This project focused on extracellular signal-regulated kinase (ERK) and focal adhesion proteins related to ERK activity, and found a novel signaling pathway contributing to aging-related defects in smooth muscle contractility. Previous members of our lab have used ERK inhibitors to demonstrate the role of ERK in smooth muscle contraction. Dr. Nicholson used the ERK inhibitor FR 18024 and noted that, in the presence of this inhibitor phenylephrine (PE) induced a higher stress increase in young mouse aortas compared to old aortas. Inhibition of the kinase ERK abolished this difference. He also quantitated ERK phosphorylation, a marker of ERK activation in PE-stimulated aortas from both young and aged mice and found a significant lower level of phosphorylated-ERK (p-ERK) in aged mouse aortas. I was interested in determining the substrate of ERK that is affected in aging. Caldesmon (CaD) is one of the known substrates of ERK in smooth muscle. More importantly, CaD, as an actin-binding protein, inhibits cross-bridge formation by blocking the interaction between actin and myosin. Thus, I tested the hypothesis that, caldesmon phosphorylation is inhibited in aged mouse aortas. To determine the mechanism by which regulation of ERK activation changes with age, the role of micro-RNAs (miRs) in the regulation ERK phosphorylation was investigated. Transfection of miR-137 and -34a into A7r5 cells resulted in a significantly lower level of p-ERK in response to the phorbol ester DPBA. Further, together with my collaborators I found that transfection of miR-137 and -34a led to significantly decreased focal adhesion protein levels in A7r5 smooth muscle cells, such as paxillin and src. To confirm whether focal adhesion proteins contribute to the impairment of agonist-induced ERK phosphorylation, paxillin siRNA and src inhibitor were used. The results showed that paxillin is required for the phosphorylation of ERK1 and ERK2 and src is required for ERK2 phosphorylation. In conclusion, age-related increases in miR-137 and -34a decrease ERK phosphorylation via downregulation of paxillin and src. The decrease in ERK phosphorylation leads to a decrease in CaD phosphorylation and inhibits contraction. Thus, the thin filament-coupled pathway in differentiated vascular smooth muscle is inhibited in the aged mouse aorta and this leads to aging-associated defects in smooth muscle contractility. / 2021-06-18T00:00:00Z

Thesis advisor: Marc-Jan Gubbels / The apicomplexan parasite <italic>Toxoplasma gondii</italic> divides rapidly and asexually through a unique process of internal daughter budding. The physical infrastructure for this process is the cytoskeleton, which is composed of subpellicular microtubules, flattened vesicles (alveoli), and a meshwork of intermediate filament-like proteins. This meshwork is composed of a family of 14 inner membrane complex (IMC) proteins that were identified based on the presence of a repeat sequence shared across the Alveolata, the alveolin-repeat. All 14 proteins were cloned as YFP fusions to study their subcellular localization and antibodies were generated against several representative IMC proteins. Each IMC displays unique spatio-temporal dynamics throughout development, but four physically distinct localizations were identified: eight IMCs localize to the alveoli, four IMCs localize to a structure known as the basal complex, IMC11 localizes to the apical cap in mature parasites, and IMC15 localizes primarily to the centrosomes and early buds. IMC15 is of particular interest because its appearance before membrane occupation and recognition nexus 1 (MORN1) in the early bud suggests that it is the first cytoskeletal component to associate with the buds. A conditional knockdown of this protein using the destabilization domain (DD) reveals IMC15 has a strong affinity for the centrosomes that overcomes targeting of the DD fusion protein to the proteasome and the presence of IMC15 in the early bud may not be necessary for the division process. Conditional knockdowns using a tetracycline repressible promoter reveal that a minimal amount of IMC15 is sufficient for parasite survival. In order to further characterize IMC15, dominant negative constructs based on mutating putative palmitoylation sites or overexpression of deletion constructs are being pursued. Collectively, the IMC family is being incorporated into the temporal and spatial dynamics of cytoskeletal development through the creation of a comprehensive timeline of daughter bud assembly. These findings are contributing unprecedented detail to the cell division process. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Process-structure-property relationships in material extrusion additive manufacturing (MEAM) are complex, non-linear, and poorly understood. Without proper characterization of the effects of each processing parameter, products produced through fused filament fabrication (FFF) and other MEAM processes may not successfully reach the material properties required of the usage environment. The two aims of this thesis were to first use an informatics approach to design a workflow that would ensure the collection of high pedigree data from each stage of the printing process; second, to apply the workflow, in conjunction with a design of experiments (DOE), to investigate FFF processing parameters. Environmental, material, and print conditions that may impact performance were monitored to ensure that relevant data was collected in a consistent manner. Acrylonitrile butadiene styrene (ABS) filament was used to print ASTM D638 Type V tensile bars. MakerBot Replicator 2X, Ultimaker 3, and Zortrax M200 were used to fabricate the tensile bars. Data was analyzed using multivariate statistical techniques, including principal component analysis (PCA). The magnitude of effect of layer thickness, extrusion temperature, print speed, and print bed temperature on the tensile properties of the final print were determined. Other characterization techniques used in this thesis included: differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), and scanning electron microscopy (SEM). The results demonstrated that printer selection is incredibly important and changes the effects of print parameters; moreover, further investigation is needed to determine the sources of these differences.

No description available.

Stem cell marker Nestin is an intermediate filament protein that plays an important role in cell integrity, migration and differentiation. Nestin expression occurs in approximately one-third of pancreatic ductal adenocarcinoma (PDAC) cases, and its expression positively correlates with tumor stage and peripancreatic invasion. Little is known of the mechanisms by which Nestin influences PDAC progression. We showed that Nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition in vitro, conversely, knockdown of endogenous Nestin expression reduced the migration rate and cells reverted to a more epithelial phenotype. In vivo mice studies showed that knockdown of Nestin significantly reduced tumor incidence and volume in xenografts. Expression of the Nestin protein was associated with Smad4 status in PDAC cells, hence Nestin expression might be regulated by the TGF-b1/SMAD4 pathway in PDAC. We examined Nestin expression after TGF-b1 treatment in human pancreatic cancer PANC-1, and PANC-1 shSmad4 cells. The TGF-b/SMAD pathway induced Nestin protein expression in PDAC cells through Smad4 in a dependent manner. Moreover, increased Nestin expression caused a positive feedback loop in the TGFb/SMAD signaling system. Finally, we demonstrated that 2 anti-microtubule inhibitors, Cytochalasin D (CD) and Withaferin A (WFA), exhibited anti-Nestin activity; these inhibitors might be potential anti-metastatic drugs. Our findings uncovered a novel role of Nestin in regulating TGF-b1-induced EMT. Anti-Nestin therapeutics are under development as a potential treatment for PDAC metastasis.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 177-196). Also available in print.

Thesis (doctoral)--Umeå Universitet, 1985. / Added t.p. with thesis statement inserted. Bibliography: p. 35-41.

Une décharge à barrière diélectrique (DBD) provenant d'un claquage de type Townsend et entretenue dans un régime luminescent peut aboutir à une décharge d'apparence homogène ou filamentaire. Grâce à une importante variété de structures filamentaires observables et à leurs similarités avec les structures observées dans d'autres domaines, les DBD sont d'un grand intérêt en dynamique des systèmes non linéaires. Numériques ou expérimentaux, les moyens d'études de systèmes complexes se doivent d'être en premier lieu fiables et simples. Une grande partie du temps de cette thèse a consisté à mettre en place un dispositif expérimental permettant d'obtenir des résultats reproductibles et à élaborer un code numérique de décharge à zéro dimension permettant des calculs rapides tout en conservant la physique essentielle des décharges. Un code fluide à deux dimensions préexistant a aussi été utilisé. Ces trois outils, par leurs complémentarités et leurs versatilités propres, ont offert des possibilités d'investigations plus poussées qu'auparavant. La question principale posée au début de la thèse concerne l'origine de l'instabilité d'une décharge menant à une structuration spatiale. Pour y répondre, une des études visa à regarder l'influence de l'ajout d'une impureté sur les paramètres expérimentaux observables grâce à la combinaison de résultats expérimentaux et numériques. Les décharges ont été réalisées dans les gaz rares tels que l'hélium, le néon et l'argon et l'impureté ajoutée est de l'azote. L'un des principaux résultats montre que dans le néon, l'effet Penning joue un rôle central dans la structuration. D'autres travaux menés en parallèle ont entre autres visé à identifier les facteurs responsables de la taille des filaments ou à aborder la spatialisation d'une décharge à partir de modèles 0D couplés. / Dielectric Barrier Discharge (DBD) starting from Townsend breakdown and used in glow discharge regime can lead to homogenous discharge or patterned discharge. Due to a large variety of observed structures and their similarity with other domains, DBD are of great scientific interest in non-linear dynamics systems. Numerical calculations or experimental studies of complex systems need to be simple and reliable. A great part of this thesis work was to improve an experimental device to obtain reproducible results and to develop a zero dimension numerical model performing faster calculations retaining the essential discharges physic. A two dimensions preexisting model was also used. These three tools, by their complementarity and their versatilities, allowed us to go further in understanding discharge mechanisms. In the beginning of this work, one of the main questions was: "What is responsible for instability in a dielectric barrier discharge leading to spatial structure?". To answer, one of the studies consisted to add an impurity in a discharge and to look the consequences combining numerical and experimental devices. Discharges were performed in rare gas such as helium, neon and argon and nitrogen was used as an impurity. One of the main results shows that Penning effect plays a central role in discharges structuring. Other studies consisted of identifying the factors responsible for the size of the filaments or to approach the spatialization of a discharge from coupled 0D models.

No description available.

Laser filamentation is a phenomenon currently being widely studied in which an ultrashort laser pulse self focuses as a result of the nonliner Kerr effect. Lim- ited data is available in terms of spatial characterization of the filament. We study the spatial distribution of molecular and atomic species generated by the filament in order to infer the relevant dynamics. We find evidence for a new impulsive vibrational excitation scheme which we introduce in this dissertation. Insight into the mechanisms of ionization is gained by consideration of the details of this process. In addition, the suitability of filaments to stimulate impulsive Raman scattering for spectroscopic purposes is evaluated. The data presented show the first measurements of ions by impulsive Raman spectroscopy. This method has advantages over previous techniques. Signal is directional and so it can be more completely collected and can be measured stand off. The energy required for detection is also provided by a probe beam rather than from the analyte molecules themselves and so there is no limit to its intensity as in the case of fluorescence spectroscopy. / Physics