CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Nosocomial infections (NI) are a serious public health problem. Knowledge about the etiology of NI is important for the development of control measures, prevention and treatment. Viruses are important etiologic agent of NI has been studied in populations considered at risk as premature, heart disease, lung disease, and immunosuppressed. Respiratory hospital infection (RHI) generate discomfort to patients, postponing medical interventions, postoperative complications, use more drugs and, in some cases, intensive care, may progress to cure or to death. Viruses are responsible for outbreaks of RHI in wards and intensive care units, with the virus as detected respiratory syncytial virus. In our country there are few data on the impact of RHI caused by respiratory viruses in children. Aims of the study were: identify and describe RHI cases in children and submit nasopharyngeal aspirates collected from January to December 2013 to search for molecular diagnosis 13 respiratory viruses [respiratory syncytial virus (RSV), adenovirus, influenza A and B, parainfluenza virus -1 ,-2 , -3 and -4 , metapneumovirus and human coronavirus OC43 , NL63 , 229E and HKU - 1]. During the study period, 120 samples were included in the study and 65 % were positive for at least one virus. A total of 114 viruses were detected (33 RSV, 32 adenovirus, 14 Parainfluenza -3, 14 influenza A , 12 Parainfluenza -4 , 5 parainfluenza -1 , 3 metapneumovirus and 1 coronavirus OC43). Co-detections occurred in 26 cases: 16 with two viruses and 10 with three viruses. No clinical differences between positive and negative RHI for viruses investigated were observed. Respiratory virus were detected in four of five deaths (5/120 4.16%) associated RHI. The knowledge about the occurrence of viral RHI in association with the period of viruses circulation in the community, as described in the study, allows to develop specific actions steps to prevent and control hospital outbreaks caused by viruses. / As infecÃÃes relacionadas à assistÃncia à saÃde (IRAS) sÃo um sÃrio problema de saÃde pÃblica. O conhecimento sobre a etiologia das IRAS à importante para o desenvolvimento de medidas de controle, prevenÃÃo e tratamento. A importÃncia dos vÃrus na etiologia das IRAS tem sido estudada em populaÃÃes consideradas de risco, como prematuros, cardiopatas, pneumopatas e imunodeprimidos. As infecÃÃes respiratÃrias hospitalares (IRH) geram aos pacientes desconforto, adiamento de intervenÃÃes mÃdicas, complicaÃÃes pÃs-cirÃrgicas, uso adicional de medicamentos e, em alguns casos, cuidados intensivos, podendo cursar para a cura ou para o Ãbito. Os vÃrus sÃo responsÃveis por surtos de IRH em enfermarias e unidades de terapia intensiva, sendo o vÃrus sincicial respiratÃrio o mais detectado. Em nosso paÃs sÃo escassos os dados sobre o impacto das IRH causadas por vÃrus respiratÃrios em pediatria. Os objetivos deste estudo foram: identificar e descrever os casos de IRH em crianÃas e submeter aspirados de nasofaringe coletados no perÃodo de janeiro a dezembro de 2013 ao diagnÃstico molecular para pesquisa de 13 vÃrus respiratÃrios [vÃrus sincicial respiratÃrio (VSR), adenovÃrus, influenza A e B, parainfluenza 1, 2, 3 e 4, metapneumovÃrus e coronavÃrus humanos OC43, NL63, 229E e HKU-1]. Para tanto, as amostras foram extraÃdas para obtenÃÃo do material genÃtico viral e, posteriormente, para os vÃrus de RNA, parte deste produto foi transformada em DNA complementar e depois a amplificado. Para detecÃÃo do adenovÃrus, foram realizados PCR e Nested PCR. No perÃodo de estudo, 120 amostras foram incluÃdas e 65% delas foram positivas para pelo menos um vÃrus. Um total de 114 vÃrus foram detectados (33 VSR; 32 adenovÃrus; 14 parainfluenza-3; 14 influenza A; 12 parainfluenza-4; 5 parainfluenza-1; 3 metapneumovÃrus e 1 coronavÃrus OC43). Co-detecÃÃes ocorreram em 26 casos: 16 com dois vÃrus e 10 com trÃs vÃrus. NÃo foram observadas diferenÃas clÃnicas entre as IRH positivas e negativas para os vÃrus pesquisados. VÃrus respiratÃrios foram identificados em quatro dos cinco casos de Ãbito (4,16%; 5/120) associados à IRH. O conhecimento sobre a ocorrÃncia da IRH virais em associaÃÃo com o perÃodo de circulaÃÃo dos vÃrus na comunidade, como descrito no estudo, permite desenvolver aÃÃes especÃficas de medidas para prevenir e controlar surtos hospitalares causados pelos vÃrus.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / O trabalho descreve o primeiro estudo sobre a atividade antimicrobiana e mecanismos de aÃÃo de concentraÃÃes sub-inibitÃrias e inibitÃrias do Ãleo essencial das folhas de Plectranthus amboinicus contra uma cepa padrÃo de Klebsiella pneumoniae e cepas obtidas de isolados clÃnicos multirresistentes. A concentraÃÃo inibitÃria mÃnima (CIM), concentraÃÃo bactericida mÃnima (CBM), a taxa de morte e o efeito do pH na aÃÃo do Ãleo essencial foram analisadas atravÃs de testes de microdiluiÃÃo em microplacas. ConcentraÃÃes sub-inibitÃrias e inibitÃrias do Ãleo essencial foram testadas para verificar sua aÃÃo sobre permeabilidade da membrana, expressÃo de cÃpsula, atividade da urease e morfologia celular de K. pneumoniae. A CIM e CBM do Ãleo essencial foram de 0,09 Â 0,01% e resultou notavelmente na inibiÃÃo completa do crescimento bacteriano apÃs 2 horas, quando as bactÃrias foram expostas a duas vezes a CIM do Ãleo essencial. A melhor CIM foi encontrada quando o pH neutro ou alcalino foi usado. CaptaÃÃo de Cristal violeta foi considerada significativa quando o dobro da CIM do Ãleo essencial foi usado (p <0,05). InibiÃÃo da atividade da urease foi observada em todas concentraÃÃes sub-inibitÃrias testadas, quando comparada ao grupo nÃo tratado (p<0,001). Verificou-se ainda alteraÃÃo na expressÃo da cÃpsula e alteraÃÃes na membrana, verificadas pelas tÃcnicas de Anthony e por microscopia de forÃa atÃmica, respectivamente. / This work describes the first study about the antimicrobial activity and mechanisms of action of the subinihibitory and inhibitory concentrations essential oil from leaves of Plectranthus amboinicus against standard strain of Klebsiella pneumoniae and multresistant clinical isolates of the bacterium. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), the rate of killing and the pH sensitivity of the essential oil were determined through microplate-microdilution tests. Subinihibitory and inhibitory concentrations of Plectranthus amboinicus essential oil were tested in order to check their action on K. pneumoniae membrane permeability, capsule expression, urease activity and cell morphology. The MIC and MBC of the essential oil were 0.09Â 0.01% and resulted remarkably in complete inhibition of the bacterial growth after 2 hours, when the bacteria were exposed to twice the MIC of the essential oil. A better MIC was found when neutral or alkaline pH broth was used. Cristal violet uptake which represented altered permeability was considered to be significant when twice the MIC of the essential oil was used compared to the untreated group (p<0.05); Reduction of the urease activity could be found when all the subinihibitory concentrations were tested compared to the untreated group (p< 0.001). Is the result was an indicative that there was alteration on the capsule expression and membrane leakage, which could be demonstrated by the AnthonyÂs method and atomic force microscopy, respectively.

Orientador : Egberto Ribeiro Turato / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-07-27T17:14:45Z (GMT). No. of bitstreams: 1 Pereira_IsabelCristina_M.pdf: 13462943 bytes, checksum: 4ebd78fddc8410dedc0c0b5c368753d9 (MD5) Previous issue date: 2000 / Resumo: A Doença de Crohn caracteriza-se por um processo inflamatório de fator etiológico não definido, que pode acometer qualquer parte do tubo digestivo. Possui caráter impactante para o paciente, principalmente devido à sua cronicidade, complexidade e quadro sintomatológico limitante e doloroso. Para TEIXEIRA, HABR-GAMA & PINOTTI (1994), a Doença de Crohn representa um aspecto importante para a saúde pública, pois, além de atingir preferencialmente adultos jovens, tem apresentado aumento de incidência, seu tratamento fteqüentemente implica um ou mais procedimentos cirúrgicos ao longo de sua evolução e pode estar associada à neoplasia. Com isso, destaca-se o valor da realização desta pesquisa, ao mesmo tempo em que se busca uma compreensão da vivência do processo de adoecer, segundo o relato das experiências de pacientes portadores dessa enfermidade, considerando os aspectos psicossociais envolvidos. Est~ estudo possibilitou a reflexão sobre a maneira de lidar com o paciente a partir de uma interação bio-psicossocial. Como método de pesquisa, optou-se pelo Método Clínico-Qualitativo. Quanto aos procedimentos da investigação, foram realizadas entrevistas semi-estruturadas de quest~es abertas, aplicadas a pacientes portadores de Doença de Crohn, acompanhados no Ambulatório de Doença Inflamatória Intestinal do Serviço de Colo-Proctologia do Hospital das Clínicas da UNICAMP. O levantamento dos prontuários médicos forneceu pados complementares à compreensão das falas dos pacientes, bem como dados clínicos. O tratamento dos dados teve como base os nos conceitos da chamada 'Análise de Conteúdo'. O referencial teórico para interpretação dos resultados foram os conceitos mais usuais na psicologia médica. A análise dos dados apóia a conclusão de que os sentidos e os significados que pacientes com doença de Crohn atribuem aos fenômenos associados a essa doença estão em relação íntima não só com aspectos psicossociais, mas também com as necessidades de cada um. Isto posto, torna-se imprescindível uma atuação interdisciplinar integrando tratamento clínico, cirúrgico, psicoterápico, . de assistência social, e outros que se :fizerem necessários / Abstract: Crohn's Disease is an inflammatory process that may involve any part of the digestive tract. It is little known and has a strong impact on the life of patients, mainly due to such characteristics as being achronic, complex disease, with a limiting and painful group of symptoms as well as an uncertain diagnosis. We would like to emphasize the importance ofthis research whi1e searching for a greater comprehension of the experience of illness from the viewpoint of patients with this infirmity, taking into account all the psychosocial aspects involved. A study was conducted which made it possible to reflect about the best way to care for the patients and the process of becoming afllicted with ill-health, from a perspective ofbio-psychosocial interaction. The Clinical-Qualitative Method was chosen as research method. As for investigational procedures Semi-Structured Interviews with Open Questions were conducted with patients diagnosed with Crohn's disease being treated at the General Clinic of the University Hospital of UNICAMP. Their medical records supplied supplementary data to the comprehension ofthe patients speech, as well as clinical data. These data were considered using as a basis referential concepts ofthe so called "Content Analysis". Results were presented in a descriptive manner accompanied by illustrative citations together with the discussion. "The theoretic reference for the interpretation of results were the most tlsual concepts in Medical Psychology. An analysis of the data obtained leads to the conclusion that the meaning and significance a Crohn disease patient attributes tê the phenomena associated with this disease are internally related, not on1y with psychosocial aspects but also with each individual' s necessities. Thus an interdiscip1inary action becomes indispensable and should integrate clinical, surgical and psychotropic treatment, social assistance, as well as any other action deemed necessary / Mestrado / Saude Mental / Mestre em Ciências Médicas

The Notch1 signaling pathway has a pivotal role in cell fate regulation and has been found to be critically deranged in different cancers, including colorectal cancer (CRC). Inflammation is known to play an important role in the pathogenesis of CRC and a prominent function of Notch1 during inflammation has been recently demonstrated. Epithelial to Mesenchymal Transition (EMT), a crucial process in the malignant transformation, is modulated by inflammation and Metalloproteinase-9 (MMP9) is involved in this interaction. Eicosapentaenoic Acid is an omega-3 polyunsaturated fatty acid (omega-3 PUFA) known for its anti-inflammatory properties as well as for its capability in preventing colon cancer development both in sporadic and in hereditary settings. In particular, our group has demonstrated that an extra-pure formulation of Eicosapentaenoic Acid as the free fatty acid (EPA-FFA) protects from CRC development in a mouse model of colitis-associated cancer (CAC) by modulating the Notch signaling pathway. In the present work, we re-created an in vitro model of inflammatory-driven CRC by exposing colon cancer cells to a cytokine-enriched conditioned medium (CM) obtained from THP-1-differentiated macrophages. We found, for the first time, that CM strongly induces Notch1 signaling and EMT markers, increasing the capability of cells to invade. Importantly we found that, upon CM exposure, Notch1 signaling is dependent on MMP9 expression. Finally, we show that a non-cytotoxic pre-treatment with 50 μM of EPA-FFA for 72h counteracts the effect of inflammation on Notch1 signaling and EMT, leading to a reduction of invasiveness. Taken together, our data demonstrate that in CRC inflammation up-regulates Notch1 signaling through MMP9 and that this mechanism can be effectively counteracted by EPA-FFA.

The increasing occurrence of CRC developing in young patients with no identified genetic predisposition, defined as sporadic early onset colorectal cancers (EOCRCs), demands maintaining a high index of suspicion when people below 50 years of age present with symptoms. To define the clinicopathological features and the stage at presentation of EOCRCs, as well as to understand whether the histological, immunohistochemical and molecular analyses were associated with particular clinicopathological parameters and oncologic outcome, a total of 94 cases of EOCRCs diagnoses between 2006 and 2014 at the Sant’Orsola University Hospital were studied. Indeed, EOCRCs appear frequently as an aggressive disease located in the sigmoid colon and rectum, and most patients are symptomatic at the time of presentation, mainly presenting with rectal bleeding, haematochezia or abdominal pain. The genetic basis in the majority of early onset colorectal carcinomas remains unknown, however, most EOCRCs, not related hereditary syndromes, appear to arise through the same pathways as sporadic CRCs, such as the classical adenoma-carcinoma sequence, but with only rare involvement of the methylator pathway. Taken together, the analyses described in this study suggest that, in the absence of screening programs for patients under 50 years of age, the risk factor of a family history and the presence of symptoms may be considered as an indication for prompt endoscopic investigation in these patients, since this may reduce the stage of disease at diagnosis and likely have an impact on improving survival.

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.

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.