Biomedical Sciences

Permanent URI for this collectionhttps://hdl.handle.net/10323/11884

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Development and functional characterization of complex in vitro models of the blood-brain barrier
(2025-01-01) Bhalerao, Aditya; Cucullo, Luca; Liu, Zijuan; Al-Shabrawey, Mohamed
The blood–brain barrier (BBB) is a specialized structure that protects the brain but also makes it difficult for many drugs to reach the central nervous system. To better understand how the BBB works and to develop ways to deliver treatments to the brain, researchers need reliable lab-based models that closely mimic how the barrier functions in the human body. This dissertation focuses on developing, characterizing and functionally testing in-vitro models of the BBB using a combination of primary cells and cells derived from stem cells. The models include co-cultures of brain endothelial cells, astrocytes, and pericytes, which are important components of the neurovascular unit (NVU). A major part of the work involved developing a microfluidic chip-based model that allows the endothelial cells to grow under flow, simulating blood flow in the brain. The model was tested for how well it could mimic natural barrier properties, including integrity, transport functions, and small molecule permeability. Compared to traditional methods, the new model showed better accuracy and could more reliably predict how drugs might behave in the human brain. We also tested our models ability to support studies on Central Nervous System (CNS) targeted therapeutic delivery via receptor- mediated transcytosis. Overall, this work presents functional and translationally relevant BBB models that can be used to study brain diseases and test potential new treatments in the lab before moving to animal or human studies.
Stimulation of Neurons in the Brain: Mechanisms and Limitations
(2025-01-01) ALZAHRANI, Mohammed J; Roth, Bradley J; Roth, Bradley J; Xia, Yang; Puwal, Steffan; Surdutovich, Eugene; Tonyushkin, Alexey
Scientists have been using electrical stimulation to monitor and treat brain disorders for years. However, the capability to target specific brain regions marked the beginning of brain stimulation. The procedure falls under a larger neuromodulation therapy category, including deep brain stimulation (DBS), transcranial magnetic stimulation (TMS), microcoil recently, and others. These therapies can target specific neurons in the brain, helping to alleviate symptoms associated with movement disorders, epilepsy, depression, and other neurodegenerative disorders. This dissertation aims to discuss different brain stimulation methods and mechanisms.The study of deep neuron stimulation in the brain involves using the Hodgkin and Huxley model when an external stimulus is applied. In response to a moderate stimulus, the membrane triggers an action potential. However, a high or low stimulus dose not excite an action potential. In other words, weak stimuli do not excite neurons, moderate strength stimuli do, but strong stimuli do not, which means you can excite deeper neurons while not exciting neurons closer to the brain surface. The electric field produced by a microcoil is too small to cause neural stimulation. The maximum value of the induced electric field due to electromagnetic induction is around 0.026 mV/m. The electric field intensity in the tissue is approximately 4 mV/m due to capacitive coupling with the same current and frequency. Therefore, the electric field produced by capacitive coupling is much larger than that produced by electromagnetic induction. Considering both large coil (TMS) and microcoil stimulation, the activating function is one source of excitation. The activating function depends on the spatial distribution of the electric field gradient in active membrane analysis and the spatial frequency in spatial-frequency analysis. Both analyses show that a microcoil (tens of microns in size) has a higher threshold than a traditional coil (tens of millimeters in size) when the spatial frequency is large, or the spatial extent of the activating function is small. Consequently, the stimulation threshold for a microcoil is much higher than that of conventional coils.
Molecular mechanisms of β-iii-spectrin in dendritic arborization and spinocerebellar ataxia type 5
(2024-01-01) Denha, Sarah; Avery, Adam W; Wu, Colin G; Westrick, Randal J
β-III-spectrin is a membrane-associated cytoskeletal protein predominantly expressed in the cerebellum, and is essential for the maintenance and development of the complex dendritic arbors extended by Purkinje neurons. Mutations in various functional domains of β-III-spectrin cause the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). My dissertation has focused on understanding the molecular mechanisms by which β-III-spectrin supports neuronal structure and function and how SCA5 mutations disrupt this function. Specifically, my work has shown that the β-III-spectrin N-terminal domain, preceding the actin-binding domain, is required for SCA5-induced high-affinity actin binding and arborization defects in vivo. Furthermore, through characterizing the molecular consequences of SCA5 mutations in the spectrin-repeat domains (SRDs) of β-III-spectrin, I have shown that SRD2- and SRD3-localized mutations disrupt the physical interaction of β-III-spectrin with actin and α-II-spectrin. My dissertation work has provided significant insights into the role of β-III-spectrin in the actin cytoskeleton and the mechanisms underlying SCA5, laying the groundwork for developing a drug compound to treat this currently untreatable disease.
The Delineation of the Fibrinogen/Triggering Receptor in Myeloid Cells (TREM) Like Transcript - (TLT) -1 Molecular Interaction
(2024-01-01) Branfield, Siobhan Laken; Washington, Anthony V; Bonney, Elizabeth A; Hosch, Sarah; Westrick, Randal J
Triggering Receptor Expressed in Myeloid Cells -1 (TLT- 1) is a platelet specific receptor that facilitates platelet aggregation and plays a role in immunohemostasis. Previous studies identified fibrinogen as a ligand for TLT-1 but little has been reported on the molecular interaction or the role this TLT-1/Fibrinogen interaction has on pathophysiological progression of disease. This dissertation research presents basic research, with potential for translational application for this interaction, highlighting the versatile functionalization and application of TLT-1 as a contributor to clot formation, as a biomarker for disease progression and, extends to understand its regulation of fibrinogen deposition in pathophysiological models of disease.
Discrete Functionalization of Resorcinarenes for Applications in Supramolecular Chemistry
(2023-01-01) Twum, Kwaku; Beyeh, Ngong K.; Sevilla, Michael; Yang, Ziming; Madlambayan, Gerard
Resorcinarenes are three-dimensional cyclic oligomers obtained by acid-catalyzed condensation between resorcinol and an appropriate aldehyde. This doctoral thesis research presents basic and translational research studies that demonstrate the versatile functionalization and applications of the resorcinarene macrocycle. In chapter one, this thesis introduces macrocyclic chemistry with a brief reflection on the history of the science and Beyeh Lab’s use of the resorcinarene macrocycle in supramolecular chemistry. Chapter two presents a fundamental study of cooperativity in non-covalent interactions using a resorcinarene as an anchor to a guest in a solution that restricts its free rotation in the solution. Chapter three explores the molecular recognition potential of functionalized resorcinarenes as sensors for ammonium cations and pyrophosphate anions. Last, chapter four presents functionalized water-soluble resorcinarenes as aggregation inhibitors for peptides that cause eye cataracts. These applications are all realized through the persistent hydrophobic cavity inherent in the basic resorcinarene cavitand and the synthetic functionalization of the macrocyclic ring, which underscores the versatile use of resorcinarene compounds in supramolecular chemistry.
Phosphorylation Patterns, Aggregation Propensities, and Morphological Studies of The Various Tau Protein Isoforms
(2022-03-25) Laryea, Erving Torgbor; Wu, Colin; Avery, Adam; Madlambayan, Gerard
Tau 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.
The Role of CD49f - Integrin α6- in Human Stem Cell Biology
(2021-11-15) Timilsina, Suraj; Villa-Diaz, Luis; K. Lal, Shailesh; Madlambayan, Gerard
Owing 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).
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 M
Articular 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.
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, Mick
Pluripotent 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.

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