This thesis provides a study of an open source software project that focuses on the software development of an e-service in a municipal context. The focus is on environmental factors that either limit or promote the motivation to participate in the open source project, the "Parent-Teacher Meeting" project, a web-based communication and information channel whose purpose is to enhance the contacts between schools and parents. The empirical context is situated at the point where traditional information systems (IS) development meets new perspectives regarding organizational structures and boundaries and, as such, provides example of ongoing cross-organizational activities that break current local organizational standards. The objective of this study is to gain a deeper understanding of motivational factors for participation and adopts a sociocultural view on the topic motivation to participate. The empirical material was collected through interviews, conversations, and meetings. Being a subproject (i.e. an initiative to develop an open source software application) within a triple helix project I found an extensive number of stakeholders. The choice was made to focus on the application development; thereby a central group of participants within the development project team was found and these became the focus within the study. Moreover, I have, in this thesis, chosen to conduct a contextual description of the participants and the course of events that lead to the start of the project of study. This has been done so as to present the context, which is the focus for this study, to the reader and to be able to use these descriptions within the analysis. I have, methodologically, approached the problem from a descriptive angle with an interpretative character using a qualitative case study design. Within the thesis, the means by which the case study has been conducted is presented; i.e. the decision regarding research focus, design, and my role as researcher. In relation to the data collection, the main source has been semi-structured interviews, which is consistent with an interpretive case study character and in which my intent is to highlight conditions and events that are important to both groups within the development team. To support the investigation of those factors that can explain and assist with the interpretation of my empirical data, my description and interpretations are built on a theoretical framework based on concepts from IS theories and theories relating to human motivation. The framework, self-determination theory (SDT), is used as a lens to direct the focus onto the situated conditions that influence how individuals experience their participation within the software development project. Given the theoretical basis of an analytical comparison of ideal types of software development constructs, together with influences from motivational theories, the analytical framework used for collecting occurrences of motivational behavior and sociocultural conditions has been constructed. After the findings and my interpretation of them with the assistance of my analytical framework have been presented, a discussion and conclusions are then detailed. The conclusions of the study are argued as being relevant as an explanation for the understanding of intrinsic and internalized extrinsic motivation to participate in a hybrid open source projects. The study contributes to our understanding of some of the challenges that are to be considered when putting together and managing systems or software development processes. In this way, the study may provide some basis for improving and meeting new demands regarding how development is adopted in a mixed scenario and this provides valuable knowledge to both practice and IS research. / Grunden för denna avhandling är en studie av ett öppen källkodsutvecklingsprojekt av en kommunal e-tjänst. Fokus ligger på ett sociokulturellt perspektiv på kontextuella faktorer vilka endera hindrar eller främjar motivation för deltagande. Öppen källkodsprojektet ”Föräldramötet” är en webbaserad kommunikations- och informationskanal vars syfte är att öka och förbättra kontakten mellan hem och skola. Denna empiriska kontext är situerad i en punkt där traditionell IS-utveckling möter nya perspektiv på organisationsstrukturer och gränser samt därigenom ger exempel på pågående tvärorganisatoriska aktiviteter vilka bryter mot nuvarande interna organisationsstandarder. Målet för denna studie är att nå en djupare förståelse för deltagandets motivationsfaktorer och anammar ett sociokulturellt perspektiv på ämnet deltagandemotivation. Det empiriska materialet samlades in genom intervjuer, konversationer samt möten. Då Föräldramötetprojektet är ett underprojekt i ett trippelhelixsammanhang visade det sig initialt finnas ett stort antal intressenter. Det gjordes ett val att fokusera på applikationsutvecklingen; genom vilket en central grupp av deltagare inom utvecklingsprojektet identifierades vilka kom att komma i fokus för denna studie. Vidare har jag i denna avhandling valt att göra en kontextuell beskrivning av deltagarna och den händelsekedja som lett fram till bildandet av det studerade projektet. Detta gjordes för att i enlighet med denna studies fokus belysa sammanhanget för läsaren samt för att använda den inom min analys. Jag har närmat mig problemet från en deskriptiv synvinkel med en tolkande karaktär användande mig av en kvalitativ fallstudiemetod. Inom avhandlingen beskrivs tillvägagångssättet för fallstudiens utförande, t.ex. hur beslut fattats angående forskningsfokus, fallstudiedesign och vad min roll som forskare inneburit. I relation till datainsamlingen, har den huvudsakliga källan varit halvstrukturerade intervjuer. Dessa har varit i konsekvens med en uttolkande fallstudiemetod och har i enlighet med min avsikt utformats för att belysa villkor och händelser som har varit betydande för bägge grupperingarna inom projektgruppen. För att stödja undersökningen av de faktorer som kan förklara och hjälpa med tolkningen av mina empiriska data, har jag byggt mina beskrivningar och tolkningar på ett teoretiskt ramverk baserat på koncept från IS-teorier och teorier relaterade till mänsklig motivation. Det teoretiska ramverket ”self-determination theory” (SDT), används som en lins för att rikta brännpunkten mot de villkor som styr hur individer upplever deras deltagande inom det studerade mjukvaruutvecklingsprojektet. Givet den teoretiska bas, som bygger på analytisk karaktärisering av idealtyper av mjukvaruutvecklingsmodeller sammantaget med influenser från motivationsteorier, har ett analytiskt ramverk, för att inhämta instanser av motiverat beteende och sociokulturella villkor, utvecklats. Efter att ha redogjort för upptäckterna och mina tolkningar av dem, med hjälp av mitt analytiska ramverk, presenteras en detaljerad diskussion med slutsatser. Jag argumenterar för att dessa slutsatser är relevanta som en förklaring för förståelsen av inre samt internaliserad yttre motivation för deltagande inom detta öppen källkodsprojekt av hybridnatur. Studien bidrar till vår förståelse för vissa av de utmaningar som måste beaktas när man ska hantera system eller utvecklingsprocesser. På detta sätt kan studien bidra med en bas för att förbättra och möta nya krav på hur utveckling bedrivs i blandade miljöer och även med kunskap för både praktik och IS-forskning. / PECOI

Les antidépresseurs actuels sont très similaires au niveau de leur mécanisme d’action et sont plus ou moins efficaces. Un des problèmes majeurs est leur long temps de latence à fournir une action thérapeutique dû aux adaptations des sites pré et post synaptiques. Dans un modèle animal, nous avons récemment découvert que l’agoniste RS67333 des récepteurs 5-HT4 était en mesure de produire en trois jours les mêmes effets antidépresseurs qui normalement prennent de deux à trois semaines à apparaître avec les antidépresseurs actuellement disponibles. De plus, nous avons constaté que les effets antidépresseurs de cet agoniste possédaient une résistance à la tolérance. Il y a d’autres agonistes du même récepteur, tel que le prucalopride qui ne produit pas d’effets antidépresseurs comme RS67333. Étant donné que l’efficacité du Prucalopride à stimuler les 5-HT4Rs est similaire sinon plus grande que celle de RS67333, nous avons énoncé l’hypothèse que le récepteur 5-HT4 pourrait adopter différentes conformations actives suite à son activation par différents agonistes. Nous avons ainsi décidé d’explorer les principales réponses fonctionnelles des récepteurs 5-HT4B en observant leurs propriétés de régulation et de signalisation. Nous avons montré que l’isoforme B du récepteur 5-HT4, étant hautement exprimé dans le système limbique, détient une signalisation et une régulation différentes dépendant du ligand activateur. Nos résultats indiquent que chacun des agonistes testés (5-HT, RS67333, ML10302, Zacopride, Prucalopride) modulent distinctivement la production d’AMPc et l’internalisation du récepteur. Les résultats nous ont clairement permis de déterminer que les agonistes possèdent une efficacité et ou puissance différentes les uns par rapport aux autres. De plus, l’ordre d’efficacité des agonistes à moduler la voie de l’AMPc était (Prucalopride > Zacopride = ML10302 = 5-HT > RS67333) et est différente de leur ordre d’efficacité à induire la régulation du récepteur par internalisation (5-HT > Zacopride > Prucalopride > ML10302 = RS67333). Ainsi, nous avons montré que les 5-HT4Rs adoptent des conformations qui sont ligand-spécifiques. Cela implique que la sélectivité fonctionnelle serait un facteur important à considérer dans les mécanismes d’action antidépresseur des agonistes de ce récepteur. / Antidepressants currently available are very similar toward their mechanism of action and are more or less effective. One major problem is their long latency to provide a therapeutic effect due to adaptations of pre and post synaptic locations. In an animal model, we recently discovered that the agonist RS67333 of the 5-HT4 receptors was able to produce in three days the same antidepressant effects that normally take two to three weeks to appear with the currently available antidepressants. In addition, we found that the antidepressant effects of this agonist had a resistance to tolerance. There are others agonists of the same receptor such as prucalopride, which does not produce antidepressant effects as RS67333. Since the effectiveness of prucalopride to stimulate 5-HT4Rs is similar if not greater than RS67333, we stated the hypothesis that the 5-HT4 receptor could adopt different active conformations following its activation by various agonists. We decided to explore the major functional responses of 5-HT4B by observing their regulatory and signaling properties. We showed that the B isoform of the 5-HT4, being highly expressed in the limbic system, has a different signaling and regulation depending on the ligand. Our results indicate that each of the agonists tested (5-HT, RS67333, ML10302, Zacopride, Prucalopride) distinctively modulate cAMP production and receptor internalization. The results have clearly identified that agonists differed in potency and efficacy. Moreover, the order of effectiveness of agonists to modulate the cAMP pathway was (prucalopride> zacopride = 5-HT = ML10302> RS67333) different from their order of effectiveness in inducing receptor regulation by internalization (5-HT> Zacopride> Prucalopride> RS67333 = ML10302). Thus, we have shown that 5-HT4Rs adopt conformations that are ligand-specific. This implies that functional selectivity is an important factor in the mechanisms of antidepressant action of this receptor agonists.

Les actions thérapeutiques des antidépresseurs, disponibles actuellement, requièrent plusieurs semaines de traitement. Ce délai est dû aux adaptations des sites pré et post-synaptiques qui, respectivement, augmentent la disponibilité synaptique des monoamines sérotonine et noradrénaline (5-HT et NA), et entraînent les changements neuroplastiques modifiant la fonction neuronales dans les régions limbiques. Il a été récemment observé, chez un modèle animal de dépression, que l’agoniste RS67333 des récepteurs sérotoninergiques de type 5-HT4 produisait des changements comportementaux, électrophysiologiques, cellulaires et biochimiques, tel qu’observé chez les antidépresseurs. Ces changements apparaissent seulement après 3 jours de traitement tandis que les antidépresseurs nécessitent souvent plusieurs semaines. De plus, l’activation des récepteurs 5-HT4 ne générait pas de tolérance, et cela pendant 21 jours de traitement. Seulement, les propriétés de signalisation et de régulation de ces récepteurs sont très loin d’êtres établies. Nous avons alors voulu mieux caractériser ces deux aspects de leur fonction, en se concentrant d’avantage sur les isoformes a et b, fortement exprimés dans le système limbique. Pour cela, nous avons voulu évaluer d’abord leur capacité de production d’AMPc dans un système hétérologue. Les essais d’accumulation d’AMPc démontrent que les deux isoformes sont capables de moduler positivement et négativement des niveaux d’AMPc en présence de 5-HT. Par contre, la stimulation au RS67333 induit seulement une augmentation du niveau d’AMPc dans les deux cas. Ensemble, ces observations indiquent que les deux isoformes sont capables de coupler à l’adénylate cyclase à travers les protéines Gαs et Gαi. La quantification des récepteurs internalisés a montré que l’isoforme b internalisait plus efficacement que l’isoforme a suite à l’incubation à la 5-HT (61 ± 3 % pour le b vs 40 ± 2 % pour le a). Les protéines kinases PKA et PKC n’étaient pas impliquées dans cette différence, toutefois, la PKC a été trouvée essentielle à l’internalisation des deux isoformes. L’internalisation de l’isoforme b par 5-HT n’a pas été affecté par la surexpression de forme inactive de GRK2 (GRK2- K220R) et a été partiellement inhibé par un mutant négative de la β-arrestine (βarr(319-418)), tandis que l’internalisation de l’isoforme a a été bloquée par les deux. Ces observations indiquent que les mécanismes d’internalisation des deux isoformes du récepteur 5-HT4 les plus abondants dans le système nerveux central sont distincts. Des comportements spécifiques à chaque isoforme ont aussi été constatés au niveau de la régulation fonctionnelle suite à l’exposition au RS67333, qui désensibilise seulement l’isoforme b. D’après nos observations, nous avons conclu que les isoformes a et b diffèrent dans leur propriétés de signalisation et de régulation. L’incapacité du RS67333 à désensibiliser l’isoforme a fournit un substrat moléculaire pour les effets antidépressifs prolongés de cet agoniste dans les études pré-cliniques. / The therapeutic actions of antidepressants, currently available, require several weeks of treatment. This delay is due to pre-and post-synaptic sites adjustments, which, respectively, increase the availability of synaptic monoamines 5-HT and NA, and induce neuroplastic changes amending neuronal function in the limbic regions. We have recently observed in animal model of depression that serotonergic 4 receptor agonist RS67333 produces behavioural, electrophysiological, cellular and biochemical changes, as seen in antidepressants. More importantly, these changes appear only after 3 days of treatment while antidepressants often require several weeks. Moreover, activation of 5-HT4 receptors does not generate tolerance and that for 21 days of treatment. However, the signalling and regulation properties of these receptors haven’t been established yet. Here, we wanted to better characterize these two aspects of their function, and in particular for isoforms a and b, strongly expressed in the limbic system. First, we assessed their ability to produce cAMP in a heterologous system. Functional assays revealed that both isoforms were capable of positive and negative modulation of cAMP levels by 5-HT. Stimulation by RS67333 induced cAMP stimulation. Together, these observations indicate that that both isoforms are able to couple adenylate cyclase through Gαs and Gαi proteins. Moreover, quantification of receptors sequestration showed that isoform b internalised more efficiently than the isoform a, following incubation with 5-HT (61 ± 3 % for b, 40 ± 2 % for a). PKA and PKC proteins, two seconds messenger kinases activated by these receptors, are not involved in producing this difference. However, PKC is essential to the internalization of both isoforms. Isoform b internalization was not affected by an inactive GRK2 mutant (GRK2-K220R) and was partially inhibited by dominant negative β-arrestin (βarr(319-418), while isoform a internalization was dependant on both. These observations indicate that internalization mechanisms of the two isoforms, most abundantly expressed in the central nervous system, are different. Functional desensitization studies showned that RS67333 selectively desensitizes the isoform b, but not isoforme a. Based on these observations, we conclude that isoform a and b differ in their signalling and regulatory properties. Isoform a incapacity to desensitization by RS67333 provides a molecular substrate for prolonged antidepressant affects of this agonist in pre-clinical studies.

科学研究費補助金 研究種目:基盤研究(C)(2) 課題番号:07610121 研究代表者:速水 敏彦 研究期間:1995-1996年度

[pt] O tema da normatividade desde sempre foi tido como misterioso. Muitas explicações foram dadas sobre o fenômeno em diversos âmbitos do saber, embora nenhuma em definitivo. Quando se trata da normatividade jurídica não é diferente. Com o objetivo de trazer novas luzes sobre o nebuloso assunto, o ponto de partida da presente investigação é o conceito de afirmações internas do direito, tal como formulado por Herbert L. A. Hart. Por meio de uma análise sociolinguística, o autor propõe que tais enunciados comprometidos com o direito sejam vistos como expressões da aceitação de certas regras. No entanto, o autor não vai muito além em pontos importantes e alguns questionamentos surgem tanto sobre a melhor leitura de certos conceitos na obra de Hart, quanto em relação a real capacidade de sua teoria dar conta do tema. Há evidências nos escritos do autor que permitem dizer que a sua proposta é bastante semelhante à ideia de expressivismo de normas, tal como formulado por Allan Gibbard no campo da metaética. Essa linha teórica aparece como uma versão sofisticada de não-cognitivismo e, portanto, entende que os termos normativos são geralmente utilizados na linguagem ordinária para expressar um estado conativo, um estado mental diferente de uma crença, e que, portanto, não possui aptidão de verdade. Pretende-se demonstrar que tal postura, expressivista, é bastante atraente para o filósofo do direito, pois consegue explicar tanto as afirmações internas do direito como o elo implícito com a ideia de normatividade. Além disso, essa perspectiva é capaz de responder às críticas que teóricos rivais (cognitivistas) formularam sobre a construção conceitual hartiana. Por meio da análise da superação por parte dos autores expressivistas de argumentos tradicionais do campo da metaética é possível deixar mais sólida a posição dentro da teoria do direito, bem como transferir o ônus argumentativo para os oponentes da posição. Por fim, será sugerida interpretação sobre o mecanismo psicológico e social por detrás do expressivismo de normas. O recente corpo de evidência científica parece fornecer uma licença para o otimismo em favor do expressivismo em relação à capacidade de se desvendar o mistério da normatividade. / [en] Normativity has Always been taken as something mysterious. Many explanations from a range of different areas were given about this phenomenon, though, no definitive one. Legal normativity is no different. Aiming to bring new lights to this cloudy subject, the starting point of the present investigation is Hebert L. A. Hart s concept of internal legal statements. Through a sociolinguistic analysis, the author claims that such statements committed with the law are to be seen as expressions of rule s acceptance. Nevertheless, Hart does not go further and a lot of relevant points and questions arise both about the best way to read his work and on the real explanatory power of his theory. There are evidences in his writings that allow us to read his theory in a very similar way to Allan Gibbard s metaethics one. This line of though seems to be a sophisticated version of a non-cognitivism and, therefore, sees normative terms as used to express conative states of mind. These mental states are different from a belief and hence cannot have truth aptness. We intend to show that such theoretical posture, expressivist, is very alluring for the legal philosopher, since it can explain the internal legal claims and its implicit relationship with normativity. Further, this perspective is capable of answering critics posed by cognitivists about Hart s conceptual work. By means of an analysis of how expressivism can answer traditional metaethical questions, it is possible to make the legal expressivist position even more solid, and to switch the argumentative burden to opponent side of the dispute. Lastly, we will indicate an interpretation of a social and psychological background mechanism to norm expressivism. The recent body of scientific evidence provides a license for optimism in favor of expressism s ability to unveil the mystery of normativity.

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Le milieu hospitalier est un monde à la fois sensible et complexe, sensible parce que la vie humaine est en jeu et complexe parce que les équipements médicaux augmentent en nombre et en complexité technique. Ainsi, afin de préserver le bon état de fonctionnement de ces équipements et à un niveau élevé de disponibilité, leur entretien est devenu l'une des préoccupations majeures des responsables de l’hôpital. L’objectif de cette thèse est de proposer, aux responsables de maintenance biomédicale dans les établissements de soins, des outils d’aide à la décision qui permettent une meilleure maitrise des coûts. Ceci en assurant la sécurité des patients et des utilisateurs et en maintenant des performances optimales de l’ensemble des équipements médicaux. Tout d’abord, une heuristique a été proposée pour le choix de l’internalisation ou de l’externalisation de la maintenance et pour la sélection du contrat adéquat. La sélection du contrat est basée sur un ensemble de critères tout en considérant la contrainte du budget disponible. Ensuite, afin d’améliorer la procédure proposée, nous avons proposé des outils d’aide à la décision multicritère pour le choix adéquat d’une stratégie de maintenance. Pour l’étude de la criticité des équipements médicaux et le choix de la maintenance, sept critères ont été étudiés en proposant un couplage de l’approche AHP « Analytical Hierarchy Process » à la technique TOPSIS « Technique for Order Performance by Similarity to Ideal Solution ». Comme les experts du service de maintenance présentaient une certaine incertitude dans leurs jugements, nous avons intégré l’évaluation linguistique floue dans l’étude de la criticité des équipements et dans la sélection de la stratégie de maintenance (Fuzzy AHP couplée avec Fuzzy TOPSIS). Un modèle mathématique MILP a été développé pour la définition des limites de la criticité afin de caractériser les trois stratégies de maintenance. Le bon choix de ces limites permet d’optimiser le coût de la maintenance en respectant le budget disponible. Enfin, un deuxième modèle mathématique MILP a été développé en se basant sur l’heuristique proposée. Ce modèle permet de sélectionner pour chaque équipement, la stratégie de maintenance, internaliser ou externaliser la maintenance et le type du contrat tout en considérant le budget disponible et la charge/capacité du service maintenance / The hospital is a world that is both sensitive and complex, sensitive because the human life is involved and complex because medical facilities are growing in number and in technical complexity. Then, the problem of the medical equipment maintenance in order to keep them in safe, reliable and with high level of availability has become a major preoccupation of the hospital. The objective of this thesis is to provide tools to help the biomedical maintenance service of the hospital to make decisions that allow a better control of costs, while ensuring patient and user safety and maintaining optimal performance of medical equipment. First, a heuristic has been proposed for the choice of internalization or outsourcing maintenance and for the selection of the appropriate contract. The selection of the contract is based on a set of criteria while considering the available budget constraint. Then, to improve the proposed procedure, we proposed multi-criteria decision-making tools to select the appropriate maintenance strategies. Seven criteria have been designed to study the criticality of medical equipment and the choice of maintenance by providing a coupling of the AHP approach "Analytical Hierarchy Process" with TOPSIS technique "Technique for Order Performance by Similarity to Ideal Solution." As the expert judgments of the maintenance department presented some uncertainty, we integrated the fuzzy language assessment of the criticality of the equipment and the selection of the maintenance strategy (Fuzzy AHP coupled with Fuzzy TOPSIS). A mixed integer linear programming model (MILP) was developed to define thresholds of criticality to characterize the three maintenance strategies. According to these thresholds, maintenance cost can be optimized within the available budget. Finally, a second mixed integer linear programming model (MILP) was developed based on the proposed heuristic. This model allows selecting for each equipment, the maintenance strategy, the internalization or the outsourcing of the maintenance and the type of contract while considering the available budget and the workload / capacity of the maintenance department

The thesis entitled “The Role of Liposomal Hybrids and Gold Nanoparticles in the Efficacious Transport of Nucleic Acids and Small Molecular Drugs for Cancer Nanomedicine” elucidates the preparation of various liposomal formulations of cationic monomeric and gemini lipids where hydrophobic domains were consisted of tocopherol, cholesterol and pseudoglyceryl backbone for the cellular transport of nucleic acids. The thesis continues while elucidating the role of various pH sensitive molecules and gold nanoparticles in liposomes to improve the delivery efficacy levels. This thesis also elucidates the role of gold nanoparticles stabilized with natural pH sensitive molecules for efficacious drug delivery applications. Additionally, the role of such pH sensitive gold nanoparticles in association with liposomes for the co-delivery of drug and gene has been discussed. The work has been divided into six chapters. Chapter 1A: Dimeric Lipids Derived from α-Tocopherol as Efficient Gene Transfection Agents. Mechanistic Insights into Lipoplex Internalization and Therapeutic Induction of Apoptotic Activity In this chapter, we present cationic dimeric (gemini) lipids for significant plasmid DNA (pDNA) delivery to different cell lines without any marked toxicity in the presence of serum. The six gemini lipids possess α-tocopherol as their hydrophobic backbone and differ from each other in terms of their spacer chain lengths. Each of these gemini lipids mixed with a helper lipid 1, 2-dioleoyl phosphatidyl ethanolamine (DOPE), was capable of forming stable aqueous suspensions. These co-liposomal systems were examined for their potential to transfect pEGFP-C3 plasmid DNA in to nine cell lines of different origins. The transfection efficacies noticed in terms of EGFP expression levels using flow cytometry were well corroborated using independent fluorescence microscopy studies. Significant EGFP expression levels were reported using the gemini co-liposomes which counted significantly better than one well known commercial formulation lipofectamine 2000 (L2K). Transfection efficacies were also analyzed in terms of the degree of intracellular delivery of labeled plasmid DNA (pDNA) using confocal microscopy which revealed an efficient internalization in the presence of serum. The cell viability assays performed using optimized formulations demonstrated no significant toxicity towards any of the cell lines used in the study. We also had a look at the lipoplex internalization pathway to profile the uptake characteristics. A caveolae/lipid raft route was attributed to their excellent gene transfection capabilities. The study was further advanced by using a therapeutic p53-EGFP-C3 plasmid and the apoptotic activity was observed using FACS and growth inhibition assay. Figure 1. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of p53-EGFP-C3 plasmid DNA that induces significant apoptotic response. Chapter 1B: Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by Cationic Gemini Tocopheryl Lipids Non-viral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding the unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We discuss in this chapter about six tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient co-liposomal formulations derived from each of these geminis and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE) were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized co-liposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz. HEK 293T, HeLa and Caco-2 significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and presence of serum (FBS). Notably, the knockdown activity of co-liposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR and western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines. Figure 2. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of siRNA against survivin that induces significant apoptotic response. Chapter 2: Efficacious in Vitro EGFP Expression and Silencing in Serum by Cationic Pseudoglyceryl Gemini Lipids To elicit the desirable efficacy levels in cationic liposome mediated nucleic acid therapeutics has been part of extensive scientific efforts. This chapter describes three cationic gemini lipids and application of their co-liposomes with DOPE as potent pDNA (plasmid DNA) and siRNA (small interfering RNA) cytofectins for remarkably advanced efficacy levels in numerous cell lines in the presence of serum. The hydrophobic structural lineament of cationic gemini lipids is made up of pseudoglyceryl backbone linked to the hydrocarbon chains via oligo-oxyethylene units. The stable aqueous co-liposomal suspensions of gemini lipids showed an efficient binding to pDNA or siRNA and their significant intracellular delivery in various cell lines. The transfection capabilities of different co-liposomal formulations were profiled based on EGFP expression (pEGFP-C3 pDNA transfection) and EGFP knockdown (anti-GFP siRNA transfections) in EGFP expressing cell lines. The cellular EGFP expression levels and intracellular delivery of labeled nucleic acids were thoroughly studied using flow cytometry (FACS), fluorescence and confocal microscopy. The MTT based cell viability assay revealed no loss in cell viabilities for all of the transfection optimized lipoplexes of siRNA or pDNA. The transfection profile of gemini co-liposomes was noted to be significantly much better than a commercial lipofection reagent, Lipofectamine 2000 used for pDNA and siRNA applications in each of the cell lines studied. The co-liposomes and their transfection optimized lipoplexes were physiochemically characterized extensively by means of zeta potential, dynamic light scattering (DLS) and atomic force microscopy (AFM). In brief, these new gemini co-liposomal formulations seem to offer a great opportunity for successful nucleic acid (DNA and siRNA) delivery in a practical scenario. Figure 3. Efficacious EGFP expression (pDNA transfection) and EGFP silencing (anti GFP siRNA transfection) mediated by co-liposomes of pseudoglyceryl gemini lipids and DOPE. Chapter 3: Efficient Elicitation of Liposomal Nucleic acid delivery through the Eminence of Gold Nanoparticles Stabilized with pH Responsive Short Tripeptide Derived from Tyrosine Kinase NGF Receptors The prerequisite in the area of gene therapy today is to serve transfection efficient formulations nullifying the enduring key issues. To this end, we discuss in this chapter, the role of hybrid liposomal formulations derived from structurally distinct cationic lipids, a neutral lipid (DOPE) and pH responsive short tripeptide (KFG, Lys-Phe-Gly) capped gold nanoparticles (PAuNPs). The hybrid liposomes are presented to be efficient enough to transfect pDNA leading to remarkably high gene expression levels in various cell lines of different origins in the presence of serum (FBS). Hybrid liposomes could deliver pDNA more effectively than the native liposomes and commercial standard lipofectamine 2000 (L2K) across the entire range of N/P ratios studied under the influence of intracellular pH response and gold nanoparticles prominence. The gene transfection capabilities are profiled based on transfections performed using two different plasmids (pGL3, luciferase activity and p-EGFP-C3, green fluorescent protein expression). pDNA cellular internalization and subsequent gene expression levels are studied using flow cytometry, fluorescence microscopy and confocal microscopic studies. The extensive physiochemical characterization of hybrid liposomal formulation and their complexes with pDNA in comparison with respective native liposomes was performed using AFM, TEM, Zeta, DLS, gel retardation assay, U.V. and fluorescence emission measurements. The hybrid liposomes are shown to possess significantly higher fusion activity at lowered pH of intracellular compartments. These hybrid liposomes are fairly biocompatible across the concentration range used in transfection experiments. Precisely, introduction of these pH responsive tripeptide capped gold nanoparticles in to liposomal formulations straightforwardly must be more advantageous for a practical application in biomedical scenario to achieve therapeutic levels. Figure 4. The hybrid of liposomes and tri-peptide capped gold nanoparticles for significantly improved gene expression levels. Chapter 4: RNA Aptamer Decorated pH Sensitive Liposomes for Active Transport of Nucleic Acids in Specific Cancer Cells This chapter describes the target specific transport of pH sensitive liposomes loaded with a RNA aptamer for promising nucleic acid therapeutics. The pH sensitive liposomes are constructed from a cationic cholesteryl gemini lipid (CGL), neutral helper lipid (DOPE) and gemini analog of a pH sensitive lipid, palmitoyl homocysteine (GPHC). The liposomes are shown to be significantly fusogenic that deliver the cargoes upon lowerin the pH (6.0). The fusogenic behaviour of the liposomes was thoroughly studied by means of dynamic light scattering (DLS), zeta potential, lipid mixing, calcein dequenching and atomic force microscopy (AFM). The facile integration of cholesterol conjugated RNA aptamer in liposomes derived from cholesteryl gemini lipids was exploited for their delivery to specific cancer cells. The RNA aptamer specifically binds to epithelial cell adhesion molecule (EpCAM) with high affinity which is a cell surface marker in various solid cancers such as colorectal and breast carcinoma. These aptamer decorated pH sensitive liposomes could efficiently enter the EpCAM expressing COLO-205, Caco-2, MCF-7 and MDA-MB-231 cell lines while no such noticeable liposome transport was observed in EpCAM negative HEK 293T cells as evidenced by flow cytometry and confocal microscopy. Additionally, the liposomes are shown to be actively transported inside the cells, i.e., receptor mediated endocytosis. These liposomes could complex the nucleic acids (pDNA) in an efficient manner. The MTT based cell viability assay accounted no noticeable loss in cell viabilities for liposome treatments. Concisely, we have formulated RNA aptamer loaded pH sensitive liposomes that would certainly be promising tool in target based cancer nanomedicine. Figure 5. (A) Cellular internalization of DY-647 labeled aptamer loaded pH sensitive liposomes. (B) The liposomes were actively internalized through receptor mediated endocytosis. Each panel (A and B) represents (from left to right) bright field image, aptamer fluorescence, DAPI stained nuclei and merge of previous three impressions. Chapter 5: Natural Tri-peptide Capped Gold Nanoparticles for Efficacious Doxorubicin Delivery in Vitro and in Vivo Nanotechnology has gained ever increasing interest for the successful implementation of chemotherapy based treatment of cancer. This chapter describes the role of gold nanoparticles (AuNPs) capped with a natural pH responsive short tri-peptide (Lys-Phe–Gly or KFG) for significant intracellular delivery of an anti-cancer drug, doxorubicin (DOX). A significantly increased apoptotic response was noted for DOX treatments mediated by KFG-AuNPs in comparison with drug alone treatments in various cell lines (BT-474, HeLa, HEK 293T and U251) in vitro. Furthermore, KFG-AuNPs mediated DOX treatment significantly decreased cell proliferation and tumor growth in BT-474 cell xenograft model in nude mice. In addition, KFG-AuNPs showed efficacious drug delivery in DOX-resistant HeLa cells (HeLa-DOXR) in comparison with drug alone treatments. Figure 6. Representative images of excised tumors after doxorubicin treatment mediated by pH responsive tri-peptide capped gold nanoparticles (DOX-KFG-AuNPs) (C) in comparison with doxorubicin alone treatments (B) and untreated tumors (A). Extensive cell death as observed under Hematoxylin/eosin (H&E) (D) and TUNEL (E) staining of DOX-KFG-AuNPs treated tumor sections. Chapter 6: Significant Apoptotic Activity Induction by Efficacious Co-delivery of p53 Gene and Doxorubicin Mediated by the Combination of Co-liposomes of Cationic Gemini lipid and pH Responsive Tri-peptide Combining chemotherapy with gene therapy has appeared as an efficient tool to treat complex biological disorder like cancer. Herein, we show efficient co-delivery of DNA and an anti-cancer drug, doxorubicin (DOX) by means of gemini cationic liposome (GCL) based lipoplex nanoaggregates that are coated with DOX encapsulated pH responsive tripeptide nanovesicles. The lipoplex, tripeptide vesicles and their association was thoroughly studied using dynamic light scattering (DLS), zeta potential, atomic force microscopy (AFM). Flow cytometry, fluorescence and confocal microscopic analysis revealed that the GCL-tripeptide association could significantly co-deliver the p53 expression plasmid (p53-EGFP-C3) and DOX in HeLa and HEK 293T cells in the presence of serum. A synergistic increase in gene expression level and DOX internalization was observed in co-delivery which was even substantially higher than individual lipoplex transfection and DOX treatment. The apoptosis induced due to p53 expression and DOX was profiled with the help of annexin-V positivity analysis under flow cytometry and nuclear damage analysis by DAPI nuclei counterstaining under confocal microscopy which noted to be significantly higher in cells during co-delivery. The MTT based cell viability assay revealed a significantly increased loss in cell viability counts for co-delivery treatments. Such a system delivering synergistically increased significant efficacy levels in combinatorial drug and nucleic acid therapeutics would be certainly advantageous for practical biomedical applications. Figure 7. The co-delivery of pDNA and drug (doxorubicin) mediated by GCL-tripeptide association as observed under (A) confocal microscopy (pDNA; green and doxorubicin; red) and (B) flow cytometry.