Comment les signalisations dynamiques et les mouvements morphogénétiques régionalisent et permettent la formation de tissus complexes durant l'embryogenèse est très peu compris. J’ai caractérise au cours de ma thèse, les évènements signalisants qui sont mis en place au cours de la myogenèse précoce chez l'embryon de poulet. J'ai montre que les progénitures musculaires présents dans les somites requièrent l'activation dynamique des voies de signalisation Wnt et Notch. L’activation transitoire de la signalisation Notch est requise pour adopter un destin myogénique. Le ligand de Notch Dll1 est exprime de manière mosaïque dans les cellules migrantes des crêtes neurales qui passent près du somite. Gain et perte de fonction de Dll1 dans les crêtes neurales modifient la signalisation Notch dans les somites, résultant en un délai ou une prématuré myogenèse. Nos résultats indiquent que les crêtes neural régulent la formation précoce du muscle par un mécanisme unique mené par la migration des cellules des crêtes neurales exprimant Dll1 qui déclenche l'activation transitoire de la signalisation Notch dans certains progénitures musculaires sélectionnes. Cette dynamique signalisation garantie une différentiation progressive du pool de progénitures musculaires. / How dynamic signalling and extensive tissue rearrangements interplay to generate complex patterns and shapes during embryogenesis is poorly understood. During my PhD, I have characterized the signalling events taking place during early morphogenesis of chick skeletal muscles. I observed that muscle progenitors present in somites require dynamic activation of Wnt and Notch signalling. I showed that a transient activation of NOTCH signalling is required to undergo terminal differentiation. The NOTCH ligand Delta1 is expressed in a mosaic pattern in neural crest cells that migrate past the somites. Gain and loss of Delta1 function in neural crest modifies NOTCH signalling in somites, which results in delayed or premature myogenesis. These results suggest that the neural crest regulates early muscle formation by a unique mechanism that relies on the migration of Delta1-expressing neural crest cells to trigger the transient activation of NOTCH signalling in selected muscle progenitors. This dynamic signalling guarantees a balanced and progressive differentiation of the muscle progenitor pool.

Cancer is a complex and dynamic disease that is difficult to treat due to its heterogeneous nature at multiple scales. Standard therapies such as surgery, radiation, and chemotherapy often fail, therefore superior therapies must be developed. Electroporation-based therapies offer an alternative to standard treatments, utilizing pulsed electric fields to permeabilize cell membranes to either enhance drug delivery (electrochemotherapy) or induce cancer cell death (irreversible electroporation). Electroporation treatments show promise in the clinic, however, are limited in the size of tumors that they can safely treat without increasing the applied voltage to an extent that induces thermal damage or muscle contractions in patients. A method to increase ablation size safely is needed. To make this advancement and to advance other cancer treatments as well, better in vitro tumor models are needed. Heterogeneity not only makes cancer difficult to treat, but also difficult to recapitulate in vitro. This dissertation addresses the complementary need to develop both better cancer therapies and more physiologically relevant in vitro tumor models. My results demonstrate that by using a calcium adjuvant with irreversible electroporation treatment, ablation size can be increased without using a higher applied voltage. Additional mechanistic studies identified signaling pathways that were differentially dysregulated under calcium and no calcium conditions, impacting cell death. Finally, I have successfully encapsulated cells in fibrin microgels which may enable the creation of more physiologically relevant and complex 3D in vitro and ex-vivo platforms to investigate IRE as well as other tumor therapies. / Doctor of Philosophy / Cancer is a complex and dynamic disease. Heterogeneity exists at the single cell, tumor, and patient levels making it difficult to establish a unified target for therapy. Standard therapies such as surgery, radiation, and chemotherapy often fail for this reason, therefore superior therapies must be developed. Electroporation-based therapies offer an alternative to standard treatments, utilizing pulsed electric fields to permeabilize cell membranes to either enhance drug delivery (electrochemotherapy) or induce cancer cell death (irreversible electroporation). Electroporation treatments show promise in the clinic, however, are limited in the size of tumors that they can safely treat without increasing the applied voltage to an extent that induces thermal damage or muscle contractions in patients. A method to increase ablation size safely is needed. To make this advancement and to advance other cancer treatments as well, better tumor models are needed. Many of the same challenges in treating cancer serve as challenges in creating physiologically relevant tumor models. In this dissertation, I have developed a simplified platform to test whether using a calcium additive with irreversible electroporation therapies enhances ablation size. My results demonstrate that by using a calcium additive with irreversible electroporation treatment, ablation size can be increased without using a higher applied voltage. In addition, the biological pathways responsible for cell death in irreversible electroporation treatment with and without calcium were studied. Finally, I have successfully encapsulated cells in fibrin microgels that can be used to create better tumor models that encompass the heterogeneity of tumors found in the body.

Brain tumors are among the most devastating types of solid tumors to treat. Standard of care for glioblastoma (GBMs), the most aggressive form of primary brain tumors, has failed to improve the current survival rates in the past decades. Despite many other solid tumors, recent advances in cancer immunotherapies have also shown disappointing outcomes in GBMs. The heterogenous nature of GBMs, the immunosuppressive tumor microenvironment and the restrictive role of blood brain barrier (BBB) are some of the main challenges faced for treating GBMs. Electroporation-based treatments have demonstrated promising results, treating preclinical models of GBMs. It has been shown that low and high frequency irreversible electroporation treatments shift the immunosuppressive tumor microenvironment and reversibly open large areas of blood brain barrier (BBB). In this dissertation, in vitro cell culture models are utilized to study electroporation-based treatments for achieving a more optimized treatment for glioblastoma. We are proposing to utilize the immunomodulatory effects of electroporation treatments to improve the outcomes of immunotherapies in the brain. / Doctor of Philosophy / Despite the current advancements in treating solid tumors, brain tumors remain among the most difficult cancers to treat. The special structure of the brain as an organ as well as tumor complexity can lead to treatment failure. It is also known that infiltration of the immune cells within the tumor mass is limited due to the tumor's immunosuppressive nature. Hence, the use of newly advancing immunotherapy techniques is limited in the brain. Local treatments have become one of the most promising tools against brain tumors. Such treatments include methods that use excessive heating of the tissue to kill the tumors. Relying on heat for tissue destruction could damage the critical structures near the tumor and will reduce the favorable immune response after the treatment. A new treatment modality known as electroporation has been introduced for non-thermal treatment of brain tumors. Due to its non-thermal nature, electroporation treatments will allow for sparing of critical structures and can lead to a more robust immune response comparing to thermal treatment modalities. In this dissertation, we utilize electroporation-based treatments to try to overcome some of the challenges associated with treating brain tumors such as tumor heterogeneity and immune suppression.

The childhood eye cancer retinoblastoma originates from the retina and its development is initiated while the foetus is in the uterus. Retinoblastoma has a reported incidence of 1 in 15-18 000 live births, and approximately 90% of all patients are diagnosed before the age of 5. The occurrence of retinoblastoma is usually detected by the parents and the most frequent symptoms are leukocoria (white pupillary reflex), strabismus (squinting) or if the child complains of visual problems. Retinoblastoma is diagnosed by examination under anaesthesia and documentation by RetCam. It is treated with various cytostatic agents, or by laser. If the treatment is unsuccessful, or there is a risk that the tumour cells will spread and form metastases, the eye is removed. Previous studies have indicated that the cell type from which the tumour arises, the cell-of-origin, may be the cone photoreceptors and/or their immediate interneuron, the horizontal cells. Determining the cell-of-origin for retinoblastoma is an important goal, however, understanding the molecular mechanisms that distinguish the photoreceptors and the horizontal cells from the other retinal cells may prove just as important for understanding this disease. The aim of my project has been to develop, optimise and validate methods to label, map and target expression to photoreceptors and horizontal cells in the chicken embryonic retina. We have successfully established several methods that test the expression pattern of conserved, regulatory DNA sequences, and have performed short- and long-term expression of various genes that have been reported to be involved in cell cycle regulation and cell fate determination. One of my most important findings was that a region from the RXRγ gene allowed us to specifically target the photoreceptors and horizontal cells. Our previous knowledge, together with the newly established tools, puts us an important step closer towards understanding the development and behaviour of the retinal photoreceptors and horizontal cells, however, further studies are of course needed.

Neoplasias são proliferações anormais do tecido. O melanoma é uma neoplasia maligna de grande pleomorfismo e apresenta baixa taxa de resposta à quimioterapia. A aplicação de pulsos elétricos aumenta a permeabilidade da membrana celular, facilitando a passagem de drogas quimioterápicas. O objetivo deste estudo foi avaliar a resposta do melanoma murino ao tratamento com uma e duas aplicações de eletroquimioterapia. Utilizou-se células de melanoma B16F10 em camundongos e realizou-se acompanhamento diário. Avaliou-se a histologia tumoral, o número de mitoses, a contagem de microvasos e o número de mastócitos. Nos animais tratados a sobrevida foi 2,3 vezes maior. Nos animais tratados com duas aplicações de eletroquimioterapia ocorreu remissão total do tumor em 60% dos casos e parcial nos demais, e apresentaram sobrevida 4,5 vezes maior. O número de mitoses nos grupos tratados com uma e duas aplicações de eletroquimioterapia e do número de mastócitos nos grupos tratados com duas aplicações de eletroporação e eletroquimioterapia foi menor que os controles. / Neoplasms are abnormal growths of tissue. Melanoma is a malignant neoplasm of large pleomorphic and has a low response rate to chemotherapy. The application of electrical pulses increases cell membrane permeability and facilitate passage of chemotherapeutic drugs. The aim of this study was to evaluate the response to treatment of murine melanoma with one and two applications of Electrochemotherapy. We used them B16F10 melanoma cells and mice held monitoring diary. We evaluated the histology of the tumor, the number of mitoses, microvessel count and the number of mast cells. In animals treated, the survival was 2.3 times higher. In animals treated with two applications of Electrochemotherapy complete tumor remission occurred in 60% of cases and partial in the other. This group had a survival 4.5 times higher. The number of mitoses in the groups treated with one or two applications of Electrochemotherapy and the number of mast cells in the groups treated with two applications of electroporation and Electrochemotherapy was lower than controls.

Diencephalic development requires the transcription factors Pax6 and Barhl2 in order to proceed correctly. Both genes are necessary for the normal development of the organizer region known as the zona limitans intrathalamica (ZLI). The ZLI goes on to pattern the diencephalon via its secretion of the morphogen Shh, which inhibits the expression of Pax6. These findings suggest that interactions between Pax6, Barhl2 and Shh may be involved in the control of diencephalic development. This project aims to characterise these interactions and investigate their roles. The expression domains of Pax6 and Barhl2 were mapped during the early development of the mouse diencephalon. Qualitative approaches were employed to confirm the high complementarity of their expression domains and obtain evidence of a mutually repressive relationship existing between the two genes. The findings from a quantitative analysis suggested that this inhibition is incomplete within the thalamus. Investigations using the Pax6-null mutant mouse confirmed that in the absence of Pax6 the thalamic Barhl2 expression domain expands beyond the ventricular zone, the site of thalamic neurogenesis. The influence of Shh signalling on the expression of Pax6 and Barhl2 was investigated via a gain-of-function approach utilising in utero electroporation to activate the Shh pathway. This led to a downregulation of both Pax6 and Barhl2 within the thalamus. In Shh loss-of-function experiments drug treatment with the Shh antagonist vismodegib led to an upregulation of Barhl2 and the loss of the GABAergic pTh-R in the Pax6-null mutant thalamus, but not in the wild type thalamus, suggesting that Pax6 and Shh may be required to inhibit Barhl2 in order for GABAergic neurogenesis to proceed. Barhl2 expression was detected in the Shh-null mutant mouse confirming that, in contrast with their homologues in Drosophila, Shh may be expressed downstream of Barhl2. Together these findings have been used to develop a novel model of thalamic development in which Barhl2 induces ZLI development, inhibition of Barhl2 by Pax6 restricts its expansion, and secretion of Shh by the ZLI then goes on to inhibit Pax6 and Barhl2 in the pTh-R while mutual repression between Pax6 and Barhl2 modulates neurogenesis in the more caudal regions of the thalamic neuroepithelium.

by Henry, Kam Yin Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 148-160). / ZEBRAFISH (BRACHYDANIO RERIO) / ACKNOWLEDGEMENTS / LIST OF CONTENTS / ABSTRACT / ABBREVIATION / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Transgenic fish --- p.1 / Chapter 1.2 --- Zebrafish --- p.4 / Chapter 1.3 --- The grass carp GH gene and protein / Chapter 1.3.1 --- The genomic sequence --- p.5 / Chapter 1.3.2 --- The cDNA sequence --- p.7 / Chapter 1.3.3 --- The grass carp GH protein --- p.7 / Chapter 1.4 --- Functional aspects of promoter regions / Chapter 1.4.1 --- PEPCK --- p.9 / Chapter 1.4.2 --- RSV-LTR --- p.10 / Chapter 1.4.3 --- hMT-IIA --- p.10 / Chapter 1.4.4 --- MMTV-LTR --- p.11 / Chapter 1.5 --- Eukaryotic gene expression in cultured cells / Chapter 1.5.1 --- COS-7 and HepG2 cells --- p.11 / Chapter 1.5.2 --- Transfection system --- p.12 / Chapter 1.5.3 --- Fate of DNA after transfection --- p.13 / Chapter 1.6 --- Electroporation and microinjection as tools for gene transfer / Chapter 1.6.1 --- Electroporation: Theory and operation --- p.13 / Chapter 1.6.2 --- Microinjection: Design of microinjector --- p.16 / Chapter 1.6.3 --- Fate of DNA after gene transfer in embryos / Transient expression --- p.16 / Stable transformation --- p.17 / Inheredity of transgene --- p.17 / Chapter 1.7 --- The aims of the present study --- p.18 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- General techniques / Chapter 2.1.1 --- Electrophoresis of DNA / Chapter 2.1.1.1 --- Agarose gel electrophoresis --- p.19 / Chapter 2.1.1.2 --- PAGE --- p.20 / Chapter 2.1.2 --- Purification of DNA --- p.21 / Chapter 2.1.3 --- Recovery of DNA fragments / Chapter 2.1.3.1 --- Electroelution --- p.22 / Chapter 2.1.3.2 --- Geneclean kit --- p.23 / Chapter 2.1.4 --- Standard recombinant DNA techniques / Chapter 2.1.4.1 --- Dephosphorylation --- p.24 / Chapter 2.1.4.2 --- Kinasing --- p.24 / Chapter 2.1.4.3 --- Ligation --- p.24 / Chapter 2.1.4.4 --- Filling in reaction --- p.25 / Chapter 2.1.4.5 --- Transformation --- p.25 / Chapter 2.1.5 --- Minipreparation of plasmids --- p.26 / Chapter 2.1.6 --- Large preparation of plasmids / Chapter 2.1.6.1 --- Qiagene kit --- p.27 / Chapter 2.1.6.2 --- CsCl density gradient centrifugation --- p.27 / Chapter 2.1.7 --- DNA sequencing --- p.29 / Chapter 2.1.8 --- "Extraction of DNA from embryos, fry and fish" / Method 1 --- p.32 / Method 2 --- p.32 / Chapter 2.1.9 --- Probe labelling / Chapter 2.1.9.1 --- End-labelling --- p.33 / Chapter 2.1.9.2 --- Random priming --- p.33 / Chapter 2.1.10 --- CAT assay --- p.33 / Chapter 2.1.11 --- Polymerase chain reaction(PCR) --- p.35 / Chapter 2.1.12 --- Radioimmunassay(RIA) of FGH --- p.36 / Chapter 2.1.13 --- Dot blotting --- p.38 / Chapter 2.1.14 --- Southern blotting --- p.39 / Chapter 2.2 --- "Linkers, primers and probes" / Chapter 2.2.1 --- Primers --- p.41 / Chapter 2.2.2 --- Linkers --- p.45 / Chapter 2.2.3 --- Probes --- p.47 / Chapter 2.3 --- Construction of chimeric growth hormone genes / Chapter 2.3.1 --- Sources of plasmids --- p.50 / Chapter 2.3.2 --- General principles --- p.50 / Chapter 2.3.3 --- PEPCKgcGHcDNA --- p.51 / Chapter 2.3.4 --- RSVgcGHcDNA --- p.54 / Chapter 2.3.5 --- hMTgcGHcDNAcDNA --- p.56 / Chapter 2.3.6 --- MMTVgcGHcDNA --- p.58 / Chapter 2.3.7 --- "PEPCKgcGH, RSVgcGH and hMTgcGH" --- p.60 / Chapter 2.4 --- Expression of chimeric genes in cultured cells / Chapter 2.4.1 --- Culturing of COS-7 and HepG2 cells --- p.66 / Chapter 2.4.2 --- Expression of chimeric genes in COS-7 and HepG2 cells --- p.67 / Chapter 2.5 --- Zebrafish / Chapter 2.5.1 --- "Culturing, Spawning and hatching" --- p.67 / Chapter 2.6 --- Electroporation and microinjection for gene transfer / Chapter 2.6.1 --- Electroporation / Chapter 2.6.1.1 --- Tuning up electroporation --- p.69 / Chapter 2.6.1.2 --- Evidence of gene transfer by electroporation / Chapter 2.6.1.2.1 --- CAT assay --- p.71 / Chapter 2.6.1.2.2 --- Dot blot --- p.71 / Chapter 2.6.1.2.3 --- PCR and Southern blotting of PCR products --- p.72 / Chapter 2.6.1.2.4 --- Southern blotting of fish total DNA --- p.73 / Chapter 2.6.2 --- Microinjection / Chapter 2.6.2.1 --- Handling of microinjection --- p.74 / Chapter 2.6.2.2 --- Evidence of gene transfer by microinjection / Chapter 2.6.2.2.1 --- CAT assay --- p.75 / Chapter 2.6.2.2.2 --- PCR and Southern blotting of PCR products --- p.75 / Chapter 2.7 --- Phenotypic alteration of fish generated from electroporated eggs / Chapter 2.7.1 --- Electroporation and handling of fish generated from electroporation --- p.75 / Chapter 2.7.2 --- Measurement of phenotypic change in fish generated from electroporation --- p.77 / Chapter 2.8 --- Detection of transgene and expression of exogenous DNA / Chapter 2.8.1 --- Transgene detection --- p.78 / Chapter 2.8.2 --- Expression of exogenous DNA --- p.79 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Construction of Chimeric growth hormone genes / Chapter 3.1.1 --- Confirmation of integrity of chimeric genes / PEPCKgcGHcDNA --- p.80 / RSVgcGHcDNA --- p.81 / hMTgcGHcDNA --- p.81 / MMTVgcGHcDNA --- p.81 / "PEPCKgcGH, RSVgcGH and hMTgcGH" --- p.82 / Chapter 3.1.2 --- Yield of chimeric genes from CsCl density gradient centrifugation --- p.82 / Chapter 3.2 --- Chimeric gene expression in COS-7 and HepG2 cells / Chapter 3.2.1 --- Expression of chimeric genes in COS-7 cells --- p.89 / Chapter 3.2.2 --- Expression of chimeric genes in HepG2 cells --- p.93 / Chapter 3.3 --- Transfer of chimeric genes into embryos / Chapter 3.3.1 --- Electroporation / Chapter 3.3.1.1 --- Monitoring of electroporation --- p.94 / Chapter 3.3.1.2 --- Evidence for gene transfer / Chapter 3.3.1.2.1 --- CAT assay --- p.98 / Chapter 3.3.1.2.2 --- Dot blotting --- p.98 / Chapter 3.3.1.2.3 --- PCR and Southern blotting of PCR product --- p.101 / Chapter 3.3.1.2.4 --- Southern blotting of DNA from fish generated from electroporation --- p.106 / Chapter 3.3.2 --- Microinjection / Chapter 3.3.2.1 --- CAT assay --- p.109 / Chapter 3.3.2.2 --- PCR --- p.109 / Chapter 3.4 --- Phenotypic alterations of fish / The first experiment --- p.112 / The second experiment --- p.113 / The third experiment --- p.113 / The fourth experiment --- p.122 / Chapter 3.5 --- Detection of transgene and expression of exogenous DNA / Chapter 3.5.1 --- Transgene --- p.128 / Chapter 3.5.2 --- Possible expression of exogenous DNA --- p.129 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Chimeric growth hormone genes --- p.132 / Chapter 4.2 --- Expression of chimeric growth hormone genes in COS-7 and HepG2 cells --- p.134 / Chapter 4.3 --- Transfer of exogenous DNA into embyros --- p.136 / Chapter 4.4 --- Phenotypic alteration of fish developed from electroporated eggs --- p.139 / Chapter 4.5 --- The possible integration and expression of exogenous DNA --- p.143 / Chapter 4.6 --- Conclusions --- p.145 / Chapter 4.7 --- Suggestions for further studies --- p.146 / REFERENCES --- p.148 / Chapter APPENDIX I --- Restriction maps / PEPCKgcGH / PEPCKgcGHcDNA / RSVgcGH / RSVgcGHcDNA / hMTgcGH / hMTgcGHcDNA / MMTVgcGHcDNA / pBH1.2 / pMSG-CAT / pUC19 / hMT-IIA / PBC12BI / RSVCAT / pUC101 / pSEl/S2 / PUCSE2/S1 / pUCS2

A eletroporação é um fenômeno que promove a formação de poros na membrana citoplasmática quando esta é submetida à exposição de um campo elétrico específico. Dentre as principais aplicações da eletroporação podemos citar a eletroquimioterapia, uma nova terapia contra o câncer, e a terapia gênica, ainda em desenvolvimento. As conexinas são unidades formadoras das junções gap, canais transmembrana responsáveis pela comunicação intercelular e que participam de uma serie de eventos fisiológicos como o crescimento e diferenciação celular. A importância desta família de genes na oncologia está cada vez mais clara dadas às evidencias de seu comportamento de supressor de tumor ou como facilitador da disseminação e metástases de acordo com a fase de em que o câncer se apresenta. A eletroporação como um fenômeno que promove perturbação em membrana poderia interferir de alguma forma na expressão das conexinas. O objetivo deste trabalho foi avaliar os possíveis efeitos de interferência na expressão gênica das células promovidos pela eletroporação em especial na família de genes das conexinas. Para tanto as células foram expostas a um campo semelhante a o utilizado para eletroquimioterapia e avaliadas em diferentes tempos após a exposição. Três linhagens, duas neoplásicas (melanoma B16/BL6 e E9) e uma linhagem não neoplásica (E10) foram utilizadas como modelo experimental para que pudesse ser feita uma analise comparativa e a avaliação dos possíveis diferentes comportamentos das mesmas frente à eletroporação. O estudo da expressão gênica foi realizado pelas técnicas de imunofluorescência, western blot e PCR em tempo real. A Cx 26 foi avaliada na linhagem B16/BL6 e a Cx 43 nas linhagens E9 e E10. Os resultados da imunofluorescência apontaram uma marcação no citoplasma em B16/BL6, predominantemente em núcleo para a linhagem E9 e em núcleo e citoplasma em E10. Somente a linhagem E9 apresentou diferentes marcações entre os grupos, com uma marcação em citoplasma no grupo t1/2 (30 minutos apos a eletroporacao) O western blot mostrou diminuição transiente para as Cxs apenas nas linhagens neoplásicas nos grupos t1/2 e t1 (1 hora apos a eletroporacao). A linhagem E10 apresentou aumento de expressão nos mesmos grupos. O PCR em tempo real apresentou diferenças significativas nos tempos t1/2 e t1 para as linhagens B16/BL6 e E10. A linhagem B16/BL6 teve diminuição de RNAm enquanto a linhagem E10 apresentou aumento de transcrição de RNAm. A linhagem E9 apresentou padrão de expressão bastante heterogêneo. Frente aos resultados apresentados podemos concluir que a eletroporação interferiu de maneira transiente na expressão de conexinas. / Electroporation is a phenomenon that promotes the formation of pores in the cytoplasmic membrane when it is exposed to a specific electric field. The main applications of electroporation include the electrochemotherapy, a new cancer therapy, and gene therapy, still in development. Connexins are forming units of gap junctions, transmembrane channels responsible for intercellular communication and participating in a series of physiological events such as cell growth and differentiation. The importance of this gene family in oncology is increasing. The evidence of its behavior as a tumor suppressor or facilitator of dissemination and metastases according to the stage where the cancer presents itself. Electroporation as a phenomenon that promotes membrane disruption could interfere somehow in the expression of connexins. The aim of this study was to evaluate the possible effects of interference in gene expression. of cells promoted by electroporation in particular gene family of connexins. For this purpose cells were exposed to a electric field similar to that used for Electrochemotherapy and evaluated at different times after exposure. Three cell lines, two neoplastic (melanoma B16/BL6 and E9) and one line non-neoplastic (E10) were used as experimental models that could provide a comparative analysis between the lines and an assessment of the possible different behaviors due to electroporation. Its expression was assessed by immunofluorescence, western blot and real-time PCR. Cx 26 was evaluated in the melanoma cell line B16/BL6 and Cx 43 in the cell lines E9 and E10. The results showed a cytoplasm immunofluorescence staining pattern in the in B16/BL6, predominantly in the nucleus in line E9 and in the nucleus and cytoplasm for cell line E10. Only the cell line E9 had different immunoflurescence stainings between the groups, with a positivity in the cytoplasm in the group t1/2. The western blot showed transient decrease of Cxs only in cell lines neoplastic in the times t1/2 and t1. The lung cell line E10 showed increased expression in the same time points. The real-time PCR showed significant differences in t1/2 and t1 for the cell lines B16/BL6 and E10. The melanoma cell line B16/BL6 had mRNA decreased while the cell line E10 showed increased transcription of mRNA. The cell line E9 showed a very heterogeneous expression pattern. According to our results, we can conclude that electroporation interfered transiently in the expression of connexins.

A eletroporação é um fenômeno que promove a formação de poros na membrana citoplasmática quando esta é submetida à exposição de um campo elétrico específico. Dentre as principais aplicações da eletroporação podemos citar a eletroquimioterapia, uma nova terapia contra o câncer, e a terapia gênica, ainda em desenvolvimento. As conexinas são unidades formadoras das junções gap, canais transmembrana responsáveis pela comunicação intercelular e que participam de uma serie de eventos fisiológicos como o crescimento e diferenciação celular. A importância desta família de genes na oncologia está cada vez mais clara dadas às evidencias de seu comportamento de supressor de tumor ou como facilitador da disseminação e metástases de acordo com a fase de em que o câncer se apresenta. A eletroporação como um fenômeno que promove perturbação em membrana poderia interferir de alguma forma na expressão das conexinas. O objetivo deste trabalho foi avaliar os possíveis efeitos de interferência na expressão gênica das células promovidos pela eletroporação em especial na família de genes das conexinas. Para tanto as células foram expostas a um campo semelhante a o utilizado para eletroquimioterapia e avaliadas em diferentes tempos após a exposição. Três linhagens, duas neoplásicas (melanoma B16/BL6 e E9) e uma linhagem não neoplásica (E10) foram utilizadas como modelo experimental para que pudesse ser feita uma analise comparativa e a avaliação dos possíveis diferentes comportamentos das mesmas frente à eletroporação. O estudo da expressão gênica foi realizado pelas técnicas de imunofluorescência, western blot e PCR em tempo real. A Cx 26 foi avaliada na linhagem B16/BL6 e a Cx 43 nas linhagens E9 e E10. Os resultados da imunofluorescência apontaram uma marcação no citoplasma em B16/BL6, predominantemente em núcleo para a linhagem E9 e em núcleo e citoplasma em E10. Somente a linhagem E9 apresentou diferentes marcações entre os grupos, com uma marcação em citoplasma no grupo t1/2 (30 minutos apos a eletroporacao) O western blot mostrou diminuição transiente para as Cxs apenas nas linhagens neoplásicas nos grupos t1/2 e t1 (1 hora apos a eletroporacao). A linhagem E10 apresentou aumento de expressão nos mesmos grupos. O PCR em tempo real apresentou diferenças significativas nos tempos t1/2 e t1 para as linhagens B16/BL6 e E10. A linhagem B16/BL6 teve diminuição de RNAm enquanto a linhagem E10 apresentou aumento de transcrição de RNAm. A linhagem E9 apresentou padrão de expressão bastante heterogêneo. Frente aos resultados apresentados podemos concluir que a eletroporação interferiu de maneira transiente na expressão de conexinas. / Electroporation is a phenomenon that promotes the formation of pores in the cytoplasmic membrane when it is exposed to a specific electric field. The main applications of electroporation include the electrochemotherapy, a new cancer therapy, and gene therapy, still in development. Connexins are forming units of gap junctions, transmembrane channels responsible for intercellular communication and participating in a series of physiological events such as cell growth and differentiation. The importance of this gene family in oncology is increasing. The evidence of its behavior as a tumor suppressor or facilitator of dissemination and metastases according to the stage where the cancer presents itself. Electroporation as a phenomenon that promotes membrane disruption could interfere somehow in the expression of connexins. The aim of this study was to evaluate the possible effects of interference in gene expression. of cells promoted by electroporation in particular gene family of connexins. For this purpose cells were exposed to a electric field similar to that used for Electrochemotherapy and evaluated at different times after exposure. Three cell lines, two neoplastic (melanoma B16/BL6 and E9) and one line non-neoplastic (E10) were used as experimental models that could provide a comparative analysis between the lines and an assessment of the possible different behaviors due to electroporation. Its expression was assessed by immunofluorescence, western blot and real-time PCR. Cx 26 was evaluated in the melanoma cell line B16/BL6 and Cx 43 in the cell lines E9 and E10. The results showed a cytoplasm immunofluorescence staining pattern in the in B16/BL6, predominantly in the nucleus in line E9 and in the nucleus and cytoplasm for cell line E10. Only the cell line E9 had different immunoflurescence stainings between the groups, with a positivity in the cytoplasm in the group t1/2. The western blot showed transient decrease of Cxs only in cell lines neoplastic in the times t1/2 and t1. The lung cell line E10 showed increased expression in the same time points. The real-time PCR showed significant differences in t1/2 and t1 for the cell lines B16/BL6 and E10. The melanoma cell line B16/BL6 had mRNA decreased while the cell line E10 showed increased transcription of mRNA. The cell line E9 showed a very heterogeneous expression pattern. According to our results, we can conclude that electroporation interfered transiently in the expression of connexins.

<p>In this project I have tried to obtain stable transformants of <em>Drosophila</em> melanogaster flies using electroporation. I have completed approximately 200 tests using different DNA concentrations, voltages and cuvettes, including a novel Petri dish cuvette which I developed and manufactured myself. I also developed new and more efficient procedures of egg collection and egg dechorionation. Although I was not  successful in obtaining true stable transformants, control experiments indicate that electroporation of DNA into embryos could be accomplished under the conditions used. The lack of stable transformants was probably due to failure of the electroporated DNA to integrate into the host genome. The reasons for why the DNA did not integrate was not further investigated in this study.</p>