Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

Alasdair Gray`s 1982 Janine (1984): a postmodernist scottish novel

Alasdair Gray is now an established figure in the Scottish literary scene and has numerous claims to be considered an important voice writing in English. First Lanark: A Life in Four Books (1981) and then 1982 Janine (1984) contributed to the recognition of Gray as one of the founding fathers of the new Scottish writing and as a figure of importance in international contemporary fiction due to his innovative, experimental and postmodernist novels. As the title of this dissertation - “Alasdair Gray’s 1982 Janine (1984): A Postmodernist Scottish Novel” - suggests, it aims at analysing the author’s second novel, 1982 Janine (1984), in a thematic and formal perspective, in order to justify the choice of the terms - Postmodernist and Scottish - to classify this novel. 1982 Janine projects a world through Jock McLeish’s mind and is a powerful stream-of-consciousness narrative. Jock is an alcoholic who lives a personal crisis and, therefore, tries to escape from his depressing reality through sexual fantasies and political diatribes. During a single night in a Scottish hotel room, he drinks and dreams, and spends the whole night alone with his fantasies and fears, his memories and hopes. In Chapter 11, the most daring experimental section of the novel, Jock attempts to commit suicide by taking an overdose of tablets with alcohol but fails. Following this, he decides to review his life and make for a new beginning; the novel thus closing with an optimistic note. Also, the narrative is based on a constant interweaving of sex fantasy with political satire, that is, it is through his protagonist that Gray manages to convey the state of Scotland as well as the concerns and aspirations of the Scottish people and then, proceed to a political and social critique. This dissertation appears structured in three chapters. In Chapter I - “Alasdair Gray: A Postmodernist Scottish Writer” - I present Gray as a powerful postmodernist writer who also sees himself as a Scottish author, and more particularly as a Glaswegian, who concentrates on Scottish subject matter in his literary work. In a first section, I offer a brief survey of the Scottish literary scene from the fourteenth to the twentieth century, in order to understand Gray’s choice of setting and themes and to check his influence or indebtedness to previous Scottish authors. As 1982 Janine is also a good example of selfconscious experimental writing, in a second section, I present various seminal fictional works that introduced and developed experimentalism in British fiction, in order to evaluate the influence of modernist developments in form and technique on recent experimental writing. The third section consists of an introduction to Gray’s work for he is not only a novelist, but also an artist, a playwright, a poet, an activist and a scholar. Chapter II - “Postmodernist Features in 1982 Janine” - aims at listing and examining the postmodernist devices that the novel includes, in what content and form are concerned. On the one hand, the use of a developed type of the modernist stream of consciousness, the presence of a protagonist who feels entrapped in a specific system, the quest for freedom, the incoherence and fragmentation of time, the nonchronological order of the narrative, the blending of fantasy and “reality”, as well as the importance of the Scottish material are definitely current aspects within postmodernist literature that can be found in Gray’s novel. On the other hand, the handling of literary self-conscious devices, such as typographical experimentation, presence of metafiction and intertextuality, and inclusion of an Epilogue, are likewise among recurrent postmodernist features. As the title - “A Narratological Analysis of 1982 Janine” - evidences, Chapter III offers a description of the mechanics of the narrative and its functioning in order to better understand the narrative technique of postmodernist fiction. This study is based primarily on Gérard Genette’s theoretical framework and terminology, presented in Narrative Discourse: An Essay in Method, an analytical tool that allows me to provide a more objective and scientific analysis. Hence, I follow the Genettian division of narrative discourse in Time, Mood and Voice while examining the novel. Finally, I proceed to a description of the intertextual relationships 1982 Janine establishes with other texts.

A narratological analysis of D. M. Thoma`s: the white hotel (1981)

“A Narratological Analysis of D. M. Thomas’s The White Hotel (1981)” originated within a seminar on British Postmodernist Literature during the first Master’s Degree in “British and North-American Culture and Literature” (2001-04) at the Universidade da Madeira set up by the Department of English and German Studies. This dissertation seeks to present a narratological analysis of Thomas’s novel. The White Hotel stands as a paradigmatic example of the kind of literature that has dominated the British literary scene in the past three decades, commonly referred to as postmodernist fiction, owing to its formal craftsmanship (multiplicity of narrative voices and perspectives, mixing of differing genres and text types, inclusion of embedded narratives) alongside the handling of what are deemed as postmodernist topoi (the distinction between truth and lies, history and fantasy, fact and fiction, the questioning of the nature of aesthetic representation, the role the author and the reader hold in the narrative process, the instability of the linguistic sign, the notion of originality and the moral responsibility the author has towards his/her work), The narratological approach carried out in this research reveals that Thomas’s text constitutes an aesthetic endeavour to challenge the teleological drive that is inherent in any narrative, i. e., the inevitable progression towards a reassuring end. Hence, the subversion of narrative telling, which is a recurrent feature in Thomas’s remaining literary output, mirrors the contemporary distrust in totalising, hierarchised and allencompassing narratives. In its handling of historical events, namely of the Holocaust, The White Hotel invites us to reassess the most profound beliefs we were taught to take for granted: progress, reality and truth. In their place the novel proposes a more flexible conception of both the world and art, especially of literary fiction. In other terms, the world appears as a brutal chaotic place the subject is forced to adjust to. Accordingly, the literary work is deemed hybrid, fragmented and open. So as to put forth the above-mentioned issues, this research work is structured in three main chapters. The initial chapter – “What is Postmodernism?” – advances a scrutiny not only of the seminal but also of more recent studies on postmodernist literary criticism. Following this, in Chapter II – “Postmodernist British Fiction” – a brief overview of postmodernist British fiction is carried out, focusing on the fictional works that, in my opinion, are fundamental for the periodising of British postmodernism. In addition, I felt the need to include a section – “D. M. Thomas as a Postmodernist Novelist” – in which the author’s remaining literary output is briefly examined. Finally, Chapter III – “A Narratological Analysis of The White Hotel” – proposes a narratological analysis of the novel according to the particular Genettian analytical model. To conclude, my dissertation constitutes an approach to D. M. Thomas’s The White Hotel as a text whose very existence is substantiated in the foregrounding of the contingency of all discourses, meeting the postmodernist precepts of openness and subversion of any narrative that claims to be true, globalising and all-inclusive.