Bilateral breast augmentation is one of the most common cosmetic surgical procedures carried out on women in the western world. Breast augmentation involves increasing the volume of a woman‘s breasts through surgery by placing a silicone implant in the subglandular or subpectoral cavity. Although a capsule forms inevitably around breast implants as a natural part of healing, it can cause significant morbidity if the capsule becomes firm and contracted, a condition known as breast capsular contracture (BCC). The aetiology of BCC remains unknown however it is characterised by dense fibrocollagenous connective tissue with a local inflammatory response. Host response is influenced by several factors including implant surface texture, chemistry and interactions between cells and the extracellular matrix. Texturing holds the implant in place, thus preventing micromotion at the host prosthesis interface. While in smooth surfaces, the implant moves inside the breast, making the fibroblasts repeatedly produce collagen in response to this host-prosthesis shearing motion. In this thesis, the effect of surface characteristics and specific coatings on the cell-surface interaction has been examined on smooth compared to textured surfaces using commercially available breast implants. The properties of breast implants shells have been characterised using confocal laser microscopy, contact angle measurements, confocal Raman spectroscopy and tensile testing. Confocal laser microscopy was used to evaluate the topographical features and surface roughness of the implant surfaces. Contact angle measurements were carried out to determine the hydrophobicity of the implant surfaces. Chemical characterisation was carried out recording Raman images and spectra of the implants using confocal Raman spectrometer. The mechanical properties of the breast implant shells were measured via tensile testing. Adhesive interactions of breast-derived fibroblasts with breast implant surfaces were examined in-vitro. For this purpose, the effect of four molecule coatings (aggrecan, collagen I, fibronectin, and hyaluronic acid) was evaluated on fibroblast attachment, proliferation, fibroblast morphology, spreading, cytotoxicity and gene expression. Results from in-vitro assays demonstrated cell susceptibility to topography and protein coatings and further showed cytoskeletal re-organisation and modification with specific cell adhesion patterns. Combination of diverse topographies and specific coatings induced differential regulation of the expression of adhesion related genes, such as focal adhesion kinase, paxillin, vinculin, and α-actinin on breast fibroblasts. In conclusion, this thesis has demonstrated the extent and strength of cell adhesion and subsequent cell proliferation and differentiation. This is based on the physical interactions between cells and the extracellular environment in the form of topography and on the chemical interactions mediated by specific coatings. Precise characterisation of the silicone breast implant surfaces was achieved. This may play an important role in the development of improved breast implant surfaces with improved qualities leading the development of surfaces that may be less prone to capsular contracture.

La membrane basale (BM) constitue une barrière physiologique entre les tissus et leur microenvironnement. Dans le cas des cancers épithéliaux, au stade de carcinome invasif, la membrane basale est compromise et les cellules cancéreuses envahissent le stroma. Dans cette thèse de doctorat, j’ai proposé d’étudier l’invasion de la membrane basale par les cellules cancéreuses et comment une population de cellules stromales, les fibroblastes, affectent cette invasion. Dans le cadre de cette étude, nous avons utilisé le modèle du cancer colorectal. En collaboration avec l’hôpital Curie, nous avons isolé des fibroblastes à partir de tumeurs de patients opérés. On a nommé les fibroblastes isolés de la partie tumorale « CAF » et ceux venant de la partie du tissu normal, à proximité de la tumeur, « NAF ». Comme modèle de BM nous avons utilisé le mésentère de souris. Afin étudier l’invasion des cellules cancéreuses à travers le mésentère et l’effet des fibroblastes, nous avons mis en place une construction en 3D in vitro. Nous avons montré que les CAFs, et rarement les NAFs, induisent l’invasion des cellules cancéreuse et que cet effet est prononcé quand les CAFs sont physiquement présents sur la membrane. En faisant une étude protéomique comparative entre CAFs et NAFs, on a montré que les CAF expriment plus de protéines composantes de la membrane basale, des protéines impliqués dans le remodelage de la matrice extracellulaire, et des protéines impliquées dans la contraction des cellules. Nous avons ensuite voulu comprendre par quel mécanisme les CAFs induisent l’invasion. Nous avons montré qu’en présence des CAFs, l’invasion ce fait de façon indépendante des métaloprotéinases mais que l’effet contractif des CAFs est nécessaire. En conclusion, l’ensemble de ces résultats mets en évidence l’effet promoteur des CAFs sur l’invasion des cellules cancéreuses et souligne l’importance de leur contractilité dans ce mécanisme. / Basement membrane represents a physiological barrier between epithelial tissues and their microenvironment. In invasive carcinomas, the membrane is breached and cancer cells disseminate in the stroma. In this PhD thesis, I investigated how cancer cells breach the BM and whether a stromal cell population, fibroblasts, assist them in that process. I used colorectal cancer as a model. In collaboration with the Institut Curie Hospital, we isolated human primary fibroblasts from human colorectal cancers, called CAFs and the adjacent normal tissue, NAFs. To study BM invasion, I developed a 3D in vitro assay based on the mouse mesentery. We showed that CAFs, and rarely NAFs, induce cancer cell invasion. This pro-invasive effect is mainly mediated when CAFs are physically present on the membrane, rather than through paracrine ways. To understand how CAFs facilitate invasion, we performed a proteomic comparison between cancer cell-stimulated CAFs and NAFs. Results showed that CAFs produced more proteins-components of the ECM, matrix remodelers and they were more contractile compared to NAFs. Further, we wished to understand the mechanism by which CAFs mediate their effect. We showed that CAFs can induce invasion in a MMP independent way. However, Inhibition of contractility abolished CAFs capacity to induce invasion. Dynamic analysis of cancer cells-fibroblasts co-cultures showed that CAFs could pull on the BM fibers. To directly test this possibility, we created holes in the BM using laser ablations. While in the presence of cancer cells alone, holes remained the same size, in the presence of CAFs, holes widen over time. We further showed that this mechanism is MMP independent but depends on contractility. Altogether, these results demonstrate that CAFs stimulate cancer cell invasion through BM by acting directly on the BM, possibly by depositing ECM components and proteins that remodel ECM and by exerting physical forces on the membrane by contraction.

Keloid scars (KS) are a type of abnormal scarring which is unique to humans. They extend beyond the confines of the original wound margins, do not regress over time and invade the surrounding unaffected skin. The mechanisms involved in the formation of KS remain largely unknown. Clinical observation has shown that in areas where increased tension occurs, such as the sternum, there is a greater propensity for developing KS. However, the precise relationship between skin tension and KS development is yet to be identified. In view of this, I hypothesize that skin tension plays a significant role in KS development by affecting tension-related biomarkers that may alter the phenotype of KS. Therefore, the objective of this research was to investigate the effect of skin tension in the formation of KS. To this end, the first aim was to identify possible targets among biomarkers that might contribute to the differentiation between KS and hypertrophic scars in tissue and cells obtained from diverse anatomical locations. The second aim was to investigate the effect of tension-related biomarkers on extracellular matrix (ECM) steady-state synthesis in keloid fibroblasts (KF) extracted from a highly tensioned body region (the sternum). The third aim was to develop a 3D in-vitro model to mimic in-vivo tension and to evaluate KF behaviour and ECM synthesis under tension. To achieve these aims 21 biomarkers were selected from published microarray and in-house microarray studies, the inclusion criteria was based on up-regulation of the genes in KS in relation to fibrosis, apoptosis and tension. For this purpose, samples from normal skin and KS were used to perform qRT-PCR screening in tissue and cells, as well as protein analysis by Western and In-cell Western blot. The siRNA knockdown technique was employed to evaluate the functional role of the tension-related markers in keloid fibroblasts. Finally, a photogrammetry technique was employed to evaluate skin tension in-vivo; the results from this evaluation were used in the development and design of a novel in-vitro 3D-model. The first biomarker screening in tissue showed convincing up-regulation of five tension-related targets (Hsp27, PAI-2 and α2β1-integrin, MMP-19 and CPRP). In addition, the expression of the above-mentioned targets was significantly higher in samples from the sternum compared to samples from other anatomical locations. To further validate these findings, the screening of the 21 biomarkers was assessed in KS and KF taken from the sternum. The results demonstrated over expression of 3 of the 5 tension-related targets (Hsp27, PAI-2 and α2β1-Integrin). It was also demonstrated that Hsp27, PAI-2 and α2β1-Integrin performed a functional role in terms of regulation of extracellular matrix production and deposition in KF when their expression was down-regulated by siRNA knockdown. Using the newly created 3D model, it was shown that mechanical tension significantly induced the expression of Hsp27, PAI-2 and α2β1-Integrin as well as ECM components such as Collagen I. Furthermore, the results showed that the knockdown of the expression of Hsp27, PAI-2 and α2β1-integrin in fibroblast populated collagen lattices subjected to tension influenced not only the ECM synthesis but also adhesion and spreading genes in keloid and normal fibroblasts. In summary, this research convincingly shows that skin tension alters keloid fibroblast behaviour, morphology, mechano-responsive gene expression and extracellular matrix production. The findings from my thesis offer insight into keloid pathobiology and provide options for targeted treatment of specific genes affected in keloids by biomechanical stress.

A form of familial sex-linked mental retardation has been associated with the expression of a fragile site near the terminal end of the long arm of the X chromosome. Previous reports on the fragile X chromosome showed expression of the fragile site to be limited to chromosome preparations from peripheral blood lymphocytes of mentally retarded males and their female relatives in families in which the disorder was segregating. Fragile site expression has also been shown to be a function of the medium employed in cell culture. The fragile X chromosome could only be demonstrated in lymphocytes cultured in medium 199 or media deprived of folic acid. This study was undertaken to develop a method for demonstrating the fragile X chromosome in cultured skin fibroblasts. Fibroblast cell lines from five patients (two mentally retarded males, two obligate carrier females, and a potential carrier female) from a family in which familial sex-linked mental retardation was known to be segregating were established and routinely maintained in a complete culture medium. Forty-three hours prior to chromosome harvest, cells from each patient were transferred to media deficient in folic acid. Under conditions of folic acid deprivation, it was possible to elicit expression of the fragile X chromosome in skin fibroblasts from all five patients studied. No fragile X chromosomes were detected in fibroblasts from three normal control subjects. In a preliminary assessment of the reliability of the fibroblast method, three patients (two mentally retarded males and a potential carrier female) from a second unrelated family in which the disorder is known to be segregating were studied with this method. The fragile X chromosome could be demonstrated in fibroblasts from both of the retarded male patients but could not be. demonstrated in fibroblast chromosome preparations from the potential carrier female. Lymphocytes for all patients studied were grown under similar folate deprived conditions for the purpose of comparing the effectiveness of fibroblast culture with lymphocyte culture in demonstrating the expression of the fragile X chromosome. Neither tissue was shown to consistently provide a higher frequency of expression of the fragile X chromosome. In addition to folate deprivation, it was shown that two other features of the fibroblast method influenced the frequency of expression of the fragile X chromosome. The fragile site was expressed at a significantly higher frequency in chromosome preparations in which the chromosomes were not severely contracted. The frequency of expression in fibroblasts was also shown to be significantly higher with a hypotonic treatment at chromosome harvest using 1% NaCitrate rather than 0.075M KC1. Because fragile site expression was shown to be a function of the degree of chromosome condensation, two agents, 5-BrdU and actinomycin-D, were studied to examine their decondensation effects on the frequency of expression. Neither BrdU nor actinomycin D proved effective in accentuating the frequency of expression. Since fibroblasts behave much like amniocytes in terms of cell culture and chromosome harvest, the development of a method for demonstrating the fragile X chromosome in cultured skin fibroblasts is a step toward the prospect of reliable antenatal diagnosis of familial sex-linked mental retardation associated with a fragile X chromosome. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Muitas complicações do Diabetes Mellitus, dentre elas a deficiência na cicatrização de feridas, estão associadas à hiperglicemia crônica, que promove estresse oxidativo e glicação protéica aleatória. O objetivo desse estudo foi avaliar os efeitos de elevada contração de glicose (ECG) sobre a migração, proliferação e morte de fibroblastos cultivados, e o potencial da terapia com laser em baixa intensidade (LILT) (660 e 780 nm; 40mW; 2, 5, 10 e 30J/cm2) na prevenção desses efeitos. A ECG reduziu a proliferação em cerca de 40%, reduziu a migração em 50% e aumentou a morte celular em 30%. A irradiação diária com LILT em 660nm e 10J/cm2 mostrou-se eficaz na prevenção desses efeitos sobre a proliferação e migração celular. A ECG tambem aumentou a produção de especies reativas de oxigênio pelas células, um efeito nao observado em células irradiadas com LILT. Dessa forma, pode-se sugerir que a terapia com LILT favorece a proliferação e migração de células cultivadas sob ECG, processos essenciais para a cicatrização, possivelmente através de um efeito antioxidante. / Diabetic complications, including wound healing deficiency, are usually associated to chronic hyperglycemia, which leads to oxidative stress and aleatory protein glycation. The aim of this study was to evaluate the effects of high glucose concentrations (HGC) on proliferation, migration and death of cultured fibroblasts, as well as the potential effect of low intensity laser therapy (LILT) (660 and 780 nm; 40mW; 2, 5, 10 and 30J/cm2) preventing these effects. HGC reduced cell proliferation in about 40%, cell migration in about 50% and increased cell death in 30%. Daily irradiation at 660nm and 10J/cm2 was efficient preventing the effects on cell proliferation and migration. HGC also increased the production of reactive oxygen species, an effect totally prevented by LILT. The results suggest that LILT favors cell proliferation and migration, processes very important for healing, possibly through an anti-oxidant effect.

O excesso de tecido cicatricial (quelóides e cicatrizes hipertróficas) é um defeito do processo de cicatrização das feridas, caracterizado por um aumento na produção da matriz extracelular. Neste estudo, fibroblastos dérmicos humanos tratados com 50 <font face=\"symbol\">mM verapamil apresentaram discreta modificação na distribuição dos microfilamentos e alteraram sua morfologia de fusiformes para estrelados/arredondados. Estes efeitos poderiam estar associados a baixos níveis de cálcio citosólico. Esta hipótese foi confirmada através marcação de fibroblastos tratados com calcium green. Observamos também, que o verapamil inibiu a proliferação celular em 64,4%, aumentou a secreção de MMP1 e diminuiu o colágeno sintetizado pelos fibroblastos, sem aparentes efeitos citotóxicos. O metabolismo celular do cálcio está aparentemente relacionado a produção da matriz extracelular e portanto as patologias hipertróficas da cicatrização (quelóides e cicatrizes hipertróficas) podem responder ao tratamento com bloqueadores do canal de cálcio (verapamil). / Excessive scar tissue (keloids and hypertrophic scars) is a defective wound healing process characterized by overproduction of extracellular matrix. In the present study human dermal fibroblasts treated with 50 <font face=\"symbol\">mM verapamil changed their normal spindle-shaped morphology to stellate/rounded and showed discrete reorganization of microfilaments We hypothesized that these effects would be associated to lower levels of cytosolic Ca2+. Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels. We also observed that verapamil decrease cellular proliferation by 64.4%, increase the secretion of MMP1 and decrease synthesis of collagen in cultured fibroblasts. This alterations induced by verapamil are not associated with cytotoxic effects. The cellular calcium metabolism appears to regulate extracellular matrix production and so those hypertrophic disorders of wound healing (keloids and hypertrophic scars) may respond to therapy with calcium antagonist drugs (verapamil).

Gastrópodes terrestres secretam muco pela superfície corporal, quando se locomovem, para proteção do corpo contra injúria mecânica, dessecação ou contato com substâncias nocivas. O muco de moluscos tem sido estudado como fonte de novos compostos naturais com diversas atividades biológicas, entre elas a capacidade de induzir proliferação celular. O presente trabalho propôs o estudo do muco produzido pelas lesmas terrestres Phyllocaulis boraceiensis como agente indutor de proliferação celular e síntese de colágeno em cultura de fibroblastos humanos normais, células endoteliais e no reparo de processos cicatriciais no modelo de ferida cirúrgica da derme de camundongos. Fibroblastos tratados com proteínas do muco de P. boraceiensis em concentrações menores que 0,012 <font face=\"Symbol\">mg/µl apresentaram aumento nas taxas de proliferação avaliadas pelo método colorimétrico do MTT e em citometria de fluxo CSFE, mostrando um efeito dose-dependente. Os resultados obtidos mostraram que fibroblastos humanos normais e células endoteliais tratados com 0,012 g/<font face=\"Symbol\">ml de proteínas do muco, aumentaram significativamente a produção e secreção de elementos da matriz extracelular, como as fibras de colágeno. Houve a redução da produção de radicais livres lipídicos poli-insaturados. O ensaio de cicatrização da derme lesionada de camundongos Balb-c tratados com proteínas do muco de P. boraceiensis na concentração de 0,012 <font face=\"Symbol\">mg/µl induziu o reparo da cicatrização com maior eficácia e em menor tempo quando comparado ao grupo controle e aos tratados com a concentração de 0,18 <font face=\"Symbol\">mg/µl. Esses resultados corroboram a premissa de que o muco de P. boraceiensis, assim como o de outros gastrópodes, apresenta propriedade proliferativa das células envolvidas nos processos de cicatrização. / Mucus of molluscs has been studied as a source of new natural compounds with diverse biological activities, as ability to induce cell proliferation. The aim of this study is the potential use of mucus produced by land slugs Phyllocaulis boraceiensis as a promoter of cell proliferation and collagen synthesis in human fibroblasts, endothelial cells and in repair processes of healing wound skin of mice. Fibroblasts treated with P. boraceiensis mucus at concentrations below 0.012 <font face=\"Symbol\">mg/µl have high rates of proliferation as evaluated by the MTT and CFSE flow cytometer assays, proliferative effect was dose-dependent. Fibroblasts and endothelial cells treated with 0.012 <font face=\"Symbol\">mlowg/µl mucus induced a significant increase in production and secretion of extracellular matrix such as collagen fibers. There was a reduction in polyunsaturated lipid production. Healing assay of lesions in mice treated with mucus at 0.012 <font face=\"Symbol\">mg/µl induce repair of the scar more effectively and in less time when compared to the control group and those treated 0.18 <font face=\"Symbol\">mg/µl. These findings support the premise that the P. boraceiensis mucus demonstrates proliferative properties in cells involved in the healing process.

The photobiomodulation therapy (PBMT) has many demonstrated applications in the health area including anti-inflammatory and wound healing effects. The main objective of this work is to verify if the PBMT causes measurable changes in the mechanical properties of cells, specifically in red blood cells, epithelial cells and fibroblasts. In addition, to contribute to the knowledge of the action mechanisms of the PBMT, this study intends to support applications of the PBMT during invasive procedures, such as the direct photo-treatment of the blood in surgical procedures with cardiopulmonary bypass, regarding security of the cellular integrity. For this analysis, three experimental techniques were used: optical magnetic twisting cytometry (OMTC), defocusing microscopy and confocal laser-scanning microscopy. Human bronchial epithelial cells were evaluated with OMTC. The epithelial cell culture was either photo-treated or not, with red laser (lambda=660 nm), and fixed power and time (power density of 153 mW/cm2, time 300 s). It was not possible to observe significant differences between photo-treated and control epithelial cells, for the hysteresivity (ratio between the cell loss and elastic shear moduli). The defocusing microscopy, similar to a phase contrast microscopy, was used to study human red blood cells from fresh blood. The red blood cells were either photo-treated or not, with red laser (lambda=660 nm), and different powers and times (power densities from 0 to 510 mW/cm2, times from 0 to 180 s). Some morphological and mechanical characteristics of individual red blood cells were evaluated, such as volume, radial profile of cell thickness, lateral and vertical membrane fluctuations, for the photo-treated and control red blood cells. It was not possible to detect differences between the two groups, for any of the parameters analyzed. For both techniques, the absence of detectable differences might be due to several factors, such as the non-action of the PBMT, with the parameters used, in the epithelial cells and red blood cells or to the small sensitivity of each technique. Confocal laser-scanning microscopy was used to evaluate the actin filaments of mouse fibroblasts. The fibroblast cell culture was either photo-treated or not, with red (lambda=625 nm) or infrared (lambda=808 nm) light and fixed power and time (power density from 113 to 158 mW/cm2, time 300 s). The nucleus and cell areas increased slightly when comparing photo-treated and control cells. On the other hand, the total actin, total actin density and the number of filaments decreased. These changes were detected for a short time after treatment, however, after 24 h they are not anymore detectable. The total branch length does not seem to suffer any modifications. In summary, with the data acquired with the three techniques, it was found that the PBMT, in the red range, with the parameters used, could not cause noticeable changes in red blood cells and epithelial cells, in vitro. On the other hand, the PBMT in the red and near-infrared range, with the power and times used, cause changes in actin filaments of fibroblasts, in vitro, in particular the decrease of the total actin density. / A terapia por fotobiomodulação tem muitas aplicações na área de Saúde devido a sua ação anti-inflamatória e de reparação tecidual. O objetivo geral desse trabalho é verificar se a terapia por fotobiomodulação provoca mudanças nas propriedades mecânicas de células, em particular em hemácias, células epiteliais e fibroblastos. Além de contribuir com o conhecimento dos mecanismos de ação da terapia por fotobiomodulação, este estudo pretende subsidiar aplicações da terapia por fotobiomodulação durante procedimentos mais invasivos, como a iluminação direta do sangue em procedimentos cirúrgicos com circulação extracorpórea, sob o ponto de vista da segurança quanto à integridade celular. Para essa análise foram utilizadas três técnicas experimentais: citometria óptica magnética de oscilação (OMTC), microscopia de desfocalização e microscopia confocal. Com a técnica de OMTC foram avaliadas células epiteliais brônquicas humanas em cultura, foto-tratadas com laser vermelho (lambda=660 nm), com potência e tempo fixos (densidade de potência de 153 mW/cm2, tempo 300 s). Não foi possível constatar diferenças significativas entre as células epiteliais foto-tratadas e as células controle, para a histerisividade (razão entre os módulos viscoso e elástico das células). Com a técnica de microscopia de desfocalização, semelhante a uma microscopia de contraste de fase, foram estudadas hemácias humanas de sangue recém coletado. As hemácias foram tratadas com laser vermelho (lambda=660 nm), com potências e tempos variados (densidade de potência de 0 a 510 mW/cm2, tempo de 0 a 180 s). Foram avaliadas algumas características morfológicas e mecânicas das hemácias individualmente, como o volume, perfil radial de espessura, flutuações lateral e vertical da membrana, tanto para hemácias foto-tratadas quanto para hemácias controle. Não foi possível detectar diferenças entre as hemácias foto-tratadas e controle para nenhum dos parâmetros avaliados. Para ambas as técnicas, a falta de mudanças observáveis poderia ser devida a diversos fatores, como a não ação da terapia por fotobiomodulação nas células epiteliais e nas hemácias, com os parâmetros aqui empregados, ou à falta de sensibilidade de cada uma das técnicas usadas. A microscopia confocal foi utilizada para avaliar os filamentos de actina de fibroblastos de camundongo em cultura, os quais foram foto-tratados com luz vermelha (lambda=625 nm) ou infravermelha (lambda=808 nm) e potência e tempo fixos (densidade de potência de 113 a 158 mW/cm2, tempo 300 s). Foi possível constatar ligeiro aumento nas áreas nuclear e celular das células foto-tratadas em relação aos fibroblastos controle. Também foi possível verificar a diminuição da quantidade total de actina, densidade de actina e do número de filamentos de actina nos fibroblastos foto-tratados. Essas mudanças são detectadas para tempos curtos após o tratamento, sendo que depois de 24 h elas desaparecem. O tamanho total dos filamentos parece não sofrer alterações. A partir dos dados coletados com as três técnicas, foi possível constatar que a terapia por fotobiomodulação, com os parâmetros utilizados, não consegue provocar mudanças perceptíveis em hemácias e em células epiteliais, in vitro. Porém, causa mudanças nos filamentos de actina de fibroblastos, in vitro, em particular a diminuição da densidade de actina total.

O linfonodo é um órgão linfoide secundário que apresenta uma arquitetura altamente organizada com diferentes compartimentos para tipos celulares específicos. Dentre as células estruturais que compõem este órgão, as células estromais como células fibroblásticas reticulares (FRCs) e células duplo negativas (DNCs) parecem ter papel importante na modulação da resposta imunológica e na tolerância periférica. As FRCs são caracterizadas pela expressão de podoplanina (gp38, PDPN) e localizam-se principalmente na zona de células T, enquanto as DNCs (gp38-) apresentam fenótipo, localização e função pouco descritos. Embora estas células tenham sido muito estudadas em modelos murinos os estudos sobre FRCs e DNCs humanas são escassos e, portanto nosso estudo deve contribuir para a compreensão da biologia e a função dessas células, podendo favorecer o conhecimento sobre a eficiência e as disfunções da resposta imune no linfonodo. Com esse intuito, isolamos e caracterizamos fenotípica e funcionalmente as FRCs e DNCs de linfonodos de pacientes com câncer, diverticulite e doadores de fígado. Nossos resultados mostraram a integridade e a distribuição celular no linfonodo. As células aderentes derivadas dos linfonodos estudados preecheram todos os critérios internacionais de caracterização de estroma, e, portanto, foram consideradas células estromais. Através da expressão de gp38 identificamos duas subpopulações de celulas estromais: FRCs (gp38+ e CD31-) e DNCs (gp38- e CD31-) e verificamos que as frequências destas células variam entre as amostras, sugerindo que a doença pode interferir na composição celular estromal dos linfonodos. As duas populações celulares foram estimuladas com citocinas inflamatórias como IFN-y ou TNF-alfa + IL-1beta por 24 e 48 horas e avaliadas quanto à expressão gênica e proteica. Em condições homeostáticas, genes relacionados com a indução e controle da proliferação foram diferencialmente expressos nas FRCs e DNCs, este dado foi confirmado in vitro, uma vez que as FRCs apresentaram maior potencial proliferativo em relação às DNCs. O estímulo com IFN-y induziu aumento de expressão nas DNCs e FRCs para citocinas, quimiocinas, moléculas de histocompatibilidade e moléculas envolvidas na regulação da resposta imunológica. Em resposta ao estímulo com TNF-alfa +IL-1beta, observamos aumento na expressão de moléculas comuns ao estímulo com IFN-?, entretanto, também observamos expressão de moléculas de citocinas, quimiocinas inflamatórias e moléculas de histocmpatibilidade especificamente relacionados a este sinal em ambas as populações. Em conjunto, nossos dados sugerem que DNCs e FRCs apresentam diferenças no perfil de resposta segundo os estímulos inflamatórios aos quais estão expostas, aumentando a expressão diferencial de moléculas envolvidas na regulação positiva e negativa da resposta imune / The lymph node is a secondary lymphoid organ that has a highly organized architecture with different compartments for specific cell types. Among the structural cells that comprise this organ, stromal fibroblastic reticular cells (FRCs) and double negative cells (DNCs) seems to play an important role in modulating the immune response and peripheral tolerance. FRCs are characterized by podoplanin (gp38, PDPN) expression and are located mainly in the T cell zone, while DNCs (gp38-) present phenotype, location and function not well described. Although these cells have been studied in murine models, studies on human FRCs and DNCs are limited and therefore our study should contribute to the understanding of biology and function of these cells and should promote knowledge of efficiency and disorders in the lymph node immune response. For this purpose, we have isolated and characterized phenotypic and functionally lymph nodes derived FRCs and DNCs from patients with cancer, diverticulitis and liver donors. Our results showed lymph node integrity and its cellular distribution. Adherent cells lymph nodes-derived fullfill the international criteria for stroma characterization, and therefore, they have been considered stromal cells. Using gp38 expression we were able to identify two stromal cells subpopulations: FRCs (gp38 + and CD31-) and DNCs (gp38- and CD31-) and found that this cells frequency varies among samples, suggesting that the disease may interfere with lymph nodes stromal cell composition. These two cells populations were stimulated with inflammatory cytokines such as IFN-y or TNF-alfa + IL-1beta for 24 and 48 hours and evaluated for gene and protein expression. In homeostatic conditions, genes involved in the induction and control of proliferation were differentially expressed by FRCs and DNCs, this data has been confirmed in vitro, since the FRCs showed higher proliferative potential compared to DNCs. IFN-y stimulation induced increase DNCs and FRCs expression for cytokines, chemokines, histocompatibility molecules and molecules involved in regulating the immune response.In response to TNF-alfa + IL-1beta stimulation, we observed common molecules expressed by the IFN-? stimulation, however, we also observed expression of cytokines, chemokines and histocompatibility molecules specifically related to this signal in both cells populations. Together, our data suggest that DNCs and FRCs differ in the response profile according to inflammatory stimuli to which they are exposed, increasing the differential expression of molecules involved in the positive and negative regulation of immune response

Cell migration involves complex mechanical interactions between cells or between cells and the underlying substrate. Using a newly developed technique, "traction force microscopy", I have been able to visualize the dynamic characteristics of mechanical forces exerted by migrating fibroblasts such as magnitude, direction, and shear. For NIH 3T3 fibroblasts, I found that the lamellipodium provides nearly all of the force necessary for cell migration. A high shear zone separates the lamellipodium from the remainder of the cell body, suggesting that they are mechanically distinct entities. The timing of the tractions at the leading edge, as well as the spatial distribution, bears no apparent relationship to concurrent local protrusive activities, yet changes in traction force patterns often precede changes in migration direction. In H-ras transformed cells I found isolated regions of weak, transient traction forces in pseudopods all along the cell that appeared to act against one another. The resulting shear pattern suggested that there were multiple disorganized mechanical domains. These results support a frontal towing model for cell migration where the dynamic traction forces at the leading edge served to actively pull the cell body forward. In H-ras transformed cells, the weak poorly coordinated traction forces coupled with weak cell substrate-adhesions were likely responsible for the abnormal motile behavior of these cells. To probe the mechanical interactions beneath various regions of migrating fibroblasts, a cell substrate inhibitor (GRGDTP peptide) was locally applied while imaging stress distribution on the substrate utilizing traction force microscopy. I found that both spontaneous and GRGDTP induced detachment of the trailing edge resulted in extensive cell shortening with no change in overall traction force magnitude or cell migration. Conversely, leading edge disruption resulted in a dramatic global loss of traction forces pnor to any significant cell shortening. These results suggested that fibroblasts transmit their contractile forces to the substrate through two distinct types of adhesions. Leading edge adhesions were unique in their ability to transmit active propulsive forces whereas trailing end adhesions created passive resistance during cell migration and readily redistributed their loads upon detachment. I have also investigated how fibroblasts regulate traction forces based on mechanical input. My results showed that stretching forces applied through the flexible substrate induced increases in both intracellular calcium concentration and traction forces in fibroblasts. Treatment with gadolinium, a well known stretch-activated ion channel inhibitor, was found to inhibit both traction forces and cell migration without inhibiting cellular spread morphology or protrusive activities. Gadolinium treatment also caused a pronounced decrease in vinculin and phosphotyrosine concentrations from focal adhesions. Local application of gadolinium to the trailing region had no detectable effect on overall traction forces or cell migration, whereas local application to the leading edge caused a global inhibition of traction forces and an inhibition of migration. These observations suggest that stretch activated entry of calcium ions in the frontal region serves to regulate the organization of focal adhesions and the output of mechanical forces. Together my experiments elucidate how fibroblasts exert mechanical forces to propel their movements, and how fibroblasts utilize mechanical input to regulate their movements.