Browsing by Subject "Microarray"
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Item The application of aptamer microarraying techniques to the detection of HIV-1 reverse transcriptase and its mutant variants(2010-08) Syrett, Heather Angel; Ellington, Andrew D.; Kitto, George B.; Willets, Katherine A.; Iyer, Vishwanath R.; Yin, Yuhui W.The work described here details the experimental progress toward an improved means of HIV-1 diagnosis and an explanation of the experimental approaches taken to advance a previously developed HIV-1 reverse transcriptase detection assay using RNA aptamers for protein capture. After characterization of the identity and function of the aptamer samples to be used, we first set about clarifying the nature of the assay and pinning down sources of variability inherent in the original Aptamer Antibody Sandwich Assay (AASA) such that through the course of this work we might bring the assay to a point of high reproducibility. In doing so, we devised a set of criteria for data analysis and filtration and established a process to examine whether modifications to the method resulted in measurable improvement. Two new methods were tested in the hope that they might later be extended to our ultimate project goal of distinguishing binding affinity variations among HIV-1 reverse transcriptase protein and its mutant variants. Both method modifications involved the addition of a fluorescently labeled Cy5 probe to the immobilized aptamer construct. The addition of a fluorescent label to each printed aptamer allowed for detection of aptamer presence in addition to protein binding, essentially serving as a simple internal control for aptamer-protein binding. After optimizing the AASA aptamer construct and experimental procedure, the AASA was extended to a multiplexed array format. Using four groups of aptamers selected against two HIV-1 RT variants (wild-type and mutant 3) we tested the hypothesis that immobilized anti-HIV-1 aptamers might be capable of binding HIV-1 RT variants and regardless of their selective target. The experiments described here are the first example of these aptamers being used in a multiplexed array format, and the results are not only a clear exemplification of the capacity of RNA aptamers for detection in this novel, immobilized assay format, but also an indicator of the utility and flexibility of RNA aptamer functionality. The promising results described in these preliminary studies are the starting block from which several interesting aptamer-protein interaction and drug-competition studies have begun.Item Comparative Performance Analysis of the Algorithms for Detecting Periodically Expressed Genes(2012-10-19) Agyepong, KwadwoThus far, a plethora of analysis on genome-wide gene expression microarray experiments on the cell cycle have been reported. Time series data from these experiments include gene expression profiles that might be periodically expressed. However, the numbers and actual genes that are periodically expressed have not been reported with consistency, analysis on similar experiments reports disparate numbers of genes that are periodically expressed with scant overlap. This work ultimately compares the performance of five spectral estimation schemes in their ability to recover periodically expressed genes profiles. Lomb-Scargle (LS), Capon, Missing-Data Amplitude and Phase Estimation (MAPES), Real Value Iterative Adaptive Approach (RIAA) and Lomb-Scargle Periodogram Regression (LSPR) are rigorously studied and pitted against each other in various simulated testing conditions. Results obtained using synthetic and microarray data reveals that RIAA is an efficient and robust method for the detection of periodically expressed genes in short time series data that might be characterized with noisy and irregularly sampled data points.Item Development of wireless DNA microarray sensors(2010-08) Chow, Kwok-Fan; Crooks, Richard M. (Richard McConnell); Bard, Allen J.; Bielawski, Christopher; Manthiram, Arumugam; Stevenson, KeithThe development of wireless DNA microelectrochemical microarray sensors is described. The operational principles of these sensors are based on bipolar electrochemistry. Bipolar electrodes are used to fabricate the wireless microarrays in this work. The systems are configured so that DNA sensing is carried out at the cathodic end of a bipolar electrode (BPE) and the result of the sensing experiment is reported at the anodic end of the BPE. There are two types of reporting platforms developed in this study. The first type relies on the emission of electrogenerated chemiluminescence (ECL). The system is configured so that ECL is emitted at the anodic end of the BPE when the target DNA is hybridized to the capture probe DNA immobilized on the cathodic end of the BPE. However, when there is no hybridization reaction occurs, there is no ECL emission on the electrode surface. The second type of reporting platform developed is based on silver electrodissolution at the anodic end of a BPE. When a reduction reaction occurs at the cathodic end of a BPE, it triggers oxidation and dissolution of silver deposited at the anodic end of the BPE. The loss of silver can easily be detected by the naked eye. This detection principle is used for DNA detection: when the target DNA is hybridized to capture probe DNA on the BPE, the BPE becomes shorter. However, if target DNA does not hybridize to the electrode surface, the length of the BPE remains the same. The BPE microarrays described in this work eliminate the need for complicated microfabrication procedures and instrumentation. For example, as many as 1000 BPEs can be simultaneously controlled using just two driving electrodes and a simple power supply. To fully utilize BPE microarrays for specific sensing tasks, a method based on robotic spotting was developed to modify the cathodic end of each BPE in the array. Because each BPE in a microarray is individually addressable, this development allows each BPE to perform a particular sensing operation.Item Functional Analyses of the Molecular mechanisms Underlying Two Equine Respiratory Diseases: Recurrent Airway Obstruction and Rhodococcus equi Pneumonia(2012-07-16) Kachroo, PriyankaRecurrent airway obstruction (RAO) and Rhodococcus equi (R. equi) pneumonia are two equine respiratory diseases. RAO is an allergic asthma like disease of the middle-aged horses while the R. equi pneumonia affects only young foals. Respiratory disease is considered among the major causes of economic loss to the equine industry and tops the priority list for research that will focus on preventative and diagnostic facets of such disease. The objective of this research was to investigate the effect of antigen exposure and remission (via allergen avoidance and/or drug) on chronically affected RAO horses. Additionally, we also wanted to understand the changes in equine neonatal immune system due to R. equi exposure and identify molecular biomarkers for early disease screening. Various biological samples (lung tissue for the RAO study and blood leukocytes and nasal epithelial cells for the R. equi study) were used to extract ribonucleic acid (RNA). Complimentary deoxyribonucleic acid (cDNA) obtained from RNA was used to perform microarray hybridization experiments. Our findings suggest that compared to control horses allergen exposure leads to an elevated protein synthesis and inflammation that contributes to aggravation of symptoms and airway changes. We found that allergen avoidance controls inflammation and causes an improvement in lung function and other chronic features of RAO. The drug administration led to an accelerated remission in the chronic RAO features; a complete remission could however not be achieved. Hence it appears that although not a complete resolution, but allergen avoidance and drugs will help in a better management of chronic RAO symptoms. Our results suggest that the neonatal immune system is capable of initiating a protective immune response through birth up to 8 weeks of age. However there are also processes present that may be counter-productive to the host. Induction of such suppressive mechanisms may be a result of bacterial modulation of the host immune response or a result of immature host immune system. We also identified molecular biomarkers that will have the potential to screen foals for R. equi pneumonia soon after birth and before the onset of clinical symptoms. The research findings of this study will improve the current understanding of the two equine diseases.Item Genetic basis for ichthyotoxicity and osmoregulation in the euryhaline haptophyte, Prymnesium parvum N. Carter(2014-05) Talarski, Aimee Elizabeth; La Claire, John W., 1951-There is limited information currently available regarding the underlying physiological responses and molecular mechanisms of osmoregulation, acetate metabolism [in relation to the synthesis of glycerolipids, polyunsaturated fatty acids (PUFA), and ichthyotoxins], and transport in Prymnesium parvum N. Carter, a microalga that causes devastating harmful algal blooms (HAB) worldwide. This dissertation examines gene expression under environmental conditions that are associated with HAB formation, including phosphate limitation and low salinity, using microarrays and RNA sequencing (RNA-Seq). A comparative fatty acid methyl ester (FAME) analysis at 30 vs. 5 practical salinity units (psu) was performed to gain additional insight into acetate metabolism. The RNA-Seq analysis included a de novo assembly of the P. parvum transcriptome, generating 47,289 transcripts, of which 35.4% were identifiable. This permitted the evaluation of the expression of many more genes compared with the microarray analysis, which examined ~3,500 genes. Relevant candidate genes identified included those whose products are involved in osmolyte production, salinity stress, and ion transport. With respect to the putative synthesis of polyketide ichthyotoxins, 32 different polyketide synthase (PKS) transcripts were identified in the transcriptome assembly, none of which were differentially expressed. Hemolysin and monogalactosyldiacylglycerol synthase were downregulated at 30 vs. 5 psu, suggesting the increased presence of additional ichthyotoxins at the lower salinity. Evidence for several PUFA synthesis pathways was also revealed. Fatty acid compositions were largely similar at the two salinities, containing relatively prominent quantities of the PUFA stearidonic acid, but compositions varied among strains. The transcription of genes whose products are associated with vesicular transport was elevated, and higher levels of extracellular prymnesins were observed in HAB-forming conditions. Thus, with regard to acetate metabolism, I have revealed evidence for the post-transcriptional regulation of the production of prymnesins and the contributory effects of hemolysin, monogalactosyldiacylglycerol, and PUFA towards ichthyotoxicity. Further, I propose that toxin transport is triggered in HAB-forming conditions, in which the toxins are actively being excreted. Collectively, these data shed light on the transcriptional responses that occur following alterations in phosphate availability and salinity, including those associated with the synthesis and delivery of a number of potential ichthyotoxins from P. parvum.Item Glycomics : integration of lectin and gene expression microarray data(2011-08) Pilobello, Kanoelani Takaishi; Mahal, Lara K.; Anslyn, Eric V., 1960-Glycomics is the systematic study of glycosylation in the context of a whole cell or organism. Glycosylated proteins are estimated to make up 50% of all proteins and cover the outside of the cell. Functional roles in glycosylation have been noted in pathogenesis, metastasis, and embryogenesis. However, the structure of these carbohydrates has been difficult to study due to the chemical nature of carbohydrates. Lectins, carbohydrate binding proteins excluding antibodies and enzymes, can be utilized to study glycosylation in a high throughput manner using a microarray format. Glycans, the carbohydrates attached to a protein or lipid, are not synthesized from a template. They are added co- or post-translationally by a concerted set of enzymes in the secretory pathway. In addition, the glycan structures may be altered by metabolism or trafficking. Cell type specific glycosylation has long been hypothesized due to observations of bacteria homing to tissues. We use lectin microarray technology to define the glycosylation in a subset of the NCI-60, a set of cell lines from different tissues. Using a customized gene expression microarray, we identify cell type dependent glycosylation genes and observe evidence of cell type dependent spliceforms for an O-glycosylated mucin. Data from the lectin microarray and a published gene expression data set were integrated using Generalized Singular Value Decomposition (GSVD), a linear matrix decomposition method. We have successfully decomposed the data into 3 cell type dependent meta patterns that segregate by glycosylation family. Correlation projection of the genes and subsequent gene ontology enrichment suggests that genes in different pathways covary with the types of glycosylation. An inverse relationship was revealed for the N- glycosylation pattern between the SVD of the lectins and the GSVD of the genes and lectins together. Whereas, the relationship was correlative for O-glycosylation, which was clearly illustrated in biplots. This work argues that types of glycosylation are regulated by different mechanisms in different cell types.Item Investigating cellular responses to mutations in the glutathione and thioredoxin pathways of Escherichia coli(2009-12) Chrysostomou, Constantine; Georgiou, George; Isaac, SanchezInhibition of disulfide bond formation in Escherichia coli implicates an intricate collaboration of proteins which comprise the glutathione and thioredoxin reducing pathways. Bioengineers have successfully engineered E. coli possessing mutated reducing pathways that promote, rather than inhibit, disulfide bond formation in the cytoplasm. The transcriptome of six such mutant E. coli strains have been characterized using Microarray technology. We find that all mutant strains, exhibit a unique response to oxidative stress, not observed in wild type. Statistical analyses revealed the expression of more than 200 genes that are affected by mutations within the reducing pathways. Significantly up-regulated biological processes include cysteine biosynthesis, histidine biosynthesis, NADH Dehydrogenase I biosynthesis, sugar catabolic processes, and activation of stress responses . The second part of this work describes the construction of an E. coli strain that promotes the complete conversion of glutathione into its seemingly dormant derivative, glutathionylspermidine. This engineered strain can be used in assays designed to evaluate the effectiveness of glutathionylspermidine as a substitute for glutathione and, hopefully, allude to its true metabolic function.Item Lurking Pathway Prediction And Pathway ODE Model Dynamic Analysis(2013-11-18) Zhang, RengjingSignaling pathway analysis is so important to study the causes of diseases and the treatment of drugs. Finding the lurking pathway from ligand to signature is a significant issue in studying the mechanism of how the cell response to the stimulation signal. However, recent literature based pathway analysis methods can only tell about highly differentially expressed pathways related to the experiment data, which may tell nothing about our interested specific ligand and signature. In this paper, we designed an approach to successfully detect the most reliable pathways for specific ligand and signature by solving multi-objective optimization problem on the bridge connecting two signaling pathways where the ligand and sig- nature locate. The pathway bridge consisted of enriched looping patterns refined the complicated entire protein interactions network with 39031 links, which made the approach time-saving. The approach was further applied to study the mod- ulator mechanism of the signal molecule, receptor, intermediate transfer proteins, transcription factor, and signature. With preliminary studied pathways, we then employed Ordinary Differential Equations(ODE) to modeling and dynamic analysis the signaling transduction. The biological reactions were represented in terms of differential equations, and the solu- tions to the group of equations were further be optimized to fit the RPPA experiment data. In order to find the potential signaling paths in specific disease and discovery the best therapy, coefficient variation analysis, system robustness study and system outcomes changes to perturbations were also utilized. Our approach successfully predicted the lurking pathway for the signal molecule T GF ?1 and the nova protein OC I AD2 in cancer microenviroment: T GF ?1 ?T GF ?R1 ? SM AD2/3 ? SM AD4/AR ? OC I AD2, and this result was verified by literature. Better than recent pathway analysis tool, our predicted pathway also took care of significant but relatively less regulated proteins in the transduction pro- cess. And by modeling the CCL2 pathway in MTB infected cells, J N K , cM Y C and P LC showed as the most significant modules. Hence, the drug treatments inhibit- ing J N K , cM Y C and P LC would effectively obstruct the increasing of MMPs and further prevent the Mtb infections.Item Multi-analyte biosensing : the integration of sensing elements into a photolithographically constructed hydrogel based biosensor platform(2005-05) Schmid, Matthew John; Willson, C. G. (C. Grant), 1939-The genome sequencing programs have identified hundreds of thousands of genetic and proteomic targets for which there are presently no ascribed functions. The challenge for researchers now is to characterize them, as well as identify and characterize their natural variants. Historically, this has meant studying each individual target separately. However, due to the recent development of multi-analyte microarray devices, these characterizations can be performed in a combinatorial manner in which a single experiment provides information on thousands of targets at a time. In the past decade, microarray technology has settled in on two major designs. The first entails spotting individual receptor types onto a functionalized glass substrate. This is a simple and inexpensive process; however, due to the limited resolution of the mechanical devices used to do the spotting, the densities of these arrays are relatively low. Moreover, receptor preparation requires substantial time and effort. The second variety of microarray uses photolithographic techniques adapted from the semi-conductor industry to chemically synthesize the receptor elements in situ on the sensing surface. Because lithographic patterning is spatially very precise, these arrays achieve very high densities, with as many as one million features per square centimeter. Although these arrays obviate the necessity for laborious "off chip" probe preparation, they are expensive to produce and are limited to two types of receptors (oligonucleotides and peptides). This dissertation presents the development work performed on a hydrogel-based biosensor platform which provides a high density and low cost alternative to the two aforementioned designs. The array features are fabricated lithographically from a liquid pre-polymer doped with biologically active sensing elements at sizes as small as 50[micrometer]. Each of the feature types is uniquely shaped, which enables the features to be mass-produced in batches, pooled together and then assembled into randomly ordered arrays using highly-parallelized self-assembly techniques. The three-dimensional hydrogel features accommodate a wide variety of sensing elements, such as enzymes, antibodies and cells, which cannot be deployed using the traditional designs. This dissertation presents methods developed to integrate cellular and oligonucleotide sensing elements into the hydrogel features which preserve their biological activity and optimize the sensor's performance.Item Studies on gene expression profiling in JB6 cells susceptible and resistant to tumor promoter induced neoplastic transformation and regulation of gene expression at the AP-1 DNA binding site(Texas A&M University, 2005-11-01) Samuel, ShaijaGene expression underlies all important biological processes in a cell and mis-regulated gene expression plays a causal or contributory role in several diseases including cancers. Towards identifying molecular determinants that confer susceptibility and resistance to tumor promoter induced neoplastic transformation, we analyzed the gene expression profile differences among tumor promoter TPA treated and untreated mouse epidermal JB6 cells by means of cDNA microarray analyses. The expression patterns for several genes were validated by real time PCR analyses. Seventy-four genes belonging to six functional categories were found to be differentially expressed. Data from this study implicate pathways which mediate cell adhesion, migration and interferon signalling, tumor suppressors, apoptotic proteins and transcription factors and includes twenty-six genes whose involvement has not been previously implicated in cancer. In a second study we used a DNA affinity chromatography based assay to purify two proteins that bound specifically to the AP-1 DNA binding site. Analyses of the purified proteins by mass spectrometric sequencing determined the identities of these proteins as nucleolin and Y-box binding protein 1 (YB-1). We tested the hypothesis that these proteins regulate transactivation at the AP-1 site. Overexpression of nucleolin and YB-1, both alone or in combination, repressed AP-1 dependent gene transactivation. To understand the mechanism of transrepression, we analyzed whether nucleolin and/or YB-1 affected the levels and/or disrupted the intracellular localization of the AP-1 protein subunits. Western blot analyses of all the AP-1 subunits revealed that the levels of AP-1 were unaffected. Cell fractionation confirmed that the AP-1 levels were not altered in the nuclear or cytoplasmic compartments. We further tested the hypothesis that nucleolin and YB-1 repressed AP-1 transactivation by competing with AP-1 proteins for the AP-1 site. The results from this experiment were inconclusive and the precise mechanism of repression is currently under investigation.Item A systems pharmacology approach to discovery of drugs to ameliorate oxidant stress in human endothelial cells(2015-05) Bynum, James Andrew, Jr.; Stavchansky, Salomon; Bowman, Phillip D; Kerwin, Sean M; Cui, Zhengrong; Williams, Robert OIschemia is characterized by reduced blood flow to an area of the body which can then cause cellular injury through the generation of reactive oxygen species (ROS), activation of inflammation, and induction of apoptosis. Although rapid reestablishment of flow is required to prevent organ death, the reperfusion phase of this injury can cause its own deleterious effects often exacerbating the initial insult. The combined action of the two injuries is termed ischemia/reperfusion (I/R) injury. Oxidative stress that results from ischemia/reperfusion injury is a common pathological condition that accompanies many human diseases including stroke, heart attack and traumatic injury. In addition, neurodegenerative diseases including Parkinson’s, Alzheimer’s, and Huntington’s disease appear to involve oxidative stress. Although actively investigated by the medical and pharmaceutical industry; limited progress has been made to ameliorate I/R injury and to date there is no drug approved for treatment for I/R injury. Therapeutic approaches to treat I/R injury have included the administration of compounds to scavenge ROS or induce protective pathways or genetic responses. It was previously reported that caffeic acid phenethyl ester (CAPE), a plant-derived polyphenol, displayed cytoprotective effects against menadione (MD)-induced oxidative stress in human umbilical vein endothelial cells (HUVEC), and the induction of heme oxygenase-1 (HMOX1), a phase II enzyme, played an important role for CAPE cytoprotection. In an effort to improve this cytoprotection, other phase II enzyme inducers were investigated and, 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (CDDO-Im) and 2-cyano-3,12-dioxooleana-1,9-dien-28-oyl methyl ester (CDDO-Me), were found to be potent inducers with a rapid onset of action. CDDO-Im and CDDO-Me, synthetic olenane triterpenoids, developed as anticancer agents were compared to CAPE revealing that CDDO-Im was a more potent inducer of Phase II enzymes including HMOX1 and provided better cytoprotection than CAPE. Gene expression profiling showed that CDDO-Im was more potent inducer of protective genes like HMOX1 than CAPE and additionally induced heat shock proteins. To better understand the mechanism of action of CDDO-IM, a gene expression time-course was undertaken to identify early initiators of the transcriptional response preceding cytoprotection. Application of systems pharmacology identified molecular networks of cell mediating processes.Item The role of neutrophil recruitment in the pathogenesis of salmonella enterica serotype typhimurium-induced enteritis in calves(2009-05-15) Nunes, Jairo SantosThe role of neutrophils in the pathogenesis of Salmonella typhimurium-induced ruminant and human enteritis and diarrhea remains incompletely understood. To address this question, the in vivo bovine ligated ileal loop model of non-typhoidal salmonellosis was used in calves with the naturallyoccurring Bovine Leukocyte Adhesion Deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from BLAD calves were compared to those from genetically normal calves negative for the BLAD mutation. Morphologic studies showed that the absence of significant tissue influx of neutrophils in intestine infected by S. typhimurium resulted in less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion compared to regular calves. Study of gene expression profile of cytokines by quantitative Real-Time PCR (qRTPCR) revealed that the massive tissue influx of neutrophils during acute infection is mainly driven by the CXC chemokine GRO- ? especially in the last stages of acute infection and to a lesser extent, IL-8. In contrast, the pro-inflammatory cytokines IL-1 ? and TNF- ? were not significantly correlated with the presence or absence of tissue neutrophils. The precise in situ localization of gene expression of these major cytokines and chemokines was investigated by qRTCPR from specific groups of intestinal cells captured by Laser Capture Microdissection in S. typhimuriuminfected ileal loops from BLAD animals. Our data confirmed that gene expression of IL-8, GRO- ?, and IL-1 ? was predominantly localized to enterocytes of crypts with less expression in enterocytes of villi tips and cells that form the domed villi were not an important source of TNF- ? gene expression. Microarray technology was used to determine the global transcriptional profile of bovine intestinal loops inoculated with S. typhimurium. The host samples were hybridized on a 13K bovine-specific oligoarray and microarray data was analyzed using a suite of gene expression analysis and modeling tools. Analysis of our data revealed that the tissue influx of neutrophils in ileal loops greatly influenced the host gene expression. Major differences in gene expression in relevant fields of Salmonella research including inflammation and immune response, Toll-like receptor signaling, cytokine profiles, apoptosis, and intracellular defense against infection are discussed.