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Item Acquired STAT4 deficiency as a consequence of cancer chemotherapy(2011-08-16) Lupov, Ivan; Chang, Hua-Chen; Randall, Stephen Karl, 1953-; Robertson, Michael J.Signal Transducer and Activator of Transcription 4 (STAT4) is an important transcription factor activated by IL-12 signaling. Activated STAT4 is essential for Th1 cell differentiation, a process characterized by increased potential for interferon (IFN)-γ production. Defective IFN-γ production due to STAT4 deficiency occurs after autologous stem cell transplantation for lymphoma. We have investigated the mechanisms of post-transplant STAT4 deficiency. The tumor-bearing state is ruled out to be the cause because STAT4 levels were not significantly different in peripheral blood mononuclear cells (PBMCs) obtained from lymphoma patients prior to treatment and healthy control subjects. The magnitude of the decrease in STAT4 levels corresponded with increasing intensity of chemotherapeutic treatment in vivo. Furthermore, treatment of normal PBMC cultures or a natural killer (NK) cell line with chemotherapy drugs in vitro also resulted in reduced STAT4 protein and reduced IL-12-induced IFN-γ production. Chemotherapy drugs are shown to have no impact on the stability of STAT4 mRNA, while steady-state levels of STAT4 transcripts are decreased in lymphoma patients. Our findings demonstrated that chemotherapeutic drugs up-regulate the ubiquitination rates of the STAT4 protein, which in turn promotes its degradation via the proteasome-mediated pathway. Treatment with the proteasome inhibitor bortezomib largely reversed the chemotherapy-induced STAT4 deficiency. Thus, acquired STAT4 deficiency in lymphoma patients is a consequence of treatment with chemotherapy. These results have important implications for design of optimal immunotherapy for lymphoma.Item Analysis of integration sites of transgenic sheep generated by lentiviral vectors using next-generation sequencing technology(2014-07-31) Chen, Yu-Hsiang; Malkova, Anna; Cornetta, Kenneth; Randall, Stephen Karl, 1953-; Atkinson, SimonThe development of new methods to carry out gene transfer has many benefits to several fields, such as gene therapy, agriculture and animal health. The newly established lentiviral vector systems further increase the efficiency of gene transfer dramatically. Some studies have shown that lentiviral vector systems enhance efficiency over 10-fold higher than traditional pronuclear injection. However, the timing for lentiviral vector integration to occur remains unclear. Integrating in different stages of embryogenesis might lead to different integration patterns between tissues. Moreover, in our previous study we found that the vector copy number in transgenic sheep varied, some having one or more copies per cells while other animals having less than one copy per cell suggesting mosaicism. Here I hypothesized that injection of a lentiviral vector into a single cell embryo can lead to integration very early in embryogenesis but can also occur after several cell divisions. In this study, we focus on investigating integration sites in tissues developing from different germ layers as well as extraembryonic tissues to determine when integration occurs. In addition, we are also interested in insertional mutagenesis caused by viral sequence integration in or near gene regions. We utilize linear amplification-mediated polymerase chain reaction (LAM-PCR) and next- generation sequencing (NGS) technology to determine possible integration sites. In this study, we found the evidence based on a series of experiments to support my hypothesis, suggesting that integration event also happens after several cell divisions. For insertional mutagenesis analysis, the closest genes can be found according to integration sites, but they are likely too far away from the integration sites to be influenced. A well-annotated sheep genome database is needed for insertional mutagenesis analysis.Item Blood on FTA™ Paper: Does Punch Location Affect the Quality of a Forensic DNA Profile?(2013-03-06) Carter, Megan Elizabeth; Picard, Christine; Siegel, Jay A.; Randall, Stephen Karl, 1953-Forensic DNA profiling is widely used as an identification tool for associating an individual with evidence of a crime. Analysis of a DNA sample involves observation of data in the form of an electropherogram, and subsequently annotating a DNA “profile” from an individual or from the evidence. The profile obtained from the evidence can be compared to reference profiles deposited in a national DNA database, which may include the potential contributor. Following a match, a random match probability is calculated to determine how common that genotype is in the population. This is the probability of obtaining that same DNA profile by sampling from a pool of unrelated individuals. Each state has adopted various laws requiring suspects and/or offenders to submit a DNA sample for the national database (such as California’s law that all who are arrested must provide a DNA sample). These profiles can then be associated with past unsolved crimes, and remain in the database to be searched in the event of future crimes. In the case of database samples, a physical sample of the offender’s DNA must be kept on file in the laboratory indefinitely so that in the event of a database hit, the sample is able to be retested. Current methods are to collect a buccal swab or blood sample, and store the DNA extracts under strict preservation conditions, i.e. cold storage, typically -20° C. With continually increasing number of samples submitted, a burden is placed on crime labs to store these DNA extracts. A solution was required to help control the costs of properly storing the samples. FTA™ paper was created to fulfill the need for inexpensive, low maintenance, long term storage of biological samples, which makes it ideal for use with convicted offender DNA samples. FTA™ paper is a commercially produced, chemically treated paper that allows DNA to be stored at room temperature for years with no costly storage facilities or conditions. Once a sample is required for DNA testing, a small disc is removed and is to be used directly in a PCR reaction. A high quality profile is important for comparing suspect profiles to unknown or database profiles. A single difference between a suspect and evidentiary sample can lead to exclusion. Unfortunately, the DNA profile results yielded from the direct addition have been unfavorable. Thus, most crime laboratories will extract the DNA from the disc, leading to additional time and cost to analyze a reference sample. Many of the profiles from the direct addition of an FTA™ disc result in poor quality profiles, likely due to an increase in PCR inhibitors and high concentrations of DNA. Currently, standardized protocols regarding the recommended locations for removal of a sample disc from a bloodspot on an FTA™ card does not exist. This study aims to validate the optimal location by comparing DNA profiles obtained from discs removed from the center, halfway, and edge locations of a bloodspot from 50 anonymous donors. Optimal punch location was first scored on the number of failed, partial or discordant profiles. Then, profile quality was determined based on peak characteristics of the resulting DNA profiles. The results for all three disc locations were 5.3% failed amplifications, 4.2% partial amplifications, and one case of a discordant profile. Profile quality for the majority of the samples showed a high incidence of stutter and the absence of non-template adenylation. Of the three disc locations, the edge of the blood stain was ideal, due to a presumably lower concentration of DNA and likely more dilute amount of the PCR inhibitor heme. Therefore, based on the results of this study, there is a greater probability of success using a sample from the edge of a blood stain spotted in FTA™ paper than any other location of the FTA™ card.Item Characterization of a cold-responsive dehydrin promoter(2014-08-27) Osadczuk, Elizabeth A.; Randall, Stephen Karl, 1953-; Watson, John C., 1953-; Picard, Christine; Atkinson, SimonDehydrins are type II LEA proteins induced in many plants during drought, low temperature, and high salinity to confer stress tolerance. AtERD14 is an Arabidopsis thaliana dehydrin that functions in part of the cold stress pathway. AtERD14 has chaperone-like capabilities that allow it to bind and protect various proteins from dehydration stresses. In order to determine the necessary components for cold induction of AtERD14, AtERD14prom::GFP/GUS and AtERD14prom::AtERD14 in AtERD14 KO constructs were created and stably transformed into A. thaliana. Analysis of the constructs showed the AtERD14 promoter alone was insufficient to respond to cold, and it was necessary to attach the AtERD14 coding region to the promoter to induce a cold response in ERD14. On the other hand, the RD29aprom::GFP/GUS promoter did respond to cold stress, indicating that RD29a does not require its coding region to support an increased amount of reporter activity after cold stress. The protoplast transformation system, while capable of transient expression of introduced constructs in protoplasts, was difficult for use for cold-inducible expression.Item Differentiation and characterization of cell types associated with retinal degenerative diseases using human induced pluripotent stem cells(2014-07-31) Gupta, Manav; Meyer, Jason S.; Belecky-Adams, Teri; Randall, Stephen Karl, 1953-Human induced pluripotent stem (iPS) cells have the unique ability to differentiate into 200 or so somatic cell types that make up the adult human being. The use of human iPS cells to study development and disease is a highly exciting and interdependent field that holds great promise in understanding and elucidating mechanisms behind cellular differentiation with future applications in drug screening and cell replacement studies for complex and currently incurable cellular degenerative disorders. The recent advent of iPS cell technology allows for the generation of patient-specific cell lines that enable us to model the progression of a disease phenotype in a human in vitro model. Differentiation of iPS cells toward the affected cell type provides an unlimited source of diseased cells for examination, and to further study the developmental progression of the disease in vitro, also called the “disease-in-a-dish” model. In this study, efforts were undertaken to recapitulate the differentiation of distinct retinal cell affected in two highly prevalent retinal diseases, Usher syndrome and glaucoma. Using a line of Type III Usher Syndrome patient derived iPS cells efforts were undertaken to develop such an approach as an effective in vitro model for studies of Usher Syndrome, the most commonly inherited disorder affecting both vision and hearing. Using existing lines of iPS cells, studies were also aimed at differentiation and characterization of the more complex retinal cell types, retinal ganglion cells (RGCs) and astrocytes, the cell types affected in glaucoma, a severe neurodegenerative disease of the retina leading to eventual irreversible blindness. Using a previously described protocol, the iPS cells were directed to differentiate toward a retinal fate through a step-wise process that proceeds through all of the major stages of neuroretinal development. The differentiation process was monitored for a period of 70 days for the differentiation of retinal cell types and 150 days for astrocyte development. The different stages of differentiation and the individually derived somatic cell types were characterized by the expression of developmentally associated transcription factors specific to each cell type. Further approaches were undertaken to characterize the morphological differences between RGCs and other neuroretinal cell types derived in the process. The results of this study successfully demonstrated that Usher syndrome patient derived iPS cells differentiated to the affected photoreceptors of Usher syndrome along with other mature retinal cell types, chronologically analogous to the development of the cell types in a mature human retina. This study also established a robust method for the in vitro derivation of RGCs and astrocytes from human iPS cells and provided novel methodologies and evidence to characterize these individual somatic cell types. Overall, this study provides a unique insight into the application of human pluripotent stem cell biology by establishing a novel platform for future studies of in vitro disease modeling of the retinal degenerative diseases: Usher syndrome and glaucoma. In downstream applications of this study, the disease relevant cell types derived from human iPS cells can be used as tools to further study disease progression, drug screening and cell replacement strategies.Item The Direct Reprogramming of Somatic Cells: Establishment of a Novel System for Photoreceptor Derivation(2013-08-22) Steward, Melissa Mary; Meyer, Jason S.; Dai, Guoli; Randall, Stephen Karl, 1953-; Atkinson, SimonPhotoreceptors are a class of sensory neuronal cells that are deleteriously affected in many disorders and injuries of the visual system. Significant injury or loss of these cells often results in a partial or complete loss of vision. While previous studies have determined many necessary components of the gene regulatory network governing the establishment, development, and maintenance of these cells, the necessary and sufficient profile and timecourse of gene expression and/or silencing has yet to be elucidated. Arduous protocols do exist to derive photoreceptors in vitro utilizing pluripotent stem cells, but only recently have been able to yield cells that are disease- and/or patient-specific. The discovery that mammalian somatic cells can be directly reprogrammed to another terminally-differentiated cell phenotype has inspired an explosion of research demonstrating the successful genetic reprogramming of one cell type to another, a process which is typically both more timely and efficient than those used to derive the same cells from pluripotent stem cell sources. Therefore, the emphasis of this study was to establish a novel system to be used to determine a minimal transcriptional network capable of directly reprogramming mouse embryonic fibroblasts (MEFs) to rod photoreceptors. The tools, assays, and experimental design chosen and established herein were designed and characterized to facilitate this determination, and preliminary data demonstrated the utility of this approach for accomplishing this aim.Item Effects of carbon nanotubes on airway epithelial cells and model lipid bilayers : proteomic and biophysical studies(2014) Li, Pin; Blazer-Yost, Bonnie; Witzmann, F. A. (Frank A.); Randall, Stephen Karl, 1953-; Petrache, Horia; Wang, XianzhongCarbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 h exposure to 10 μg/mL and 100 ng/mL of two common carbon nanoparticles, singleand multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatics analysis of proteins identified by LFQMS. Interestingly, after exposure to a high concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT, respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins common to both kinds of nanotubes are associated with the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The decrease in expression of the majority proteins suggests a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure. To examine the effect of nanotubes on the plasma membrane, we investigated the interaction of short purified MWCNT with model lipid membranes using a planar bilayer workstation. Bilayer lipid membranes were synthesized using neutral 1, 2-diphytanoylsn-glycero-3-phosphocholine (DPhPC) in 1 M KCl. The ion channel model protein, Gramicidin A (gA), was incorporated into the bilayers and used to measure the effect of MWCNT on ion transport. The opening and closing of ion channels, amplitude of current, and open probability and lifetime of ion channels were measured and analyzed by Clampfit. The presence of an intermediate concentration of MWCNT (2 μg/ml) could be related to a statistically significant decrease of the open probability and lifetime of gA channels. The proteomic studies revealed changes in response to CNT exposure. An analysis of the changes using multiple databases revealed alterations in pathways, which were consistent with the physiological changes that were observed in cultured cells exposed to very low concentrations of CNT. The physiological changes included the break down of the barrier function and the inhibition of the mucocillary clearance, both of which could increase the risk of CNT’s toxicity to human health. The biophysical studies indicate MWCNTs have an effect on single channel kinetics of Gramicidin A model cation channel. These changes are consistent with the inhibitory effect of nanoparticles on hormone stimulated transepithelial ion flux, but additional experiments will be necessary to substantiate this correlation.Item Evaluation of storage conditions on DNA used for forensic STR analysis(2014) Beach, Lisa Renae; Picard, Christine; Goodpaster, John V. (John Vincent); Randall, Stephen Karl, 1953-Short tandem repeat (STR) analysis is currently the most common method for processing biological forensic evidence. STRs are highly polymorphic and allow for a strong statistical power of discrimination when comparing deoxyribonucleic acid (DNA) samples. Since sample testing and court proceedings occur months, if not years apart, samples must be stored appropriately in the event additional testing is needed. There are generally accepted methods to store DNA extracts long-term; however, one universally recognized method does not exist. The goal of this project was to examine various methods of storage and make recommendations for a universal storage method that maintained DNA integrity over time. Four variables were evaluated: storage buffer, storage temperature, initial storage concentration and the effects of repeated freeze-thaw cycles. DNA quantity was assessed using real-time polymerase chain reaction and DNA quality was evaluated using STR genotyping. Overall, the Tris-EDTA (TE) buffer outperformed nuclease free water as a long-term storage buffer for DNA extracts. Stock tubes stabilized concentration better than single use aliquots when eluted with TE while tube type was not significant when water was the buffer. For samples stored in TE, temperature had no effect on DNA integrity over time, but samples stored in water were largely affected at room temperature. Additionally, the greater the initial DNA concentration, the less likely it was to degrade in water. As a result of this research, DNA extracts from forensic samples should be stored long-term in TE buffer with a minimum concentration of 0.1 ng/μL. When water is the buffer, frozen storage is recommended.Item Evaluation of the IrisPlex DNA-based eye color prediction tool in the United States(2014-07-31) Dembinski, Gina M.; Picard, Christine; Randall, Stephen Karl, 1953-; Goodpaster, John V. (John Vincent)DNA phenotyping is a rapidly developing area of research in forensic biology. Externally visible characteristics (EVCs) can be determined based on genotype data, specifically from single nucleotide polymorphisms (SNPs). These SNPs are chosen based on their association with genes related to the phenotypic expression of interest, with known examples in eye, hair, and skin color traits. DNA phenotyping has forensic importance when unknown biological samples at a crime scene do not result in a criminal database hit; a phenotype profile of the sample can therefore be used to develop investigational leads. IrisPlex, an eye color prediction assay, has previously shown high prediction rates for blue and brown eye color in a European population. The objective of this work was to evaluate its utility in a North American population. We evaluated the six SNPs included in the IrisPlex assay in an admixed population sample collected from a U.S.A. college campus. We used a quantitative method of eye color classification based on (RGB) color components of digital photographs of the eye taken from each study volunteer and placed in one of three eye color categories: brown, intermediate, and blue. Objective color classification was shown to correlate with basic human visual determination making it a feasible option for use in future prediction assay development. In the original IrisPlex study with the Dutch samples, they correct prediction rates achieved were 91.6% for blue eye color and 87.5% for brown eye color. No intermediate eyes were tested. Using these samples and various models, the maximum prediction accuracies of the IrisPlex system achieved was 93% and 33% correct brown and blue eye color predictions, respectively, and 11% for intermediate eye colors. The differences in prediction accuracies is attributed to the genetic differences in allele frequencies within the sample populations tested. Future developments should include incorporation of additional informative SNPs, specifically related to the intermediate eye color, and we recommend the use of a Bayesian approach as a prediction model as likelihood ratios can be determined for reporting purposes.Item Expression and Function of the PRL Family of Protein Tyrosine Phosphatase(2012-12) Dumaual, Carmen Michelle; Stauffacher, Cynthia; Randall, Stephen Karl, 1953-; Malkova, Anna; Sandusky, George Earl, 1945-The PRL family of enzymes constitutes a unique class of protein tyrosine phosphatase, consisting of three highly homologous members (PRL-1, PRL-2, and PRL-3). Family member PRL-3 is highly expressed in a number of tumor types and has recently gained much interest as a potential prognostic indicator of increased disease aggressiveness and poor clinical outcome for multiple human cancers. PRL-1 and PRL-2 are also known to promote a malignant phenotype in vitro, however, prior to the present study, little was known about their expression in human normal or tumor tissues. In addition, the biological function of all three PRL enzymes remains elusive and the underlying mechanisms by which they exert their effects are poorly understood. The current project was undertaken to expand our knowledge surrounding the normal cellular function of the PRL enzymes, the signaling pathways in which they operate, and the roles they play in the progression of human disease. We first characterized the tissue distribution and cell-type specific localization of PRL-1 and PRL-2 transcripts in a variety of normal and diseased human tissues using in situ hybridization. In normal, adult human tissues we found that PRL-1 and PRL-2 messages were almost ubiquitously expressed. Only highly specialized cell types, such as fibrocartilage cells, the taste buds of the tongue, and select neural cells displayed little to no expression of either transcript. In almost every other tissue and cell type examined, PRL-2 was expressed strongly while PRL-1 expression levels were variable. Each transcript was widely expressed in both proliferating and quiescent cells indicating that different tissues or cell types may display a unique physiological response to these genes. In support of this idea, we found alterations of PRL-1 and PRL-2 transcript levels in tumor samples to be highly tissue-type specific. PRL-1 expression was significantly increased in 100% of hepatocellular and gastric carcinomas, but significantly decreased in 100% of ovarian, 80% of breast, and 75% of lung tumors as compared to matched normal tissues from the same subjects. Likewise, PRL-2 expression was significantly higher in 100% of hepatocellular carcinomas, yet significantly lower in 54% of kidney carcinomas compared to matched normal specimens. PRL-1 expression was found to be associated with tumor grade in the prostate, ovary, and uterus, with patient gender in the bladder, and with patient age in the brain and skeletal muscle. These results suggest an important, but pleiotropic role for PRL-1 and PRL-2 in both normal tissue function and in the neoplastic process. These molecules may have a tumor promoting effect in some tissue types, but inhibit tumor formation or growth in others. To further elucidate the signaling pathways in which the PRLs operate, we focused on PRL-1 and used microarray and microRNA gene expression profiling to examine the global molecular changes that occur in response to stable PRL-1 overexpression in HEK293 cells. This analysis led to identification of several molecules not previously associated with PRL signaling, but whose expression was significantly altered by exogenous PRL-1 expression. In particular, Filamin A, RhoGDIalpha, and SPARC are attractive targets for novel mediators of PRL-1 function. We also found that PRL-1 has the capacity to indirectly influence the expression of target genes through regulation of microRNA levels and we provide evidence supporting previous observations suggesting that PRL-1 promotes cell proliferation, survival, migration, invasion, and metastasis by influencing multi-functional molecules, such as the Rho GTPases, that have essential roles in regulation of the cell cycle, cytoskeletal reorganization, and transcription factor function. The combined results of these studies have expanded our current understanding of the expression and function of the PRL family of enzymes as well as of the role these important signaling molecules play in the progression of human disease.