Browsing by Subject "Gene Expression"
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Item B Cell Signaling and Bioinformatics: Revealing Components of the MHC Class II Antigen Processing and Presentation Pathway(2005-05-11) Lee, Jamie Anne; Scheuermann, Richard H.Stimulation of mature B lymphocytes by extracellular ligands induces phenotypic changes through complex signal transduction pathways. Gene expression is altered as a result of these changes and re-programs the cell to undergo differentiation, activation, effecter function, anergy, and/or apoptosis. Gene expression microarrays are used to determine expression levels of a large number (tens of thousands) of genes simultaneously, resulting in a gene expression profile of the experimental sample. Microarray data must be appended with biological information in order to be interesting, and this field of microarray bioinformatics is rapidly expanding. These studies prompted the development of a bioinformatics tool termed CLASSIFI (Cluster Assignment for Biological Inference), which identifies statistically significant co-clustering of genes with similar Gene Ontology annotation within microarray gene clusters. CLASSIFI was used to analyze microarray results from two B cell projects from the Alliance for Cellular Signaling (AfCS): 1) the BAFF/CD40L project, which evaluates the effects of BAFF and CD40L on primary mouse B cells in long-term cultures, and 2) the B cell single ligand screen project, which evaluates the effects of 32 single ligands on primary mouse B cells in short-term cultures. CLASSIFI was able to identify significant overrepresentation of related genes within gene clusters for both of these data sets and facilitates hypothesis generation as to the biological process affected by a specific ligand. As CLASSIFI is strictly a statistical tool that aids in hypothesis generation, experimental validation of hypotheses was performed. The B cell single ligand screen microarray and CLASSIFI analysis followed by experimental validation revealed a biological process specific to B cell antigen receptor stimulation but not LPS or CD40L stimulation - antigen processing and presentation - and provides the groundwork for new discoveries in this field. As a result, several putative components were identified that are not currently known to play a role in antigen processing and presentation in B cells.Item Characterization and Development of Strategies for Altering Protein Expression in JSL1 Cells(2007-08-08) Senitko, Annette Nelson; Lynch, KristenAlternative splicing is a common mechanism for regulating gene expression in eukaryotic cells. This process of differentially including or excluding variable exons provides a means for increasing proteome complexity. Alternative gene splicing occurs in a cell specific manner and may be influenced by changes in the extracellular environment. Despite the importance of this method for regulating gene expression, little is known about the factors involved in regulating its function. The T cell tyrosine phosphatase CD45 provides a valuable model for investigating the factors involved in regulating alternative splicing. The CD45 gene contains three variable exons whose splicing is regulated in response to T cell activation. Studies of this gene have revealed the presence of an exonic silencer sequence within variable exon 4 that is capable of influencing exon skipping under both resting and stimulated conditions. Biochemical assays have shown that the regulatory protein hnRNP L binds to this silencer sequence and results in basal exon repression during resting conditions and undergoes modifications which further influence exon skipping upon stimulation. Furthermore, in vitro assays indicate that upon stimulation, an additional regulatory protein, PSF, binds to the regulatory complex associated with the silencer sequence. Although these studies have provided novel information regarding the regulation of splicing, biochemical assays are unable to fully mimic the signaling pathways inside a cell, thus creating a need for a cell culture system. A Jurkat derived cell line, JSL1 cells, has been identified as being able to recapitulate the signal induced alternative splicing of the CD45 gene as seen in primary human T cells. This cell line presents a cell based system for evaluating the factors involved in splicing. However, in order to conduct in vivo experiments one must be able to modify protein expression. JSL1 cells present limitations due to difficulties in being able to alter protein expression. A strong promoter, EF1-alpha , has been employed to drive the expression of candidate proteins in JSL1 cells. Transient transfections and stable cell lines expressing cDNAs driven by this promoter have shown little if any overexpression of candidate proteins normally expressed at high levels within the cell; however, significant overexpression has been achieved with the transfection of at least one protein that exists at a lower concentration. Initial experiments indicate that stably expressed flag-tagged proteins, driven by the EF1-alpha promoter, may be easily purified from JSL1 cells during resting and stimulated conditions and analyzed. Such data suggests that this promoter may afford more flexibility in altering and analyzing protein expression in JSL1 cells, thereby facilitating the investigation of signaling pathways involved in regulating alternative splicing. Furthermore, strategies for regulating protein expression, through the use of a Tet-suppressor system, are in initial stages of being developed and hold the potential for providing an additional tool for evaluating the factors involved in regulating alternative splicing.Item Effects of Acute and Chronic Stress on Immune- and Inflammatory-response Gene Expression in Beef Calves(2012-02-14) Terrill, CooperTransport stress research has shown correlations among stress, morbidity, and mortality in calves subjected to the traditional U.S. market system, indicating the possibility of compromised immune function. The objective of this study was to determine if expression of specific immune and inflammatory response genes differed between calves that were subjected to either an acute stress (AS, handled and weaned for 1.5 h) or a chronic stress (CS, weaned, handled and transported for 3 to 4 d). Two groups of forty calves, Bos taurus (n = 20) and crossbred calves (n = 20), weighing 181 kg to 250 kg were used in each of two trials. Jugular veni-puncture blood samples (9 ml) were collected from AS calves 1.5 h after the start of handling and separation from their dam. Samples were collected from CS calves during processing after arrival at a north Texas feed lot. RNA for gene expression analysis was extracted from peripheral blood leukocytes obtained from blood samples by a filtration method. During the second trial, the filtrate was centrifuged for measurement of plasma cortisol. A diagonal covariance mixed model ANOVA was used to determine effects of treatment, breed, and breed by treatment interaction on cortisol concentrations. Expression values for each gene were analyzed using linear models that considered the effects of treatment (AS and CS) and breed (Bos taurus and crossbred calves) comparing each trial separately. Mean plasma cortisol concentrations did not differ between AS (16.40 +/- 1.08 ng/ml) and CS calves (18.06 +/- 1.14 ng/ml) (P > 0.296). The interaction of effects was detected for 2 genes in Trial 1, and 3 genes in Trial 2 (P < 0.029). Breed was influential for 5 genes in both Trial 1 and 2 (P < 0.046). Significant differences were found in relative quantification for 30 genes in Trial 1 and 36 genes in Trial 2, in which CS calves had greater expression than AS calves (P < 0.047). Fifteen of those genes were common between the two trials with mean treatment differences of RQ values from the 15 genes ranging from 0.309 to 913.19, excluding outliers. Similar elevated cortisol concentrations in both the AS and CS calves indicated that both groups experienced significant stress. However, changes in gene expression differences were greater in the calves subjected to CS, indicating that gene expression may be more useful than cortisol for identifying detrimental long-term stress.Item Gene Expression in the Stallion Testes(2011-08-08) Laughlin, Andy M.Understanding the genes that regulate spermatogenesis and steroidogenesis in the testis is critical for enhancement of stallion fertility. Stallion testicular samples were used to identify candidate genes by cDNA microarrays that simultaneously assessed expression levels of 9132 genes. First, gene expression was compared between light (spermatogenically active) and dark (spermatogenically inactive) testis tissue of 1.5-year-old horses (n = 3). Ninety-three genes were differentially expressed (35 light specific, 58 dark specific) in matched paired samples. Second, gene expression was compared between testicular tissue of two mature stallions, one with normal quality semen (fertile) and one with poor quality semen (subfertile). A total of 233 genes were differentially expressed (122 in fertile tissue, 111 in subfertile tissue). Of these, phosphodiesterase 3B (PDE3B), steroidogenic acute regulatory (StAR) protein, and outer dense fiber of sperm tails 2 (ODF2) mRNAs, were localized and quantified by in situ hybridization (ISH) in mature stallions and/or in unilateral cryptorchids. ISH revealed differences (P < 0.05) among mature stallions (n = 10) for PDE3B (localized to seminiferous tubules) and StAR protein (localized to interstitial spaces) mRNAs. A positive correlation coefficient (r = .556, p = .025) was found between StAR protein mRNA and plasma concentration of testosterone. Additionally, both gene products were evaluated in 1-year-old (n = 3) and 3-year-old (n = 3) unilateral cryptorchid stallions. Expression of both PDE3B and StAR protein gene was significantly higher in mature, descended testes compared to mature, retained testes and the descended and retained testes of immature, cryptorchid stallions. StAR protein gene demonstrated significantly higher expression in immature retained testes compared to immature descended testes. A precision-cut tissue slice (PCTS) in vitro culture system was evaluated as a potential tool to study equine testes function. Testes from immature stallions (n = 3) were cut into slices (mean slice weight = 13.85 +/- 0.20 mg; mean slice thickness = 515.00 +/- 2.33 ?m) and exposed to medium containing ovine luteinizing hormone (oLH) at concentrations of 0, 5, 50 and 500 ng/ml for 6 h at 32 degrees C. Medium content of testosterone and estradiol was increased 500% and 120%, respectively, by addition of oLH versus that observed for the testis tissue slices treated with 0 ng oLH (control). An oLH concentration-dependent increase in StAR protein mRNA in tissue slices was detected by in situ hybridization; whereas, differences for PDE3B and ODF2 mRNAs were not observed. Collectively, these results demonstrate that the stallion is an excellent model for studying male fertility due to the initiation of spermatogenesis, frequency of cryptorchidism, and routine castration providing useful tissue to use for studying gene expression.Item Gene expression of beta-defensins in chicken white blood cells(2009-06-02) Supak, Tiffany MarieInfectious agents such as bacteria or viruses can grow rapidly. If a microorganism invades a host, it must be recognized rapidly and destroyed before it overwhelms the immune system. Limiting infection to a minimum in the early stage is critical for the outcome and the recovery from infection. The innate immune system has evolved to recognize a few highly conserved, constitutive structures present only in microorganisms, such as bacterial lipopolysaccharide (LPS), called pathogen-associated molecular patterns (PAMP). Toll-like receptors are the host receptors that recognize PAMP, ultimately activating a variety of transcription factors to induce expression of a wide spectrum of immune related genes, e.g. defensins. Defensins are antimicrobial peptides that play an important role in innate defense against microorganisms in plants and animals. Beta-defensins are the largest family of antimicrobial peptides, which can directly kill microorganisms and have regulatory effects on the immune system. Thirteen beta-defensins have been identified; however, the regulation of these genes has not been well-investigated in the chicken. The objective of this research was to understand constitutive and inducible gene expression of beta-defensins in chicken white blood cells. Real-time RT-PCR was used to quantify gene expression level before and after LPS stimulation. Transcription factor binding sites in the genes were identified to understand the gene expression regulation. From the expression profile results, most chicken beta-defensins had induced gene expression by LPS stimulation in the early phase (0- to 3-hour) and reduced gene expression in the late phase (3- to 8-hour). As for the level of gene expression, the results show that the induced gene expression in the early phase corresponded to the higher levels of expression at 3-hours after LPS stimulation, and the reduced gene expression in the late phase corresponded to the lower levels of gene expression at 8-hours after LPS stimulation.Item Histone Deacetylase 7 and Transcriptional Regulatory Networks of the Vascular Endothelium(2010-05-14) Young, Bryan Daniel; Olson, EricCells respond to stimuli in part through the modulation of gene expression. Signal transduction from the environment to the nucleus culminates in the activation of factors that modify chromatin structure to either facilitate or inhibit gene transcription. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are two such classes of enzymes that regulate the epigenetic code. Their opposing actions – to activate transcription by histone acetylation and to inhibit transcription by deacetylation – are tightly regulated to coordinate the vast gene programs required for cellular growth and differentiation. The class II HDACs are restricted in their expression patterns, and each have unique developmental and physiological functions. The studies described here focus on HDAC7, a class II HDAC that is expressed in vascular endothelial cells and whose function is essential for the maintenance of vascular integrity during embryogenesis. Mice lacking HDAC7 die by e11.5 with complex cardiovascular malformations including endothelial, vascular smooth muscle, and myocardial defects. By generating HDAC7 conditional knockout mice, it was observed that all of these defects are recapitulated in mice bearing an endothelial-specific deletion of HDAC7, but no defects are observed upon deletion of HDAC7 in the other cell types that were affected in the HDAC7 nulls. This in vivo evidence demonstrated that HDAC7 acts cell autonomously to maintain normal vascular development, and lead to the identification of the genetic abnormalities and mechanism leading to cardiovascular failure in the HDAC7 knockout. Further, this work begins the investigation of HDAC7 in adult vascular physiology, the findings of which will reveal new mechanisms whereby the vasculature responds to stress signals or disease. To this end, methods have been developed for the deletion of HDAC7 in the adult mouse using an inducible cre recombinase system together with the HDAC7 conditional allele. Additionally, these studies present progress toward the identification of the enhancer elements driving the endothelial-specific expression pattern of HDAC7. Detailed characterization of this enhancer is likely to implicate new signaling pathways as being involved in the genetic regulation of vascular development and maintenance. Finally, this work investigates the role of microRNAmediated gene silencing in the vascular system by identifying microRNAs involved in MEF2-dependent signaling in endothelial cells.Item Molecular profiling in early stage IB1 squamous cell carcinomas of the uterine cervix.(2007-03-07) Kendra Leann Stisser; Concepcion Diaz-Arrastia, M.D.; Kathleen O’Connor, Ph.D.; Golda A. Leonard, Ph.D.; Elizabeth R. Unger, Ph.D., M.D.; Bruce A. Luxon, Ph.D.Treatment of tumors of the cervix is based on clinical staging of disease. The International Federation of Gynaecology and Obstetrics system (FIGO staging) has established a system whereby cervical cancers are staged on the basis of anatomical extent of the tumor. Microinvasive tumors most often require non-radical surgical resection and pose little risk of recurrence and metastasis. On the other extreme, cancers that are inoperable, due to spread beyond the cervix (Stage IIB to IVB) require concomitant chemotherapy and radiation therapy. Currently, of all women undergoing radical hysterectomies and pelvic lymphadenectomy for FIGO stage IB1 carcinomas, forty to fifty percent are deemed at risk for recurrence and require radiation therapy and chemotherapy 1. Much research has focused on linking histologic phenotype to outcome. However, tumor heterogeneity has made predictions of recurrence risk difficult. Ten percent of patients deemed having a low risk of recurrence by histological criteria will present with recurrent tumors within 5 years. The problem is that women may not receive optimal radiation therapy. If under treated, the cancer comes back. If over-treated there is a risk of toxicity associated with the adjuvant therapy. \r\nThe overall purpose of this research was to clarify the molecular mechanisms of progressive IB1 cervical disease in order to stratify risk to better match multi-modality therapy to minimize morbidity, mortality, and costs. The goal is to improve characterization of the tumor to individualize therapy. \r\nMolecular profiling was used to identify a group of 98 genes that differentiate early cancers with a low risk of recurrence from those with a high risk of recurrence. Tumor heterogeneity is an important consideration when using molecular profiling to characterize outcome. There are location-specific genes that can be used for elucidating the mechanism of disease and individualizing patient care. \r\nGeneChip technology is a powerful tool for teasing out the orchestration of molecular processes leading to progressive disease. As the molecular signature technology develops, we can move beyond improved diagnosis to improved therapy. This preliminary analysis may help to identify improved markers for predicting outcome so we can offer patients more precisely tailored treatment regimens.\r\nItem Regulation of Insulin and CHOP Gene Expression in Pancreatic Beta Cells(2009-01-14) Shao, Chunli; Cobb, MelanieInsulin is a major hormone in maintaining glucose homeostasis. It is essential to understand the mechanisms by which insulin gene expression is regulated in pancreatic beta cells. In addition to examining histone modifications on the insulin gene promoter, I focused on the effect of MafA modification on insulin expression. MafA is a transcriptional activator of the insulin gene via binding to the RIPE3b/C1 (rat insulin promoter element 3b) element. Mutagenesis showed that MafA was post-translationally modified by SUMO-1/2 (small ubiquitin-like modifier) mainly at lysine 32. Low glucose starvation or hydrogen peroxide stimulation increased sumoylation of MafA. Forced sumoylation of MafA reduced its transcriptional activity towards the insulin gene promoter and increased its suppression of the CHOP (C/EBP homologous protein) gene promoter. However, sumoylation of MafA did not alter its nuclear localization, protein stability, or apparently its DNA binding to the insulin promoter in beta cells. These studies suggest that MafA sumoylation modulates gene transcription in beta cells. In type I diabetes, beta-cell apoptosis is the major reason for immune-mediated pancreatic beta-cell death. IL-1beta (interleukin 1beta), a proinflammatory cytokine, induces ER (endoplasmic reticulum) stress and activates proapoptotic networks in beta cells, such as NF-kappaB (nuclear factor-kappaB) and JNK (c-Jun N-terminal kinase) signaling pathways. The second project focused on the mechanisms by which JNK and NF-kappaB regulate the expression of CHOP, a mediator of ER stress-induced apoptosis, upon IL-1beta stimulation. Exposure of beta cells to IL-1beta markedly increased CHOP messenger RNA and protein. Electrophoretic mobility shift assays showed that IL-1beta-activated NF-kappaB bound to the CHOP promoter. Furthermore, immunoblot data indicated that expression of c-Jun was strongly increased, and that multiple residues on c-Jun were phosphorylated after IL-1beta treatment. IL-1beta also increased c-Fos expression in beta cells. These data suggest that IL-1beta-induced activation of NF-kappaB and JNK controls CHOP gene expression in pancreatic beta cells, and that IL-1beta influences beta-cell function through a variety of signaling pathways.Item Structural and functional characterization of the polled interval on bovine chromosome 1(Texas A&M University, 2008-10-10) Wunderlich, Kris RakowitzThe horned condition in cattle is believed to be the wild type with morphogenesis primarily occurring after birth. The polled condition has existed since domestication and has been selected for its economic importance. The polled locus has previously been mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. In order to help us eventually identify the causative mutation, the objective of the study was to structurally and functionally characterize the polled interval from IFNAR1 to SOD1 on BTA1. Our hypothesis was that the polled locus is a tissue specific transcription factor that is expressed in the developing horn buds and acts directly or indirectly upon SOX9. A 2.5 Mb BAC contig and STS content map of the polled interval was constructed. Three candidate genes encoding transcription factors were identified within this region but only C21orf66 was expressed in the horn buds from 1 d old Bos indicus influenced calves. The C21orf66 gene has 18 exons, spans 30,976 bp of genomic DNA, and 144 SNP were identified. No single SNP discovered in C21orf66 can be attributed as the causative mutation. None of the genes from the polled interval were differentially expressed in skin and horn from 1 d old Bos indicus influenced calves. However, there were significant differences in the levels of expression of RUNX2, SOX9, BMP4, PRKCA, and FOXL2 in these samples. Expression of RUNX2 was localized to the osteoblasts, and both RUNX2 and SOX9 were expressed in sebaceous glands of the horn at 1 d of age. Histological examination of horns and scurs from newborn, 5 to 6 mo, and ~1.5 yr old Bos indicus influenced cattle suggest that horns form through intramembranous ossification. Based on the data presented herein, we propose that the polled locus is upstream of RUNX2 and SOX9 in the osteogenic pathway, and could have its primary effect on the differentiation of mesenchymal condensations. The genes IL10RB, SFRS15, C21orf66, OLIG1, OLIG2 and HUNK remain candidates for the polled locus and warrant further investigation.Item A Structural/Behavioral Analysis of the Regulation of Dopamine Signaling by Striatal RGS Proteins(2005-08-11) Waugh, Jeffrey Lynn; Gold, Stephen J.The regulators of G-protein signaling (RGS) proteins negatively modulate heterotrimeric G protein signaling by acting as GTPase activating proteins for Galpha subunits. In the striatum and nucleus accumbens, brain regions critical for control of movement, motivation and reward, overlapping RGS expression profiles suggested that functional specificity could not be explained by anatomical localization alone. We set out to assess striatal specificity within two distinct RGS pools, the R7 RGS subfamily and RGS10. The highly striatal-specific splice form RGS9-2 is a negative modulator of dopamine D2 receptor signaling, and has been shown to inhibit drug stimulated (cocaine or direct dopamine receptor agonists) locomotor activity. RGS9-2 is a member of the R7 subfamily, comprised of RGS6, -7, -9, and -11, which share highly similar subdomain structure. We analyzed the specificity of R7 modulation of dopamine receptor signaling using a novel behavioral assay. R7 RGS proteins were virally-overexpressed in rat or mouse accumbens via a stereotaxic injection of an engineered HSV virus. Following this surgery, drug-stimulated locomotor responses were assayed. We found that in rats and RGS9 KO mice, overexpression of R7 RGS proteins produces distinct locomotor and drug sensitization phenotypes, each of which occurs only during the period of RGS overexpression. Moreover, studies using truncation and chimeric RGS mutants demonstrated that while all tested subdomains were necessary for activity, only the C-terminus of RGS9-2 was sufficient to convey activity to RGS7. Lastly, RGS overexpression leads to distinct acute changes in weight: loss (RGS9-2) or gain (RGS7, RGS11). To elucidate RGS10 function in the brain, we mapped RGS10 protein in rodent brain using light microscopic and electron microscopic immunohistochemical techniques. Light microscopic analyses showed that RGS10 immunoreactivity labels all subcompartments of neurons and microglia, including their nuclei. Electron microscopy confirmed the presence of dense RGS10 immunoreactivity in euchromatin and resolved dense staining on terminals at symmetric synapses onto pyramidal cell somata. Dual-labeling histochemistry showed that RGS10 is expressed in specific neuronal cell types and circuits. Taken together, these data support a role for RGS10 in diverse processes including modulation of pre- and postsynaptic G-protein signaling and a potential role in modulating gene expression.Item Study of the Mechanisms Underlying Hippocampal Neuron Synaptogenesis: The Roles of Neurotrophin Signaling and Micrornas(2010-11-02T18:20:38Z) Zhang, Wei; Parada, LuisSynapse formation requires contacts between dendrites and axons. Although this process is often viewed as axon mediated, dendritic filopodia may be actively involved in mediating synaptogenic contacts. Brain-derived neurotrophic factor (BDNF) increases the density of dendritic filopodia and the conditional deletion of tyrosine receptor kinase B (TrkB) reduces synapse density in vivo (Luikart et al., 2005). Here, we report that TrkB associates with dendritic growth cones and filopodia, mediates filopodial motility, and does so via the phosphoinositide 3 kinase (PI3K) pathway. We used genetic and pharmacological manipulations of mouse hippocampal neurons to assess signaling downstream of TrkB. Conditional knock-out of two downstream negative regulators of TrkB signaling, Pten (phosphatase with tensin homolog) and Nf1 (neurofibromatosis type 1), enhanced filopodial motility. This effect was PI3K-dependent and correlated with synapse density. Phosphatidylinositol 3,4,5- trisphosphate (PIP3) was preferentially localized in filopodia and this distribution was enhanced by BDNF application. Thus, intracellular control of filopodial dynamics converged on PI3K activation and PIP3 accumulation, a cellular paradigm conserved for chemotaxis in other cell types. Our results suggest that filopodial movement is not random, but responsive to synaptic guidance cues. In order to further elucidate the mechanisms of BDNF-TrkB-PI3K pathway downstream signaling involved in regulating dendritic filopodial motility, we used a pharmacological approach as well as a gene expression approach to show that Rac1 and RhoA may play a role in this pathway. Rac1 positively regulated dendritic filopodial motility while RhoA had a negative effect. Our data suggest that BDNF-TrkB signaling might function to regulate the balance between Rac1 and RhoA, thus controlling dendritic filopodial motility. The developing nervous system is shaped by highly orchestrated programs of gene expression. This tight regulation is regulated by various transcriptional and post-transcriptional events that control individual gene expression. The recent discovery of small, non-coding RNAs has greatly expanded our understanding of the mechanisms that regulate gene expression at the post-transcriptional level. Here, I characterized the expression pattern of one neuronal microRNA, miR-381, and used in vitro cultured hippocampal neurons to show that miR-381 regulates neurite growth, as overexpression of miR-381 promotes neuronal dendritic branching. The effect of miR-381 on neuronal dendritic branching might be through a net regulation of multiple target genes.Item Studying physiological functions of APP using mice models(2008-09-18) Li, Hongmei; Sudhof, Thomas C.Beta-amyloid precursor protein (APP) is sequentially cleaved by alpha /beta - secrease and gamma-secrease into three pieces: a soluble ectodomain (sAPPalpha or sAPPbeta ), a p3 or Abeta peptide, and an APP intracellular domain (AICD). Mounting evidence indicates the neuroprotective and neurotrophic effects of sAPP domain. In order to find out whether sAPP domain carries out the major physiological function of APP, we have generated knock-in mice that express truncated ectodomain of APP at beta-cleavage site (sAPPbeta ) with FLAG tag at C-terminus. The knock-in mice were viable and fertile, with no obvious phenotype. However, when sAPPbeta - FLAG knock-in mice were bred to APLP2 knockout background ("knockin/ knockout" mice), the expression of sAPPbeta -FLAG failed to fully rescue the postnatal lethal phenotype of APP/APLP2 double knockout pups, suggesting sAPPbeta alone cannot substitute for the function of full length APP. We quantified the expression levels of a series of synaptic proteins and AICD-interacting proteins in the brains of new born APP/APLP2 double knockout (DKO) pubs, as well as in the "knock-in/knockout" (KI/KO) pubs, and found that Fe65 protein expression level is upregulated in brains from DKO pubs but not the KI/KO pubs. Collaborating with Dr. Yi Sun's lab investigating the DNA sequences in genome that potentially bind to AICD binding partners has shown that various promoters of a broad set of genes can bind to Fe65 and their expressions might be influenced by Fe65/AICD.Item To develop a small interfering Rna (siRNA) design and information resource to facilitate genetic manipulaton of human cells.(2004-05-25) Shah, Jyoti Khetsi; Minna, John D.Part I: Small interfering RNAs (siRNAs) have revolutionized our ability to study the effects of altering the expression of single genes in mammalian (and other) cells through targeted knockdown of gene expression. In the past, there were a set of rules designed to develop siRNA which worked efficiently in most cases. There was further refinement performed in these rules in some modern research analyses which attempted to address the question of what most closely determines siRNA functionality. I have designed and implemented a new software tool siRNA Information Resource ('sIR') that incorporates the most recent refinements in the design algorithm in order to provide fast and efficient siRNA design. sIR is a web-based computational tool which takes these existing rules for designing synthetic siRNAs and puts them in a software architecture that allows the researcher to design siRNAs for every gene. It also provides a database containing information about already developed siRNA and thus allows the researcher to access the siRNA information database consisting of siRNA information from literature and various other sources. This will ultimately help in future siRNA related discoveries. It also includes a scoring system which helps in rational selection of efficient siRNA. sIR was successfully validated using already designed and developed target siRNA sequences. Part II: One of the major problems in using chemotherapy to treat cancer is whether patients, whose tumors do not respond to one drug, would respond to another. Thus, it would be very useful if one could rationally select the appropriate chemotherapy for each patient's tumor. We are asking is whether tumor gene "expression signatures" detected by microarray analysis could identify a set of genes correlating with sensitivity or resistance to a particular drug. A large panel of breast cancer cell lines was tested with cisplatin, paclitaxel, vinorelbine, doxorubicin and gemcitabine, in vitro using a colorimetric assay to determine the concentration of drug that gives 50% growth inhibition (IC50). Gene expression profiles were also performed using Affymetrix chips and the two data sets were merged. It was found that a panel of ~100 genes were significantly up regulated (4 fold or more) for each drug in resistant cells. As an alternative approach, Pearson correlations between each gene expression data and each drug IC50 across all cell lines analyzed were determined. A positive correlation for a pair of gene and drug indicates the gene may be associated with resistance to the drug whereas a negative correlation would associate that gene with sensitivity to the drug. Some of these genes might be associated with the drug mechanism of action. We conclude that gene expression signatures do exist for individual breast tumor cell chemosensitivity and these could be of clinical significance.Item Transcriptional Regulation of Cardiac Ventricular Development(2004-05-04) Pierce, Stephanie Angelo; Srivastava, DeepakCongenital heart disease is the leading non-infectious cause of death in children. Disruption of cardiac gene expression during development can result in congenital heart defects. Numerous transcription factors regulate specific temporo-spatial events during cardiac differentiation and morphogenesis, however the mechanisms that regulate such events are largely unknown. Using a novel modified subtractive hybridization method to identify early cardiac-specific genes, we found Bop, a histone deacetylase-dependent transcriptional repressor. Bop was expressed specifically in the myocardium of the heart and somites during development. Targeted deletion of Bop in mouse resulted in hypoplasia of the right ventricle and expansion of the extracellular matrix between the myocardium and endocardium of the embryonic heart, suggesting a persistence of immature ventricular cardiomyocytes. Expression of dHAND, the evolutionarily conserved bHLH factor necessary for proper right ventricle development, was downregulated in the heart of the Bop-null embryo. In an effort to understand the mechanism by which Bop functions in cardiac differentiation and morphogenesis, the yeast two-hybrid assay was used to identify factors that interact with m-Bop in the embryonic heart. The DNA-binding factor, skNAC, and the inhibitor of mitosis, TRB3, were identified. TRB3 enhanced repression of SV40-driven luciferase activity by m-Bop. Disruption of this interaction resulted in the inability of TRB3 to enhance m-Bop's repressive activity, suggesting a novel function for TRB3 as a corepressor of m-Bop in the developing heart. skNAC was expressed in the myocardium of the heart and somites, strikingly similar to the expression pattern of Bop. While early in vitro attempts to study m-Bop and skNAC failed, in vivo efforts to study a genetic interaction are ongoing.