Browsing by Subject "Gene expression"

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The Effect of Including Capsaicin and Gut Microbiota Feed Additives on Growth Performance of Nursery Pigs
(2020-12) Rosa Medina, Eduardo
Feed additives such as capsaicin, prebiotics, and microbiota feed additives can increase postweaning growth performance of pigs while decreasing antibiotic use. Therefore, we conducted two experiments to evaluate the growth performance of newly weaned pigs fed capsaicin, prebiotics, and microbiota feed additives. On the first experiment, we observed that feeding Capsaicin to sows during lactation and the corresponding offspring during weaning was more effective at increasing feed efficiency of weaning pigs than feeding Capsaicin only in lactation or only to nursery pigs. Likewise, there was a greater number of genes differentially expressed when sows and their offspring consumed Capsaicin than feeding to sows or nursery pigs alone. On the second experiment, we observed that the offspring of multiparous sows had greater post-weaning growth performance than those of primiparous sows and that feeding four different microbial feed additives did not increase the growth performance of the offspring of either group of sows.
Gene and protein expression in canine follicular thyroid carcinoma.
(2011-08) Metivier, Kristy Stacy
The major goal of this study was to investigate the molecular characteristics of canine follicular thyroid carcinoma (FTC). This is a rapidly growing and highly aggressive tumor in dogs, and many patients present with evidence of invasion or metastasis. Some smaller independent studies have attempted to evaluate the role of single molecules such as p53 and thyroid transcription factor-1 in tumor development, often with inconclusive results. In the present study, a genome-wide approach was employed to achieve the first objective of determining the gene expression profile of FTC compared to normal thyroid tissue. Microarray analysis was performed in a pilot study using five FTC tissues and four normal thyroid gland tissues, and this showed 489 transcripts as differentially expressed between the two groups; 242 were down-regulated and 247 were up-regulated. Some important biological functions that were affected include regulation of cell shape, cell adhesion, regulation of MAP kinase activity, angiogenesis, and regulation of cell migration. Osteopontin was a gene of interest as tumors consistently expressed it at high levels while it was expressed at low levels in all of the healthy samples. One of its up-stream regulators, VEGFA, was also differentially expressed but with a smaller fold change. The expression of osteopontin was validated by quantitative PCR using three groups: non-invasive FTC (tumors with capsular invasion only), invasive FTC (tumors with capsular and vascular invasion), and normal thyroid tissue. Both non-invasive FTC and invasive FTC had higher osteopontin gene expression than normal thyroid tissue but the two tumor groups were not different from each other. The second objective was to determine the protein expression of osteopontin and VEGFA in the same cases using semi-quantitative scoring of tissues stained by immunohistochemistry. The results were similar, with non-invasive and invasive FTC having higher osteopontin protein expression than normal thyroid tissue, but showing no difference from each other. With respect to VEGFA, there was no difference in gene or protein expression among the three groups. The final objective was to determine the plasma concentration of VEGFA and osteopontin in dogs diagnosed with FTC compared to clinically healthy dogs using a commercially available canine-specific ELISA. In this case, both VEGFA and osteopontin had higher plasma concentrations in dogs with FTC compared to healthy dogs. A small number of FTC cases were also measured two weeks after surgical removal of the tumor. Some cases showed a post-surgical decrease in VEGFA and osteopontin while others either remained the same or increased; however, the sample size for this comparison was small. The consistent expression of osteopontin in both tissues and blood suggest that it is a promising marker for identification of canine FTC. As in human studies of osteopontin in aggressive carcinomas, it is also possible to investigate it as a means of monitoring response to therapy, recurrence, and clinical outcome.
Immunological responses during the incubation period and acute phase of naturally acquired primary Epstein-Barr virus infection
(2015-07) Dunmire, Samantha
Epstein-Barr virus (EBV) in a human herpesvirus. It infects about 90% of the human population, and is the main causative agent of infectious mononucleosis. The incubation period, the time between viral acquisition and onset of symptoms, is unusually long in patients presenting with infectious mononucleosis, lasting about six weeks. In addition to causing acute illness, there can also be long-term consequences as the result of acquisition of the virus, including nasopharyngeal carcinoma and lymphoma. Nevertheless, there remains a surprising dearth of knowledge regarding the establishment of and immune response to persistent EBV infection in its natural hosts, especially during the incubation period. We sought to address many of these gaps by studying the incubation period, acute phase, and convalescence of undergraduates experiencing infectious mononucleosis during primary natural EBV infection in a cohort of prospectively studied volunteers. Particular attention was paid to the previously uncharacterized incubation period. Our findings have focused on understanding the immune response that occurs in young adults presenting with infectious mononucleosis, via gene expression changes as observed in peripheral blood mononuclear cells and innate and adaptive immune cells. Using a systems biology approach we discovered that important gene expression changes occur during the immune response to primary EBV infection. A typical antiviral type I interferon response was not observed at onset of infectious mononucleosis symptoms, but rather up to two weeks prior. The gene expression signature at symptom onset was dominated by cell cycle related genes, probably due to the CD8 T cell lymphocytosis, and type II interferon regulated genes. Interestingly, comparison of the EBV signature with other acute viral infections revealed very little similarity. The EBV signature showed the greatest similarity with hemophagocytic syndromes. This result is consistent with the view that infectious mononucleosis is an immunopathologic disease, and is supported by evidence that EBV can cause hemophagocytic lymphohistiocytosis. As an extension of this work, we carefully examined changes in cellular phenotypes and population frequencies to determine if there were significant alterations to certain compartments during the response to primary EBV infection. We observed a type I interferon signature in a larger subset of study participants during the incubation period. This response was concurrent with the transition of virally infected B cells from the oral cavity to the blood, a decline in plasmacytoid dendritic cells from the circulation, and a polyclonal CD8 T cell activation. No EBV specific CD8 T cells activation was observed until the onset of infectious mononucleosis symptoms. A major obstacle to understanding EBV related sequelae has been the lack of an efficient animal model for EBV infection, although progress in primate and mouse models has recently been made. Taken together, the data compiled in this thesis provide important first descriptions of the immune responses that occur during the establishment of a natural persistent infection in humans. Key future challenges are to develop protective vaccines and effective treatment regimens.
MicroRNAs As Predictors of Nucleoside Analog Sensitivity in Acute Myeloid Leukemia
(2015-12) Bhise, Neha
Acute myeloid leukemia (AML) is the most aggressive form of hematological malignancies. Despite advances in treating AML, development of resistance to nucleoside analog therapy remains one of the major obstacles in AML treatment. Various factors, including SNPs in genes, epigenetics, etc. play a role in mediating variability in response to nucleoside analog therapy. Recent studies have shown that microRNAs can also serve as regulators of gene expression that can contribute to variability in response to therapeutic agents. Thus the main objective of the thesis was to determine the role of microRNAs as predictors of variability in nucleoside analog response. To our knowledge no studies have been reported that identify microRNAs as predictors of response to cytarabine therapy in AML patients. In chapter II we used a translational approach of conducting in vitro and clinical study to identify microRNAs that were predictive of overall survival in AML patients. Our study conclusively identified that miR107-Myb, miR-378-granzyme B and miR10a-MAP4K4 as miRNA-mRNA pairs that can be used as predictors of overall survival in AML patients. Additionally, we also showed that the miRNAs mechanistically regulate the expression of these mRNAs by binding to the 3’- untranslated region of these mRNAs. miRNAs can also cause variability in response to cytarabine therapy by regulating the expression of the genes involved in disposition of cytarabine. In chapter III we identified that miR-24 and miR-34a as regulator of DCTD (an enzyme involved in inactivation of cytarabine) and DCK (activating enzyme), respectively. These miRNAs along with other miRNAs can be used as part of biomarker signature that can be used to predict the overall survival in AML patients. In chapter IV, we determined the impact of cytarabine treatment on in vivo cytarabine-induced changes in leukemia cell transcriptome and miRNA expression, to evaluate their impact on clinical outcome. In the first part of this chapter, we identified key genes (such as tumor suppressors DKK3, TRIM33, PBRM1, an oncogene SET, cytidine-deaminase family members APOBEC2 and APOBEC3G) influenced by cytarabine infusion that were also predictive of response. In the second part of this chapter, using data from clinical studies, we identified several miRNAs that were altered by cytarabine treatment. The changes in the expression of these miRNAs resulted in alteration in gene expression that correlated with the overall survival in AML patients. Additionally, using cell lines we were able to identify various miRNA – mRNAs that were altered by drug treatment, indicating that the therapy itself can influence the predictive ability of miRNAs as biomarkers. Significant data has been published on cytarabine as it is the standard of care in AML patients, however, there is limited knowledge about newer nucleoside analogs such as clofarabine. In chapter V, using in vitro methods, we identified several microRNAs, such as miR-16, miR-515 cluster, etc., that can be used as predictors of response for clofarabine therapy. Our data clearly suggests that there are several distinct microRNAs that can be used as predictors of response to clofarabine therapy in AML patients. We propose doing a clinical study to evaluate the clinical utility of these miRNAs as predictors of response. In summary, using a translational approach, we were able to identify miRNAs and several miRNA-mRNA pairs that can be used as biomarkers of response to cytarabine therapy. Additionally, we also identified that drug therapy itself can influence the outcome in AML patients. These findings are clinically important as they will help provide a new strategy to optimize dosing of nucleoside analogs in AML patients which in turn would lead to better overall survival while reducing the side effects.
Network-based support vector machines for classification of microarray gene expression data.
(2009-09) Zhu, Yanni
The importance of network-based approach to identifying biological markers for diag- nostic classification and prognostic assessment in the context of microarray has been increasingly recognized. Standard methods treat all genes independently and identically a priori and ignore the biological observation that genes function together in biological processes. For binary classification, we are motivated to improve predictive accuracy and gene selection by developing novel network-based classification tools that explicitly incorporate interrelationships of genes as described by gene networks. We propose three network-based support vector machines (SVM) by suitably forming the penalty term. The neighboring-gene (NG) penalty groups pairwise gene neighbors and sums up the L1-norm of each group over the entire network, leading to NG-SVM. NG-SVM tends to select pairs of neighboring genes. The disease-gene-centric (DGC) penalty is constructed on groups defined on an upper-lower hierarchy imposed on the undirected network. DGC-SVM aims to detect collectives of genes clustering together and around some key disease genes. The truncated L1-norm (TL1) penalty intends to correct bias induced by penalization through a threshold parameter C > 0 built into the L1-norm as used in NG-SVM and DGC-SVM. Simulation studies and real data applications demonstrate that the proposed methods are able to capture more disease genes and less noise genes than the existing popular methods, standard SVM and L1-SVM. We conclude that the proposed methods have the potential to be effective classification tools for microarrays and other high-dimensional data.
Phosphorylated and SUMO-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression.
(2012-07) Knutson, Todd Philip
Introduction: Progesterone receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by MAPK and CDK2. Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (i.e. a repressive modification). Antagonism of PR SUMOylation by mitogenic protein kinases provides a mechanism for derepression (i.e. transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression. Methods: Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild-type (SUMOylation-capable) or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Gene sets were validated by RT-qPCR. Recruitment of coregulators and histone methylation levels were determined by chromatin immunoprecipitation. Changes in cell proliferation and survival were determined by MTT and western blotting. Finally, human breast tumor cohort datasets were probed to identify PR-associated gene signatures; metagene analysis was employed to define survival rates in patients whose tumors express a PR gene signature. Results: “SUMO-sensitive” PR target genes (i.e. repressed by PR SUMOylation) primarily include genes required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors are preferentially recruited to enhancer regions of derepressed genes (i.e. MSX2, RGS2, MAP1A, and PDK4) along with the steroid receptor coactivator, CBP, and MLL2, a histone methyltransferase mediator of nucleosome remodeling. PR SUMOylation blocks these events, suggesting that SUMO modification of PR prevents interactions with mediators of early chromatin remodeling at “closed” enhancer regions. SUMO-deficient (phospho-Ser294) PR gene signatures are significantly associated with ERBB2-positive luminal breast tumors and predictive of early metastasis and shortened survival. Treatment with antiprogestin or MEK inhibitor abrogated expression of SUMO-sensitive PR targetgenes and inhibited proliferation in BT-474 (ER+/PR+/ERBB2+) breast cancer cells. Conclusions: We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters target gene selection in breast cancer cells. Phosphorylation-induced PR deSUMOylation favors a permissive chromatin environment via recruitment of CBP and MLL2. Patients whose ER+/PR+ tumors are driven by hyperactive (i.e. derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin. Supplementary files: The supplementary files presented in this dissertation are fully described in the appendices. They include: (A) Antibodies used in this study, (B) PCR primer sets used in this study, (C) Genes differentially regulated by wild-type and SUMO-deficient PR, (D) Overlapping lists of PR-dependent target genes from previously described gene expression microarrays, (E) The PR ligand-dependent (LD) and ligand-independent (LI) KR>WT gene signatures, (F) Breast tumor Oncomine concepts associated with the LD KR>WT gene signature.
Proliferative Enteropathy: Pathogenesis and Host Adaptation
(2013-03) Vannucci, Fabio Augusto
Proliferative enteropathy (PE) is an infectious disease caused by an obligate intracellular bacterium, Lawsonia intracellularis, and characterized by thickening of the intestinal epithelium due to enterocyte proliferation. The overall goals of this thesis were to improve the understanding of the pathogenesis of PE by evaluating phenotypic traits, genome variations and transcriptome patterns of L. intracellularis infection and to evaluate the adaptation of the bacterium to porcine and equine hosts. In the first section, the susceptibility of pigs to a homologous porcine L. intracellularis isolate continuously grown in vitro was assessed. A loss of virulence after 40 passages of the bacteria in culture was established. The comparative whole genome analysis of the pathogenic (low passage) and non-pathogenic (high passage) isolates identified the loss of a prophage-associated genomic island in the non-pathogenic variant. This chromosomal island proved not to be essential for the virulent phenotype, since it was not identified in horses clinically affected with PE. However, this genetic element was associated with host-adapted L. intracellularis variants. While pathogenic porcine isolates harbor this genetic element, it was absent in equine isolates and PE-affected horses. Gene expression profiling of a porcine pathogenic isolate showed a wider transcriptional landscape compared with the non-pathogenic variant. In addition, genes highly activated in vitro by the pathogenic variant also were significantly expressed in vivo. However, genes identified in the genomic island were not expressed by intracellular bacteria either in vitro or in vivo. The proliferative changes exhibited by the infected enterocytes in vivo were associated with deregulation of the G1 phase of the host cell cycle and repression of membrane transporters related to nutrient acquisition, characterizing a malabsorptive syndrome as the major mechanism involved in the poor performance and growth of affected animals. Finally, an alternative method for cultivation of L. intracellularis was developed in order to perform a cross-species experimental study evaluating the susceptibility of pigs and horses to a porcine and an equine L. intracellularis isolate. Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates indicating that host susceptibility is driven by the origin of the L. intracellularis strain.
The Road From Variants To Traits: How Regulatory Variants Affect Gene Expression & Organismal Phenotypes
(2024-03) Renganaath, Kaushik
Nature hosts an incredible amount of diversity and beneath such diversity lies fascinating genetics that we have spent years trying to decode. Differences in our DNA sequences lead to variation in organismal traits. Most of these variants have been found to reside in noncoding portions of the genome, implying that a lot of organismal trait variation arises from variation in gene expression levels. Advances in sequencing technology have over the years allowed us to map hundreds of genomic loci underlying gene expression variation, and these loci are called expression quantitative trait loci (eQTLs). These eQTLs are of two types, local and trans, depending on their proximity to the genes they regulate. Local eQTLs regulate expression of genes in close genomic proximity while trans eQTLs regulate distant genes. Today, we possess a vast catalog of eQTLs across multiple taxa. Yet, we don’t fully understand the mechanisms by which eQTLs affect organismal traits. In this dissertation, I computationally dissect the mechanisms connecting genetic variation, gene expression and organismal traits in yeast Saccharomyces cerevisiae. As the first eukaryotic organism to have its genome fully sequenced, S.cerevisiae has over the years been a workhorse for understanding the genetics underlying complex traits. We today have comprehensive sets of QTLs underlying traits like gene expression and growth in yeast that account for most of heritable variation in these traits, allowing us to investigate the mechanisms by which eQTLs lead to organismal trait variation. In this dissertation, I characterize causal variants underlying local eQTLs in yeast (Chapter II) and the mechanisms by which eQTLs influence growth in different conditions (Chapter III). My work unravels fundamental principles by which eQTLs influence complex organismal traits.
A study of Fusarium graminearum virulence factors.
(2011-05) Menke, Jon R.
The plant pathogen F. graminearum (Gibberella zeae) presents a two-fold threat to farmers and consumers. Not only does this filamentous fungus cause the disease Fusarium head blight (FHB) that results in significant yield loss in infected grains, it also taints these grains with potent mycotoxins harmful to humans, animals, and plants alike. Equally alarming is the evidence that grain can appear to be physically sound while still being significantly contaminated with trichothecene mycotoxins. Tri12 encodes a predicted major facilitator superfamily transporter protein suggested to play a role in the export of trichothecene mycotoxins produced by the Fusarium species. However, the role of Tri12p in toxin sensitivity and plant pathogenicity of Fusarium graminearum was previously unknown. In this study, the correct intron positions for Tri12 in F. graminearum (FgTri12) were established using cDNA sequencing, EST data, and comparative genomics. Reverse genetics was used to establish that FgTri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta. To identify the subcellular location of FgTri12p during toxin production in culture, FgTri12p was tagged with eGFP. FgTri12p::eGFP was localized in small motile vesicles, the plasma membrane, and the lumen of vacuoles within fungal cells. Treatment of cells with latrunculin A resulted in the absence of motile vesicles labeled with FgTri12p::eGFP, suggesting their formation relies upon actin polymerization. To determine if FgTri12p co-localizes with enzymes involved in trichothecene metabolism, its cellular fate was compared with FgTri1p::eGFP, a fluorescently tagged oxygenase catalyzing a key intermediate step in trichothecene biosynthesis. While FgTri1p::eGFP initially localizes to small motile vesicles and later accumulates in the vacuole, during the period of initial trichothecene biosynthesis it is targeted to the periphery of intermediate sized vesicles, presumed to be the site of toxin synthesis. These results indicate FgTri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta. The interactions between F. graminearum and its hosts – wheat, rice, or barley – differ in disease severity and the levels of trichothecenes that accumulate in response to infection. The transcriptome of the fungus in rice and wheat was examined in order to identify genes expressed in planta. The hypothesis that fungal genes expressed in planta, but not during growth in culture, could include those that determine the plant infection phenotype was tested by reverse genetics: Four genes expressed exclusively in planta were deleted and two of these were determined to significantly alter disease phenotype. FGSG_03539, also called Tri9, is a previously uncharacterized gene in the major trichothecene biosynthetic gene cluster. A mutant with a tri9 deletion has attenuated virulence and lower trichothecene levels in wheat compared to wild type or a mutant strain complemented with the intact Tri9 gene. FGSG_11164 encodes a predicted trypsin protease and deletion of this gene also results in a small but significant reduction in pathogenicity toward wheat. The results demonstrate that a reverse genetic approach using in planta gene expression data may supplement forward genetic screens for identifying genes encoding virulence factors.

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