M.S. - Cell and Molecular Biology

Permanent URI for this collectionhttps://hdl.handle.net/10125/20028

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Now showing 1 - 12 of 12

IASO: Precise genome editing idealizing backbone healing and tale-guided piggyBac
(University of Hawai'i at Manoa, 2025) Errard, Clara; Owens, Jesse B.; Cell and Molecular Biology
Gene editing, which is the alteration of the genetic material of a living organism by inserting, replacing or deleting a DNA sequence, could provide a long-term solution for patients suffering from genetic diseases. A major challenge in the field is the efficient and precise insertion of DNA sequences without off-target insertions. While CRISPR/Cas9 has revolutionized genome engineering, its clinical applicability is limited due to potential off-target effects, limited efficiency in dividing cells, and having a limited DNA cargo capacity. Thus, this thesis aims to optimize a novel approach that would result in a specific and efficient method of transferring a gene of interest to a particular target in the genome. We hypothesize that the piggyBac transposon (pB), TALE and non-homologous end joining (NHEJ) can be idealized to target and integrate at a specific DNA target site.
INVESTIGATION OF THE PGM5 KNOCKOUT MOUSE AND ITS EFFECT ON MUSCLE FUNCTION
(University of Hawai'i at Manoa, 2024) Wright, Ryan D.; Hoffmann, Peter R.; Cell and Molecular Biology
Abstract The phosphoglucomutase (PGM) family of proteins is composed of five members that have a similar predicted structure containing four distinct domains. In humans and mice, this protein family includes PGM1, PMG2, PGM2L1, PGM3, and PGM5, many of which have been implicated in catalyzing reactions involved in glycogenesis. PGM5 is most closely related to PGM1 in terms of amino acid sequence. However, PGM5 appears to lack enzymatic activity and instead has been shown to localize to cytoskeletal structural complexes. A PGM5 whole animal (with CMV-Cre) knockout (KO) mouse model was developed to determine the biological role of this protein, and we found that mice developed similar to wild-type (WT) controls with no apparent phenotype in development. A Western blot of different tissues showed particularly high levels of PGM5 in cardiac, aortic, and skeletal muscle tissues and a confirmed absence of protein in KO tissues. Given the high expression in these muscle tissues, we subjected the KO and WT control mice to a treadmill critical speed test to evaluate potential differences in exercise tolerance. We found that KO mice exhibit a significantly lower critical speed compared to age matched WT mice, suggesting an impairment in muscle oxidative metabolism. The PGM5 heterozygous mice appear to exhibit a critical speed closer to the WT mice than that of the KO mice, suggesting that one functional PGM5 allele was sufficient for mice to maintain muscle function. These experiments suggest that PGM5 expression is not required for embryogenesis in mice but may impair muscle tissue in the heart for optimal physical activity. Further investigations into heart muscle strength and tolerance to exercise needs to be undertaken along with cell biology experiments to determine the precise role within muscle cells of this PGM family member.
Epigenetic Targets And Downstream Effects Of Noni Aqueous Extract On Anti-cancer Activity
(University of Hawaii at Manoa, 2022) Head, Tony; Maunakea, Alika K.; Cell and Molecular Biology
This study aims to analyze methylation levels of genomic loci in HCT116 colon cancer cells altered by exposure to aqueous Noni extract for 48 hours. A Polynesian ethnobotanical with anti-cancer properties, Noni is a complex assembly of chemical constituents that influence DNA methylation states in cancer cells; we hypothesize that by analyzing and correlating DNA methylation and RNA expression pattern changes induced by Noni treatment, we will identify signaling cascades or pathways that explain decreases in cell viability, membrane integrity, and adhesion. Our focus is on the combined effects of Noni fruit in aqueous solution that contribute to the upregulation of gene expression and the downstream pathways potentially leading to the observed morphological and cell inhibitory effects on HCT116 cells. We identified multiple differentially expressed genes involved in proliferation/survival pathways, plasma membrane integrity, and cell adhesion. However, the direct influences of DNA methylation and expression were more complex; we conclude that differential DNA methylation and other factors likely interact indirectly to induce observed effects on expression, and consequently to anti-cancer activity. The identification of specific molecular pathways modulated by Noni has wide implications with respect to commercial production and harvesting, in addition to altering treatment options for a wide array of illnesses and diseases including cancer. Noni fruit is relatively abundant in Hawaii and can be prepared using simple methods. In addition, continued studies may further elucidate effects of variations in commercialized Noni products.
Characterizing the morphological and cellular effects of amyloid-beta on oligodendroglia and myelination
(University of Hawaii at Manoa, 2022) Delgado, Donovan David; Nichols, Robert A.; Cell and Molecular Biology
Loss of white matter integrity is a feature of natural aging and Alzheimer’s disease (AD).Notably, the spread of AD pathology reflects the pattern of myelination during normal development but in reverse (neuropathologic retrogenesis), where later myelinated brain regions develop AD pathology before early myelinated regions. Classical neuropathologic changes in AD (i.e. accumulation of soluble oligomeric amyloid-β) are correlated with progressive neuronal death, but may also induce changes in the cells of the oligodendroglia lineage and damage myelin. This project sought to elucidate the cellular responses of the oligodendroglia cells to amyloid-β (Aβ) and the subsequent impact on white matter structure. Using neonatal C57BL/6J mice, we were able to establish a variety of primary glial cultures: oligodendrocyte precursor cells (OPC) and oligodendrocytes cultures. We assessed the effects of Aβ on OPC survival as well as their proliferation using TUNEL and EdU detection assays. These preliminary results indicate that exposure to 1 μM Aβ1-42 for 5 days is sufficient to reduce OPC proliferation but not induce apoptosis. Interestingly, we found that a small population of OPCs seemingly switch glial fates to type II astrocytes indicated by the loss of typical OPC morphology concomitant with the induction of expression of the glial fibrillary acid protein (GFAP). We further investigated the effects Aβ may have on the mature oligodendrocyte, where cultures treated with Aβ displayed decreased MBP intensity and changes in the number of processes. Using ex vivo organotypic brain slices, we were able to corroborate the changes in MBP intensity seen in the in vitro cultures, but unable to discern direct changes in axon myelination. However, immunoblots of hippocampal lysates from 5XFAD mice compared to age-matched wild-type mice demonstrated alterations in myelin protein levels. Our preliminary results showcasing the effects of Aβ on cells of the oligodendroglia lineage and CNS white matter suggest that impaired myelin maintenance leading to demyelination may be more prominent in AD than previously thought. Hence, characterizing changes in white matter alterations due to AD-related pathogenesis beyond the normal aging process is key to furthering our understanding of AD pathology.
Beta Amyloid-induced Dysregulation Of The Exocyst Complex Impedes Postsynaptic Receptor Trafficking And Alters Dendritic Spines
(University of Hawaii at Manoa, 2022) Ormsbee, Kendra Marie; Nichols, Robert A.; Cell and Molecular Biology
Synaptic dysfunction and loss are hallmarks of the early pathogenesis of Alzheimer’s disease (AD), leading to impaired cognitive function, including disruption of memory processing. Accumulation of neurotoxic oligomeric beta amyloid (Aβ) in the prodromic period leading up to AD is thought to contribute to dendrite atrophy, altered dendritic spine dynamics resulting in synaptic dysfunction and spine loss, and compromised postsynaptic receptor trafficking, but the molecular mechanisms for these Aβ-associated changes are still yet to be fully understood. As the exocyst complex has been implicated in vesicle tethering and trafficking preceding SNARE protein-mediated vesicle exocytosis in yeast and mammalian cells, and preliminary work in our laboratories has found evidence of exocyst involvement in amyloid precursor protein (APP) trafficking in murine primary neurons, I investigated the impact of neurotoxic levels of Aβ on the octameric exocyst complex in relation to altered dendrite and spine integrity and postsynaptic receptor trafficking in spines. Specifically, I utilized Actin-GFP expression to examine dendritic arborization as a measure of neuronal complexity and dendritic spine dynamics, which are driven by cytoskeletal changes, and immunocytochemistry and proximity ligand assay (PLA) of key exocyst subunits and AMPA GluA1 receptors in conjunction with select exocyst subunit deletion to correlate Aβ -induced changes in the exocyst with altered postsynaptic receptor trafficking. The results were consistent with our central hypothesis that Aβ induces dysregulation of the exocyst complex, obstructing major postsynaptic neurotransmitter receptor AMPAR trafficking and, consequently, contributing to neurodegeneration through dendrite atrophy and loss of dendritic spine density in AD. The findings provide new insights into the molecular mechanisms underlying synapse dysfunction in AD pathogenesis, potentially revealing novel targets for therapeutic intervention.
Intracellular Trafficking of Amyloid Precursor Protein by the Exocyst: Mechanisms in Alzheimer's Disease and Insulin Signaling
(University of Hawaii at Manoa, 2021) Sachs, Rachel; Fogelgren, Benjamin C.; Cell and Molecular Biology
Alzheimer’s disease is a devastating progressive neurodegenerative disease that causes memory loss, personality changes, and impaired reasoning. It currently affects approximately 6 million people in the US, and is America’s 6th leading cause of death. Research on the disease has been extensive for decades, however there are still no effective therapies and the pathogenic mechanisms are not fully understood. Two of the pathways implicated in causing the damage seen in Alzheimer’s disease focus around amyloid beta peptide aggregates, that form plaques, and hyperphosphorylated tau, which forms tangles. Studies on the Amyloid hypothesis focus on the generation and aggregation of the peptide, which is cleaved from the amyloid precursor protein (APP). How APP is cut and whether it will lead formation of the amyloid peptide has been shown to be influenced by the trafficking of APP bringing it into contact with different secretases. Based on the intracellular trafficking patterns of APP, and hints from the literature, we hypothesized that the eight-protein exocyst complex regulates APP trafficking and amyloid beta generation in neurons. For our research, we used two cell models: the SH-SY5Y cell line, a human neuroblastoma cell line able to be differentiated into neuronal cells using retinoic acid, and mouse primary hippocampal neurons. For our experiments, we generated several transgenic SH-SY5Y lines, including a mutant APP overexpressing line that secretes high levels of amyloid beta, as well as lines that co-express fluorescent-tagged mutant APP and exocyst proteins for use in live cell imaging. We found that when several members of the exocyst family are knocked down using siRNA in our mutAPP overexpressing line, the amount of amyloid beta released in the media significantly decreases. Using the proximity ligation assay (PLA), we also discovered that Exoc5 and APP closely co-localize (within 40nm) in mouse primary hippocampal neurons. Since the exocyst regulates the insulin-induced trafficking of Glut4 glucose transporters in adipocytes and muscle cells, we tested the effect of insulin signaling in neurons on the exocyst and its relationship to APP and Glut4. When insulin was present, the exocyst holocomplex assembly increased significantly, as did its association with Glut4, however APP association with the exocyst was largely abolished. The data suggests that insulin signaling is able to switch the exocyst complex away from APP-containing vesicles and potentially reduce amyloid beta generation. These findings point to the regulation of APP trafficking by the exocyst complex and a direct connection between the exocyst’s influence on the production of the amyloid beta peptide and insulin signaling effects. Understanding the involvement of the exocyst in Alzheimer’s disease could provide further targets for drug development and help bridge the gap in our understanding of connections between insulin signaling in the brain and Alzheimer’s disease.
Role Of Tcf21 In Perinatal Cardiac Fibroblasts Proliferation And Gene Expression
(University of Hawaii at Manoa, 2020) Riggsbee, Kara; Tallquist, Michelle D.; Cell and Molecular Biology
Cardiac fibroblasts play a dominant role in heart disease, but our understanding of the signals that control these cells during heart development is inadequate. Tcf21 is a coronary artery disease associated gene whose function in the heart is not well understood. Previously using Tcf21 null animals, we have demonstrated that this basic helix loop helix transcription factor is essential for the formation of epicardial-derived cardiac fibroblasts. Lack of Tcf21 resulted in a failure of cardiac fibroblast progenitor migration from the epicardium. Recently, we have found that Tcf21 continues to be expressed in mature cardiac fibroblasts suggesting a continued role for this transcription factor. To evaluate the function of Tcf21 after embryonic development, we generated animals which lacked Tcf21 in the fibroblast lineage. When Tcf21 is removed at birth, we find a 30 percent reduction in the number of fibroblasts. Using EdU, we determined that loss of Tcf21 leads to reduced fibroblast proliferation in the perinatal period. To identify transcriptional targets of Tcf21, we have compared fibroblast transcriptomes in control and Tcf21 deficient fibroblasts. We have found an increase in lipid genes in the Tcf21 deficient fibroblasts. Taken together our results demonstrate that Tcf21 continues to play important roles in fibroblast biology beyond the initial formation of cardiac fibroblasts from the epicardium.
Arc In AMPA Receptor Endocytosis: Direct Evidence Indicating Arc As A Scaffolding Protein
(University of Hawaii at Manoa, 2017-05) Goo, Brandee; Cell and Molecular Biology
Long-term potentiation (LTP) and long-term depression (LTD) are processes thought to underlie learning and memory, which require proper regulation of activity-regulated cytoskeletonassociated protein (Arc; also known as Arg3.1). Abnormal expression levels of Arc have been associated with Alzheimer’s disease, schizophrenia, Fragile-X syndrome, Angelmann’s syndrome and substance abuse. Arc is an immediate early gene product that is transcribed in dendritic spines and is a positive regulator of AMPA receptor (AMPAR) endocytosis during LTD. Verified protein-protein interactions between Arc and proteins involved in endocytosis, including dynamin and endophilin, support the role of Arc as a regulator of AMPAR endocytosis. Nevertheless, the mechanism by which Arc specifically targets AMPARs for endocytosis is currently unknown. This is key to understanding the mechanisms of learning and memory and how they are affected by Arc associated human conditions. Here we show evidence of a novel interaction between Arc and protein interacting with C kinase 1 (PICK1), a protein known to bind to the GluR2 subunit of AMPARs and associated with AMPAR trafficking. Cross-correlation raster image correlation spectroscopy and Förster resonance energy transfer (FRET) with Arc-mCherry and EGFP-PICK1 demonstrate this interaction. FRET is more apparent in the projections of transfected SH-SY5Y cells and is enhanced by depolarization. Interestingly, TIRF imaging shows PICK1 aggregates that are only present when cotransfected with Arc that colocalize with Arc aggregates. These findings show an interaction of Arc and PICK1, which may answer how Arc directs endocytic machinery to AMPARs during LTD.
Insertion of a functional copy of Six2 to generate a transgenic mouse via piggyBac transposase
(University of Hawaii at Manoa, 2013-08) Chang, Cara
Identification of gene function based on known mutations remains an integral objective in the field of basic science. The adult Br heterozygous mouse displays a reduced number of nephrons in association with chronic renal failure, hypertension and reduced Six2 expression during embryonic renal morphogenesis. The purpose of this study was to determine whether a functional copy of Six2 could be inserted into the Br mouse genome in an effort to overexpress Six2. After processing and isolation by restriction enzyme digestion and pulse field gel electrophoresis, 26kb Six2 BAC DNA fragment was then cloned into the mGENIE-3-BAC transposon vector via the in vitro Gibson Assembly method. The mGENIE-3-BAC-Six2 construct was subsequently confirmed by colony PCR, restriction enzyme digestion; the construct was also tested for functionality and expression in human cell lines and transgenic mice. Our data indicates that our single plasmid transposase-mediated approach to transgenesis requires fewer embryos, while capable of incorporating 42kb of exogenous DNA into the mouse genome.
Novel insights on the role of selenoprotein P in sperm viability
(University of Hawaii at Manoa, 2012-08) Nguyen-Wu, Elizabeth Q M Dao
Selenium (Se) is a micronutrient essential for life in many organisms. Selenium is incorporated into selenoproteins as the twenty-first amino acid, selenocysteine (Sec), and has antioxidant properties. One member of the family of twenty-five selenoproteins in humans is Selenoprotein P (Sepp1). This protein is synthesized primarily in liver and is proposed to transport selenium throughout the body, particularly to the brain and testes. Sepp1 knockout (KO) mice on (normal) diets without selenium supplementation have decreased selenoprotein expression in brain and testes. Previous studies have suggested that Sepp1 male KO mice are infertile due to kinks in the flagellum of spermatozoa, greatly reducing sperm motility, therefore leading to dramatically decreased fertility. In this two-part study, our first objective was to further understand the role of Sepp1 on sperm viability. We hypothesize that Sepp1 plays a critical role in sperm DNA viability independent of motility, potentially through modulating glutathione peroxidase 4 (GPx4) biosynthesis. GPx4 is another selenoprotein known to protect cells from membrane lipid peroxidation and has been implicated in development and fertility [71]. The second objective of this study was to introduce a novel application to rescue Sepp1 global expression in knockout animals (Sepp1r/r CMV+) using Cre recombinase transgenic mice. We addressed the role of Sepp1 in sperm DNA viability with intracytoplasmic spermatozoa injections (ICSI) of Sepp1 KO sperm into wild type oocytes. Surrogate female mice carrying embryos resulting from injection of Sepp1-/-sperm resulted in a 72.3% reduction in live pups born compared to Sepp1 heterozygous control sperm. Our results from the ICSI experiments, in which sperm were directly injected into oocytes without flagella, suggest that Sepp1 is critical for sperm DNA viability independent of motility. We show through western blot analysis that GPx4 levels are significantly decreased in the testes and epididymides of Sepp1 KO mice, whereas Sepp1r/r CMV+ rescue mice had restored expression levels comparable to the Sepp1 wild type (Sepp1 WT). Immunohistochemistry studies using an antibody against GPx4 further confirmed that GPx4 levels were undetectable in fresh Sepp1 KO mouse sperm, while GPx4 levels in Sepp1r/r CMV+ rescue mice were similar to those of wild type controls. Cre-Lox recombination is a commonly used genetic tool for site-specific gene deletion. However, we demonstrate that this system can be used to rescue gene expression as well, restoring the expression of Sepp1 in KO mice. We show that this approach produced viable progeny of the systemic Sepp1r/r CMV+ (rescue) mice that express the CMV-Cre driven Sepp1 gene in all tissues. We confirmed through the Morris Water Maze (MWM) and other behavior assays that in contrast to Sepp1 KO mice, Sepp1r/r CMV+ mice had normal neuromotor function and memory compared to Sepp1+/+. Successful implementation of this method can further be utilized to restrict gene expression to specific cells. Our study presents new data showing that Sepp1 is crucial for viability of sperm DNA, potentially through regulation of GPx4 levels. Furthermore, we demonstrate an innovative method for restoration of gene expression using the Cre recombinase transgenic system, which can be applied to restrict gene expression to specific cells.
Investigation of selenoprotein k function and associated proteins
(University of Hawaii at Manoa, 2012-05) Fay, Jeffrey D.
Selenoprotein K (SelK) is a small (16kD) single pass transmembrane protein localized to the ER. An Src Homology 3 (SH3)-binding domain within the amino acid sequence was exploited to identify potential protein-protein interactions. The effects of SelK deficiency in immune cells and effector calcium flux served as a model system to elucidate a function for SelK. Immunoprecipitation and mass spectrophotometry identification of potential binding partners returned Arf-GAP2 and pyruvate kinase as possible binding partners. Plasmid DNA vectors were designed for use in the Two-Hybrid system to confirm preliminary data of suspected interactions with the creation of fusion proteins coding for Vav1 or Vav2 SH3 domain and the SelK SH3 binding domain. Protein-protein interaction could not be confirmed in this system however SelK seems to play a role in receptor mediated calcium flux, and subsequent low-level production of nitric oxide by way of neuronal nitric oxide synthase.
Characterization of the interaction between TRPA1 and creatine kinase
(University of Hawaii at Manoa, 2007) Cochrane, Kimberly Eileen Maile

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