Matching Items (15)
Description
Specific dendritic morphologies are a hallmark of neuronal identity, circuit assembly, and behaviorally relevant function. Despite the importance of dendrites in brain health and disease, the functional consequences of dendritic shape remain largely unknown. This dissertation addresses two fundamental and interrelated aspects of dendrite neurobiology. First, by utilizing the genetic power of Drosophila melanogaster, these studies assess the developmental mechanisms underlying single neuron morphology, and subsequently investigate the functional and behavioral consequences resulting from developmental irregularity. Significant insights into the molecular mechanisms that contribute to dendrite development come from studies of Down syndrome cell adhesion molecule (Dscam). While these findings have been garnered primarily from sensory neurons whose arbors innervate a two-dimensional plane, it is likely that the principles apply in three-dimensional central neurons that provide the structural substrate for synaptic input and neural circuit formation. As such, this dissertation supports the hypothesis that neuron type impacts the realization of Dscam function. In fact, in Drosophila motoneurons, Dscam serves a previously unknown cell-autonomous function in dendrite growth. Dscam manipulations produced a range of dendritic phenotypes with alteration in branch number and length. Subsequent experiments exploited the dendritic alterations produced by Dscam manipulations in order to correlate dendritic structure with the suggested function of these neurons. These data indicate that basic motoneuron function and behavior are maintained even in the absence of all adult dendrites within the same neuron. By contrast, dendrites are required for adjusting motoneuron responses to specific challenging behavioral requirements. Here, I establish a direct link between dendritic structure and neuronal function at the level of the single cell, thus defining the structural substrates necessary for conferring various aspects of functional motor output. Taken together, information gathered from these studies can inform the quest in deciphering how complex cell morphologies and networks form and are precisely linked to their function.
ContributorsHutchinson, Katie Marie (Author) / Duch, Carsten (Thesis advisor) / Neisewander, Janet (Thesis advisor) / Newfeld, Stuart (Committee member) / Smith, Brian (Committee member) / Orchinik, Miles (Committee member) / Arizona State University (Publisher)
Created2013
Description
In somatic cells, the mitotic spindle apparatus is centrosomal and several isoforms of Protein Kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is unclear. Other protein kinases such as, Glycogen Synthase Kinase 3â (GSK3â) also have been shown to be associated with the mitotic spindle. In the study in chapter 2, we show the enrichment of active (phosphorylated) PKCæ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases, PKC and GSK3â are associated with the mitotic spindle, first, the co-localization and close molecular proximity of PKC isoforms with GSK3â was studied in metaphase cells. Second, the involvement of inactive GSK3â in maintaining an intact mitotic spindle was shown. Third, this study showed that addition of a phospho-PKCæ specific inhibitor to cells can disrupt the mitotic spindle microtubules. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCæ acting through GSK3â. The MAPK pathway has been implicated in various functions related to cell cycle regulation. MAPKK (MEK) is part of this pathway and the extracellular regulated kinase (ERK) is its known downstream target. GSK3â and PKCæ also have been implicated in cell cycle regulation. In the study in chapter 3, we tested the effects of inhibiting MEK on the activities of ERK, GSK3â, PKCæ, and á-tubulin. Results from this study indicate that inhibition of MEK did not inhibit GSK3â and PKCæ enrichment at the centrosomes. However, the mitotic spindle showed a reduction in the pixel intensity of microtubules and also a reduction in the number of cells in each of the M-phase stages. A peptide activation inhibitor of ERK was also used. Our results indicated a decrease in mitotic spindle microtubules and an absence of cells in most of the M-phase stages. GSK3â and PKCæ enrichment were however not inhibited at the centrosomes. Taken together, the kinases GSK3â and PKCæ may not function as a part of the MAPK pathway to regulate the mitotic spindle.
ContributorsChakravadhanula, Madhavi (Author) / Capco, David G. (Thesis advisor) / Chandler, Douglas (Committee member) / Clark-Curtiss, Josephine (Committee member) / Newfeld, Stuart (Committee member) / Arizona State University (Publisher)
Created2012
Description
Cells live in complex environments and must be able to adapt to environmental changes in order to survive. The ability of a cell to survive and thrive in a changing environment depends largely on its ability to receive and respond to extracellular signals. Initiating with receptors, signal transduction cascades begin translating extracellular signals into intracellular messages. Such signaling cascades are responsible for the regulation of cellular metabolism, cell growth, cell movement, transcription, translation, proliferation and differentiation. This dissertation seeks to dissect and examine critical signaling pathways involved in the regulation of proliferation in neural stem cells (Chapter 2) and the regulation of Glioblastoma Multiforme pathogenesis (GBM; Chapter 3). In Chapter 2 of this dissertation, we hypothesize that the mTOR signaling pathway plays a significant role in the determination of neural stem cell proliferation given its control of cell growth, metabolism and survival. We describe the effect of inhibition of mTOR signaling on neural stem cell proliferation using animal models of aging. Our results show that the molecular method of targeted inhibition may result in differential effects on neural stem cell proliferation as the use of rapamycin significantly reduced proliferation while the use of metformin did not. Abnormal signaling cascades resulting in unrestricted proliferation may lead to the development of brain cancer, such as GBM. In Chapter 3 of this dissertation, we hypothesize that the inhibition of the protein kinase, aPKCλ results in halted GBM progression (invasion and proliferation) due to its central location in multiple signaling cascades. Using in-vitro and in-vivo models, we show that aPKCλ functions as a critical node in GBM signaling as both cell-autonomous and non-cell-autonomous signaling converge on aPKCλ resulting in pathogenic downstream effects. This dissertation aims to uncover the molecular mechanisms involved in cell signaling pathways which are responsible for critical cellular effects such as proliferation, invasion and transcriptional regulation.
ContributorsKusne, Yael (Author) / Sanai, Nader (Thesis advisor) / Neisewander, Janet (Thesis advisor) / Tran, Nhan (Committee member) / Hammer, Ronald (Committee member) / Narayanan, Vinodh (Committee member) / Shapiro, Joan (Committee member) / Arizona State University (Publisher)
Created2014
Description
Rett syndrome is a genetically based, X-linked neurodevelopmental disorder that affects 1 in 10,000 live female births. Approximately 95-97% of Rett syndrome cases are attributed to a mutation in the MECP2 gene. In the laboratory setting, key neuropathological phenotypes of Rett syndrome include small neuronal soma and nuclear size, increased cell packing density, and abnormal dendritic branching. Our lab previously created and characterized the A140V mouse model of atypical Rett syndrome in which the males are viable. Hippocampal and cerebellar granule neurons in A140V male mice have reduced soma and nuclear size compared to wild type. We also found that components of the mTOR pathway including rictor, 4E-BP-1, and mTOR, were reduced in A140V mutant mice. Quantitative PCR analysis also showed reduced IGFPB2 expression in A140V mice along with an upward trend in AKT levels that did not meet statistical significance. The objective of this study is i) to characterize the down regulation of AKT-mTOR pathway, and ii) to examine the effect of a genetic strategy to rescue mTOR pathway deficiencies in Mecp2 mutant mouse model. Genetic rescue of the mTOR pathway downregulation was done by crossing heterozygous female A140V mice with heterozygous male Tsc2 mice. Quantitative PCR analysis of A140V_Tsc2 RNA expression supported genetic rescue of mTOR pathway components, however, more testing is needed to fully characterize the rescue effect. Western blot analysis also showed reduction in phosphorylated AKT in Mecp2 A140V and T158A mutant mice, however, more testing is still needed to characterize the mTOR pathway in A140V_Tsc2 mice. Finally, other methods, such as a pharmacological approach, or transfection to increase mTOR pathway activity in cell lines, will be tested to determine if rescue of mTOR pathway activity ameliorate the Rett syndrome phenotype.
ContributorsGerald, Brittany Madison (Author) / Newbern, Jason (Thesis director) / Narayanan, Vinodh (Committee member) / Rangasamy, Sampath (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
One of the fundamental questions in molecular biology is how genes and the control of their expression give rise to so many diverse phenotypes in nature. The mRNA molecule plays a key role in this process as it directs the spatial and temporal expression of genetic information contained in the DNA molecule to precisely instruct biological processes in living organisms. The region located between the STOP codon and the poly(A)-tail of the mature mRNA, known as the 3′Untranslated Region (3′UTR), is a key modulator of these activities. It contains numerous sequence elements that are targeted by trans-acting factors that dose gene expression, including the repressive small non-coding RNAs, called microRNAs.
Recent transcriptome data from yeast, worm, plants, and humans has shown that alternative polyadenylation (APA), a mechanism that enables expression of multiple 3′UTR isoforms for the same gene, is widespread in eukaryotic organisms. It is still poorly understood why metazoans require multiple 3′UTRs for the same gene, but accumulating evidence suggests that APA is largely regulated at a tissue-specific level. APA may direct combinatorial variation between cis-elements and microRNAs, perhaps to regulate gene expression in a tissue-specific manner. Apart from a few single gene anecdotes, this idea has not been systematically explored.
This dissertation research employs a systems biology approach to study the somatic tissue dynamics of APA and its impact on microRNA targeting networks in the small nematode C. elegans. In the first aim, tools were developed and applied to isolate and sequence mRNA from worm intestine and muscle tissues, which revealed pervasive tissue-specific APA correlated with microRNA regulation. The second aim provides genetic evidence that two worm genes use APA to escape repression by microRNAs in the body muscle. Finally, in aim three, mRNA from five additional somatic worm tissues was sequenced and their 3′ends mapped, allowing for an integrative study of APA and microRNA targeting dynamics in worms. Together, this work provides evidence that APA is a pervasive mechanism operating in somatic tissues of C. elegans with the potential to significantly rearrange their microRNA regulatory networks and precisely dose their gene expression.
Recent transcriptome data from yeast, worm, plants, and humans has shown that alternative polyadenylation (APA), a mechanism that enables expression of multiple 3′UTR isoforms for the same gene, is widespread in eukaryotic organisms. It is still poorly understood why metazoans require multiple 3′UTRs for the same gene, but accumulating evidence suggests that APA is largely regulated at a tissue-specific level. APA may direct combinatorial variation between cis-elements and microRNAs, perhaps to regulate gene expression in a tissue-specific manner. Apart from a few single gene anecdotes, this idea has not been systematically explored.
This dissertation research employs a systems biology approach to study the somatic tissue dynamics of APA and its impact on microRNA targeting networks in the small nematode C. elegans. In the first aim, tools were developed and applied to isolate and sequence mRNA from worm intestine and muscle tissues, which revealed pervasive tissue-specific APA correlated with microRNA regulation. The second aim provides genetic evidence that two worm genes use APA to escape repression by microRNAs in the body muscle. Finally, in aim three, mRNA from five additional somatic worm tissues was sequenced and their 3′ends mapped, allowing for an integrative study of APA and microRNA targeting dynamics in worms. Together, this work provides evidence that APA is a pervasive mechanism operating in somatic tissues of C. elegans with the potential to significantly rearrange their microRNA regulatory networks and precisely dose their gene expression.
ContributorsBlazie, Stephen M (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Josh (Committee member) / Lake, Doug (Committee member) / Newfeld, Stuart (Committee member) / Arizona State University (Publisher)
Created2016
Description
Systems biology studies complex biological systems. It is an interdisciplinary field, with biologists working with non-biologists such as computer scientists, engineers, chemists, and mathematicians to address research problems applying systems’ perspectives. How these different researchers and their disciplines differently contributed to the advancement of this field over time is a question worth examining. Did systems biology become a systems-oriented science or a biology-oriented science from 1992 to 2013?
This project utilized computational tools to analyze large data sets and interpreted the results from historical and philosophical perspectives. Tools deployed were derived from scientometrics, corpus linguistics, text-based analysis, network analysis, and GIS analysis to analyze more than 9000 articles (metadata and text) on systems biology. The application of these tools to a HPS project represents a novel approach.
The dissertation shows that systems biology has transitioned from a more mathematical, computational, and engineering-oriented discipline focusing on modeling to a more biology-oriented discipline that uses modeling as a means to address real biological problems. Also, the results show that bioengineering and medical research has increased within systems biology. This is reflected in the increase of the centrality of biology-related concepts such as cancer, over time. The dissertation also compares the development of systems biology in China with some other parts of the world, and reveals regional differences, such as a unique trajectory of systems biology in China related to a focus on traditional Chinese medicine.
This dissertation adds to the historiography of modern biology where few studies have focused on systems biology compared with the history of molecular biology and evolutionary biology.
This project utilized computational tools to analyze large data sets and interpreted the results from historical and philosophical perspectives. Tools deployed were derived from scientometrics, corpus linguistics, text-based analysis, network analysis, and GIS analysis to analyze more than 9000 articles (metadata and text) on systems biology. The application of these tools to a HPS project represents a novel approach.
The dissertation shows that systems biology has transitioned from a more mathematical, computational, and engineering-oriented discipline focusing on modeling to a more biology-oriented discipline that uses modeling as a means to address real biological problems. Also, the results show that bioengineering and medical research has increased within systems biology. This is reflected in the increase of the centrality of biology-related concepts such as cancer, over time. The dissertation also compares the development of systems biology in China with some other parts of the world, and reveals regional differences, such as a unique trajectory of systems biology in China related to a focus on traditional Chinese medicine.
This dissertation adds to the historiography of modern biology where few studies have focused on systems biology compared with the history of molecular biology and evolutionary biology.
ContributorsZou, Yawen (Author) / Laubichler, Manfred (Thesis advisor) / Maienschein, Jane (Thesis advisor) / Creath, Richard (Committee member) / Ellison, Karin (Committee member) / Newfeld, Stuart (Committee member) / Arizona State University (Publisher)
Created2016
Description
A central task for historians and philosophers of science is to characterize and analyze the epistemic practices in a given science. The epistemic practice of a science includes its explanatory goals as well as the methods used to achieve these goals. This dissertation addresses the epistemic practices in gene expression research spanning the mid-twentieth century to the twenty-first century. The critical evaluation of the standard historical narratives of the molecular life sciences clarifies certain philosophical problems with respect to reduction, emergence, and representation, and offers new ways with which to think about the development of scientific research and the nature of scientific change.
The first chapter revisits some of the key experiments that contributed to the development of the repression model of genetic regulation in the lac operon and concludes that the early research on gene expression and genetic regulation depict an iterative and integrative process, which was neither reductionist nor holist. In doing so, it challenges a common application of a conceptual framework in the history of biology and offers an alternative framework. The second chapter argues that the concept of emergence in the history and philosophy of biology is too ambiguous to account for the current research in post-genomic molecular biology and it is often erroneously used to argue against some reductionist theses. The third chapter investigates the use of network representations of gene expression in developmental evolution research and takes up some of the conceptual and methodological problems it has generated. The concluding comments present potential avenues for future research arising from each substantial chapter.
In sum, this dissertation argues that the epistemic practices of gene expression research are an iterative and integrative process, which produces theoretical representations of the complex interactions in gene expression as networks. Moreover, conceptualizing these interactions as networks constrains empirical research strategies by the limited number of ways in which gene expression can be controlled through general rules of network interactions. Making these strategies explicit helps to clarify how they can explain the dynamic and adaptive features of genomes.
The first chapter revisits some of the key experiments that contributed to the development of the repression model of genetic regulation in the lac operon and concludes that the early research on gene expression and genetic regulation depict an iterative and integrative process, which was neither reductionist nor holist. In doing so, it challenges a common application of a conceptual framework in the history of biology and offers an alternative framework. The second chapter argues that the concept of emergence in the history and philosophy of biology is too ambiguous to account for the current research in post-genomic molecular biology and it is often erroneously used to argue against some reductionist theses. The third chapter investigates the use of network representations of gene expression in developmental evolution research and takes up some of the conceptual and methodological problems it has generated. The concluding comments present potential avenues for future research arising from each substantial chapter.
In sum, this dissertation argues that the epistemic practices of gene expression research are an iterative and integrative process, which produces theoretical representations of the complex interactions in gene expression as networks. Moreover, conceptualizing these interactions as networks constrains empirical research strategies by the limited number of ways in which gene expression can be controlled through general rules of network interactions. Making these strategies explicit helps to clarify how they can explain the dynamic and adaptive features of genomes.
ContributorsRacine, Valerie (Author) / Maienschein, Jane (Thesis advisor) / Laubichler, Manfred D (Thesis advisor) / Creath, Richard (Committee member) / Newfeld, Stuart (Committee member) / Morange, Michel (Committee member) / Arizona State University (Publisher)
Created2016
Description
Methyl-CpG binding protein 2 (MECP2) is a widely abundant, multifunctional regulator of gene expression with highest levels of expression in mature neurons. In humans, both loss- and gain-of-function mutations of MECP2 cause mental retardation and motor dysfunction classified as either Rett Syndrome (RTT, loss-of-function) or MECP2 Duplication Syndrome (MDS, gain-of-function). At the cellular level, MECP2 mutations cause both synaptic and dendritic defects. Despite identification of MECP2 as a cause for RTT nearly 16 years ago, little progress has been made in identifying effective treatments. Investigating major cellular and molecular targets of MECP2 in model systems can help elucidate how mutation of this single gene leads to nervous system and behavioral defects, which can ultimately lead to novel therapeutic strategies for RTT and MDS. In the work presented here, I use the fruit fly, Drosophila melanogaster, as a model system to study specific cellular and molecular functions of MECP2 in neurons. First, I show that targeted expression of human MECP2 in Drosophila flight motoneurons causes impaired dendritic growth and flight behavioral performance. These effects are not caused by a general toxic effect of MECP2 overexpression in Drosophila neurons, but are critically dependent on the methyl-binding domain of MECP2. This study shows for the first time cellular consequences of MECP2 gain-of-function in Drosophila neurons. Second, I use RNA-Seq to identify KIBRA, a gene associated with learning and memory in humans, as a novel target of MECP2 involved in the dendritic growth phenotype. I confirm bidirectional regulation of Kibra by Mecp2 in mouse, highlighting the translational utility of the Drosophila model. Finally, I use this system to identify a novel role for the C-terminus in regulating the function of MECP in apoptosis and verify this finding in mammalian cell culture. In summary, this work has established Drosophila as a translational model to study the cellular effects of MECP2 gain-of-function in neurons, and provides insight into the function of MECP2 in dendritic growth and apoptosis.
ContributorsWilliams, Alison (Author) / Duch, Carsten (Thesis advisor) / Orchinik, Miles (Committee member) / Gallitano, Amelia (Committee member) / Huentelman, Matthew (Committee member) / Narayanan, Vinodh (Committee member) / Newfeld, Stuart (Committee member) / Arizona State University (Publisher)
Created2015
Description
Mitochondrial methionyl-tRNA-formyltransferase (MTFMT) is essential for mitochondrial protein translation. The MTFMT gene encodes for an enzyme of the same name, which acts to formylate the methionine of mitochondrial Met-tRNA(Met). In Homo sapiens, MTFMT-formylated-tRNA is an initiator and elongator for the synthesis of 13 mitochondrially-encoded proteins in complexes I, III and IV of the ETC. To understand this mechanism, it is necessary to perform a comprehensive analysis of energy metabolism and oxidative phosphorylation (OXPHOS) among impacted patients. Alterations to this gene vary, with the most documented as a single-splice-site mutation (c.626C>T). Here, we discuss MTFMT involvement in mitochondrial protein translation and neurodegenerative disorders, such as Leigh Syndrome and combined OXPHOS deficiency, in two families. We aim to delineate the impact of OXPHOS dysfunction in patients presenting with MTFMT mutation.
ContributorsChain, Kelsey (Author) / Chen, Qiang (Thesis director) / Rangasamy, Sampathkumar (Committee member) / Narayanan, Vinodh (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Gene expression patterns assayed across development can offer key clues about a gene’s function and regulatory role. Drosophila melanogaster is ideal for such investigations as multiple individual and high-throughput efforts have captured the spatiotemporal patterns of thousands of embryonic expressed genes in the form of in situ images. FlyExpress (www.flyexpress.net), a knowledgebase based on a massive and unique digital library of standardized images and a simple search engine to find coexpressed genes, was created to facilitate the analytical and visual mining of these patterns. Here, we introduce the next generation of FlyExpress resources to facilitate the integrative analysis of sequence data and spatiotemporal patterns of expression from images. FlyExpress 7 now includes over 100,000 standardized in situ images and implements a more efficient, user-defined search algorithm to identify coexpressed genes via Genomewide Expression Maps (GEMs). Shared motifs found in the upstream 5′ regions of any pair of coexpressed genes can be visualized in an interactive dotplot. Additional webtools and link-outs to assist in the downstream validation of candidate motifs are also provided. Together, FlyExpress 7 represents our largest effort yet to accelerate discovery via the development and dispersal of new webtools that allow researchers to perform data-driven analyses of coexpression (image) and genomic (sequence) data.
ContributorsKumar, Sudhir (Author) / Konikoff, Charlotte (Author) / Sanderford, Maxwell (Author) / Liu, Li (Author) / Newfeld, Stuart (Author) / Ye, Jieping (Author) / Kulathinal, Rob J. (Author) / College of Health Solutions (Contributor) / Department of Biomedical Informatics (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor)
Created2017-06-30