Cutaneous melanoma is an increasingly common aggressive malignancy. The molecular mechanisms responsible for melanoma’s initiation and progression are still unclear, but new evidence suggests microRNAs (miRNAs) may be involved. MicroRNAs are small non-coding RNAs that have been shown to act as either oncogenes or tumour suppressors. These short, ~22 nucleotide long, single stranded RNA molecules regulate gene expression post-transcriptionally, through complementary binding to target messenger RNA (mRNA), and mediate mRNA degradation and translational repression. Our laboratory has previously shown that miRNA expression levels are altered through the different stages of melanoma tumourigenesis and has identified numerous significantly dysregulated miRNAs. miR-205 expression is significantly decreased in both primary and metastatic melanoma. Because of this decrease in miR-205 level with increasing cancer aggressiveness, we originally hypothesized that miR-205 may act as a tumour suppressor in melanoma. Unexpectedly, miR-205 re-expression in metastatic melanoma cells has shown oncogenetic potential. Through functional assays, we determined that miR-205 plays a primary role in promoting cellular migration and invasion, and in repressing adhesion. A gene expression analysis was conducted and the target prediction algorithm TargetScan was utilized to determine potential mRNA targets for miR-205. CADM1, PTPRJ and SHIP2 were three of the targets investigated, because of their known functional role in migration and cellular adhesion. CADM1 and PTPRJ were both verified to be directly targeted by miR-205 in an in vitro melanoma system using a luciferase reporter assay. In summary, we have demonstrated a surprising functional role for miR-205 in melanoma. The re-expression of miR-205 promotes malignant phenotypes and therefore is functioning with oncogenic potential within our metastatic melanoma cell culture system. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2012-07-09 12:32:35.235

The thesis deal with a model of electronically controlled assembly minicell MAP-205 based on pneumatic drives and its debugging. Assembly minicell simulates assembling and disassembling of simple four item assembly. Minicell is controlled by Phoenix Contact ILC 150ETH programmable logic controller. The minicell control program is composed of several subroutines/subprograms providing various functionalities to assembly minicell. There is integrated HMI created in Control Web application too. It allows to display actual status of each component and to control each drive in manual mode. Communication between HMI based on PC and PLC controller is supported by Bluetooth module and OPC server.

A paracoccidioidomicose (PCM) é a micose sistêmica mais frequente no Brasil. Na última década, foi demonstrado que é possível enviar antígenos diretamente para as células dendríticas utilizando o anticorpo αDEC205 e na presença de um estímulo de maturação, o resultado é a indução de uma resposta imunológica. Verificamos que o anticorpo αDEC fusionado ao peptídeo P10 induziu uma resposta por células produtoras de IFN-γ após uma única dose em relação à administração de P10, mesmo tendo sido administrado em uma concentração menor. Entretanto, essa resposta não se manteve após segunda dose do anticorpo. Após desafio dos animais com P. brasiliensis, imunizados com duas doses do anticorpo quimérico, detectamos níveis de IFN-γ e IL-4 no tecido pulmonar estatisticamente maiores no grupo αDEC/P10 e ISO/P10 em relação à administração de P10, todos em presença de Poly I:C. Em ensaios de terapia, verificamos no pulmão de camundongos tratados com o anticorpo quimérico, principal órgão envolvido em modelo animal de PCM, baixa concentração de IFN-γ e IL-10 em relação aos controles. Em adição, ficou evidente que nos animais tratados com o anticorpo αDEC/P10 o tecido pulmonar está compatível com o tecido de animais não infectados, enquanto que na ausência de tratamento adequado encontramos aglomerados de leveduras e um tecido com aumento no infiltrado celular. Esses achados indicam uma boa evolução clínica em animais tratados e indicam que o direcionamento do P10 através do anticorpo quimérico αDEC/P10, na presença de Poly I:C, é uma estratégia promissora para terapia contra P. brasiliensis. / Paracoccidioidomycosis (PCM) is the most common systemic mycosis in Brazil. In the last decade, it was demonstrated that antigens can be directly target to the dendritic cells using the antibody αDEC205 in the presence of a maturation stimulus, resulting in the induction of a strong immune response. We found that αDEC205 antibody fused to peptide P10 induced great response by IFN-γ producing cells after a single dose in relation to the administration of P10, although it has been administered in a lower concentration. However, this response was not maintained after second dose of antibody. Animals challenge with P. brasiliensis, after immunization with two doses of the chimeric antibody, produced high levels IFN-γ and IL-4 in lung tissue significantly higher in αDEC/P10 group in relation to the administration of P10, all in the presence of Poly I:C. In therapy assays, we found in the lungs of mice treated with the chimeric antibody, the main organ involved in an animal model of PCM, low concentration of IFN-γ and IL-10 compared to controls. In addition, it became evident that animals treated with αDEC/P10 antibody have a lung tissue much closer to that of non-infected tissue, while in the absence of suitable treatment we find clusters of yeasts and tissue filled with cellular infiltrates. Altogether, these findings show a clinical improvement in treated animals and indicate that targeting of P10 through the chimeric antibody αDEC/P10 in the presence of Poly I:C, is a promising strategy for therapy against P. brasiliensis.

作者何志華. / 書名原題 : 《淮南子》、《呂氏春秋》、《戰國策》三書高誘《注》斠證. / 論文(博士) -- 香港中文大學硏究院中國語言及文學學部, 1995. / 參考文献 : leaves 399-406. / zuo zhe He Zhihua. / Chapter 第一章 --- 高《注》釋例 --- p.1 / Chapter 1 --- 循文立訓例 --- p.1 / Chapter 1.1 --- 訓解單字例 --- p.1 / Chapter 1.2 --- 訓解兩字詞語例 --- p.5 / Chapter 1.3 --- 訓解多字例 --- p.7 / Chapter 2 --- 稱引師說例 --- p.8 / Chapter 2.1 --- 高誘稱引師說例 --- p.8 / Chapter 2.2 --- 高誘不采師說例 --- p.13 / Chapter 3 --- 互文例 --- p.15 / Chapter 4 --- 闕疑例 --- p.17 / Chapter 4.1 --- 音律 --- p.18 / Chapter 4.2 --- 地理 --- p.18 / Chapter 4.3 --- 時令 --- p.21 / Chapter 4.4 --- 夸誕之說 --- p.22 / Chapter 4.5 --- 鳥獸 --- p.23 / Chapter 4.6 --- 古人姓名事跡 --- p.24 / Chapter 4.7 --- 與書傳所記不合者 --- p.24 / Chapter 4.8 --- 文義未明者 --- p.25 / Chapter 5 --- 高誘聲訓例 --- p.26 / Chapter 5.1 --- 擬音例 --- p.27 / Chapter 5.1.1 --- 讀如 --- p.27 / Chapter 5.1.2 --- 讀若 --- p.28 / Chapter 5.1.3 --- 讀近 --- p.29 / Chapter 5.1.4 --- 讀似 --- p.30 / Chapter 5.1.5 --- 讀與「某」同 --- p.30 / Chapter 5.2 --- 改讀例 --- p.31 / Chapter 5.2.1 --- 據字書訓詁 --- p.33 / Chapter 5.2.1.1 --- 《爾雅》 --- p.33 / Chapter 5.2.2 --- 據傳注舊詁 --- p.34 / Chapter 5.2.2.1 --- 毛《傳》 --- p.34 / Chapter 5.2.2.2 --- 《毛詩》鄭《箋》 --- p.34 / Chapter 5.2.2.3 --- 《周禮》鄭《注》 --- p.35 / Chapter 5.2.3 --- 別書重文 --- p.35 / Chapter 5.3 --- 改字例 --- p.36 / Chapter 5.4 --- 附論：《淮南子》高《注》音讀斠證 --- p.40 / Chapter 6 --- 考文例 --- p.50 / Chapter 第二章 --- 《淮南子》、《呂氏春秋》、《戰國策》三書高《注》引斠證 --- p.57 / Chapter 1 --- 高誘據《文子》爲《注》證 --- p.57 / Chapter 1.1 --- 《文子》因襲《淮南》證 --- p.57 / Chapter 1.2 --- 古人據重文爲注證 --- p.61 / Chapter 1.3 --- 高《注》與《文子》重文相合例 --- p.64 / Chapter 1.4 --- 高《注》與《文子》重文不合例 --- p.71 / Chapter 1.5 --- 結語 --- p.74 / Chapter 1.6 --- 附論 --- p.76 / Chapter 1.6.1 --- 附論一：《淮南子》高誘《注》雖與《文子》相同而非出於 《文子》例 --- p.76 / Chapter 1.6.2 --- 附論二 --- p.81 / Chapter 2 --- 高誘引《老子》考 --- p.82 / Chapter 3 --- 高誘引《莊子》考 --- p.85 / Chapter 4 --- 高誘引《淮南子》考 --- p.89 / Chapter 5 --- 高誘引《呂氏春秋》考 --- p.107 / Chapter 6 --- 高誘引《戰國策》考 --- p.108 / Chapter 7 --- 高誘引《周易》考 --- p.110 / Chapter 8 --- 高誘引¯《‘ة書》考 --- p.113 / Chapter 9 --- 高誘引《逸周書》考 --- p.118 / Chapter 10 --- 高誘用《詩》考 --- p.118 / Chapter 11 --- 高誘引《周禮》考 --- p.151 / Chapter 12 --- 高誘引《禮記》考 --- p.166 / Chapter 13 --- 高誘引《春秋經》考 --- p.172 / Chapter 14 --- 高誘引《春秋左傳》考 --- p.173 / Chapter 15 --- 高誘引《國語》考 --- p.208 / Chapter 16 --- 高誘引《公羊傳》考 --- p.216 / Chapter 17 --- 高誘本《穀梁傳》爲《注》考 --- p.219 / Chapter 18 --- 高誘引《史記》考 --- p.220 / Chapter 19 --- 高誘引《漢書》考 --- p.221 / Chapter 20 --- 高誘引《山海經》考 --- p.224 / Chapter 21 --- 高誘引《論語》考 --- p.230 / Chapter 22 --- 高誘引《孟子》考 --- p.243 / Chapter 23 --- 高誘引《孝經》考 --- p.251 / Chapter 24 --- 高誘據《爾雅》爲訓詁證 --- p.253 / Chapter 25 --- 高誘據毛《傳》爲訓詁證 --- p.271 / Chapter 26 --- 高誘引兵書考 --- p.278 / Chapter 26.1 --- 《孫子》 --- p.278 / Chapter 26.2 --- 《司馬法》 --- p.279 / Chapter 26.3 --- 《尉繚子》 --- p.279 / Chapter 27 --- 高誘引《說苑》考 --- p.280 / Chapter 28 --- 高誘引《楚辭》考 --- p.281 / Chapter 29 --- 高誘引《世本》考 --- p.282 / Chapter 30 --- 高誘引緯書考 --- p.282 / Chapter 30.1 --- 《河圖括地象》 --- p.282 / Chapter 30.2 --- ¯《‘ة書五行傳》 --- p.283 / Chapter 30.3 --- 《洪範五行傳》 --- p.284 / Chapter 第三章 --- 《淮南子》、《呂氏春秋》、《戰國策》三書高《注》互異集證 --- p.287 / Chapter 第四章 --- 《淮南子》、《呂氏春秋》、《戰國策》三書高《注》斠證 --- p.307 / 徵引書目 --- p.399-406

A paracoccidioidomicose (PCM) é a micose sistêmica mais frequente no Brasil. Na última década, foi demonstrado que é possível enviar antígenos diretamente para as células dendríticas utilizando o anticorpo αDEC205 e na presença de um estímulo de maturação, o resultado é a indução de uma resposta imunológica. Verificamos que o anticorpo αDEC fusionado ao peptídeo P10 induziu uma resposta por células produtoras de IFN-γ após uma única dose em relação à administração de P10, mesmo tendo sido administrado em uma concentração menor. Entretanto, essa resposta não se manteve após segunda dose do anticorpo. Após desafio dos animais com P. brasiliensis, imunizados com duas doses do anticorpo quimérico, detectamos níveis de IFN-γ e IL-4 no tecido pulmonar estatisticamente maiores no grupo αDEC/P10 e ISO/P10 em relação à administração de P10, todos em presença de Poly I:C. Em ensaios de terapia, verificamos no pulmão de camundongos tratados com o anticorpo quimérico, principal órgão envolvido em modelo animal de PCM, baixa concentração de IFN-γ e IL-10 em relação aos controles. Em adição, ficou evidente que nos animais tratados com o anticorpo αDEC/P10 o tecido pulmonar está compatível com o tecido de animais não infectados, enquanto que na ausência de tratamento adequado encontramos aglomerados de leveduras e um tecido com aumento no infiltrado celular. Esses achados indicam uma boa evolução clínica em animais tratados e indicam que o direcionamento do P10 através do anticorpo quimérico αDEC/P10, na presença de Poly I:C, é uma estratégia promissora para terapia contra P. brasiliensis. / Paracoccidioidomycosis (PCM) is the most common systemic mycosis in Brazil. In the last decade, it was demonstrated that antigens can be directly target to the dendritic cells using the antibody αDEC205 in the presence of a maturation stimulus, resulting in the induction of a strong immune response. We found that αDEC205 antibody fused to peptide P10 induced great response by IFN-γ producing cells after a single dose in relation to the administration of P10, although it has been administered in a lower concentration. However, this response was not maintained after second dose of antibody. Animals challenge with P. brasiliensis, after immunization with two doses of the chimeric antibody, produced high levels IFN-γ and IL-4 in lung tissue significantly higher in αDEC/P10 group in relation to the administration of P10, all in the presence of Poly I:C. In therapy assays, we found in the lungs of mice treated with the chimeric antibody, the main organ involved in an animal model of PCM, low concentration of IFN-γ and IL-10 compared to controls. In addition, it became evident that animals treated with αDEC/P10 antibody have a lung tissue much closer to that of non-infected tissue, while in the absence of suitable treatment we find clusters of yeasts and tissue filled with cellular infiltrates. Altogether, these findings show a clinical improvement in treated animals and indicate that targeting of P10 through the chimeric antibody αDEC/P10 in the presence of Poly I:C, is a promising strategy for therapy against P. brasiliensis.

Recognition memory - a type of episodic memory in long term memory - is known in the literature to be affected by emotion, aging and the modality of the presented stimuli. The major aim of this study was to investigate whether emotional stimuli enhances recognition memory. Another goal was to observe whether modality and aging effects are present and differentiable in a non-Western subject sample. In literature, emotion studies were based on mainly two dimensions of emotions: valence and arousal. However, the contribution of these two dimensions to the enhancement of recognition memory still needs clarification. The present study investigated specifically the effect of valence on recognition memory. Moreover, the experimental manipulations of this study allowed observing the effect of valence on recognition memory due to normal aging. Since modality of the presented stimuli is a major confounding factor on recognition, separate experiments involving visual and verbal stimuli were designed. Pictures and words were selected on the basis of valence and arousal ratings. The stimulus set of the visual recognition memory task consisted of the pictures selected from the International Affective Picture System (IAPS) (Lang et al., 2005). The stimulus set of the verbal recognition memory task was constructed from partially standardized material for affective norms of Turkish emotional words (METU TEW), which was developed as a part of this study. METU TEW allowed selecting words with positive, neutral and negative valence while controlling arousal. The results replicated two findings reported in the literature: (1) younger adults recognized more accurately than older adults / (2) recognition memory was enhanced for visual items regardless of age and valence. Interestingly, this study revealed that recognition memory was not enhanced for emotional stimuli varying only on the valence dimension. More specifically, there was a decline in recognition memory for positive items and no change was observed for negative items, regardless of age. Further analysis also revealed that there may be differential effects of abstractness and concreteness on verbal recognition memory in aging.

Προκειμένου να παρεμποδιστεί διάδοση του σεξουαλικά μεταδιδόμενου HIV είναι απαραίτητη η ανάπτυξη κατάλληλης κολπικής φαρμακομορφής για τοπική χορήγηση η οποία θα αποτρέπει την μετάδοση του ιού σε υγιείς ανθρώπους. Το κολπικό αυτό σκεύασμα είναι σημαντικό να αποτελείται από κατάλληλη βιολογικώς δραστική ουσία και ως τέτοια μπορεί να θεωρηθεί το MC-1220 και το δραστικό (R)-εναντιομερές του (MC-εναντιομερές). Πρόκειται για μικροβιοκτόνα της κατηγορίας των DABOs (3,4-Dihyro-2-Alkoxy-6-Benzyl-4-OxopyrimidineS–DABOs) αναστολέων και ανήκουν στην κατηγορία των μη-νουκλεοτιδικών αναστολέων της αντίστροφης μεταγραφάσης (NNRTI). Η εξαιρετικά χαμηλή διαλυτότητα του MC σε υδατικά διαλύματα (< 3 ppm) μας οδήγησε στην ανάπτυξη μικρογαλακτωμάτων των MC και MC-εναντιομερούς. Τα μικρογαλακτώματα αυτά χρησιμοποιήθηκαν για την παρασκευή φαρμακομορφών (απλές γέλες) στις οποίες η συγκέντρωση του MC και MC-εναντιομερούς ήταν ~1.000 και 7.000 ppm αντίστοιχα. Αντιθέτως η ανάπτυξη κατάλληλης λιποσωμικής μορφής του MC (DRV_HPC/Chol(2:1) λιποσώματα) με υψηλές τιμές εγκλωβισμού MC, μας έδωσε την δυνατότητα παρασκευής τελικών σκευασμάτων (σύνθετες - λιποσωμικές γέλες) με εξαιρετικά υψηλές συγκεντρώσεις MC (~15.000 ppm). Επιπλέον και προκειμένου να αυξηθεί η συγκέντρωση του MC στις τελικές φαρμακομορφές (λιποσωμικές γέλες), αναπτύχθηκαν σύμπλοκα υδροξυπροπυλ-β-κυκλοδεξτρίνης/MC (HP-β-CD/MC), τα οποία επίσης ενσωματώθηκαν κατάλληλα στις τελικές λιποσωμικές φαρμακομορφές. Τέλος αναπτύχθηκαν κατάλληλες in vitro μέθοδοι, προκειμένου να γίνει συγκριτική αξιολόγηση των νέων φαρμακομορφών, ως προς την δυνατότητα σταδιακής αποδέσμευσης του MC στην περιοχή ενδιαφέροντος. Οι νέες φαρμακομορφές μελετήθηκαν in vivo σε πειραματόζωα (μακάκους), με ιδιαίτερα επιτυχή αποτελέσματα ως προς την πρόληψη και θεραπεία από τον HIV. / -

Le cancer du sein est marqué par une grande hétérogénéité. C´est une maladie complexe, fortement influencée par l´environnement pourtant, elle dépend aussi d´une accumulation de mutations génétiques associées à la dérégulation épigénétique des voies clés. Les altérations présentes dans le profil d´expression génique observées dans la tumeur, peuvent être le résultat de mécanismes de régulation des gènes à différent niveaux, comme des modifications post-transcriptionnelles menées par le mécanisme d´ARN d´interférence sous forme de microARN (miARN). Ce mécanisme peut conduire au début et développement du cancer aussi bien qu’à la résistance aux thérapies. Les miARN font partie d’une classe d´ARN non-codants qui ont émergé ces dernières années comme l'un des principaux régulateurs de l'expression des gènes par sa capacité à réguler négativement l'activité des ARN messagers (ARNm). L´importance de cette régulation a été observée par la présence de ce type de contrôle dans plusieurs processus biologiques, parmi eux, des voies liées à la prolifération, différentiation et apoptose. Afin de mieux comprendre les mécanismes d’initiation et progression tumorale dans le cancer du sein, nous avons fait une analyse globale de l´expression des miARN, par la technique de microarray, dans la série de lignées cellulaires 21T. Cette série est un modèle in vitro de la progression tumorale, comprenant la lignée 16N, obtenue à partir de l’épithélium normal infecté par des virus HPV-16, les lignées 21PT et 21NT, qui correspondent au carcinome in situ et les lignées 21MT1 et 21MT2 obtenues à partir d´une effusion pleurale métastatique à l’endroit de la métastase. Etant donné que les miARN jouent un rôle dans la régulation de l´apoptose et d´autres mécanismes de réponse aux dommages fait à l´ADN et que l´irradiation dans des formes différentes est couramment utilisée comme outil diagnostique, par exemple dans des mammographies, nous avons évalué l´expression de miARN après avoir soumis les cellules à des irradiations de haute et basse énergie, et au traitement avec de la doxorubicine. Les tests ont été faits sur les lignées non tumorales (MCF-10A et HB-2) et sur les lignées tumorales (MCF-7 et T-47D). On a pu observer que le rayon X de basse énergie est capable de causer des cassures double brin à l´ADN et de conduire les cellules à l´apoptose. Une légère altération dans les profils d´expression des miARN impliqués dans cette voie, comme let-7a, miR-34a et miR-29b, a aussi été remarquée. En ce qui concerne la réponse aux dommages fait à l´ADN, une upregulation dans l´expression de miR-29b, qui sous des conditions physiologiques normales est régulée négativement, a été observée après les traitements. Le microARNome de la série 21 montre une importante sous-expression de miR-205, un enrichissement du facteur pro-métastatique ZEB-1 et une réduction conséquente dans les niveaux d’e-cadherine, observée par western blot, seulement dans les lignées métastatiques (21MT). L´ensemble des résultats, suggèrent que miR-29b peut être un bio-marqueur potentiel du stress génotoxique et que miR-205 peut participer du processus de transition épithélium-mésenchyme et, en outre, quand il est sous-exprimé, peut augmenter le potentiel métastatique des cellules de la série 21T. / Breast tumors are characterized by their high heterogeneity. Breast cancer is a complex disease, which has its development strongly influenced by environmental factors, combined with a progressive accumulation of genetic mutations and epigenetic dysregulation of critical pathways. Changes in gene expression patterns may be a result of a deregulation in epigenetic events as well as in post-transcriptional regulation driven by RNA interference endogenously represented by microRNA (miRNA). These mechanisms are capable to promote the initiation, maintenance and progression of carcinogenesis and are also implicated on the development of therapy resistance. miRNAs form a class of non-coding RNAs, which have emerged in recent years as one of the major regulators of gene expression through its capacity to silence messenger RNAs (mRNAs) containing a partially complementary sequence. The importance of regulation mediated by miRNAs was observed on their ability to regulate a wide range of biological processes, including cell proliferation, differentiation and apoptosis.To gain insights into the mechanisms involved in breast cancer initiation and progression we conducted a miRNA global expression on 21T series that are an in vitro model of breast cancer progression, comprising cell lines derived from the same patient, which include a normal epithelia (16N), primary in situ ductal carcinoma (21PT and 21NT) and cells derived from pleural effusion of lung metastasis (21MT-1 and 21MT-2). Considering the importance of miRNAs in the regulation of apoptosis, and that irradiation in different spectra is commonly used in diagnostic procedures, as mammography and on radiotherapy, we evaluated the miRNA expression after cell low and high energy irradiation and doxorubicin treatment to determine whether miRNAs are useful biomarkers to detect cell response after DNA damage. The experiments were done on the non-tumoral cell lines MCF-10A and HB-2 and on the breast carcinoma derived cell lines MCF-7 and T-47D. We observed that low energy X-rays is able to promote DNA strand breaks and apoptosis and to slightly change the expression of miRNAs involved on this pathway, such as let-7a, miR-34a and miR-29b. Regarding DNA stress response pathways, an upregulation on miR-29b expression, that in normal conditions is downregulated in tumor cell lines could be observed after all treatments. The microRNAome of 21T series revealed a significant downregulation of miR-205, an enrichment of the pro-metastatic factor ZEB-1, potential target for miR-205 and the consequent reduction of e-cadherin levels in 21MT cells checked by western blot. Our results indicate that miR-29b is a possible biomarker of genotoxic stress and that miR-205 can participate on the metastatic potential of 21T cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os tumores de mama são caracterizados pela sua alta heterogeneidade. O câncer de mama é uma doença complexa, que possui o seu desenvolvimento fortemente influenciado por fatores ambientais, combinada a uma progressiva acumulação de mutações genéticas e desregulação epigenética de vias críticas. Alterações nos padrões de expressão gênica podem ser resultado de uma desregulação no controle de eventos epigenéticos, assim como, na regulação pós-transcricional pelo mecanismo de RNA de interferência endógeno via microRNA (miRNA). Estes eventos são capazes de levar à iniciação, à promoção e à manutenção da carcinogênese, como também ter implicações no desenvolvimento da resistência à terapia Os miRNAs formam uma classe de RNAs não codificantes, que durante os últimos anos surgiram como um dos principais reguladores da expressão gênica, através da sua capacidade de regular negativamente a atividade de RNAs mensageiros (RNAms) portadores de uma seqüencia parcialmente complementar. A importância da regulação mediada por miRNAs foi observada pela capacidade destas moléculas em regular uma vasta gama de processos biológicos incluindo a proliferação celular, diferenciação e a apoptose. Para avaliar a expressão de miRNAs durante a progressão tumoral, utilizamos como modelo experimental a série 21T que compreende 5 linhagens celulares originárias da mesma paciente diagnosticada com um tumor primário de mama do tipo ErbB2 e uma posterior metástase pulmonar. Essa série é composta pela linhagem obtida a partir do tecido normal 16N, pelas linhagens correspondentes ao carcinoma primário 21PT e 21NT e pelas linhagens obtidas um ano após o diagnóstico inicial, a partir da efusão pleural no sítio metastatico 21MT1 e 21MT2. O miRNAoma da série 21T revelou uma redução significativa nos níveis de miR-205 e nos níveis da proteina e-caderina e um enriquecimento do fator pró-metastático ZEB-1 nas células 21MT. Considerando a importância dos miRNAs na regulação da apoptose, e que a irradiação em diferentes espectros é comumente usada em procedimentos de diagnóstico como mamografia e na radioterapia, avaliamos a expressão de miRNAs após irradiação de alta e baixa energia e do tratamento doxorrubicina. Para os ensaios foram utilizados as linhagens não tumorais MCF-10A e HB-2 e as linhagens de carcinoma da mama MCF-7 e T-47D. Observou-se que raios-X de baixa energia são capazes de promover quebras na molécula do DNA e apoptose assim como, alterar sensivelmente miRNAs envolvidos nessas vias como o let-7a, miR-34a e miR-29b. No que diz respeito à resposta a danos genotóxicos, uma regulação positiva sobre a expressão de miR-29b, o qual em condições normais é regulado negativamente foi observada uma regulação positiva sobre miR-29b expressão após todos os tratamentos em células tumorais. Nossos resultados indicam que miR-29b é um possível biomarcador de estresse genotóxico e que miR-205 pode participar no potencial metastático das células 21T. / Breast tumors are characterized by their high heterogeneity. It is a complex disease, which has its development strongly influenced by environmental factors, combined with a progressive accumulation of genetic mutations and epigenetic dysregulation of critical pathways. Changes in gene expression patterns may be a result of a deregulation in epigenetic events as well as in post-transcriptional regulation driven by RNA interference endogenously represented by microRNA (miRNA) these mechanisms are capable to promote the initiation, maintenance and progression of carcinogenesis; they are also implicated on the development of therapy resistance. miRNAs form a class of non-coding RNAs which have emerged in recent years as one of the major regulators of gene expression through its capacity to silence messenger RNAs (mRNAs) containing a partially complementary sequence. The importance of regulation mediated by miRNAs was observed on their ability to regulate a wide range of biological processes including cell proliferation, differentiation and apoptosis.To gain insights into the mechanisms involved in breast cancer initiation and progression conducted a miRNA global expression on 21T series that are an in vitro model of breast cancer progression comprising cell lines derived from the same patient which include a normal epithelia (16N), primary in situ ductal carcinoma (21PT and 21NT) and cells derived from pleural effusion of lung metastasis (21MT-1 and 21MT-2). Considering the importance of miRNAs in the regulation of apoptosis, and that irradiation in different spectra is commonly used in diagnostic procedures as mammography and on radiotherapy, we evaluate the miRNA expression after cell low and high energy irradiation and doxorubicin treatment to determine whether miRNAs are useful biomarkers to detect cell response after DNA damage. The experiments were done on the non-tumoral cell lines MCF-10A and HB-2 and on the breast carcinoma derived cell lines MCF-7 and T-47D. We observed that of low energy X-rays is able to promote DNA strand breaks and apoptosis and to slightly change the expression of miRNAs involved on this pathway such as let-7a, miR-34a and miR-29b. Regarding DNA stress response pathways an upregulation on miR-29b expression, that in normal conditions is downregulated in tumor cell lines could be observed after all treatments. The microRNAome of 21T series revealed a significant downregulation of miR-205, an enrichment of the prometastatic factor ZEB-1, potential target for miR-205 and the consequent reduction of ecadherin levels in 21MT cells checked by western blot. Our results indicate that miR-29b is biomarkers of genotoxic stress and that miR-205can participate on the metastatic potential of 21T cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os tumores de mama são caracterizados pela sua alta heterogeneidade. O câncer de mama é uma doença complexa, que possui o seu desenvolvimento fortemente influenciado por fatores ambientais, combinada a uma progressiva acumulação de mutações genéticas e desregulação epigenética de vias críticas. Alterações nos padrões de expressão gênica podem ser resultado de uma desregulação no controle de eventos epigenéticos, assim como, na regulação pós-transcricional pelo mecanismo de RNA de interferência endógeno via microRNA (miRNA). Estes eventos são capazes de levar à iniciação, à promoção e à manutenção da carcinogênese, como também ter implicações no desenvolvimento da resistência à terapia Os miRNAs formam uma classe de RNAs não codificantes, que durante os últimos anos surgiram como um dos principais reguladores da expressão gênica, através da sua capacidade de regular negativamente a atividade de RNAs mensageiros (RNAms) portadores de uma seqüencia parcialmente complementar. A importância da regulação mediada por miRNAs foi observada pela capacidade destas moléculas em regular uma vasta gama de processos biológicos incluindo a proliferação celular, diferenciação e a apoptose. Para avaliar a expressão de miRNAs durante a progressão tumoral, utilizamos como modelo experimental a série 21T que compreende 5 linhagens celulares originárias da mesma paciente diagnosticada com um tumor primário de mama do tipo ErbB2 e uma posterior metástase pulmonar. Essa série é composta pela linhagem obtida a partir do tecido normal 16N, pelas linhagens correspondentes ao carcinoma primário 21PT e 21NT e pelas linhagens obtidas um ano após o diagnóstico inicial, a partir da efusão pleural no sítio metastatico 21MT1 e 21MT2. O miRNAoma da série 21T revelou uma redução significativa nos níveis de miR-205 e nos níveis da proteina e-caderina e um enriquecimento do fator pró-metastático ZEB-1 nas células 21MT. Considerando a importância dos miRNAs na regulação da apoptose, e que a irradiação em diferentes espectros é comumente usada em procedimentos de diagnóstico como mamografia e na radioterapia, avaliamos a expressão de miRNAs após irradiação de alta e baixa energia e do tratamento doxorrubicina. Para os ensaios foram utilizados as linhagens não tumorais MCF-10A e HB-2 e as linhagens de carcinoma da mama MCF-7 e T-47D. Observou-se que raios-X de baixa energia são capazes de promover quebras na molécula do DNA e apoptose assim como, alterar sensivelmente miRNAs envolvidos nessas vias como o let-7a, miR-34a e miR-29b. No que diz respeito à resposta a danos genotóxicos, uma regulação positiva sobre a expressão de miR-29b, o qual em condições normais é regulado negativamente foi observada uma regulação positiva sobre miR-29b expressão após todos os tratamentos em células tumorais. Nossos resultados indicam que miR-29b é um possível biomarcador de estresse genotóxico e que miR-205 pode participar no potencial metastático das células 21T. / Breast tumors are characterized by their high heterogeneity. It is a complex disease, which has its development strongly influenced by environmental factors, combined with a progressive accumulation of genetic mutations and epigenetic dysregulation of critical pathways. Changes in gene expression patterns may be a result of a deregulation in epigenetic events as well as in post-transcriptional regulation driven by RNA interference endogenously represented by microRNA (miRNA) these mechanisms are capable to promote the initiation, maintenance and progression of carcinogenesis; they are also implicated on the development of therapy resistance. miRNAs form a class of non-coding RNAs which have emerged in recent years as one of the major regulators of gene expression through its capacity to silence messenger RNAs (mRNAs) containing a partially complementary sequence. The importance of regulation mediated by miRNAs was observed on their ability to regulate a wide range of biological processes including cell proliferation, differentiation and apoptosis.To gain insights into the mechanisms involved in breast cancer initiation and progression conducted a miRNA global expression on 21T series that are an in vitro model of breast cancer progression comprising cell lines derived from the same patient which include a normal epithelia (16N), primary in situ ductal carcinoma (21PT and 21NT) and cells derived from pleural effusion of lung metastasis (21MT-1 and 21MT-2). Considering the importance of miRNAs in the regulation of apoptosis, and that irradiation in different spectra is commonly used in diagnostic procedures as mammography and on radiotherapy, we evaluate the miRNA expression after cell low and high energy irradiation and doxorubicin treatment to determine whether miRNAs are useful biomarkers to detect cell response after DNA damage. The experiments were done on the non-tumoral cell lines MCF-10A and HB-2 and on the breast carcinoma derived cell lines MCF-7 and T-47D. We observed that of low energy X-rays is able to promote DNA strand breaks and apoptosis and to slightly change the expression of miRNAs involved on this pathway such as let-7a, miR-34a and miR-29b. Regarding DNA stress response pathways an upregulation on miR-29b expression, that in normal conditions is downregulated in tumor cell lines could be observed after all treatments. The microRNAome of 21T series revealed a significant downregulation of miR-205, an enrichment of the prometastatic factor ZEB-1, potential target for miR-205 and the consequent reduction of ecadherin levels in 21MT cells checked by western blot. Our results indicate that miR-29b is biomarkers of genotoxic stress and that miR-205can participate on the metastatic potential of 21T cells.