LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there is no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In this study, we found increased LIV-1 expression in a progresssive manner in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives prostate cancer EMT in an androgen-refractory human prostate cancer cell (ARCaP) bone metastasis model. LIV-1, when overexpressed in ARCaPE cells (derivative cells of ARCaP with epithelial phenotype), promoted EMT irreversibly. LIV-1 overexpressed ARCaPE cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaPE cells, eliciting constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. Further investigation of the HB-EGF promoter revealed that both Stat3 and AP-1 controlled HB-EGF promoter activity. Ectopic LIV-1 overexpression induced AP-1 and Stat3 activation. Blockade of both Stat3 and AP-1 by specific inhibitors or dominant negative expression vectors diminished the HB-EGF promoter activity induced by LIV-1 overexpression. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promotes EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases.

Pathological changes to tumor extracellular matrix (ECM) composition, mechanics, and architecture promote cancer progression and metastasis. Exploration of tumor-ECM interactions using in vitro matrix-mimetic culture systems has largely been restricted to naturally-derived matrix materials that permit limited experimental control. Such study of a novel lung adenocarcinoma model in Matrigel™ (MG) has suggested key matrix cues that mediate epithelial-mesenchymal transition (EMT) and metastasis. In this thesis work, synthetic hydrogel scaffolds based on poly(ethylene glycol) (PEG) featuring high experimental control and modular bioactivity were used to study matrix influences on the EMT-prone model line 344SQ. Encapsulation of 344SQ cells in PEG hydrogels modified for cell adhesivity and cell-mediated enzymatic degradability induced formation of lumenized, polarized spheres mimicking the epithelial phenotype observed in three-dimensional MG. Tuning matrix stiffness, adhesive ligand concentration, and ligand spatial presentation altered epithelial morphogenesis. Exploration of the EMT phenotype of PEG-encapsulated 344SQ cells revealed TGFβ-initiated changes in morphology, polarity, expression levels of EMT marker genes and their epigenetic controller, and the organization of cell-secreted ECM. Notably, a potent role for adhesive ligand was illuminated as matrices with low PEG-RGDS concentration even in the absence of TGFβ induced formation of spheres with a post-EMT phenotype by several of these measures. A matrix-invasive phenotype was also revealed by altering matrix structural parameters and tuned with incorporation of an alternative protease-cleavable sequence. Finally, the influence of cell-cell contacts was explored by covalent incorporation of cadherin proteins into the matrix. Matrix-tethered E- and -N-cadherin affected 344SQ sphere development in otherwise non-cell-adhesive matrices and modulated polarity and the degree of TGFβ response. Further, in 344SQ with a knockdown of the essential polarity-determining protein Scribble, matrix-tethered cadherin influenced the formation of a phenotype with partially normalized epithelial polarity with corresponding differences in membrane localization of cell-expressed E-cadherin. Overall, this thesis demonstrates the utility of the more experimentally controllable PEG system in studying ECM influences on cancer progression with findings providing greater insight into stromal biomechanical, biochemical, and cell-cell factors that mediate lung adenocarcinoma epithelial morphogenesis and EMT. These contributions help advance the state of the field towards a goal of developing new metastasis-targeting cancer therapeutics.

Single minded 2 (Sim2) is a member of the basic helix-loop-helix Per-ARNT-Sim (Period-Arylhydrocarbon Nuclear Translocator-Single minded) family. Human SIM2 is involved in the etiology of the Down’s phenotype. In addition to the physical and mental deficiencies associated with DS, it has become apparent that women with DS are 10-25 times less likely to develop breast cancer in comparison to age-matched normal populations. Such significant effects on breast cancer susceptibility are thought to result from gene dosage effects of one or more tumor suppressor genes on chromosome 21. Here we report the identification and transcriptional characterization of mouse Sim2s, a splice variant of Sim2, which is missing the carboxyl Pro/Ala-rich repressive domain. Similar to full-length Sim2, Sim2s interacts with ARNT and to a lesser extent, ARNT2. The effects of Sim2s on transcriptional regulation through hypoxia-, dioxin- and central midline response elements are different than that of full length Sim2. Specifically, Sim2s exerts a less repressive effect on hypoxia-induced gene expression than full length Sim2, but is just as effective as Sim2 at repressing TCDD-induced gene expression from a dioxin response element. Interestingly, Sim2s binds to and activates expression from a central midline response element-controlled reporter through an ARNT transactivation domain-dependent mechanism. Forced expression of SIM2s in MDA-MB-435 breast cancer cells significantly inhibited proliferation, reduced anchorage-independent growth, and decreased invasive potential. SIM2s directly decreased expression of matrix metalloprotease-3, a known mediator of breast cancer metastasis. In addition, loss of Sim2 in the mouse mammary gland increased ductal branching, accelerated lobuloalveolar-like precocious hyperplasia, and decreased cell apoptosis, suggesting that SIM2s is a mammary tumor suppressor. Sim2-/- mammary glands lose E-cadherin expression, suggesting that Sim2s plays a role in regulating E-cadherin/beta-catenin signaling. Loss of Sim2 in the mammary glands also resulted in dramatically increased MMP3 expression. The mechanism of SIM2smediated repression of MMP3 was found to be due to its ability to inhibit AP-1 binding to the MMP3 promoter. These results suggest that SIM2s contributes to the breast cancer protective effects observed in DS individuals.

Breast cancer is a heterogeneous disease with vastly different tumor progression kinetics and survival outcomes depending upon the differentiation state and gene expression patterns of the tumor. Effective treatments exist for patients with endocrine therapy sensitive or HER2 overexpressing tumors, but targeted treatments are not available for other tumor types. The mechanisms governing mammary tumor phenotype generation could prove critical to finding treatments. The c-Jun N-terminal kinase (JNK) pathway has recently been implicated in the inhibition of breast tumor luminal differentiation (1, 2) and JNK2, in particular, is important in mammary tumorigenesis and tumor progression (3-8). Therefore, the involvement of JNK2 in inhibition of mammary luminal cell differentiation was investigated in normal glands and tumors. Studies found that JNK2 inhibits luminal cell populations in normal mammary ducts. Additionally, JNK2 suppresses Notch activity in stem cell niche of the developing mammary gland. In vitro assays show that control over differentiation by JNK2 is due to suppression of p53-dependent Notch1 expression. Inhibition of luminal cell populations by JNK2 is also apparent in tumor cell models regardless of p53 expression. In the p53-competent Polyoma Middle T-antigen model, Notch1 expression is suppressed by JNK2. In the absence of p53, JNK2 suppresses luminal populations independent of Notch1. In this model, decreased luminal marker expression is accompanied by increased epithelial to mesenchymal transition. It was also found that JNK2-dependent epithelial to mesenchymal transition inhibits luminal populations and is driven by JNK2-dependent suppression of Brca1. JNK2 also confers resistance to estrogen signaling inhibition, and increases the metastatic ability of tumor cells in vivo. These data establish the importance of JNK2 in mammary epithelial cell differentiation in normal glands and tumors. They also suggest that JNK2 may be an effective prognostic marker or treatment target. / text

Epithelial-mesenchymal transition (EMT) is a fundamental developmental program, which is believed to be reactivated during the progression of in situ carcinoma to aggressive metastatic cancers. Ras-ERK pathway has been shown to play a crucial role in EMT. We have previously shown that ERK2, but not ERK1, is necessary for RasV12-induced EMT and overexpression of ERK2 is sufficient to promote EMT. ERK2 promotes EMT by regulating several factors, including the upregulation of transcription factors ZEB1/2. ZEB1/2 repress expression of E-cadherin, which is necessary for polar epithelial tissue formations.

Low to moderate level of chronic arsenic exposure contributes to cardiovascular ailments including heart disease and aneurysms. Current research on the etiology and progression of cardiovascular disease focuses mainly on adult which fails to capture the developmental origins of cardiovascular disease. Thus, disruption in morphogenetic events during early development may initiate and pattern the molecular programming of cardiovascular ailments in adulthood. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. Coronary vessel development is initiated by precursor cells that are derived from the epicardium. Epicardial derived cells undergo proliferate, migrate, and differentiate into several cardiac cell types which are the cellular components of the coronary vessels. The key cellular event occurs in this process is the epithelial to mesenchymal transition (EMT), which can also be utilized by endocardial cushion cells to form aortic and pulmonary valves. The TGFβ family of ligands and receptors are essential for developmental cardiac EMT and coronary smooth muscle cell differentiation. Whether arsenic has any impact on TGFβ mediated cardiovasculogenesis is not known. Monomethylarsonous acid [MMA(III)] is the most potent metabolite of inorganic arsenic and has been shown to partly account for arsenic induced toxicity. The fetus is exposed to relatively higher levels of MMA (III) as compared to adults probably due to deficiency in methylation of transferred inorganic arsenic from the placenta. However, the developmental toxicity of MMA (III) has not yet been studied. In this study, we exploit a novel cardiac progenitor cell line to recapitulate epicardial EMT in vitro and to study developmental toxicity caused by arsenicals. We show that chronic exposure to low level of arsenite and MMA (III) disrupts developmental EMT programming in epicardial cells causing deficits in cardiac mesenchyme production. The expression of EMT program genes is also decreased in a dose-dependent manner following exposure to arsenite and MMA (III). Smad-dependent TGFβ2 canonical signaling and the non-canonical Erk signaling pathways are abrogated as detected by decreases in phosphorylated Smad2/3, Erk1/2 and Erk5 proteins. There is also loss of nuclear accumulation of p-Smad and p-Erk5 due to arsenical exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenicals do not block TGFβ2 stimulated p38 activation. Additionally, smooth muscle cell differentiation, which is proven to be governed by p38 signaling in epicardial cells, also remains intact with arsenical exposure. Overall these results show that arsenite and MMA (III) are strong and selective cardiac silencers. The molecular mechanisms of arsenical toxicity on TGFβ-Smad signaling in epicardial cells is further explored. A relatively high level of acute arsenical exposure rapidly depletes phosphorylated nuclear Smad2/3. Restoration of the nuclear accumulation of Smads can be achieved by inhibiting the expression or activation of Smad specific exportins suggesting that arsenicals augment Smad nuclear exportation. Abrogated Smad signaling caused by arsenicals is associated with severe deficits in EMT during mouse epicardium and chick endocardial cushion development. Thus progenitor cell outgrowth, migration, invasion and vimentin filament reorganization are significantly inhibited in response to arsenical exposure. Disrupted Smad nuclear shuffling is probably caused by zinc displacement on the MH-1 DNA binding domain of Smad2/3. Thus zinc supplementation restores both nuclear content and transcriptional activities of Smad2/3. Rescued TGFβ-Smad signaling by zinc also contributes to cellular transformation and mesenchyme production in embryonic heart explants. LINE1 (L1) retrotransposons are a group of mobile DNA elements that shape the genome via novel epigenetic controls. Although expression of L1 is required for early embryo implantation and development, abnormally elevated L1 is shown to inhibit embryonic cells from transforming and differentiating during organogenesis. Cellular redox signaling, which is regulated by antioxidant responsive elements (AREs), has been shown to play a key role in L1 activation and retrotransposition. However, whether L1 can be induced by the cellular oxidative stress caused by arsenic is not known. We provide evidence showing that L1 ORF-1 and ORF-2 mRNA levels are both up-regulated by arsenic. Nuclear accumulation of L1 ORF-2 is observed in response to 30 min arsenic exposure, which may lead to active retrotransposition events in the genome. Transcriptional activity of L1 is regulated by Nrf2 as mutations in ARE regions within the L1 promoter and Nrf2 silencing using siRNA both significantly inhibit L1 transcriptional activity. Nrf2 overexpression together with arsenic exposure creates synergic induction in L1 promoter activity suggesting that arsenic mediated L1 activation is partially Nrf2 dependent. Taken together, these findings reveal a molecular mechanism responsible for arsenic cardiac toxicity and define a novel genetic toxic effect of arsenic during embryonic heart development.

Το βασικοκυτταρικό καρκίνωμα του δέματος αποτελεί τον συχνότερο τύπο καρκίνου του δέρματος. Η επιθήλιο-μεσεγχυματική μετάβαση (ΕΜΤ) είναι η διαδικασία κατά την οποία τα επιθηλιακά κύτταρα χάνουν τις μεσοκυττάριες εξειδικευμένες συνάψεις αναδιοργανώνουν τον κυτταροσκελετό τους και αποκτούν μεσεγχυματικό φαινότυπο και αυξημένη ικανότητα μετανάστευσης. Το φαινόμενο της ΕΜΤ παίζει σημαντικό ρόλο τόσο στην εμβρυική ανάπτυξη όσο και στην διήθηση και μετάσταση του καρκίνου. Κύρια χαρακτηριστικά της διεργασίας της επιθήλιο-μεσεγχυματικής μετάβασης είναι απώλεια των ζωνών πρόσφυσης λόγω μείωσης της έκφρασης της Ε-κατχερίνης, καθώς και η αναδιοργάνωση του κυτταροσκελετού η οποία συμβάλλει στην αυξημένη ικανότητα μετανάστευσης και μεταξύ των άλλων χαρακτηρίζεται από την υπερέκφραση μεσεγχυματικών συστατικών του κυτταροσκελετού όπως η βιμεντίνη και η ακτίνη των λείων μυϊκών ινών. Η ενεργοποίηση επίσης του μονοπατιού Wnt/β-catenin και η υπερέκφραση της ILK είναι γνωστό ότι παίζουν σημαντικό ρόλο στην επιθήλιο-μεσεγχυματική μετάβαση. Στην παρούσα μελέτη εξετάσαμε ανοσοϊστοχημικά την έκφραση των μεσεγχυματικών δεικτών βιμεντίνης και a-SMA, την έκφραση του επιθηλιακού δείκτη Ε-κατχερίνη καθώς και την έκφραση της β-κατενίνης και της ILK σε 100 βασικοκυτταρικά καρκινώματα Επιπλέον, εξετάσαμε την πιθανή συσχέτιση μεταξύ των μορίων αυτών αλλά και την σχέση της έκφρασης τους με κλινικο-παθολογοανατομικές παραμέτρους της νόσου όπως ο ιστολογικός τύπος και το βάθος διήθησης. Οι μεσεγχυματικοί δείκτες βιμεντίνη και α- SMA εκφράστηκαν σε 99% και 97% των περιπτώσεων αντίστοιχα και η έκφραση τους ήταν σημαντικά μεγαλύτερη στα διηθητικού τύπου βασικοκυτταρικά καρκινώματα και επιπλέον συσχετίστηκε στατιστικώς σημαντικά με το βάθος διήθησης των όγκων. Απώλεια της Ε-κατχερίνης από τις μεμβράνες παρατηρήθηκε σε 71% των όγκων ενώ σε 92% και 90% των περιπτώσεων αντίστοιχα παρατηρήθηκε κυτταροπλασματική και πυρηνική εντόπιση της Ε-κατχερίνης. Το προφίλ αυτό έκφρασης της Ε-κατχερίνης συσχετίστηκε τόσο με το διηθητικό ιστολογικό τύπο όσο και με το βάθος διήθησης των όγκων. Πυρηνική εντόπιση της β-κατενίνης ενδεικτική ενεργοποίησης του μονοπατιού αναδείχθηκε επίσης σε 99% των περιπτώσεων και συσχετίστηκε με το αυξανόμενο βάθος διήθησης των όγκων. Σε αντίθεση με το φυσιολογική επιδερμίδα όπου η έκφραση της ήταν αρνητική θετική έκφραση ILK διαπιστώθηκε σε 100% των βασικοκυτταρικών καρκινωμάτων και συσχετίστηκε τόσο με το διηθητικό ιστολογικό τύπο όσο και με το βάθος διήθησης των όγκων. Επιπλέον η έκφραση της ILK συσχετίστηκε με την έκφραση των βιμεντίνης, α-SMA πυρηνικής β-κατενίνης και πυρηνικής Ε-κατχερίνης Συμπερασματικά τα ευρήματα αυτά υποστηρίζουν ότι η διεργασία της επιθηλιο-μεσεγχυματικής μετάβασης παρατηρείται στο βασικοκυτταρικό καρκίνωμα και φαίνεται να παίζει σημαντικό ρόλο στην διηθητική του ικανότητα και επιθετική βιολογική του συμπεριφορά. Επιπλέον η υπερέκφραση της ILK φαίνεται να παίζει σημαντικό ρόλο στην επιθετική συμπεριφορά των βασικοκυτταρικών καρκινωμάτων και στο φαινόμενο της επιθηλιομεσεγχυματικης μετάβασης. Τέλος η πυρηνική εντόπιση της Ε-κατχερίνης στα βασικοκυτταρικά αποτέλεσε ένα πρωτότυπο εύρημα με πιθανή προγνωστική σημασία. / The process of EMT has been known to play a significant role during embryonic development and tumour progression. Integrin-linked kinases (ILK) has been recently added to the growing list of EMT regulators that control some aspect of oncogenesis. However the expression of ILK and its relevance to EMT markers has not been previously studied in human basal cell carcinoma (BCC). In this study, we examined the expression pattern of EMT related markers in a series of cutaneus BCC. Formalin-fixed paraffin embedded tissue sections from 100 cases of BCC were processed by immunohistochemistry for the expression of vimentin a-SMA, E-cadherin, b-catenin and ILK. Expression of mesenchymal markers vimentin and a-SMA in tumour cells was observed in 99% and 97% of cases. In several cases vimentin and a-sma expressing tumour cells excibited a spindle shaped mesenchymal like phenotype. Downregulation of membranous E-cadherin was found in 71% while nuclear b-catenin and ilk overexpression was observed in 99% and 100% of cases respectively. Interestingly nuclear E-cadherin was also observed in 90% cases and it was significantly higher in infiltrative/sclerosing BCC variant. ILK expression correlated significantly with vimentin, a-sma nuclear b-catenin, loss of membranous E-cadherin as well as with nuclear E-cadherin. ILK overexpression was also significantly higher in infiltrative/morphaic variant of BCC and correlated with increased depth of invasion. EMT seems to underlie invasive and locally aggressive features of BCC. ILK overexpression correlates with EMT markers and highly invasive aggressive features of human BCC. Nuclear localization of E-cadherin probably suggests novel nuclear fuctions of E-cadherin in BCC.

Epithelial mesenchymal/-myofibroblast transition (EMT/EMyT) has emerged as one of the central mechanisms in wound healing and tissue fibrosis. The main feature of EMyT is the activation of a myogenic program, leading to the induction of the α-smooth-muscle actin (SMA) gene in the transitioning epithelium. Recent research suggests that intercellular contacts are not merely passive targets, but are active contributors to EMT/EMyT. Indeed, our group showed previously that contact uncoupling or injury is necessary for TGFβ to induce EMyT (two-hit paradigm). Further, our previous work also revealed that Smad3, the main TGFβ-regulated transcription factor, binds to the Myocardin Related Transcription Factor (MRTF), the prime driver of SMA promoter, and inhibits MRTF’s transcriptional activity. During EMyT, Smad3 eventually degrades, which liberates the MRTF-driven myogenic program. However the mechanisms whereby cell contacts regulate the fate of Smad3 and MRTF during EMyT are poorly understood. Accordingly, the central aim of my studies was to explore the role of intercellular contacts, in particular that of Adherens Junction (AJs) in the induction of the myogenic reprogramming of the injured epithelium. This thesis describes two novel molecular mechanisms through which AJs impact EMyT. In the first part, we show β-catenin, an AJs component and transcriptional co-activator counteracts the inhibitory action of Smad3 on MRTF. Moreover we reveal that β-catenin is necessary to maintain MRTF stability via protecting MRTF from proteasomal degradation. Thus, β-catenin is an indispensable permissive factor for SMA expression. In the second part, we demonstrate that contact injury and TGFβ suppress the expression of the phosphatase PTEN. EMyT-related reduction or absence of PTEN potentiates Smad3 degradation. EMyT is associated with enhanced phosphorylation of the T179 residue in Smad3 linker region, and this event is necessary for Smad3 degradation. PTEN silencing increases the stimulatory effect of contact uncoupling and TGFβ on SMA promoter activity and SMA protein expression. Thus, the integrity of intercellular contacts regulates the level of PTEN, which in turn controls Smad3 stability through impacting on T179 phosphorylation. This new knowledge holds promises for targeted therapies and more effective prevention of the currently incurable fibroproliferative and fibrocontractile diseases.

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2007. / Submitted by Camila Duarte (camiladias@bce.unb.br) on 2017-01-26T15:45:45Z No. of bitstreams: 1 2007_DoraluciaPedrosaDeAraujo.pdf: 2379583 bytes, checksum: 2d6e1962c469416850b707a044238fef (MD5) / Approved for entry into archive by Patrícia Nunes da Silva(patricia@bce.unb.br) on 2017-01-30T13:51:13Z (GMT) No. of bitstreams: 1 2007_DoraluciaPedrosaDeAraujo.pdf: 2379583 bytes, checksum: 2d6e1962c469416850b707a044238fef (MD5) / Made available in DSpace on 2017-01-30T13:51:13Z (GMT). No. of bitstreams: 1 2007_DoraluciaPedrosaDeAraujo.pdf: 2379583 bytes, checksum: 2d6e1962c469416850b707a044238fef (MD5) / No presente estudo, realizamos a rEMT em 5 pacientes com epilepsia farmacologicamente intratável, registrando o número de crises por dia durante 3 meses pré-tratamento, 3 meses de tratamento com rEMT duas vezes por semana, e 3 meses pós-tratamento. Observamos uma redução média no número de crises de 22,0% (variação de 9,53% a 43,09%), a qual foi estatisticamente significativa, embora 2 dos 5 pacientes não tenham apresentado qualquer redução do número de crises. Este estudo demonstrou que há um significativo efeito de redução da hiper-excitabilidade cortical em epilépticos com o uso da rEMT, principalmente em pacientes com displasia cortical focal. O segundo experimento procurou avaliar o potencial terapêutico da estimulação magnética transcraniana repetitiva (EMTr) de baixa freqüência em pacientes deprimidos. Foram estudadas 3 pacientes, consideradas de difícil tratamento por seus psiquiatras clínicos. Durante toda a pesquisa, os pacientes permaneceram em uso dos antidepressivos que foram prescritos anteriormente pelos seus clínicos, com as dosagens inalteradas. A paciente 1 apresentava quadro de depressão psicótica grave (38 pontos), não se observando melhora significativa. A paciente 2 também apresentava sintomas psicóticos, igualmente classificada como grave (34 pontos). Se, por um lado, esta paciente teve melhora clínica que pode ser considerada significativa (diminuição de mais de 50% na pontuação de da escala de depressão HAM - D), por outro lado sua pontuação ao final da pesquisa ainda era de depressão leve. A paciente 3 não apresentava sintomas psicóticos e sua depressão era classificada como moderada (22 pontos); ela apresentou melhora com o tratamento, terminando com 2 pontos. Sugere-se que a EMTr de baixa freqüência é uma forma segura e eficaz de tratamento para a depressão quando associada às medicações antidepressivas. No terceiro experimento o nosso estudo teve por objetivo usar a EMT para comparar a destreza manual da mão “sadia” de 18 pacientes hemiplégicos e avaliar a excitabilidade do córtex motor não envolvido pela lesão cerebral. Os pacientes foram comparados com 18 controles normais pareados por idade e sexo. Verificamos que existe um déficit motor significativo no membro superior “sadio” dos hemiplégicos; mais ainda, esse déficit se faz acompanhar de um aumento do limiar motor do córtex não afetado. / In the present study, we performed rTMS in five patients with intractable epilepsy, recording the number of daily seizures during 3 months before treatment, 3 months of treatment with bi-weekly rTMS sessions, and for 3 months after treatment. We have found a mean reduction of 22% in the number of daily seizures, during the period of treatment (range: 9.23 % to 43.09%). This reduction was statistically significant, although 2 of the 5 patients did not experience any reduction in the mean daily number of seizures. These findings demonstrate that there is a significant effect of rTMS in reducing cortical hyper-excitability in epileptics. In another study, we used rTMS to treat intractable depression in 3 patients. Patient 1 had severe psychotic depression (Hamilton score=38), and did not benefit from rTMS. Patient 2 also had psychotic symptoms, and there was a 50% reduction in her depression score, although she still had mild depression after treatment. The third patient, without psychotic symptoms, had moderate depression (score=22) and had significant improvement (final score=2). We suggest that slow-frequency rTMS is a safe adjunctive treatment for depression. Our third study aimed at using TMS to compare manual dexterity and motor cortex excitability for the “healthy” hand of hemiplegic patients. We have found significant motor deficits in the unaffected hand of hemiplegics; moreover, this was associated with an increased motor threshold of the healthy cerebral hemisphere.

In the UK, 12,000 patients die from metastatic breast cancer annually. There is therefore an urgent need to identify the molecules that cause metastasis. Recent work has revealed a role for the RUNX family of transcription factors in the development of metastatic breast cancer. The RUNX proteins form active transcription factor complexes when bound by the heterodimeric partner CBFβ to regulate the expression of metastatic genes. Previous work from our laboratory has demonstrated that knockdown of CBFβ resulted in a decreased invasion capacity of the metastatic breast cancer cell line MDA-MB-231. Three-dimensional culture of MDA-MB-231 cells revealed that loss of CBFβ induces a mesenchymal to epithelial transition (MET). The aim of this project was to determine the role of the RUNX/CBFβ complex in maintaining the mesenchymal phenotype of metastatic breast cancer cells. The data presented show that the phenotype changes were accompanied by changes in EMT marker-gene expression, including Snai2, MMP9, and MMP13. Induction of CBFβ in the CBFβ-knockdown cells remarkably restored both the invasive capacity and the mesenchymal phenotype. Further analysis revealed that maintenance of the mesenchymal phenotype was dependent upon both CBFβ-partner proteins, RUNX1 and RUNX2. Taken together the data presented in this thesis demonstrate that RUNX/CBFβ complexes drive the epithelial to mesenchymal transition (EMT) in breast cancer cells. These findings are likely to be important in the development of potential therapies to inhibit the metastatic spread of breast cancer.