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.

Argilas modificadas como catalisadores ácidos sólidos

Important bentonitic deposits are present in Porto Santo Island, part of the Madeira Archipelago. Several locations were selected and samples were collected and characterised. The bentonite obtained at Serra de Dentro (SD) was selected for further laboratorial work. The fine fraction of SD bentonite was purified using several methods and the sodium homoionic form was prepared. This was the starting material used in the three generic types of modifications: metal exchange, acid activation and pillaring. These modifications produce materials with markedly different acidic (e.g. Brönsted and/or Lewis acidity), textural (e.g. increase of the surface area and active site accessibility) or structural (e.g. creation of permanent porous structures) properties. The wide range of materials obtained (including reference clays counterparts) was characterised in terms of chemical, structural, textural and catalytic properties. Limonene is an important raw material produced in Portugal, and its aromatisation reaction was chosen for the catalytic characterisation of the clay catalysts prepared.

Preservação digital: o estado da arte

CUNHA, Jacqueline; GALINDO, Marcos. Preservação digital: o estado da arte. In:ENCONTRO NACIONAL DE PESQUISA EM CIÊNCIA DA INFORMAÇÃO, 8., Savador, 2007. Anais... Salvador: ANCIB, 2007. Disponível em:

A utilização de serviços odontológicos entre crianças e fatores associados em Sobral, Ceará, Brasil

NORO, L. R. A. et al. A utilização de serviços odontológicos entre crianças e fatores associados em Sobral, Ceará, Brasil. Cad. Saúde Pública, v. 24, n. 7, p. 1509-1516. 2008. ISSN 0102-311X.

Fatores críticos de sucesso das empresas familiares: uma abordagem competitiva

The present objective study to inside identify the critical factors of success of the local family companies of a competitive boarding. For in such a way the same it understands the culture and the management of the family companies of success, identifies the essential areas of performance, it establishes the restrictive factors of the success, and analyzes the level of influence of the critical factors of success in the competitiveness of this type of company. In function of the subject little to be explored, and of this study to provide a general vision concerning the factors that take the family companies to get success, this research is explorer. On the other hand, for describing characteristic of the familiar companies in prominence in the local scene and for being worried about the practical performance, the same one also is descriptive. The sample in turn is the not-probabilist one of the intentional type, for accessibility. For operacionalization of the collection of data, the direct contact was used, being the composed instrument of research for variable as management, culture, critical factors of success and competitiveness. The study it evidences that in regards to the management and the culture of the family companies of success, some variable are turned aside from the standard of the conventional family companies cited by literature. Of general form in the familiar companies of success it has a bigger level of professionalization of the management. As for the value given to the knowledge, the study sample that the conventional family companies give little importance to it, in contrast of the family companies in prominence, who value of significant form the search for the knowledge. He is demonstrated despite the family companies of success, even so total are not professionalized, possess a bigger level of professionalization of the management, ratifying of certain forms the reason for which the majority develops the Strategical Planning formal periodically. In short, the results point 17 critical factors of success with respect to the family companies, in special factors as the product quality and services, and the use of the technology

Auto-eficácia de alunos de administração: um estudo com concluintes do curso de graduação em Administração no Estado do Pará

The current study presents the characteristics of self-efficacy of students of Administration course, who work and do not work. The study was conducted through a field research, descriptive, addressed quantitatively using statistical procedures. Was studied a population composed of 394 students distributed in three Higher Education Institutions, in the metropolitan region of Belém, in the State of Pará. The sampling was not probabilistic by accessibility, with a sample of 254 subjects. The instrument for data collection was a questionnaire composed of a set of questions divided into three sections: the first related to sociodemographic data, the second section was built to identify the work situation of the respondent and the third section was built with issues related to General Perceived Self-Efficacy Scale proposed by Schwarzer and Jerusalem (1999). Sociodemographic data were processed using methods of descriptive statistics. This procedure allowed characterizing the subjects of the sample. To identify the work situation, the analysis of frequency and percentage was used, which allowed to classify in percentage, the respondents who worked and those that did not work, and the data related to the scale of self-efficacy were processed quantitatively by the method of multivariate statistics using the software of program Statistical Package for Social Sciences for Windows - SPSS, version 17 from the process of Exploratory Factor Analysis. This procedure allowed characterizing the students who worked and the students who did not worked. The results were discussed based on Social Cognitive Theory from the construct of self-efficacy of Albert Bandura (1977). The study results showed a young sample, composed the majority of single women with work experience, and indicated that the characteristics of self-efficacy of students who work and students who do not work are different. The self-efficacy beliefs of students who do not work are based on psychological expectations, whereas the students who work demonstrated that their efficacy beliefs are sustained by previous experiences. A student who does not work proved to be reliant in their abilities to achieve a successful performance in their activities, believing it to be easy to achieve your goals and to face difficult situations at work, simply by invest a necessary effort and trust in their abilities. One who has experience working proved to be reliant in their abilities to conduct courses of action, although know that it is not easy to achieve your goals, and in unexpected situations showed its ability to solve difficult problems

Democratização do acesso ao ensino superior: os cursos de graduação à distância na UFRN

This thesis pursuits to contextualize the theoretical debate between the implementation of public education policy of the Federal Government focused in a distance learning and legal foundations for its enforcement, in order to raise questions and comments on the topic in question. Its importance is back to provide scientific input and can offer to the academy, particularly in the UFRN, and elements of society to question and rethink the complex relationship between the socio-economic and geographic access to higher education. It consists of a descriptive study on the institutionalization of distance education in UFRN as a mechanism for expanding access to higher education, for both, the research seeks to understand if the distance undergraduate courses offered by the UAB system and implemented at UFRN, promote expanding access to higher education, as it is during implementation that the rules, routines and social processes are converted from intentions to action. The discussion of this study lasted between two opposing views of Implementation models: Top-down and Bottom-up. It is worth noting that the documents PNE, PDE and programs and UAB MEETING reflect positively in improving the educational level of the population of the country It is a qualitative study, using the means Bibliographic, Document and Field Study, where they were performed 04 (four) in 2010 interviews with the management framework SEDIS / UAB in UFRN. The data were analyzed and addressed through techniques: Document Analysis and Content Analysis. The results show that the process of implementation of distance education at UFRN is in progress. According to our results, the research objective is achieved, but there was a need to rethink the conditions of the infrastructure of poles, the structure of the academic calendar, the management of the SEDIS UFRN, regarding the expansion of existing vacancies and the supply of new courses by the need for a redesign as the Secretariat's ability to hold the offerings of undergraduate courses offered by the Federal Government to be implemented in the institution. It was also found that levels of evasion still presents a challenge to the teaching model. Given the context, we concluded that the greatest contribution of UAB and consequently UFRN by distance learning for undergraduate courses (Bachelor in Mathematics, Physics, Chemistry, Geography and Biological Sciences, beyond the bachelor's degrees in Business and Public Administration ) is related to increasing the number of vacancies and accessibility of a population that was previously deprived of access to university

A cidade e o idoso

This thesis concerns the study of the city and impacts that will be caused in its structure due to the aging process of the human society, mainly in Brazil. The most important focus will be those related to problems of accessibility, leisure, housing, health and labor, issues that most affect people over 60 years of age. Beyond the analysis of inherent problems to the subject, proposals will be made for urban intervention that the cities become more suitable for the living of the elderly. To support this study, a review of different theories about the city was carried out, then a panel about the presence of the elderly in society, including Brazil, and in the cities, aiming at a vision, as broad as possible, on how the elderly were treated throughout history. In order to establish paradigms and parameters in the approach to the subject, a series of systematic observations on the urban space in different cities proceeded, in Brazil and abroad, with works aiming the inclusion of the elderly in urban areas, such as Amsterdam, Barcelona, Brasilia, Luanda and Rio de Janeiro, a city considered by the UN as physical and territorial urban laboratory suitable for the elderly. From there we tried to obtain, in addition to literature and observation of other countries experiences, the conduct of field research and official standards analysis, the theoretical basis for establishing guidelines on how to plan and design a more appropriate urban space for the elderly

Turismo cultural na ZEPH-Ribeira: possibilidades e limitações

Considering that urban interventions, planned and executed recently at Natal, RN, should put Ribeira - one of the town centers - in the new limits of the expanded center of the city, this work examines the use of this new accessibility for the benefit of the development of an infrastructure to support a type of tourism currently seen as an alternative to diversification into areas dominated by entertainment tourism (the case of Natal). A local with city's traditional cultural facilities and holder of a set built that is a synthesis of the images of the neighborhood, it fears that this increase in the potential for movement and flows of use stimulate speculation and deployment of a bulk type of activity incompatible with the preservation the architectural heritage. Important economy for the city, tourism has been showing a factor propellant processing urban related changes in accessibility. It is argued that cultural tourism, often used in plans and projects of revitalization across the country, may be factor for sustainable development and conserve the assets, and contributes to the process of revitalization of the historic cultural center as Ribeira. Intended to illustrate the need to consider other information that might help in the revitalization of the neighborhood, this research presents the offer cultural tourism of Ribeira and identifies and maps certain physical attributes that, combined with functional attributes, can stimulate or inhibit the diversity and vitality in the region

A forma do privilégio: renda, acessibilidade e densidade em Natal-RN

The aim of this work is to understand the morphological expression of ground occupation by the higher income population, by focusing on population distribution in accordance with income layers and demographical density, as well as topological accessibility (HILLIER and HANSON, 1984) resulting from the urban grid structure. It endeavors to identify a functional organizing principle regarding the intra-urban space of Natal capital city of the state of Rio Grande do Norte, the research focus. In order to achieve this, census data as well as syntactic data were utilized for mapping and spatial analysis of income patterns, topological accessibility and demographical density using Geographical Information System GIS. The organizing principle was named as the Form of Privilege, a pattern that concentrates or tends to concentrate wealth, topological accessibility and low demographical density. Attempting to assess its extent, beyond Natal, this principle was applied to other Brazilian northeastern capitals such as: Fortaleza, CE; Teresina, PI; Aracaju, SE; Recife, PE; and João Pessoa, PB. Findings point out that although the urban structures of these cities are not immune to the Form of Privilege, Natal is emblematic of this phenomenon, a fact that demonstrates the perverse character of its spatial process, which historically creates privileged areas within the city, by means of the appropriation of accessibility as well as of the many urban benesses that are related to it by higher income groups at the expense of the major part of the population, which though being the people mostly in need of the benefits originating from the urban form are excluded from them

A cidade sem barreiras é para todos?: avaliação das condições de deslocamento no bairro da Cidade Alta, Natal/RN

In this present work, the conditions of displacements and the behaviors of the users are evaluated, face to the physical adequacies in accessibility in the inserted area in the central quadrilateral of the Quarter of Cidade Alta, in the City of Natal/RN, space clipping established by its great meaning historical and cultural. For this reason such area was a target of an integrated actions plan during the years of 1993 and 1998, with the implantation of part of the Project CIDADE SEM BARREIRAS (City without Barriers) and, later, the implementation of the Project CIDADE PARA TODOS's proposals (City for All), having as supports the constant Brazilian norms inserted into the NB9050/1994 and in the Municipal law number 4.090/92, effective ones in the period of the interventions. Considering that the carried through actions at the time were directed to the elimination of the architectural barriers to guarantee the right to go and to come, the research received a human universe formed by all the people in situation of displacement in the central area of the interventions in the quarter of the Cidade Alta, independently of their locomotive or sensorial conditions, emphasizing the aspects most excellent how much to the accessibility of the sidewalk, as promotional of mobility, integration and urban organization, as well as of the public squares of that one quadrilateral, conceived, originally, as spaces of aggregation and social inclusion. The work appealed to the direct comment and the gotten results had been collated with the ex port facto law and technique norms, of the year of 2004, and with the legal devices contained in the Federal Decree number 5.296/2004, as way to certify the levels of efficiency of these adaptations in that it says respect to the current conditions of demanded accessibility and urban mobility

Bibliotecas digitais em Arquitetura e urbanismo: um estudo sobre a arquitetura da informação digital

The goal of this paper was to search the state of the art from the Digital Libraries in Architecture and Urbanism in the Higher Education Institutions (IES) through conceptualizations and showing the importance of Digital Libraries in the disclosure and easing of information transferring. Questions about digital information architecture, usability, digital preservation and accessibility were approached. The research was made in the websites of Brazilian Universities, firstly to identify the institutions which offered the Architecture and Urbanism course, focusing on postgraduate education. After identifying the offering, the research was done by analyzing the contents, storage and dissemination and access to information, these libraries. It was found that the digital libraries are increasingly and taking part of organizations and educational institutions focusing on the knowledge dissemination releasing digitally information that may be needed for institution or the individual. A monitoring was done over of the physical and computational restructuring of the Board of Studies and Research in Architecture and Urbanism (Câmara de Estudos e Pesquisa em Arquitetura e Urbanismo, CEPAU), from the Architecture and Urbanism Course of the Federal University of Rio Grande do Norte (UFRN), showing the need of installing a Digital Library to integrate the databases of PPGAU s research groups, which today remain independent, with no interface among themselves. The research chosen area was Architecture and Urbanism, because there is a gap and little documentation about digital libraries in this area