Diss. (sammanfattning) Stockholm : Stockholms universitet, 2008. / Härtill 7 uppsatser.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciencias Fisicas e Matematicas / Made available in DSpace on 2012-10-15T19:58:03Z (GMT). No. of bitstreams: 0Bitstream added on 2016-01-08T21:54:30Z : No. of bitstreams: 1 107316.pdf: 1424783 bytes, checksum: 9dfd2d6cc051a8261700e586a06de4b3 (MD5) / A estrutura da matéria nuclear é estudada no contexto de aproximações relativísticas. Inicialmente, reproduzimos os cálculos com a aproximação de campo médio nos modelos de Walecka e de Zimanyi-Moszkowski (ZM). O modelo de Zimanyi-Moszkowski difere do modelo de Walecka na forma dos acoplamentos do nucleon com os mésons. Também aplicamos a estes modelos um método conhecido como expansão d. A expansão d é uma aproximação não perturbativa para modelos de teoria de campo, que combina técnicas de teoria de perturbação e método variacional. Nossos resultados para ZM são, então, comparados com os obtidos de cálculos de campo médio e também com os resultados do modelo de Walecka.

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This thesis makes an original contribution to the field of formulation development by providing new experimental data and insights into the effect of processing and formulation conditions on the quality of lyophilised biopharmaceuticals. The quality attributes of lyophilised products include: a quick reconstitution time, product elegance and protein stability, which are known to be affected by processing and formulation parameters. However, choice of formulation excipients or processing conditions often relies on previous experience rather than mechanistic insight. The motivation of this thesis was therefore to provide a greater understanding of how process variables andexcipient choice affect these quality attributes. Bovine serum albumin (BSA) and immunoglobulin G (IgG) were used as model proteins to investigate formulation conditions, which included the excipient, the lyophilisation cooling profile and duration of the annealing step. BSA was also used as a model protein to explore the effects of sucrose and arginine as lyoprotectants. Unique to this study was the investigation of arginine salts as lyoprotectants, wherein the counterions were dicarboxylic acids with increasing chain length. Two key results regarding quality attributes were observed. Firstly, characterisation of the lyophilised structure established that there was an optimal annealing time, beyond which there was an increase in primary drying time, batch heterogeneity and variable moisture content. Secondly, a relationship was found between decreasing dicarboxylic acid chain length and improved protein stability. To explain these findings, two mechanisms are proposed that account for ice crystal growth during annealing and the observed changes in protein stability at the molecular level. Significantly, this research provides insights for future formulation development studies.

Global pharmacy education and pharmacy practice continue to face remarkable changes. Many countries are undergoing major transformations in the field of pharmacy education. In developing countries, there is an increasing trend towards adopting the PharmD degree. Thai pharmacy education has transitioned from having two entry-level degrees – a 5-year BPharm (with three main tracks: pharmaceutical care, pharmaceutical sciences, social and administrative pharmacy) and a traditional 6-year PharmD (pharmaceutical care) programme – to a single national 6-year PharmD programme or ‘an all-PharmD programme’ (including industry pharmacy and pharmaceutical care tracks). Similar to other countries that have also been transitioning to ‘an all-PharmD programme’, there was limited evidence for the merit of the transition in Thailand. Although opinions and questions put forth on social media networks triggered debates about the need for the transition, there is a lack of an evidence-based and in-depth investigation about the reasons for this transition. This thesis explores the experiences and perceptions held by stakeholders concerning the transition towards an all-PharmD programme in Thailand. The study used a mixed methods approach through a quantitative analysis of surveys (Phase 1 and Phase 2) and interviews with stakeholders who were involved in pharmacy education (Phase 3). Findings from the three phases and other resources were triangulated and validated (by comparing and confirming them) to provide a better picture of the transition of pharmacy education in Thailand. Phase 1: A survey of the status of Thai pharmacy education. This study aimed to explore the status of pharmacy education in Thailand by using a questionnaire survey. The surveys were distributed to the deans of all 19 faculties of pharmacy in Thailand. The response rate was 84% (n = 16). Characteristics of the Faculties of Pharmacy, the teaching staff, types of PharmD programme, the number of training sites and quality assurance mechanisms were reported. The results showed that the Thai PharmD curriculum includes industrial pharmacy and clinical pharmacy tracks that differentiate it from the traditional US PharmD programme, which only focuses on patient care. There was a shortage of academic staff in the pharmaceutical care area and some faculties needed to better prepare for their training sites. Phase 2: A survey of the pharmacists’ perceptions towards the suitability of the PharmD graduates employed in hospital and community pharmacy settings and the competencies difference between the BPharm and the PharmD graduates. This study aimed to explore Thai pharmacists’ perceptions regarding the suitability of the PharmD graduates employed in hospital and community pharmacy settings, as well as the competency differences between the BPharm and PharmD graduates. A cross-sectional survey questionnaire was distributed to 180 hospital pharmacists and 200 community pharmacists during two conferences. The response rate was 55.6% among hospital pharmacists and 20% in the community pharmacists group. The findings highlighted that the PharmD graduates were suited for large hospital settings as they were well coordinated with the health care team. However, there were concerns regarding the suitability of the PharmD graduates for primary care settings, because of their lack of training in health promotion. Half of the respondents perceived PharmD graduates as having higher competencies in clinical activities and being more prepared to work than BPharm graduates. However, the other half of the respondents perceived the competencies of both pharmacy qualifications as being similar; PharmD graduates provide non-clinical activities similar to BPharm graduates, due to numerous barriers (e.g., high workload in dispensing services and the shortage of pharmacists) preventing PharmD pharmacists from providing direct pharmaceutical care services. Phase 3: Thai stakeholder’s perceptions of the introduction of the PharmD programme: a qualitative study. This qualitative study aimed to understand the experiences and perceptions of stakeholders, regarding the transition to an all-PharmD programme in Thailand. Semi-structured interviews were conducted with 130 stakeholders (e.g., policy makers, educators, health care providers, patients, students, and parents). The data were audio recorded, transcribed verbatim and analysed using an inductive thematic analysis. Three main themes were derived from the findings: 1) influences on the transition (e.g., the US-Thai consortium for the development of pharmacy education); 2) perceived benefits (e.g., improved pharmacy competencies from generalist to specialists); and 3) concerns (e.g., the higher cost of a longer period of study, and insufficient preceptors and training sites). This PhD study carries important implications for both universities and policy makers. Faculties of pharmacy should consider a long-term plan to develop sufficient qualified academic staff and preceptors. Policy makers should prepare a strategic plan for the future workforce supply and requirements, increase the flexibility of the PharmD curriculum during its transition stage, and prepare a supportive and enabling system for PharmD graduates to provide advanced practice at their full potential. Close coordination between faculties, the Pharmacy Council of Thailand and pharmacy professional organisations is needed to ensure that pharmacy education provides the necessary competencies for graduates to offer a high level of needed pharmacy services. Further research focussing on the outcome, impact and efficiency of the PharmD programme is also needed. To conclude, in this thesis, the issues surrounding the transition to an all-PharmD programme in Thailand were carefully investigated. This study reflects the influences and the requirements of the transition that it was initiated, in order to meet the need for higher levels of competency for the nation’s pharmacists and is influenced by many factors. The stakeholders perceived benefits from the transition. They thought that the PharmD graduates will have higher competencies and be ready to work as pharmacists compared to graduates from the previous pharmacy curriculum. The findings also addressed the following issues concerning curriculum change: the higher costs of a longer period of study, the mismatch between the pharmacy graduates’ competency and the job market’s needs and the shortage of qualified preceptors.

Lung cancer is considered a major health concern and is responsible for most cancer-related deaths. Nasopharyngeal carcinoma (NPC) is another type of cancer that is predominantly in China and has a low survival rate, which makes it a serious health issue. There is currently no cure for lung cancer and NPC, so it was decided to investigate derivatives of the highly bioactive natural product, cardamonin, for a potential drug candidate. 19 analogues of cardamonin were synthesised and tested against A549 (lung) and HK1 (NPC) cell lines. The techniques employed in synthesising the analogues were one-step reactions which included alkylation, acylation, reduction, condensation, cyclisation and complexation reactions. The analogues were fully characterised. MTS assay showed that several derivatives, such as the allyl derivative of cardamonin (2) and cardamonin’s Cu (II) complex (19), had more potent cytotoxic activities than cardamonin. Furthermore, the active analogues have generally demonstrated lower toxicity towards normal MRC5 cells. Structure-activity relationship (SAR) analysis showed the importance of the ketone and alkene groups for bioactivity, while substituting cardamonin’s phenolic groups with more polar moieties resulted in activity enhancement. As part of the SAR study and further exploration of chemical space, the effect of metal coordination on cytotoxicity was also investigated, but it was only possible to successfully obtain the Cu (II) complex of cardamonin (19), and the metal ion enhanced bioactivity. 19 was the most potent analogue possessing IC50 values of 13.2 μM and 0.7 μM against A549 and HK1 cells, corresponding to a 5- and 32-fold increase in activity, respectively. It was also able to inhibit the migration of A549 and HK1 cells. Mode of action studies revealed that 19 induced DNA damage in both cell lines resulting in G2/M-phase arrest, which further led to apoptosis via the activation of caspase-9 and caspase-3/7. Moreover, qPCR analysis showed that 19 inhibited the expression of the mammalian target of rapamycin (mTOR) by >50% in A549 and HK1 cells which indicated that it exerted its anticancer activity, at least in part, via inhibition of the mTOR signalling pathway. So molecular docking of cardamonin and 19 to mTOR was performed and the study showed that the higher activity of 19 might be due to formation of further hydrogen bond interactions with the receptor resulting in a higher binding free energy of -9.8 kcal/mol. Therefore, all these assays have further proven the high bioactivity of 19. However, further in vivo and animal model studies would have to be conducted in order to confirm the potential of 19 as an anticancer agent.

Os estoques de carbono orgânico do solo (ECOS) nos ecossistemas naturais normalmente podem se manter em equilíbrio por longos períodos, porém as atividades antrópicas, especialmente a expansão da agricultura, usualmente provoca diminuição nos ECOS, com implicações nas funções ecossistêmicas e produtivas dos solos. Assim, a estimativa dos ECOS nos solos sob condições naturais e em agroecossistemas e a distribuição desses estoques na paisagem são questões científicas atuais. Este trabalho teve como objetivo amostrar solos para estimar os ECOS, compilar estudos sobre ECOS já publicados e avaliar comparativamente ECOS nas regiões dos Campos de Cima de Serra e na parte central do Planalto do Rio Grande do Sul. Para estabelecer os ECOS nestas áreas foram utilizados dados primários (coleta de amostras a campo e análises laboratoriais) e dados secundários (levantamentos, estudos e projetos de pesquisa já realizados), prioritariamente em camadas superficiais (0-30cm). A consolidação de dos resultados obtidos do campo e de estudos possibilitaram comparar os estudos existentes de forma tabular e visualizar em mapas considerando classes de solos e usos das terras. Considerando a camada mais superficial de 0-30 cm, os estoques médios (média geral dos valores medidos) na região dos Campos de Cima da Serra (173,5 Mg C ha-1) foram três vezes maiores que na região central do Planalto do Rio Grande do Sul (59,5 Mg C ha-1). De maneira similar, os ECOS nas camadas mais superficiais (0-10 e 0-20cm) também consideradas neste estudo também foram pelo menos duas vezes maiores nos Campos de Cima da Serra. Os mapas produzidos permitem visualizar a distribuição espacial destes estoques nas regiões. Estas informações podem ser usadas para definir políticas públicas no âmbito de agricultura conservacionista e auxiliar na atualização dos inventários nacionais de gases de efeito estufa. / Organic carbon stocks in soils under natural conditions are usually in equilibrium, but human intervention specially with the expansion of agriculture and silviculture leads to SOC (soil organic C) stocks losses. Thus, SOC stocks estimation and their distribution in the landscape is a crucial scientific topic. This study aimed to measure and benchmark current SOC stocks in fields from the Campos de Cima da Serra and the central part of the Planalto do Rio Grande do Sul. To update the SOC stocks in the study areas, primary data were assessed derived from field sampling and laboratory analysis) and secondary data (obtained from published surveys and research projects). Data from 0-30cm soil layer were primarily considered in this study. The consolidation of results from field and studies allowed to compare SOC stocks in tabular form and view maps considering soil types and land of uses. Considering the upper soil layer (0-30 cm), the average SOC stock (173.5 Mg C ha-1 - overall means of the measured values) in the region of Campos de Cima da Serra was three times higher than in the central section of the Planalto of Rio Grande do Sul (59.5 Mg C ha- 1). Similarly, SOC stocks in the shallower layers (0-10 and 0-20 cm) were at least twice as high in the Campos de Cima da Serra. The maps produced display the spatial distribution of measured or calculated SOC stocks. This information can be used to guide public policy for conservation agriculture programs and assist in conducting national greenhouse gas inventories.

The blood-brain barrier (BBB) is the most extensive and restrictive barrier to brain delivery for therapeutic agents. A low proportion of low molecular-weight agents can cross into the CNS. This decreases further as the molecular weight increases, meaning therapeutic antibodies, oligonucleotides and other supramolecular entities effectively cannot reach therapeutic levels within the CNS. Targeting ligands against receptors thought to undergo transcytosis across the brain microvascular endothelial cells (BMECs), can boost CNS delivery of therapeutics. Understanding these mechanisms, in an in-vitro setting, has proved challenging, due to the constraints of cell culture systems and the difficulty to replicate the in-vivo environment. With even the most extensively studied targeting receptor, transferrin receptor, not producing clear evidence to suggest the occurrence of transcytosis. To understand in-vitro trafficking of brain targeting ligands a pulse-chase assay, in combination with sub-cellular localisation microscopy was developed and compared to the current permeability-based assay method. The characterisation was done by comparison of transferrin receptor ligands; native holo-transferrin, the 8D3 antibody and a low-affinity variant; with the non-specific uptake probe, dextran. The method could distinguish between the two endocytosis methods, with concentration-dependent efflux efficiency observed with the targeted probes. The combination of techniques was then applied to the novel targeting ligand, Rabies-Virus Glycoprotein (RVG) peptide, to assess its suitability as a brain delivery. Studies were performed to confirm the target receptor of the RVG peptide, including competitive uptake, siRNA knockdown methods. The RVG peptide demonstrated desirable delivery characteristics, and the target receptor was confirmed as the α7 nicotinic acetylcholine receptor. Finally, attempts were made to develop a total internal reflection fluorescence (TIRF) microscopy assay for the assessment of ligand arrival at the basolateral membrane of BMECs. Initial work for this was performed with the transferrin receptor and transferrin, using both labelled ligand and photoswitchable receptor constructs. In summary, the pulse-chase assay provides a complementary technique to permeability assays for the assessment of brain targeting ligand trafficking in BMEC cell-lines in-vitro.

The potential applications of transition metals-based nanoparticles are expanding in the biomedical field. Oxides of iron are the matter of investigation in this study where various preparations of ultrafine superparamagnetic iron oxide nanoparticles (USPIONs) were fabricated using flow injection technology with spinning disc reactor. Basically, two types of preparation parameters were examined; first, instrument-related (physical) parameters and, second, chemistry-related parameters. USPIONs fabricated by this instrument showed fine-tuning size adjustment. Subsequent surface modification of these nanoparticles produced hydrophobic, hydrophilic and neutral amino acids modified surface, whereby aminoacid ‘monomers’, rather than polymeric materials were used. Transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements, thermogravimetric analysis (TGA) and fourier-transform infrared-attenuated total reflection spectroscopy (FTIR-ATR) were employed to characterise the coated nanoparticles. The data show that ultrafine, 4-9 nm sized coated nanoparticles show good dispersion upon TEM imaging. Measurement of number of monomers molecules effectively associated with USPIONs suggest formation of multilayer of amino acid adsorbed on nanoparticles (NPs). Prediction of NPs- amino acid association mechanism by FTIR-ATR study reveals presence of either monodentate or bidentate molecular adsorption on the surface of USPIONs. In the second stage of the project, interactions of differently modified USPIONs with epithelial cell layer (model of intestinal epithelium) are now investigated. An intestinal adenocarcinoma cell line (Caco-2) is used for performing the in vitro studies. The toxicity of three types of USPIONs (Asp-, His-, and Phe-USPIONs) reveals that these particles have potential toxic effect on biological system. Relatively long term exposure to these particles (24 hours) with high concentration 250 μg/ml and more was found to enhance apoptotic mode of cell death. Cell-NPs interaction study displayed presence of different forms of cellular interaction which are supposed to be related to USPIONs surface chemistry. While some of Phe-USPIONs are found internalised and accumulated inside some cells, Asp-USPIONs exhibit different interaction mode where the cell membrane of most cells is covered with thin layer of NPs without significant cell penetration. This gives an indication that metal oxide NPs (USPIONs) that are associated by their surface with small molecules could render these NPs with aggravated toxicity and cell-NPs interaction and hence long term effect.

Many different nanoparticle delivery systems have been reported as potential cancer therapeutics, however, the tumour penetration and uptake characteristics have been determined for very few systems. Animal models are effective for assessing tumour localisation of nanosystems, but difficult to use for studying penetration beyond the vasculature. In this work, defined HCT 116 colorectal cancer spheroids were used to study the effect of nanoparticle size and surface modifications on their penetration and uptake. Incubation of spheroids with Hoechst 33342 resulted in a dye gradient which facilitated discrimination between the populations of cells in the core and at the periphery of spheroids by flow cytometry based on the degree of Hoechst staining. This model was used to compare doxorubicin and Doxil, a range of model polystyrene nanoparticles in different sizes (30 nm, 50 nm, 100 nm) and with different surface chemistry (50 nm unmodified, carboxylated, aminated) and polyethylene glycol modified NPs prepared from a promising new functionalized biodegradable polymer (poly(glycerol-adipate), PGA). Unmodified polystyrene nanoparticles (30 nm/50 nm) were able to penetrate to the core of HCT 116 spheroids more efficiently than larger polystyrene nanoparticles (100 nm). Penetration was also dependent on surface charge. PGA NPs of 100 nm showed similar penetration into spheroids as 50 nm polystyrene nanoparticles, and PEG surface modification significantly improved penetration into the spheroid core. The new spheroid model with Hoechst staining is shown to be a useful model for assessing NPs penetration and demonstrates the importance of controlling physical properties when designing nanomedicine.