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- All Subjects: Amyloid beta-protein
- All Subjects: Phage
- Creators: Sierks, Michael
- Resource Type: Text
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
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
Alzheimer's Disease (AD) is a debilitating neurodegenerative disease. The disease leads to dementia and loss of cognitive functions and affects about 4.5 million people in the United States. It is the 7th leading cause of death and is a huge financial burden on the healthcare industry. There are no means of diagnosing the disease before neurodegeneration is significant and sadly there is no cure that controls its progression. The protein beta-amyloid or Aâ plays an important role in the progression of the disease. It is formed from the cleavage of the Amyloid Precursor Protein by two enzymes - â and ã-secretases and is found in the plaques that are deposits found in Alzheimer brains. This work describes the generation of therapeutics based on inhibition of the cleavage by â-secretase. Using in-vitro recombinant antibody display libraries to screen for single chain variable fragment (scFv) antibodies; this work describes the isolation and characterization of scFv that target the â-secretase cleavage site on APP. This approach is especially relevant since non-specific inhibition of the enzyme may have undesirable effects since the enzyme has been shown to have other important substrates. The scFv iBSEC1 successfully recognized APP, reduced â-secretase cleavage of APP and reduced Aâ levels in a cell model of Alzheimer's Disease. This work then describes the first application of bispecific antibody therapeutics to Alzheimer's Disease. iBSEC1 scFv was combined with a proteolytic scFv that enhances the "good" pathway (á-secretase cleavage) that results in alternative cleavage of APP to generate the bispecific tandem scFv - DIA10D. DIA10D reduced APP cleavage by â-secretase and steered it towards the "good" pathway thus increasing the generation of the fragment sAPPá which is neuroprotective. Finally, treatment with iBSEC1 is evaluated for reduced oxidative stress, which is observed in cells over expressing APP when they are exposed to stress. Recombinant antibody based therapeutics like scFv have several advantages since they retain the high specificity of the antibodies but are safer since they lack the constant region and are smaller, potentially facilitating easier delivery to the brain
ContributorsBoddapati, Shanta (Author) / Sierks, Michael (Thesis advisor) / Arizona State University (Publisher)
Created2011
Description
This project aims to address the current protocol regarding the diagnosis and treatment of traumatic brain injury (TBI) in medical industries around the world. Although there are various methods used to qualitatively determine if TBI has occurred to a patient, this study attempts to aid in the creation of a system for quantitative measurement of TBI and its relative magnitude. Through a method of artificial evolution/selection called phage display, an antibody that binds highly specifically to a post-TBI upregulated brain chondroitin sulfate proteoglycan called neurocan has been identified. As TG1 Escheria Coli bacteria were infected with KM13 helper phage and M13 filamentous phage in conjunction, monovalent display of antibody fragments (ScFv) was performed. The ScFv bind directly to the neurocan and from screening, phage that produced ScFv's with higher affinity and specificity to neurocan were separated and purified. Future research aims to improve the ScFv characteristics through increased screening toward neurocan. The identification of a highly specific antibody could lead to improved targeting of neurocan post-TBI in-vivo, aiding researchers in quantitatively defining TBI by visualizing its magnitude.
ContributorsSeelig, Timothy Scott (Author) / Stabenfeldt, Sarah (Thesis director) / Ankeny, Casey (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05