Matching Items (29)
Description
Alzheimer's Disease (AD) is a progressive neurodegenerative disease accounting for 50-80% of dementia cases in the country. This disease is characterized by the deposition of extracellular plaques occurring in regions of the brain important for cognitive function. A primary component of these plaques is the amyloid-beta protein. While a natively unfolded protein, amyloid-beta can misfold and aggregate generating a variety of different species including numerous different soluble oligomeric species some of which are precursors to the neurofibrillary plaques. Various of the soluble amyloid-beta oligomeric species have been shown to be toxic to cells and their presence may correlate with progression of AD. Current treatment options target the dementia symptoms, but there is no effective cure or alternative to delay the progression of the disease once it occurs. Amyloid-beta aggregates show up many years before symptoms develop, so detection of various amyloid-beta aggregate species has great promise as an early biomarker for AD. Therefore reagents that can selectively identify key early oligomeric amyloid-beta species have value both as potential diagnostics for early detection of AD and as well as therapeutics that selectively target only the toxic amyloid-beta aggregate species. Earlier work in the lab includes development of several different single chain antibody fragments (scFvs) against different oligomeric amyloid-beta species. This includes isolation of C6 scFv against human AD brain derived oligomeric amyloid-beta (Kasturirangan et al., 2013). This thesis furthers research in this direction by improving the yields and investigating the specificity of modified C6 scFv as a diagnostic for AD. It is motivated by experiments reporting low yields of the C6 scFv. We also used the C6T scFv to characterize the variation in concentration of this particular oligomeric amyloid-beta species with age in a triple transgenic AD mouse model. We also show that C6T can be used to differentiate between post-mortem human AD, Parkinson's disease (PD) and healthy human brain samples. These results indicate that C6T has potential value as a diagnostic tool for early detection of AD.
ContributorsVenkataraman, Lalitha (Author) / Sierks, Michael (Thesis advisor) / Rege, Kaushal (Committee member) / Pauken, Christine (Committee member) / Arizona State University (Publisher)
Created2013
Description
Specificity and affinity towards a given ligand/epitope limit target-specific delivery. Companies can spend between $500 million to $2 billion attempting to discover a new drug or therapy; a significant portion of this expense funds high-throughput screening to find the most successful target-specific compound available. A more recent addition to discovering highly specific targets is the application of phage display utilizing single chain variable fragment antibodies (scFv). The aim of this research was to employ phage display to identify pathologies related to traumatic brain injury (TBI), particularly astrogliosis. A unique biopanning method against viable astrocyte cultures activated with TGF-β achieved this aim. Four scFv clones of interest showed varying relative affinities toward astrocytes. One of those four showed the ability to identify reactive astroctyes over basal astrocytes through max signal readings, while another showed a statistical significance in max signal reading toward basal astrocytes. Future studies will include further affinity characterization assays. This work contributes to the development of targeting therapeutics and diagnostics for TBI.
ContributorsMarsh, William (Author) / Stabenfeldt, Sarah (Thesis advisor) / Caplan, Michael (Committee member) / Sierks, Michael (Committee member) / Arizona State University (Publisher)
Created2013
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
Alzheimer's disease (AD) is the most common type of dementia, affecting one in nine people age 65 and older. One of the most important neuropathological characteristics of Alzheimer's disease is the aggregation and deposition of the protein beta-amyloid. Beta-amyloid is produced by proteolytic processing of the Amyloid Precursor Protein (APP). Production of beta-amyloid from APP is increased when cells are subject to stress since both APP and beta-secretase are upregulated by stress. An increased beta-amyloid level promotes aggregation of beta-amyloid into toxic species which cause an increase in reactive oxygen species (ROS) and a decrease in cell viability. Therefore reducing beta-amyloid generation is a promising method to control cell damage following stress. The goal of this thesis was to test the effect of inhibiting beta-amyloid production inside stressed AD cell model. Hydrogen peroxide was used as stressing agent. Two treatments were used to inhibit beta-amyloid production, including iBSec1, an scFv designed to block beta-secretase site of APP, and DIA10D, a bispecific tandem scFv engineered to cleave alpha-secretase site of APP and block beta-secretase site of APP. iBSec1 treatment was added extracellularly while DIA10D was stably expressed inside cell using PSECTAG vector. Increase in reactive oxygen species and decrease in cell viability were observed after addition of hydrogen peroxide to AD cell model. The increase in stress induced toxicity caused by addition of hydrogen peroxide was dramatically decreased by simultaneously treating the cells with iBSec1 or DIA10D to block the increase in beta-amyloid levels resulting from the upregulation of APP and beta-secretase.
ContributorsSuryadi, Vicky (Author) / Sierks, Michael (Thesis advisor) / Nielsen, David (Committee member) / Dai, Lenore (Committee member) / Arizona State University (Publisher)
Created2014
Description
Spatiotemporal processing in the mammalian olfactory bulb (OB), and its analog, the invertebrate antennal lobe (AL), is subject to plasticity driven by biogenic amines. I study plasticity using honey bees, which have been extensively studied with respect to nonassociative and associative based olfactory learning and memory. Octopamine (OA) release in the AL is the functional analog to epinephrine in the OB. Blockade of OA receptors in the AL blocks plasticity induced changes in behavior. I have now begun to test specific hypotheses related to how this biogenic amine might be involved in plasticity in neural circuits within the AL. OA acts via different receptor subtypes, AmOA1, which gates calcium release from intracellular stores, and AmOA-beta, which results in an increase of cAMP. Calcium also enters AL interneurons via nicotinic acetylcholine receptors, which are driven by acetylcholine release from sensory neuron terminals, as well as through voltage-gated calcium channels. I employ 2-photon excitation (2PE) microscopy using fluorescent calcium indicators to investigate potential sources of plasticity as revealed by calcium fluctuations in AL projection neuron (PN) dendrites in vivo. PNs are analogous to mitral cells in the OB and have dendritic processes that show calcium increases in response to odor stimulation. These calcium signals frequently change after association of odor with appetitive reinforcement. However, it is unclear whether the reported plasticity in calcium signals are due to changes intrinsic to the PNs or to changes in other neural components of the network. My studies were aimed toward understanding the role of OA for establishing associative plasticity in the AL network. Accordingly, I developed a treatment that isolates intact, functioning PNs in vivo. A second study revealed that cAMP is a likely component of plasticity in the AL, thus implicating the AmOA-beta; receptors. Finally, I developed a method for loading calcium indicators into neural components of the AL that have yet to be studied in detail. These manipulations are now revealing the molecular mechanisms contributing to associative plasticity in the AL. These studies will allow for a greater understanding of plasticity in several neural components of the honey bee AL and mammalian OB.
ContributorsProtas, Danielle (Author) / Smith, Brian H. (Thesis advisor) / Neisewander, Janet (Committee member) / Anderson, Trent (Committee member) / Tyler, William (Committee member) / Vu, Eric (Committee member) / Arizona State University (Publisher)
Created2014
Description
Tobacco and alcohol are the most commonly abused drugs worldwide. Many people smoke and drink together, but the mechanisms of this nicotine (NIC) -ethanol (EtOH) dependence are not fully known. EtOH has been shown to affect some nicotinic acetylcholine receptors (nAChRs), which potentially underlies NIC-EtOH codependence. Ventral Tegmental Area (VTA) dopamine (DA) and γ-aminobutyric acid (GABA) neurons express different nAChR subtypes, whose net activation results in enhancement of DA release in the Prefrontal Cortex (PFC) and Nucleus Accumbens (NAc). Enhancement of DA transmission in this mesocorticolimbic system is thought to lead to rewarding properties of EtOH and NIC, clarification of which is relevant to public health and clinical diseases. The aim of this study was to elucidate pharmacological mechanisms of action employed by both NIC and EtOH through nAChRs in VTA neurons by evaluating behavioral, network, synaptic and receptor functions therein. It was hypothesized that VTA GABA neurons are controlled by α7 nAChRs on presynaptic GLUergic terminals and α6 nAChRs on presynaptic GABAergic terminals. NIC and EtOH, via these nAChRs, modulate VTA GABA neuronal function. This modulation may underlie NIC and EtOH reward and reinforcement, while pharmacological manipulation of these nAChRs may be a therapeutic strategy to treat NIC or EtOH dependence. This data demonstrates that in VTA GABA neurons, α7 nAChRs on GLUergic terminals play a key role in the mediation of local NIC-induced firing increase. α6*-nAChRs on GABA terminals enhances presynaptic GABA release, and leads to greater inhibition to VTA GABA neurons, which results in an increase VTA DA neuron firing via a disinhibition mechanism. Genetic knockout of these nAChRs significantly prevents EtOH-induced animal conditioned place preference (CPP). Furthermore, levo-tetrahydropalmadine (l-THP), a compound purified from natural Chinese herbs, blocks nAChRs, prevents NIC-induced DA neuronal firing, and eliminates NIC CPP, suggesting it as a promising candidate in a new generation of interventions for smoking cessation. Improved understanding of underlying mechanisms and development of new drugs will increase the number of successful quitters each year and dramatically improve the quality of life for millions suffering from addiction, as well as those around them.
ContributorsTaylor, Devin (Author) / Wu, Jie (Committee member) / Olive, M F (Committee member) / Whiteaker, Paul (Committee member) / Vu, Eric (Committee member) / Hammer, Ronald (Committee member) / Arizona State University (Publisher)
Created2015
Description
Patients with Alzheimer's disease (AD) exhibit a significantly higher incidence of unprovoked seizures compared to age-matched non-AD controls, and animal models of AD (i.e., transgenic human amyloid precursor protein, hAPP mice) display neural hyper-excitation and epileptic seizures. Hyperexcitation is particularly important because it contributes to the high incidence of epilepsy in AD patients as well as AD-related synaptic deficits and neurodegeneration. Given that there is significant amyloid-β (Aβ) accumulation and deposition in AD brain, Aβ exposure ultimately may be responsible for neural hyper-excitation in both AD patients and animal models. Emerging evidence indicates that α7 nicotinic acetylcholine receptors (α7-nAChR) are involved in AD pathology, because synaptic impairment and learning and memory deficits in a hAPPα7-/- mouse model are decreased by nAChR α7 subunit gene deletion. Given that Aβ potently modulates α7-nAChR function, that α7-nAChR expression is significantly enhanced in both AD patients and animal models, and that α7-nAChR play an important role in regulating neuronal excitability, it is reasonable that α7-nAChRs may contribute to Aβ-induced neural hyperexcitation. We hypothesize that increased α7-nAChR expression and function as a consequence of Aβ exposure is important in Aβ-induced neural hyperexcitation. In this project, we found that exposure of Aβ aggregates at a nanomolar range induces neuronal hyperexcitation and toxicity via an upregulation of α7-nAChR in cultured hippocampus pyramidal neurons. Aβ up-regulates α7-nAChRs function and expression through a post translational mechanism. α7-nAChR up-regulation occurs prior to Aβ-induced neuronal hyperexcitation and toxicity. Moreover, inhibition of α7-nAChR or deletion of α7-nAChR prevented Aβ induced neuronal hyperexcitation and toxicity, which suggests that α7-nAChRs are required for Aβ induced neuronal hyperexcitation and toxicity. These results reveal a profound role for α7-nAChR in mediating Aβ-induced neuronal hyperexcitation and toxicity and predict that Aβ-induced up-regulation of α7-nAChR could be an early and critical event in AD etiopathogenesis. Drugs targeting α7-nAChR or seizure activity could be viable therapies for AD treatment.
ContributorsLiu, Qiang (Author) / Wu, Jie (Thesis advisor) / Lukas, Ronald J (Committee member) / Chang, Yongchang (Committee member) / Sierks, Michael (Committee member) / Smith, Brian (Committee member) / Vu, Eric (Committee member) / Arizona State University (Publisher)
Created2011
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 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
Description
Gene therapy is a promising technology for the treatment of various nonheritable and genetically acquired diseases. It involves delivery of a therapeutic gene into target cells to induce cellular responses against diseases. Successful gene therapy requires an efficient gene delivery vector to deliver genetic materials into target cells. There are two major classes of gene delivery vectors: viral and non-viral vectors. Recently, non-viral vectors such as cationic polymers have attracted more attention than viral vectors because they are versatile and non-immunogenic. However, cationic polymers suffer from poor gene delivery efficiency due to biological barriers. The objective of this research is to develop strategies to overcome the barriers and enhance polymer-mediated transgene expression. This study aimed to (i) develop new polymer vectors for gene delivery, (ii) investigate the intracellular barriers in polymer-mediated gene delivery, and (iii) explore new approaches to overcome the barriers. A cationic polymer library was developed by employing a parallel synthesis and high-throughput screening method. Lead polymers from the library were identified from the library based on relative levels of transgene expression and toxicity in PC3-PSMA prostate cancer cells. However, transgene expression levels were found to depend on intracellular localization of polymer-gene complexes (polyplexes). Transgene expression was higher when polyplexes were dispersed rather than localized in the cytoplasm. Combination treatments using small molecule chemotherapeutic drugs, e.g. histone deacetylase inhibitors (HDACi) or Aurora kinase inhibitor (AKI) increased dispersion of polyplexes in the cytoplasm and significantly enhanced transgene expression. The combination treatment using polymer-mediated delivery of p53 tumor-suppressor gene and AKI increased p53 expression in PC3-PSMA cells, inhibited the cell proliferation by ~80% and induced apoptosis. Polymer-mediated p53 gene delivery in combination with AKI offers a promising treatment strategy for in vivo and clinical studies of cancer gene therapy.
ContributorsBarua, Sutapa (Author) / Rege, Kaushal (Thesis advisor) / Dai, Lenore (Committee member) / Meldrum, Deirdre R. (Committee member) / Sierks, Michael (Committee member) / Voelkel-Johnson, Christina (Committee member) / Arizona State University (Publisher)
Created2011
Description
Cancer diseases are among the leading cause of death in the United States. Advanced cancer diseases are characterized by genetic defects resulting in uncontrollable cell growth. Currently, chemotherapeutics are one of the mainstream treatments administered to cancer patients but are less effective if administered in the later stages of metastasis, and can result in unwanted side effects and broad toxicities. Therefore, current efforts have explored gene therapy as an alternative strategy to correct the genetic defects associated with cancer diseases, by administering genes which encode for proteins that result in cell death. While the use of viral vectors shows high level expression of the delivered transgene, the potential for insertion mutagenesis and activation of immune responses raise concern in clinical applications. Non-viral vectors, including cationic lipids and polymers, have been explored as potentially safer alternatives to viral delivery systems. These systems are advantageous for transgene delivery due to ease of synthesis, scale up, versatility, and in some cases due to their biodegradability and biocompatibility. However, low efficacies for transgene expression and high cytotoxicities limit the practical use of these polymers. In this work, a small library of twenty-one cationic polymers was synthesized following a ring opening polymerization of diglycidyl ethers (epoxides) by polyamines. The polymers were screened in parallel and transfection efficacies of individual polymers were compared to those of polyethylenimine (PEI), a current standard for polymer-mediated transgene delivery. Seven lead polymers that demonstrated higher transgene expression efficacies than PEI in pancreatic and prostate cancer cells lines were identified from the screening. A second related effort involved the generation of polymer-antibody conjugates in order to facilitate targeting of delivered plasmid DNA selectively to cancer cells. Future work with the novel lead polymers and polymer-antibody conjugates developed in this research will involve an investigation into the delivery of transgenes encoding for apoptosis-inducing proteins both in vitro and in vivo.
ContributorsVu, Lucas (Author) / Rege, Kaushal (Thesis advisor) / Nielsen, David (Committee member) / Sierks, Michael (Committee member) / Arizona State University (Publisher)
Created2011