Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 1996. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 1996. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2001. / Spine title: Yersinia uptake by mammalian cells. Includes bibliographical references (leaves 214-240). Also available online through Digital Dissertations.

Made available in DSpace on 2014-06-11T19:31:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-28Bitstream added on 2014-06-13T20:21:58Z : No. of bitstreams: 1 mamede_fv_dr_jabo.pdf: 467286 bytes, checksum: f692f69fc0d5d4f1a73028aaad9d672a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Fundação para o Desenvolvimento da UNESP (FUNDUNESP) / Mioplastias extra-oculares podem ensejar aderências entre o músculo operado e os tecidos adjacentes, produzindo, não raro, estrabismos cicatriciais. Com intuito de se minimizar a ocorrência de aderências, investigaram-se os efeitos da mitomicina C (MMC), um antifibrótico, em concentrações ascendentes de 0,008, 0,02 e 0,04%, aplicada no per-operatório de mioplastias do reto dorsal do bulbo do olho de coelhos. Para tal, foram operados 56 animais, os quais foram divididos em sete grupos. Para o pós-operatório, instituíram-se limpeza com solução fisiológica das áreas operadas, profilaxia antimicrobiana e antiinflamatória, na forma de colírio. Procederam-se avaliações clínica, histológica, morfométrica e quanto à imunoistoquímica, em que se estudou o Fator de Crescimento Fibroblástico-básico (FGF-2). Encontraram-se, clinicamente, mais aderências nos olhos controle, comparativamente aos tratados, no entanto, sem significação estatística (p>5%). À histologia, verificou-se que a MMC ensejou retardo da cicatrização junto às áreas das mioplastias, notadamente no grupo que a recebeu, à concentração de 0,02%. A imunoistoquímica revelou marcação do FGF-2 em fibroblastos e macrófagos, indistintamente, entre os grupos. Com base nos resultados, permite-se admitir que a MMC, nas concentrações em que fora empregada, foi capaz de retardar a cicatrização e, por conseguinte, o estrabismo secundário, sem ensejar efeitos colaterais. / Extraocular myoplasties may cause adhesions to adjacent tissues, resulting cicatricial strabismus. With the purpose of reducing to a minimum the occurrence of adhesion, we studied the effects of mitomycin C (MMC), an antifibrotic, in concentrations of 0,008%, 0,02%, and 0,04% applied during intraoperative of myoplasties of the superior rectus muscle of laboratory albine rabbits. Fifty six animals were operated on and were divided in seven groups. During the postoperative the operated areas were cleaned with physiological solution. Eyedrop instillation to prevent inflammation and bacterial infection were used. The method to analyze the results consisted of clinical and histological evaluation and statistical analyzes. We also evaluated at the same time the amount of basic fibroblast growth factor (FGF-2) by immunohistochemical study. Clinically we found more adhesions in the eyes of the control group than in the groups of treated eyes. However there was no significant statistics difference between the two groups (p>5%). Histologically we found that MMC caused a delayed cicatrization in the mioplastic areas, specially in the group who received the 0,02% concentration. The immunohistochemical showed FGF-2 marking in fibroblasts and macrophages, but between the groups there wasn’t no difference. Based on those results we conclude that MMC in the utilized concentrations was capable of delaying the cicatrization and consequently avoid the secondary strabismus without undesirable side effects.

Proteins represent major constituent of the extracellular matrix which plays an important role in the formation, maintenance and remodeling of tissues, this project focuses on adsorption of two specific serum proteins fibronectin (FN) and vitronectin (VTN) responsible for mediating cell matrix interaction through integrin binding, tripeptide Arg-Gly-Asp (RGD) sequence found in these protein features are recognized by αβV3 integrin which ultimately helps in clot formation.

Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle cells (VSMCs). However, it is often assumed that FAs in non-migratory, differentiated vascular smooth muscle cells (dVSMCs) embedded in the wall of healthy blood vessels are static structures. Recent work from our lab has demonstrated agonist-induced actin polymerization and Src-dependent focal adhesion phosphorylation in dVSMCs, suggesting that agonist-induced FA remodeling occurs. However, the mechanisms and extent of FA remodeling are largely unknown in dVSM. Here we show, for the first time, that a distinct subpopulation of dVSM FA proteins, but not the entire FA, remodels in response to the alpha-agonist phenylephrine. VASP and Zyxin displayed the largest redistributions while beta-integrin and FAK showed undetectable redistribution. Vinculin, metavinculin, Src, CAS, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and EEA1. Endosomal disruption with the small molecule inhibitor primaquine inhibits agonist-induced redistribution of FA proteins, confirming endosomal recycling. FA recycling was also inhibited by cytochalasin D, latrunculin B and colchicine, indicating that the redistribution is actin and microtubule-dependent. Furthermore, inhibition of endosomal recycling causes a significant inhibition of the rate of development of agonist-induced dVSM contractions. Thus, these studies are consistent with the concept that FAs in dVSMCs, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor.

The cornea forms the anterior surface of the eye and is responsible for most of the eye’s refractive power. Injury to the outermost layer of the cornea, a non-keratinized stratified squamous epithelium, triggers a transient rise in intracellular calcium concentration that propagates radially from the wound. This calcium mobilization is initiated by the binding of nucleotides such as adenosine triphosphate (ATP), which are released from cells ruptured by the injury, to purinergic receptors (purinoreceptors) on undamaged cells near the wound. Downstream effects of this injury-induced "calcium wave" are generally thought to include the activation of signaling pathways that promote wound healing. However, the specific contributions of individual purinergic receptors to the overall wound response have in most cases not been well characterized. Purinoreceptors are classified into two broad categories: the P2Y class of G protein-coupled receptors, which act through second messengers to release calcium into the cytosol from the endoplasmic reticulum, and the P2X class of ligand-gated ionotropic receptors, which release calcium into the cytosol from the extracellular environment. Previously, our lab established the importance of the P2Y2 receptor to corneal epithelial wound healing by showing that P2Y2 activation makes a substantial contribution to the overall wound-induced calcium response, particularly in cells back from the leading edge, and promotes cell migration after injury. P2Y2 activation was also found to promote the phosphorylation of proteins involved in focal adhesions, which are multi-protein complexes that facilitate cell migration by transmitting the forces generated by the actin cytoskeleton to the extracellular environment. More recently, our lab has begun to demonstrate that P2X7 may play an equally important, yet distinct and perhaps complementary role in corneal epithelial wound healing. For instance, P2X7 was found to strongly influence the intensity of the injury-induced calcium response in cells immediately adjacent to the wound, and treatment with the P2X7 inhibitor oxidized ATP (oxATP) was shown to impair migration after injury both in vitro and in ex vivo rat corneas. Additionally, immunofluorescence of cells fixed eight hours after injury revealed an altered actin cytoskeletal architecture and localization of the focal adhesion proteins talin and vinculin in oxATP-treated cells compared to control cells. The goal of the present study was to further characterize P2X7’s role in the overall response to injury by using live cell imaging to examine actin cytoskeletal rearrangements and focal adhesion dynamics after injury under both control conditions and conditions of P2X7 inhibition. Human corneal limbal epithelial (HCLE) cells were transduced to express either actin or talin tagged with green fluorescent protein (GFP), grown into confluent monolayers, and scratch wounded in the presence or absence of oxATP. Cells at the leading edge of the wound were imaged using confocal microscopy every 10 minutes for 4 hours beginning 0.5 hours after injury. Analysis of the resulting actin-GFP movies revealed trends toward delayed extension of filopodia in oxATP-treated cells relative to control cells, as well as complex changes in the number of filopodia per cell over time. Additionally, while both groups formed lamella containing thick actin bundles that were oriented perpendicularly to the direction of migration, in oxATP-treated cells the formation of these structures was delayed. Furthermore, in oxATP-treated cells these actin bundles tended to persist once formed. This was in contrast to control cells, in which they tended to turn over to be replaced by thinner and shorter actin bundles that were oriented more obliquely relative to the direction of migration. Finally, analysis of talin-GFP movies demonstrated that focal adhesion lifespan was extended in oxATP-treated cells compared to control cells. Focal adhesions in oxATP-treated cells also exhibited a greater propensity to merge together or split apart, further suggesting impaired focal adhesion turnover. Overall, these findings suggest that P2X7 plays a critical role in promoting migration after corneal epithelial injury by coordinating rapid rearrangements of the actin cytoskeleton and turnover of focal adhesions at the leading edge.

The focal adhesion kinase (FAK) and the protein tyrosine kinase 2-beta (PYK2) are two closely related non-receptor tyrosine kinases that link cell adhesion, migration and proliferation, and thus also promote cancer cell invasiveness. FAK and PYK2 have the same domain structure (comprising the FERM, kinase and FAT domains) and possess several overlapping functions, however their cellular roles can be different or even opposing. In particular, PYK2 can be activated by calcium, and has important functions in the brain and neurodegenerative disease. The molecular basis for calcium-based activation of PYK2 is unclear and controversial. In this work we combined biophysical and structural methods to determine the molecular basis for calcium-sensing in PYK2. For this, we investigated the least-studied region of these kinases, namely the long linker (KFL) region between the kinase and FAT domains. This linker is only ~20% conserved between FAK and PYK2, and, therefore, is a prime candidate for causing their differential properties. We find that the linker harbors a helical segment, which is conserved in both FAK and PYK2, and contributes to their dimerization (an important step in their activation). Helix-flanking regions differ between both proteins, and we show that these of PYK2 create a non-canonical dimeric binding site for calcium-bound calmodulin. Calmodulin-binding is synergistic with linker dimerization in PYK2, explaining how calcium influx can be translated into activation of PYK2. Collectively, our work clarifies the capacities for FAK and PYK2 to receive, process and transduce cellular signals, and may provide new opportunities for targeted therapeutic intervention.