Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease with no cure. TAR-DNA binding protein 43 (TDP-43) is the major component of the cytoplasmic inclusions characteristic of ALS. Transgenic Drosophila lines expressing wild-type, mutant and splice variants of human TDP-43 were generated. I find that ubiquitous expression of all TDP-43 transgenes, except for TDP-43∆C-term, is sufficient to cause lethality. I also show that eye-specific expression of a TDP-43∆N-term splice variant, which localizes diffusely to the cytosol, results in increased cell toxicity suggesting an association between cytosolic localization and toxicity. Consistent with this model, I find that the TDP-43∆N-term splice variant is capable of recruiting full length TDP-43 into the cytoplasm, and I suggest this may represent an initiating event in TDP-43-linked ALS. Altogether, my results seem to indicate that exclusion of TDP-43 from the nucleus rather than its presence in aggregates is linked to increased cytotoxicity and lethality in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease with no cure. TAR-DNA binding protein 43 (TDP-43) is the major component of the cytoplasmic inclusions characteristic of ALS. Transgenic Drosophila lines expressing wild-type, mutant and splice variants of human TDP-43 were generated. I find that ubiquitous expression of all TDP-43 transgenes, except for TDP-43∆C-term, is sufficient to cause lethality. I also show that eye-specific expression of a TDP-43∆N-term splice variant, which localizes diffusely to the cytosol, results in increased cell toxicity suggesting an association between cytosolic localization and toxicity. Consistent with this model, I find that the TDP-43∆N-term splice variant is capable of recruiting full length TDP-43 into the cytoplasm, and I suggest this may represent an initiating event in TDP-43-linked ALS. Altogether, my results seem to indicate that exclusion of TDP-43 from the nucleus rather than its presence in aggregates is linked to increased cytotoxicity and lethality in ALS.

No description available.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2000. / Includes bibliographical references (leaves 174-192). Also available online through Digital Dissertations.

Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias / La hembra canina presenta características reproductivas particulares comparado con las otras especies de mamíferos domésticos, donde destaca el arresto meiótico prolongado del ovocito reanudando la meiosis posterior a la ovulación. El desarrollo del ovocito es regulado por señales entre el ovocito y las células foliculares que le rodean mediante conexones constituidos por conexinas, de ellas la Conexina 43 es la proteína con mayor expresión en el ovario. El objetivo de este trabajo fue estudiar la expresión génica de Cx43 y de su proteína en células foliculares de perra durante el desarrollo folicular y a lo largo del ciclo estral. Usando un lupa estereoscópica se aislaron células de folículos preantrales y antrales de ovarios de perra en anestro, proestro, estro y diestro. Se utilizó q-PCR para la evaluación de los niveles de mARN y Western Blot e lnmunohistoquímica para la cuantificación y localización de la proteína respectivamente. Los valores de expresión génica y densidad óptica de las bandas de Western Blot se analizaron con ANOVA; además, se hicieron correlaciones de Pearson. La Cx43 se encontró en todos los folículos desde folículo primordial hasta preovulatorio, la expresión del gen de Cx43 y su proteína codificante varió a lo largo del ciclo estral, presentando los mayores niveles de mARN en anestro (P<0,05) y los mayores niveles de Cx43 en diestro (P<0,05), adicionalmente la expresión de mRNA y de Cx43 no disminuyeron en folículos preovulatorios, sugiriendo que la comunicación entre el ovocito y las células foliculares se mantienen, lo que podría explicar el arresto meiótico prolongado de la perra. La correlación entre mARN y proteína fue negativa en la mayoría de etapas del ciclo, lo que se podría asociar a los mecanismos de control traduccional a las que son sometidas las conexinas. / The female canine has particular reproductive characteristics compared to the other species of domestic mammals, where it stands out the prolonged meiotic arrest of the oocyte that resumes meiosis after ovulation. Oocyte development is regulated by signals between the oocyte and follicular cells that sorround it by connexons constituted of connexins, of these, Connexin 43 is the protein with the highest expression in the ovary. The objective of this work was to study the gene expression of Cx43 and its protein in dog follicular cells during follicular development and throughout the estrous cycle. Using a stereoscopic magnifying glass, cells were isolated from preantral and antral follicles of the ovaries of bitches in anestrus, proestrus, estrus and diestrus. q-PCR was used for the evaluation of mRNA levels and Western Blot and lmmunohistochemistry for the quantification and localization of the protein respectively. The values of gene expression and optical density of the Western Blot bands were analyzed with ANOVA; in addition , Pearson correlations were made. The Cx43 was found in all the follicles from primordial to preovulatory, the expression of the Cx43 gene and its coding protein varied throughout the estrous cycle, presenting the highest levels of mRNA in anoestrus (P <0.05) and the highest levels of Cx43 in diestrus (P <0.05), additionally the expression of mRNA and Cx43 did not decrease in preovulatory follicles, suggesting that communication between the oocyte and follicular cells is maintained, which could explain the prolonged meiotic arrest of the dog. The correlation between mRNA and protein was negative in most stages of the cycle, which could be associated with the mechanisms of translational control to which connexins are subjected. / Financiamiento: Proyecto Fondecyt 1171670

Indiana University-Purdue University Indianapolis (IUPUI) / Old age is associated with increased bone fragility and risk of fracture as a result of skeletal alterations, including low bone density and cortical thinning. Further, apoptotic osteocytes accumulate in old mice and humans. We have previously shown that mice lacking osteocytic connexin (Cx) 43 (Cx43ΔOt) exhibit a phenotype similar to that of the aging skeleton, with elevated osteocyte apoptosis and an associated increase in osteoclastogenesis. These findings suggest that osteocyte apoptosis results in the release of factors that recruit osteoclasts to bone surfaces close to areas that contain apoptotic osteocytes. However, the specific chemotactic signals, the events mediating their release, and the mechanisms of their action remain unknown. Consistent with this notion, we also found that HMGB1 released by Cx43-deficient (Cx43def) MLO-Y4 osteocytic cells enhances osteoclastogenesis in part by increasing osteocytic RANKL, which promotes osteoclastogenesis, and, at the same time, directly stimulating osteoclastogenesis. Further, expression of the pro-survival microRNA (miR), miR21, is low in Cx43def cells and bones from old female mice, and low miR21 levels increase osteocyte apoptosis. However, surprisingly, mice lacking miR21 (miR21ΔOt) have decreased osteoclast number and activity even under conditions of elevated osteocyte apoptosis; suggesting that osteocytic miR21 may mediate osteoclast precursor recruitment/survival induced by apoptotic osteocytes. However, whether HMGB1/miR21 are released by osteocytes, and if the HMGB1 receptors, receptor for advanced glycation end products (RAGE) and/or tolllike receptor (TLR4) are involved in osteoclast recruitment in Cx43ΔOt and old mice is unknown. The overall objectives of this series of studies were to elucidate the mechanisms

No description available.

Yes / Motor neuron disease (MND) is a progressive neurodegenerative disease with no effective treatment. One of the principal pathological hallmarks is the deposition of TAR DNA binding protein 43 (TDP-43) in cytoplasmic inclusions. TDP-43 aggregation occurs in both familial and sporadic MND; however, the mechanism of endogenous TDP-43 aggregation in disease is incompletely understood. This study focused on the induction of cytoplasmic accumulation of endogenous TDP-43 in the motor neuronal cell line NSC-34. The endoplasmic reticulum (ER) stressor tunicamycin induced casein kinase 1 (CK1)-dependent cytoplasmic accumulation of endogenous TDP-43 in differentiated NSC-34 cells, as seen by immunocytochemistry. Immunoblotting showed that induction of ER stress had no effect on abundance of TDP-43 or phosphorylated TDP-43 in the NP-40/RIPA soluble fraction. However, there were significant increases in abundance of TDP-43 and phosphorylated TDP-43 in the NP-40/RIPA-insoluble, urea-soluble fraction, including high molecular weight species. In all cases, these increases were lowered by CK1 inhibition. Thus ER stress signalling, as induced by tunicamycin, causes CK1-dependent phosphorylation of TDP-43 and its consequent cytosolic accumulation. / Funded by a biomedical research grant from the Motor Neurone Disease Association (ref Rattray/Apr15/837-791). The Bioimaging Facility microscopes used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund.

Die vorgelegte Dissertationsschrift dient der Identifizierung des Mechanismus, über welchen neonatale Kardiomyozyten der Ratte biaxialen zyklischen Stretch wahrnehmen. Angriffspunkt dafür bildeten sowohl Stretch Activated Ion Channels (SAIC) als auch die mit Integrinen assoziierte Focal Adhesion Kinase (FAK). Die Inhibition der SAIC erfolgte mit Gadolinium und eine Blockade der FAK konnte durch den FAK-Inhibitor II erreicht werden. Eigens angelegte Zellkulturen wurden unter definierten Parametern für 24 Stunden unter Einwirkung genannter Stoffe gestretcht und anschließend einer Analyse mittels Immunfluoreszenz und Western Blot unterzogen. Gestretchte Kardiomyozyten richteten sich schräg zur Achse der einwirkenden Kraft aus und wiesen eine Polarisierung von Connexin 43 auf, außerdem zeigte sich dessen gesteigerte Expression. Durch die Blockade der FAK konnte lediglich eine aufgehobene Polarisierung von Connexin 43 bei unveränderter Expression und Ausrichtung der Kardiomyozyten festgestellt werden. Auch die Mikrotubuli veränderten nach Stretch ihre Orientierung bezogen auf die Zellachse. Die zunächst annähernde Parallelität der Fasern zeigte sich nach Inhibition der FAK deutlich aufgelockert. Für die Aktinfilamente konnte dies nicht nachgewiesen werden. Die Integrine dienen demnach der Wahrnehmung von Stretch und vermitteln die Polarisierung von Connexin 43 sowie die Orientierung der Mikrotubuli über die FAK. Für die anderen genannten Prozesse ist die Aktivierung eines FAK-unabhängigen Integrin-Signalweges denkbar. Gadolinium hatte insgesamt keinen Effekt auf beschriebene Veränderungen, sodass ein Einfluss der SAIC auf Stretch-induzierte Veränderungen in Kardiomyozyten ausgeschlossen werden konnte. Gleichzeitig konnte durch die Beobachtung der Polarisierung von Microtubule Organizing Center, Kinesin und Golgi-Apparat die Hypothese der sich durch Stretch orientierenden neonatalen Kardiomyozyte unterstützt werden, welche die Voraussetzung für die Anordnung von Connexin 43 an den Zellpolen darstellen könnte. Sowohl die Selbstorganisation des Myokards im Rahmen der Embryogenese als auch die Pathophysiologie verschiedener kardialer Erkrankungen könnte dadurch womöglich besser verstanden werden.