One of the characteristics that distinguishes living things from non-living things is motility. On the cellular level, the motility or non-motility of different types of cells can be life building, life-saving or life-threatening. A thorough study of cell motion is needed to help understand the underlying mechanisms that enhance or prohibit cell motion. We introduce a discrete centroid model of cell motion in the context of a generalized random walk. We find an approximation for the theoretical mean square displacement (MSD) that uses a subset of the state space to estimate the MSD for the entire space. We give some intuition as to why this is an unexpectedly good estimate. A lower and upper bound for the MSD is also given. We extend the centroid model to an ODE model and use it to analyze the distribution of focal adhesion (FA) lifetimes gathered from experimental data. We found that in all but one case a unimodal, non-symmetric gamma distribution is a good match for the experimental data. We use a detach-rate function in the ODE model to determine how long a FA will persist before it detaches. A detach-rate function that is dependent on both force and time produces distributions with a best fit gamma curve that closely matches the data. Using the data gathered from the matching simulations, we calculate both the cell speed and mean FA lifetime and compare them. Where available, we also compare this relationship to that of the experimental data and find that the simulation reasonably matches it in most cases. In both the simulations and experimental data, the cell speed and mean FA lifetime are related, with longer mean lifetimes being indicative of slower speeds. We suspect that one of the main predictors of cell speed for migrating cells is the distribution of the FA lifetimes.

The very large G-protein-coupled receptor 1 (VLGR1/ADGRV1) is the largest member of the adhesion G-protein-coupled receptor (ADGR) family. Mutations in VLGR1/ADGRV1 cause human Usher syndrome (USH), a form of hereditary deaf-blindness, and have been additionally linked to epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying the development of these diseases are still unknown. Our study aimed to identify novel, previously unknown protein networks associated with VLGR1 in order to describe new functional cellular modules of this receptor. Using affinity proteomics, we have identified numerous new potential binding partners and ligands of VLGR1. Tandem affinity purification hits were functionally grouped based on their Gene Ontology terms and associated with functional cellular modules indicative of functions of VLGR1 in transcriptional regulation, splicing, cell cycle regulation, ciliogenesis, cell adhesion, neuronal development, and retinal maintenance. In addition, we validated the identified protein interactions and pathways in vitro and in situ. Our data provided new insights into possible functions of VLGR1, related to the development of USH and epilepsy, and also suggest a possible role in the development of other neuronal diseases such as Alzheimer’s disease.

This thesis describes a comprehensive study of understanding the mechanism of rigidity sensing by quantitative analysis using submicron pillar array substrates. From mechanobiology perspective, we explore and study molecular pathways involved in rigidity and force sensing at cell-matrix adhesions with regard to cancer, regeneration, and development by quantification methods. In Chapter 2 and 3, we developed fabrication and imaging techniques to enhance the performance of a submicron pillar device in terms of spatial and temporal measurement ability, and we discovered a correlation of rigidity sensing forces and corresponding proteins involved in the early rigidity sensing events. In Chapter 2, we introduced optical effect arising from submicron structure imaging, and we described a technique to identify the correct focal plane of pillar tip by fabricating a substrate with designed-offset pillars. From calibration result, we identified the correct focal plane that was previously overlooked, and verified our findings by other imaging techniques. In Chapter 3, we described several techniques to selectively functionalize elastomeric pillars top and compared these techniques in terms of purposes and fabrication complexity. Techniques introduced in this chapter included direct labeling, such as stamping of fluorescent substances (organic dye, nano-diamond, q-dot) to pillars top, as well as indirect labeling that selectively modify the surface of molds with either metal or fluorescent substances. In Chapter 4, we examined the characteristics of local contractility forces and identified the components formed a sarcomere like contractile unit (CU) that cells use to sense rigidity. CUs were found to be assembled at cell edge, contain myosin II, α-actinin, tropomodulin and tropomyosin (Tm), and resemble sarcomeres in size (~2 μm) and function. Then we performed quantitative analysis of CUs to evaluate rigidity sensing activity over ~8 hours time course and found that density of CUs decrease with time after spreading on stiff substrate. However addition of EGF dramatically increased local contraction activity such that about 30% of the total contractility was in the contraction units. This stimulatory effect was only observed on stiff substrate not on soft. Moreover, we find that in the early interactions of cells with rigid substrates that EGFR activity is needed for normal spreading and the assembly of local contraction units in media lacking serum and any soluble EGF. In Chapter 5, we performed high temporal- and spatial-resolution tracking of contractile forces exerted by cells on sub-micron elastomeric pillars. We found that actomyosin-based sarcomere-like CUs simultaneously moved opposing pillars in net steps of ~2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing.

A incontinência urinária na mulher é uma afecção crônica que altera a sua qualidade de vida. Um dos tratamentos propostos é o exercício da musculatura do assoalho pélvico. A eficácia desse tipo de terapêutica depende da adesão da paciente a ele. Aderir a um tratamento é seguir as orientações dadas; no caso dos exercícios da musculatura do assoalho pélvico, envolve a incorporação da terapêutica ao cotidiano. O objetivo deste estudo é descrever três aspectos relacionados à adesão ao tratamento: satisfação, expectativa e repetição ou não do tratamento. Para a sua realização foram entrevistadas 50 mulheres que aderiram ao tratamento, atendidas no setor de Uroginecologia da Disciplina de Ginecologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Os resultados mostraram que 68% das mulheres estavam satisfeitas com o tratamento, 62% tinham expectativa de cura e apenas 12% não o repetiriam. Quando questionadas sobre os motivos para a não repetição do tratamento, a maior parte das mulheres referiu a não melhora do quadro clínico. Concluiu-se que a satisfação é alta nas mulheres que aderiram ao tratamento, a expectativa mais comum entre as entrevistadas é de cura e que o fator mais importante para repetir a terapêutica foi a melhora do quadro clínico. / The female urinary incontinence is a chronic disease which affects life quality. One of the treatments suggested is the pelvic floor exercises. The therapy depends on the patient’s adhesion. Adhesion to treatment implies to follow all medical instructions; in case of pelvic floor exercises, it involves its incorporation to daily routine. The aim of this study is to describe tree aspects regarding to the treatment adhesion – satisfaction, expectation and repetition (or not). Fifty women who have followed the treatment were interviewed. They were recruited from the Urogynecology Service of Clínicas Hospital of São Paulo University Medical School. The results demonstrated that 68% of women were satisfied with the treatment, 62% expected to be cured, and only 12% hadn’t undergone the treatment appropriately. When inquired about the reasons for not repeating the treatment, most of the women have mentioned that they haven’t seen positive results. In conclusion, patient’s satisfaction level was high, cure was the main expectation, and the good response to clinical treatment was the most important reasons to repeat the treatment.

A incontinência urinária na mulher é uma afecção crônica que altera a sua qualidade de vida. Um dos tratamentos propostos é o exercício da musculatura do assoalho pélvico. A eficácia desse tipo de terapêutica depende da adesão da paciente a ele. Aderir a um tratamento é seguir as orientações dadas; no caso dos exercícios da musculatura do assoalho pélvico, envolve a incorporação da terapêutica ao cotidiano. O objetivo deste estudo é descrever três aspectos relacionados à adesão ao tratamento: satisfação, expectativa e repetição ou não do tratamento. Para a sua realização foram entrevistadas 50 mulheres que aderiram ao tratamento, atendidas no setor de Uroginecologia da Disciplina de Ginecologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Os resultados mostraram que 68% das mulheres estavam satisfeitas com o tratamento, 62% tinham expectativa de cura e apenas 12% não o repetiriam. Quando questionadas sobre os motivos para a não repetição do tratamento, a maior parte das mulheres referiu a não melhora do quadro clínico. Concluiu-se que a satisfação é alta nas mulheres que aderiram ao tratamento, a expectativa mais comum entre as entrevistadas é de cura e que o fator mais importante para repetir a terapêutica foi a melhora do quadro clínico. / The female urinary incontinence is a chronic disease which affects life quality. One of the treatments suggested is the pelvic floor exercises. The therapy depends on the patient’s adhesion. Adhesion to treatment implies to follow all medical instructions; in case of pelvic floor exercises, it involves its incorporation to daily routine. The aim of this study is to describe tree aspects regarding to the treatment adhesion – satisfaction, expectation and repetition (or not). Fifty women who have followed the treatment were interviewed. They were recruited from the Urogynecology Service of Clínicas Hospital of São Paulo University Medical School. The results demonstrated that 68% of women were satisfied with the treatment, 62% expected to be cured, and only 12% hadn’t undergone the treatment appropriately. When inquired about the reasons for not repeating the treatment, most of the women have mentioned that they haven’t seen positive results. In conclusion, patient’s satisfaction level was high, cure was the main expectation, and the good response to clinical treatment was the most important reasons to repeat the treatment.

Made available in DSpace on 2015-03-26T13:47:22Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1054251 bytes, checksum: 89af20b09635b80159627f67aeb85cad (MD5) Previous issue date: 2006-01-31 / Resection and anastomosis of the small intestine is commonly performed in horses to treat lesions produced by strangulated obstructions and other diseases. However this procedure is associated with several complications such as peritoneal adhesions, intestinal stenosis at the site of the anastomosis, necrosis of the preserved intestinal stumps, leakage of intestinal contents and septic peritonitis. The present study was conducted to evaluate a single- layer appositional end-to-end anastomosis technique covered by mesenteric flaps with respect to adhesion formation, distortion and stomal diameter. To evaluate the use of n-butyl cyanoacrylate surgical glue to fix a mesenteric flap over the jejunum serosa and to perform skin closure in horses that underwent laparotomy and end-to-end anastomosis in the jejunum. Six healthy horses, without evidence of previous abdominal surgery were used. Midline laparotomy was performed and the jejunum was exteriorized. Two jejunal segments (one aboral and other oral and each corresponding to 2 mesenteric arches) were delimited, 2 and 4 meters proximal to the ileocecal junction, respectively. In a crossover design, each one of the jejunal segments underwent one of the following treatments: resection and end-to-end anastomosis performed with a simple continuous pattern (simple anastomosis) or the same type of anastomosis covered by two mesenteric flaps (conserved from the removed segment) and fixed with n-butyl cyanoacrylate to the anastomosis site. Tissue adhesive was also used to perform skin closure. The animals were euthanized 14 days after surgery and peritoneal adhesions were assigned grades of 0 to 3 according to BAXTER, et al. (1993). A 40 cm segment of jejunum containing the anastomotic site was harvested and distended by barium sulfate solution 0,1% to 20 mmHg and radiographed. Stomal diameter reduction and stenosis length were determined by measuring the barium column visible on the radiographs. Was concluded that the single layer appositional end-to-end anastomosis technique covered by mesenteric flaps was not advantageous and must not be used in equines. Prospective studies are needed to evaluate the use of n-butyl cyanoacrylate in the intestine. The skin suture was simpler and faster when performed with n-butyl cyanoacrylate glue. / Ressecção e anastomose do intestino delgado são procedimentos comumente empregados em eqüinos para tratar lesões produzidas por obstruções estrangulantes e outras doenças. No entanto, este procedimento é altamente associado com uma grande quantidade de complicações como aderências peritoneais, estenose no local da anastomose, necrose continuada do segmento intestinal preservado, vazamento do conteúdo intestinal e peritonite séptica. Este trabalho têm como objetivos avaliar uma técnica de anastomose término-terminal no jejuno de eqüinos com uma sutura simples contínua e coberta com retalhos do mesentério quanto à formação de aderências peritoneais, à deformação e à estenose do intestino. Avaliar a utilização do adesivo cirúrgico n-butil cianoacrilato para a fixação do mesentério na serosa do jejuno e para a síntese da pele em eqüinos submetidos a laparotomia e anastomose término-terminal do jejuno. Foram utilizados 6 equinos sadios. Através de uma laparotomia mediana préumbilical, dois segmentos do jejuno (um oral e o outro aboral e correspondentes a dois arcos mesentericos) localizados a 2 metros um do outro e a 2 metros da extremidade oral da prega ileocecal foram demarcados. Num desenho experimental cross-over , cada um desses segmentos foi submetido a um dos seguintes tratamentos: ressecção seguida de enteroanastomose término-terminal com uma sutura simples contínua ou o mesmo tipo de anastomose coberta com dois retalhos mesentéricos pedic ulados criados de cada uma das extremidades do segmento intestinal removido e fixados com n-butil cianoacrilato sobre o local da enteroanastomose. O adesivo cirúrgico também foi utilizado para a síntese da pele. Após 14 dias da cirurgia, os animais foram sacrificados e as aderências peritoneais foram classificadas em graus de 0 a 3 segundo BAXTER, et al. (1993). Segmentos de aproximadamente 40 cm de jejuno com o local da anastomose no centro foram colhidos e distendidos pela administração de uma solução de sulfato de bário 0,1%, até alcançar 20mmHg de pressão interna e radiografados. A redução da luz intestinal foi determinada por medições da coluna de bário visível na radiografia. Concluiu-se que a anastomose término-terminal do jejuno coberta com retalho mesentérico aderido à serosa pelo n-butil cianoacrilato não apresenta nenhuma vantagem e não é recomendado seu uso em eqüinos. O uso do adesivo n-butil cianoacrilato no intestino precisa ser melhor investigado. O uso desse adesivo na pele parece não ter nenhuma desvantagem e torna a síntese mais simples e rápida.

Introduction: Actually 0.7-1 percent of all deliveries can be followed by secondary uterine bleeding. There is a residual trophoblastic tissue diagnosed in most of these cases and it is often managed by repeat intrauterine interventions. These operations are connected with high risk of formation of intrauterine adhesions and their early diagnosis and management can be important for next fertility. Material and methods: There were generally 188 patients included into the study. All patients underwent ultrasound examination in 6 weeks after delivery and ambulant hysteroscopy after next 2 months without anesthesia. Described intrauterine pathologies (residual tissue and adhesions) were managed in one step. Results: In cases with suspect ultrasound finding, the retained trophoblastic tissue was diagnosed by hysteroscopy in 66 percent vs. in 96 percent with sensitivity 85 percent and specificity 85 percent. Patients with intrauterine adhesions had normal ultrasound finding in 74 percent and it did not recognized patients with severe adhesions in 94 percent. Clinical signs had generally 72 percent of patients with diagnosed left residual tissue. Number of severe residual tissue is increasing with delay of instrumental evacuation from delivery (10 vs. 30 percent). When is necessary to repeat the operation...

Afin d’améliorer la performance et l’intégration des biomatériaux dans le tissu hôte, l’intérêt actuel est d’exploiter des approches de nanotechnologie pour produire des biomatériaux possédant des surfaces bioactives. Il est connu que l’interaction des cellules avec la surface des biomatériaux détermine la réponse du tissu hôte et le succès d’un implant. La topographie est l'un des principaux facteurs influençant l'activité fonctionnelle des cellules en contact avec des biomatériaux. Cependant, les mécanismes impliqués demeurent imprécis. Notre groupe a exploité un traitement chimique simple afin de créer des surfaces de titane nanoporeuses uniques qui expriment une influence cellulaire sélective, favorisant ainsi la formation osseuse in vivo et in vitro. Dans ce travail, nous avons réduit la durée du traitement afin d’obtenir une surface nanotopographique mono-planaire, puis évaluer l’influence d’une telle surface sur la formation par des cellules pré-ostéoblastiques MC3T3-E1 d’adhésions focales et de filopodes, ainsi que sur l’expression de gènes codant pour différentes protéines associées à l’adhésion et la signalisation cellulaire. Des disques de titane commercialement pur (cp-Ti) ont été traités avec un mélange d’acide sulfurique et de peroxyde d’hydrogène (50/50 v/v) pendant 1.5 heures. La caractérisation par microscopie électronique à balayage à haute résolution et pour microscopie à force atomique a confirmé la formation d’une surface effectivement mono-planaire caractérisée par des nanopores d’une taille moyenne de 20 ± 5 nm. Les cellules ont été mises en culture pour des périodes de 6, 24 et 72 heures sur des disques contrôles polis et avec une surface nanoporeuse. L'analyse de l’expression de la vinculine par immunofluorescence a révélé un plus grand nombre d’adhésions focales par les cellules sur la surface traitée. Le PCR quantitatif a également montré une augmentation significative de l'expression des gènes pour différents marqueurs d'adhésions focales, telles que paxilline, taline, et différentes intégrines comme par exemple les intégrines α1, β1 et α5. Par microscopie électronique à balayage, les cellules sur la surface nanoporeuse révèlent une présence plus importante de filopodes vis à vis des surfaces contrôles. Ces structures affichent de manière unique de très petites protrusions membranaires latérales d’entre 10-15 nm qui suivent les bords des nanopores. L’augmentation des adhésions focales, l'abondance des filopodes et de leurs petites protrusions pourraient engendre interaction accrue avec la surface et modifier les relations biomécaniques à l’échelle nanométrique pour déclencher des cascade régulant la prolifération cellulaire. / To improve the performance and integration of biomaterials in the host tissue, the focus is presently on exploiting nanotechnology approaches to produce biomaterials with bioactive surfaces. It is known that the cell-biomaterial interactions determine the response of the host tissue and therefore the success of implants. Topography is a key factor that influences the functional activity of cells; however, the mechanisms implicated remain unclear. Our group has exploited a simple chemical treatment to create unique nanoporous titanium surfaces that selectively influence cell behaviour and favor osteogenic activity both in vitro and in vivo. In this work, we have reduced treatment time in order to obtain a monoplanar nanostructured surface, and we have evaluated its influence on the formation of focal adhesions, filopodia, and on gene expression for different cell adhesion and signaling proteins by MC3T3-E1 pre-osteoblastic cells. Commercially pure titanium (cp-Ti) was treated with a mixture of H2SO4/H2O2 (50/50 v/v) for 1.5h. Characterization by high-resolution field-emission scanning electron microscopy and atomic force microscopy characterization showed the formation of a nanoporous surface with a mean pore diameter of 20 ± 5 nm. Cells were cultured and plated on polished (control) and nanotextured discs for periods of 6, 24 and 72 hours. Immunofluorescence analysis of vinculin expression revealed the formation of more focal adhesions by cells seeded on nanostructured surfaces. Quantitative PCR likewise showed significant increase of gene expression for various focal adhesion markers, including paxillin, talin, and different integrins such as integrin α1, β1 and α5. As compared to controls, scanning electron microscopy of cells on the treated surface revealed the presence of more filopodia. These uniquely displayed very small lateral membrane protrusions between 10-15 nm that appeared to follow the walls of the nanopores. Together with the increase in focal adhesions, the abundance of filopodia and associated protrusions could contribute to the adhesive interaction with the surface and modify the nanoscale biomechanical relationships to trigger cellular cascades regulating cell proliferation.

Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...

Protein CAS is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes. It is a multidomain adaptor protein, which serves as a scaffold for assembly of signalling complexes which are important for migration and invasiveness of Src-transformed cells. A novel phosphorylation site in N-terminal SH3 domain was identified - tyrosine 12 located on binding surface of CAS SH3 domain. To study biological importance of tyrosine 12 phosphorylation, non-phosphorylable (Y12F) and phosphomimicking ( Y12E) mutant of CAS were prepared. We found that phosphomimicking mutation Y12E leads to decreased interaction of CAS SH domain with kinase FAK a phosphatase PTP-PEST and also reduce tyrosine phosphorylation of FAK. Using GFP-tagged CAS protein, we show that Y12E mutation caused delocalization of CAS from focal adhesion but has no effect on localization of CAS to podosome-type adhesion. Non-phosphorylable mutation Y12F cause hyperphosphorylation of CAS substrate domain and decrease turnover of focal adhesion and associated cell migration of mouse embryonal fibroblasts (MEFs) independent to integrin singalling. Analogically to migration, CAS Y12F decrease invasiveness of Src-transformed MEF. The results of this diploma thesis show that phosphorylation of Tyr12 in CAS SH3 domain is...