Notre équipe a récemment développé une méthode polyvalente permettant de préparer des dérivés complexes de C60 hexa-adduits fonctionnalisés. Cette méthodologie permet d'obtenir des produits aux caractéristiques originales. Le C60 central agit comme un support central peu réactif, autour duquel des fonctionnalités sont réparties dans un espace octaédrique parfaitement défini. La première partie de ce travail de thèse a consisté à exploiter cette méthodologie pour créer des C60 hexa-adduits polycationiques aux propriétés de transfection remarquables. Dans un second temps, les dendrons polyamines synthétisés ont été mis à profit pour créer des structures supramoléculaires de C60 hexa-adduits, sous forme micellaire. Par la suite, l'étude de ces assemblages a orienté nos investigations vers l'élaboration de dérivés de C60 hexa-adduits mannosylés multivalents résultant d'un assemblage supramoléculaire, dont leurs possibles applications biologiques sont actuellement à l'étude. En parallèle une synthèse covalente a permis d'obtenir un "équivalent dendritique" de C60 hexa-adduit multimannosylé. Partant du constat que notre méthodologie est efficace principalement pour des dérivés de C60 hexa-adduits qui ont une régio-sélectivité particulière, la dernière partie a été consacrée au développement de nouvelles voies de synthèses qui pourront permettre de créer des dérivés de C60 avec un contrôle régio-sélectif original.

La dualité hydrophobe/hydrophile des molécules amphiphiles est à l'origine de leur autoassemblage en solution, sous forme de nombreuses structures supramoléculaires, telles que les micelles. Ce travail de thèse présente la formation, la caractérisation et l'étude de nouvelles micelles diacétylèniques photopolymérisables. Une première partie décrit ainsi lasynthèse de nouvelles micelles cationiques et l'étude de leur utilisation en tant qu'agent de transfert de gènes. Dans une seconde partie, nos travaux présentent l'étude de micelles polydiacétylèniques, porteuses de têtes polaires octaéthylèneglycol, comme potentiel système de délivrance de médicament. Les propriétés d'encapsulation de ces micelles ont été évaluées en présence d'un dérivé fullerène fluorescent. Puis, l'incorporation d'une sonde membranaire dans leur couronne lipophile a permis de réaliser une étude de leurs propriétés de délivrance in vitro. Une étude préliminaire de leur biodistribution in vivo a également été réalisée par tomographie à émission monophotonique grâce à la chélation d'un isotoperadioactif sur la surface des micelles. Enfin une dernière partie présente l'analyse de deux nouveaux auto-assemblages tubulaires obtenus au cours de nos travaux.

Abstract Peptide surfactants developed in the Centre for Biomolecular Engineering at The University of Queensland are engineered to combine the advantages of traditional surfactants with biodegradability, biocompatibility, formation of a mechanically strong interfacial cohesive network, and reversible stimuli-responsiveness. In this project, the potential of peptide-stabilised emulsions as delivery systems for small interfering RNA (siRNA) was explored. In recent years, the potential of siRNA as a new class of therapeutics has attracted great attention. The ubiquitous nature of RNA interference (RNAi) implies that siRNA can be used to silence any disease-causing gene to treat any disease. The hurdle that needs to be overcome to turn siRNA therapy into clinical reality is its delivery into the cytosol, where gene silencing by siRNA occurs. Although numerous systems have been developed for the delivery of siRNA, safety and efficiency are major concerns associated with current formulations. Therefore this project aimed to prepare a stable peptide emulsion formulation and to conduct initial tests of its ability to deliver siRNA in vitro. The human tumour suppressor gene p53 and the human breast cancer MCF-7 cell line were used as the model gene and model cell line, respectively. The commercially available lipid-based transfection reagent Lipofectamine™ 2000 was used as the benchmark control. Sonication and membrane extrusion were used to formulate emulsions with droplet size (d=120 nm) suitable for intravenous applications using peptide surfactant in the presence of Zn(II). Although these peptide emulsions are stable by themselves and in bovine serum, emulsion stability was found to be strongly affected by the presence of salt, EDTA, and proteins. The instability of AM1 emulsion in cell culture media has been a concern when it was subjected to in vitro cell culture tests. AM1-stabilised emulsion droplets were shown to be taken up by MCF-7 cells. However, siRNA when coupled with AM1 emulsion was not delivered into cells. Cytotoxicity studies showed that peptide surfactants did not exhibit high-level toxicity to CHO cells at the tested concentrations (0.25-2 mg mL-1). AM1 peptide-stabilised emulsions were mildly toxic to CHO cells but no toxicity was observed with MCF-7 cells. Future work could include evaluation of peptide emulsion-siRNA complex formation, and exploring the effects of different cell culture media compositions on emulsion stability and their relation to cytotoxicity.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 110-125). Also available online via the World Wide Web.

Theses (Ph. D.)--Marshall University, 2004. / Title from document title page. Includes abstract. Document formatted into pages: contains xiv, 192 p. including illustrations. Bibliography: p. 157-192.

Thesis (MSc (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: Haliotis midae is the most important aquaculture species in South Africa, with abalone farming contributing 80% of the Rand value of the aquaculture industry. Although genetic research has benefited the abalone industry, several issues still hinder increases in abalone production. Progress towards an increase in H. midae growth rate by utilizing conventional genetic studies and selective breeding has been relatively slow. Gene transfer has therefore become a plausible option to address this problem. Genes that code for certain desirable traits, such as increased growth rate, could be incorporated into the genome of commercial abalone. The current study undertook the optimization of a chemically-mediated gene transfer technique using Polyethylenimine (PEI) as transfection reagent and fluorescent proteins as reporter genes. Before gene transfer could be undertaken, several complementary studies also needed to be undertaken due to the novel nature of the study. The auto fluorescence of H. midae, the suitability of several H. midae tissues as targets for gene transfer and the cytotoxic effect of transfection reagents and selection antibiotics were assessed before gene transfer optimization could be attempted. Also, genes linked to an increase in growth rate were characterized for differential expression in different abalone age-groups to determine the suitability of these genes for incorporation into a homologous gene construct in future transfection studies. The auto fluorescence of ova, embryos and larvae were found to be comparable to that of the fluorescent reporter genes, EGFP and DsRed. A PCR-based transfection validation method was therefore employed to confirm the presence of internalized transgenes. It was established that sperm, ova, larvae and haemocyte cell culture were the most suitable target tissues for transfection. The transfection reagents, a 25kDa PEI and ExGen 500, were not cytotoxic to sperm, embryos and haemocyte cell cultures. The minimum lethal concentration of the selection antibiotics, neomycin and zeocin, was determined for larvae and haemocytes. After transfection treatment of sperm and fertilization of untreated ova, the presence of internalized transgenes could be verified for larvae. The presence of internalized transgenes could not be detected after transfection treatment of ova and larvae. Fluorescent flow cytometry and microscopy analysis of haemocytes could not detect the expression of the fluorescent reporter genes. Expression of two of the growth related genes was found to differ between age-groups. The perlustrin gene was upiv regulated in older animals, while the insulin related peptide receptor gene was down regulated in older animals. The third gene, a thrombospondin-1 precursor was stably expressed in all age-groups. This study represents the first report of transfection studies carried out on H. midae. Future studies will benefit from the groundwork established in H. midae transfection. / AFRIKAANSE OPSOMMING: Haliotis midae is die belangrikste akwakultuur spesie in Suid-Afrika met perlemoen boerdery wat 80% van die Rand waarde van die akwakultuur industrie bydrae. Alhoewel genetiese studies die perlemoen industrie ‘n hupstoot gegee het, is daar steeds sekere struikelblokke wat verdere toename in produksie verhoed. Vooruitgang ten opsigte van ‘n toename in H. midae se groei tempo deur gebruik te maak van konvensionele genetiese studies en selektiewe teling was tot dusver relatief stadig. Genetiese transformasie het daarom ‘n wesenlike alternatief geword wat moontlik hierdie probleem kan oplos. Gene wat kodeer vir sekere eienskappe, soos ‘n toename in groeitempo, kan in die genoom van kommersiële perlemoen inkorporeer word. Die huidige studie het onderneem om ‘n chemies-gemedieerde genetiese transfeksie tegniek te optimiseer en van Polyethylenimine (PEI) as transfeksie reagens en fluoresserende proteine as verklikkers gebruik te maak. As gevolg van die oorspronklikheid van die studie moes verskeie bykomende ondersoeke ook aangepak word voordat genetiese transfeksie uitgevoer kon word. Die outofluoressensie van H. midae, die geskiktheid van verskeie H. midae teiken weefsels en die sitotoksiese effek van die transfeksie reagense en seleksie antibiotika is ondersoek voordat transfeksie uitgevoer is. Gene gekoppel aan ‘n toename in groeitempo is ook gekarakteriseer vir verskille in uitdrukking in verskillende perlemoen ouderdoms-groepe om te bepaal of hierdie gene moontlik in ‘n homoloë geen konstruk ingesluit kan word vir toekomstige transfeksie studies. Dit is gevind dat die outofluoressensie van ova, embrios en larwes vergelykbaar is met die fluoressensie van die verklikker proteïene, EGFP en DsRed. ‘n PKR-baseerde metode om die internalisering van die transgeen te kontroleer is daarom gebruik. Dit is vasgestel dat sperm, ova, larwes en haemosiete die mees geskikte teiken vir transfeksie sou wees. Die transfeksie reagense, ‘n 25kDa PEI en Exgen 500, is nie sitotoksies vir sperm, embrios of haemosiete nie. Die minimum dodelike konsentrasie van die seleksie antibiotika, neomycin en zeocin, is bepaal. Na transfeksie behandeling van sperm en bevrugting van onbehandelde ova, kon die teenwoordigheid van internaliseerde transgene bevestig word vir larwes. Die teenwoordigheid van internaliseerde transgene kon nie bevestig word na transfeksie behandeling van ova en larwes nie. Fluoressente vloei sitometrie en mikroskopiese analise kon nie die uitdrukking van die fluoressente verklikker gene bevestig in haemosiete nie. Die uitdrukking van twee van die gene gekoppel aan groei het verskil tussen ouderdomsgroepe. Die perlustrin geen is meer uitgedruk in ouer diere terwyl die insulien geassosieerde peptied reseptor geen minder uitgedruk is in ouer diere. Die thrombospondin-1 voorloper geen is stabiel uitgedruk in al die ouderdomsgroepe. Hierdie studie verteenwoordig die eerste verslag van transfeksie studies uitgevoer op H. midae. Toekomstige studies sal baat vind by die grondslag wat deur hierdie projek gelê is.

Embora a imunização gênica apresente vantagens sobre as vacinas convencionais, nem sempre a produção de anticorpos e a conseqüente proteção conferida pelo sistema imune contra o antígeno de interesse são satisfatórias. As causas da baixa eficácia da imunização gênica, nestes casos, ainda são pouco conhecidas, porém um dos pontos mais relevantes em todo o processo é a eficiência da transfecção do DNA. O sucesso desta metodologia depende do estabelecimento de um sistema de entrega de gene eficiente e seguro. De acordo com a teoria proposta de Kuhn et al. (1999), baixas concentrações de moléculas anfifílicas podem ser usadas para neutralizar a carga do DNA, sem a formação de lipossomos. Este trabalho teve como objetivo avaliar o uso do lipídio catiônico dimetildioctadecil brometo de amônio (DDAB) como neutralizador da carga do DNA para a imunização de camundongos. Para a realização dos experimentos, um plasmídeo expressando a proteína do capsídeo (CA) do vírus da artrite encefalite caprina (CAEV), sob o comando do promotor citomegalovírus humano (CMV), foi utilizado nas imunizações. Os camundongos foram divididos em cinco grupos e receberam, via subcutânea, três inoculações de 10 μg de DNA linear (grupo 1), DNA linear mínimo (grupo 2), DNA circular (grupo 3) associados a 2,7 μg de DDAB ou DNA circular nu (grupo 4). O grupo controle foi inoculado com 2,7 μg de DDAB em solução fisiológica. Os camundongos imunizados com o plasmídeo na forma linear associado ao DDAB apresentaram maiores títulos de anticorpos CA que os inoculados com a forma circular ou linear mínima. Os dados sugerem que o lipídio catiônico DDAB tem potencial de uso como veículo de transfecção gênica em animais. / Genetic immunization has several advantages over conventional vaccines. However, antibody production and protection against the antigen of interest are often not satisfactory. In these cases, the causes of the low effectiveness of the genetic immunization are still poorly understood. One of the most relevant points in the immunization process is the DNA delivery. A successful DNA vaccine depends on the establishment of an efficient and safe gene delivery system. In accordance with Kuhn et al. (1999), low concentrations of amphiphilic molecules can be used to neutralize the DNA charge without the formation of liposomes. The aim of this study was to examine the effect of cationic lipid dimethildioctadecyl-ammonium bromide (DDAB) as a charge neutralizer for DNA for the immunization in mice. A plasmid expressing the capsid protein (CA) of caprine arthritis encephalitis virus (CAEV) under the control of the human cytomegalovirus immediate early promoter (hCMVie1) was used in the immunization. Mice were grouped and immunized with three injections of either 10 μg of linear DNA (group 1), minimal linear DNA (group 2) or circular DNA (group 3) associated with 2,7 μg of DDAB or naked circular DNA (group 4). The control group received 2,7 μg of DDAB diluted in physiologic solution. Mice immunized with DNA in linear form associated with DDAB developed higher titers of antibodies against CA than the animals immunized with the circular or minimal linear form. The data suggest that lipidic cationic DDAB could be a useful delivery system for genetic immunization.

Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Dajana Mihaličoková Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ.Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: In vitro assays for investigating nucleic acid assay Keywords: transfection, splice correction, BCA assay, polyplexes One of the most important tasks of biochemical research is to find out the right way how to cure cancer, genetic disorders and other illnesses which are still not curable. Towards this, gene therapy is emerging as a potential treatment owing to its ability to deliver genetic material inside the cell. Reporer gene based transfection process can be used to study gene expression. Transfection is mediated by vectors, either of viral or non-viral origin. Non-viral vectors offer several advantages over the viral counterparts like easier to synthesize, relatively cheap and the most important is their non-immunogenicity. Cationic polymers based on polyethylenimine form complexes with plasmid DNA reffered to as...

Embora a imunização gênica apresente vantagens sobre as vacinas convencionais, nem sempre a produção de anticorpos e a conseqüente proteção conferida pelo sistema imune contra o antígeno de interesse são satisfatórias. As causas da baixa eficácia da imunização gênica, nestes casos, ainda são pouco conhecidas, porém um dos pontos mais relevantes em todo o processo é a eficiência da transfecção do DNA. O sucesso desta metodologia depende do estabelecimento de um sistema de entrega de gene eficiente e seguro. De acordo com a teoria proposta de Kuhn et al. (1999), baixas concentrações de moléculas anfifílicas podem ser usadas para neutralizar a carga do DNA, sem a formação de lipossomos. Este trabalho teve como objetivo avaliar o uso do lipídio catiônico dimetildioctadecil brometo de amônio (DDAB) como neutralizador da carga do DNA para a imunização de camundongos. Para a realização dos experimentos, um plasmídeo expressando a proteína do capsídeo (CA) do vírus da artrite encefalite caprina (CAEV), sob o comando do promotor citomegalovírus humano (CMV), foi utilizado nas imunizações. Os camundongos foram divididos em cinco grupos e receberam, via subcutânea, três inoculações de 10 μg de DNA linear (grupo 1), DNA linear mínimo (grupo 2), DNA circular (grupo 3) associados a 2,7 μg de DDAB ou DNA circular nu (grupo 4). O grupo controle foi inoculado com 2,7 μg de DDAB em solução fisiológica. Os camundongos imunizados com o plasmídeo na forma linear associado ao DDAB apresentaram maiores títulos de anticorpos CA que os inoculados com a forma circular ou linear mínima. Os dados sugerem que o lipídio catiônico DDAB tem potencial de uso como veículo de transfecção gênica em animais. / Genetic immunization has several advantages over conventional vaccines. However, antibody production and protection against the antigen of interest are often not satisfactory. In these cases, the causes of the low effectiveness of the genetic immunization are still poorly understood. One of the most relevant points in the immunization process is the DNA delivery. A successful DNA vaccine depends on the establishment of an efficient and safe gene delivery system. In accordance with Kuhn et al. (1999), low concentrations of amphiphilic molecules can be used to neutralize the DNA charge without the formation of liposomes. The aim of this study was to examine the effect of cationic lipid dimethildioctadecyl-ammonium bromide (DDAB) as a charge neutralizer for DNA for the immunization in mice. A plasmid expressing the capsid protein (CA) of caprine arthritis encephalitis virus (CAEV) under the control of the human cytomegalovirus immediate early promoter (hCMVie1) was used in the immunization. Mice were grouped and immunized with three injections of either 10 μg of linear DNA (group 1), minimal linear DNA (group 2) or circular DNA (group 3) associated with 2,7 μg of DDAB or naked circular DNA (group 4). The control group received 2,7 μg of DDAB diluted in physiologic solution. Mice immunized with DNA in linear form associated with DDAB developed higher titers of antibodies against CA than the animals immunized with the circular or minimal linear form. The data suggest that lipidic cationic DDAB could be a useful delivery system for genetic immunization.

Embora a imunização gênica apresente vantagens sobre as vacinas convencionais, nem sempre a produção de anticorpos e a conseqüente proteção conferida pelo sistema imune contra o antígeno de interesse são satisfatórias. As causas da baixa eficácia da imunização gênica, nestes casos, ainda são pouco conhecidas, porém um dos pontos mais relevantes em todo o processo é a eficiência da transfecção do DNA. O sucesso desta metodologia depende do estabelecimento de um sistema de entrega de gene eficiente e seguro. De acordo com a teoria proposta de Kuhn et al. (1999), baixas concentrações de moléculas anfifílicas podem ser usadas para neutralizar a carga do DNA, sem a formação de lipossomos. Este trabalho teve como objetivo avaliar o uso do lipídio catiônico dimetildioctadecil brometo de amônio (DDAB) como neutralizador da carga do DNA para a imunização de camundongos. Para a realização dos experimentos, um plasmídeo expressando a proteína do capsídeo (CA) do vírus da artrite encefalite caprina (CAEV), sob o comando do promotor citomegalovírus humano (CMV), foi utilizado nas imunizações. Os camundongos foram divididos em cinco grupos e receberam, via subcutânea, três inoculações de 10 μg de DNA linear (grupo 1), DNA linear mínimo (grupo 2), DNA circular (grupo 3) associados a 2,7 μg de DDAB ou DNA circular nu (grupo 4). O grupo controle foi inoculado com 2,7 μg de DDAB em solução fisiológica. Os camundongos imunizados com o plasmídeo na forma linear associado ao DDAB apresentaram maiores títulos de anticorpos CA que os inoculados com a forma circular ou linear mínima. Os dados sugerem que o lipídio catiônico DDAB tem potencial de uso como veículo de transfecção gênica em animais. / Genetic immunization has several advantages over conventional vaccines. However, antibody production and protection against the antigen of interest are often not satisfactory. In these cases, the causes of the low effectiveness of the genetic immunization are still poorly understood. One of the most relevant points in the immunization process is the DNA delivery. A successful DNA vaccine depends on the establishment of an efficient and safe gene delivery system. In accordance with Kuhn et al. (1999), low concentrations of amphiphilic molecules can be used to neutralize the DNA charge without the formation of liposomes. The aim of this study was to examine the effect of cationic lipid dimethildioctadecyl-ammonium bromide (DDAB) as a charge neutralizer for DNA for the immunization in mice. A plasmid expressing the capsid protein (CA) of caprine arthritis encephalitis virus (CAEV) under the control of the human cytomegalovirus immediate early promoter (hCMVie1) was used in the immunization. Mice were grouped and immunized with three injections of either 10 μg of linear DNA (group 1), minimal linear DNA (group 2) or circular DNA (group 3) associated with 2,7 μg of DDAB or naked circular DNA (group 4). The control group received 2,7 μg of DDAB diluted in physiologic solution. Mice immunized with DNA in linear form associated with DDAB developed higher titers of antibodies against CA than the animals immunized with the circular or minimal linear form. The data suggest that lipidic cationic DDAB could be a useful delivery system for genetic immunization.