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- Genre: Academic theses
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
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