There is currently no solution for imaging fibrin-rich adhesions following surgery, yet the condition costs healthcare providers upwards of $2 billion annually. Over the past decade the development of ultrasound contrast agents has seen an increase in commercialization of generic microbubble formulations for standard diagnostic applications such as echocardiography. To enhance diagnostic power, molecularly targeted microbubbles are formulated with the addition of a ligand to the outer shell. The microbubble formulation must be modified so that the contrast agents are stable over time and targeted with the appropriate ligand while maintaining their echogenicity relative to surrounding soft tissue. We used a dual approach to look at microbubbles optically to predict their relative signal enhancement in vivo given their size distribution and concentration. An ImageJ macro script was developed based off BubblesizerJ, a previously developed open-source program. To confirm that modified microbubbles maintain acoustic properties relative to soft tissue, an agarose phantom model was developed that allows for high throughput testing of multiple microbubble formulations. / 2026-09-03T00:00:00Z

Yes / Cells migrating over 2D substrates are required to polymerise actin at the leading edge to form lamellipodia protrusions and nascent adhesions to anchor the protrusion to the substrate. The major actin nucleator in lamellipodia formation is the Arp2/3 complex, which is activated by the WAVE regulatory complex (WRC). Using inducible Nckap1 floxed mouse embryonic fibroblasts (MEFs), we confirm that the WRC is required for lamellipodia formation, and importantly, for generating the retrograde flow of actin from the leading cell edge. The loss of NCKAP1 also affects cell spreading and focal adhesion dynamics. In the absence of lamellipodium, cells can become elongated and move with a single thin pseudopod, which appears devoid of N-WASP. This phenotype was more prevalent on collagen than fibronectin, where we observed an increase in migratory speed. Thus, 2D cell migration on collagen is less dependent on branched actin.

Despite recent advances in surgical technique and the development of numerous therapeutic agents, the formation post surgical adhesions (PSA) continues to cause complications for many patients. In this research, we have employed a rational system to develop a novel treatment to address this clinical need. Based on an understanding of the biochemical events that lead to PSA formation, a series of targeted polymeric biomaterials was designed to interrupt the fibrin gel matrix propagation and suppress PSA formation. Using group transfer polymerization, a series of well controlled block copolymers of polyacrylic acid and poly(ethylene glycol-methacrylate) based materials was synthesized. Subsequent functionalization with the pentapeptide Cys-Arg-Glu-Lys-Ala (CREKA) was employed to target the materials to fibrin as a marker of pro-adhesive sites. While preliminary testing of the untargeted materials verified their ability to suppress non-specific protein adsorption to model surfaces, numerous in vitro tests were conducted to study the ability to inhibit fibrin gel propagation. The ability to inhibit both the rate and quantity of fibrinogen deposition to a fibrin coated surface has been demonstrated. In addition, the rate of fibrin gel propagation and the degree of cellular attachment can modulated. Taking advantage of the systematic variation in structure facilitated by the robust synthetic methodology employed, statistical analysis was used to elucidate the structureproperty relationships governing the performance of these materials. The most important factors that lead to enhanced performance in in vitro tests are the length of PEG chain and number of peptide units conjugated to the polymer: increasing PEG chain length and increasing the number of peptides conjugated to the polymer both improve performance in all tests. The synthetic methods that have been developed, in conjunction with the experimental results, will be used to direct future studies, including cytotoxicity and animal studies.

A montagem e desmontagem das adesões focais (AF) desempenham um papel fundamental em diversos processos celulares, incluindo migração celular e sobrevivência. Resultados prévios do nosso laboratório mostram que fibroblastos nulos ou silenciados para miosina-Va sofrem um atraso na desmontagem das adesões, sugerindo um papel para a miosinaVa neste processo. Neste trabalho, visamos analisar a dinâmica de montagem das AF em fibroblastos murinos imortalizados NIH3T3, utilizando sondas fluorescentes para visualização de componentes de adesão focal. A formação das AF foi analisada após estímulo por soro de células quiescentes, o que leva a intensa polimerização de actina, reorganização do citoesqueleto e montagem das AF. A cinética de montagem das AF foi observada em ensaios ao longo do tempo, de células fixadas em 0, 5, 15, 30, 120 minutos após estímulo, e marcadas para miosina-Va fosforilada (p-miosina-Va, S1650), FAK fosforilada (p-FAK, Y397), vinculina, dinamina-2, integrina-?1, faloidina, Ki67 e DAPI. Os nossos resultados mostraram um aumento de fluorescência de p-miosina-Va por todo o citoplasma após a estímulo com soro, e revelaram que a p-miosina-Va co-localiza com pFAK nas AF logo após o estímulo, essa localização da p-miosina-Va nas AF diminui ao passar do tempo e retorna após 120 minutos. Isto é consistente com os resultados anteriores de um papel da miosina-Va na dinâmica das AF. Também é possível perceber uma maior concentração de p-miosina-Va e dinamina-2 na região perinuclear, 5 minutos após estímulo, e o espalhamento de ambas as proteínas pelo citoplasma com o passar do tempo. Demonstramos, por Western blotting, que o estímulo por soro não causa alteração na quantidade total de miosina-Va em nenhum dos tempos analisados em relação à condição de quiescência, mas induz, após 5 e 15 minutos, um aumento apreciável de p-miosina-Va, que sofre queda e variações nos tempos posteriores. Para nosso conhecimento, esta é a primeira demonstração de que a fosforilação da miosina-Va aumenta em resposta ao soro e estamos investigando se este evento está ligado à dinâmica das adesões focais em fibroblastos / The assembly and disassembly of focal adhesions (FA) play a critical role in several cellular process, including cell migration and survival. Previous work from our laboratory showed that fibroblasts without myosin-Va show a delay in focal adhesion disassembly, suggesting a role for myosin-Va in this process. In this work, we aim at imaging the dynamics of focal adhesion disassembly and reassembly in cells, with fluorescent probes for visualization of focal adhesion components. Here, we used murine NIH3T3 fibroblasts to analyze FA formation after serum stimulation of quiescent cells, which leads to intense polymerization of actin and reorganization of the cytoskeleton and FA assembly. The kinetics of FA assembly was observed in a time-course assay of cells fixed at 0, 5, 15, 30 and 120 min after serum stimulation, and stained for phosphorylated myosin-Va (p-myosin-Va, S1650), phosphorylated FAK (p-FAK, Y397), vinculin, phalloidin and DAPI. Our results showed an increase of pmyosin-Va staining throughout the cytoplasm upon serum stimulation, and revealed that pmyosin-Va does not colocalize with FAK in FA at early time points. However, colocalization is observed after 30 to 120 min. This is consistent with previous results of a role for myosin-Va in FA disassembly. It is also possible to observe a higher concentration of p-myosin-Va and dynamin-2 in the perinuclear region 5 minutes after stimulation, and the spreading of both proteins in the cytoplasm over time. We demonstrate by Western blotting that serum stimulation does not cause change in total amount of myosin-Va, in any of the times analyzed in relation to the quiescent condition, but induces, after 5 and 15 minutes, an appreciable increase of pmyosin-Va suffering drop and variations in the later times. To our knowledge, this is the first demonstration that phosphorylation of myosin-Va increases in response to serum and we are investigating whether this event is connected to the dynamics of focal adhesions in fibroblasts

Objetivo: Avaliar a eficiência da Triancinolona Acetonida como agente modulador da resposta inflamatória e cicatricial em coelhos submetidos a cirurgia de músculo extra-ocular. Método: Foi realizado estudo prospectivo, mascarado, em dois estádios. No primeiro estádio, dez coelhos foram submetidos a retrocesso do músculo reto superior em ambos os olhos. Aplicouse, em um deles, 0,15 cc de Triancinolona Acetonida (40mg/cc) nos tecidos circunjacentes ao local de reinserção muscular e, como controle, 0,15cc de solução de cloreto de sódio a 0,9% no local equivalente no olho contra-lateral. Quinze dias após, cinco coelhos foram submetidos a exenteração das órbitas e os restantes dos animais tiveram o mesmo procedimento realizado após trinta dias. O material do sítio de reinserção muscular foi avaliado por meio de análise histopatológica qualitativa e quantitativa (morfometria). No segundo estádio, com incrementação da agressão cirúrgica, dezesseis coelhos foram submetidos aos mesmos procedimentos com exenteração das órbitas após quinze dias, e posterior análise histopatológica dos tecidos. Resultados: Houve efeito inibitório sobre a intensidade da resposta inflamatória nos olhos tratados em comparação com os olhos controle. Conclusão: Nas condições de realização do presente estudo o uso per-operatório da triancinolona acetonida foi efetivo no controle da resposta inflamatória em olhos de coelhos submetidos a cirurgia de músculo extra-ocular. / Purpose: To evaluate the efficiency of triamcinolone acetonide (TRI) in limiting the postoperative inflammatory response and scarring following strabismus surgery. Methods: A prospective, two-stage, masked, controlled trial was conducted. In the first stage, the inflammatory response at the extraocular reattachment site was analyzed following superior rectus recession in ten rabbits. One eye had 0,15 cc of triamcinolone acetonide (40mg/cc) applied around the new insertion site and, similarly, 0,15 cc of isotonic saline solution (0,9%) was applied to the fellow eye following the same procedure, thus serving as a control. Fifteen days later, orbital exenteration was performed in five rabbits and the remaining five were exenterated thirty days later. The reattachment site tissues were submitted to qualitative and quantitative histological examinations. In the second stage 16 rabbits were submitted to amplified surgical trauma, after which the aforementioned steps were also carried out. Granuloma total area at the extraocular muscle reattachment sites of control and treated eyes were compared. Results: There was an inhibitory effect of TRI on the inflammatory response of treated eyes as compared to control eyes. Conclusions: TRI was effective in controlling the postoperative inflammatory response in rabbit eyes submitted to traumatic recession of the superior rectus muscle.

Submitted by Biblioteca Central (biblioteca@unicap.br) on 2019-01-24T18:42:29Z No. of bitstreams: 2 ane_caroline_melo_silva.pdf: 4163065 bytes, checksum: c02af54b1eca2ea275982d3c4353e8a7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2019-01-24T18:42:29Z (GMT). No. of bitstreams: 2 ane_caroline_melo_silva.pdf: 4163065 bytes, checksum: c02af54b1eca2ea275982d3c4353e8a7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-07-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The detachment of ceramic tiles in buildings is a problem that still persists in several modern buildings, despite the various advances achieved in the sector of façade technology and an increasingly demanding consumer public. Such detachments may occur by adhesive rupture at the interfaces of the system or by cohesive rupture within any of its layers. Studies have shown that most ceramic façades in facades occur in the interface mortar and ceramic plaque and this is because it is the region of the most requested coating system by shear stresses. In Brazil, the most used methodology to evaluate the adhesion of ceramic coatings is the adhesion strength test to direct traction, however, such evaluation may be insufficient to verify the adhesion quality of the coatings to the substrate, since the adherence of the system is resulting not only from the tensile strength, but also from the shear bond strength and the extent of adhesion of the mortar. This dissertation aims to contribute to the study and understanding of the influence of the contact area on the adhesion of the interface between adhesive mortar and ceramic plate by means of the evaluation of the adhesion resistance from the crack propagation test in mixed mode of stress (MMF - Mixed Mode Flexure) simulates tensile and shear forces simultaneously. The systems produced in the laboratory reproduce conditions where the failure in the extension of the mortar contributes to the decrease of the resistance of adhesion. Through a universal mechanical testing machine, a punctual progressive load was applied to the 120 specimens produced in order to promote internal tensile and shear forces. The variables incorporated into the experiment were the type of mortar used, the thickness of the mortar in the specimen and the size of the glue failure induced in the set. The experimental results show that the adhesion strength at the interface between the adhesive mortar and the ceramic plate decreases as the adhesion failure increases, with a loss of adhesion of up to 44.2% and 51.4% (AC II and AC III, respectively). The adhesion strength values found by the direct drive test were 60.8% (AC II) and 58.1% (AC III) higher than the results obtained by the mechanical test in mixed mode, indicating an oversized system result of the facade cladding when the direct tensile adhesion strength was verified alone. / O descolamento de placas de revestimento em edificações é um problema que ainda persiste em várias construções modernas, apesar dos diversos avanços alcançados no setor de tecnologia de fachadas e de um público consumidor cada vez mais exigente. Tais descolamentos podem ocorrer por ruptura adesiva nas interfaces do sistema ou, ainda, por ruptura coesiva no interior de qualquer uma de suas camadas. Estudos mostraram que a maioria dos descolamentos cerâmicos em fachadas acontecem na interface argamassa colante e placa cerâmica e isso se dá em razão que é a região do sistema de revestimento mais solicitada por esforços de cisalhamento. No Brasil, a metodologia mais utilizada para avaliação da aderência de revestimentos cerâmicos é o ensaio de resistência de aderência à tração direta, contudo, tal avaliação pode ser insuficiente para constatação da qualidade da adesão dos revestimentos ao substrato, visto que a aderência do sistema é resultante não somente da resistência de aderência à tração, mas também da resistência de aderência ao cisalhamento e da extensão de aderência da argamassa. Esta dissertação objetiva contribuir para o estudo e compreensão da influência da área de contato na aderência da interface entre argamassa colante e placa cerâmica por meio da avaliação da resistência de aderência a partir do ensaio de propagação de fissura no modo misto de tensões (MMF – Mixed Mode Flexure) que simula esforços de tração e cisalhamento simultaneamente. Os sistemas produzidos em laboratório reproduzem condições onde a falha na extensão da argamassa contribui para a diminuição da resistência de aderência. Através de uma máquina de ensaios mecânicos universais, aplicou-se uma carga progressiva pontual nos 120 corpos de prova produzidos, de modo a promover esforços internos de tração e cisalhamento. As variáveis incorporadas ao experimento foram o tipo de argamassa utilizada, a espessura da argamassa no corpo de prova e o tamanho da falha de colagem induzida no conjunto. Os resultados experimentais mostram que a resistência de aderência na interface entre a argamassa colante e placa cerâmica decresce à medida que a falha na colagem aumenta, apresentando perda de aderência de até 44,2% e 51,4% (AC II e AC III, respectivamente). Os valores de resistência de aderência encontrados por meio do ensaio de tração direta são 60,8% (AC II) e 58,1% (AC III) maiores que os resultados obtidos pelo ensaio mecânico em modo misto, indicando um resultado superdimensionado do sistema de revestimento da fachada quando verificada a resistência de aderência à tração direta isoladamente.

Physical factors in the environment of a cell affect its function and behavior in a variety of ways. There is increasing evidence that, among these factors, the geometric arrangement of receptor ligands plays an important role in setting the conditions for critical cellular processes. The goal of this thesis is to develop new techniques for probing the role of extracellular ligand geometry, with a focus on T cell activation. In this work, top-down molecular-scale nanofabrication and bottom-up selective self-assembly were combined in order to present functional nanomaterials (primarily biomolecules) on a surface with precise spatial control and single-molecule resolution. Such biomolecule nanoarrays are becoming an increasingly important tool in surface-based in vitro assays for biosensing, molecular and cellular studies. The nanoarrays consist of metallic nanodots patterned on glass coverslips using electron beam and nanoimprint lithography, combined with self-aligned pattern transfer. The nanodots were then used as anchors for the immobilization of biological ligands, and backfilled with a protein-repellent passivation layer of polyethylene glycol. The passivation efficiency was improved to minimize nonspecific adsorption. In order to ensure true single-molecule control, we developed an on-chip protocol to measure the molecular occupancy of nanodot arrays based on fluorescence photobleaching, while accounting for quenching effects by plasmonic absorption. We found that the molecular occupancy can be interpreted as a packing problem, with the solution depending on the nanodot size and the concentration of self-assembly reagents, where the latter can be easily adjusted to control the molecular occupancy according to the dot size. The optimized nanoarrays were used as biomimetic architectures for the study of T cell activation with single-molecule control. T cell activation involves an elaborate arrangement of signaling, adhesion, and costimulatory molecules organized into a stereotypic geometric structure, known as the immunological synapse, between T cell and antigen-presenting cell. Novel bifunctionalization schemes were developed to better mimic the antigen-presenting surfaces. Nanoarrays were functionalized by single molecules of UCHT1 Fab', and served as individual T cell receptor binding sites. The adhesion molecule ICAM-1 was bound to either static PEG background, or a mobile supported lipid bilayer. The minimum geometric requirements (receptor clustering, spacing and stoichiometry) for T cell activation was probed by systematic variation of the nanoarray spacing and cluster size. Out-of-plane spatial control of the two key molecules by way of nanopillar arrays was used to adjust the membrane bending and steric effects, which were essential for the investigation of molecular segregation in T cell activation. The results provide insights into the complicated T cell activation mechanism, with translational implications toward adoptive immunotherapies for cancer and other diseases. This single-molecule platform serves as a novel and powerful tool for molecular and cellular biology, e.g., receptor-mediated signaling/adhesion, especially when multiple ligands or membrane deformation are involved.

The cytoskeleton is a diverse, multi-protein framework that plays a fundamental role in many cellular activities including mitosis, cell division, intracellular transport, cell motility, muscle contraction, and the regulation of cell polarity and organization. Furthermore, cytoskeletal filaments have been implicated in the pathogenesis of a wide variety of diseases including cancer, blood disease, cardiovascular disease, inflammatory disease, neurodegenerative disease, and problems with skin, nail, cornea, hair, liver and colon. Increasing evidence suggests that the distribution and organization of the cytoskeleton in living cells are affected by mechanical stresses and the cytoskeleton determines cell stiffness. We developed a fully nonlinear, constrained mixture model for adherent cells that allows one to account separately for the contributions of the primary structural constituents of the cytoskeleton and extended a prior solution from the finite elasticity literature for use in a sub-class of atomic force microscopy (AFM) studies of cell mechanics. The model showed that the degree of substrate stretch and the geometry of the AFM tip dramatically affect the measured cell stiffness. Consistent with previous studies, the model showed that disruption of the actin filaments can reduce the stiffness substantially, whereas there can be little contribution to the overall cell stiffness by the microtubules or intermediate filaments. To investigate the effect of mechanical stretching on cytoskeletal remodeling and cell stiffness, we developed a simple cell-stretching device that can be combined with an AFM and confocal microscopy. Results demonstrate that cyclic stretching significantly and rapidly alters both cell stiffness and focal adhesion associated vinculin and paxillin, suggesting that focal adhesion remodeling plays a critical role in cell stiffness by recruiting and anchoring F-actin. Finally, we estimated cytoskeletal remodeling by synthesizing data on stretch-induced dynamic changes in cell stiffness and focal adhesion area using constrained mixture approach. Results suggest that the acute increase in stiffness in response to an increased cyclic stretch was probably due to an increased stretch of the original filaments whereas the subsequent decrease back towards normalcy was consistent with a replacement of the highly stretched original filaments with less stretched new filaments.

The cytoskeleton is a diverse, multi-protein framework that plays a fundamental role in many cellular activities including mitosis, cell division, intracellular transport, cell motility, muscle contraction, and the regulation of cell polarity and organization. Furthermore, cytoskeletal filaments have been implicated in the pathogenesis of a wide variety of diseases including cancer, blood disease, cardiovascular disease, inflammatory disease, neurodegenerative disease, and problems with skin, nail, cornea, hair, liver and colon. Increasing evidence suggests that the distribution and organization of the cytoskeleton in living cells are affected by mechanical stresses and the cytoskeleton determines cell stiffness. We developed a fully nonlinear, constrained mixture model for adherent cells that allows one to account separately for the contributions of the primary structural constituents of the cytoskeleton and extended a prior solution from the finite elasticity literature for use in a sub-class of atomic force microscopy (AFM) studies of cell mechanics. The model showed that the degree of substrate stretch and the geometry of the AFM tip dramatically affect the measured cell stiffness. Consistent with previous studies, the model showed that disruption of the actin filaments can reduce the stiffness substantially, whereas there can be little contribution to the overall cell stiffness by the microtubules or intermediate filaments. To investigate the effect of mechanical stretching on cytoskeletal remodeling and cell stiffness, we developed a simple cell-stretching device that can be combined with an AFM and confocal microscopy. Results demonstrate that cyclic stretching significantly and rapidly alters both cell stiffness and focal adhesion associated vinculin and paxillin, suggesting that focal adhesion remodeling plays a critical role in cell stiffness by recruiting and anchoring F-actin. Finally, we estimated cytoskeletal remodeling by synthesizing data on stretch-induced dynamic changes in cell stiffness and focal adhesion area using constrained mixture approach. Results suggest that the acute increase in stiffness in response to an increased cyclic stretch was probably due to an increased stretch of the original filaments whereas the subsequent decrease back towards normalcy was consistent with a replacement of the highly stretched original filaments with less stretched new filaments.

No description available.