Hannover, University, Diss., 2002.

Thesis (doctoral)--Technische Hochschule, Aachen, 2000.

Universiẗat, Diss.--Jena, 2007.

Tenascin X (TNX) is a matricellular protein involved in regulating cellular functions by interacting with other extracellular matrix (ECM) proteins within the cell matrix and has anti-adhesive properties evidenced in tumours and wound healing. TNX is the only member of the tenascin family that is lost in Joint Hypermobility Syndrome (JHS) and exerts a crucial architectural function. Of importance, TNX deficient and JHS patients have gastrointestinal (GI) dysfunction. Despite this association no study has described the role of TNX in the GI tract. Thus, the aim of this thesis was to characterise the expression of TNX in the stomach and colon in mouse and human tissue. Second, we aimed to elucidate the functional role of TNX using TNX knockout (TNX KO) mice. Expression studies revealed TNX in vagal afferent endings in the mouse, and myenteric cell bodies in human stomach. In colon, TNX strongly associated with cholinergic submucous and myenteric neurons in both species, however, was not found in CGRP positive fibres. Cell bodies in nodose ganglia, dorsal root ganglia, ventral and dorsal horn were also TNX positive. Functional studies in stomach, using single fibre electrophysiology showed TNX KO mice had increased vagal afferent mechanoreceptor sensitivity. Octanoic acid breath test revealed rapid gastric emptying in TNX KO. Colonic manometry showed the amplitude and frequency of colonic contractions were reduced in TNX KO mice, particularly in the distal colon. Ussing chamber studies measuring changes in ion flux (indirect measure of secretion) showed no major difference between TNX KO and wild type (WT) mice. The specific localisation of TNX with neuronal structures in the gut is shown here for the first time suggesting that TNX is more than just an architectural protein. Indeed, its role in specific GI functions supports this observation and provides a mechanism for GI symptoms in JHS.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 149-179)

No description available.

Die vorliegende Arbeit befasst sich mit der Entwicklung und Anwendung elektrochemischer Biosensoren zur Erweiterung oder zum Ersatz herkömmlicher Diagnostikverfahren. Als Basis für die Biosensoren wurden Elektrodenarraychips entworfen und im Reinraum gefertigt. Die als 9WPtE bezeichneten Elektrodenarrays waren aus 3 x 3 Elektrodenpaaren im 96-well-Maßstab (ANSI-Standard) aufgebaut. Jedes Elektrodenpaar bestand aus einer kreisrunden Arbeitselektrode mit einem Durchmesser von 1,9 mm und einer Gegenelektrode als offenem Kreisring um die Arbeitselektrode mit einem Durchmesser von 7 mm. Außerhalb des Reinraums wurden separate Messkammern und Ag/AgCl-Referenzelektroden integriert. Sowohl das Referenzsystem als auch die Signalqualität der 9WPtE-Elektrodenarraychips wurden mittels Zyklovoltammetrie, Impedanzspektroskopie und Rasterkraftmikroskopie analysiert und anhand dieser Untersuchungen optimiert. Das Augenmerk lag hierbei auf den Produktionsprozessen zur Herstellung der Elektrodenarraychips, auf den Elektrolytbedingungen für die elektrochemischen Messungen und auf der Recyclebarkeit der Chips. Die Funktionalisierung der Arbeitselektroden der 9WPtE-Chips erfolgte mit sich selbst-organisierenden Schichten aus Thiolen. An die Thiole wurden mittels Chemoligation die biologischen Erkennungskomponenten kovalent gekoppelt. Mit dem 9WPtE-Elektrodenarray wurde auf diese Weise ein funktionsfähiger kompetitiver Immunosensoren gegen den Tumormarker Tenascin C entwickelt. Außerdem wurden der 9WPtE-Chip und ein zusätzlich entwickelter Durchflusssensor, basierend auf dem Prinzip des 9WPtE, genutzt, um die Möglichkeit der Detektion ganzer eukaryotischer Zellen zu untersuchen.

Thesis (Ph.D. in Cell Biology, Stem Cells, and Development) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 102-114). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Toll-like receptor (TLR)-4 is a pattern recognition receptor (PRR) that recognises the pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) produced by Gram-negative bacteria. LPS binds to Myeloid differentiation 2 (MD-2)/TLR-4 heterodimers, driving their dimerisation and inducing a conformational change of the intracellular TLR-4 toll/interleukin-1 receptor (TIR) domains. The adaptor protein Myeloid differentiation primary response gene 88 (MyD88)-adaptor-like (Mal)/TIR domain-containing adaptor protein (TIRAP) then binds to the TIR domains of TLR-4 and acts as a bridge for MyD88 which goes on to form the myddosome, a large protein complex of six to eight MyD88 molecules and four Interleukin-1 receptor- associated kinase (IRAK) 4 and four IRAK1/2 molecules. This triggers a signalling cascade which results in nuclear factor (NF)-κB transcription factor activation and production of pro-inflammatory effector molecules such as the cytokine Tumour Necrosis Factor (TNF)-α. Upon activation TLR-4 is also endocytosed where it interacts with a second set of adaptor proteins TIR-domain-containing adaptor- inducing interferon (IFN)-β (TRIF)-related adaptor molecule (TRAM) and TRIF to initiate the type I IFN response. How TLR-4 dimerisation results in the formation of the oligomeric myddosome is not fully understood, but it is possible that the stoichiometry of Mal/TIRAP may be important in the formation of this protein complex. The aim of my thesis was to determine the stoichiometry of Mal/TIRAP at the plasma membrane of immortalised bone marrow derived macrophages (iBMDMs) and whether this stoichiometry changes upon stimulation with different TLR-4 ligands. To investigate Mal/TIRAP stoichiometry I first developed a viral transduction experimental cell model to visualise fluorescently labelled Mal/TIRAP. Mal/TIRAP-/- iBMDMs were lentivirally transduced with a Mal/TIRAPHALO construct. The halotag was fluorescently labelled then the cells were stimulated with TLR-4 ligands, such as LPS, fixed at different time points, then imaged. Total internal reflection fluorescence (TIRF) microscopy was used to image the plasma membrane and photobleaching experiments performed to determine Mal/TIRAP stoichiometry. I developed a computer-based analysis pipeline to analyse the resulting photobleaching data. Under resting conditions, Mal/TIRAP is present at the plasma membrane in clusters of approximately ten Mal/TIRAP molecules per cluster. After five minutes of stimulation with 10 ng/ml LPS Mal/TIRAP redistributes into cluster sizes of approximately six, twelve and much larger. After ten and fifteen minutes stimulation with 10 ng/ml LPS the clusters return to the resting size of approximately ten Mal/TIRAP molecules per cluster with a few much larger clusters remaining present. This confirms the rapid time frame within which TLR-4 signalling occurs at the plasma membrane and is consistent with myddosome stoichiometry of six MyD88 molecules or proposed super myddosomes of twelve MyD88 molecules. The computer-based analysis pipeline developed can be used to analyse any protein of interest at the plasma membrane. Protein ligands have also been found to activate TLR-4; for example allergens, such as Fel d 1 and Der p 2, as well as endogenous damage associated molecular patterns (DAMPs), such as extracellular matrix (ECM) proteins, for example fragments of fibronectin and tenascin-C. The mechanism by which these proteins interact with TLR-4 and induce signalling is unclear. Proteins from the ECM (fragments FNIII1c, FNIII13-14, FNIII9-E and FNIII9-E-14 from fibronectin and the fibrinogen-like globe (FBG) domain of tenascin-C) were tested using a transient transfection assay in HEK293 cells and shown to activate TLR-4. In conclusion, I have developed new tools and methodology to investigate how TLR-4 signals in response to LPS and DAMPs in living cells. Whether DAMP- activated TLR-4 forms similar signalling complexes to those induced by LPS will form part of a future study.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Recentemente, nosso grupo demonstrou que a matriz extracelular de astrocitomas promove a seleçãode células endoteliais altamente proliferativas, porém com reduzida capacidade tubulogênica, além de determinar a morte de uma segunda sub-população endotelial, por desaderência ou anoikis. Estratégias de simulação dos teores de tenascina-C (TN-C) e fibronectina (FN) nas matrizes de astrocitomas, realizados com ambas as proteínas purificadas na forma de substratos definidos, sugeriram que o balanço TN-C:FN estava relacionado com os fenótipos endoteliais observados. No entanto, este procedimento não permitia abordar a participação de outros componentes da matriz tumoral nativa neste processo. Com objetivo de estudar a modulação do fenótipo angiogênico das células endoteliais por matrizes de astrocitoma, realizamos o silenciamento da expressão de TN-C na linhagem de astrocitoma U-373 MG. O silenciamento foi confirmado por western blotting, PCR em tempo real e ELISA, que permitiram concluir que, no período pós-transfecção (120h) necessário para se obter matrizes tumorais nativas para ensaios funcionais com células endoteliais, as células U-373 MG mantiveram-se silenciadas em índices superiores a 90%. A diminuição de TN-C nas matrizes tumorais resultou em um pequeno (≅18%, em média), porém significativo aumento na taxa de adesão endotelial. HUVECs incubadas com a matriz secretadas por células silenciadas apresentaram uma redução de ≅35% do número de núcleos picnóticos, quando comparadas a HUVECs incubadas com a matriz de células U-373 MG (selvagens ou transfectadas com siRNA controle). O silenciamento da expressão da TN-C na matriz nas células U-373 MG restaurou ainda o defeito tubulogênico das células endoteliais, que passaram a apresentar formação de tubos comparável à obtida quando HUVECs foram incubadas com sua matriz autóloga, rica em FN. Tais resultados apoiam observações anteriores do grupo, que já sugeriam que a maior proporção de FN na matriz autóloga, comparada a matriz do astrocitoma, seria o fator principal para a seleção dos fenótipos angiogênicos observados, demonstrando mais uma vez a importância do balanço FN:TN-C na regulação de processos angiogênicos. Dados anteriores sugeriam ainda que a sub-população endotelial que morre por anoikisapós contato prolongado (24 horas) com matrizes de astrocitomas corresponde a células que já haviam entrado na fase S do ciclo celular, no início da incubação. A fim de nos aprofundarmos sobre a participação do ciclo celular neste processo, a expressão da proteína p27, um inibidor de quinases dependentes de ciclinas (CKI), também foi analisada. HUVECs incubadas com a matriz de astrocitoma apresentaram um aumento de 2 a 3 vezes na expressão de p27, quando comparada com HUVECs provenientes de sua matriz autóloga. No entanto, células endoteliais incubadas com matriz secretada por células U-373 MG silenciadas apresentaram um nível de expressão de p27 comparável ao das HUVECs incubadas com matriz secretada por células selvagens, indicando que a expressão de TN-C não modula, ou não está diretamente correlacionada à expressão da proteína p27. Este resultado sugere que outros componentes da matriz tumoral devam estar envolvidos na modulação do ciclo celular endotelial. / We have previously shown that extracellular matrices secreted by high-grade astrocitoma promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells, while also leading to the death, by anoikis, of another endothelial subpopulation. The use of defined adhesion substrata containing various levels of tenascin-C (TN-C) and fibronectin (FN) allowed us to confirm that the balance between TN-C and FN was a major cause of the selection of both endothelial phenotypes. However, this strategy did not allow us to address the potential role of other molecular components present in tumor matrices. In the present work, we studied the effect of a matrix produced by U-373 MG cells previously silenced for TN-C expression in endothelial cell adhesion, survival and tubulogenic differentiation, as compared to the matrix secreted by wild-type astrocitoma cells. U-373 MG cells silencing was confirmed by Western blotting, real time RT-PCR and ELISA, and cells remained silenced (≥ 90 %) throughout the 120 hours period necessary for generating immobilized native matrices required for endothelial cell function assays. Human umbilical vein endothelial cells (HUVECs) adhesion to the extracellular matrix secreted by astrocitoma cells silenced for TN-C expression was significantly increased by ≅18 %, as compared to wild-type tumor matrix. The number of HUVECs exhibiting picnotic nuclei a hallmark of advanced apoptosis has also been significantly decreased by ≅35 %, when endothelial cells were allowed to incubate with TN-C-depleted tumor matrix, as compared to the wild-type tumor matrix. Concerning angiogenic differentiation, endothelial cells incubated with the matrix produced by silenced U-373 cells were strongly attenuated for their tubulogenesis defect, as compared to HUVECs incubated with the TN-C-rich wild-type matrix. Thus, these data corroborated our previous observations that TN-C in astrocitoma matrices crucially interferes with endothelial cell differentiation. Besides adhesion, survival and tubulogenic differentiation, the responses of endothelial cells to astrocitomas matrices are also affected by cell cycle transitions. We have previously shown that endothelial cells undergoing anoikis had already transitioned through the S-phase of cell cycle at the moment of seeding. Thus, we decided to investigate the expression of p27 protein, an inhibitor of ciclin-dependent kinases (CKI) that has been already implicated in glioma angiogenesis. We found that HUVECs incubated with the matrix secreted by U-373 MG wild-type cells for 24 hours exbibited a 2 to 3-fold increase in p27 expression. In contrast to the other results discussed herein, these differences were not correlated with the expression of TN-C by U-373 MG cells, since the matrix produced by tumor cells silenced for TN-C did not alter the expression of p27 in endothelial cells. Overall, the present data suggest that, although TN-C in native tumor matrix does play a major role in endothelial cell angiogenic differentiation, other matrix components may act in concert with TN-C to modulate endothelial cell proliferation in tumor contexts.