Orientadores: Benedicto de Campos Vidal, Sergio Rocha Piedade / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T11:12:09Z (GMT). No. of bitstreams: 1 Paoli_Flaviade_D.pdf: 810753 bytes, checksum: 47e246e2b853e83471c2956a0fbadee2 (MD5) Previous issue date: 2007 / Resumo: A relação estrutura-função tem sido abordada em diversos estudos de tecidos colagenosos, como o tendão. É conhecido, que dependendo do tipo de força aplicada, uma composição e organização molecular específica, com características morfológicas peculiares podem ser observadas. Tendões comportam-se viscoelasticamente, sendo esta propriedade estudada através de análise biomecânica, na qual está diretamente relacionada com a organização dos componentes teciduais. Com isso, os objetivos do presente trabalho foram descrever a importância dos componentes não-colagênicos na propriedade viscoelástica de tendões e verificar possíveis alterações na organização e no arranjo dos componentes da matriz extracelular nos tendões após os ensaios de tensão. Tendões de frangos foram escolhidos como modelo experimental já que apresentam um comprimento viável para ensaios de tensão, além de possuírem peculiaridades em sua composição e organização molecular. Tendões flexores digitais superficiais foram dissecados e distribuídos em: grupo I (não-tratados), grupo II (tendões tratados com hialuronidase testicular) e grupo III (tendões tratados com papaína). Parâmetros dimensionais como peso, espessura, largura e área seccional transversa foram mensurados. Os tendões foram preservados em solução fisiológica até sua utilização nos ensaios. Após os ensaios, eles foram fixados, incluídos em parafina e encaminhados para análises morfológicas, histoquímicas e microespectrofotométricas. Os ensaios do grupo I mostraram maiores valores de tensão, energia elástica e módulo de elasticidade do que os dos grupos II e III. Ao contrário, valores de deformação foram maiores para o grupo II, indicando que, após o tratamento enzimático, os tendões perderam sua capacidade de resistir a forças tensionais e sugere a participação de componentes não-colagênicos nesta função. Análises histoquímicas confirmaram a remoção dos componentes não-colagênicos nos grupos II e III. Ainda, curvas de absorção espectral foram obtidas em cortes de tendões não submetidos a ensaios e do grupo I (não-tratado com enzimas, porém estirado) corados com azul de toluidina e ponceau SS. Em cortes de tendões do grupo I corados com azul de toluidina, dois picos de absorção foram observados, enquanto em cortes de tendões não submetidos a ensaios foi observado apenas um pico. Com isso, pode-se considerar que o grupo negativo dos glicosaminoglicanos disponíveis quando o tendão encontrava-se não-tensionado tornaram-se menos disponíveis quando o tendão foi estirado. Cortes de tendões corados com ponceau SS demonstraram um pico de absorção e cortes do grupo I apresentaram altos valores de absorbâncias. A deformação de materiais envolve diversos mecanismos, como o desaparecimento de crimps. Essa alteração morfológica explicaria tanto a não disponibilidade dos grupos negativos de glicosaminoglicanos quanto o alto valor de absorbância detectado em cortes do grupo I corados com ponceau SS. Em conclusão, os componentes dos tendões são estruturas dinâmicas, altamente organizadas e orientadas, que são capazes de se rearranjarem quando tensionados. Mais ainda, esses componentes não-colagênicos são fundamentais para a integridade funcional dos tendões / Abstract: The structure-function relationship has been shown in several works regarding collagenous tissues, such as tendon. It is known that different loads can alter composition, molecular organization and morphological characteristics. Tendons present viscoelastic behavior and this characteristic is studied by biomechanical analyses and is directly related to tissue component organization. The aim of this work was to describe the importance of noncollagenous components on tendon viscoelasticity by analyzing changes in the organization and arrangement of extracellular matrix constituents and, consequently, changes in tendon morphology after mechanical tests. Chicken tendons were chosen due to their organization and composition; besides presenting a good length for mechanical tests. Superficial digital flexor chicken tendons were dissected and distributed into 3 groups: group I, non-treated; group II, tendons treated with testicular hyaluronidase; and group III, tendons treated with papain. Then, dimensional parameters, such as weight, width, thickness and cross-sectional area, were measured. The tendons were preserved in physiological solution until use in the mechanical tests. During the mechanical tests, they were tensioned and immediately afterwards were fixed and processed for morphological and histochemical analyses. The mechanical test results of groups II and III showed decreased ultimate tensile strength, elastic energy and elasticity modulus values, when compared with group I. In contrast, the ultimate strain values were highest for group II, indicating that after enzymatic treatment the tendons lost their capacity to resist tensile forces. Groups II and III presented different deformations in comparison with group I. Histochemical analysis confirmed the removal of the noncollagenous components in groups II and III. Spectral absorption curves were obtained for the non-stretched and group I tendon sections (non-treated to enzyme, but stretched) stained with toluidine blue and Ponceau SS. In group I sections stained with toluidine blue, two absorption peaks was observed, while in the non-stretched sections only one absorption peak was observed. Negative groups of GAGs available in non-stretched tendon sections most likely became unavailable in stretched tendons sections. Sections stained with Ponceau SS showed one absorption peak and group I sections showed the highest absorbancy values. Deformation of tendinous materials could involve a number of mechanisms, like collagen uncrimping. Collagen uncrimping could be involved in the unavailable of negative GAG groups and the highest absorbancy values in group I sections stained with Ponceau SS. In conclusion, tendon component constituents are dynamic, highly organized and oriented structures capable of rearranging during stretching. Moreover, the noncollagenous components are fundamental to tendon functions / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

Ageing poses one of the largest risk factors for the development of cardiovascular disease (CVD). The increased propensity towards vascular pathology with advancing age maybe explained, in part, by a reduction in the ability of circulating endothelial progenitor cells (EPCs) to contribute to vascular repair and regeneration. Among all current putative EPC populations, outgrowth endothelial cells (OECs) display the most features consistent with a human postnatal vasculogenic cell. Cell-surface heparan sulfate (HS) proteoglycans, by virtue of specific sulfated domains within the glycosaminoglycan chain, are able to bind and modulate the activities of a variety of proteins important for EPC mobilisation, homing and function at sites requiring neovascularization. This study aimed to determine if human OEC function is impaired with age, and to ascertain whether this is accompanied by changes in the fine structure of OEC HS.Using in vitro cell culture methods, OECs were isolated from healthy subjects across an age range and cell phenotype was verified by the demonstration of numerous endothelial, but not hematopoietic, cell characteristics. The functional capacity of peripheral blood derived OECs from young and old subjects, and comparative cord blood derived OECs, was assessed in terms of their susceptibility to apoptosis, proliferative, migratory and tube-forming capabilities. In vitro scratch and transwell migration assays revealed that the migratory capacity of peripheral blood derived OECs isolated from old subjects was impaired in comparison to those from young subjects and cord blood derived OECs. Structural analysis of HS by high performance liquid chromatography (HPLC) demonstrated a significant reduction in the relative percentage of the trisulfated disaccharide, 2-O-sulfated-uronic acid, N, 6-O-sulfated-glucosamine (UA[2S]-GlcNS[6S]), within OEC HS with age (r = -0.847, p=<0.01). Moreover, a decline in the migratory response of OECs towards a gradient of VEGF significantly correlated with the percentage expression of this disaccharide (r = 0.840, p<0.01). Disruption of cell surface HS by pre-treatment with heparinase I and III was found to significantly reduce the VEGF-induced migratory response of peripheral blood derived OECs isolated from young subjects to levels similar to that observed for OECs from older individuals. Understanding the role of HS in regulating the directional migration of EPCs to sites requiring neovascularization and developing approaches to facilitate EPC migration may aid in the design of more successful strategies to optimise the regenerative capacity of these cells in the ageing vasculature.

Chronic peritonitis contributes to technique failure in peritoneal dialysis (PD), an effective replacement therapy for chronic kidney failure. Staphylococcus aureus infection is one of the most common causes of peritonitis in PD. Interestingly, mice deficient in the cell surface heparan sulfate proteoglycan, syndecan-1, were reported to clear S. aureus corneal infection more effectively than wild-type mice. The objectives of this study were to examine the protein expression and role of syndecan-1 in leukocyte recruitment, chemokine presentation and S. aureus infection in the microcirculation underlying the parietal peritoneum in wild-type and syndecan-1-/- mice. Immunofluorescence intravital microscopy (IVM) of the parietal peritoneum microcirculation revealed that syndecan-1 was localized to the subendothelial region of venules and the mesothelial layer but does not regulate leukocyte recruitment and is not necessary for presentation of the chemokine MIP-2 in peritoneal venules. IVM was also used to study the effects of a conventional PD solution injected through a peritoneal catheter in a mouse PD model. After 6 weeks of dialysis, the peritoneal catheter implant increased leukocyte rolling and extravasation, fibrosis and angiogenesis in the parietal peritoneum independently from the dialysis solution treatment. Furthermore, the role of syndecan-1 was examined using a 4 week PD model. Four hours after infection with S. aureus through the dialysis catheter or intraperitoneal injection, the dialyzed syndecan-1-/- mice were more susceptible to S. aureus infection than undialyzed syndecan-1-/- controls and wild-type animals. IVM showed that in S. aureus infection, syndecan-1 was removed from the subendothelial surface of peritoneal venules but syndecan-1 deficiency did not affect leukocyte recruitment during S. aureus infection. This study indicates that syndecan-1 in the peritoneum and microcirculation is not a regulator of inflammatory responses but is crucial for providing a barrier to S. aureus infection, which may have important implications for susceptibility to S. aureus infections in PD. / Dissertation / Doctor of Philosophy (Medical Science)

La myostatine est un membre de la superfamille du TGF-β (transforming growth factor-β) impliqué dans la régulation négative de la masse musculaire. En effet, l’absence de myostatine (MSTN) chez la souris est responsable d’un phénotype hypermusclé. Depuis, il a été confirmé qu’une baisse de l’activité de la MSTN conduit à une augmentation de la masse musculaire chez d’autres espèces, y compris l’Homme. L’identification de la MSTN et des conséquences de son invalidation sur le développement musculaire ouvre de nombreuses perspectives en médecine humaine. Il existe de nombreuses situations pathologiques qui conduisent à une fonte musculaire importante : c’est le cas pour des maladies génétiques telles que les dystrophies musculaires ou pour d’autres pathologies comme le cancer et le sida. Différentes approches anti-MSTN ont été développées au cours des dernières années, par exemple un anticorps anti-MSTN ou des ligands de la MSTN. L’objectif majeur de ce projet de recherche a consisté à identifier de nouveaux inhibiteurs de la MSTN, en particulier appartenant à la famille de protéines appelées SLRP (Small Leucine Rich Proteoglycans). Il a été mis en évidence que des membres de cette famille, notamment la décorine (DCN) ainsi que des fragments issus de la DCN dont le peptide 31-71, sont capables de se lier à la MSTN en présence de zinc. La DCN peut alors empêcher l’activité de la MSTN en s’opposant à la liaison de cette dernière à son récepteur. Dans ce contexte, nous avons étudié des séquences peptidiques plus restreintes de la DCN murine pouvant interagir efficacement avec la MSTN et des peptides dérivés d’autres SLRP pour leur aptitude à lier la MSTN. Afin de faciliter le criblage in vitro de ces composés, nous avons tout d’abord créé une lignée cellulaire HEK293T exprimant stablement une cassette inductible par la MSTN fusionnée au gène de la luciférase (pCAGA-Luc). Parmi les candidats testés, le peptide mDCN48-71 a été le plus intéressant de par sa forte activité anti MSTN in vitro comparée aux autres, avec un IC50 de 7 µM. Notons également que le peptide mDCN48-71 n’a pas inhibé d’autres membres de la superfamille du TGF-β : TGF-β2, activine A et GDF-11 – ce qui suggère une spécificité d’action du peptide. En outre, des études d’anisotropie de fluorescence ont permis de prouver l’interaction directe du peptide mDCN48 71 avec la MSTN et la dépendance au zinc de cette liaison. Pour finir, nous avons montré que des injections intramusculaires répétées de ce peptide chez le modèle murin dystrophique mdx, conduisent à une augmentation significative de la masse des muscles tibiaux antérieurs injectés de l’ordre de 21 % par rapport aux muscles contrôles. / Myostatin is a member of the transforming growth factor-β (TGF-β) superfamily and a negative regulator of skeletal muscle growth. In 1997, lack of myostatin (MSTN) was related to increased muscle mass in mice. Since then, MSTN has been found in other species including humans. Inhibition of this protein offers opportunities in human medicine for many pathological conditions leading to a significant muscle loss: genetic disorders such as muscular dystrophy as well as other diseases like cancer and AIDS. Recently, several anti-MSTN approaches have been developed such as antibodies against MSTN or naturally occurring proteins that bind to and inactivate MSTN. The aim of this research was to identify novel inhibitors of MSTN, especially belonging to the SLRP (Small Leucine Rich Proteoglycans) family of proteins. Members of this family, including decorin (DCN) and fragments thereof (murine derived peptide mDCN31-71) can bind to MSTN in a zinc-dependent manner. In this context, smaller peptide sequences of mouse DCN and peptides from other SLRP have been studied for their ability to bind MSTN. First, we created a HEK293T stable cell line expressing the luciferase gene under control of a MSTN-inducible promoter (pCAGA-Luc) so as to screen these compounds in vitro. Here we report that the peptide mDCN48-71 shows the stronger activity anti MSTN in vitro among all the peptides tested (IC50 = 7 µM). Furthermore, other members of the TGF β superfamily: TGF β2, activin A and GDF-11 are not inhibited by the mDCN48-71 peptide - which suggests a specificity of its action. By performing fluorescence anisotropy studies, we proved the direct and zinc dependent interaction between peptide mDCN48-71 and MSTN. Finally, we showed that repeated intramuscular injections of this peptide in the dystrophic mdx mouse model led to a significant increase of the injected tibialis anterior muscle mass (21 %) compared to control muscles.

Nervous system function is closely tied to its structure, which ensures proper connectivity and neural activity. Neuronal architecture is assembled by a series of morphogenetic events, including the coordinated migrations of neurons and axons during development. Subsequently, the neuronal architecture established earlier must persist in the face of further growth, maturation of the nervous system, and the mechanical stress of body movements. In this work, we have shed light on the molecular mechanisms governing both the initial assembly of the nervous system and the long-term maintenance of neural circuits. In particular, we identified heparan sulfate proteoglycans (HSPGs) as regulators of neuronal migrations. Our discovery and analysis of viable mutations in the two subunits of the heparan sulfate co-polymerase reveals the importance of the coordinated and dynamic action of HSPGs in neuronal and axon guidance during development. Furthermore, we uncovered that the HSPG LON-2/glypican functions as a modulator of UNC-6/netrin signaling through interactions with the UNC-40/DCC receptor. During larval and adult life, molecules such as the protein SAX-7, homologous to mammalian L1CAM, function to protect the integrity of nervous system architecture. Indeed, loss of sax-7 leads to progressive disorganization of neuronal architecture. Through a forward genetic screen, we identified LON-1 as a novel maintenance molecule that functions post-embryonically with SAX-7 to maintain the architecture of the nervous system. Together, our work highlights the importance of extracellular interactions to modulate signaling events during the initial development of the nervous system, and to subsequently maintain neuronal architecture for the long-term.

Abstract not available

Alzheimer’s disease (AD) is a neurodegenerative disorder, with extensive evidence implicating the misfolding, aggregation and deposition of the amyloid-β (Aβ) peptide as central to the pathogenesis. Heparan sulfate (HS) is an interactive glycosaminoglycan, attached to core proteins as HS proteoglycans (HSPGs). HSPGs are present on cell surfaces and in the extracellular matrix where they facilitate multiple signaling functions, but HS is also consistently present in all amyloid deposits, including those of AD. In amyloidosis HS has been studied as an aggregation template, promoting fibril formation and serving a scaffold function in the resulting deposits. The objective of this thesis was to assess how cell surface HS is potentially implicated in Aβ amyloidosis and the associated neuroinflammation of AD.   In AD brain we determined that HS predominantly accumulated in Aβ deposits with dense cores and found glial-expressed HSPGs within these deposits. Aβ elevated HSPG levels in primary glial cultures, implicating activated glia as one source of the Aβ-associated HS. Next, we determined that microglial HSPGs are critical for the upregulation of interleukin-1β and tumor necrosis factor-α following exposure to lipopolysaccharide, an established inflammatory insult. Together these results raise the possibility that Aβ-induced expression of microglial HSPGs may promote neuroinflammation.   Multiple mechanisms of Aβ toxicity have been proposed and different Aβ assemblies exert their toxicity through alternative routes. We found that three different preparations of Aβ aggregates all exhibited HS-dependent cytotoxicity, which in part correlated with Aβ internalization. Furthermore, heparin treatment attenuated Aβ cytotoxicity and uptake. In Aβ-positive AD microvasculature, HS deposited with Apolipoprotein E (ApoE) and its receptor, the low density lipoprotein receptor-related protein 1 (LRP1). In cell culture, HS and LRP1 co-operated in Aβ interactions and the addition of ApoE increased the levels of cell-associated Aβ in a HS- and LRP1-dependent manner. This ApoE-mediated increase in cell-associated Aβ may promote toxicity and vascular degeneration, but equally HS-mediated internalization of Aβ could represent a clearance route across the blood-brain-barrier. The findings presented here illustrate multiple roles for cell-surface HSPGs in interactions relevant to the pathogenesis of AD.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

L’épiderme est un tissu épithélial stratifié et kératinisé, majoritairement composé de kératinocytes. La cohésion de l’épiderme, élémentaire à la fonction-barrière et donc à la protection de l’organisme, est assurée grâce à des systèmes de jonctions intercellulaires, notamment les desmosomes. Comme l’indiquent nos résultats d’étude de la desmosealine, un protéoglycanne épidermique présent dans la partie extracellulaire des desmosomes, la composition de ces jonctions n’est pas encore entièrement élucidée. Les éléments matriciels issus des espaces extracellulaires de l’épiderme peuvent être incorporés au sein des desmosomes et participer ainsi à la régulation de la différenciation et la cohésion épidermiques. Nous avons mis au point une méthode permettant d’isoler les desmosomes épidermiques humains utilisables pour créer de nouveaux anticorps et favorisant la caractérisation biochimique de ces structures. Un nouvel anticorps monoclonal reconnaissant un antigène situé à la surface des kératinocytes, dont l’expression varie en fonction du degré de différenciation kératinocytaire, a été crée. A l’aide de cet anticorps, nous avons entrepris la caractérisation biochimique et par spectrométrie de masse de l’antigène associé. Nous avons ainsi développé de nouveaux outils biologiques et techniques utilisables pour l’étude des desmosomes et de leurs éléments issus de la matrice extracellulaire épidermique / Epidermis is a stratified, keratinized epithelial tissue, mostly composed of keratinocytes. Epidermal barrier function provided by epidermis is essential for protection of the organism and largely depends on cell cohesion. Desmosomes constitute the most prominent cell-to-cell junction system involved in this function. As indicated by our results of studies on desmosealin, an epidermal proteoglycan present in the extracellular parts of desmosomes, the composition of these junctions is not yet completely resolved. Elements of the intercellular matrix can be incorporated into desmosomes and thus participate in the regulation of the epidermal differentiation and cohesion. We established a method to isolate human epidermal desmosomes in order to create new antibodies allowing the biochemical characterization of new desmosomal components. A new monoclonal antibody has been generated. It recognizes an antigen located at the keratinocyte surface with an expression pattern depending on the level of keratinocyte differentiation. Using this antibody, we have engaged the biochemical and mass spectrometry characterization of the corresponding antigen. This work contributes to the development of new biological and technical tools useful for studies of desmosomes and of their components issued from the epidermal extracellular matrix

O biglicam é um proteoglicano é altamente expresso em células trofoblásticas de patologias placentárias com invasividade exacerbada. No entanto, as funções do biglicam no trofoblasto ainda não foram elucidadas. Sendo assim, verificamos a expressão e as funções de biglicam e seus receptores Toll-like (TLR)-2 e TLR-4 nas células trofoblásticas durante a gestação. As células do citotrofoblasto extraviloso (CTEV) foram positivas para todas as moléculas, menos para o biglicam em placentas a termo. Adição exógena de biglicam promoveu migração e invasão das células trofoblásticas. O biglicam estimulou a fosforilação de AKT nos sítios Thr308 e Ser473 nas células trofoblásticas. A migração e a invasão biglicam-dependentes e as fosforilações de AKT foram inibidas após a adição de anticorpos bloqueadores anti-TLR-2 e anti-TLR-4. O silenciamento gênico de AKT1 em células SGHPL-5 aboliu os efeitos do biglicam na motilidade. Em conclusão, o biglicam aumenta a motilidade de células trofoblásticas após sinalização por AKT através da ativação de TLR-2 e TLR-4. / Biglycan is a highly expressed proteoglycan in trophoblast cells from invasiveness-changed placental pathologies. However, biglycan functions in the trophoblast were not yet identified. Therefore, it was verified the expression and functions of biglycan and its receptors Toll-like (TLR)-2 and TLR-4 in trophoblast cells throughout pregnancy. The extravillous cytotrophoblast cells (EVT) were positive to all the molecules, although biglycan was negative in term placentas. Exogenous biglycan promoted migration and invasion of trophoblast cells. Biglycan stimulated AKT phosphorilation at Thr308 and Ser473 sites in trophoblast cells. The biglycan-dependent migration, invasion and AKT phosphorilation were inhibited upon addiction of anti-TLR-2 and anti-TLR-4 blocking antibodies. AKT1 genic silencing in SGHPL-5 cells abolished the motility effects. In conclusion, biglycan increases the motility of trophoblast cells after AKT signaliing throughout TLR-2 and TLR-4 activation.