Matching Items (25)
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Description
Gene manipulation techniques, such as RNA interference (RNAi), offer a powerful method for elucidating gene function and discovery of novel therapeutic targets in a high-throughput fashion. In addition, RNAi is rapidly being adopted for treatment of neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease, etc. However, a major challenge

Gene manipulation techniques, such as RNA interference (RNAi), offer a powerful method for elucidating gene function and discovery of novel therapeutic targets in a high-throughput fashion. In addition, RNAi is rapidly being adopted for treatment of neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease, etc. However, a major challenge in both of the aforementioned applications is the efficient delivery of siRNA molecules, plasmids or transcription factors to primary cells such as neurons. A majority of the current non-viral techniques, including chemical transfection, bulk electroporation and sonoporation fail to deliver with adequate efficiencies and the required spatial and temporal control. In this study, a novel optically transparent biochip is presented that can (a) transfect populations of primary and secondary cells in 2D culture (b) readily scale to realize high-throughput transfections using microscale electroporation and (c) transfect targeted cells in culture with spatial and temporal control. In this study, delivery of genetic payloads of different sizes and molecular characteristics, such as GFP plasmids and siRNA molecules, to precisely targeted locations in primary hippocampal and HeLa cell cultures is demonstrated. In addition to spatio-temporally controlled transfection, the biochip also allowed simultaneous assessment of a) electrical activity of neurons, b) specific proteins using fluorescent immunohistochemistry, and c) sub-cellular structures. Functional silencing of GAPDH in HeLa cells using siRNA demonstrated a 52% reduction in the GAPDH levels. In situ assessment of actin filaments post electroporation indicated a sustained disruption in actin filaments in electroporated cells for up to two hours. Assessment of neural spike activity pre- and post-electroporation indicated a varying response to electroporation. The microarray based nature of the biochip enables multiple independent experiments on the same culture, thereby decreasing culture-to-culture variability, increasing experimental throughput and allowing cell-cell interaction studies. Further development of this technology will provide a cost-effective platform for performing high-throughput genetic screens.
ContributorsPatel, Chetan (Author) / Muthuswamy, Jitendran (Thesis advisor) / Helms Tillery, Stephen (Committee member) / Jain, Tilak (Committee member) / Caplan, Michael (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2012
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Description
Dendrites are the structures of a neuron specialized to receive input signals and to provide the substrate for the formation of synaptic contacts with other cells. The goal of this work is to study the activity-dependent mechanisms underlying dendritic growth in a single-cell model. For this, the individually identifiable adult

Dendrites are the structures of a neuron specialized to receive input signals and to provide the substrate for the formation of synaptic contacts with other cells. The goal of this work is to study the activity-dependent mechanisms underlying dendritic growth in a single-cell model. For this, the individually identifiable adult motoneuron, MN5, in Drosophila melanogaster was used. This dissertation presents the following results. First, the natural variability of morphological parameters of the MN5 dendritic tree in control flies is not larger than 15%, making MN5 a suitable model for quantitative morphological analysis. Second, three-dimensional topological analyses reveals that different parts of the MN5 dendritic tree innervate spatially separated areas (termed "isoneuronal tiling"). Third, genetic manipulation of the MN5 excitability reveals that both increased and decreased activity lead to dendritic overgrowth; whereas decreased excitability promoted branch elongation, increased excitability enhanced dendritic branching. Next, testing the activity-regulated transcription factor AP-1 for its role in MN5 dendritic development reveals that neural activity enhanced AP-1 transcriptional activity, and that AP-1 expression lead to opposite dendrite fates depending on its expression timing during development. Whereas overexpression of AP-1 at early stages results in loss of dendrites, AP-1 overexpression after the expression of acetylcholine receptors and the formation of all primary dendrites in MN5 causes overgrowth. Fourth, MN5 has been used to examine dendritic development resulting from the expression of the human gene MeCP2, a transcriptional regulator involved in the neurodevelopmental disease Rett syndrome. Targeted expression of full-length human MeCP2 in MN5 causes impaired dendritic growth, showing for the first time the cellular consequences of MeCP2 expression in Drosophila neurons. This dendritic phenotype requires the methyl-binding domain of MeCP2 and the chromatin remodeling protein Osa. In summary, this work has fully established MN5 as a single-neuron model to study mechanisms underlying dendrite development, maintenance and degeneration, and to test the behavioral consequences resulting from dendritic growth misregulation. Furthermore, this thesis provides quantitative description of isoneuronal tiling of a central neuron, offers novel insight into activity- and AP-1 dependent developmental plasticity, and finally, it establishes Drosophila MN5 as a model to study some specific aspects of human diseases.
ContributorsVonhoff, Fernando Jaime (Author) / Duch, Carsten J (Thesis advisor) / Smith, Brian H. (Committee member) / Vu, Eric (Committee member) / Crook, Sharon (Committee member) / Arizona State University (Publisher)
Created2012
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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

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
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Description
Abstract: The RAS/RAF/MEK/ERK (RAS signaling cascade) pathway is a highly conserved biochemical signaling cascade that exists in every mammalian cell. The pathway is highly versatile in functionality due to hundreds of substrates that regulate metabolism, apoptosis, and proliferation in both adult and developing tissues. The RAS signaling cascade has been

Abstract: The RAS/RAF/MEK/ERK (RAS signaling cascade) pathway is a highly conserved biochemical signaling cascade that exists in every mammalian cell. The pathway is highly versatile in functionality due to hundreds of substrates that regulate metabolism, apoptosis, and proliferation in both adult and developing tissues. The RAS signaling cascade has been examined in the context of cancers since mutations can lead to the disruption of the cell cycle and unregulated cellular proliferation. In addition, germline mutations in the pathway have been shown to cause a group of syndromes known as RASopathies. RASopathies are marked by facial defects, seizures, developmental delays, and cognitive dysfunction often due to enhanced activation of the RAS signaling cascade. Although there are noted factors that play roles in neurological disease, such as a hyperactivated RAS signaling cascade, the pathogenesis of neurological defects is not fully understood. The Newbern lab uses conditional mutagenesis to examine how hyperactivating the RAS/MAPK pathway affects GABAergic neurons in a cortical microcircuit, especially during development. Inhibitory neurons are implicated in seizures and epilepsy is common in RASopathies, thus GABAergic neurons are of particular interest (Rauen, 2013). Gain-of-function ERK was not found to significantly alter global locomotion or anxiety-like behaviors. Interestingly, the mutant mice exhibited freezing behavior in the first twenty-two seconds of the open field assay that appeared to be consistent with absence seizures. Direct EEG recordings confirmed spontaneous seizure activity and mutants had a reduced seizure threshold. We hypothesized that these deficits were due to altered GABAergic neuron number. Indeed, mutant mice exhibited a 30% reduction in total cortical GABAergic neuron number. This effect appeared to be cell subtype specific, where neurons expressing somatostatin (SST) existed in similar numbers among controls and mutants but a significant decrease in the number of those expressing parvalbumin (PV) was observed. I hypothesized that a recently identified GABAergic neuron expressing vasoactive intestinal polypeptide (VIP) would also be affected in such a manner that fewer VIP neurons exist in the mutants than the wildtype. Subsequent histological studies in these mice found there to be no significant difference in VIP populations. Selective affects seem to only have an effect on the development of PV neurons in the cortex. Further studies are underway to define the mechanism responsible for aberrant GABAergic neuron development.
ContributorsGonzalez, Javier (Author) / Newbern, Jason (Thesis director) / Neisewander, Janet (Committee member) / Barrett, The Honors College (Contributor)
Created2016-05
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Unmanned aerial vehicles have received increased attention in the last decade due to their versatility, as well as the availability of inexpensive sensors (e.g. GPS, IMU) for their navigation and control. Multirotor vehicles, specifically quadrotors, have formed a fast growing field in robotics, with the range of applications spanning from

Unmanned aerial vehicles have received increased attention in the last decade due to their versatility, as well as the availability of inexpensive sensors (e.g. GPS, IMU) for their navigation and control. Multirotor vehicles, specifically quadrotors, have formed a fast growing field in robotics, with the range of applications spanning from surveil- lance and reconnaissance to agriculture and large area mapping. Although in most applications single quadrotors are used, there is an increasing interest in architectures controlling multiple quadrotors executing a collaborative task. This thesis introduces a new concept of control involving more than one quadrotors, according to which two quadrotors can be physically coupled in mid-flight. This concept equips the quadro- tors with new capabilities, e.g. increased payload or pursuit and capturing of other quadrotors. A comprehensive simulation of the approach is built to simulate coupled quadrotors. The dynamics and modeling of the coupled system is presented together with a discussion regarding the coupling mechanism, impact modeling and additional considerations that have been investigated. Simulation results are presented for cases of static coupling as well as enemy quadrotor pursuit and capture, together with an analysis of control methodology and gain tuning. Practical implementations are introduced as results show the feasibility of this design.
ContributorsLarsson, Daniel (Author) / Artemiadis, Panagiotis (Thesis advisor) / Marvi, Hamidreza (Committee member) / Berman, Spring (Committee member) / Arizona State University (Publisher)
Created2016
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Description
Recent new experiments showed that wide-field imaging at millimeter scale is capable of recording hundreds of neurons in behaving mice brain. Monitoring hundreds of individual neurons at a high frame rate provides a promising tool for discovering spatiotemporal features of large neural networks. However, processing the massive data sets is

Recent new experiments showed that wide-field imaging at millimeter scale is capable of recording hundreds of neurons in behaving mice brain. Monitoring hundreds of individual neurons at a high frame rate provides a promising tool for discovering spatiotemporal features of large neural networks. However, processing the massive data sets is impossible without automated procedures. Thus, this thesis aims at developing a new tool to automatically segment and track individual neuron cells. The new method used in this study employs two major ideas including feature extraction based on power spectral density of single neuron temporal activity and clustering tree to separate overlapping cells. To address issues associated with high-resolution imaging of a large recording area, focused areas and out-of-focus areas were analyzed separately. A static segmentation with a fixed PSD thresholding method is applied to within focus visual field. A dynamic segmentation by comparing maximum PSD with surrounding pixels is applied to out-of-focus area. Both approaches helped remove irrelevant pixels in the background. After detection of potential single cells, some of which appeared in groups due to overlapping cells in the image, a hierarchical clustering algorithm is applied to separate them. The hierarchical clustering uses correlation coefficient as a distance measurement to group similar pixels into single cells. As such, overlapping cells can be separated. We tested the entire algorithm using two real recordings with the respective truth carefully determined by manual inspections. The results show high accuracy on tested datasets while false positive error is controlled within an acceptable range. Furthermore, results indicate robustness of the algorithm when applied to different image sequences.
ContributorsWu, Ruofan (Author) / Si, Jennie (Thesis advisor) / Sadleir, Rosalind (Committee member) / Crook, Sharon (Committee member) / Arizona State University (Publisher)
Created2016
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Samuel Randall Detwiler was an embryologist who studied neural development in embryos and vertebrate retinas. He discovered evidence for the relationship between somites and spinal ganglia, that transplanted limbs can be controlled by foreign ganglia, and the plasticity of ganglia in response to limb transplantations. He also extensively

Samuel Randall Detwiler was an embryologist who studied neural development in embryos and vertebrate retinas. He discovered evidence for the relationship between somites and spinal ganglia, that transplanted limbs can be controlled by foreign ganglia, and the plasticity of ganglia in response to limb transplantations. He also extensively studied vertebrate retinas during and after embryonic development. Detwiler's work established many principles studied in later limb transplantation experiments and was identified by Viktor Hamburger as an important bridge between his and Ross Granville Harrison's research.

Created2007-11-01
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In 2011, Sonja Vernes and Simon Fisher performed a series of experiments to determine which developmental processes are controlled by the mouse protein Foxp2. Previous research showed that altering the Foxp2 protein changed how neurons grew, so Vernes and Fisher hypothesized that Foxp2 would affect gene networks that involved in

In 2011, Sonja Vernes and Simon Fisher performed a series of experiments to determine which developmental processes are controlled by the mouse protein Foxp2. Previous research showed that altering the Foxp2 protein changed how neurons grew, so Vernes and Fisher hypothesized that Foxp2 would affect gene networks that involved in the development of neurons, or nerve cells. Their results confirmed that Foxp2 affected the development of gene networks involved in the growth of neurons, as well as networks that are involved in cell specialization and cell communication. The researchers determined that Foxp2 is important for a variety of developmental processes such as motor control, language acquisition, and cognition.

Created2017-05-30
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Camillo Golgi studied the central nervous system during the late nineteenth and early twentieth centuries in Italy, and he developed a staining technique to visualize brain cells. Called the black reaction, Golgi’s staining technique enabled him to see the cellular structure of brain cells, called neurons, with much greater precision.

Camillo Golgi studied the central nervous system during the late nineteenth and early twentieth centuries in Italy, and he developed a staining technique to visualize brain cells. Called the black reaction, Golgi’s staining technique enabled him to see the cellular structure of brain cells, called neurons, with much greater precision. Golgi also used the black reaction to identify structures within animal cells like the internal reticular apparatus that stores, packs, and modifies proteins, later named the Golgi apparatus in his honor. Golgi, along with Santiago Ramón y Cajal, received the Nobel Peace Prize in 1906 for their independent work on the structure of the nervous system. Golgi’s discovery of the black reaction enabled other scientists to better study the structure of the nervous system and its development.

Created2017-02-23
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Scientists use cerebral organoids, which are artificially produced miniature organs that represent embryonic or fetal brains and have many properties similar to them, to help them study developmental disorders like microcephaly. In human embryos, cerebral tissue in the form of neuroectoderm appears within the first nine weeks of human development,

Scientists use cerebral organoids, which are artificially produced miniature organs that represent embryonic or fetal brains and have many properties similar to them, to help them study developmental disorders like microcephaly. In human embryos, cerebral tissue in the form of neuroectoderm appears within the first nine weeks of human development, and it gives rise to the brain and spinal cord. In the twenty-first century, Juergen Knoblich and Madeleine Lancaster at the Institute of Molecular Biotechnology in Vienna, Austria, grew cerebral organoids from pluripotent stem cells as a model to study developmental disorders in embryonic and fetal brains. One such disorder is microcephaly, a condition in which brain size and the number of neurons in the brain are abnormally small. Scientists use cerebral organoids, which they've grown in labs, because they provide a manipulable model for studying how neural cells migrate during development, the timing of neural development, and how genetic errors can result in developmental disorders.

Created2017-05-12