The Transforming Growth Factor (TGF) superfamily of cytokines and their serine/threonine kinase receptors play an important role in the regulation of cell division, differentiation, adhesion, migration, organization, and death. Smad proteins are the major intracellular signal transducers for the TGF receptor superfamily that mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 (BMP-4) is a representative of the TGF superfamily, which regulates the formation of teeth, limbs and bone, and also plays a role in fracture repair. Binding of BMP-4 to its receptor stimulates phosphorylation of Smad1, which subsequently recruits Smad4. A hetero-oligomeric complex consisting of Smad1 and Smad4 then translocates into the nucleus and regulates transcription of target genes by interacting with transcription factors. Although the individual steps of the signaling cascade from the receptor to the nucleus have been identified, the exact kinetics and the rate limiting step(s) have remained elusive. Standard biochemical techniques are not suitable for resolving these issues, as they do not offer sufficiently high sensitivity and temporal resolution. In this study, advanced optical techniques were used for direct visualization of Smad signaling in live mammalian cells. Novel fluorescent biosensors were developed by fusing cyan and yellow fluorescent proteins to the signaling molecules Smad1 and Smad4. By measuring Fluorescence Resonance Energy Transfer (FRET) between the two fluorescent proteins, the kinetics of BMP/Smad signaling was unraveled. A rate-limiting delay of 2 - 5 minutes occurred between BMP receptor stimulation and Smad1 activation. A similar delay was observed in the complex formation between Smad1 and Smad4. Further experimentation indicated that the delay is dependent on the Mad homology 1 (MH1) domain of Smad1. These results give new insights into the dynamics of the BMP receptor – Smad1/4 signaling process and provide a new tool for studying Smads and for testing inhibitory drugs. / Die Transforming Growth Factor" (TGF)-Superfamilie der Cytokine und ihrer Serin/Threonin-Kinase-Rezeptoren spielt eine bedeutende Rolle bei der Regulierung der Zellteilung, -differenzierung, -adhäsion, -migration, -organisation, und beim Zelltod. Die Smad-Proteine sind die wichtigsten intrazellulären Signalüberträger für die TGF-Rezeptor-Familie, da sie das Signal von der Zellmembran zum Kern übermitteln. Das ,,Bone Morphogenetic Protein4" (BMP-4) ist ein Vertreter der TGF-Familie, der die Bildung von Zähnen, Gliedmaßen und Knochen reguliert und darüber hinaus eine Rolle bei der Frakturheilung spielt. Das Binden von BMP-4 an seinen Rezeptor stimuliert die Phosphorylierung von Smad1, welches in der Folge Smad4 rekrutiert. Ein hetero-oligomerer Komplex bestehend aus Smad1 und Smad4 verlagert sich dann in den Zellkern, wo er durch Interaktion mit Transkriptionsfaktoren die Transkription von Zielgenen reguliert. Obwohl die einzelnen Schritte der Signalkaskade vom Rezeptor bis in den Zellkern bereits identifiziert wurden, blieben die Kinetik und die geschwindigkeitsbegrenzenden Schritte bisher unbekannt. Gängige biochemische Methoden eignen sich nicht um diese Fragen zu lösen, da sie nicht über ausreichende Empfindlichkeit und zeitliches Auflösungsvermögen verfügen. In der vorliegenden Arbeit wurden hochentwickelte optische Techniken angewandt, um die Smad-vermittelte Signaltransduktion direkt in lebenden Zellen sichtbar zu machen. Neue fluoreszierende Biosensoren wurden konstruiert, indem gelb- und cyan-fluoreszierende Proteine mit den Signalmoleküle Smad1 und Smad4 fusioniert wurden. Durch Messung des "Fluorescent Resonance Energy Transfer" (FRET) zwischen den zwei fluoreszierenden Proteinen konnte die Kinetik der BMP-Smad-Signalkaskade bestimmt werden. Zwischen der Stimulation des Rezeptors und der Aktivierung von Smad1 trat eine geschwindigkeitsbegrenzende Verzögerung von 2-5 Minuten auf. Eine ähnliche Verzögerung wurde bei der Bildung des Komplexes aus Smad1 und Smad4 beobachtet. Weitere Experimente zeigten, dass die Verzögerung von der Mad-Homologie-Domäne 1 (MH1) von Smad1 abhängt. Die Ergebnisse dieser Arbeit geben neue Einblicke in die Dynamik der BMP-Rezeptor-Smad1/4 Signaltransduktion und stellen neue Werkzeuge zur Untersuchung von Smads und zur Austestung inhibitorischer Wirkstoffe zur Verfügung.

OBJETIVO: Investigar o efeito da expressão de RNAm dos SMADs e de microRNAs (miRNAs) que possuem o TGFB1 como alvo na expressão gênica (RNAm e proteína) de TGF-β1 e seu papel na fisiopatologia da angiogênese em pacientes com mielofibrose (MF) e trombocitemia essencial (TE). MÉTODOS: Foram incluídos 21 pacientes com MF primária (MFP), 21 com MF pós-TE (MFPTE) e 24 com TE, além de 98 indivíduos controles pareados de acordo com gênero e idade com os pacientes. As análises realizadas no sangue periférico foram: quantificação das concentrações plasmáticas e de RNAm de TGFB1, VEGFA e FGF2; quantificação de RNAm de SMADs 1 a 7 e de miRNAs 193a-5p, 369-5p, 542-5p, 590-3p, e 590- 5p; e detecção das mutações JAK2V617F (com quantificação alélica), MPLW515K/L e CALR. Em 26 biópsias de medula óssea dos pacientes, foram determinados o grau de microvasculatura (angiogênese estimada - CD34), a imunoexpressão de TGF-b1 ativo, TGF-β1 latente e c-MPL. RESULTADOS: As concentrações de TGF- β1 plasmático foram semelhantes entre os pacientes e controles, enquanto o VEGFA plasmático foi maior em todos os grupos de pacientes comparados aos seus controles. O FGF2 plasmático também foi maior em todos os grupos de pacientes, e a expressão de seu RNAm foi maior nos pacientes com TE do que em seus controles. As expressões de SMADs e de miRNAs foram semelhantes entre pacientes e controles. TGF-β1 e FGF2 plasmáticos apresentaram correlações positivas nos pacientes com MFP, e correlações negativas nos seus controles, assim como nos controles de MFPTE. Em todos os grupos estudados foi observada correlação positiva entre TGF-β1 e VEGFA plasmáticos. Além disso, foram demonstrados diferentes perfis de correlações entre a expressão gênica de TGF-β1 e os diversos SMADs e miRNAs em cada grupo de pacientes e controles. Os pacientes com MFP com maior angiogênese (de acordo com a mediana da concentração plasmática de VEGFA e FGF2) apresentaram maiores concentrações plasmáticas de TGF-β1 do que aqueles com menor angiogênese. A angiogênese medular estimada (CD34) não foi diferente entre os três grupos de pacientes estudados. Além disso, não foram encontradas correlações entre a imunoexpressão de CD34 e as expressões de RNAm de TGFB1, VEGFA e FGF2 medulares nem em leucócitos de sangue periférico, ou a concentrações plasmáticas de TGF-β1, VEGFA e FGF2. As imunoexpressões de TGF-b1 ativo, TGF-β1 latente e c-MPL foram semelhantes entre os três grupos de pacientes. As frequências das mutações avaliadas foram similares às descritas na literatura. Os pacientes com MFPTE portadores de mutação CALR apresentaram menores concentrações plasmáticas de VEGFA e FGF2 do que os JAK2V617F positivos, enquanto os pacientes com TE portadores de mutação CALR exibiram menores concentrações plasmáticas de TGF-β1 do que os portadores de JAK2V617F. CONCLUSÕES: O presente trabalho permitiu confirmar a correlação positiva entre o TGF-β1 com outros dois marcadores de angiogênese (VEGFA e FGF2). As expressões de SMADs e de miRNAs estudados foram semelhantes entre pacientes e controles, visto não haver diferenças na expressão gênica de TGF-β1. Entretanto, disparidades encontradas nas correlações entre a expressão gênica de TGF-β1 e diferentes SMADs e miRNAs nos pacientes e controles poderiam indicar que a regulação da expressão gênica de TGF-β1 nas doenças estudadas seja distinta da apresentada nos indivíduos sem essas doenças. / AIM: To investigate the effects of the expression of SMADs mRNA and microRNAs (miRNAs) that target TGFB1 in TGF-β1 gene expression (mRNA and protein) and its role in the angiogenesis pathophysiology in myelofibrosis (MF) and essential thrombocythemia (ET) patients. METHODS: Twenty-one primary MF (PMF), twenty-one MF post-ET (MPET) and twenty-four ET patients were included, besides 98 controls matched for gender and age with patients. In peripheral blood were assessed: TGF-β1, VEGFA and FGF2 plasmatic levels and mRNA quantification; SMADs 1 to 7 mRNA quantification and miRNAs 193a-5p, 369-5p, 542-5p, 590-3p, and 590-5p quantification; and detection of JAK2V617F (and allele burden), MPLW515K/L and CALR mutations. Estimated angiogenesis (microvessel grade - CD34), active TGF-b1, latent TGF-β and c-MPL immunoexpression were determined in 26 bone marrow biopsies. RESULTS: Plasmatic TGF-β1 levels were similar in patients and controls, while all the patients groups had higher plasmatic VEGFA than controls. Plasmatic FGF2 was higher in all the patients groups, and its mRNA expression was higher in ET patients than in controls. No differences in SMADs and miRNAs expression were found between patients and controls. There was a positive correlation between plasmatic TGF-β1 and FGF2 in PMF, and a negative correlation between these variables in their controls, as well as in MPET controls. In all studied groups, there was a positive correlation between plasmatic TGF-β1 and VEGF. In addition, different profiles of correlations were demonstrated between TGF-β1 gene expression and the several SMADs and miRNAs studied in each group of patients and controls. PMF patients with higher angiogenesis (according to the median of VEGFA and FGF2 plasma levels) had higher plasmatic TGF-β1 levels than those with lower angiogenesis. Estimated angiogenesis (CD34) in bone marrow biopsies were not different among PMF, MPET and ET patients. Moreover, there were no correlation between CD34 immunoexpression and TGFB1, VEGFA and FGF2 mRNA bone marrow or peripheral blood expression or plasmatic levels, as well as latent TGF-β1, active TGF-b1, and c-MPL immunoexpression were similar in patients studied groups. The frequencies of evaluated mutations were similar to previously reported. MPET patients harboring CALR mutations had lower plasmatic VEGFA and FGF2 than JAK2V617F mutated, while ET patients carrying CALR mutations had lower plasmatic TGF-β1 than JAK2V617F mutated. CONCLUSIONS: This study confirmed the positive correlation among TGF-β1 and two other markers of angiogenesis (VEGFA and FGF2). SMADs and miRNAs expressions were similar between patients and controls, since there were no differences in TGF-β1 gene expression between patients and controls. However, disparities found in the correlations between TGF-β1 gene expression and different SMADs and miRNAs in patients and controls may indicate that TGF-β1 gene expression regulation in studied diseases is distinct from those presented by individuals without these diseases.

Les lamines A et C sont deux composants majeurs de la lamina nucléaire, réseau de filaments intermédiaires situé sous la membrane nucléaire interne. Les mutations du gène LMNA, codant les lamines A/C, ont été associées à de nombreuses pathologies humaines, appelées laminopathies, et affectant un ou plusieurs tissus dont le muscle. Les mécanismes physiopathologiques sous-jacents ne sont encore que partiellement élucidés. Les lamines A/C jouant un rôle crucial dans l’architecture nucléaire et l’organisation de la chromatine, l’hypothèse d’une altération de l’expression de facteurs de transcription ou de gènes tissus-spécifiques a été formulée. De plus, au niveau musculaire, il a été décrit que les lamines A/C jouent un rôle majeur dans la mise en place d’une différenciation musculaire efficace.Afin d’identifier des altérations potentielles au sein des voies de signalisation régulant la différenciation musculaire, nous avons utilisés un modèle de myoblastes murins conditionnellement immortalisés et comparés le profil d’expression entre les myoblastes sauvages et inactivés pour le gène Lmna (Lmna-/-). Nous avons donc identifiés deux altérations majeures de la voie BMP (Bone Morphogenetic Pathway) : la diminution de l’expression du ligand Bmp4 et l’augmentation de celle de Smad6, un inhibiteur intracellulaire de la voie. Cette surexpression de Smad6 est responsable d’une séquestration cytoplasmique des Smads 1, 5 et 8 phosphorylées et d’une diminution de l’expression des gènes cibles, Id1 et Id2. Les myoblastes Lmna-/- montrent une différenciation myogénique prématurée, phénotype réversible par des expériences d’ARN interférent ciblant Smad6. Enfin, nous avons montré que ces défauts sont retrouvés dans des myoblastes humains porteurs hétérozygotes de la mutation LMNA R310X.Ces résultats apportent un nouveau mécanisme physiopathologique des laminopathies musculaires et identifient une nouvelle cible thérapeutique potentielle / LMNA gene encodes lamins A and C, two major components of the nuclear lamina, a network of intermediate filaments underlying the inner nuclear membrane. LMNA mutations have been associated with a wide spectrum of human diseases collectively called “laminopathies” affecting one or several tissues, such as muscles. The physiopathological mechanisms underlying laminopathies remain unclear. Given the crucial role of lamins A/C in nuclear architecture and chromatin organization, the “gene regulation” hypothesis have been proposed. It suggests that LMNA mutations could alter in a tissue-specific manner transcription factors and/or genes expression. Moreover, lamins A/C have been described as important regulators in muscle differentiation regulation.To identify potential alterations in signaling pathways regulating muscle differentiation in LMNA-mutated myoblasts, we used a previously described model of conditionally immortalized murine myoblasts and compared gene expression profiles in wild-type and Lmna-/- H-2K myoblasts. We identified two major alterations of the Bone Morphogenetic Protein (BMP) pathway in Lmna-/- myoblasts: Bmp4 downregulation and Smad6 overexpression. We demonstrated that Smad6 overexpression lead to Smad1/5/8 sequestration in the cytoplasm and to the downregulation of their target genes, Id1 and Id2. As a consequence, Lmna-null myoblasts displayed a premature differentiation which could be rescued by downregulating Smad6 expression. Finally, we showed that these defects are relevant for human laminopathies as they are also present in myoblasts from a human patient carrying a LMNA+/Q310X mutation.Taken together, these results provide a potential mechanism for the muscle stem cell exhaustion and muscle atrophy observed in muscle laminopathies and identify a new therapeutical target likely to reverse pathological phenotypes

<p>During the course of breast cancer progression, normally dormant tumor-promoting effects of transforming growth factor-beta (TGF-beta) including migration, invasion, and metastasis are unmasked. Although this switch or gain of TGF-beta function has been modeled extensively in in-vivo and in-vitro breast cancer systems, the signaling mechanisms that control this TGF-beta switch are poorly understood. Indeed, the precise role of canonical TGF-beta signaling through the type I TGF-beta receptor, ALK5, and its intracellular effectors, Smad2 and Smad3, is still poorly understood. In an effort to identify mechanisms that regulate the ability of TGF-beta to stimulate mammary epithelial cell migration in-vitro, we found that TGF-beta stimulates the phosphorylation of Smad1 and Smad5, intracellular effectors that are typically associated with bone morphogenetic protein (BMP) signaling. As this phosphorylation response has not been reported extensively, little is known about the prevalance, mechanism, function, or pathological relevance of TGF-beta-stimulated Smad1/5 phosphorylation.</p><p>Herein, we use pharmacologic inhibition, RNA interference, and additional biochemical and cell-based approaches to identify a novel mechanism and function for non-canonical TGF-beta signaling through an ALK5-Smad1/5 axis. We show that TGF-beta stimulates Smad1/5 phosphorylation in an ALK5 dependent manner in cells of epithelial, endothelial, and embryonic origin. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of ALK5. Functionally, this phosphorylation event is essential to the initiation and promotion of TGF-beta-stimulated migration in mammary epithelial cells. Interestingly, this phosphorylation event may promote migration by regulating TGF-beta target gene expression, as evidenced by the identification of putative Smad1/5-dependent TGF-beta target genes using microarray analysis. Finally, of particular relevance to mammary tumor progression, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or HER2 oncogene activation.</p><p>Taken together, our data provides evidence that TGF-beta-stimulated Smad1/5 phosphorylation, which occurs through a non-canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro-migratory TGF-beta switch in mammary epithelial cells.</p> / Dissertation

Το μονοπάτι του Transforming- Growth Factor beta είναι ένα από τα πιο πολύπλοκα και πιο καλά μελετημένα σε μια σειρά παθήσεων και έχει βρεθεί να δρα άλλοτε ως ογκοκατασταλτικό και άλλοτε ως προαγωγό της κακοήθους εξαλλαγής. Πρόσφατα έγινε η ανακάλυψη των υποστρωμάτων του, της οικογένειας των Smad πρωτεϊνών, που μεταφέρουν το σήμα στον πυρήνα του κυττάρου, με τη συμμετοχή πολλαπλών παραγόντων, συν –ενεργοποιητών ή συν – καταστολέων. Η Ski πρωτεΐνη έχει ταυτοποιηθεί τελευταία ως ένας σημαντικός συν-καταστολέας του εν λόγω μονοπατιού. Επιπλέον, ο καρκίνος του μαστού είναι ένας από τους πιο συχνούς καρκίνους στις γυναίκες και η ανακάλυψη καινούριων μορίων στόχων μοριακής θεραπείας αποτελεί μια μεγάλη πρόκληση, ιδίως σε ασθενείς με νόσο αρχικού σταδίου. Σκοπός της παρούσας διδακτορικής διατριβής ήταν η μελέτη της έκφρασης του υποδοχέα του TGF-β, των πρωτεϊνών Smad2/3, Smad4 και Ski σε καρκινώματα μαστού σταδίου Τ1 και Τ2 με απουσία λεμφαδενικών μεταστάσεων, και η συσχέτιση της έκφρασης τους με ποικίλες κλινικοεργαστηριακές παραμέτρους, κυριότερες των οποίων ήταν ο βαθμός κακοηθείας των όγκων, η έκφραση ορμονικών υποδοχέων, η εμφάνιση απομακρυσμένων μεταστάσεων καθώς και ο θάνατος των ασθενών. Υλικά και μέθοδος: Σε 146 δείγματα από πορογενή καρκινώματα μαστού (εκ των οποίων 21 in situ και 125 διηθητικά) μελετήθηκε με τη μέθοδο της ανοσοϊστοχημείας (έμμεση μέθοδος βιοτίνης- στρεπταβιδίνης) η έκφραση των προαναφερθέντων μορίων. Η εκτίμηση της ανοσοθετικότητας ήταν τόσο ποιοτική (θετικη – αρνητική), όσο και ποσοτική (αρνητική, μέτρια, έντονη) ανάλογα με το ποσοστό των θετικών νεοπλασματικών κυττάρων και την ένταση της χρώσης. Η στατιστική ανάλυση των αποτελεσμάτων έγινε με το στατιστικό πρόγραμμα SPSS 13 for windows. Αποτελέματα: Η έκφραση του υποδοχέα του TGF-β είχε στατιστικώς σημαντική, αντίστροφη συσχέτιση με το βαθμό κακοηθείας των όγκων, καθώς και με την εμφάνιση αιματογενών μεταστάσεων και θανάτου των ασθενών. Η έκφραση της Smad2/3 πρωτεΐνης αποδείχτηκε ανεξάρτητος προγνωστικός παράγοντας στα Grade I διηθητικά καρκινώματα και η Smad4 βρέθηκε να αποτελεί ισχυρό προγνωστικό παράγοντα στους ER (Estrogen Receptor) θετικούς όγκους. Η έκφραση της Smad2/3 και της Smad4 συσχετίστηκαν σημαντικά τόσο μεταξύ τους όσο και με την έκαραση του TGF-β υποδοχέα. Η έκφραση της πρωτεΐνης Ski συσχετίστηκε με το βαθμό κακοηθείας των όγκων, την παρουσία αιματογενών μεταστάσεων και αποδείχτηκε ανεξάρτητος προγνωστικός παράγοντας για την επιβίωση των ασθενών. Επίσης σημαντική ήταν η παρατήρηση της ενδοκυττάριας μετακίνησης της Ski από τον πυρήνα προς το κυτταρόπλασμα του κυττάρου, αυξανομένου του βαθμού κακοηθείας των όγκων και η στατιστικώς σημαντική συσχέτιση της παρουσίας κυτταροπλασματικής Ski ανοσοχρώσης και απώλειας έκφρασης της πρωτεΐνης Smad2/3. Συμπεράσματα: Οι Smad πρωτεΐνες αποδεικνύεται για άλλη μια φορά να αποτελούν τα ενδοκυττάρια υποστρώματα του TGF-β και φαίνεται να διαδραματίζουν ρόλο ογκοκατασταλτικών πρωτεϊνών στην εξέλιξη της νεοπλασίας του μαζικού αδένα, ενώ η Ski πρωτεϊνη δρα ως ένα ισχυρό ογκογονίδιο καταστέλλοντας τη δράση του TGF-beta μονοπατιού. Όλες οι μελετηθείσες πρωτεΐνες αποτελούν δυνητικά ενδιαφέροντες στόχους μελλοντικής μοριακής θεραπείας. / Transgorming Growth Factor beta signaling pathway is thoroughly studied in a series of diseases and this molecule has been proved to act either as an ancogene or as a tumor suppressor molecule in human carcinogenesis. Recently, the Smad proteins’ family has been identified as TGF-b’s intracellular substrates, which transfer the signal in the cell’s nucleus. A lot of molecules have also been found to act as co-repressors or co-activators in this procedure. Ski protein is one of the most well known Smad proteins’ co-repressors. On the other hand, breast cancer is one of the most common cancers in women, and the fi of new predictive factors, especially in the early stages of the disease is very alluring. The purpose of the present study was the investigation of the expression of TGF-β receptor, as well as the expression of Smad2/3 and Ski protein in T1, T2, -node negative breast cancer specimens and theirs potent correlation with several clinicopathological parameters The most important of these were tumor Grade, hormone receptors’ positivity as well as the patients’ outcome (blood – borne metastases or death of their disease). Materials and methods: 146 breast cancer specimens were used, among which 21 in situ and 125 invasive. The proteins’ expression was studied using immunohistochemistry (biotin – streptabidin indirect method). The evaluation of the immunopositivity was not only qualitative (negative versus positive) but also quantitative (negative, weakly positive and strongly positive) depending on the number of positive tumor cells and the staining’s intensity). The statistical analysis of the results was implemented using the SPSS13 for windows. Results: TGF-β receptor’s expression was inversely correlated with tumor Grade as well as with the presence of blood- borne metastases and patients’ death. The expression of Smad2/3 protein was proved to be an independent prognostic factor in Grade I invasive carcinomas while loss of Smad4 expression was strongly correlated with poor patients’ outcome in ER (Estrogen Receptor) –positive tumors. All three proteins’ positivity had statistically significant correlation with each other. Ski proteins’ expression was strongly correlated with tumors’ Grade, the presence of distant metastases and was also an independent prognostic factor for patients’ survival. An other important observation was the intracellular metatopisi of Ski’ s expression from the nucleus to the cytoplasm in high Grade tumors and the strong relationship between cytoplasmic Ski and loss of expression of Smad2/3. Conclusions: Smad proteins seem to be TGF-beta intracellular substrates and, according to our results, play a tumor suppressor role in mammary gland tumorigenesis. On the other hand, Ski protein acts as an oncogene in breast carcinogenesis procedure modulating the TGF-beta pathways’ effect. All the studied proteins are potent targets of a future molecular therapy.

OBJETIVO: Investigar o efeito da expressão de RNAm dos SMADs e de microRNAs (miRNAs) que possuem o TGFB1 como alvo na expressão gênica (RNAm e proteína) de TGF-&#946;1 e seu papel na fisiopatologia da angiogênese em pacientes com mielofibrose (MF) e trombocitemia essencial (TE). MÉTODOS: Foram incluídos 21 pacientes com MF primária (MFP), 21 com MF pós-TE (MFPTE) e 24 com TE, além de 98 indivíduos controles pareados de acordo com gênero e idade com os pacientes. As análises realizadas no sangue periférico foram: quantificação das concentrações plasmáticas e de RNAm de TGFB1, VEGFA e FGF2; quantificação de RNAm de SMADs 1 a 7 e de miRNAs 193a-5p, 369-5p, 542-5p, 590-3p, e 590- 5p; e detecção das mutações JAK2V617F (com quantificação alélica), MPLW515K/L e CALR. Em 26 biópsias de medula óssea dos pacientes, foram determinados o grau de microvasculatura (angiogênese estimada - CD34), a imunoexpressão de TGF-b1 ativo, TGF-&#946;1 latente e c-MPL. RESULTADOS: As concentrações de TGF- &#946;1 plasmático foram semelhantes entre os pacientes e controles, enquanto o VEGFA plasmático foi maior em todos os grupos de pacientes comparados aos seus controles. O FGF2 plasmático também foi maior em todos os grupos de pacientes, e a expressão de seu RNAm foi maior nos pacientes com TE do que em seus controles. As expressões de SMADs e de miRNAs foram semelhantes entre pacientes e controles. TGF-&#946;1 e FGF2 plasmáticos apresentaram correlações positivas nos pacientes com MFP, e correlações negativas nos seus controles, assim como nos controles de MFPTE. Em todos os grupos estudados foi observada correlação positiva entre TGF-&#946;1 e VEGFA plasmáticos. Além disso, foram demonstrados diferentes perfis de correlações entre a expressão gênica de TGF-&#946;1 e os diversos SMADs e miRNAs em cada grupo de pacientes e controles. Os pacientes com MFP com maior angiogênese (de acordo com a mediana da concentração plasmática de VEGFA e FGF2) apresentaram maiores concentrações plasmáticas de TGF-&#946;1 do que aqueles com menor angiogênese. A angiogênese medular estimada (CD34) não foi diferente entre os três grupos de pacientes estudados. Além disso, não foram encontradas correlações entre a imunoexpressão de CD34 e as expressões de RNAm de TGFB1, VEGFA e FGF2 medulares nem em leucócitos de sangue periférico, ou a concentrações plasmáticas de TGF-&#946;1, VEGFA e FGF2. As imunoexpressões de TGF-b1 ativo, TGF-&#946;1 latente e c-MPL foram semelhantes entre os três grupos de pacientes. As frequências das mutações avaliadas foram similares às descritas na literatura. Os pacientes com MFPTE portadores de mutação CALR apresentaram menores concentrações plasmáticas de VEGFA e FGF2 do que os JAK2V617F positivos, enquanto os pacientes com TE portadores de mutação CALR exibiram menores concentrações plasmáticas de TGF-&#946;1 do que os portadores de JAK2V617F. CONCLUSÕES: O presente trabalho permitiu confirmar a correlação positiva entre o TGF-&#946;1 com outros dois marcadores de angiogênese (VEGFA e FGF2). As expressões de SMADs e de miRNAs estudados foram semelhantes entre pacientes e controles, visto não haver diferenças na expressão gênica de TGF-&#946;1. Entretanto, disparidades encontradas nas correlações entre a expressão gênica de TGF-&#946;1 e diferentes SMADs e miRNAs nos pacientes e controles poderiam indicar que a regulação da expressão gênica de TGF-&#946;1 nas doenças estudadas seja distinta da apresentada nos indivíduos sem essas doenças. / AIM: To investigate the effects of the expression of SMADs mRNA and microRNAs (miRNAs) that target TGFB1 in TGF-&#946;1 gene expression (mRNA and protein) and its role in the angiogenesis pathophysiology in myelofibrosis (MF) and essential thrombocythemia (ET) patients. METHODS: Twenty-one primary MF (PMF), twenty-one MF post-ET (MPET) and twenty-four ET patients were included, besides 98 controls matched for gender and age with patients. In peripheral blood were assessed: TGF-&#946;1, VEGFA and FGF2 plasmatic levels and mRNA quantification; SMADs 1 to 7 mRNA quantification and miRNAs 193a-5p, 369-5p, 542-5p, 590-3p, and 590-5p quantification; and detection of JAK2V617F (and allele burden), MPLW515K/L and CALR mutations. Estimated angiogenesis (microvessel grade - CD34), active TGF-b1, latent TGF-&#946; and c-MPL immunoexpression were determined in 26 bone marrow biopsies. RESULTS: Plasmatic TGF-&#946;1 levels were similar in patients and controls, while all the patients groups had higher plasmatic VEGFA than controls. Plasmatic FGF2 was higher in all the patients groups, and its mRNA expression was higher in ET patients than in controls. No differences in SMADs and miRNAs expression were found between patients and controls. There was a positive correlation between plasmatic TGF-&#946;1 and FGF2 in PMF, and a negative correlation between these variables in their controls, as well as in MPET controls. In all studied groups, there was a positive correlation between plasmatic TGF-&#946;1 and VEGF. In addition, different profiles of correlations were demonstrated between TGF-&#946;1 gene expression and the several SMADs and miRNAs studied in each group of patients and controls. PMF patients with higher angiogenesis (according to the median of VEGFA and FGF2 plasma levels) had higher plasmatic TGF-&#946;1 levels than those with lower angiogenesis. Estimated angiogenesis (CD34) in bone marrow biopsies were not different among PMF, MPET and ET patients. Moreover, there were no correlation between CD34 immunoexpression and TGFB1, VEGFA and FGF2 mRNA bone marrow or peripheral blood expression or plasmatic levels, as well as latent TGF-&#946;1, active TGF-b1, and c-MPL immunoexpression were similar in patients studied groups. The frequencies of evaluated mutations were similar to previously reported. MPET patients harboring CALR mutations had lower plasmatic VEGFA and FGF2 than JAK2V617F mutated, while ET patients carrying CALR mutations had lower plasmatic TGF-&#946;1 than JAK2V617F mutated. CONCLUSIONS: This study confirmed the positive correlation among TGF-&#946;1 and two other markers of angiogenesis (VEGFA and FGF2). SMADs and miRNAs expressions were similar between patients and controls, since there were no differences in TGF-&#946;1 gene expression between patients and controls. However, disparities found in the correlations between TGF-&#946;1 gene expression and different SMADs and miRNAs in patients and controls may indicate that TGF-&#946;1 gene expression regulation in studied diseases is distinct from those presented by individuals without these diseases.

Bone morphogenetic proteins (BMPs) can induce osteoblast differentiation during bone formation and repair. BMP-2 is currently the most used BMP in delivery systems (DSs) for growth factors. Approved by the US Food and Drug Administration, BMP-2 and type I collagen are already used in orthopaedic clinical applications. Recently, studies demontrated that BMP-9 has a higher osteogenic potential than BMP-2. However, high purification costs of BMPs limit their use. So, alternatives such as peptides derived from BMPs are studied. We have developped a peptide derived from the knuckle epitope of human BMP-9 (pBMP-9) which inhibits the proliferation of murine preosteoblasts MC3T3-E1 and increases their differentiation when used at 400 ng/mL. This study first compares in vitro the effects of equimolar concentrations (1.92 nM) of BMP-2, BMP-9 or pBMP-9 on the differentiation of MC3T3-E1 in serum-free medium. Like BMP-2, BMP-9 and pBMP-9 both activate Smads signaling pathway within 1h. In contrary to BMP-2, the Smad phosphorylation induced by BMP-9 and pBMP-9 is not prevented by noggin, an extracellular antagonist of BMP-2. Moreover, BMP-9 and pBMP-9 increase dose dependently alkaline phosphatase activity, an early marker of osteoblast differentiation within 1 day. Quantitative real-time polymerase chain reaction (qPCR) analysis demonstrates that BMP-2, BMP-9 and pBMP-9 all activate the transcription of Runx2, Osterix, type I collagen a1 chain and Osteocalcin within 6 days. Osteocalcin is the only truly osteoblast-specific gene that encodes for a protein required for Ca[superscript 2+] deposition in the extracellular matrix and subsequent mineralization. The peptide pBMP-9 allows a slight deposition of calcium ions (Ca[superscript 2+]) in the extracellular matrix of cells within 18 days. To favor efficiency of these molecules, DSs for BMP-9 or pBMP-9 using type I collagen gel or chitosan matrix have been studied. Collagen and chitosan DSs release in vitro within 1h about 35% and 80% of the initial dose of BMP-9 respectively. A slower release of pBMP-9 is observed in both DSs. The release of BMP-9 and pBMP-9 from both DSs follows Korsmeyer-Peppas kinetics. Collagen DS containing 3.84 nM BMP-2, BMP-9 or pBMP-9 activates in vitro the expression of osteogenic genes in MC3T3-E1 cells within 6 days. 6.35 [micro]g BMP-9 or 100 [micro]g pBMP-9 incorporated into chitosan DS can induce in vivo bone formation in C57BL/6 mouse quadriceps muscles within 24 days. Furthermore, collagen DS is less efficient than chitosan DS. Since BMPs can also influence adipogenic cell lineages, the effects of 3.84 nM BMP-2, BMP-9 or pBMP-9 have been studied on human white preadipocytes (HWP). pBMP-9 dose dependently reduces the proliferation of HWP without affecting the number of apoptotic cells. Incubation for 1h with BMPs or pBMP-9 activates the Smad pathway. These molecules also enhance the levels of the mRNA of the adipogenic markers aP2 and adipoQ and increase the number of lipid vesicles within 7 days in adipogenic differentiation (AD) serum-free medium containing ciglitazone. Thus, pBMP-9 seems a promising replacement for costly BMP in tissue engineering applications since this molecule can favor osteogenic differentiation of preosteoblasts and adipogenic differentiation of preadipocytes in serum-free medium.

The v-rel oncogene is the most efficiently transforming member of the Rel/NF-κB family of transcription factors. Identification of genes or signal transduction pathways that contribute to v-Rel transformation provide insight into the mechanisms of tumorigenesis by Rel/NF-κB proteins. In these studies, the contribution of TGF-β/Smad signaling to v-Rel transformation was assessed. TGF-β/Smad signaling regulates several cellular processes, including growth, differentiation, and apoptosis and has been implicated in a number of different cancers. Using microarray technology and Northern blot analysis, key components of the TGF-β/Smad pathway (tgf-β2 and tgf-β3 ligands, TGF-β type II receptor, and receptor-activated smad3) were identified with upregulated mRNA expression in v-Rel-transformed fibroblasts and lymphoid cells relative to control cells. A corresponding change in their protein levels was also observed. Further analysis revealed elevated levels of the phosphorylated, active form of Smad3, which correlated with its increased DNA-binding activity in v-Rel transformed cells. In contrast, the overexpression of c-Rel resulted in little to no alteration in the RNA and protein expression of members of the TGF-β/Smad pathway. Further studies demonstrated that elevated TGF-β/Smad signaling is required for the transforming ability of v-Rel. Blocking TGF-β signaling with a kinase inhibitor of TGF-β type I receptor inhibited the activation of Smad3 and dramatically reduced the ability of v-Rel transformed cells to form colonies in soft agar. Overexpression of a constitutively active form of Smad3 in the inhibitor-treated cells restored their ability to form colonies in soft agar close to the levels seen in untreated cells. Additional experiments with dominant negative Smad3 also revealed its ability to hinder the oncogenic potential of v-Rel. In complementary experiments, a stimulatory effect on v-Rel transformation was observed with cells treated with recombinant TGF-β2 ligand or overexpressed with wild-type Smad3. Taken together, these studies demonstrate that TGF-β signaling is crucial for the transformation potential of v-Rel and is primarily mediated by Smad3 activity. / text

Master of Science / Department of Chemistry / Kenneth J. Klabunde / The motivation for the preparation of gold nanoparticles includes their potential utility in sensors, nanoelectronics, and the vast basic knowledge we can gain from these novel materials. Colloids of gold nanoparticles are also one of the most stable and easiest to manipulate. Synthesizing gold nanoparticles with narrow size distribution, uniform shape, and good crystalline nature represents a significant challenge. Thiols were found to be very efficient capping ligands for the digestive-ripening process in our research group, during which a colloidal suspension in a solvent is refluxed at the solvent boiling temperature in the presence of a capping ligand to convert a highly polydispersed colloid into a nearly monodispersed one. The current thesis research focuses on using amines instead of thiols as the capping ligands, which were also found to have similar efficiency for this purpose. The major part of the work is devoted to understanding the digestive ripening of gold-amine colloids system, and the effect of the nature of the amine ligands. A noteworthy achievement of the current work is the ability to synthesize stable gold colloids with different sizes by using different amine ligands. A diverse set of instrumental techniques is used for the characterization of the gold nanoparticles.

<p>Transforming growth factor–β (TGF–β) signaling regulates a range of processes in a variety of cell types. Consequently, TGF–β plays a complex role in the progression of several types of cancers; it acts as a tumor suppressor in normal cells and early in tumor progression, yet it can promote tumor progression in later stages of cancer.</p><p>Among the cancers that TGF–β has been implicated in is glioblastoma multiforme (GBM), the most common primary brain neoplasm and one of the most lethal types of cancer. Because of its high mortality rate and the lack of effective treatments, discovering the molecular mechanisms that underlie GBM formation and growth is of great clinical interest. To this end, we investigated the function of a TGF–β target gene — the putative tumor suppressor N‐Myc downstream‐regulated gene 4 (NDRG4) — in GBM cell viability, proliferation and tumor formation. Contrary to the established roles of other NDRG family members, we found that NDRG4 expression is elevated in GBM and that NDRG4 is required for the survival of established GBM cell lines and primary GBM xenograft cells enriched for highly tumorigenic GBM cancer stem cells. Knockdown of NDRG4 expression results in G₁ cell cycle arrest followed by apoptosis that is associated with a decrease in the expression of XIAP and survivin. Finally, knockdown of NDRG4 expression in established GBM cell lines and GBM cancer stem cells results in decreased tumorigenicity following intracranial implantation of these cells into immunocompromised mice. Collectively, these data indicate that NDRG4 does not function as a tumor suppressor like other NDRG family members, but rather it is essential for GBM tumorigenicity and may represent a potential therapeutic target for this devastating disease.</p><p>In the second portion of this dissertation, we examine the TGF–β cytostatic signaling pathway in B lymphocytes. TGF–β–induced growth inhibition is the most extensively studied biological response to a TGF–β signal. Although in most cell types this response is mediated by Smad3– dependent regulation of c–Myc, p15<super>Ink4B</super>, and p21<super>Cip1</super> transcription, studies from Smad3 null mice suggest that TGF–β–induced growth inhibition in B lymphocytes occurs regardless of Smad3 status. We prove that this response does indeed occur independently of Smad3 in purified primary B lymphocytes and WEHI–231 cells. Consistent with this, p15<super>Ink4B</super> and p21<super>Cip1</super> are not noticeably induced by TGF–β in these cells, whereas Id3 and cyclin G2 are induced in a Smad3–independent manner. Finally, unlike the MAPK pathways we tested, the BMP–specific Smads 1 and 5 are activated in response to TGF–β in these cells, and this activation is dependent on ALK5 kinase activity. Collectively, these data indicate that TGF–β induces growth inhibition in B lymphocytes through a novel signaling pathway, and Smads 1 and 5 may help mediate this response.</p> / Dissertation