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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
Alzheimer's disease (AD) is the leading neurodegenerative disease, affecting roughly 8% of people 65 years of age or older. There exists an imperative need to develop a non-invasive test for the earlier detection of AD. The use of biomarkers is a promising option that examines the toxic mechanisms and metabolic pathways that cause Alzheimer's disease, eventually leading to an early diagnostic method. This thesis presents the use of oligomeric beta-amyloid as a biomarker to detect Alzheimer's disease via a specialized enzyme-linked protein assay. Specifically, this paper details the optimization and development of a novel phage capture enzyme-linked immunosorbent assay (ELISA) that can detect the relative quantity of beta-amyloid oligomers in samples from a mouse model of AD. The objective of this thesis was to optimize a phage capture ELISA using the A4 single-chain variable fragment (scFv) to quantify the amount of beta-amyloid oligomers in various mice samples. A4 selectively recognizes a toxic oligomeric form of beta-amyloid. The level of A4-reactive oligomeric beta-amyloid was measured in triplicate in homogenized mouse brain tissue samples from eight transgenic (TG) and eight nontransgenic (NTG) animals aged five, nine, and thirteen months. There was a significant difference (p < 0.0005) between the five month TG and NTG mice. A decrease in beta-amyloid levels with the aging of the TG mice suggested that the beta-amyloid oligomers may be aggregating to form beta-amyloid fibrils. Conversely, the quantity of beta-amyloid increased with the aging of the NTG mice. This indicated that beta-amyloid oligomers may develop with normal aging.
ContributorsBrownlee, Taylor (Author) / Sierks, Michael (Thesis advisor) / Williams, Stephanie (Committee member) / Xin, Wei (Committee member) / Arizona State University (Publisher)
Created2013
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 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
Description
The use of electromyography (EMG) signals to characterize muscle fatigue has been widely accepted. Initial work on characterizing muscle fatigue during isometric contractions demonstrated that its frequency decreases while its amplitude increases with the onset of fatigue. More recent work concentrated on developing techniques to characterize dynamic contractions for use in clinical and training applications. Studies demonstrated that as fatigue progresses, the EMG signal undergoes a shift in frequency, and different physiological mechanisms on the possible cause of the shift were considered. Time-frequency processing, using the Wigner distribution or spectrogram, is one of the techniques used to estimate the instantaneous mean frequency and instantaneous median frequency of the EMG signal using a variety of techniques. However, these time-frequency methods suffer either from cross-term interference when processing signals with multiple components or time-frequency resolution due to the use of windowing. This study proposes the use of the matching pursuit decomposition (MPD) with a Gaussian dictionary to process EMG signals produced during both isometric and dynamic contractions. In particular, the MPD obtains unique time-frequency features that represent the EMG signal time-frequency dependence without suffering from cross-terms or loss in time-frequency resolution. As the MPD does not depend on an analysis window like the spectrogram, it is more robust in applying the timefrequency features to identify the spectral time-variation of the EGM signal.
ContributorsAustin, Hiroko (Author) / Papandreou-Suppappola, Antonia (Thesis advisor) / Kovvali, Narayan (Committee member) / Muthuswamy, Jitendran (Committee member) / Arizona State University (Publisher)
Created2012
Description
Sensitivity is a fundamental challenge for in vivo molecular magnetic resonance imaging (MRI). Here, I improve the sensitivity of metal nanoparticle contrast agents by strategically incorporating pure and doped metal oxides in the nanoparticle core, forming a soluble, monodisperse, contrast agent with adjustable T2 or T1 relaxivity (r2 or r1). I first developed a simplified technique to incorporate iron oxides in apoferritin to form "magnetoferritin" for nM-level detection with T2- and T2* weighting. I then explored whether the crystal could be chemically modified to form a particle with high r1. I first adsorbed Mn2+ ions to metal binding sites in the apoferritin pores. The strategic placement of metal ions near sites of water exchange and within the crystal oxide enhance r1, suggesting a mechanism for increasing relaxivity in porous nanoparticle agents. However, the Mn2+ addition was only possible when the particle was simultaneously filled with an iron oxide, resulting in a particle with a high r1 but also a high r2 and making them undetectable with conventional T1-weighting techniques. To solve this problem and decrease the particle r2 for more sensitive detection, I chemically doped the nanoparticles with tungsten to form a disordered W-Fe oxide composite in the apoferritin core. This configuration formed a particle with a r1 of 4,870mM-1s-1 and r2 of 9,076mM-1s-1. These relaxivities allowed the detection of concentrations ranging from 20nM - 400nM in vivo, both passively injected and targeted to the kidney glomerulus. I further developed an MRI acquisition technique to distinguish particles based on r2/r1, and show that three nanoparticles of similar size can be distinguished in vitro and in vivo with MRI. This work forms the basis for a new, highly flexible inorganic approach to design nanoparticle contrast agents for molecular MRI.
ContributorsClavijo Jordan, Maria Veronica (Author) / Bennett, Kevin M (Thesis advisor) / Kodibagkar, Vikram (Committee member) / Sherry, A Dean (Committee member) / Wang, Xiao (Committee member) / Yarger, Jeffery (Committee member) / Arizona State University (Publisher)
Created2012
Description
Infertility has become an increasing problem in developed countries and in many cases can be attributed to compromised sperm quality. Assessment of male fertility typically utilizes semen analysis which mainly examines sperm morphology, however many males whose sperm appear normal are sub- or infertile, suggesting that sperm from these males may be deficient in a protein or suite of proteins. To date, very little is known about the composition of sperm or the complex maturation process that confers motility and fertilization competency to sperm. Chapter 1 discusses the use of whole cell mass spectrometry to identify 1247 proteins comprising the Rhesus macaque (Macaca mulatta) sperm proteome, a commonly used model of human reproduction. This study provides a more robust proxy of human sperm composition than was previously available and facilitates studies of sperm using the rhesus macaque as a model. Chapters 2 & 3 provide a systems level overview of changes in sperm proteome composition that occurs during epididymal transit. Chapter 2 reports the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. Chapter 3 reports the sperm proteome from four distinct segments of the Rhesus macaque epididymis, including the caput, proximal corpus, distal corpus and cauda, identifying 1951, 2014, 1764 and 1423 proteins respectively. These studies identify a number of proteins that are added and removed from sperm during epididymal transit which likely play an important role in the sperm maturation process. To date no comparative evolutionary studies of sperm proteomes have been undertaken. Chapter 4 compares four mammalian sperm proteomes including the human, macaque, mouse and rat. This study identified 98 proteins common to all four sperm proteomes, 82 primate and 90 rodent lineage-specific proteins and 494, 467, 566, and 193 species specific proteins in the human, macaque, mouse and rat sperm proteomes respectively and discusses how differences in sperm composition may ultimately lead to functional differences across species. Finally, chapter 5 uses sperm proteome data to inform the preliminary design of a rodent contraceptive vaccine delivered orally using recombinant attenuated Salmonella vaccine vectors.
ContributorsSkerget, Sheri Jo (Author) / Karr, Timothy L. (Thesis advisor) / Lake, Douglas (Committee member) / Petritis, Konstantinos (Committee member) / Arizona State University (Publisher)
Created2013
Description
Advances in implantable MEMS technology has made possible adaptive micro-robotic implants that can track and record from single neurons in the brain. Development of autonomous neural interfaces opens up exciting possibilities of micro-robots performing standard electrophysiological techniques that would previously take researchers several hundred hours to train and achieve the desired skill level. It would result in more reliable and adaptive neural interfaces that could record optimal neural activity 24/7 with high fidelity signals, high yield and increased throughput. The main contribution here is validating adaptive strategies to overcome challenges in autonomous navigation of microelectrodes inside the brain. The following issues pose significant challenges as brain tissue is both functionally and structurally dynamic: a) time varying mechanical properties of the brain tissue-microelectrode interface due to the hyperelastic, viscoelastic nature of brain tissue b) non-stationarities in the neural signal caused by mechanical and physiological events in the interface and c) the lack of visual feedback of microelectrode position in brain tissue. A closed loop control algorithm is proposed here for autonomous navigation of microelectrodes in brain tissue while optimizing the signal-to-noise ratio of multi-unit neural recordings. The algorithm incorporates a quantitative understanding of constitutive mechanical properties of soft viscoelastic tissue like the brain and is guided by models that predict stresses developed in brain tissue during movement of the microelectrode. An optimal movement strategy is developed that achieves precise positioning of microelectrodes in the brain by minimizing the stresses developed in the surrounding tissue during navigation and maximizing the speed of movement. Results of testing the closed-loop control paradigm in short-term rodent experiments validated that it was possible to achieve a consistently high quality SNR throughout the duration of the experiment. At the systems level, new generation of MEMS actuators for movable microelectrode array are characterized and the MEMS device operation parameters are optimized for improved performance and reliability. Further, recommendations for packaging to minimize the form factor of the implant; design of device mounting and implantation techniques of MEMS microelectrode array to enhance the longevity of the implant are also included in a top-down approach to achieve a reliable brain interface.
ContributorsAnand, Sindhu (Author) / Muthuswamy, Jitendran (Thesis advisor) / Tillery, Stephen H (Committee member) / Buneo, Christopher (Committee member) / Abbas, James (Committee member) / Tsakalis, Konstantinos (Committee member) / Arizona State University (Publisher)
Created2013
Description
Mathematical modeling of infectious diseases can help public health officials to make decisions related to the mitigation of epidemic outbreaks. However, over or under estimations of the morbidity of any infectious disease can be problematic. Therefore, public health officials can always make use of better models to study the potential implication of their decisions and strategies prior to their implementation. Previous work focuses on the mechanisms underlying the different epidemic waves observed in Mexico during the novel swine origin influenza H1N1 pandemic of 2009 and showed extensions of classical models in epidemiology by adding temporal variations in different parameters that are likely to change during the time course of an epidemic, such as, the influence of media, social distancing, school closures, and how vaccination policies may affect different aspects of the dynamics of an epidemic. This current work further examines the influence of different factors considering the randomness of events by adding stochastic processes to meta-population models. I present three different approaches to compare different stochastic methods by considering discrete and continuous time. For the continuous time stochastic modeling approach I consider the continuous-time Markov chain process using forward Kolmogorov equations, for the discrete time stochastic modeling I consider stochastic differential equations using Wiener's increment and Poisson point increments, and also I consider the discrete-time Markov chain process. These first two stochastic modeling approaches will be presented in a one city and two city epidemic models using, as a base, our deterministic model. The last one will be discussed briefly on a one city SIS and SIR-type model.
ContributorsCruz-Aponte, Maytee (Author) / Wirkus, Stephen A. (Thesis advisor) / Castillo-Chavez, Carlos (Thesis advisor) / Camacho, Erika T. (Committee member) / Kang, Yun (Committee member) / Arizona State University (Publisher)
Created2014
Description
The explicit role of soil organisms in shaping soil health, rates of pedogenesis, and resistance to erosion has only just recently begun to be explored in the last century. However, much of the research regarding soil biota and soil processes is centered on maintaining soil fertility (e.g., plant nutrient availability) and soil structure in mesic- and agro- ecosystems. Despite the empirical and theoretical strides made in soil ecology over the last few decades, questions regarding ecosystem function and soil processes remain, especially for arid areas. Arid areas have unique ecosystem biogeochemistry, decomposition processes, and soil microbial responses to moisture inputs that deviate from predictions derived using data generated in more mesic systems. For example, current paradigm predicts that soil microbes will respond positively to increasing moisture inputs in a water-limited environment, yet data collected in arid regions are not congruent with this hypothesis. The influence of abiotic factors on litter decomposition rates (e.g., photodegradation), litter quality and availability, soil moisture pulse size, and resulting feedbacks on detrital food web structure must be explicitly considered for advancing our understanding of arid land ecology. However, empirical data coupling arid belowground food webs and ecosystem processes are lacking. My dissertation explores the resource controls (soil organic matter and soil moisture) on food web network structure, size, and presence/absence of expected belowground trophic groups across a variety of sites in Arizona.
ContributorsWyant, Karl Arthur (Author) / Sabo, John L (Thesis advisor) / Elser, James J (Committee member) / Childers, Daniel L. (Committee member) / Hall, Sharon J (Committee member) / Stromberg, Juliet C. (Committee member) / Arizona State University (Publisher)
Created2014
Description
The basal ganglia are four sub-cortical nuclei associated with motor control and reward learning. They are part of numerous larger mostly segregated loops where the basal ganglia receive inputs from specific regions of cortex. Converging on these inputs are dopaminergic neurons that alter their firing based on received and/or predicted rewarding outcomes of a behavior. The basal ganglia's output feeds through the thalamus back to the areas of the cortex where the loop originated. Understanding the dynamic interactions between the various parts of these loops is critical to understanding the basal ganglia's role in motor control and reward based learning. This work developed several experimental techniques that can be applied to further study basal ganglia function. The first technique used micro-volume injections of low concentration muscimol to decrease the firing rates of recorded neurons in a limited area of cortex in rats. Afterwards, an artificial cerebrospinal fluid flush was injected to rapidly eliminate the muscimol's effects. This technique was able to contain the effects of muscimol to approximately a 1 mm radius volume and limited the duration of the drug effect to less than one hour. This technique could be used to temporarily perturb a small portion of the loops involving the basal ganglia and then observe how these effects propagate in other connected regions. The second part applied self-organizing maps (SOM) to find temporal patterns in neural firing rate that are independent of behavior. The distribution of detected patterns frequency on these maps can then be used to determine if changes in neural activity are occurring over time. The final technique focused on the role of the basal ganglia in reward learning. A new conditioning technique was created to increase the occurrence of selected patterns of neural activity without utilizing any external reward or behavior. A pattern of neural activity in the cortex of rats was selected using an SOM. The pattern was then reinforced by being paired with electrical stimulation of the medial forebrain bundle triggering dopamine release in the basal ganglia. Ultimately, this technique proved unsuccessful possibly due to poor selection of the patterns being reinforced.
ContributorsBaldwin, Nathan Aaron (Author) / Helms Tillery, Stephen I (Thesis advisor) / Castaneda, Edward (Committee member) / Buneo, Christopher A (Committee member) / Muthuswamy, Jitendran (Committee member) / Si, Jennie (Committee member) / Arizona State University (Publisher)
Created2014