Orientador: Renée Laufer Amorim / Resumo: As alterações na expressão de E-caderina, -catenina, APC e Caveolina-1 nas células epiteliais prostáticas têm sido estudados em humanos como mecanismos relacionados com progressão tumoral, invasão e metástase. Estas proteínas estão envolvidas no processo de adesão celular e ativação da via WNT canônica. No cão, a perda da expressão proteica de E-caderina e a translocação da -catenina da membrana para o citoplasma/núcleo foram descritas anteriormente em lesões prostáticas caninas. No entanto, nenhum estudo correlacionou alterações de expressão dessas proteínas com as proteínas APC e Caveolina-1. Devido ao prognóstico desfavorável do carcinoma prostático (CaP) no cão e a importância da identificação de novos marcadores prognósticos e preditivos, o presente estudo visou avaliar as expressões gênica e proteica de E-caderina, -catenina, APC e Caveolina-1 em diferentes lesões prostáticas caninas, além de avaliar o padrão de metilação do gene APC. Foram utilizados neste estudo 10 CaPs, 4 metástases de carcinoma prostático, 10 amostras de atrofia inflamatória proliferativa (PIA) e 10 próstatas normais de cães para análise imuno-histoquímica. Para a técnica de RT-qPCR forma utilizados 11 próstatas normais, 11 PIA, 17 CaPs e 3 metástases. Para análise de metilacão foram utilizadas seis próstatas normais, seis próstatas com PIA e 12 CaPs. Este estudo revelou aumento de expressão gênica e proteica de Caveolina-1 nas amostras de CaP e metástases, além de menor expressão nas amostras de ca... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Altered expression of E-cadherin, -catenin, APC and Caveolin-1 in prostate epithelial cells has been studied in humans as mechanisms related with tumor progression, invasion and metastasis. These proteins are envolved in cell-cell cohesion and participate in the activation of the canonical WNT pathway. In dogs, membranous E-cadherin loss and translocation of -catenin from the membrane to cytoplasm/nucleus were described previously in canine prostatic lesions. However, studies correlating these genes have not been reported their proteins with APC and Caveolin-1 expressions in canine prostatic lesions. Due to poor prognosis in canine prostate carcinoma (PC) and the need to develop new prognostic and predictive biomarkers, this study aimed to evaluate gene and protein expression of E-cadherin, -catenin, APC and Caveolin-1 in different canine prostatic lesions, and the methylation status of APC gene. We used 10 PC, 4 prostate metastasis, 10 proliferative inflammatory atrophy(PIA) and 10 canine normal prostates tissues for immunohistochemistry. For RT-qPCR we used 11 normal prostate, 11 PIA, 17 PC and 4 metastasis. For methylation analysis, we used six normal prostates, 6 PIA and 13 PC. This study revealed increased Caveolin-1 gene and protein expression in PC and metastasis and lower expression were found in tumors with lower Gleason score. Membranous E-cadherin and - catenin staining was observed in normal prostate samples whereas heterogenous loss was detected in other samples.... (Complete abstract click electronic access below) / Mestre

Meckel-Gruber syndrome (MKS) is a universally lethal heritable human disease characterised by CNS malformations, cystic kidney, polydactyly, and liver fibrosis. MKS is classed as one of the ciliopathies due to its association with dysfunctional primary cilia, signalling organelles found on most cells in the human body. Some of the symptoms of MKS can be explained as a consequence of disrupted developmental signalling through the primary cilium, other defects are harder to explain, and evidence now exists for non-ciliary influences on ciliopathies. The nature of these influences, and the implications they may have for our understanding of ciliary function and the aetiology of MKS, remain unclear. In this thesis, defects in cell-extracellular matrix (ECM) interaction in MKS are investigated to determine whether MKS proteins have a role in this process, and if so, whether this role may be involved in MKS pathology. A combination of transcriptomic, proteomic, and cell imaging approaches are used to demonstrate that MKS patient cells produce a defective extracellular matrix, and that the MKS protein TMEM67 is present at the cell surface at sites of cell-ECM interaction. It is shown that the full-length TMEM67 protein is required for correct ECM morphology, and it is further shown that the abnormal extracellular matrix morphology in MKS cells underlies other defects, including failure to build cilia and alterations to the actin cytoskeleton. This represents the first set of causal relationships identified between the cellular defects in this complex disease. It is further shown that treatment with developmental signalling pathway antagonists can rescue these defects, potentially revealing a new avenue of therapeutic intervention for MKS. Finally, possible upstream defects are investigated that might underlie the ECM defect, including alterations to cell spreading behaviour and cell deformation resistance.

Melanoma primário da mucosa oral (MPMO) é um tumor raro e agressivo. Estudos recentes demonstraram uma correlação entre o aumento da invasão tumoral e o fenótipo metastático com uma alteração no padrão de expressão das moléculas de adesão. Neste estudo analisamos a expressão de integrinas e imunoglobulinas nos melanomas primários da mucosa oral e relacionamos os resultados com os parâmetros clínicos. As análises imunoistoquímicas dos padrões de expressão destas moléculas foram realizadas em 35 casos de melanomas primários da mucosa oral, e os resultados foram correlacionados com características clínicas e histológicas. Observou-se que a subunidade beta-4 de integrina foi negativa em casos com invasão vascular. A presença de integrina beta-3 e de CD166 (ALCAM) estavam estatisticamente associadas à extensa invasão vascular (p < 0,05). A menor expressão de CD54 (ICAM) foi marginalmente relacionada a casos com necrose extensa, enquanto a maioria dos casos com doença metastática foi negativa para CD66 (CEACAM). Conclusão: padrões alterados de expressão de moléculas de adesão, principalmente integrinas e imunoglobulinas, podem participar da patogênese e do desenvolvimento dos melanomas primários da mucosa oral / Primary oral mucosal melanoma is a rare and an aggressive tumor. Recent studies have demonstrated the correlation among increased tumor invasion, the metastatic phenotype and altered adhesion molecule expression profiles. The present study analyzed the expression of integrins and immunoglobulin-like adhesion molecules in oral mucosal melanomas and correlated results with clinical parameters. Immunohistochemical analyses of their expression patterns were performed on thirty-five cases of primary oral mucosal melanomas. The results were correlated with clinical and histological features of the cohort. The beta-4 subunit of integrin was negative and this was related with vascular invasion. Positivity of integrin beta-3 and CD166 (ALCAM) was statistically associated with extensive vascular invasion (p < 0.05). Lower expression of CD54 (ICAM) was associated with cases with extensive necrosis. Most cases with metastatic disease were negative for CD66 (CEACAM). Conclusion: Altered patterns of adhesion molecule expression, mainly integrins and immunoglobulin-like proteins, may participate in the pathogenesis and outcome of primary oral mucosal melanomas

Bacteria adhere despite severe mechanical perturbations. In Gram-positive bacteria, this adhesion is dependent upon a set of extracellular proteins, most notably pili, that have a unique abundance of internal disulfide, isopeptide, and thioester bonds. How these cell adhesion proteins manage to withstand such mechanical assaults, and what role these internal covalent bonds play to that end, remain open questions. Herein, we apply single-molecule force spectroscopy to delve into the mechanical behavior of three Gram-positive pilus proteins. We find that structural components of the Actinomyces oris and Corynebacterium diphtheriae pili have isopeptide-delimited extensions at extreme mechanical forces. This behavior enables efficient energy dissipation under high mechanical loads. Meanwhile, the pilus tip adhesin of Streptococcus pyogenes can covalently bind to targets via its internal thioester bond. We find that reactions with this internal thioester bond are reversible, and that both the nucleophilic bond cleavage and its spontaneous reformation are negatively force-dependent, inhibited at forces above ~30 pN and above ~7 pN, respectively. Based on these observations, we propose a model of shear-enhanced covalent adhesion for Gram-positive bacteria. Finally, we move from single-molecule characterization to application as we explore the potential of a peptide competitors to modulate the folding and function of bacterial virulence factors.

The mammalian visual system develops to perform many complex tasks that allow us to perceive the natural world. These tasks rely on a dense network of synaptic connections transporting visual information both to and within visual cortex (V1). The laminar organization and functional properties of visual cortical neurons are largely conserved across mammals, and the mouse has been adopted as a model organism to study the development of this cortical circuit. Neurons in each cortical layer must find the correct synaptic partners for the optimal receipt, transfer, and processing of information. The molecular cues guiding the development of these connections, however, are largely unknown. In this thesis, I identify and then examine the role of molecular factors important for synapse formation in layer 2/3 (L2/3) of visual cortex. L2/3 neurons are highly interconnected and fire selectively to a refined set of visual stimuli. The developmental refinement of these visual preferences has been shown to occur in the week following eye opening, corresponding with a period of intense synapse formation and dynamic gene expression in mouse V1. In Chapters II–IV, I use the TU-tagging technique to identify molecular factors enriched L2/3 neurons before and after eye opening and identify several candidate genes with potential functions in synapse formation. In Chapter V, I examine the function of cell adhesion molecules nectin-1 and nectin-3, identified here as enriched in L2/3 visual cortex at eye opening, and previously shown to interact across synaptic junctions. I focus mainly on the effect of nectin-3 (having post-synaptic localization in hippocampus) on post-synaptic dendritic spine densities in developing L2/3 cortical neurons. I show that nectin-3 knockdown further increases spine densities after eye opening, while overexpressing a full length or truncated nectin-3 protein reduces spine densities. I conclude that nectin-3 may have a role in synapse formation following eye opening, and propose a mechanism describing the effects observed. Here, I describe a unique approach for understanding how cell-type specific connections are formed in visual cortex, beginning with the spatiotemporal examination gene expression and followed by the spatiotemporal manipulation of a single gene. This dissertation includes previously published co-authored material.

Polysialylated NCAM (polySia-NCAM) is re-expressed in a number of tumours, including small cell lung carcinoma and neuroblastoma and is strongly associated with aggressive, invasive and metastatic tumours in the clinic. SiRNA knockdown of the polysialyltransferases (polySTs), the enzymes responsible for polysialylation of neural cell adhesion molecule (NCAM), has been shown to abolish cell migration. PolySia-NCAM is thus a highly attractive novel therapeutic target. A library of potential polyST inhibitors has been synthesised, using substrate-based design and computational chemistry. Compounds synthesised include N-acylmannosamine analogues, thio-linked CMP-sialic acid analogues, N-acyl modified sialic acids and compounds incorporating elements of both approaches. Novel methodology development in the synthesis of many of the compounds is described, notably a novel route to N-acyl sialosides. In addition, compounds identified from in silico screening were considered. Routes to synthesis and isolation of analogues of biologically active compounds are described. Using an enzyme assay, compounds were evaluated for their ability to reduce polySia synthesis through polyST inhibition. Effects of agents on polySia expression in cells, and the ability of compounds to reduce cell migration in vitro was studied using a wound healing ‘scratch assay’. The data from these experiments revealed a number of potent modulators of polySia assembly and their efficacy in reducing cell migration, as well as the limits of the biosynthetic pathway to accept unnatural sialic acid precursors. This is the first example of polyST inhibition modulating tumour cell migration, and points to the potential of the polysialyltransferases as a therapeutic target in metastatic tumours.

Orientador: Willian Fernando Zambuzzi / Resumo: As tendências na terapia com implantes têm incluído a modificação de suas superfícies utilizando ferramentas de nanotecnologia e princípios de bioengenharia, aumentando seu desempenho quando implantado. Embora muito se tenha alcançado em ferramentas para desenvolvimento destes materiais, metodologias de avaliação biológicas não avançaram nesta velocidade. Amparados por ferramentas de bioinformática e utilizando conceitos de biologia sistêmica, o objetivo deste trabalho foi produzir uma metodologia computacional, alternativa ao uso de experimentação animal, capaz de detectar e analisar o quinoma da resposta da interação célula-biomaterial, obtida com ensaio de microarranjo de peptídeos. Estes dados servirão para a construção de um banco de dados para guiar a produção de biomateriais para a área médico-odontológica. Batizaremos este de OsteoBLAST. Para tanto, fizemos uso de superfícies de titânio com diferentes superfícies (maquinado e duplo ataque ácido), as quais foram desafiadas com o cultivo de células tronco indiferenciadas. As amostras biológicas foram utilizadas para avaliar quinases diferencialmente ativadas através de substratos sintéticos, cuja metodologia é conhecida como PamGene, as quais foram reunidas em um banco de dados chamado OsteoBLAST, o qual fora constituído através de um algoritmo em quatro etapas que selecionou os resultados confiados de PamGene, em seguida obetendo o quinoma diferencial e um nível de similaridade com superfícies amplamente usadas na roti... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Current trends in implant therapy have included the modification of their surfaces using nanotechnology tools and principles of bioengineering, increasing their performance when deployed. Although much has been achieved in tools for developing these materials, biological analysis methodologies did not advance at this speed. Supported by bioinformatics tools and using concepts of systemic biology, the objective of this work was to produce a computational methodology, alternative to the use of animal experimentation, capable of detecting and analyzing the kinome of the response of the cell-biomaterial interaction, obtained with microarray peptides. These data will serve to build a database to guide the production of biomaterials for the medical-dental area. We will baptize this one from OsteoBLAST. To do so, we made use of titanium surfaces with different surfaces (machined and double acid etched), which were challenged with the cultivation of undifferentiated stem cells. Biological samples were used to evaluate differentially activated kinases through synthetic substrates, a methodology known as PamGene, as the banks were assembled in a database called OsteoBLAST, which consists of a four-step algorithm that selected the results confident of PamGene, then obtaining the differential kinome and a level of similarity with surfaces widely used in the routine. Our results showed that the EGRF, ENO2, EPHA4, FRK, KRT6B, NCF1, PDPK1, PDGFRB and KDR as proteins involved in this molecul... (Complete abstract click electronic access below) / Mestre

Orientador: Francisco de Assis Mollo Junior / Resumo: A longevidade dos implantes dentários depende da manutenção do tecido peri-implantar e da ausência de inflamação nessa área. Semelhante aos dentes, as bactérias patogênicas podem aderir nas superfícies dos abutments dos implantes e causar doença peri-implantar. As propriedades físico-químicas intrínsecas a cada material ao longo do tempo podem sofrer alterações e assim afetar a adesão devido a constante renovação celular e ao desenvolvimento do biofilme. Avaliar a influência do envelhecimento de materiais de abutment de titânio (Ti) e zircônia (ZrO2) na viabilidade, morfologia, adesão e proliferação de biofilmes de ceranócitos orais normais (Nok-si) e biofilme multiespécie Porphyromonas gingivalis, Streptococcus sanguinis e Fusobacterium nucleatum. Nos discos de ZrO2 e Ti foram analisados rugosidade superficial (Ra), ângulo de contato com a água e difração de raios X (DRX), antes e após o envelhecimento em autoclave. A viabilidade celular de Nok-si foi avaliada pelo ensaio de MTT e a morfologia foi analisada por microscopia eletrônica de varredura (MEV). A proliferação celular e a adesão foram analisadas usando microscópio confocal. Biofilme multiespécies foram analisados quantitativamente por Log (UFC/mL) e qualitativamente por MEV. Para Ti, diferença estatisticamente significante foram encontradas entre as medidas de rugosidade antes e depois do processo de envelhecimento, entretanto o processo de envelhecimento não afetou a rugosidade média dos discos de ZrO2. O processo d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The longevity of dental implants depends on the maintenance of the peri-implant tissue and the absence of inflammation in that area. Similar to teeth, pathogenic bacteria may adhere to the surfaces of implant abutments and cause peri-implant disease and consequently implant loss. The physico-chemical properties intrinsic to each material over time may undergo changes and thus affect adhesion due to constant cell turnover and biofilm development. To evaluate the influence of aging of titanium and zirconia abutment materials on the viability, morphology, adhesion and proliferation of normal oral ceranocytes biofilms (Nok-si) and multispecies biofilm Porphyromonas gingivalis, Streptococcus sanguinis and Fusobacterium nucleatum. Surface roughness (Ra), water contact angle and X-ray diffraction (XRD) were analyzed on the zirconia (ZrO2) and titanium (Ti) discs before and after autoclaving aging. Nok-si cell viability was assessed by the MTT assay and the morphology was analyzed by scanning electron microscopy (SEM). Cell proliferation and adhesion were analyzed using confocal microscopy. Multispecies biofilm were quantitatively analyzed by Log (CFU/mL) and qualitatively by SEM. For Ti, a statistically significant difference was found between the roughness measurements before and after the aging process, however the aging process did not affect the average roughness of the zirconia discs. The aging process affected the wettability of the Ti surface when in contact with water and po... (Complete abstract click electronic access below) / Doutor

L’objectif du travail présenté dans cette thèse était de développer des nanoparticulesfonctionnelles ayant la capacité d’induire l’adhésion et la migration de kératinocyteshumains normaux. L’utilisation de systèmes particulaires pour favoriser l’adhésion etla migration cellulaire dans les processus de cicatrisation constitue une nouvelleapproche de l’ingéniérie tissulaire. Dans cette optique, un dérivé hydrosoluble du chitosan rendu fonctionnel par l’ajoutde peptides RGD a été développé. Les nanoparticules furent développées parcoacervation complexe entre le dérivé cationique du chitosan et le sulfate dechondroïtine anionique. La capacité du système particulaire à induire unchangement cellulaire phénotypique a été évaluée in vitro.Lors de l’évaluation de ce nouveau polymère, le succès de la synthèse a été montrépar l’absence de cytotoxicité et par la préservation de son activité biologique médiéepar les séquences RGD. Aussi bien les polymères que les nanoparticules ont induitl’adhésion et la mobilité de fibroblastes dermiques humains, confirmant le conceptde nanoparticules bio-actives. Cependant, concernant l’étude des interactions entreles nanoparticules et les kératinocytes, aucune conclusion n’a pu être tirée etd’autres travaux sont nécessaires. Pour résumer, un système particulaire bio-actif a été développé. Le choix despeptides RGD pour induire la migration des kératinocytes doit être réévalué, etl’utilisation de concentrations plus importantes, de mélange de peptides d’adhésionou l’utilisation de peptides d’adhésion différents doit être envisagée pour laréalisation d’études ultérieures. / The aim of the work presented in this thesis, was to develop functionalizednanoparticles with the ability to induce adhesion and migration in normal humankeratinocytes. Using particulate systems to promote and support cell adhesion andmigration in epidermal restoration is a novel approach of tissue engineering.In this view, a water-soluble chitosan derivative functionalized with RGD peptideswas developed. Nanoparticles were formed through complex coacervation betweenthe cationic chitosan derivative and the anionic chondroitin sulfate. The particulatesystem was evaluated in vitro for its ability to change phenotype in cells.In the evaluation of the novel hybrid polymer, the successful synthesis wasconfirmed by the absence of cytotoxicity and a preserved bioactivity specific to theRGD-moieties. Both the polymer and the particles formed thereof induced celladhesion and spreading in human dermal fibroblasts, proving the concept ofbioactive nanoparticles. However, when investigating the interaction between thenanoparticles and keratinocytes, no clear conclusion could be drawn and furtherassays are required. To summarize, a bioactive particulate system was developed. The choice of RGDpeptides to induce migration in keratinocytes needs to be re-evaluated and higherconcentrations, mixtures of adhesion peptides or other adhesion peptides might beconsidered for further investigations.

Melanoma primário da mucosa oral (MPMO) é um tumor raro e agressivo. Estudos recentes demonstraram uma correlação entre o aumento da invasão tumoral e o fenótipo metastático com uma alteração no padrão de expressão das moléculas de adesão. Neste estudo analisamos a expressão de integrinas e imunoglobulinas nos melanomas primários da mucosa oral e relacionamos os resultados com os parâmetros clínicos. As análises imunoistoquímicas dos padrões de expressão destas moléculas foram realizadas em 35 casos de melanomas primários da mucosa oral, e os resultados foram correlacionados com características clínicas e histológicas. Observou-se que a subunidade beta-4 de integrina foi negativa em casos com invasão vascular. A presença de integrina beta-3 e de CD166 (ALCAM) estavam estatisticamente associadas à extensa invasão vascular (p < 0,05). A menor expressão de CD54 (ICAM) foi marginalmente relacionada a casos com necrose extensa, enquanto a maioria dos casos com doença metastática foi negativa para CD66 (CEACAM). Conclusão: padrões alterados de expressão de moléculas de adesão, principalmente integrinas e imunoglobulinas, podem participar da patogênese e do desenvolvimento dos melanomas primários da mucosa oral / Primary oral mucosal melanoma is a rare and an aggressive tumor. Recent studies have demonstrated the correlation among increased tumor invasion, the metastatic phenotype and altered adhesion molecule expression profiles. The present study analyzed the expression of integrins and immunoglobulin-like adhesion molecules in oral mucosal melanomas and correlated results with clinical parameters. Immunohistochemical analyses of their expression patterns were performed on thirty-five cases of primary oral mucosal melanomas. The results were correlated with clinical and histological features of the cohort. The beta-4 subunit of integrin was negative and this was related with vascular invasion. Positivity of integrin beta-3 and CD166 (ALCAM) was statistically associated with extensive vascular invasion (p < 0.05). Lower expression of CD54 (ICAM) was associated with cases with extensive necrosis. Most cases with metastatic disease were negative for CD66 (CEACAM). Conclusion: Altered patterns of adhesion molecule expression, mainly integrins and immunoglobulin-like proteins, may participate in the pathogenesis and outcome of primary oral mucosal melanomas