Fibronectin (fn) and integrin α5 (itgα5) are both key players in cell adhesion and intracellar signaling, however the specific in vivo role of these proteins has never been analyzed in the vertebrate lens. The results presented here indicate that Fn1 and Itgα5 proteins are essential for the proper development of the lens. The loss of Fn1 protein in the zebrafish embryo results in distinct adhesion defects, defects in lens fiber morphogenesis, and cataracts. These results were phenocopied in zebrafish itga5 mutants, thereby indicating an essential role for Fn1 and Itgα5 during lens development. Furthermore, embryos with reduced levels of ptk2.1 (focal adhesion kinase – FAK) also phenocopied the defective fn1 and itgα5 lens, suggesting that FAK is a major player in the intracellular signaling mediated by Fn1/Itgα5 interactions in the lens. / text

Cancer metastasis is a multi–step process that initiates with a tumor cell obtaining the ability to migrate. A multitude of changes occur in such a cell including changes to cell adhesion molecules such as integrins. In cancer cells, integrins are known to be involved in migration, invasion and metastasis. Investigation by our group of the α6 integrin led to the discovery of a cleaved form of the integrin lacking the ligand binding domain, called α6p. While it is known that the integrin is cleaved by urokinase plasminogen activator (uPA) little is known about how this process is regulated. There is a need to better understand the players involved in regulation of α6 cleavage as inhibiting this event from occurring may contribute to prolonged or increased patient survival or ultimately a cure.The existence of the integrin–actin complex has been known for many years. In this study actin was identified as a potential regulator of α6 cleavage. Using a diverse set of tumor cell lines (DU145, PC3 and MDA–MB–231) and a number of actin modifing compounds (latrunculin A, jasplakinolide and siRNA) it is reported here that disassembling actin filaments leads to an increase in α6p production. Although the increase in cleavage product did not always correlate with an increase in uPA receptor, an increase in uPAR was observed when actin was complexed by small molecule inhibitors. Taken together the results demonstrate a potential role for actin filaments to protect α6 integrin from uPA–uPAR induced cleavage via a multi–protein complex.

This thesis presents a simple force-based model of moving and interacting cells that incorporates a realistic description of cell adhesion and applies it to a system of spherical cells. In addition, several results in matrix theory are proved with the end of showing that the equations produced by the model uniquely determine the motion of the system or cells.

<p>Integrins are a protein family of cell surface receptors, expressed in all cell types in the human body, except the red blood cells. Besides their importance in mediating physical connections with the surrounding environment, the integrin family members are also vital signalling mediators. They have no intrinsic kinase activity; instead the signals are transduced through conformational changes. </p><p>In this thesis, work is presented which is focused on molecular mechanisms of integrin signal transduction. The signal transduction was first studied from a structural point of view, determining the transmembrane domain borders of a few selected integrin family members and ruling out a signalling model involving a “piston-like” movement. </p><p>Then, downstream signalling events involved in the beta1 integrin-induced activation of Akt via the PI3kinase family were characterized. Our results identify a novel pathway for PI3K/Akt activation by beta1 integrins, which is independent of focal adhesion kinase (FAK), Src and EGF receptor. Furthermore, both beta1 integrins and EGF receptors induced phosphorylation of Akt at the regulatory sites Thr308 and Ser473, but only EGF receptor stimulation induced tyrosine phosphorylation of Akt.</p><p>Finally, signals from beta1 integrins underlying the morphologic changes during cell spreading were studied. A rapid integrin-induced cell spreading dependent on actin polymerisation was observed by using total internal reflection fluorescence (TIRF) microscopy. This integrin-induced actin polymerisation was shown to be dependent on PI3K p110alpha catalytic subunit and to involve the conserved Lys756 in the beta1-integrin membrane proximal part.</p>

Integrins are a protein family of cell surface receptors, expressed in all cell types in the human body, except the red blood cells. Besides their importance in mediating physical connections with the surrounding environment, the integrin family members are also vital signalling mediators. They have no intrinsic kinase activity; instead the signals are transduced through conformational changes. In this thesis, work is presented which is focused on molecular mechanisms of integrin signal transduction. The signal transduction was first studied from a structural point of view, determining the transmembrane domain borders of a few selected integrin family members and ruling out a signalling model involving a “piston-like” movement. Then, downstream signalling events involved in the beta1 integrin-induced activation of Akt via the PI3kinase family were characterized. Our results identify a novel pathway for PI3K/Akt activation by beta1 integrins, which is independent of focal adhesion kinase (FAK), Src and EGF receptor. Furthermore, both beta1 integrins and EGF receptors induced phosphorylation of Akt at the regulatory sites Thr308 and Ser473, but only EGF receptor stimulation induced tyrosine phosphorylation of Akt. Finally, signals from beta1 integrins underlying the morphologic changes during cell spreading were studied. A rapid integrin-induced cell spreading dependent on actin polymerisation was observed by using total internal reflection fluorescence (TIRF) microscopy. This integrin-induced actin polymerisation was shown to be dependent on PI3K p110alpha catalytic subunit and to involve the conserved Lys756 in the beta1-integrin membrane proximal part.

Tip growth, the mechanism by which hyphae, pollen tubes, root hairs, and algal rhizoids extend, is a complex and dynamic process that is characterised by localised extension at the extreme apex of the cell and morphological polarity. Its complexity suggests that high degree of regulation is needed to ensure that the characteristics of a particular cell type are maintained during growth. Regulation is likely to come about through bidirectional interplay between the cell wall and cytoplasm, although the mechanisms by which such cross-talk might occur are unknown. Results of this thesis present immunocytochemical data that indicate the presence of, and a close association between β4 integrin subunit-like proteins and proteins containing phosphorylated tyrosine residues in the oomycete Achlya bisexualis. When hyphae were plasmolysed, these proteins were present in wall-membrane attachment sites where there was also F-actin. A combination of immunoblots, ELISA, and a coupled enzyme assay suggest that phosphorylation may occur by both autophosphorylation and through the possible action of a tyrosine kinase. Tyrphostins, which are inhibitors of tyrosine kinases, abolished the anti-phosphotyrosine staining, inhibited the kinase activity, slowed tip growth and affected the organisation of the actin cytoskeleton, in a dose-dependent manner. In addition, results show A. bisexualis contains proteins epitopically similar to the rod domain of animal talin. However, these proteins do not co-localise with F-actin, and mainly locate at the sub-apical region in hyphae. For comparative purposes, Saccharomyces cerevisiae was also used to investigate the presence of β4 integrin subunit-like proteins and tyrosine phosphorylation. Immunoblotting showed that S. cereviaise contains a protein, which is found in the microsomal pellet fraction, that cross reacts with anti-β4 integrin subunit antibody. Furthermore, there are a number of proteins containing phosphotyrosine residues. Immunocytochemistry shows that this anti-β4 integrin staining is at the cortical site but anti-phosphotyrosine residues are distributed throughout cells. On the basis of an ELISA and a coupled enzyme assay, it is suggested that a soluble fraction of S. cerevisiae contains tyrosine kinase activity. This activity is strongly inhibited by tyrphostins.

Laminin binding integrins 6 (CD49f) and 3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, 6 integrin internalized with a rate constant (k(actual)) of 3.25min(-1), threefold faster than 3 integrin (1.0min(-1)), 1.5-fold faster than the vitronectin binding v integrin (CD51) (2.2min(-1)), and significantly slower than the unrelated transferrin receptor (CD71) (15min(-1)). Silencing of 3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in k(actual) for 6 integrin. The internalized 6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of 3 integrin expression resulted in redistribution of 64 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of 3 integrin increased cell migration by 1.8-fold, which was dependent on 61 integrin. Silencing of 6 integrin expression however, had no significant effect on the k(actual) of 3 integrin or its distribution in early endosomes. These results indicate that 3 and 6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017.

Thesis advisor: Eric S. Folker / Thesis advisor: David R. Burgess / During muscle development, myonuclei undergo a complex set of movements that result in evenly spaced nuclei throughout the muscle cell. In many muscle diseases mispositioned myonuclei have been used as a hallmark phenotype of disease. A number of studies over the last decade have started to piece together the cytoskeletal elements that govern these movements. In Drosophila, two separate pools of Kinesin and Dynein work in synchrony to drive nuclear movement. However, it is still not clear how these two pools of microtubule motors become specified. In addition, it is not clear how nuclear position impacts the other defining feature of the muscle cell, which is the highly organized contractile network of sarcomeres. Previously, mispositioned myonuclei have been correlated with improper muscle function, yet no direct link between nuclear position and sarcomere development or function has been demonstrated. In this thesis, we show a role for Aplip1 (the Drosophila homolog of JIP1), a known regulator of both Kinesin and Dynein, in myonuclear positioning. Aplip1 localizes to the myotendinous junction and has genetically separable roles in myonuclear positioning and muscle stability. Furthermore, we show that a number of sarcomeric proteins, including ZASP, Actin and β-integrin localize to the nucleus prior to being incorporated into the sarcomere, regardless of nuclear position. Finally, we show that the LINC complex is required for nuclear dependent sarcomere assembly and that disruption of nuclear dependent sarcomere assembly or nuclear position resulted in a compromised sarcomeric network. Together, this thesis adds to the mechanisms that are important in positioning nuclei and shows the first direct link between the nucleus and sarcomere assembly. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Intestinal immune cells remain tolerant to the trillions of commensal bacteria present in the gut, with perturbations of this process implicated in development of inflammatory bowel disease (IBD). The cytokine TGF-β is a key factor promoting intestinal immune tolerance, but is secreted in a latent state that requires activation to function. Binding of TGF-β to the integrin αvβ8 is a principal mechanism of TGF-β activation, with mouse models demonstrating a crucial role for αvβ8 expression by dendritic cells and regulatory T cells in intestinal immune regulation. Despite this evidence, very little is known regarding the importance of this activating integrin in human intestinal homeostasis. Utilising flow cytometry here we find that integrin αvβ8 is highly expressed on peripheral blood monocytes with highest levels on intermediate CD14++CD16+ monocytes. Upon monocyte to macrophage differentiation high β8 expression is observed on anti-inflammatory M-CSF differentiated macrophages versus pro-inflammatory GM-CSF macrophages. In monocytes, expression of β8 is upregulated by specific bacterial TLR ligands. Utilising a TGF-β reporter cell line both monocytes and M-CSF MDM display an enhanced ability to activate TGF-β in an αvβ8-dependent manner. Data presented here indicate that macrophage αvβ8-dependent TGF-β activation does not alter expression of surface markers associated with a tolerogenic macrophage phenotype, phagocytosis, or production of the anti-inflammatory cytokine IL-10; nor does TGF-β appear to influence the metabolic profile of macrophages, key differences of which are associated with pro- or anti-inflammatory phenotype. However, the previously undescribed finding of integrin αvβ8 expression on human monocytes and macrophages, which was subsequently confirmed in intestinal populations and found to be downregulated in inflamed IBD mucosa, may highlight an important functional pathway in intestinal immune homeostasis and represent a potential future therapeutic target in IBD.

A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.