Matching Items (13)
Filtering by
- All Subjects: Molecular Biology
- Creators: Newbern, Jason
Description
Stroke remains the leading cause of adult disability in developed countries. Most survivors live with residual motor impairments that severely diminish independence and quality of life. After stroke, the only accepted treatment for these patients is motor rehabilitation. However, the amount and kind of rehabilitation required to induce clinically significant improvements in motor function is rarely given due to the constraints of our current health care system. Research reported in this dissertation contributes towards developing adjuvant therapies that may augment the impact of motor rehabilitation and improve functional outcome. These studies have demonstrated reorganization of maps within motor cortex as a function of experience in both healthy and brain-injured animals by using intracortical microstimulation technique. Furthermore, synaptic plasticity has been identified as a key neural mechanism in directing motor map plasticity, evidenced by restoration of movement representations within the spared cortical tissue accompanied by increase in synapse number translating into motor improvement after stroke. There is increasing evidence that brain-derived neurotrophic factor (BDNF) modulates synaptic and morphological plasticity in the developing and mature nervous system. Unfortunately, BDNF itself is a poor candidate because of its short half-life, low penetration through the blood brain barrier, and activating multiple receptor units, p75 and TrkB on the neuronal membrane. In order to circumvent this problem efficacy of two recently developed novel TrkB agonists, LM22A-4 and 7,8-dihydroxyflavone, that actively penetrate the blood brain barrier and enhance functional recovery. Findings from these dissertation studies indicate that administration of these pharmacological compounds, accompanied by motor rehabilitation provide a powerful therapeutic tool for stroke recovery.
ContributorsWarraich, Zuha (Author) / Kleim, Jeffrey A (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Tillery, Stephen-Helms (Committee member) / Santello, Marco (Committee member) / Arizona State University (Publisher)
Created2013
Description
Parkinson’s disease (PD) is a progressive neurodegenerative disorder, diagnosed late in
the disease by a series of motor deficits that manifest over years or decades. It is characterized by degeneration of mid-brain dopaminergic neurons with a high prevalence of dementia associated with the spread of pathology to cortical regions. Patients exhibiting symptoms have already undergone significant neuronal loss without chance for recovery. Analysis of disease specific changes in gene expression directly from human patients can uncover invaluable clues about a still unknown etiology, the potential of which grows exponentially as additional gene regulatory measures are questioned. Epigenetic mechanisms are emerging as important components of neurodegeneration, including PD; the extent to which methylation changes correlate with disease progression has not yet been reported. This collection of work aims to define multiple layers of PD that will work toward developing biomarkers that not only could improve diagnostic accuracy, but also push the boundaries of the disease detection timeline. I examined changes in gene expression, alternative splicing of those gene products, and the regulatory mechanism of DNA methylation in the Parkinson’s disease system, as well as the pathologically related Alzheimer’s disease (AD). I first used RNA sequencing (RNAseq) to evaluate differential gene expression and alternative splicing in the posterior cingulate cortex of patients with PD and PD with dementia (PDD). Next, I performed a longitudinal genome-wide methylation study surveying ~850K CpG methylation sites in whole blood from 189 PD patients and 191 control individuals obtained at both a baseline and at a follow-up visit after 2 years. I also considered how symptom management medications could affect the regulatory mechanism of DNA methylation. In the last chapter of this work, I intersected RNAseq and DNA methylation array datasets from whole blood patient samples for integrated differential analyses of both PD and AD. Changes in gene expression and DNA methylation reveal clear patterns of pathway dysregulation that can be seen across brain and blood, from one study to the next. I present a thorough survey of molecular changes occurring within the idiopathic Parkinson’s disease patient and propose candidate targets for potential molecular biomarkers.
the disease by a series of motor deficits that manifest over years or decades. It is characterized by degeneration of mid-brain dopaminergic neurons with a high prevalence of dementia associated with the spread of pathology to cortical regions. Patients exhibiting symptoms have already undergone significant neuronal loss without chance for recovery. Analysis of disease specific changes in gene expression directly from human patients can uncover invaluable clues about a still unknown etiology, the potential of which grows exponentially as additional gene regulatory measures are questioned. Epigenetic mechanisms are emerging as important components of neurodegeneration, including PD; the extent to which methylation changes correlate with disease progression has not yet been reported. This collection of work aims to define multiple layers of PD that will work toward developing biomarkers that not only could improve diagnostic accuracy, but also push the boundaries of the disease detection timeline. I examined changes in gene expression, alternative splicing of those gene products, and the regulatory mechanism of DNA methylation in the Parkinson’s disease system, as well as the pathologically related Alzheimer’s disease (AD). I first used RNA sequencing (RNAseq) to evaluate differential gene expression and alternative splicing in the posterior cingulate cortex of patients with PD and PD with dementia (PDD). Next, I performed a longitudinal genome-wide methylation study surveying ~850K CpG methylation sites in whole blood from 189 PD patients and 191 control individuals obtained at both a baseline and at a follow-up visit after 2 years. I also considered how symptom management medications could affect the regulatory mechanism of DNA methylation. In the last chapter of this work, I intersected RNAseq and DNA methylation array datasets from whole blood patient samples for integrated differential analyses of both PD and AD. Changes in gene expression and DNA methylation reveal clear patterns of pathway dysregulation that can be seen across brain and blood, from one study to the next. I present a thorough survey of molecular changes occurring within the idiopathic Parkinson’s disease patient and propose candidate targets for potential molecular biomarkers.
ContributorsHenderson, Adrienne Rose (Author) / Huentelman, Matthew J (Thesis advisor) / Newbern, Jason (Thesis advisor) / Dunckley, Travis L (Committee member) / Jensen, Kendall (Committee member) / Wilson, Melissa (Committee member) / Arizona State University (Publisher)
Created2019
Description
Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known.
MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.
I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.
MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.
I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.
ContributorsKotagama, Kasuen Indrajith Bandara (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Newbern, Jason (Committee member) / Rawls, Alan (Committee member) / Arizona State University (Publisher)
Created2019
Description
Alternative polyadenylation (APA) is the biological mechanism in which the same gene can have multiple 3'untranslated region (3'UTR) isoforms due to the presence of multiple polyadenylation signal (PAS) elements within the pre mRNAs. Because APA produces mRNA transcripts that have different 3'UTR isoforms, certain transcripts may be subject to post-transcriptional regulation by regulatory non-coding RNAs, such as microRNAs or RNA binding proteins defects of which have been implicated in diseases such as cancer. Despite the increasing level of information, functional understanding of the molecular mechanisms involved in transcription is still poorly understood, nor is it clear why APA is necessary at a cell or tissue-specific level. To address these questions I wanted to develop a set of sensor strain plasmids capable of detecting cleavage and polyadenylation in vivo, inject the complete sensor strain plasmid into C. elegans and prepare stable transgenic lines, and perform proof-of-principle RNAi feeding experiments targeting genes associated with the cleavage and polyadenylation complex machinery. I demonstrated that it was possible to create a plasmid capable of detecting cleavage and polyadenylation in C. elegans; however, issues arose during the RNAi assays indicating the sensor strain plasmid was not sensitive enough to the RNAi to effectively detect in the worms. Once the problems involved with sensitivity and variability in the RNAi effects are resolved, the plasmid would be able to better address questions regarding the functional understanding of molecular mechanisms involved in transcription termination.
ContributorsWilky, Henry Patrick (Author) / Mangone, Marco (Thesis director) / Newbern, Jason (Committee member) / Blazie, Stephen (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
One of the most prominent biological challenges for the field of drug delivery is the blood-brain barrier. This physiological system blocks the entry of or actively removes almost all small molecules into the central nervous system (CNS), including many drugs that could be used to treat diseases in the CNS. Previous studies have shown that activation of the adenosine receptor signaling pathway through the use of agonists has been demonstrated to increase BBB permeability. For example, regadenoson is an adenosine A2A receptor agonist that has been shown to disrupt the BBB and allow for increased drug uptake in the CNS. The goal of this study was to verify this property of regadenoson. We hypothesized that co-administration of regadenoson with a non-brain penetrant macromolecule would facilitate its entry into the central nervous system. To test this hypothesis, healthy mice were administered regadenoson or saline concomitantly with a fluorescent dextran solution. The brain tissue was either homogenized to measure quantity of fluorescent molecule, or cryosectioned for imaging with confocal fluorescence microscopy. These experiments did not identify any significant difference in the amount of fluorescence detected in the brain after regadenoson treatment. These results contradict those of previous studies and highlight potential differences in injection methodology, time windows, and properties of brain impermeant molecules.
ContributorsWohlleb, Gregory Michael (Author) / Sirianni, Rachael (Thesis director) / Stabenfeldt, Sarah (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
Pantothenate kinase-associated neurodegeneration, PKAN, is a neurological disease that is caused by biallelic mutations in the PANK2 gene, which codes for a pantothenate kinase. Some PANK2 mutations that cause PKAN retain enzymatic activity. A possible explanation for the mutations that have residual activity but still cause the disease is that they do not have the correct cellular localization. The localization of PANK2 was studied through cellular fractionation. We found the precursor form of PANK2, pPANK2, appears to be anchored to the inner membrane of the mitochondria, and the mature form, mPANK2, is located in the inter-membrane space, IMS. However, the IMS of the PKAN causing mutants is completely devoid of mPANK2 which suggests some disease-causing mutations may be mislocalized. In addition, PANK2 catalyzes the first and rate limiting step in Coenzyme A biosynthesis, and in other studies, it has been shown that the CoA biosynthesis enzymes form a complex in yeast. Therefore, we also considered the possibility that PKAN-causing mutations that retain activity have altered interactions with the other CoA biosynthesis enzymes. Coimmunoprecipitation of the proteins in the pathway was done to determine if there were any interactions with PANK2. The results indicate that PANK2 does not directly interact with either PPCS or CoASY, the second and final enzymatic activities in the CoA biosynthesis pathway.
ContributorsHadziahmetovic, Una (Author) / Newbern, Jason (Thesis director) / Kruer, Michael (Thesis director) / Padilla-Lopez, Sergio (Committee member) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The pathophysiology of neurodegenerative diseases, such as Alzheimer’s disease (AD), remain difficult to ascertain in part because animal models fail to fully recapitulate the complex pathophysiology of these diseases. In vitro models of neurodegenerative diseases generated with patient derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) could provide new insight into disease mechanisms. Although protocols to differentiate hiPSCs and hESCs to neurons have been established, standard practice relies on two dimensional (2D) cell culture systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment.
I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics.
I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics.
ContributorsPetty, Francis (Author) / Brafman, David (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Nikkhah, Mehdi (Committee member) / Arizona State University (Publisher)
Created2016
Description
Neurological disorders are difficult to treat with current drug delivery methods due to their inefficiency and the lack of knowledge of the mechanisms behind drug delivery across the blood brain barrier (BBB). Nanoparticles (NPs) are a promising drug delivery method due to their biocompatibility and ability to be modified by cell penetrating peptides, such as transactivating transciptor (TAT) peptide, which has been shown to increase efficiency of delivery. There are multiple proposed mechanisms of TAT-mediated delivery that also have size restrictions on the molecules that can undergo each BBB crossing mechanism. The effect of nanoparticle size on TAT-mediated delivery in vivo is an important aspect to research in order to better understand the delivery mechanisms and to create more efficient NPs. NPs called FluoSpheres are used because they come in defined diameters unlike polymeric NPs that have a broad distribution of diameters. Both modified and unmodified 100nm and 200nm NPs were able to bypass the BBB and were seen in the brain, spinal cord, liver, and spleen using confocal microscopy and a biodistribution study. Statistically significant differences in delivery rate of the different sized NPs or between TAT-modified and unmodified NPs were not found. Therefore in future work a larger range of diameter size will be evaluated. Also the unmodified NPs will be conjugated with scrambled peptide to ensure that both unmodified and TAT-modified NPs are prepared in identical fashion to better understand the role of size on TAT targeting. Although all the NPs were able to bypass the BBB, future work will hopefully provide a better representation of how NP size effects the rate of TAT-mediated delivery to the CNS.
ContributorsCeton, Ricki Ronea (Author) / Stabenfeldt, Sarah (Thesis director) / Sirianni, Rachael (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The WNT signaling pathway plays numerous roles in development and maintenance of adult homeostasis. In concordance with it’s numerous roles, dysfunction of WNT signaling leads to a variety of human diseases ranging from developmental disorders to cancer. WNT signaling is composed of a family of 19 WNT soluble secreted glycoproteins, which are evolutionarily conserved across all phyla of the animal kingdom. WNT ligands interact most commonly with a family of receptors known as frizzled (FZ) receptors, composed of 10 independent genes. Specific interactions between WNT proteins and FZ receptors are not well characterized and are known to be promiscuous, Traditionally canonical WNT signaling is described as a binary system in which WNT signaling is either off or on. In the ‘off’ state, in the absence of a WNT ligand, cytoplasmic β-catenin is continuously degraded by the action of the APC/Axin/GSK-3β destruction complex. In the ‘on’ state, when WNT binds to its Frizzled (Fz) receptor and LRP coreceptor, this protein destruction complex is disrupted, allowing β-catenin to translocate into the nucleus where it interacts with the DNA-bound T cell factor/lymphoid factor (TCF/LEF) family of proteins to regulate target gene expression. However in a variety of systems in development and disease canonical WNT signaling acts in a gradient fashion, suggesting more complex regulation of β-catenin transcriptional activity. As such, the traditional ‘binary’ view of WNT signaling does not clearly explain how this graded signal is transmitted intracellularly to control concentration-dependent changes in gene expression and cell identity. I have developed an in vitro human pluripotent stem cell (hPSC)-based model that recapitulates the same in vivo developmental effects of the WNT signaling gradient on the anterior-posterior (A/P) patterning of the neural tube observed during early development. Using RNA-seq and ChIP-seq I have characterized β-catenin binding at different levels of WNT signaling and identified different classes of β-catenin peaks that bind cis-regulatory elements to influence neural cell fate. This work expands the traditional binary view of canonical WNT signaling and illuminates WNT/β-catenin activity in other developmental and diseased contexts.
ContributorsCutts, Joshua Patrick (Author) / Brafman, David A (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Nikkhah, Mehdi (Committee member) / Wang, Xiao (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2019
Description
Skeletal muscle injury, whether acute or chronic, is characterized by influxes of pro- and anti-inflammatory cells that coordinate with muscle to precisely control the reparative process. This intricate coordination is facilitated by a signaling feedback loop between satellite cells and extravasated immune cells. Regulation of the cytokines and chemokines that mediate healthy repair is critical for the overall success of fiber regeneration and thus provides a prospective direction for the development of therapeutics aimed at fine-tuning the local inflammatory response. This work describes (1) the contribution of non-myogenic cells in skeletal muscle regeneration, (2) the role of the transcription factor Mohawk (Mkx) in regulating inflammation following acute muscle injury and the identification of an overarching requirement for Mkx in the establishment of a pro-inflammatory response, and (3) characterization of eosinophils in acute and chronic muscle damage. Mice deficient for Mkx exhibited delayed muscle regeneration, accompanied by impaired clearance of necrotic fibers and smaller regenerated fibers. This diminished regenerative capacity was associated with a reduction in the recruitment of pro-inflammatory macrophages to the site of damage. In culture, Mkx-/- bone marrow-derived macrophages displayed reduced proliferative capacity but retained the ability to polarize in response to a pro-inflammatory stimulus. The necessity of Mkx in mounting a robust immune response was further confirmed by an immunological challenge in which Mkx-/- mice exhibited increased susceptibility to Salmonella enterica serovar Typhimurium infection. Significant downregulation of key cytokine and chemokine expression was identified throughout the course of muscle repair in Mkx-/- mice and represents one mechanism in which Mkx regulates the establishment of an inflammatory response. Previous research discovered that Mkx is highly expressed in eosinophils, a type of innate immune cell that participates in disease-fighting and inflammation, however the role of eosinophils in muscle repair is not well described. This work outlines the contribution of eosinophils in muscle repair following acute and chronic injury. In healthy mice, eosinophils were found to inhibit efficient muscle repair following acute injury. Utilizing the mdx-/-utrn-/- muscular dystrophy mouse model, eosinophil depletion via administration of anti-IL-5 antibody significantly improved diaphragm fiber diameter and increased the survival rate during the course of treatment.
ContributorsLynch, Cherie Alissa (Author) / Rawls, Alan (Thesis advisor) / Wilson-Rawls, Jeanne (Committee member) / Newbern, Jason (Committee member) / Lake, Douglas (Committee member) / Allen, Ronald (Committee member) / Arizona State University (Publisher)
Created2020