation that affects demethylase activity, kdm5A512P. mRNA-seq from kdm5A512P mutant heads revealed a significant enrichment for genes required for ribosomal assembly and function. Comparison with ChIP-seq data available from whole adults revealed that a significant number of these affected genes are also direct targets of KDM5. Consistent with the downregulation of this class of gene, incorporation of the tRNA analog puromycin revealed a 2-fold reduction in translation specifically in head tissue but not in thoraces. Because translation is critical for neuronal function, we utilized association based appetitive learning and/or memory tests of kdm5A5I2P adults. This revealed both short-term and long-term memory defects compared to wildtype animals. KDM5A512P is likely to abolish enzymatic activity, since H3K4me3 levels are increased to a similar extent to the demethylase dead strain (kdm5JmjC* ). Indeed, consistent with the primary defect of kdm5AS12P being the loss of histone demethylase function, the transcriptional and behavioral changes in kdm5AS12P flies were indistinguishable from kdm5JmjC* flies. Based on striking similarities between kdm5A512P and kdm5JmjC*, we extended our transcriptional analyses to include three additional ID mutants: kdm5A224T which affects demethylase activity, and also kdm5 F765L and kdm5L854F that lie in an inter-domain region and in the C5HC2 domain, respectively. Comparison of all four ID genotypes uncovered a significant number of genes that were similarly dysregulated in all mutant strains. Consistent with our previous analyses of kdm5A512P these were also enriched for ribosomal function genes. These studies also revealed that the four ID mutants examined, regardless of their demethylase activity status, have significant overlap with the kdm5JmjC* mutant. We propose that the mutation in the kdm5JmjC* strain that disrupts the structure of the Jumonji C domain may also affect the adjacent C5HC2 motif suggesting that this motif of unknown function is involved in KDM5's and neuronal functions. In conclusion, we have generated a Drosophila model to examine the molecular, cellular and behavioral phenotypes caused by mutations in KDM5. Our study has revealed an exciting link between KDM5 and the activation of genes required for ribosome structure and function. Defects in learning and memory are a hallmark of ID. We have recapitulated this ID defect in Drosophila. By investigating the transcriptional and cellular deficits, we also uncovered translation as a key biological process that may contribute to ID in patients with KDM5 mutations. It is therefore conceivable that restoring the reduced translation may ameliorate the cognitive dysfunction observed with these mutations. This possibility warrants a more in-depth study of the translation link as a significant underlying cause of ID caused by KDM5 mutations.
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    Consequences of Chromosome Instability in Mammalian Cells In Vitro and In Vivo
    (ProQuest Dissertations & Theses Global, 2018) Andriani, Grasiella Angelina; Montagna, Cristina
    Chromosomal instability (CIN) is a cellular state with high propensity for chromosome mis-segregation. Ploidy changes, a direct consequence of CIN, have been correlated with impaired cellular fitness and a transcriptional signature associated with stress. Mounting evidence suggests that CIN plays a role in the etiology of neurological and neurodegenerative diseases and in the aging process, however the precise mechanisms involved are not completely elucidated. The general goal of the present work was to shed light into the cellular consequences of CIN, both in vitro and in vivo. In the first part of this thesis we asked whether the age-related accumulation of aneuploidy observed during normative aging in the mammalian cerebral cortex was recapitulated in murine models of accelerated aging. To this end, we collected brain and cerebellum samples from BUB1BH/H mice, which have a faulty mitotic checkpoint and ERCC1-/Δ7 mice, defective in nucleotide excision and interstrand crosslink repairs. Both murine cohorts, which exhibit features of premature aging, were analyzed at their maximum life span time point. Ploidy analysis carried on by Fluorescent In Situ Hybridization revealed no difference in the levels of not-diploid cells between progeroid and WT age-matched control mice during adulthood, suggesting that not all aging phenotypes are recapitulated in these mice models. Surprisingly, we found significantly high levels of aneuploidy in BUB1B H/H mice during embryonic development, which was accompanied by increased apoptosis and microglia staining, suggesting that not-diploid cells are eliminated, likely through immune-mediated processes. Therefore, the lack of aneuploid cells in the adult brain of progeroid mice possibly reflects the efficient clearance of these cells at earlier time points. However, exacerbated depletion of aneuploid cells within the brain is likely detrimental, as both mice models present markers of age-related neurodegeneration. In the second part of this thesis we investigated the fate of cells undergoing CIN in vitro. For this purpose, we knocked down genes important for faithful chromosome segregation (BUB1 and SMC1A) in human fibroblasts to induce CIN, and then followed growth, morphology and cellular outcomes. While apoptosis was a minor consequence of CIN under these experimental conditions, significant high levels of premature senescence were observed upon CIN generation, as assessed by decreased BrdU incorporation and expression of proliferation marker MKI67, increased SA- Rgalactosidase staining and cellular size and up-regulation of CDKN1A and CDKN2A. Genomic instability, through the generation of micronuclei and DNA double strand breaks, was observed in not-diploid cells. Oxidative stress was also a confirmed feature of CIN cells, as assessed by increased production of reactive oxygen species (ROS), elevated cellular autofluorescence and oxidative damage to DNA and RNA. In addition, we showed that CIN-induced senescent cells activate a unique SASP, which includes a novel growth factor CLEC1IA, which has not been associated with senescence before. These results showed that CIN is sufficient to trigger premature senescence in vitro , which is accompanied by SASP activation, suggesting that accumulation of CIN cells during aging potentially contributes to age-related inflammation through secretion of SASP components. Collectively, the findings presented in this thesis contribute to the field of aneuploidy and CIN because they demonstrate how cellular responses to ploidy changes can vary, depending on the experimental system, the cellular type and function and the genetic background.
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    Neuropsychiatric Lupus: Pathogenic Mechanisms and Novel Therapeutic Strategies
    (ProQuest Dissertations & Theses Global, 2018) Mike, Elise V.; Putterman, Chaim
    Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder that predominantly affects women of color. About 20 to 40% of lupus patients suffer from diffuse neuropsychiatric manifestations of SLE (NPSLE), including cognitive impairment and depression. The pathogenesis of NPSLE is not well understood, and current treatment strategies are suboptimal and generally noncurative. Brain barrier disruption, which allows peripheral mediators to enter the central nervous system (CNS), and proinflammatory signaling within the brain parenchyma are both thought to play an important role in disease development. Modulation of neuroinflammation is a potential therapeutic strategy. Here, we investigated the effects of altering the brain microenvironment through genetic and pharmacological means. Lipocalin-2, an acute phase reactant protein upregulated systemically in SLE, promotes neuroinflammation through inducing brain barrier disruption, glial activation, and neurotoxicity. We hypothesized that the absence of LCN2 would ameliorate NPSLE. To test this hypothesis, lupus-prone B6.Slel.S1e3 (Sle1,3) mice, Sle1,3-LCN2 knockout (KO), B6.LCN2KO, and B6 mice were evaluated. Sle1,3 mice exhibited significant impairment in spatial and recognition memory and depression-like behavior when compared with B6 mice, and these deficits were significantly attenuated in Sle1,3LCN2KO mice. Flow cytometry of hemisected brain lysates showed a significant increase in brain infiltrating cells, including CD4+ and CD8+ T cells, that was not reduced with LCN2 deficiency. Immunofluorescence staining of the hippocampus revealed a preponderance of apoptotic microglia and neurons in Sle1,3 mice that was reduced with LCN2 deficiency. Transcriptome profiling of sorted microglia showed that several genes, upregulated in Sle1,3 mice and involved in inflammation and memory, were normalized in Sle1,3-LCN2KO mice. Additionally, in two independent large cohorts, NPSLE patients displayed high CSF levels of LCN2. These demonstrate that LCN2 deficiency attenuates neurobehavioral deficits, reduces apoptosis in the brain, and modulates microglia morphology. Moreover, our results show that LCN2 regulates microglial expression of genes essential to NPSLE development. LCN2 secretion can be stimulated by sphingosine-1-phosphate (S1P), a bioactive sphingolipid involved in several immune processes. Fingolimod, approved for the treatment of multiple sclerosis, functionally antagonizes S1P receptors and reduces the circulation of autoreactive lymphocytes. In the CNS, fingolimod exerts a neuroprotective role, reducing pro-inflammatory cytokines and promoting brain barrier integrity. We investigated the effects of fingolimod in MRL-1pr/lpr mice, a classic spontaneous NPSLE model, and we hypothesized that fingolimod treatment would attenuate disease. Fingolimod-treated mice exhibited significantly improved visual and recognition memory and displayed a reduction in depression-like behavior, demonstrating an amelioration of the NPSLE phenotype. A significant reduction of CD4+ and CD8+ T cell infiltration of the choroid plexus in the lateral ventricles was also observed by immunofluorescence staining and flow cytometry. There was also a significant decrease in the number and/or activation of parenchymal cells, including microglia and astrocytes. These results highlight the important role of SIP in the pathogenesis of neurocognitive manifestations of SLE. Taken together, our results highlight the LCN2 pathway and the SIP signaling axis as novel therapeutic targets and elucidate two potential determinants of disease development.
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    BAF180 Bromodomains Differentially Affect PBAF Targeting to Chromatin Subcompartments and Behave Oncogenically in U2-OS Cells
    (ProQuest Dissertations & Theses Global, 2018) Kenworthy, Charles Andrew; Coleman, Robert A
    Chromatin remodeling is a widespread nuclear function that impacts almost every aspect of nuclear physiology. Thus, the action of SWI/SNF chromatin remodelers can impact a broad range of cellular processes. SWI/SNF remodelers traditionally have been thought to localize to euchromatin via bromodomains to activate transcription. However, SWI/SNF remodelers are also involved in a broad range of other functions including transcriptional repression, the DNA damage response, and specification of boundary elements. We developed a method of examining the binding of the mammalian SWI/SNF remodeler, PBAF, to chromatin with single particle precision. This involves tagging the BAF180 subunit within PBAF with a modified bacterial haloalkane dehalogenase: the halo tag. This allows BAF180 to be conjugated to bright organic fluorophores. When imaged at slow acquisition times, molecules that are freely diffusing will blur into the background and stationary molecules are thus able to be resolved with single particle precision. We were able to further localize PBAF binding activity by performing two-color imaging between Halo-BAF180 and chromatin structural proteins tagged with the SNAP tag: HP1a and H3.3. We added the SNAP tag to H3.3, which is associated with transcriptionally active euchromatin, and HP1a, which is associated with transcriptionally repressed heterochromatin. We also found that PBAF resided in the transcriptionally active euchromatin for less time than it did in transcriptionally repressed heterochromatin, which we think is linked with chromatin stability. Furthermore, we found that for PBAF, the BAF180 bromodomains didn't have an appreciable effect on the amount of time that PBAF resided within euchromatin. Surprisingly, we found that the BAF180 bromodomains did affect the amount of time that PBAF resided in heterochromatin, suggesting that it targets an unknown acetylated factor in heterochromatin. Apart from its extensive role in various aspects of cellular physiology, PBAF is also highly mutated across a range of cancers. These PBAF mutations were thought to be associated exclusively with tumor suppressive functions. However, recent literature has shown that certain PBAF subunits can also be over-expressed in neoplastic processes and that this overexpression can drive tumorigenesis. We found that overexpression of a BAF180WT transgenic construct in U2-OS cells leads to increased cellular migration in a wound healing assay, decreased cellular adhesion, and increased colony size in colony formation assays. These are all traits that are associated with tumor progression. We examined expression of a cancer-derived missense mutation within the second bromodomain of BAF180 (BAF180-T232P) and found that it leads to truncation of the protein after the third bromodomain, which likely inactivates PBAF function. We find that Halo-fBAF180-T232P didn't appear to have these same effects on wound healing, cellular adhesion, and colony formation. Thus ectopic expression of a Halo-fBAF180WT construct appeared to increase tumorigenic behavior in U2-OS cells and expression of a point mutation in a bromodomain (Halo-fBAF180-T232P) reversed this tumorigenic function. We further performed ChIP-exo of both of these constructs and found that Halo-fBAF180WT localized to genomic regions associated with focal adhesion, WNT signaling, and TGF(3 signaling. Finally, we performed dynamic fluorescent cellular imaging of cells expressing either Halo-fBAF180WT or HalofBAF180-T232P. We found that in FRAP assays, Halo-fBAF180-T232P exhibited rapid recovery dynamics when compared to Halo-fBAF180WT. When performing single particle slow-diffusion imaging however, we found that the amount of time that HalofBAF180WT attached to chromatin was comparable to Halo-fBAF180-T232P. Thus, Halo-fBAF180-T232P appears to have a defect in association with chromatin, but once bound it doesn't appear to have a deficit in residence on chromatin. Our work thus describes the development of a system that can survey chromatin residence times and bromodomain-dependent interactions. We have also defined a potential tumorigenic function of BAF180 in U2-OS cells, and determined that mutations within a single bromodomain could reduce this tumorigenic function.
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    Generation of an In Situ Tumor Vaccine Using Focused Ultrasound-Induced Immune Priming
    (ProQuest Dissertations & Theses Global, 2018) Skalina, Karin A.; Guha, Chandan
    The goal of cancer treatment is to destroy all malignant cells within the patient's body, thereby preventing metastases or recurrence. This destruction can be achieved, in broad terms, by direct killing of the cells with a chemical or mechanical agent or by harnessing the power of the patient's immune system, using immunotherapy. Immune activation against tumors often fails because of a dearth of antigenic stimulus for the immune system and defective antigen presentation. How the innate antigen presenting cells interact with dying tumor cells and the mechanism of tumor cell death determines whether the immune system will be activated after tumor ablation to potentiate tumor immunotherapy. Through a combination of targeted therapies, we can achieve immunogenic cell death, which invigorates the immune system to attack the malignant cells and prevent dissemination. Low intensity focused ultrasound (LoFU) is a noninvasive, nontoxic, confocal therapy, delivering sonic stress. LoFU significantly alters expression and localization of heat shock proteins, which are highly conserved molecules that are rapidly upregulated in times of stress to encourage cell survival or promote a robust immune response and enhance cross-presentation to CD8+ T cells. Here, we demonstrate that treatment of murine solid tumors, including breast and prostate tumors, with LoFU followed by radiation therapy results in primary tumor cure and upregulation of the immune response. Through these noninvasive therapies, we are able to generate an in situ vaccine, generating CD8+ T cells against tumor-associated antigens and providing a viable oncologic treatment option for other tumor models.
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    Therapeutic Targeting of the Interleukin 1 Receptor Accessory Protein (IL1 RAP) Reveals Novel Oncogenic Functions in Acute Myeloid Leukemia
    (ProQuest Dissertations & Theses Global, 2018) Mitchell, Kelly; Steidl, Ulrich
    Leukemia stem cells (LSC) are the defective counterparts of hematopoietic stem and progenitor cells (HSPC) that can give rise to acute myeloid leukemia (AML). LSC have been implicated as drivers of relapse in AML as they are generally chemotherapy resistant and thus are not eliminated. Our lab has previously performed gene expression profiling to identify genes dysregulated in HSPCs from AML patient samples compared to immunophenotypically matched cells from healthy control samples. The interleukin-1 receptor accessory protein (IL1 RAP) was one of the few genes whose expression was commonly upregulated across multiple hematopoietic stem and progenitor compartments and AML subtypes, whereas expression was minimal or absent in healthy HSPC. The IL1RAP protein was validated to be overexpressed in AML HSPC, and since it spans the plasma membrane, it has potential to be an easily targetable molecule. However, the functions of IL1RAP in AML have yet to be described. Previous investigation by our lab showed that higher expression of IL1 RAP correlates with poor prognosis in AML. Additionally, shRNA-mediated knockdown of IL1RAP significantly decreased clonogenic growth and increased apoptosis in AML cell lines. These studies indicate a functional role for IL1RAP in AML. In this study, we characterized previously undescribed roles of IL1RAP in AML and healthy hematopoiesis as well as its efficacy as a therapeutic target in AML. As the hematopoietic system is compromised in AML, it is often the case that patients do not have a fully functioning immune system. Therefore, our study aimed to apply therapeutic strategies that do not rely on the assistance of immune effectors for elimination of malignant cells, but rather that exploit functional dependence of AML cells on IL1RAP. These therapeutic strategies served also as a tool for determining cell-intrinsic functions of IL1RAP. We found that targeting of IL1RAP via RNA interference, genetic deletion, or antibodies directed at its extracellular domain inhibited AML cell growth and viability in vitro and in vivo, with minimal perturbation of healthy hematopoietic function or viability. These effects were seen in particular mutational subsets of AML, specifically those lacking activating mutations in the receptor tyrosine kinase FLT3. Incubation of FLT3 wildtype AML cell lines and primary human patient samples with IL1RAP shRNAs and antibodies inhibited growth, promoted differentiation and led to apoptosis in vitro. IL1 RAP shRNAs and in vivo antibody treatment were effective in eliminating human AML cells in xenograft mouse models, and Ill rap deletion inhibited progression of AML in a mouse genetic model of the disease. Using phospho-immunoblotting, phospho-flow cytometry analysis, and cytokine-stimulated growth of AML cells, we investigated the signaling pathways affected by shRNAmediated targeting, antibody-mediated targeting, and genetic deletion of IL1 RAP in AML cells. We found that the role of IL1RAP is not restricted to the canonical IL-1 receptor pathway, but that IL1RAP mediates signaling and proliferation through FLT3 and c-KIT, two receptor tyrosine kinases with known key roles in AML pathogenesis. Coimmunoprecipitation and Fbrster resonance energy transfer (FRET) experiments demonstrated interactions between IL1RAP and these receptors in AML cell lines, indicating a mechanism for how targeting IL1RAP dysregulates the FLT3 and c-KIT signaling pathways and leads to AML cell death. Overall, our study provides a new mechanistic basis for the efficacy of IL1RAP targeting in AML and reveals novel roles for this protein in the pathogenesis of the disease.