5 Abstract Definitive diagnosis of inherited metabolic disorders commonly depends on the measurement of enzyme activity (which is often complicated) and/or molecular genetic testing. Yet even the standard mutation analysis can bring false negative results in the case of gross chromosomal rearrangements or incorrect regulation of gene expression due to the mutations in regulatory regions. In the present study I focused on characterization of complex mutations affecting the gene encoding ornithin transcarbamylase (OTC) followed by studies of regulatory regions of OTC and GBA (the gene encoding β-glucocerebrosidase). In the first study we identified 14 novel mutations including three large deletions in a cohort of 37 patients with OTC deficiency (OTCD). Subsequently we evaluated clinical significance of all these mutations. We also found a heterozygote carrying a hypomorphic mutation and manifesting OTCD most likely due to unfavorable X-inactivation which was observed independently in three different peripheral tissues. In order to evaluate the clinical significance of a promoter variation c.-366A>G found in a family with mild OTCD we identified three alternative transcription start sites (TSSs) of human OTC and delimited the promoter. We also found a distal enhancer and performed functional analysis of both...

Indiana University-Purdue University Indianapolis (IUPUI) / The LHX3 transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie combined pituitary hormone deficiency (CPHD) disease featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved 7.9 kilobase (kb) enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. In this study, I characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in αGSU-expressing cells secreting the TSHβ, LHβ or FSHβ hormones and expressing the GATA2 and SF1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module for expression specifically in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression in other cell types. Further, these studies demonstrate significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. Furthermore understanding the regulation of human LHX3 gene will help develop tools to better diagnose and treat pituitary CPHD disease.

There is a lack of low toxicity, specific anticancer therapies and in many cancer types there are limited effective treatments. Enhancer RNAs are noncoding RNA transcripts transcribed from enhancer regions. Increasing evidence of the function of eRNA in gene regulation suggests the possibility of eRNA involvement in cancer development. This report examines literature on enhancer RNA as a potent component in transcription control specifically in cancer development. Therefore, I conducted a systematic literature review to further clarify the involvement of eRNAs in cancer. There is strong evidence of eRNA upregulating oncogenes. For instance, the eRNA (CCAT1) upregulates the oncogene MYC in colorectal cancer. Other eRNAs were also found to be required for p53-dependent cell-cycle arrest and tumour inhibition. A study showed the interplay of a long noncoding RNA with eRNAs in p53-regulated enhancers, while another showed p53-bound enhancer regions transcribing an eRNA which mediates G1 arrest, DNA repair, and tumorigenesis through its interaction with the (BRCA2) gene. Finally, a study across numerous cancer patient samples revealed a cancer/lineage specificity of eRNAs and explored the clinical feasibility of eRNA-targeted therapy. These studies demonstrate how eRNAs can be a link in cancer signalling pathways both as a regulator of oncogenes and tumour suppressor genes, as well as suggest a promising future of eRNA-targeted cancer therapy.

MicroRNAs (miRNAs) sind endogenene kurze RNA-Moleküle, die zentrale Aufgaben bei der Regulation der eukaryotischen Zellhomöostase erfüllen. MiRNAs wurden bereits als potente Immunregulatoren beschrieben. Trotz dieser Erkenntnisse blieb die Rolle dieser kurzen RNA Moleküle in Infektionen mit Mycobacterium tuberculosis weitgehend unerforscht. Im Rahmen dieser Arbeit wurde ein miRNA-Expressionsprofil von Makrophagen generiert, die mit Mycobacterium tuberculosis infiziert waren. Dies ermöglichte die Identifizierung von miRNAs, welche bei der Infektion differenziell reguliert waren. Anhand eines ex-vivo-Modells von Todesrezeptor-induzierter Apoptose konnte gezeigt werden, dass miRNA-133b apoptoseresitente Zellen empfindlich gegen Tumornekrosefaktor-alpha (TNFalpha), TNF-related apoptosis-inducing ligand (TRAIL) oder CD95 ligand (Fas/APO1 ligand) induzierte Zytotoxizität machte. Eine umfassende Studie führte zur Identifizierung der anti-apoptotischen Proteine Fas apoptosis inhibitory molecule (FAIM) und glutathione-S-transferase pi (GSTP1) als direkte Zielgene für miRNA-133b. Desweiteren zeigte sich die Expression von Osteoprotegerin (OPG) und Fettsäuresynthase (FASN), als miRNA-133b abhängig. Dies unterstrich die pleiotrope Art der pro-apoptotischen Aktivität dieser miRNA. Die Expression von miRNA-133b wurde durch Mitglieder der Toll-like Rezeptor (TLR)-Familie aktiviert. MiRNA-133b Transfektion führte zu einer verstärkten Aktivierung des Transkriptionsfaktors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB). Diese resultierte in erhöhten Mengen an Interleukinen 6 und 8 (IL6/8). Diese Ergebnisse stellen die erste detaillierte Charakterisierung von miRNA-133b im Zusammenhang der Todesrezeptor-vermittelten Apoptose und der angeborenen Immunität dar. Die erforschten molekularen Wechselwirkungen ergänzen und bereichern das Verständnis über die regulatorischen molekularen Mechanismen, die mit der Tumorentstehung und Entzündung verbunden sind. / MicroRNAs (miRNAs) are endogenous short RNA molecules which perform essential tasks in the regulation of eukaryotic cell homeostasis. During the past few years miRNAs have emerged as very potent immune regulators. Despite the consequences of this discovery for our understanding of immune response regulation hitherto virtually nothing is known about miRNA function during innate immunity to Mycobacterium tuberculosis. Herein, a miRNA expression profile of human macrophages infected with Mycobacterium tuberculosis was generated. This led to the identification of miRNAs being differentially regulated during infection. By using an experimental ex-vivo model of death receptor (DR)-induced apoptosis it could be demonstrated that miRNA-133b rendered apoptosis-resistant cells sensitive to tumor necrosis factor-alpha (TNFalpha)-, TNF-related apoptosis-inducing ligand (TRAIL)- or CD95 ligand (Fas/APO1 ligand)-activated cytotoxicity. Comprehensive analysis led to the discovery of the anti-apoptotic proteins Fas apoptosis inhibitory molecule (FAIM) and glutathione-S-transferase pi (GSTP1) as direct miRNA-133b targets. Moreover, underlining the pleiotropic and synergistic nature of miRNA activity, the expression of osteoprotegerin (OPG) and fatty acid synthase (FASN) could be further proven as miRNA-133b dependent. Expression of miRNA-133b increased following innate immune activation by members of the Toll-like receptor (TLR) family. MiRNA-133b enhanced the activity of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB). This translated into increased levels of the pro-inflammatory interleukins 6 and 8 (IL6/8). The results presented in this work represent the first detailed characterization of miRNA-133b in the context of DR-mediated apoptosis and innate immunity. The molecular interactions dissected herein improve the understanding of the regulatory processes associated with tumorigenesis and the immune response.

The study of cis-regulatory elements (CREs) will allow for increased understanding of regulation and lead to insight regarding the mechanisms governing growth, development, health, and disease. The aim of this study was to characterize the de novo in silico predictions of Arabidopsis CREs. Eight synthetic and 30 native promoter-constructs containing an eGFP/GUS reporter protein were generated for cold, genotoxic, heat, osmotic, and salt stress; the circadian clock; ABA signaling; root and epidermis tissue. Constructs were stably transformed into A. thaliana Col-0 and the effects of the CREs were evaluated by in planta stress or tissue assays using GUS expression levels. Results reveal a novel genotoxic element that specifically directs GUS expression in rosette leaves during genotoxic stress. Results also look promising for novel epidermis and root-specific elements. Results of these assays validate the de novo prediction pipeline's ability to identify novel and known CREs related to abiotic stress.

The study of cis-regulatory elements (CREs) will allow for increased understanding of regulation and lead to insight regarding the mechanisms governing growth, development, health, and disease. The aim of this study was to characterize the de novo in silico predictions of Arabidopsis CREs. Eight synthetic and 30 native promoter-constructs containing an eGFP/GUS reporter protein were generated for cold, genotoxic, heat, osmotic, and salt stress; the circadian clock; ABA signaling; root and epidermis tissue. Constructs were stably transformed into A. thaliana Col-0 and the effects of the CREs were evaluated by in planta stress or tissue assays using GUS expression levels. Results reveal a novel genotoxic element that specifically directs GUS expression in rosette leaves during genotoxic stress. Results also look promising for novel epidermis and root-specific elements. Results of these assays validate the de novo prediction pipeline's ability to identify novel and known CREs related to abiotic stress.

Background: Recent evidence suggests that the prostate cancer (PCa)-specific up-regulation of certain genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 could be associated with an aberrant expression of non-coding microRNAs (miRNA). Methods: In silico analyses were used to search for miRNAs being putative regulators of PCa-associated genes. The expression of nine selected miRNAs (hsa-miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660) as well as of the aforementioned PCa-associated genes was analyzed by quantitative PCR using 50 malignant (Tu) and matched non-malignant (Tf) tissue samples from prostatectomy specimens as well as 30 samples from patients with benign prostatic hyperplasia (BPH). Then, correlations between paired miRNA and target gene expression levels were analyzed. Furthermore, the effect of exogenously administered miR-26a on selected target genes was determined by quantitative PCR and Western Blot in various PCa cell lines. A luciferase reporter assay was used for target validation. Results: The expression of all selected miRNAs was decreased in PCa tissue samples compared to either control group (Tu vs Tf: -1.35 to -5.61-fold; Tu vs BPH: -1.17 to -5.49-fold). The down-regulation of most miRNAs inversely correlated with an up-regulation of their putative target genes with Spearman correlation coefficients ranging from -0.107 to -0.551. MiR-186 showed a significantly diminished expression in patients with non-organ confined PCa and initial metastases. Furthermore, over-expression of miR-26a reduced the mRNA and protein expression of its potential target gene AMACR in vitro. Using the luciferase reporter assay AMACR was validated as new target for miR-26a. Conclusions: The findings of this study indicate that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction.

No description available.

Trippelnegativ bröstcancer (TNBC) är den mest maligna formen av bröstcancer utan någon framträdande behandlingsbar biomarkör. Således har den djupa återfallsfrekvensen och bristen på behandlingsalternativ öppnat behovet av att förstå TNBC: s etiopatogenes och molekylära mekanism. Huvudsyftet är att kartlägga det genomomfattande differentiella uttrycket av den förmodade cancergenen (en prenyleringsgen) och dess betydelse vid trippelnegativ bröstcancer. Hi-Cap (Capture Hi-C) är en teknik som genererar högupplösta promotor-förstärkare interak- tioner med nästan en-förstärkare upplösning.Vi arbetade med cancercellinjer, MDA-MB_231, med och utan den förmodade genen. Hi-C-tekniken optimerades i enlighet därmed för cancer- cellinjen för att generera en högupplöst berikad region. Resultaten kan vidare användas för att utföra biblioteksförberedelser och bioinformatikanalys. Dessa fynd kommer att ägnas åt att upptäcka nya vägar involverade i prenylering och TNBC. / Triple-negative Breast cancer (TNBC) is the most malignant form of breast cancer with no prominent treatable biomarker. The profound recurrence rate and lack of treatment options have opened the need to understand the etiopathogenesis and molecular mechanism of TNBC. The main objective of this study is to map the genome-wide differential expression of the putative cancer gene (a prenylation gene) and its importance in Triple-Negative Breast cancer. Hi-Cap (Capture Hi-C) is a technique which generates high-resolution promoter-enhancer interactions with almost single-enhancer resolution. We worked with cancer cell lines, MDA-MB_231, with and without the putative gene. The Hi-C technique was optimized accordingly for the cancer cell line to generate a high-resolution enriched region. The results can be further used to perform library prep and bioinformatics analysis. These findings will devote to discovering novel pathways involved with prenylation and TNBC.

Das Lipopolysaccharid Bindende Protein (LBP) ist ein überwiegend in der Leber synthetisiertes Akutphaseprotein. Es bindet den Zellwandbestandteil Lipopolysaccharid (LPS) Gram-negativer Bakterien und transportiert es zu zellulären Rezeptoren, wodurch das angeborene Immunsystem aktiviert wird. In dieser Arbeit wird die Regulation der LBP-Expression in Interleukin (IL)-1, IL-6 und Dexamethason (Dex) stimulierten humanen Hepatomzelllinien HuH-7 und HepG2 untersucht. Der wichtigste Stimulator ist dabei IL-6, dessen Wirkung über die Transkriptionsfaktoren (TF) Stat-3, C/EBP-beta und AP-1 vermittelt wird. Für alle 3 TF konnten aktive Bindungsstellen auf dem LBP-Promotor nachgewiesen werden. Für IL-1-Effekte die u. a. über den TF NF-kappaB vermittelt werden, konnten ebenfalls aktive Bindungsstellen nachgewiesen werden. Die Wirkung von Dex wird über Glucocorticoid Responsive Elements (GREs) vermittelt. Auf dem LBP-Promotor befinden, sich wie gezeigt werden konnte, mehrere aktive GREs, wobei einige verstärkend und einige hemmend wirken. Eine zu beobachtende Synergiewirkung von Dex und IL-6 wird durch die Aufregulation des IL-6-Rezeptors durch Dex verursacht. Die LBP-Expression kann durch TGF (Transforming Growth Factor)-beta gehemmt werden. Der TGF-beta-Signalweg über Smads ist in den Hepatomzellen aktiv, vermittelt aber nicht den TGF-beta-Hemmeffekt, sondern eine geringe stimulierende Wirkung, die bei alleiniger TGF-beta-Inkubation auftritt. Die inhibierende Wirkung von TGF-beta wird durch Gfi-1- und AP-1-Bindungsstellen vermittelt. Die Gfi-1-Bindungsstelle nimmt dabei, wie hier erstmals gezeigt werden konnte, eine herausragende Stellung ein. Die Aufklärung der LBP-Regulation und dabei besonders die Hemmung der LBP-Expression kann mittelfristig dazu beitragen, den klinischen Verlauf von inflammatorischen und infektiösen Erkrankungen zu beeinflussen und bietet daher Potenzial für neue Therapieansätze. / Lipopolysaccharide (LPS) binding protein (LBP) is an acute phase protein with the ability to bind and transfer LPS of Gram-negative bacteria. This soluble pattern recognition molecule represents an important defense principle of the host. Regulation of the hepatic acute phase response and its termination are important mechanisms for limiting systemic inflammatory activity of the host. Here were analyze the cooperation of Interleukin (IL)-1, IL-6, and Dexamethasone (Dex) at LBP expression in the hepatoma cell lines HuH-7 and Hep G2. The major inducer of LBP expression is IL-6. Within the LBP promoter numerously highly consensus binding sites such as AP-1, C/EBP-beta? and STAT3 are present, that confer transcriptional activity as shown by truncation and mutation experiments. Additionally, activate NF-kappaB sites activated by IL-1 were detected at the LBP promoter. By mutation experiments of the promoter furthermore were found differentially active glucocorticoid response elements (GREs). The promoter contains GREs enhancing the activity as well as inhibitory ones. The enhancing effect towards LBP expression by Dex was mediated by IL-6. Dex stimulated the expression of the IL-6 receptor and therefore upregulated the IL-6 pathway. Transforming Growth Factor (TGF)-beta is able to inhibit LBP expression in stimulated cells. An AP-1 binding site was identified mediating inhibitory TGF-beta effects towards LBP promoter activity. Furthermore it was shown that a growth factor independence (Gfi)-1 binding site localized near the AP-1 site is essential for mediating the TGF-beta inhibitory effect. The relevancy of the Gfi-1 site fore mediating TGF-beta effects indicates a novel mechanism for understanding inhibitory TGF-beta effects at the transcriptional level. In summary the complex regulation of LBP were elucidate which may help to eventually develop novel intervention strategies for acute phase, sepsis, and septic shock.