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Item PHOSPHORYLATION PATTERNS, AGGREGATION PROPENSITIES, AND MORPHOLOGICAL STUDIES OF THE VARIOUS TAU PROTEIN ISOFORMS(2022-03-25) Laryea, Erving Torgbor; Wu, Colin; Avery, Adam; Madlambayan, GerardTau protein is a microtubule-binding protein as well as a biomarker of neurodegeneration. Its core function is to stabilize microtubules for proper neuronal communication. When hyperphosphorylated, it detaches itself from microtubules and self-assembles into cytotoxic structures. However, little is known about how phosphorylation, the commonest posttranslational modification process found in eukaryotic cells regulate Tau protein structure, conformation, and function. Herein, the role of specific kinases or kinase combinations from the three main classes of proteins kinases that phosphorylate Tau: Proline-directing protein kinase (Glycogen synthase kinase (GSK-3β)) and non-proline directing protein kinase (Microtubule associated regulating kinase (MARK4)) and Tyrosine kinase (Fyn) were systematically evaluated in vitro. After the expression and purification of all the six Tau isoforms from E. Coli cells, Tau 441, also known as full-length Tau, which comprises of all the domains found in the other isoforms was extensively investigated. Phosphorylation of Tau 441 by GSK-3β, MARK4 and Fyn was detected by immunostaining using phosphospecific antibodies. With Tau protein been identified to co-localize with sulfated aminoglycans such as heparin and heparin sulfates, single and multi-kinase phosphorylated aggregation studies of Tau 441 were conducted in the presence or absence of heparin. Functional assays including proteostat assays, turbidity assays and SDS-PAGE were used to evaluate the aggregation properties of Tau 441 after phosphorylation.The phosphosites on all the single and multi-kinase phosphorylated Tau 441 samples were characterized by Tandem mass spectrometry. Tau 441 protein structure and conformational changes after phosphorylation was also determined by Hydrogen Deuterium Exchange mass spectrometry (HDX-MS). The flexibility and accessibility to the first and second hexapeptide repeats (H1 and H2), a repeat motif found in the MTBR of Tau protein identified to increase the aggregation tendencies of Tau protein were used to describe the single kinase or multi-kinase combinations evaluated ability to promote Tau fibrillization.Item PRECLINICAL STUDIES: TREATMENT OF MULTIPLE SCLEROSIS AND RETINAL DEGENERATIVE DISEASE USING STEM CELLS(2021-11-15) Cormier, Christina Frances; Chaudhry, G.; Govind, Chhabi; Svinarich, David; Perez-Cruet, MickPluripotent stem cells (PSCs) isolated from an embryo or generated by ectopic expression of transcription factors can self-renew indefinitely and differentiate into all cell types found in the body. PSCs have the highest potential for cell therapies, but they face ethical concerns and technical and safety challenges, including teratoma formation. In contrast, mesenchymal stem cells (MSCs) isolated from adult and perinatal sources do not pose ethical and moral dilemmas. While MSCs isolated from adult sources, such as bone marrow, require invasive procedures, their use may also cause graft verse host disease. Therefore, we have focused on MSCs isolated from perinatal sources such as the umbilical cord (UC). These cells have advantages over adult MSCs in that they are highly proliferative, do not display HLA-DR markers, and thus are not likely to be immunogenic. We tested the therapeutic efficacy of UC-derived MSCs and their derivatives in preclinical studies to treat multiple sclerosis (MS) and retinal degenerative disease (RDD). The specific aims were to 1. production of MSCs for preclinical studies; 2. treatment of MS using MSCs and NSCs; and 3. treatment of RDD using MSCs and RPC. Our results showed that MSC-derived neural stem cells (NSCs) countered the inflammatory response, provided neural protection, and induced neurogenesis in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Likewise, MSC-derived retinal progenitor cells (RPCs) survived, integrated, and migrated into various neural layers of the retina in the rd12 mouse model of retinitis pigmentosa (RP). RPCs promoted retinal structure, function, neural protection, and regeneration of the retina resulting in vision improvement. These highly promising findings are likely to facilitate clinical studies for treating MS and RDD.Item MULTI-RESOLUTION STUDY OF TOPOGRAPHICAL AND ZONAL PROPERTIES OF OSTEOARTHRITIS IN ARTICULAR CARTILAGE USING MICROSCOPIC MRI AND POLARIZED LIGHT MICROSCOPY(2022-03-22) Badar, Farid-Ahmed Wajihuddin; Xia, Yang; Roth, Bradley J; Khain, Evgeniy; Jiang, Quan; Bowyer, Susan MArticular cartilage is a thin layer of connective tissue found in diarthrodial joints that overlay the opposing ends of bones and acts as lubricating surfaces to distribute stress and reduce friction with the help of synovial fluid. Hyaline cartilage contains an abundance of water molecules that are essential to the behavior and function of cartilage, the negatively charged proteoglycans that influence the biomechanical properties of the tissue, and the collagen fibers that act as the rebar or reinforcement bars to preserve the structural integrity of the tissue. Cartilage supports the applied load and distributes stress based on its intrinsic material properties, together with its underlying bone. At different surface locations of any single diarthrodial joint, the properties of cartilage are prone to have many morphological and molecular variations topographically, which are mainly due to the patterns of mechanical loading for any specific joint. Any change in the morphological and molecular properties of the cartilage and bone is likely to directly impact the clinical diagnoses of joint diseases such as osteoarthritis (OA). In addition to the topographical variations, cartilage also has a number of depth-dependent variations over its thin thickness, which begins in the non-calcified cartilage with articular laminae and the unequal thickness of sub-tissue zones. Conceptually, the non-calcified cartilage is commonly subdivided based on the orientations of collagen fibers and chondrocytes, into three histological zones. They are the superficial (SZ), transitional (TZ), and radial (RZ) zones. The non-calcified cartilage interfaces with the calcified cartilage and subchondral bone plate (SBP) through the tidemark (TM). This dissertation has seven chapters, which describe a number of multi-resolution projects that use high-resolution imaging to determine the topographical and depth-dependent variations in cartilage, in order to diagnose OA at its earliest stages. The dissertation begins with a brief introduction of background and literature review (Chapters 1-2), continues with the description of the materials and methods (Chapter 3), and summarizes three published peer-reviewed journal articles (Chapters 4-6). The dissertation ends with a summary and comments on future directions (Chapter 7). Among the three research projects described in this dissertation, the first project (Chapter 4) investigates the improvement in the OA detection in cartilage by the interpolation of T2 images, in the situation when the native MRI resolution is insufficient to resolve the depth-dependent T2 characteristics in articular cartilage. The second project (Chapter 5) establishes the topographical and zonal T2 patterns of multi-resolution MRI in medial tibial cartilage in a canine model of OA, initiated by an anterior cruciate ligament (ACL) transection surgery, which was studied after 8-weeks and 12-weeks post-surgery. The third project (Chapter 6) quantifies the interface region between the non-calcified cartilage and the subchondral bone plate, which includes the deep portion of the non-calcified articular cartilage and the zone of calcified cartilage (ZCC) using a dual-modality microscopic imaging study.Item THE ROLE OF CD49f - INTEGRIN α6- IN HUMAN STEM CELL BIOLOGY(2021-11-15) Timilsina, Suraj; Villa-Diaz, Luis; K. Lal, Shailesh; Madlambayan, GerardOwing to the intrinsic capability for unlimited self-renewal and the ability to make all the cells in the body, pluripotent stem cells (PSC) are an ideal candidate to be used as starting material for cell therapies. The development of a standard human PSC (hPSC), which includes embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC), culture methods using completely defined and xeno-free culture environment will advance our knowledge of hPSC biology, and also increase the effectiveness of hPSC expansion on defined conditions for potential human applications. It has been shown that stem cell fates are controlled by their specialized microenvironment, the stem cell niche, via direct cell-cell interactions, cell-extracellular matrix (ECM) contact- largely by surface proteins known as integrins- and the molecular signals emitting from the niche. Activation of integrins by binding to their ligands triggers signal transduction mechanisms involved in cell fate determination. In addition, because of their cell surface localization, integrins are used as biological markers to identify cell populations, and in this regard, integrin α6 (ITGA6), also known as CD49f, is a key biomarker identifying stem cells as it is commonly expressed in all identified stem cell types. Although, numerous findings strongly suggest that CD49f plays important functions in stem cell biology, the underlying molecular mechanisms by which CD49f sustain stem cell’s self-renewal have been only partially described. In this project I have established a chemically defined and xeno-free culture condition for long-term maintenance and derivation of hPSC using chemically defined and xeno-free culture conditions. I also described a novel molecular mechanism involved in the maintenance of self-renewal and proliferation of hPSC using simulated microgravity (sμg). Moreover, my results highlighted CD49f as a reliable biomarker in identifying and characterizing functional status of human mesenchymal stem cells (hMSC).