No description available.

No description available.

Alternatives Spleißen ist von großer Bedeutung für die Steuerung der Genexpression und Proteindiversität. Die Cdc2-like Kinasen (Clk) und DNA Topoisomerase I (DNA topo I) steuern alternatives Spleißen über die Phosphorylierung von Serine/Arginin-reichen (SR) Proteinen. Tissue Factor (TF) und die endotheliale Stickstoffmonoxidsynthase (eNOS) beeinflussen maßgeblich die Endothelfunktion. Alternatives Spleißen führt zur Bildung von Isoformen von TF („full length“ (fl)TF, alternativ gespleißter humaner (asH)TF) und eNOS (eNOS, eNOS 13A, B und C). Wie das alternative Spleißen von TF und eNOS gesteuert wird und welchen Einfluss dies auf die Endothelfunktion hat ist unbekannt und wurde in dieser Arbeit untersucht. Die TNF-a-Stimulation der HUVEC erhöhte die Expression beider TF-Isoformen. Die Clk-Inhibition reduzierte die mRNA-Expression von flTF und asHTF. Die DNA topo I-Inhibition erhöhte die asHTF-Expression und verringerte flTF. Die Inhibition der DNA topo I reduzierte ebenfalls die Expression des flTF-Proteins aber nicht von asHTF, verbunden mit einer reduzierten TF-Aktivität. Die Clk-Inhibition reduzierte das flTF-Protein und die TF-Aktivität nur leicht aber asHTF vollständig. Die Inhibition der Kinasen beeinflusste das SR Protein-Phosphorylierungsmuster. Die Inhibition von SF2/ASF und SRp75 mittels siRNAs veränderte die Expression und die Aktivität von TF in HUVEC. Die TNF-a-Stimulation von HUVEC induzierte ferner die mRNA-Expression von eNOS 13A, B und C, nicht aber von eNOS. Dies führte zu einer Reduktion der eNOS-Aktivität. Die Inhibition der DNA topo I, nicht aber der Clks hob diese Effekte auf. Diese Daten zeigen, dass die DNA topo I sowie die Clks an der Regulation der endothelialen TF-Expression und Aktivität beteiligt sind, während die eNOS-Isoformexpression und Aktivität nur durch die DNA topo I beeinflusst wurde.Diese Ergebnisse eröffnen neue Einblicke in die Regulation des alternativen Spleißens von TF und eNOS sowie dessen Einfluss auf die Endothelfunktion. / Alternative splicing is an important mechanism to control gene expression and protein diversity. The Cdc2-like kinases (Clk) and DNA topoisomerase I (DNA topo I) control alternative splicing by regulating the phosphorylation state of serine/arginine-rich (SR) proteins. Tissue Factor (TF) and the endothelial nitric oxide synthase (eNOS) are crucial for the endothelial function. Alternative splicing lead to the formation of different isoforms of TF („full length“ (fl)TF, alternatively spliced human (asH)TF) and eNOS (eNOS, eNOS 13A, B and C). How alternative splicing of TF and eNOS is regulated as well as the impact on endothelial function is still unknown and was investigated in this study. The stimulation of HUVEC with TNF-a induced the expression of both TF isoforms. The inhibition of Clks reduced the mRNA expression of flTF and asHTF. DNA topo I inhibition increased asHTF and reduced flTF mRNA expression. Inhibition of DNA topo I also reduced flTF but not asHTF on protein level in stimulated cells leading to a reduced TF activity. Clk-inhibition slightly reduced flTF protein and TF activity, whereas asHTF was completely blocked. The inhibition of both kinases altered the phosphorylation pattern of SR proteins. The siRNA-mediated inhibition of SF2/ASF and SRp75 modified the TF isoform expression and TF activity in TNF-a-induced HUVEC. Moreover, stimulation of HUVEC with TNF-a induced the mRNA expression of eNOS 13A, B and C, but not of eNOS. This led to a reduction of eNOS activity. The inhibition of DNA topo I but not of Clks abolished these TNF-a-mediated effects in HUVEC. These data indicate DNA topo I and the Clks to be involved in the regulation of endothelial TF expression and activity, whereas, eNOS isoform expression and activity was influenced by DNA topo I, but not Clks in TNF-a-stimulated HUVEC. In conclusion, this study reveals new insights into the regulation of alternative splicing of TF and eNOS and the impact of these processes on endothelial function.

In einem RT-PCR Ansatz aus neuronalen post mortem Gewebe des Menschen konnten EPB41 (Erythrozytäres Protein Bande 4.1) Spleißformen in verschiedenen Hirnregionen nachgewiesen werden. In einem weiteren RT-PCR Ansatz wurden höhermolekulare p4.1R-Spleißormen generiert, kloniert und zwei der erhaltenen Spleißformen (Klon 9 und Klon 13) charakterisiert. In einer In-situ-Hybridisierungsstudie an humanen Temporalkortex und Hippocampus konnten EPB41-Isoformen in fast allen Neuronen nachgewiesen werden. In immunhistochemischen Untersuchungen mit selbstgenerierten p4.1R spezifischen Antikörper wurden ebenfalls ausschließlich Neurone markiert. In proteinbiochemischen Untersuchungen konnte in verschiedenen humanen Hirnareale mit den p4.1R spezifischen Antikörpern eine 110 kDa und 120 kDa immunreaktive Bande nachgewiesen werden. In Experimenten an Primärkulturen von Rattenneuronen konnte eine Herunterregulation der p4.1R Proteine sowie der mRNA von p4.1R durch Verarmung des funktionellen Pools an G-Proteinen der Rho-Familie in der Zelle gezeigt werden. Die GTPasen der Rho-Familie regulieren unter anderem die Plastizität des Dendritenbaumes von Neuronen. / In a RT-PCR approach using human postmortem cerebral tissue from different brain regions several EPB41 (erythrocyte protein band 4.1) spliceforms could be generated. The amplificates were cloned and two of the highmolecular EPB41 spliceforms Klon 9 and Klon 13 were characterized. Klon 9 is a new spliceform, Klon13 is identical with EPB41 (accesion number AF156225). In an in situ hybridization study the EPB41 spliceforms were detected in almost all neurons of the temporal cortex and the hippocampus. Immunhistochemical localization of the p4.1R immunreactive proteins in human temporal cortex using p4.1R specific peptide antibodies, confirmed these results. The stning pattern of soma and dendrites of the neurones was punctuated. In Western Blot experiments a 110 kDa and 120 kDa p4.1R immunreactive proteinband was detected. A regulation of the protein 4.1R immunreactive proteins as well as the mRNA of protein 4.1 was found in experiments in which the functional pool of Rho GTPases in hippocampal primary neurones of the rat was manipulated.

Chronic Obstructive Pulmonary Disease (COPD) is the 3rd leading cause of death in the US, and idiopathic pulmonary fibrosis (IPF), a type of Interstitial Lung Disease (ILD), is a fast acting, irreversible disease that leads to mortality within 3-5 years. RNA-sequencing provides the opportunity to quantitatively examine the sequences of millions mRNAs, and offers the potential to gain unprecedented insights into the structure of chronic non-malignant lung disease transcriptome. By identifying changes in splicing and novel loci expression associated with disease, we may be able to gain a better understanding of their pathogenesis, identify novel disease-specific biomarkers, and find better targets for therapy. Using RNA-seq data that our group generated on 281 human lung tissue samples (47=Control, 131=COPD, 103=ILD), I initially defined the transcriptomic landscape of lung tissue by identifying which genes were expressed in each tissue sample. I used a mixture model to separate genes into reliable and not reliable expression. Next, I employed reads that overlapped splice junctions in a linear model interaction term to identify disease-specific differential splicing. I identified alternatively spliced genes between control and disease tissues and validated three (PDGFA, NUMB, SCEL) of these genes with qPCR and nanostring (a hybridization-based barcoding technique used to quantify transcripts). Finally, I implemented and improved a pipeline to perform transcriptome assembly using Cufflinks that led to the identification of 1,855 novel loci that did not overlap with UCSC, Vega, and Ensembl annotations. The loci were classified into potential coding and non-coding loci (191 and 1,664, respectively). Expression analysis revealed that there were 120 IPF-associated and 10 emphysema-associated differentially expressed (q < 0.01) novel loci. RNA-seq provides a high-resolution transcript-level view of the pulmonary transcriptome and its modification in lung disease. It has enabled a new understanding of the lung transcriptome structure because it measures not only the transcripts we know but also the ones we do not know. The approaches and improvements I have employed have identified these novel targets and make possible further downstream functional analysis that could identify better targets for therapy and lead to an even better understanding of chronic lung disease pathogenesis. / 2999-01-01T00:00:00Z

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Anna Zolkiewska / The ADAM (A Disintegrin and Metalloprotease) family of multi-domain proteins modulates a number of cellular signaling pathways in both normal and cancerous cells. ADAM12 has been shown to be a candidate cancer gene for breast cancer and its expression is up-regulated in breast tumors. The human ADAM12 transcript is alternatively spliced. One of these splice variants encodes a transmembrane ADAM12 isoform, ADAM12-L, which has been demonstrated to release cell signaling molecules from the cell surface. Another variant encodes a secreted protease, ADAM12-S, which cleaves extracellular matrix proteins and other secreted proteins. Although these variants are expressed from the same promoter, their relative expression levels are highly discordant. Here, I demonstrate variant-specific regulation of ADAM12 transcripts by microRNAs. Members of the microRNA-29 and microRNA-200 families target the unique 3’UTR of the ADAM12-L transcript and cause transcript degradation. Additionally, I show the presence of a novel ADAM12 splicing event in which 9 additional nucleotides are inserted in the region encoding the autoinhibitory pro-domain. I demonstrate that this novel variant is expressed in breast epithelial cells and breast cancer cell lines. The resulting protein isoform does not undergo proteolytic processing to activate the protease. Additionally, trafficking of the novel isoform to the cell surface is impaired and this isoform is localized to the endoplasmic reticulum. Finally, I determined a role for ADAM12-L in the progression of triple negative breast cancers (TNBCs). These tumors are lacking expression of hormone receptors and the HER2 receptor. HER2 is a member of the epidermal growth factor receptor (EGFR) family and the loss of the HER2 receptor causes tumors to rely on EGFR for propagating pro-growth signals. I show here that, in TNBC tumors, ADAM12-L expression is strongly correlated with poor patient prognosis and increased activation of EGFR. These data suggest that in TNBCs, ADAM12-L enhances tumor growth via EGFR activation. Collectively, the data presented here demonstrate (a) transcript-specific regulation of ADAM12 in breast cancer, (b) the existence of a novel splice variant and protein isoform with impaired cellular trafficking, and (c) an important role of the ADAM12-L isoform in EGFR activation in TNBC.

No description available.

X-linked myopathy with excessive autophagy (XMEA) is a skeletal muscle disorder inherited in recessive fashion, affecting boys and sparing carrier females. Onset is in childhood with weakness of the proximal muscles of the lower extremities, progressing slowly to involve other muscle groups. Pathological analysis of skeletal muscle biopsies shows no inflammation, necrosis or apoptosis. Instead, forty to 80% of fibers exhibit giant autophagic vacuoles with heterogeneous degradative content. Numerous critical functions of all cells are compartmentalized in particular pH environments established by the intracellular transmembrane V-ATPase proton pump complex. Assembly of this complex, directed by the Vma21p chaperone, is well-studied in yeast but completely unknown in other organisms. The aim of my project was a better understanding of XMEA pathogenesis, with a focus on finding the disease-causing gene. In this thesis, I identify mutations in XMEA patients in a novel, previously uncharacterized gene, which we name VMA21. Most of the mutations are located in splicing-relevant positions and decrease splicing efficiency. After establishing that XMEA is caused by hypomorphic alleles of the VMA21 gene, I show that VMA21 is the diverged human orthologue of the yeast Vma21p protein, and that like Vma21p, it is an essential assembly chaperone of the V-ATPase. Decreased VMA21 reduces V-ATPase activity, resulting in altered lysosomal pH and a blockage at the degradative step of autophagy. Towards understanding disease pathogenesis, I show evidence of compensatory autophagy upregulation consecutive to the impaired clearance. Accumulated autolysosomes due to increased autophagy continue to face the degradative block and are slow to disappear. Instead, they merge to each other and form the characteristic giant XMEA vacuoles. These results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy. This work describes the clinical outcome at the cusp of tolerable reduction in V-ATPase, with implications on common diseases like osteoporosis and cancer metastasis, where increased V-ATPase activity is an important component. Our XMEA patients show that the safety margin of reducing V-ATPase activity in humans is wide, increasing the potential to utilize chemical or biological V-ATPase inhibitors as possible therapies.

Cytochrome P450 enzymes (P450s) are involved in cancer development and treatment due to their roles in the oxidative metabolism of various endogenous (e.g. oestrogen) and exogenous (e.g. tamoxifen) compounds. It is well-known that intermediate P450 metabolites derived from oestrogen metabolism are associated with breast carcinogenesis. The main aim of this project was to profile the cytochrome P450 and P450-regulatory nuclear receptor mRNAs in a series of breast cancer cell lines (BCCs) and compare this profile with normal breast cells. This study used the qualitative reverse transcriptasepolymerase chain reaction (RT-PCR) to detect mRNA expression of target genes. Results showed CYP1B1, CYP2D6, CYP2J2, CYP2R1, CYP2U1 and CYP4X1 mRNA to be present in all cell lines. CYP2A6, CYP2C8, CYP2C18, CYP2F1 and CYP4Z1 mRNA were expressed in oestrogen receptor (ER)-positiveCaucasian and ER-negative Afro- Caribbean BCCs. Although no differences in P450 mRNA were observed between the different ethnic groups, these preliminary findings suggest potential similarities in the ERpositive Caucasian and ER-negative Afro-Caribbean BCCs which warrant further investigation The CYP4Z1 PCR product was identified as two distinct bands. Specific primer sets were used to demonstrate potential intron retention in CYP4Z1. Using established in vitro models for the study of regulatory mechanisms of CYP4Z1, T47D and ZR-75-1 breast cancer cell lines were used to determine the appropriate nuclear receptors (i.e. progesterone receptor, glucocorticoid receptor or peroxisome proliferator-activated receptor alpha ). These findings suggest that there may be an alternative receptor mechanism involved in CYP4Z1 mRNA induction in these cells. In conjunction, pre-treatment of these two cell lines with the RNA synthesis inhibitor actinomycin D followed by the agonists showed a significant reduction (p < 0.05) of CYP4Z1 mRNA levels and inhibited CYP4Z1 induction by either progesterone, dexamethasone or pirinixic acid, indicating that these agonists have effects on CYP4Z1 mRNA transcription or stability. In contrast, cycloheximide differentially affected the level of CYP4Z1 mRNA induction by these agonists. Taken together, these results suggest that CYP4Z1 mRNA induction in T47D and ZR-75-1 is mediated through differential cell type specific regulatory mechanisms and there is evidence for differential regulation of the splice variants.

Nous montrons l’utilisation de la puce exon d’Affymetrix pour l’analyse simultanée de l’expression des gènes et de la variation d’isoformes. Nous avons utilisé les échantillons d’ARN du cerveau et des tissus de référence qui ont été antérieurement utilisés dans l’étude du consortium MicroArray Quality Control (MAQC). Nous démontrons une forte concordance de la quantification de l’expression des gènes entre trois plateformes d’expression populaires à savoir la puce exon d’Affymetrix, la puce Illumina et la puce U133A d’Affymetrix. Plus intéressant nous montrons que la majorité des discordances entre les trois plateformes résulterait des positions différentes des sondes à travers les plateformes et que les variations d’isoforme exactes ne peuvent être identifiées que par la puce exon. Nous avons détecté avec succès, entre les tissus de référence et ceux du cerveau, une centaine de cas d’évènements d’épissage alternatif. La puce exon est requise dans l’analyse de l’épissage alternatif associé aux pathologies telles que les cancers et les troubles neurologiques. Comme application de cette technologie, nous avons analysé les variations d’épissage dans la métastase du cancer de sein développé dans le model de la souris. Nous avons utilisé une gamme bien définie de trois lignées de tumeur mammaire ayant différents potentiels métastatiques. Par des analyses statistiques, nous avons répertorié 2623 transcripts présentant des variations d’expression et d’isoformes entre les types de tumeur. Une analyse du réseau de gènes montre qu’environ la moitié d’entre eux est impliquée dans plusieurs activités cellulaires, ainsi que dans nombreux cancers et désordres génétiques. / We demonstrate how the Affymetrix Exon Array, can be used to simultaneously profile gene expression level, and detect variations at the isoform level. We use a well studied set of brain and reference RNA samples previously used by the MicroArray Quality Control (MAQC) consortium study. We demonstrate a high concordance of gene expression measurements among three popular expression platforms – Affymetrix Exon Array, Illumina, and Affymetrix 3’ targeted array (U133A). More interestingly, we show that in many cases of discordant results, the effect can be explained by differential probe placements across platforms, and that the exact isoform change can only be captured by the Exon Array. Finally, we are able to detect hundreds of cases of splicing, transcript initiation, and termination differences between the brain and reference tissue samples. We propose that the Exon Array is a highly effective tool for transcript isoform profiling, and that it should be used in a variety of systems where such changes are known to be associated with diseases, such as neurological disorders and cancer. As application, we used the Affymetrix Exon Array to identify metastatis-specific alternative splicing in mouse model of breast cancer at the whole genome level. We utilize a well characterized series of three mouse mammary tumor lines exhibiting varying levels of metastatic potential. We catalogued 2623 transcripts which exhibit splicing aberrations during the progression of cancer. A genetic pathway analysis shows the half of them implicated in several cell activities, cancers and genetic disorders.