Avaliamos a capacidade de indução de migração, invasão e secreção de protease pelo peptídeo derivado da laminina, C16 (KAFDITYVRLKF, cadeia g1) em linhagem celular (CAC2) de carcinoma adenóide cístico humano. Laminina g1 foi imunolocalizada no carcinoma adenóide in vivo e in vitro. Ensaio de ferida, em câmara bipartite e em vídeo microscopia (time-lapse) mostraram que C16 estimula migração em células CAC2. C16 também estimulou invasão em ensaio com câmaras bipartites cobertas com Matrigel. Invasão depende de atividade de protease. Zimografia mostrou que C16 aumentou secreção de MMPs 2 e 9. Diferentes vias de sinalização podem estar relacionadas com os efeitos de C16. Immunoblot revelou que C16 aumentou a fosforilação de AKT e ERK. Para o estudo de possíveis receptores do peptídeo, preparações de membrana foram passadas em colunas de afinidade com C16 acoplado. Banda de 40kDa foi eluída e analisada por espectrometria de massa (LC-MS/MS) que identificou a cadeia a1 do colágeno. O fragmento de colágeno eluído poderia ser parte de um complexo protéico envolvendo C16. Integrinas são receptores de colágeno e candidatas a fazerem parte desse complexo. Células CAC2 expressaram as integrinas av, a5, b3 and b1. Silenciamento dessas integrinas promoveu redução da migração e secreção de protease induzidas por C16. Sugerimos que C16 estimularia migração, invasão e secreção de protease em células de carcinoma adenóide cístico através de integrinas a5b1 e avb3. O sinal gerado por C16 seria transduzido pelas vias AKT e ERK1/2. / We studied induction of migration, invasion and protease activity by laminin-derived peptide C16 (KAFDITYVRLKF, g1 chain) in a cell line (CAC2) from adenoid cystic carcinoma. Laminin g1 was immunolocalized in adenoid cystic carcinoma in vivo and in vitro. C16 increased migratory activity of CAC2 cells, as shown by monolayer wound assay, Transwell migration assay and time-lapse video microscopy. This peptide also stimulated cell invasion in Transwell chambers coated with Matrigel. Invasion depends on protease activity. Zymograms showed that C16 increased secretion of MMPs 2 and 9. Different signaling pathways could be related to C16 regulation in CAC2 cells. Immunoblot showed that C16 increased phosphorylation of both AKT and ERK compared to controls. To study putative receptors of this peptide we used affinity chromatography. Membrane preparations were run through C16-affinity columns. A 40kDa band was eluted and analyzed by mass spectrometry (LC-MS/MS) identifying a collagen a1 chain. The collagen fragment eluted could be part of a protein complex involving C16. This protein complex may include integrins, which are collagen receptors. CAC2 cells exhibited av, a5, b3 and b1 integrins. siRNA knockdown of these integrins inhibited both C16-induced migration and protease activity. We propose that C16 increases migration, invasion and protease activity of a human salivary gland adenoid cystic carcinoma cell line through a5b1 and avb3 integrins. The signal generated by C16 is transduced by AKT and ERK1/2 signaling pathways.

As funções fisiológicas da proteína prion (PrPc) estão sob ampla investigação e caracterização, especialmente as funções associadas ao desenvolvimento cerebral. Destaca-se que a associação de PrPc com Stress Inducible Protein 1 (STI1), induz neuritogênese e neuroproteção via proteína cinase extracelular reguladora (ERK) e proteína cinase dependente de AMPc (PKA) respectivamente. O presente estudo avaliou como a expressão de PrP cem astrócitos pode modular a interação neurônioglia e o papel de STI1 como um fator autócrino em astrócitos. PrPc modula a interação neurônio-glia, a produção de fatores tróficos solúveis e a organização da laminina secretada na matriz extracelular pelos astrócitos. Desta forma, a expressão de PrP ctanto em astrócitos quanto em neurônios é essencial para a neuritogênese e sobrevivência neuronal. O papel autócrino de STI1 em astrócitos também foi demonstrado. A interação PrPc-STI1 previne a morte celular por ativação da via de PKA, e ativa a diferenciação astrocitária, de uma forma protoplasmática para uma fibrosa pela indução de ERK1/2. De acordo com estes resultados, um menor grau de diferenciação é encontrado em camundongos deficientes para PrPc. Estes apresentam uma expresão reduzida GFAP (proteína fibrilar acídica glial) e aumentada de vimentina e nestina em comparação com aqueles derivados de animais tipo-selvagem. STI1 promove ainda parada da proliferação astrocitária ativando a via de PKC de maneira independente de PrPc. O mecanismo pelo qual STI1 é secretada por astrócitos também foi avaliado e verificou-se que este é independente da via clássica mediada pelo complexo de Golgi. STI1 secretada é encontrada numa forma solúvel e em outra associada a componentes lipídicos e foram caracterizados por microscopia eletrônica como vesículas que variam entre 20-200nm. Dentre as vias de secreção não clássicas dependentes de lipídeos, a via de \"shedding\" de membrana foi descartada visto que STI1 não é secretada em associação com lipoproteínas. STI1 está presente em frações positivas para o receptor de transferrina, Hsp70, Hsp90 e PrPc, sugerindo composição exossomal. Esses resultados indicam que STI1 pode ser classificada como um fator trófico que associado ao seu \"receptor\" ou \"co-receptor\", PrPc, modula a sobrevivência e diferenciação tanto de neurônios quanto de astrócitos / The physiological functions of PrPc are under intense investigation and characterization, particularly those associated with brain development. In neurons, the association of PrPc with its ligand, STI1, induces neuritogenesis and neuroprotection via ERK and PKA signaling pathways, respectively. The present study evaluated whether PrPc expression in astrocytes modulates neuron-glia crosstalk and the autocrine role of STI1 in astrocytes. PrPc modulates neuron-glia interaction, the production and secretion of soluble factors, and the organization of the laminin in the extracellular matrix. PrPc expression in neurons and astrocytes is essential to neuritogenesis and neuronal survival. The autocrine role of STI1 in astrocytes was also demonstrated. The PrPc-STI1 interaction prevents cell death in a PKA-dependent manner, and induces astrocyte differentiation, from a flat to a process-bearing morphology in an ERK1/2 dependent manner. We showed that PrPccnull astrocytes presented a slower rate of astrocyte maturation than wild-type ones, with reduced expression of GFAP and increased vimentin and nestin expression. STI1 inhibited proliferation of both wild-type and PrPCnull astrocytes in a PKC-dependent manner. The mechanisms by which STI1 can be secreted by astrocytes was avaliated and we demonstrated that this secretion is independent on the classical secretory pathway mediated by the Golgi apparatus. Secreted STI1 is found in a soluble form and associated with lipidic compartments and we characterized by electron microscopy as vesicles that range from 20-200nm. Among the non-classical lipid-dependent secretory pathways, STI1 secretion by shedding was ruled out since STI1 was not secreted with lipoprotein fractions. On the other hand, STI1 is present in fractions that are positive for transferrin receptor, Hsp70, Hsp90 and PrPc, suggesting an exosome identity. Taken together, these data indicate that STI1 acts as a neurotrophic factor whose activity is dependent on the expression of PrP c at the neuronal surface, modulating differentiation and survival of both neurons and astrocytes

Οι εξωκυττάριες ουσίες παράγονται από τα κύτταρα, εκκρίνονται από αυτά και αλληλεπιδρώντας τόσο μεταξύ τους όσο και με τα κύτταρα ρυθμίζουν πολλές αναπτυξιακές πορείες. Μελετήσαμε τη χρονική και τοπική εμφάνιση των mRNAs της εντακτίνης και της λαμινίνης, γλυκοπρωτεϊνών των εξωκυττάριων ουσιών, στο πρώϊμο έμβρυο όρνιθας. Χρησιμοποιήσαμε την τεχνική της in situ υβριδοποίησης, που κατέδειξε διαφορική έκφραση της εντακτίνης και των αλυσίδων της λαμινίνης σε ξεχωριστούς κυτταρικούς πληθυσμούς και όργανα σε διαφορετικά στάδια ανάπτυξης του εμβρύου. Επίσης μελετήσαμε την χρονική και τοπική κατανομή του πολυπεπτι- δίου της εντακτίνης από το στάδιο του μοριδίου ως την αρχή της οργανογένεσης με τις τεχνικές του ανοσοφθορισμού και της ανοσοκατακρήμνισης. Ανιχνεύσαμε την παρουσία της εντα-κτίνης ήδη από το στάδιο του όψιμου μοριδίου. Με τη χρήση αντισωμάτων για την εντακτίνη μελετήσαμε το ρόλο της κατά την ανάπτυξη του πρώϊμου εμβρύου. Στα αποτελέσματά μας φάνη-κε οτι η εντακτίνη είναι απαραίτητη για το σωστό προσανατο-λισμό των κυττάρων καθώς μεταναστεύουν κατά την γαστριδίω-ση. Αυτό έχει ως αποτέλεσμα την αναστολή του σχηματισμού του εμβρυϊκού άξονα. / The extracelular matrix is a complex cell product which regulates many developmental processes. We used specific antisense RNA probes for the laminin α1, β1 and γ1 chains and for entactin, two extracellular matrix glycoproteins, to study the tissue specific and temporal patterns of their mRNAs in the early chick embryo. Our work employing in situ hybridization, showed strong signals of the laminin and of entactin mRNAs at the morula stage and differential express-ion of these mRNAs in the forming embryonic tissues and organs in the developing chick embryo. We also studied the time of appearance and subsequent distribution of the entactin polypeptide using immunofluorescence and immunopre-cipitation from the morula stage up to the early organogene-sis in the chick embryo. The first presence of entactin was detected at the late morula stage and showed differential expression in the various cell populations in the develop- ing embryo. To study the role of entactin in the major cell-ular migrations during gastrulation we used blocking anti- bodies in set of functional studies. Entactin seemed to be essential for the directional migrations of cells during gastulation and the embryonic axis was not formed in the embryos treated with the anti-entactin antibodies.

Traumatic brain injury (TBI) results in over 50,000 deaths and 80,000 disabilities each year. Current treatment strategies aim to alleviate acute disturbances, but are not able to address the chronic disorders associated with TBI. Neural transplantation is one potential treatment that will provide multifaceted sustained therapy to degenerating injured tissue. Transplantation of multipotent neural stem cells (NSCs) has been shown to enhance functional recovery in TBI models; however, poor cell survival and integration with host tissue potentially restrict the efficacy of such transplants. This limitation may be due to the absence of inherent NSC pro-survival cues (e.g., cell-ECM interactions). Furthermore, the neural injury environment presents cell death factors to transplanted NSCs. It is hypothesized that a 3-D scaffold presenting specific CNS adhesive moieties will enhance donor cell survival and promote differentiation and migration. This project encompassed material development and in vitro characterization. Results highlighted the importance of ligand tethering chemistry and density and also the mechanical integrity of cell scaffold systems. Furthermore, the developed scaffold provides a controlled microenvironment to assess the influence of LN on NSC survival, migration, and differentiation. Lastly, co-delivering NSC with the MC-LN tissue engineered scaffold into a mechanically injured neural co-culture test-bed or in vivo TBI model confirmed the importance of ECM cues for NSC survival and migration, respectively.

As funções fisiológicas da proteína prion (PrPc) estão sob ampla investigação e caracterização, especialmente as funções associadas ao desenvolvimento cerebral. Destaca-se que a associação de PrPc com Stress Inducible Protein 1 (STI1), induz neuritogênese e neuroproteção via proteína cinase extracelular reguladora (ERK) e proteína cinase dependente de AMPc (PKA) respectivamente. O presente estudo avaliou como a expressão de PrP cem astrócitos pode modular a interação neurônioglia e o papel de STI1 como um fator autócrino em astrócitos. PrPc modula a interação neurônio-glia, a produção de fatores tróficos solúveis e a organização da laminina secretada na matriz extracelular pelos astrócitos. Desta forma, a expressão de PrP ctanto em astrócitos quanto em neurônios é essencial para a neuritogênese e sobrevivência neuronal. O papel autócrino de STI1 em astrócitos também foi demonstrado. A interação PrPc-STI1 previne a morte celular por ativação da via de PKA, e ativa a diferenciação astrocitária, de uma forma protoplasmática para uma fibrosa pela indução de ERK1/2. De acordo com estes resultados, um menor grau de diferenciação é encontrado em camundongos deficientes para PrPc. Estes apresentam uma expresão reduzida GFAP (proteína fibrilar acídica glial) e aumentada de vimentina e nestina em comparação com aqueles derivados de animais tipo-selvagem. STI1 promove ainda parada da proliferação astrocitária ativando a via de PKC de maneira independente de PrPc. O mecanismo pelo qual STI1 é secretada por astrócitos também foi avaliado e verificou-se que este é independente da via clássica mediada pelo complexo de Golgi. STI1 secretada é encontrada numa forma solúvel e em outra associada a componentes lipídicos e foram caracterizados por microscopia eletrônica como vesículas que variam entre 20-200nm. Dentre as vias de secreção não clássicas dependentes de lipídeos, a via de \"shedding\" de membrana foi descartada visto que STI1 não é secretada em associação com lipoproteínas. STI1 está presente em frações positivas para o receptor de transferrina, Hsp70, Hsp90 e PrPc, sugerindo composição exossomal. Esses resultados indicam que STI1 pode ser classificada como um fator trófico que associado ao seu \"receptor\" ou \"co-receptor\", PrPc, modula a sobrevivência e diferenciação tanto de neurônios quanto de astrócitos / The physiological functions of PrPc are under intense investigation and characterization, particularly those associated with brain development. In neurons, the association of PrPc with its ligand, STI1, induces neuritogenesis and neuroprotection via ERK and PKA signaling pathways, respectively. The present study evaluated whether PrPc expression in astrocytes modulates neuron-glia crosstalk and the autocrine role of STI1 in astrocytes. PrPc modulates neuron-glia interaction, the production and secretion of soluble factors, and the organization of the laminin in the extracellular matrix. PrPc expression in neurons and astrocytes is essential to neuritogenesis and neuronal survival. The autocrine role of STI1 in astrocytes was also demonstrated. The PrPc-STI1 interaction prevents cell death in a PKA-dependent manner, and induces astrocyte differentiation, from a flat to a process-bearing morphology in an ERK1/2 dependent manner. We showed that PrPccnull astrocytes presented a slower rate of astrocyte maturation than wild-type ones, with reduced expression of GFAP and increased vimentin and nestin expression. STI1 inhibited proliferation of both wild-type and PrPCnull astrocytes in a PKC-dependent manner. The mechanisms by which STI1 can be secreted by astrocytes was avaliated and we demonstrated that this secretion is independent on the classical secretory pathway mediated by the Golgi apparatus. Secreted STI1 is found in a soluble form and associated with lipidic compartments and we characterized by electron microscopy as vesicles that range from 20-200nm. Among the non-classical lipid-dependent secretory pathways, STI1 secretion by shedding was ruled out since STI1 was not secreted with lipoprotein fractions. On the other hand, STI1 is present in fractions that are positive for transferrin receptor, Hsp70, Hsp90 and PrPc, suggesting an exosome identity. Taken together, these data indicate that STI1 acts as a neurotrophic factor whose activity is dependent on the expression of PrP c at the neuronal surface, modulating differentiation and survival of both neurons and astrocytes

Avaliamos a capacidade de indução de migração, invasão e secreção de protease pelo peptídeo derivado da laminina, C16 (KAFDITYVRLKF, cadeia g1) em linhagem celular (CAC2) de carcinoma adenóide cístico humano. Laminina g1 foi imunolocalizada no carcinoma adenóide in vivo e in vitro. Ensaio de ferida, em câmara bipartite e em vídeo microscopia (time-lapse) mostraram que C16 estimula migração em células CAC2. C16 também estimulou invasão em ensaio com câmaras bipartites cobertas com Matrigel. Invasão depende de atividade de protease. Zimografia mostrou que C16 aumentou secreção de MMPs 2 e 9. Diferentes vias de sinalização podem estar relacionadas com os efeitos de C16. Immunoblot revelou que C16 aumentou a fosforilação de AKT e ERK. Para o estudo de possíveis receptores do peptídeo, preparações de membrana foram passadas em colunas de afinidade com C16 acoplado. Banda de 40kDa foi eluída e analisada por espectrometria de massa (LC-MS/MS) que identificou a cadeia a1 do colágeno. O fragmento de colágeno eluído poderia ser parte de um complexo protéico envolvendo C16. Integrinas são receptores de colágeno e candidatas a fazerem parte desse complexo. Células CAC2 expressaram as integrinas av, a5, b3 and b1. Silenciamento dessas integrinas promoveu redução da migração e secreção de protease induzidas por C16. Sugerimos que C16 estimularia migração, invasão e secreção de protease em células de carcinoma adenóide cístico através de integrinas a5b1 e avb3. O sinal gerado por C16 seria transduzido pelas vias AKT e ERK1/2. / We studied induction of migration, invasion and protease activity by laminin-derived peptide C16 (KAFDITYVRLKF, g1 chain) in a cell line (CAC2) from adenoid cystic carcinoma. Laminin g1 was immunolocalized in adenoid cystic carcinoma in vivo and in vitro. C16 increased migratory activity of CAC2 cells, as shown by monolayer wound assay, Transwell migration assay and time-lapse video microscopy. This peptide also stimulated cell invasion in Transwell chambers coated with Matrigel. Invasion depends on protease activity. Zymograms showed that C16 increased secretion of MMPs 2 and 9. Different signaling pathways could be related to C16 regulation in CAC2 cells. Immunoblot showed that C16 increased phosphorylation of both AKT and ERK compared to controls. To study putative receptors of this peptide we used affinity chromatography. Membrane preparations were run through C16-affinity columns. A 40kDa band was eluted and analyzed by mass spectrometry (LC-MS/MS) identifying a collagen a1 chain. The collagen fragment eluted could be part of a protein complex involving C16. This protein complex may include integrins, which are collagen receptors. CAC2 cells exhibited av, a5, b3 and b1 integrins. siRNA knockdown of these integrins inhibited both C16-induced migration and protease activity. We propose that C16 increases migration, invasion and protease activity of a human salivary gland adenoid cystic carcinoma cell line through a5b1 and avb3 integrins. The signal generated by C16 is transduced by AKT and ERK1/2 signaling pathways.

Prions são definidos como partículas protéicas infecciosas compostas, quase que exclusivamente, por uma proteína conhecida como prion scrapie (PrPsc). O envolvimento dessas partículas na etiologia de doenças neurodegenerativas, tanto em homens como em animais, já está bem determinado. Acredita-se que o PrPsc seja sintetizado através de modificações pós-traducionais que ocorreriam na isoforma celular da proteína prion (PrPc), uma glicoproteína expressa constitutivamente na superfície de vários tipos celulares, principalmente de neurônios, ancorada na membrana plasmática por glicosil-fosfatidil inositol (GPI). Apesar de ser uma molécula conservada em várias espécies, a função de PrPc ainda permanece desconhecida. Interessados nos possíveis papéis fisiológicos desempenhados pelo PrPc, decidimos investigar certas interações que o PrPc poderia realizar com outras moléculas, na tentativa de se encontrar pistas sobre a função dessa proteína em células normais. Assim, nosso grupo identificou e vem caracterizando duas interações nas quais o PrPc está envolvido: com o seu receptor de 60-66 kDa e com a principal proteína não colagênica da matriz extracelular, a laminina (LN). Grande parte da caracterização dessas duas interações vem sendo desenvolvida graças ao uso de PrPc recombinante produzido em sistema heterólogo (em E.coli). O trabalho em questão trata principalmente da produção de PrPc recombinantes em sistema heterólogo e a utilização destes como importantes ferramentas para a melhor caracterização das interações identificadas. Alguns trabalhos na literatura vinham sugerindo a existência de um receptor para prions. Através da teoria da hidropaticidade complementar dos aminoácidos, nosso grupo foi capaz de identificar uma proteína de 60-66 kDa como sendo esse provável receptor. Ensaios de ligação in vitro utilizando PrPc recombinante, ou PrPc nativo (ancorado na superfície de células) confirmaram que a forma desse receptor presente na membrana (de 66 kDa) é capaz de se ligar ao PrPc. Com a ajuda de PrPc recombinante também foi possível verificar uma ligação específica, saturável e de alta afinidade (Kd da ordem de 10-8 M) entre PrPc e a LN. Através de ensaios de competição, utilizando peptídeos sintéticos correspondentes a domínios da LN já bem caracterizados e de funções estabelecidas, fomos capazes de mapear o sítio dessa molécula que se liga ao PrPc. A sequência identificada (RNIAEIIKDI) encontra-se na região C-terminal da cadeia γ1 da LN e, como demonstrado na literatura, esse domínio é responsável por estimular tanto a adesão celular, quanto o crescimento de neuritos em neurônios de cerebelo em cultura primária. De fato, resultados obtidos pelo nosso grupo indicam que a interação PrPc/LN participa no processo de neuritogênese. Experimentos de esquiva inibitória, realizados em colaboração com o grupo do Prof. Dr. Ivan Izquierdo (UFRGS) indicaram que a ligação PrPc/LN também desempenha um importante papel nos processos de memória e aprendizado. / Prions are defined as proteinaceous infectious particles that mediate the pathogenesis of certain neurodegenerative diseases, in humans and in animals. The prion particle is composed largely, if not entirely, by PrPsc (prion scrapie), a posttranslationaly modified isoform of the cellular host-encoded prion protein (PrPc). PrPc is a glycosylphosphatidylinositol anchored protein that is constitutively expressed by several cell types, mainly on neuronal cell surface. However, the physiological role of this conserved protein remains unclear. ínterested in this normal function of PrPc, our group decided to investigate some interactions that PrPc could be done with other molecules in order to find some clues about it. We have been identified and characterized two interactions that PrPc is involved: with its putative 60-66 kDa receptor and with laminin. In order to better characterize these interactions it was necessary to produce purified PrPc. In this work we will report the expression and the purification of mouse PrPc protein in heterologous system and its use as important tools to investigate the PrPc interactions. A specific cell surface receptor for PrPc has been predicted. Using the concept of complementary hydropathy, our group has identified a 60-66 kDa membrane protein in mouse brain, which seems to be a putative PrPc receptor. ln vitro binding assays using recombinant and native PrPc were able to confirm that the membrane receptor (66 kDa) binds PrPc. Recombinant PrPc was also useful to demonstrate a specific and high affinity (Kd around 10-8M) interaction between PrPc and laminin. In an attempt to map the PrPc binding site in this molecule, laminin peptides with established physiological functions were used in cornpetition binding assays. A peptide derived frorn C-terminal γ-1 chain of mouse laminin, RNIAEIIKDI, was the only one that was able to block the binding of laminin to PrPc, suggesting that this region comprises the PrPc binding site. It was reported in the literature that this peptide simulates the neurite outgrowth and cellular adhesion. In collaboration with Dr. Izquierdo\'s group (UFRGS) we demonstrated that the interaction characterized between this laminin\'s domain and PrPc is involved in the neuritogenesis process, as well as in learning and memory.

O câncer de mama é a doença maligna mais comum e a principal causa de morte entre as mulheres. Sua complexa etiologia envolve múltiplos fatores de risco, a maioria deles relacionada aos níveis cumulativos de exposição da mama aos estrógenos. A maioria de suas ações é mediada pela ligação a seus receptores ER e ER que são fatores de transcrição. Outro fator que exerce um controle extraordinário no comportamento celular, regulando a transcrição gênica e influenciando diversos processos biológicos, e que, quando alterado, é associado ao processo de tumorigênese da mama é a matriz extracelular. A laminina, um dos principais componentes da matriz extracelular, interage com as células através das integrinas e está relacionada ao fenótipo maligno, atuando na adesão, migração, proliferação, diferenciação e sobrevivência celular. Nosso grupo identificou diversos genes diferencialmente expressos em células de câncer de mama ER+ na presença ou ausência de uma monocamada de laminina utilizando a técnica DDRT-PCR. Dois dos genes identificados, DDEF2 e PHLDA1, estão associados à adesão; DDEF2 envolvido na sinalização das integrinas e PHLDA1 relacionado com apoptose por perda de adesão. Nosso objetivo foi investigar os efeitos do 17-estradiol e da laminina na regulação da expressão dos genes DDEF2 e PHLDA1 nas linhagens celulares MCF-7, MDA-MB-231 e posteriormente S30, utilizando a técnica RT-PCR em tempo real. O gene PHLDA1 foi induzido pelo E2 via ER nas células MCF-7 e pela laminina nas células S30, e o gene DDEF2 foi reprimido pelo E2 e induzido pela laminina nas células S30 / The breast cancer is the most common malignant disease and the leading cause of death among women. Its complex etiology involves multiple risk factors, most of them related to the levels of cumulative breast exposure to estrogen. Most of its actions is mediated by binding to its receptor ER and ER that are transcription factors. Another factor that has a tremendous control in cell behavior, regulating the gene transcription and influencing various biological processes, which when altered, is attached to the process of tumorigênese of the breast is the extracellular matrix (ECM). The laminin, one of the main components of the ECM, interacts with the cells through integrins and is related to the malignant phenotype, acting in adhesion, migration, proliferation, differentiation and cell survival. Our group identified several genes diferentialy expressed in breast cancer cells ER + in the presence or absence of a laminin monolayer using the technique DDRT-PCR. Two of the genes identified, DDEF2 and PHLDA1, are associated with adhesion; DDEF2 is involved in the integrins signaling and PHLDA1 is related with apoptosis by loss of adhesion. Our goal was to investigate the effects of 17-estradiol and laminin in regulating the expression of the genes DDEF2 and PHLDA1 in MCF-7, MDA-MB-231 and later S30 cell lines, using the real time RT-PCR technique. The gene PHLDA1 was induced by E2 via ER in MCF-7 cells and the laminin in S30 cells, and the gene DDEF2 was suppressed by E2 and induced by laminin in S30 cells

Abstract This thesis focuses on mammalian kidney development and in particular on the question of how two Wnt signalling pathway genes, an antagonistic Dkk1 and an agonistic ligand Wnt5a, regulate the process. Wnts are secreted ligands that are involved in many developmental processes, including gonadal differentiation and kidney development, but also in various diseases and malformations. Wnts form a large signalling family containing 19 different glycoprotein ligands in mammals. Wnt signalling occurs via two different intracellular pathways. A canonical pathway proceeds via beta-catenin, and a non-canonical pathway utilizes other signalling molecules. Dkk1 is an antagonist of the canonical pathway and Wnt5a is considered a ligand that activates the non-canonical signalling pathway. As part of the thesis, I have studied the role of Dkk1 in kidney morphogenesis using a conditional mouse model, in which the gene is deleted in a cell specific manner from the collecting ducts. Dkk1 deficiency increased renal papilla growth and the risk of hydronephrosis. Research pointed out that the lack of Dkk1 in the collecting ducts increased cell proliferation and disturbed the balance of canonical Wnt signalling, which led to an overgrowth of renal papilla. This led to functional phenotypes including increased water reabsorption and changes in ion secretion/absorption. These changes are most likely due to altered Wnt7b signalling. The second part of the thesis examines the role of the non-canonical Wnt5a gene in kidney development with a conventional knock out mouse model. At the time work began on the thesis, no corresponding kidney phenotype had been published. The primary finding in kidneys lacking Wnt5a was an altered basement membrane organization of the collecting ducts and glomeruli. The phenotype is most likely the reason behind morphological phenotypes which vary from bilateral kidney agenesis to duplex collecting system. Notably, during the course of this study we found a mutation in the human WNT5A gene of a CAKUT patient. This is the first time Wnts have been shown to organize kidney development via basement membrane formation. / Tiivistelmä Tämän väitöskirjan tarkoituksena on ollut tutkia munuaisen kehitystä ja kuinka kaksi Wnt-signalointireitin geeniä, signalointia estävä Dkk1 ja signalointia edistävä Wnt5a säätelevät sitä. Wnt ligandit ovat eritettäviä signaalimolekyylejä, jotka ovat osallisina monissa kehitysbiologissa prosesseissa kuten sukupuolen määräytymisessä ja munuaisen kehityksessä. Myös monissa taudeissa on havaittu muuntuneita Wnt geenien tuottotasoja. Wnt-geenit muodostava suuren signalointimolekyyliperheen, johon lukeutuu 19 jäsentä nisäkkäillä ja Wnt-signointi on jaettu perinteisesti kahteen signalointiryhmään. Dkk1 on kanonisen Wnt-signaloinnin estäjä ja Wnt5a:ta pidetään pääsaantiöisesti ei-kanonisena Wnt-ligandina. Väitöskirjassani olen tutkinut Dkk1 geenin toimintaa kohdennetussa Dkk1-poistogeenisessä hiiressä, jossa geenin toiminta on poistettu spesifisesti munuaisen kokoojaputkista. Dkk1:n puutos johtaa munuaisen papillan kasvuun ja lisää riskiä hydronefroksen muodostumiseen. Tutkimukset osoittivat että Dkk1:n puutos aiheuttaa lisääntynyttä solujakautumista kokoojaputkissa, jolloin Wnt-signaloinnin muutos aiheuttaa papillan ylikasvua. Ylikasvusta seuraa lisääntynyttä veden takaisin imeytymistä ja muutoksia ionien erittämisessä ja takaisin imeytymisessä. Todennäköisimmin muutokset johtuvat muuntuneesta Wnt7b signaloinnista, jota Dkk1 normaalisti säätelee. Väitöskirjan toisessa osassa tutkittiin ei-kanonisen reitin Wnt5a ligandin roolia munuaisen kehityksessä käyttäen poistogeenistä hiirimallia, jossa Wnt5a:n roolia munuaisenkehityksessä ei ollut julkaistu työn aloituksen aikaan. Wnt5a:n puutoksen havaittiin vaikuttavan tyvikalvon järjestymiseen kokoojaputkissa ja munuaiskeräsessä. Tyvikalvon häiriö on todennäköisin syy morfologisiin muutoksiin, jotka vaihtelevat molempien munuaisen puuttumisesta kaksois-kokoojatiehyen muodostumiseen. Työssä osoitetaan ensimmäistä kertaa kuinka Wnt-signalointireitin proteiinit säätelevät munuaisen kehitystä tyvikalvon muodostuksen kautta.

Abstract Collagen XV is a proteoglycan localized in the outermost layer of the basement membrane (BM) and in the fibrillar matrix. Although it is widely distributed in many tissues, its amount is generally low. It is characterized by a highly interrupted collagenous domain flanked by large globular domains and attached glycosaminoglycan chains, thus also being identified as a chondroitin sulphate proteoglycan. The C-terminal end of collagen XV, termed restin, has proved to have antiangiogenic effects. In view of its location in the outermost layer of the lamina densa and in association with interstitial collagen fibrils near BMs, it has been suggested that collagen XV may form a bridge between the fibrillar collagen matrix and the BMs. In vivo studies of mice lacking collagen XV have demonstrated that collagen XV is needed as a structural component to stabilize the skeletal muscle cells and capillaries. The role of collagen XV in the extracellular matrix (ECM) was studied using a mouse model lacking the gene of interest (Col15a1-/-). The matrix of peripheral nerves, skeletal muscle, heart and the uterus during pregnancy was analysed at several time points, and peripheral nerve development was evaluated in a mouse model lacking collagen XV and the laminin α4 chain simultaneously as well as in Col15a1-/- mice. The function of collagen XV in the heart was analysed in young, adult and old mice separately. The results indicate that collagen XV is needed for organizing collagen fibrils into proper bundles in the cardiac ECM. In the developing nerve it regulates collagen fibril size and the organization of the fibrils within the collagen bundle. In mature nerves and skeletal muscles it structures the BM–fibrillar matrix interphase, and in the uterus of pregnant mice it participates in fibrillar collagen remodelling, affecting the lateral fusion of collagens into thick fibrils. Even delicate changes in the matrix can lead to an alteration in the functioning of certain organs. Abnormal C-fibrils, polyaxonal myelination and decreased sensory conduction velocity were observed in the Col15a1-/- peripheral nerves, and a simultaneous lack of collagen XV and the laminin α4 chain resulted in more severe and permanent impairment of the segregation and myelination of the nerve axons. A complex cardiac phenotype was observed in the Col15a1-/- mice, predisposing them to cardiomyopathy. / Tiivistelmä Kollageeni XV on proteoglykaani, jonka lokalisaatio on tyvikalvon uloimmassa kerroksessa ja soluväliaineen säikeissä. Vaikka sitä tuotetaan laajalti useissa eri kudoksissa, sitä on määrällisesti vähän kudoksissa. Kollageenin XV ominaisuuksiin kuuluvat useat katkokset kollageenisilla alueilla, globulaariset alueet proteiinin päissä, sekä siihen kiinnittyneet glykosaminoglykaaniketjut, jotka on tunnistettu kondroiittisulfaatiksi. Kollageenin XV C-terminaalisella osalla, jota kutsutaan restiniksi, on osoitettu olevan antiangiogeneettisiä vaikutuksia. Ottaen huomioon kollageeni XV lokalisaation tyvikalvon uloimmassa kerroksessa, ja sen liittymisen säikeisiin kollageeneihin lähellä tyvikalvoa, on ehdotettu että sen tehtävänä olisi toimia yhteys-siltana säikeisten kollageenien ja tyvikalvon välillä. In vivo tutkimuksissa kollageeni XV poistogeenisillä hiirillä on osoitettu, että kollageenia XV tarvitaan rakenteellisena osana stabiloimaan luurankolihasta sekä hiussuonia. Kollageenin XV merkitystä sidekudoksessa tutkittiin käyttäen hiirimallia, jolta puuttuu kyseinen geeni (Col15a1-/-). Ääreishermojen, luurankolihaksen, sydämen ja kohdun soluväliainetta analysoitiin eri-ikäisillä hiirillä. Lisäksi ääreishermojen kehitystä tutkittiin hiirimallilla, jolta puuttuu sekä kollageeni XV että laminiini α4-ketju samanaikaisesti, sekä Col15a1-/- hiirillä. Kollageenin XV tehtävää sydämessä analysoitiin nuorilla, aikuisilla ja ikääntyneillä Col15a1-/- hiirillä. Tulokset viittaavat kollageeni XV merkittävään rooliin soluväliaineen järjestäytymisessä, erityisesti kollageenisäikeiden järjestäytymisessä kimpuiksi sydämen soluvälitilassa. Kehittyvässä hermossa sen tehtävä on säädellä säikeisten kollageenien läpimittaa sekä säikeiden järjestäytymistä säiekimpun sisällä. Täysin kehittyneessä hermossa ja luurankolihaksessa se strukturoi tyvikalvon ja säikeisen soluväliaineen välitilaa, kun taas raskaana olevien hiirien kohdussa se osallistuu säikeisten kollageenien muokkaukseen, vaikuttamalla kollageenien paksuuntumiseen lateraalisen fuusion kautta. Jopa hienoiset muutokset soluväliaineen rakenteessa voivat johtaa muutoksiin elinten toiminnassa. Epämuodostuneita C-säikeitä, polyaksonaalista myelinisaatiota ja hidastunutta sensorista johtonopeutta todettiin Col15a1-/- perifeerisissä hermoissa. Samanaikainen kollageenin XV ja laminiini α4-ketjun puutos johtivat vaikeampaan ja pysyvämpään haittaan hermon aksonien segregaatiossa ja myelinisaatiossa. Col15a1-/- hiirillä todettiin monimuotoinen sydämen ilmiasu, joka altistaa hiiret kardiomyopatialle.