Des progrès récents dans l’utilisation préclinique et clinique des petits ARN interférents (siRNA) ont montré leur potentiel en tant qu’inhibiteur de la synthèse protéique dans de nombreuses pathologies comme le cancer. L’administration des siRNA rencontre un certain nombre de problèmes liés à leur dégradation rapide dans les milieux biologiques, ainsi qu’à leur difficulté à pénétrer au sein des cellules cibles en raison de leur hydrophilie et de leur charge négative. Une des clés de l’amélioration de l’efficacité thérapeutique de ces molécules repose sur l’emploi de vecteurs. Au cours de cette thèse, des lipoplexes capables de protéger les siRNA contre la dégradation et de favoriser leur transport jusqu’aux cellules cibles ont été développés et optimisés. Pour ce faire, des lipoplexes recouverts d’HA ont été formulés pour la vectorisation active de siRNA vers des cellules tumorales surexprimant le récepteur CD44. Dans la première partie de cette thèse, la formation des lipoplexes a été étudiée, ainsi que les paramètres influençant leur organisation supramoléculaire. L'insertion de l'HA dans la structure du liposome au moment de la formation des vésicules a entraîné une augmentation de la taille des liposomes en fonction de la concentration d’HA. Leur complexation avec les siRNA a encore augmenté la taille des particules obtenues. L’ajout des acides nucléiques lors de la formation des lipoplexes a provoqué un déplacement d'une partie du conjugué HA-DOPE de la structure des lipoplexes, comme montré par électrophorèse capillaire. La titration calorimétrique isotherme et les études de diffraction des rayons X ont démontré que, sous l’effet des interactions électrostatiques avec les siRNA, un réarrangement des bicouches lipidiques a lieu, conduisant à la formation de vésicules oligolamellaires, confirmé visuellement par cryo-microscopie. Enfin, le positionnement convenable de l’HA sur la surface des lipoplexes et sa capacité de se lier spécifiquement aux récepteurs de CD44 ont été démontrés par la technique de résonance plasmonique de surface. Dans la deuxième partie de cette thèse, la capture cellulaire et localisation intracellulaire des lipoplexes-HA ont été évalués par cytométrie en flux et microscopie de fluorescence, et ont montré que les lipoplexes modifies par l’HA sont internalisés plus rapidement que les lipoplexes non modifiés, et une fois dans des cellules, ils sont localisés principalement à l’intérieur des endosomes. La capacité des lipoplexes à transporter des molécules de siRNA intactes au cytoplasme a été confirmée par 81 % d'inhibition d'expression de luciferase in vitro sur la lignée cellulaire de cancer du poumon A549-luc. In vivo, le traitement avec les lipoplexes-HA transportant un siRNA anti-luciferase a mené à une diminution statistiquement significative de l’expression de luciferase, ce qui a été confirmé par la réduction de l'expression d’ARNm de la luciferase dans le poumon des animaux traités avec les lipoplexes-HA. L'analyse de la distribution des lipoplexes dans les poumons a montré que les lipoplexes modifiés par l’HA sont distribués de façon plus importante et plus homogène dans le tissu pulmonaire que les lipoplexes non modifiés. Dans la troisième partie de cette thèse, la diffusion des lipoplexes-HA de siRNA dans le mucus a été étudié, afin d’évaluer la faisabilité de l’administration pulmonaire de ces particules. Les études utilisant la technique de « multiple particle tracking (MPT) » ont montré que la présence d’HA combinée à l'ajout de siRNA ont permis l’obtention de deux formulations de lipoplexes présentant une pénétration efficace dans le mucus, les lipoplexes-HA and les lipoplexes-PEG/HA. En conclusion, un système efficace de lipoplexes utilisant siRNA pour l'inhibition de l'expression génique ciblant les récepteurs CD44 a été développé. Les résultats obtenus confirment que les HA-lipoplexes sont capables de libérer efficacement le siRNA dans le cytoplasme des cellules in vitro et in vivo. / Recent progresses in the preclinical and clinical use of small interfering RNA (siRNA) have shown their potential as an inhibitor of protein synthesis in many diseases such as cancer. The administration of siRNA encounters a number of problems related to their rapid degradation in biological media, and their difficulty in penetrating targeted cells due to their hydrophilicity and negative charge. A key to improving the therapeutic efficacy of these molecules is based on the use of vectors. In this thesis, lipoplexes that can protect siRNA against degradation and facilitate their transport into target cells were developed and optimized. To do this, lipoplexes covered with HA were formulated for active vectorization of siRNA to tumor cells overexpressing the receptor CD44.In the first part of this thesis, the formation of lipoplexes was studied, and the parameters influencing their supramolecular organization. Insertion of HA within the liposome structure during vesicle formation resulted in the increase in liposome size as a function of HA concentration. Their complexation with siRNA has further increased the size of the particles obtained. The addition of siRNAs when forming lipoplexes caused a displacement of a portion of the HA-DOPE conjugate from the lipoplexes structure, as shown by capillary electrophoresis. The isothermal titration calorimetry and X-ray diffraction studies showed that a rearrangement of the lipid bilayers occur under the effect of electrostatic interactions with siRNAs, leading to the formation of oligolamellar vesicles, which was visually confirmed by cryo-microscopy. Finally, the proper positioning of the HA on the surface of the lipoplexes and its ability to specifically bind to the CD44 receptors has been demonstrated by the surface plasmon resonance technique.In the second part of this thesis, cellular uptake and intracellular localization of HA-lipoplexes were assessed by flow cytometry and fluorescence microscopy, and showed that lipoplexes modified by HA are internalized more rapidly than unmodified lipoplexes, and once in the cells, they are mainly localized within the endosomes. The ability of lipoplexes to transport intact siRNA molecules to the cytoplasm was confirmed by 81% of luciferase in vitro expression inhibition on the lung cancer cell line A549-luc. In vivo, treatment with HA-lipoplexes carrying anti-luciferase siRNA led to a statistically significant decrease in expression of luciferase, which was confirmed by reducing the mRNA expression of luciferase in lungs of animals treated with HA-lipoplexes. The analysis of the distribution of lipoplexes in the lungs showed that lipoplexes modified with HA are distributed more evenly in the lung tissue than unmodified lipoplexes.In the third part of this thesis, the movement of siRNA HA-lipoplexes in the mucus was studied to assess the feasibility of administering these particles directly to the lungs. Studies using the technique of "multiple particle tracking (MPT)" showed that the presence of HA combined with the addition of siRNA allowed the preparation of two lipoplexes formulations with efficient mucus-penetration, HA-lipoplexes and PEG/HA-lipoplexes.In conclusion, an efficient siRNA lipoplex system for inhibiting gene expression targeted TO the CD44 receptorS has been developed. The results confirm that the HA-lipoplexes are able to effectively release in vitro and in vivo the siRNA molecules in the cytoplasm of cells.

O objetivo deste estudo consistiu em avaliar a expressão imuno-histoquímica da podoplanina e do CD44v6 pelas células malignas, verificando a associação destas proteínas com as variáveis clínicas, microscópicas, com o índice histopatológico de malignidade e com a sobrevivência livre de doença de 91 pacientes portadores de carcinomas espinocelulares (CEC) de lábio inferior, tratados no Centro de Tratamento e Pesquisa do Hospital do Câncer A.C.Camargo, São Paulo. Os tumores foram corados, separadamente, com os anticorpos anti-podoplanina e anti-CD44v6, sendo avaliada a imunoexpressão destas proteínas pelas células neoplásicas, no front de invasão tumoral, por meio de um método semi-quantitativo de escores. A associação da expressão da podoplanina e do CD44v6 com as variáveis demográficas, clínicas e microscópicas foi feita pelo teste do qui-quadrado ou exato de Fisher. As taxas de sobrevivência livre de doença, acumuladas em cinco e dez anos, foram calculadas pelo teste de Kaplan-Meier e a influência das variáveis clínicas e microscópicas no prognóstico avaliadas pelo modelo de regressão de Cox. A correlação entre a podoplanina e o CD44v6 foi analisada pelo teste de Spearman. Em todos os testes estatísticos utilizou-se um nível de significância de 5%. Os resultados mostraram uma predominância da forte expressão membranosa e citoplasmática da podoplanina pelas células malignas. Verificou-se uma associação significativa da podoplanina citoplasmática com a recidiva locorregional (p=0,028) e da podoplanina membranosa com o índice histopatológico de malignidade tumoral (p=0,026). O CD44v6 foi fortemente expresso pelas células neoplásicas de 95,4% dos CECs e significativamente, associado com o estadiamento clínico T (p=0,034). Não houve correlação entre a podoplanina e o CD44v6 nos CECs de lábio inferior. A forte expressão de podoplanina membranosa (p=0,016) e citoplasmática (p=0,030) pelas células malignas foi fator de prognóstico favorável independente na sobrevivência livre de doença. Concluímos que a podoplanina e o CD44v6 são fortemente expressos pelas células neoplásicas e que a forte imunoexpressão membranosa e citoplasmática da podoplanina pode auxiliar na identificação do risco de recidiva locorregional nos pacientes portadores de carcinoma espinocelular de lábio inferior. / The aim of this study was evalute the podoplanin and CD44v6 immunohistochemical expression by malignant cells and its association with the clinical and microscopic variables, tumor histopathological grading and disease-free survival of 91 patients with lip squamous cell carcinomas (SCC), submitted to surgical treatment at Research and Treatment Center of the Cancer Hospital A.C. Camargo, São Paulo. The tumors were stained separately, with the antibodies anti-podoplanin and anti-CD44v6, and the immunoexpression of these proteins, by the neoplastic cells in the invasion front, was evaluated by a semi-quantitative scores method. Chi-square test or Fishers exact test was used to analyze the association of podoplanin and CD44v6 expression with demographic, clinical, and microscopic variables. Disease-free survival in five and ten years, were calculated by the Kaplan-Meier method and the influence of clinical and microscopic variables on prognosis were evaluated by the Cox regression model. The correlation between podoplanin and CD44v6 expression was analyzed by Spearman\'s test and a significance level of 5% was used in all statistical tests. The results showed a predominance of strong membranous and cytoplasmic podoplanin expression by malignant cells. An association between cytoplasmic podoplanin and locorregional recurrence (p=0,028) and membranous podoplanin with tumor histopathological grading (p=0,026). CD44v6 was strongly expressed in 95.4% of the SCCs neoplastic cells and significantly associated with the clinical staging T (p=0,034). There was no correlation between podoplanin and CD44v6 expression in the lower lip SCC. The strong expression of membranous (p=0.016) and cytoplasmic (p=0.030) podoplanin by malignant cells was a favorable independent prognostic factor in disease-free survival. Concluding, the podoplanin and CD44v6 are strongly expressed by neoplastic cells and the strong membranous and cytoplasmic immunoexpression of podoplanin can help the identification of locoregional recurrence risk in patients with squamous cell carcinoma of the lower lip.

O objetivo deste estudo consistiu em avaliar a expressão imuno-histoquímica da podoplanina e do CD44v6 pelas células malignas, verificando a associação destas proteínas com as variáveis clínicas, microscópicas, com o índice histopatológico de malignidade e com a sobrevivência livre de doença de 91 pacientes portadores de carcinomas espinocelulares (CEC) de lábio inferior, tratados no Centro de Tratamento e Pesquisa do Hospital do Câncer A.C.Camargo, São Paulo. Os tumores foram corados, separadamente, com os anticorpos anti-podoplanina e anti-CD44v6, sendo avaliada a imunoexpressão destas proteínas pelas células neoplásicas, no front de invasão tumoral, por meio de um método semi-quantitativo de escores. A associação da expressão da podoplanina e do CD44v6 com as variáveis demográficas, clínicas e microscópicas foi feita pelo teste do qui-quadrado ou exato de Fisher. As taxas de sobrevivência livre de doença, acumuladas em cinco e dez anos, foram calculadas pelo teste de Kaplan-Meier e a influência das variáveis clínicas e microscópicas no prognóstico avaliadas pelo modelo de regressão de Cox. A correlação entre a podoplanina e o CD44v6 foi analisada pelo teste de Spearman. Em todos os testes estatísticos utilizou-se um nível de significância de 5%. Os resultados mostraram uma predominância da forte expressão membranosa e citoplasmática da podoplanina pelas células malignas. Verificou-se uma associação significativa da podoplanina citoplasmática com a recidiva locorregional (p=0,028) e da podoplanina membranosa com o índice histopatológico de malignidade tumoral (p=0,026). O CD44v6 foi fortemente expresso pelas células neoplásicas de 95,4% dos CECs e significativamente, associado com o estadiamento clínico T (p=0,034). Não houve correlação entre a podoplanina e o CD44v6 nos CECs de lábio inferior. A forte expressão de podoplanina membranosa (p=0,016) e citoplasmática (p=0,030) pelas células malignas foi fator de prognóstico favorável independente na sobrevivência livre de doença. Concluímos que a podoplanina e o CD44v6 são fortemente expressos pelas células neoplásicas e que a forte imunoexpressão membranosa e citoplasmática da podoplanina pode auxiliar na identificação do risco de recidiva locorregional nos pacientes portadores de carcinoma espinocelular de lábio inferior. / The aim of this study was evalute the podoplanin and CD44v6 immunohistochemical expression by malignant cells and its association with the clinical and microscopic variables, tumor histopathological grading and disease-free survival of 91 patients with lip squamous cell carcinomas (SCC), submitted to surgical treatment at Research and Treatment Center of the Cancer Hospital A.C. Camargo, São Paulo. The tumors were stained separately, with the antibodies anti-podoplanin and anti-CD44v6, and the immunoexpression of these proteins, by the neoplastic cells in the invasion front, was evaluated by a semi-quantitative scores method. Chi-square test or Fishers exact test was used to analyze the association of podoplanin and CD44v6 expression with demographic, clinical, and microscopic variables. Disease-free survival in five and ten years, were calculated by the Kaplan-Meier method and the influence of clinical and microscopic variables on prognosis were evaluated by the Cox regression model. The correlation between podoplanin and CD44v6 expression was analyzed by Spearman\'s test and a significance level of 5% was used in all statistical tests. The results showed a predominance of strong membranous and cytoplasmic podoplanin expression by malignant cells. An association between cytoplasmic podoplanin and locorregional recurrence (p=0,028) and membranous podoplanin with tumor histopathological grading (p=0,026). CD44v6 was strongly expressed in 95.4% of the SCCs neoplastic cells and significantly associated with the clinical staging T (p=0,034). There was no correlation between podoplanin and CD44v6 expression in the lower lip SCC. The strong expression of membranous (p=0.016) and cytoplasmic (p=0.030) podoplanin by malignant cells was a favorable independent prognostic factor in disease-free survival. Concluding, the podoplanin and CD44v6 are strongly expressed by neoplastic cells and the strong membranous and cytoplasmic immunoexpression of podoplanin can help the identification of locoregional recurrence risk in patients with squamous cell carcinoma of the lower lip.

Le cancer gastrique (CG) est une maladie multifactorielle, fréquemment associée à l’infection chronique par des souches CagA+ d’Helicobacter pylori. La transition épithélio-mésenchymateuse (EMT) est un processus réversible dans lequel une cellule épithéliale polarisée acquiert un phénotype mésenchymateux. L’EMT est à l’émergence de cellules souches cancéreuses (CSC) qui expriment CD44 et présentent une activité ALDH élevée. L’infection des cellules épithéliales gastriques humaines (CEGs) par CagA+ H. pylori induit des cellules CD44+ avec des propriétés des CSCs via une EMT. La voie Hippo est composée par les kinases MST et LATS, et leurs cibles, les YAP1 et TAZ. Suite à la phosphorylation, YAP1 et TAZ sont inhibés. YAP1 et TAZ activés lient les facteurs TEAD pour promouvoir la croissance cellulaire et l’inhibition de l’apoptose.Notre premier objectif était de rechercher si H. pylori change l’état d’activation de la voie Hippo et l'effet sur l’EMT et les CSC in vitro et in vivo. Le deuxième but est la caractérisation du rôle de YAP1/TEAD dans les propriétés de CSCs gastriques in vitro et les conséquences de son inhibition dans la croissance tumorale in vivo.Pour étudier la régulation de la voie Hippo pendant l’infection par H. pylori, LATS2, YAP1 et CD44 ont été évalués dans la muqueuse gastrique de sujets non-infectés et infectés par H. pylori, qui ont été augmentés avec l’infection et leur surexpression a été associée avec la gastrite et la métaplasie intestinale. Dans les CEGs l’expression de gènes de la voie Hippo a été altérée par l’infection. La régulation de la voie Hippo par H. pylori a une cinétique diphasique et dépendante de CagA. Dans l’infection précoce, H. pylori déclenche l’activité transcriptionelle de YAP1. Cette période d’inactivité de la voie Hippo est suivi de son activation progressive, soutenue par l’accumulation de LATS2 et la phosphorylation inhibitrice de YAP1. La répression de LATS2 avec siRNAs a accéléré l’acquisition du phénotype mésenchymateux après l’infection, l’augmentation de marqueurs de l’EMT (Zeb1 et Snail1), et la diminution des miR-200 épithéliaux. Les CSC induites par H. pylori ont été potentialisées par l’inhibition de LATS2, ce qui suggère que LATS2 limite l’EMT et le phénotype de CSC acquis pendant l’infection. L’inhibition de LATS2 ou YAP1 diminue l’expression de ces deux protéines, révélant ainsi une boucle de régulation positive. Dans des coupes de tissu de CG, l’expression de LATS2 et YAP1 est hétérogène et positivement corrélée, fait qui a été confirmé dans 38 CEGs de la CCLE. L’expression LATS2 est fortement corrélée à celle de CTGF et CYR61, ce qui suggère que LATS2 peut aussi être un gène cible de YAP1/TEAD.La verteporfine (VP) est capable d’interrompre l’interaction YAP1/TEAD, et donc d’inhiber son activité transcriptionelle. In vitro, utilisant CEGs et des cellules de tumeurs de patients amplifiées chez la souris (patient-derived xenograft PDX), le traitement à la VP a diminué la croissance cellulaire, l’expression de gènes cible de YAP1/TAZ/TEAD, l’activité du rapporteur TEAD-luciférase et la capacité de formation de sphères. L’activité de la VP a été testée in vivo par injection péri-tumorale dans un modèle de greffe sous-cutanés des CEGs MKN45 et MKN74 et le PDX GC10 chez la souris NSG. La croissance tumorale a été diminuée. Le poids des tumeurs, l’analyse par IHC (CD44, ALDH, Ki67) et la capacité de formation de sphères des CSCs résiduelles ont été diminuées. Ces résultats montrent une activité inhibitrice de la VP sur les CSCs gastriques in vitro et in vivo.Ce travail montre pour la première fois que l’axe LATS2/YAP1/TEAD est précocement activé pendant l’infection chronique avec H. pylori et que celui-ci contrôle l’EMT et les propriétés de CSC. Le ciblage de la voie Hippo a été montré comme étant efficace dans la prévention de la croissance tumorale, mettant en évidence le potentiel de son inhibition dans le traitement du cancer gastrique. / Gastric cancer (GC) is a multifactorial disease, most frequently associated to chronic infection with CagA-positive Helicobacter pylori strains. Epithelial-to-mesenchymal transition (EMT) is reversible process in which polarized epithelial cells acquire a mesenchymal phenotype. EMT is at the origin of cancer stem cells (CSC). In GC, CSCs express CD44 and high aldehyde-dehydrogenase (ALDH) activity. Infection with H. pylori of human gastric cancer cell lines (hGECs) in vitro induces the emergence of a population of CD44+ cells with CSC-properties through an EMT process in a CagA-dependent manner. The Hippo pathway is composed by the kinases MST and LATS, and their phosphorylation targets,YAP1 and TAZ. Upon phosphorylation by LATS, YAP1 and TAZ are inhibited. Active YAP1 and TAZ bind to TEAD transcription factors to promote the expression of genes that regulate cell growth and apoptosis.The first aim of this work was to investigate whether H. pylori affects the activation state of the Hippo pathway, and its effect on the EMT process and the CSCs. Second, we intended to characterize the role of YAP1/TEAD in gastric CSC properties in vitro and the consequences of its pharmacological inhibition on tumor growth in vivo.To study the Hippo pathway regulation during infection, LATS2, YAP1 and CD44 were evaluated in gastric mucosae of non-infected or H. pylori-infected patients. They were upregulated in infected mucosae and were associated to pathology. Hippo pathway regulation by H. pylori infection has biphasic kinetics and is CagA-dependent. Early in infection, H. pylori transiently triggered YAP1 expression and co-transcriptional activity, along with LATS2. This period of Hippo pathway inactivity is followed by a progressive activation, sustained by LATS2 accumulation and inhibitory YAP1Ser127-phosphorylation. LATS2 siRNA-mediated repression accelerated the acquisition of the EMT-phenotype upon infection, the up-regulation of EMT-markers ZEB1 and Snail1, and the decrease of the epithelial miR-200. H. pylori-induced CD44 upregulation, invasion and sphere-forming capacity were further enhanced upon LATS2 knockdown, suggesting that LATS2 restricts the EMT and CSC-like phenotype in hGECs upon H. pylori infection. Inhibition of either LATS2 or YAP1 reduced the expression of both proteins, revealing a positive feedback loop. In tissue sections of GC, LATS2 and YAP1 were heterogeneous and co-expressed. The positive correlation between LATS2 and YAP1 was confirmed in the 38 hGECs of the CCLE. The expression of CTGF and CYR61 was also strongly correlated to LATS2, suggesting that LATS2 could also be a YAP1/TEAD target gene.hGECs of the CCLE. The expression of CTGF and CYR61 was also strongly correlated to LATS2, suggesting that LATS2 could also be a YAP1/TEAD target gene.Verteporfin (VP) disrupts the YAP1/TEAD interaction inhibiting its transcriptional activity. In vitro, using hGECs and cells from patient derived primary tumor xenogratfs (PDXs), we showed that treatment with VP decreased cell growth, expression of YAP1/TAZ/TEAD target genes, TEAD-luciferase reporter activity and sphere-forming capacity. The activity of VP was tested in vivo, by peritumoral injection in a model of subcutaneous graft of hGECs (MKN45 and MKN74) and PDX (GC10) in NGS mice. Tumor growth was followed and a decrease was observed. Tumor weight measurement, IHC analysis (CD44, ALDH and Ki67), and CSCs were decreased in treated tumors. These results show the CSC-inhibitory activity of VP both in vitro and in vivo.We showed for the first time that the LATS2/YAP1/TEAD axis is early activated during the carcinogenesis process induced by chronic H. pylori infection and controls the subsequent EMT and CSC-like features. Targeting the Hippo pathway efficiently prevented tumor growth in a PDX model, highlighting the potential of its inhibition to be implemented in gastric cancer therapy.

Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-01Bitstream added on 2014-06-13T20:57:22Z : No. of bitstreams: 1 magalhaes_gm_me_jabo.pdf: 1621150 bytes, checksum: b5a01c9394af10e93a8a4a2595f1190d (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As neoplasias mamárias caninas são as mais frequentes nessa espécie e também são comuns em mulheres. Um dos principais fatores prognósticos dessa enfermidade é a presença ou ausência de metástase em linfonodos. A metástase é um mecanismo complexo que envolve vários fatores. Em mulheres sabe-se que a molécula de adesão CD44 está relacionada com invasão e metástase. Este trabalho teve como objetivos: avaliar a imunomarcação de CD44 nas neoplasias mamárias malignas da cadela, com e sem metástase em linfonodos regionais; associá-la como fator prognóstico; na detecção precoce de metástase; relacioná-la com a imunomarcação de MMP-9, E-caderina e VEGF e associar com a sobrevida das cadelas. Compuseram os grupos experimentais, cadelas com tumor mamário, com (Grupo M) ou sem metástase (Grupo N) detectável em linfonodos e um grupo controle composto por tecido mamário normal. Não houve predileção por localização mamária, mas utilizou-se mais as mamas inguinais e linfonodos inguinais. As neoplasias mamárias foram classificadas de acordo com a Organização Mundial de Saúde. Para isso utilizou-se a técnica de imuno-histoquímica, em amostras incluídas em parafina. Para a determinação da porcentagem de imunomarcação considerou-se somente as células epiteliais neoplásicas. Para o anticorpo CD44 contou-se as marcações em linfócitos T nos linfonodos dos dois grupos Observou-se aumento significativo na marcação do CD44 do sítio primário do tumor para a metástase, assim como nas marcações de MMP-9 e E-caderina. As marcações em linfócitos T foram maiores no grupo N e menores no grupo M. O tipo histopatológico mais comum foi o carcinoma simples túbulo papilífero. A raça predominante no grupo das metástases foi Teckel, com idade média de 9,4 anos, e com tempo de sobrevida de sete meses. Concluiu-se... / Mammary neoplasia are the most frequently type of cancer in bitches and also in women. One of the main prognostic factors of this desease is the presence or absent of lymphonodes metastasis. Metastasis is a complex mechanism which involve multiple factors. At women, researchs have shown that the CD44, a adhesion molecule, is related with invasion and metastasis. This research looked for as objectives: Evaluation of CD44 in malignant mammary neoplasia at bitches with and without lymphonodes metastasis; link CD44 and prognosis; CD44 and early detection of metastasis; link CD44 with MMP-9; E-cadherin and VEGF; and associate with the survival of bitches. Experimental groups were arranged with bitches suffering from mammary neoplasia and divided into three groups: Group one(M) were compounded by bitches with visible metastasis in the lymphonodes; Group two(N) were compounded by bitches without visible metastasis in the lymphonodes;Group three or control compounded by bitches with normal mammary tissue. During the research did not have predilection about the mammary localization, despite inguinal breast and lymphonodes were more collected. Mammary neoplasias were classified according to the OMS norms. For this we used the technique of immunohistochemistry in paraffin sections and counted only epithelial cancer cells by method of percentage of labeled cells. Only antibody to CD44 told that the markings on T lymphocytes in lymph nodes of the two groups observed a significant increase in CD44 marking the site of the primary tumor to metastasis, as well as the markings of MMP-9 and E-cadherin. The markings on T lymphocytes were higher in group N and lower in group M. The most common pathological type was the simple tubular papillary carcinoma. The xiii predominant race in the group of metastasis was Daschshund, mean age 9,4 years, and survival... (Complete abstract click electronic access below)

No description available.

Hyaluronic acid (HA) is one of the major components of the extracellular matrix; and may exhibit different biological functions, dependent on polymer molecular weight (MW). The signalling events performed by HA are mediated through interactions with its main cell receptors: CD44 and RHAMM. However, the direct effect between the HA MW and the expression of CD44 and RHAMM remains unclear. This study aimed to investigate whether different HA polymer MW alters the proliferation of tumour-derived cell lines, and whether different HA-sized has an effect on the regulation of the expression of CD44 and RHAMM. In order to determine size-specific responses of tumour cells of defined fragment MW, this investigation was undertaken using HA-tethered culture surfaces. Four surfaces were constructed, coated with polymers of different MWs. HA (4, 234, 2590 kDa) and an oligomer mixture were tethered onto an aminosilane (AHAPTMS)-treated glass surfaces using a carbodiimide reaction. Surfaces were analysed using a toolbox of in situ characterisation techniques, including wettability measurements, QCM, AFM and confocal microscopy. Using the constructed surfaces was demonstrated that HA-polymer MW modulates cell proliferation of human bladder (RT112 and T24) and prostate (PC3 and PNT1A) cell lines, with low HA MW (HA4) increasing proliferation, whereas a decrease is seen in the presence of medium (HA234) and high MW fragments (HA2590). The proliferation stimulus performed by HA was found to be phenotype dependent, with HA4 surfaces stimulating an increased proliferation in those less invasive cell lines (T24 and PNT1A), while HA234 and HA2590 inducing a sharper decrease in the most malignant tumour cell lines (RT112 and PC3). It was also demonstrated that the regulation of CD44 and RHAMM transcripts expression appears to be phenotype dependent but not HA-MW dependent. HA down-regulates CD44 and RHAMM in the most malignant cell lines; with up-regulation of the expression of the cell receptors in the less invasive cell lines. In addition, the presence of exogenous HA was shown to be involved in the regulation of the expression of CD44 variants expression. The results obtained for the CD44 and RHAMM protein expression were also found to be correlated with the obtained transcripts expression. However, the significance of these findings in tumourigenesis remains unclear. Findings from this investigation may help in the design and development of biocompatible implants with controlled surface properties to be used in cancer therapeutics; with medium and large HA polysaccharides being potential biopolymer candidates, useful for the development of novel therapies for highly invasive cancer. In addition, implications from this work can serve as a base for future research, and can lead to ideas for drugs and methods to be used in cancer therapeutic approaches.

Lymphocyte activation and trafficking are indispensable to the immune system. CD44, an adhesion molecule, plays important roles in T cell activation, lymphocyte homing/trafficking, and tumor metastasis. Although the functions of CD44 have been shown in T cells and other leukocytes, little is known about its role in B cells. The effects of CD44 cross-linking on murine B cell activation via CD40L/IL-4 was explored using the anti-CD44 mAbs RK3G9 and IM7. Immobilized RK3G9 and IM7 could strongly inhibit B cell proliferation and Ig production, with IgE inhibition being prominent. Soluble anti-CD44 had no effect. The inhibitory effect of RK3G9 was not influenced by addition of anti-FCγRII, indicating no role for the inhibitory receptor. The effects of delayed addition of immobilized anti-CD44 mAbs were studied, and the results indicated no inhibition after 96 hrs of culture. B cells were also activated by either LPS or anti-IgM F(ab')2. While LPS-induced B cell activation was inhibited by immobilized anti-CD44 mAbs, anti-IgM activation was refractory. Interestingly, addition of both anti-IgM and CD40L or LPS resulted in some modulation of the inhibitory activity. Additionally, FACS and Elispot revealed that RK3G9-treated cells had reduced numbers of plasma cells. Taken together, these results suggest that CD44 cross-linking could control polyclonal B cell activation by CD40L, but allow sIgM/CD40L activation to continue.

No description available.

CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored. The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development. The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading. The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRbeta, and CCR7 expression. There were also increased thymic NK and gamma/delta T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified. Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated.