Dissertations / Theses on the topic 'Nano medicine'

La combinaison de biomatériaux et cellules souches, a pour but de protéger des cellules endommagées et de ralentir la progression des maladies neurodégénératives, comme la maladie de Huntington (MH). Les cellules souches mésenchymateuses et particulièrement une sous-population, les cellules MIAMI, ont déjà démontré leur efficacité dans la maladie de Parkinson. Il est cependant essentiel d’améliorer leur différenciation neuronale, leur survie et évaluer leur sécrétome. L’objectif principal de ce travail fut de proposer une stratégie innovante de médecine régénératrice pour la MH associant cellules souches, nano et micro médecines. Pour l’évaluer, un nouveau modèle animale ex vivo de la MH a été mis en place. Nous avons ensuite développé et optimisé deux nano-vecteurs, des nanocapsules lipidiques et des nanoparticules solides de SPAN, et les avons associés à un inhibiteur de REST qui est un facteur de transcription qui empêche la différenciation neuronale. La transfection de ce siREST a montré une amélioration du phénotype neuronal. Ces cellules ainsi modifiées furent ensuite induites vers un phénotype GABAergic grâce à des facteurs de croissance. Puis elles ont été associées à un support 3D, les microcarriers pharmacologiquement actif (MPA) permettant une meilleure intégration des cellules après greffe. Les MPA sont des microsphères ayant une surface biomimétique de laminine et libérant de façon contrôlée un facteur trophique le « brain derived neurotrophic factor » (inducteur d’un phénotype neuronal et neuro-protecteur). Des résultats prometteurs ont été obtenus, encourageant à continuer l’évaluation de cette stratégie in vivo dans des modèles génétiques de la MH
The combination of biomaterials and stem cells aims to protect damaged cells and slow the progression of neurodegenerative diseases such as Huntington's disease(HD). Mesenchymal stem cells, particularly a subpopulation known as MIAMI cells, have already demonstrated their effectiveness in Parkinson's disease. However, it is essential to improve their neuronal differentiation, survival, and to assess their secretome. The main objective of this work was to propose an innovative regenerative medicine strategy for HD by combining stemcells, micro and nano medicines. To perform this assessment, a new ex vivo animal model of HD has been set up. We then developed and optimized two nanovectors,lipid nanocapsules and solid SPAN nanoparticles,carrying an inhibitor of REST a transcription factor, which prevents neuronal differentiation. The transfection of this siREST showed an improvement in the neuronal phenotype. These modified cells were then induced into a GABAergic phenotype through growth factors. They were then associated with a 3D support, the pharmacologically active microcarriers (PAM) allowing a high rate of engraftment. The PAM are microspheres which have a biomimetic surface of laminin and release a trophic factor BDNF, brain derived neurotrophic factor (inducer of a neural phenotype and neuroprotective) in a controlled manner. Promising results were obtained, further encouraging continuing the evaluation of this strategy in vivo in genetic models of HD

Objectives: Orthopaedic and dental implants are prone to frequent infections. This can lead to detrimental and often irreversible outcomes for many patients. The objective of this study was to develop a novel system using zinc oxide nanoparticles (nZnO) as a coating material that inhibits both bacterial adhesion / growth and promotes osteoblast growth. Methods and Results: Initially bacteria (S. aureus, E. coli, S. epidermidis and P. aeruginosa) were exposed to different concentrations of zinc oxide nanoparticulate suspensions (250 μg/mL, 500 μg/mL, 1000 μg/mL and 2500 μg/mL); with the higher concentrations of the suspensions demonstrating significant bactericidal effects. A novel electrohydrodynamic atomization coating technique (EHDA) was used to deposit mixtures of nZnO and nano-hydroxyapatite (nHA) onto the surface of glass samples (1 cm2). Exposure of the coated samples to phosphate buffered saline (PBS) and adult bovine serum (ABS) and measurement of bactericidal activity demonstrated superior antimicrobial activity for 100% and 75% nZnO composite coated samples. Lactate dehydrogenase (LDH) release from osteoblast-like cells (UMR-106 and MG-63) exposed to both nano-TiO2 and nano-ZnO nanoparticulate suspension supernatants indicated minimal toxicity. Nano-ZnO coated samples did not elicit LDH release with an increase in proliferation and viability of cells was observed. Scanning electron microscopy (SEM) and optical microscopy indicated that all cell types used (mesenchymal stem cells and osteoblast-like cells) were able to maintain their normal morphological state when adhered to the surface of the nano-coated material. Further studies as regards to patterned coated samples showed an exclusive adhesion selection by osteoblast-like cells to nZnO patterned regions that needs to be further investigated. Conclusion: ZnO NPs provide an antimicrobial and biocompatible coating material for medical and dental bone implants.

Cancer is the second leading cause of death and is responsible for approximately 9.6 million deaths worldwide in 2018. Among all oncological diseases, lung cancer claims the highest mortality (male: 23.5%; female: 22%) and the second most new cases (male: 13%; female: 12%) in the US. Approximately 40% of newly diagnosed lung cancer patients are in the advanced stage IV, for which platinum-based chemotherapy is the first-line treatment, either by itself or in combination with surgery or radiotherapy. Cisplatin, the first-generation platinum-based anticancer chemotherapeutic agent, has the highest potency against lung cancer but carries many severe adverse effects. Cisplatin also induces drug resistance during long-term chemotherapy. Many more platinum complexes have been investigated as better alternatives, which led to the approval of carboplatin and oxaliplatin by Food and Drug Administration (FDA). In addition, miriplatin suspended in iodolipds (lipiodolization) was approved in Japan for the treatment of hepatocellular carcinoma (HCC) in 2009. Miriplatin has the same non-leaving group as oxaliplatin but different leaving groups of two myristate chains, which make it highly lipophilic. Several characteristics of solid tumors in lung cancer constitute a physiochemical barrier to the homogenous distribution and deep penetration of chemotherapy agents. Nanocarriers provide a promising platform to overcome the physiochemical barrier and to reduce the systemic toxicity of anticancer chemotherapy. In this study, miriplatin is formulated with various lipid-based nanocarriers including micelles and solid lipid nanoparticles (SLNs) thanks to its highly lipophilic structure. The goal of this thesis is to develop and evaluate miriplatin-loaded nano formulations against lung cancer. Miriplatin-loaded formulations were prepared by different methods, including thin film hydration and several scale-up methods including chloroform dripping, chloroform injection, chloroform evaporation, co-solvent evaporation, chloroform slow evaporation and co-solvent slow evaporation. Between the two types of nano formulations under this study, micelles were much smaller (~10 nm in diameter) and more homogeneous (PDI < 0.3), while SLNs were bigger (~ 100 nm in diameter) and more heterogeneous (PDI ~0.8). A quantification method of miriplatin was established using inductively coupled plasma-optical emission spectrometry (ICP-OES). The quantification of platinum recovery from different miriplatin-loaded nano formulations was facilitated by digestion with 70% nitric acid and heating. The co-solvent slow evaporation method to prepare miriplatin-loaded nano formulations improved the platinum recovery prominently from 10% to 70%. Thus, co-solvent slow evaporation has been established as a pharmaceutically viable scale-up method to prepare nano formulations of miriplatin. Miriplatin-loaded nano formulations of different compositions were negatively stained with uranyl acetate and then imaged by transmission electron microscopy (TEM), which showed the formulations’ size and morphology that were consistent with the size and PDI data from dynamic light scattering studies by the Malvern Zetasizer. In the TEM studies, micelles showed a morphology of spherical dots at around 10 nm in diameter while SLNs showed both spherical and rod structures with a size distribution from 50 to 150 nm. A three-dimensional multicellular spheroid (3D MCS) model of A549-iRFP cells was used for in vitro evaluation of the nano formulations’ activity against lung cancer. A549-iRFP cells were engineered from the common lung cancer cell line A549 to stably express the near-infrared fluorescent protein (iRFP). The viability of A549-iRFP 3D MCS after exposure to cisplatin or nano formulations was similar to A549 3D MCS. The anticancer activity of miriplatin-loaded nano formulations against 3D MCS was positively associated with the platinum recovery as quantified by ICP-OES. The miriplatin-loaded nano formulations that had been prepared by the co-solvent slow evaporation method showed substantial anticancer activities against A549 3D MCS and A549-iRFP 3D MCS, which were comparable to cisplatin. Taken together, miriplatin-loaded nano formulations were successfully prepared by co-solvent slow evaporation. The formulations were developed to carry favorable physiochemical properties to enhance the activities of platinum drugs against lung cancer.

Multifunctional polymeric nanoparticles (NP) represent a promising alternative for the treatment of neurodegenerative diseases using the intravenous route (i.v.). In current treatments, the effects of the intravenously injected drugs are systemic, requiring high drug doses to achieve therapeutic effects, thus causing severe side effects. NP can act specifically in a tissue provided that they are properly designed. An interesting approach is the preparation of NPs by nano-emulsion templating. Nano-emulsions (NE) are fine emulsions, with droplet sizes typically between 20 – 200nm. They can be prepared by the phase inversion composition (PIC) method, a low-energy emulsification method appropriate for pharmaceutical applications, since it can be performed at mild process conditions. Nanoparticles are obtained from nano-emulsions by solvent evaporation. To target the central nervous system (CNS), a specific targeting moiety on the nanoparticle surface is required to cross the blood-brain barrier (BBB), which is a current key goal under intense investigations. The aim of this work was to obtain multifunctional polymeric NP as advanced delivery systems able to cross the BBB. O/W polymeric NE were prepared by the PIC method and polymeric NPs were obtained by solvent evaporation. Polymeric NP with appropriate sizes for the i.v. administration (<1milimicron) were obtained. With the aim to design imaging systems, a model fluorescent dye and magnetic nanoparticles were encapsulated in polymeric NPs. An analgesic and an antiapoptotic drugs were also encapsulated into PLGA NP for therapeutic purposes. High encapsulation efficiencies were found for all tested compounds, attributed to the method of nanoparticle preparation as well as to low solubility of the components in the aqueous dispersion media. In addition, a sustained and controlled release of fluorescent dyes / drugs was achieved. NP surface was functionalized using various elements. On the one hand, it was functionalized with a monoclonal antibody against the transferring receptor, overexpressed in the BBB, to achieve an active targeting to the BBB. On the other hand, NPs were functionalized with oligonucleotides, to be used as non-viral gene delivery systems. Firstly, carbosilane cationic dendrons were covalently attached to nanoparticle surface to achieve a cationic surface. In a further step, antisense oligonucleotides, siRNA and plasmids were electrostatically bound to cationized nanoparticles. In vitro tests showed that the formulated NP did produce neither cytoxicity nor hemolysis. In addition, they were weak activators of the immune system and produced only a slight adsorption of blood proteins. Therefore, they are appropriate to be used by the i.v. route. NPs functionalized with oligonucleotides enhanced gene transfection in cell cultures, up to values comparable to those of commercial values (up to 90%). These NPs are advantageous in terms of toxicity issues over the commercial formulations. Therefore, they represent promising non-viral gene delivery systems. In vivo tests, which measured the central analgesia produced by an encapsulated drug into nanoparticles (loperamide) that is not able to cross the BBB, confirmed a central analgesic effect, reaching a potency of analgesia comparable to positive controls when nanoparticles were functionalized with the antibody. Therefore, the antibody functionalized nanoparticles efficiently crossed the BBB. In conclusion, the designed polymeric nanoparticles, functionalized with the antitransferrin receptor antibody, are able to cross the BBB with high efficiency. These nanoparticles represent promising nanosystems to deliver actives to the central nervous system.
Les nanopartícules polimèriques multifuncionals (NPs) representen una alternativa prometedora pel tractament de malalties neurodegeneratives, a través de l’administració intravenosa (i.v.), ja que els tractaments actuals provoquen molts efectes secundaris. Les NPs, en canvi, si estan correctament dissenyades, poden actuar específicament en el teixit diana. Ja que l’òrgan diana és el cervell, és necessari un element de vectorització per poder creuar la barrera hemato-encefàlica (BBB). En aquest context, l’objectiu de la present tesi és l’obtenció de NPs com a sistemes avançats d’alliberament de principis actius que travessin la BBB. Es van obtenir NPs a partir de nano-emulsions (NE) plantilla, emprant l’àcid poli-(làctic-co-glicòlic) com a polímer i el mètode d’inversió de fases a temperatura constant per emulsionar, seguit d’evaporació de solvent per obtenir NPs. Les NPs obtingudes tenen mides apropiades per l’administració i.v.. Es va aconseguir encapsular un fluorescent i NPs magnètiques dins les NPs polimèriques, per fer-les servir com a sistemes d’imatge. També es van encapsular fàrmacs per usar-les com a sistemes terapèutics. En tots els casos, es van aconseguir eficiències d’encapsulació molt elevades i un alliberament del fàrmac controlat i prolongat en el temps. A més, es va aconseguir funcionalitzar la superfície de les NPs amb diferents elements. Es van unir covalentment dendrons catiònics per posteriorment unir oligonucleòtids electrostàticament. També es va afegir una coberta exterior de polietilenglicol per protegir el material genètic. Per altra banda, es va funcionalitzar la superfície de les NPs amb un anticòs específic contra el receptor de la transferrina, sobreexpressat a la BBB. A continuació, es van fer assajos in vitro, que van posar de manifest que les NPs no són citotòxiques ni hemolítiques. També es va estudiar l’eficiència de transfecció cel•lular del material genètic, arribant a eficiències de transfecció equivalents a les dels vectors comercials. Assajos in vivo van permetre confirmar el pas a través de la BBB, sobretot de les NPs funcionalitzades amb l’anticòs. Els resultats obtinguts permeten concloure que s’ha aconseguit dissenyar noves NPs polimèriques a partir de NE, apropiades per l’administració i.v. i amb capacitat de travessar la BBB.

There is an urgent need for improvements to existing vaccine delivery technologies to run parallel with the development of new-generation vaccines. The burdens of needle-based immunisation strategies are exacerbated by poor resource provision in such areas as sub-Saharan Africa, where annual malaria mortality stands at 860,000. Needle-free delivery of vaccine to the skin holds promise for improved immunogenicity with lower doses of vaccine, in addition to significant logistical advantages. Various methods have been described for the transcutaneous delivery of vaccines, including the use of microneedles to overcome the outer stratum corneum of the skin for efficient delivery of liquid or solid, microneedle-coated vaccines into underlying strata rich in antigen-presenting cells. This thesis aims to evaluate two transcutaneous silicon microneedle and microprojection patch technologies for the delivery of live recombinant Adenovirus and Modified Vaccinia Ankara-vectored vaccines encoding pre-erythrocytic malaria antigens in mice. Cellular immunogenicity directed against a well-documented epitope of the Plasmodium berghei circumsporozoite protein is evaluated, as is protection against lethal P. berghei sporozoite challenge. Immunological and logistical benefits of each technology are assessed, as well as mechanisms underlying differences in the generation of a patch-induced immune response to vaccination. These data inform the future development of transcutaneous microneedle patches for the delivery of live vaccine.

Tumour-associated vasculature provides an accessible target for systemic gene therapy using targeted adenoviruses. The aim of this thesis is to develop strategies for targeting adenovirus infection to tumour-associated endothelium. Adenovirus expressing luciferase (Adluc) was coated with an amino-reactive polymer based on poly [N-(2-hydroxypropyl) methacrylamide] [pHPMA] to ablate normal infection pathways¬. This was a pre-requisite to redirecting virus tropism to infect endothelial cells via specific receptors. Direct attachment to the pHPMA-adenovirus (pcAdluc) of ligands including vascular endothelial growth factor (VEGF165) and a monoclonal antibody (RAFL) recognising VEGF receptor 2 (VEGFR-2) retargeted infectivity to VEGFR-2-positive endothelial cells and not to receptor-negative cells. Specificity of transduction in vitro was shown by competition with excess antibody. In vivo however, the VEGF165-retargeted virus failed to transduce tumour-associated endothelia following systemic administration. Similarly, direct linkage of a monoclonal antibody against E-Selectin (MHES) demonstrated E-Selectin-specific transduction of tumour necrosis factor-α (TNF-α)-activated endothelial cells, although overall levels of infection were not increased compared to unmodified Adluc. A two-component targeting system using protein A or protein G as ‘bridging’ agents was developed to ensure the required orientation of targeting antibodies. Using this system MHES mediated greater transduction of TNF-α-activated endothelial cells than Adluc. Conjugation using protein A also gave non-specific effects which were not seen with protein G. Whereas the unmodified Adluc virus failed to transduce TNF-α-activated endothelium in an umbilical vein model ex vivo, the MHES-protein G-pHPMA-adenovirus (MHES-StrepGpcAdluc) mediated good transduction. Similarly, StrepGpcAdluc retargeted with a chimeric P-Selectin Glycoprotein Ligand-1 (PSGL-1)-Fc fusion protein, showed good circulation kinetics and significant uptake into HepG2 xenografts following intravenous administration. Histological studies suggested selective targeting to tumour-associated endothelial cells. Overall these findings support the assertion that tumour-associated vasculature is an accessible target for systemic gene delivery, and the use of protein G as bridging agent facilitates rapid screening of Fc-bearing ligands for retargeting pcAd infection to tumour-associated endothelium.

Orientador: Alessandro Moura Zagatto
Banca: Marcelo Papoti
Banca: Guilherme Giannini Artioli
Resumo: O objetivo geral do presente trabalho foi verificar o potencial ergogênico da suplementação por 4 semanas de β-alanina sobre a potência anaeróbia, habilidade de esforços repetidos e desempenho no polo aquático. 22 jogadores de elite do sexo masculino (média±dp: idade = 18±4 anos, peso = 78,5±9,5 kg e altura = 1,79±0,06 m) participaram do estudo, que foi conduzido de modo randomizado, duplo cego e placebo controlado. Os participantes foram divididos em dois grupos (β-alanina e placebo) de 11 atletas cada e foram submetidos a testes específicos (teste de habilidade de esforços repetidos (RSA) e teste máximo de 30s de salto sob o gol (30CJ)) e semi-específicos (teste de 30s máximo em nado atado (30ATADO), teste máximo de 3 minutos (All Out 3min), teste incremental máximo (GXTATADO) e performance de 200m em nado crawl (P200m)) para a modalidade e um jogo simulado para possibilitar o rastreamento das atividades realizadas por meio de filmagem. As avaliações ocorreram pré e após o período de suplementação (4 semanas). Não foram encontrados efeitos significativos de interação entre os grupos para nenhuma variável do presente estudo. No entanto, alguns ligeiros indícios de melhora com a suplementação de β-alanina foram encontrados como: (1) melhora significativa entre os momentos (pré × pós) no número total de sprints durante o jogo simulado de polo aquático; (2) efeito provavelmente benéfico (análise de inferência baseada na magnitude) para o tempo médio, pior tempo e tempo total na... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The overall aim of this study was to investigate the ergogenic effect of 4 weeks β-alanine supplementation on the anaerobic power, ability to performed repeated efforts and performance of water polo. 22 male elite players (mean±SD age = 18±4 years, weight = 78.5±9.5 kg and height = 1.79±0.06 m) participated in the study, which was conducted in order randomized, double blind and placebo controlled. Participants were divided into two groups (β-alanine and placebo) of 11 athletes each and were subjected to specific tests (repeated sprint ability test (RSA) and maximum 30s jump under the goal test (30CJ)) and semi-specific (30s maximal test in tethered swimming (30TS), maximal 3 min effort (AllOut-3min), tethered swimming graded exercise test (GXTTS) and 200m in front crawl (P200m)) for the modality and a simulated game to enable tracking of the activities carried out by video record. Assessments occurred before and after the supplementation period (4 weeks). There were no significant interaction effects between the groups for any variable of this study. However, some slight improvement indications with β-alanine supplementation were found to: (1) significant improvement between moments (pre × post) the total number of sprints during the simulated game water polo; (2) probably beneficial effect (magnitude-based inference analysis) for the mean time, worst time and total time in the first series of the RSA test (RSA1); (3) significant improvement between moments for mean force and integral of force during the 30TS and P200m; (4) significant improvement between moments for peak power at GXTTS. Therefore, it is concluded that supplementation for 4 weeks of β-alanine can promote only slightly improve some parameters related to swimming ability in water polo as total number of sprints in simulated game, mean time,... Complete abstract electronic access below)
Mestre

>Magister Scientiae - MSc
Nanotechnology has emerged as an elementary division of modern science and stemmed directly from green chemistry twelve basic concepts, it receives global attention due to its unique character and ample applications. It also has great potential to mitigate the challenges they face in various fields, especially medical sector. Nanodrugs are increasingly considered as a potential candidate to carry therapeutic agents safely into a targeted compartment in an organ, particular tissue or cell. In this study, twenty (20) Libyan plants were selected and evaluated for their potential to synthesis gold and silver nanoparticles. The screening of the different plant extracts was performed using 96 well plate method at 25 °C and 70 °C. The NPs formation was confirmed and characterized using UV- Vis, DLS, HR-TEM and EDX. A well-defined NPs were obtained at high temperature (70 °C). The Au NPs had an average diameter of 92 nm at 25 °C and 66 nm at 70 °C. The zeta potential values were observed to be negative (-14 to -24) and indicate the stability of the Au NPs. The HR-TEM showed polydispersity, which decreased at higher temperature (70 °C). The stability of Au NPs in nutrient broth prior was conducted as well. All the Au NPs under study showed stability, only minimal changes in the UV-Vis spectra can be observed. Two plant extract viz Pistacia atlantica, Junipers phoenicea showed consistent results and forming stable and smaller NPs compared to others, both of the plant extracts and the corresponding NPs were tested against Streptococcus mutans and showed MIC value ~ 49 g/mL. In case of silver NPs, two plant extracts viz J. phoenicea, Rosmarinus officinalis, showed superior results than the others; both plants produced stable and small Ag NPs. The antibacterial activity against S. mutans demonstrated MIC valus ~ 50 g/mL. The synthesised NPs showed a promising bioactivity for developments of new antibacterial agents against S. mutans strains. Dose-dependent activity was observed for the tested NPs.

Background: Dysphagia is common after stroke, so feeding through a naso-gastric (NG) tube may be necessary. NG tubes are frequently dislodged, potentially causing feed or fluids to enter the lungs. Interventions to prevent this include taping NG tubes to the face, hand mittens and nasal bridles. Overall Aim The aim of this study was to explore the opinions of staff, patients and relatives about the maintenance of NG tube feeding for stroke patients while investigating current clinical practice. Research Design and Methods: A three-phased mixed method design was used. Phase 1 involved focus groups with multidisciplinary stroke unit staff (n=17); one-to-one interviews, with stroke patients (n=4) and relatives (n=6). Phase 2 incorporated a postal survey sent to a convenience sample (n=528) registered nurses working in the field of stroke across the UK. Phase 3 involved interviews with nurses (n=5) outside the speciality of stroke. Findings: Phase 1 highlighted many categories, including: lack of protocols; ethical and legal concerns; training to insert NG tubes; patient dignity; patient autonomy and potential harms and benefits of interventions used. There were variations in the opinions of staff, patients and relatives concerning the effectiveness and acceptability of methods for securing NG tubes. Phase 2 achieved a response rate of 59% (n=314/528); 22% (n=68/312) of nurses used hand mittens, only 11% (n=34/312) used a protocol; 56% (n=176/314) of nurses had received formal training to insert an NG feeding tube, more senior nurses had been formally trained than junior nurses (p<0.005). Acceptability and effectiveness ratings for tube securing interventions varied: 50% (n=158/312) considered hand mittens to be unacceptable. However, from a total of n=92 responses about their effectiveness, 66% (n=61/92) felt they were effective. Phase 3 produced more detailed results about fear associated with NG feeding; inconsistent approaches to training and ethical and legal issues of patient restraint. Conclusions: Overall this study demonstrates differences in opinion about what constitutes acceptable, effective and legal practice when maintaining NG feeding for stroke patients. It also suggests that the lack of consistent nurse training affects the standards of care patients receive. Furthermore, there is a need for more robust evidence to inform clinical practice. This study culminates in a model of nursing related to the insertion and maintenance of NG feeding for stroke patients.

Life Cycle Assessment (LCA) is a chain-oriented tool to evaluate the environment performance of products focussing on the entire life cycle of these products: from the extraction of resources, via manufacturing and use, to the final processing of the disposed products. Through all these stages consumption of resources and pollutant releases to air, water, soil are identified and quantified in Life Cycle Inventory (LCI) analysis. Subsequently to the LCI phase follows the Life Cycle Impact Assessment (LCIA) phase; that has the purpose to convert resource consumptions and pollutant releases in environmental impacts. The LCIA aims to model and to evaluate environmental issues, called impact categories. Several reports emphasises the importance of LCA in the field of ENMs. The ENMs offer enormous potential for the development of new products and application. There are however unanswered questions about the impacts of ENMs on human health and the environment. In the last decade the increasing production, use and consumption of nanoproducts, with a consequent release into the environment, has accentuated the obligation to ensure that potential risks are adequately understood to protect both human health and environment. Due to its holistic and comprehensive assessment, LCA is an essential tool evaluate, understand and manage the environmental and health effects of nanotechnology. The evaluation of health and environmental impacts of nanotechnologies, throughout the whole of their life-cycle by using LCA methodology. This is due to the lack of knowledge in relation to risk assessment. In fact, to date, the knowledge on human and environmental exposure to nanomaterials, such ENPs is limited. This bottleneck is reflected into LCA where characterisation models and consequently characterisation factors for ENPs are missed. The PhD project aims to assess limitations and challenges of the freshwater aquatic ecotoxicity potential evaluation in LCIA phase for ENPs and in particular nanoparticles as n-TiO2.
L’LCA è una metodologia standardizzata volta alla valutazione delle performance ambientali di un prodotto lungo il suo intero ciclo di vita. Il consumo di risorse e le emissioni di sostanze inquinanti in aria, acqua, suolo sono quantificate nella fase di inventario (LCI).La fase successiva di Life Cycle Impact Assessment ha lo scopo di convertire i consumi di risorse e le emissioni di inquinanti in impatti ambientali. classificate in categorie di impatto.Grazie all’ utilizzo di specifici fattori di caratterizzazione i risultati della fase d’inventario vengono convertiti in un unità comune ed espressi in termini di impact score per ogni categoria d’impatto. L’importanza di studi di LCA nel settore dei nanomateriali (ENM) è stata sottolineata da diversi studi. Nonostante le nanotecnologie, sembrano offrire enormi potenzialità per lo sviluppo di nuovi prodotti e applicazioni con migliori prestazioni energetiche o un ridotto uso di materiali ed energia, vi sono ancora forti incertezze legate ai loro possibili impatti ambientali e sanitari. Negli ultimi dieci anni l'aumento della produzione, l'uso e il consumo di nanoprodotti, ha accentuato l'obbligo di garantire che i rischi potenziali siano adeguatamente compresi per proteggere sia la salute umana e l'ambiente. Grazie al suo approccio olistico l’LCA è stato identificata come uno strumento essenziale per valutare, gli effetti sull'ambiente e sulla salute. Solo pochi studi di LCA sono stati condotti sui nanoprodotti e tra questi solo pochi considerando gli impatti ambientali di tipo tossicologico. La scarsa applicazione della metodologia LCA nel settore della nanotecnologia è principalmente dovuta alle frammentarie conoscenze scientifiche correlati alla valutazione del rischio ambientale.Ad oggi, nella metodologia LCA si risente della completa mancanza di opportuni modelli di caratterizzazione per gli impatto tossicologici. Il progetto di dottorato si propone di valutare i limiti e le sfide nella fase di LCIA per la valutazione degli impattiecotossicologi per nanoparticelle di n-TiO2.

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Trata-se de uma pesquisa qualitativa que teve como objetivo principal identificar a influência e participação do parceiro na decisão de abortar através das percepções e experiências de mulheres que referiram situações de aborto induzido e de relacionamentos estáveis com estes parceiros. Foram realizadas 16 entrevistas semiestruturadas com moradoras de duas favelas da zona norte do Rio de Janeiro, das quais analisamos 20 casos de abortos. Utilizamos a perspectiva relacional de gênero como categoria de análise.Incluímos como temas de análise: as representações de gênero, a contracepção e gravidez indesejada, a tomada da decisão de abortar e a participação masculina na decisão de abortar. Observamos que estas mulheres conciliam significados de uma ideologia de gênero tradicional com outra mais igualitária, principalmente em relação à identidade feminina e ao trabalho da mulher, o que denotaria um processo de transição de gênero. A gravidez indesejada foi construída a partir da visão e interação de ambos os parceiros e condicionada fortemente por fatores de contexto, entre eles, o desemprego. A participação masculina na decisão de abortar é variada (o parceiro se exclui, promove ou recusa o aborto); mas o desejo de não assumir a paternidade parece refletir as características da natureza e qualidade do relacionamento, assim como a situação do parceiro como trabalhador ou provedor da família.

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Cutaneous melanoma is a type of skin cancer that originates in melanocytes, predominantly affecting young and middle-aged adults. Several evidence suggests that cancer stem cells exist for melanoma. The POU5F1 / OCT4, NANOG genes have been studied as cancer stem markers. OCT4 and NANOG are involved in the maintenance of pluripotency and self-renewal of undifferentiated embryonic stem cells. This study aims to evaluate the epidemiological, clinical-pathological and expression of the OCT4 and NANOG proteins in cutaneous melanoma samples. We selected 102 cases for the epidemiological, clinical and pathological study, diagnosed between the years 2004 to 2008. For survival study, patients with a followup of up to 60 months were selected, with a recorded death. Of the 102 cases evaluated, 62.7% were female and 37.3% male; With mean age of 57.2 years and 63.1 years respectively (p= 0.0026). The most prevalent age at diagnosis of melanoma was between 51 and 70 years (44.1% p= 0.023). In this study, there was a predominance of lesions located in the trunk (32.3%). Histopathological examination of the type of tumor growth showed a predominance of the superficial extensive type in 52.9% of the cases. According to the Breslow index, lesions with ≤1.0 mm predominated in 39.2% of the individuals, followed by lesions> 4.0 mm in 23.5% of the cases. According to the Clark level 29.4% of the cases were classified in level IV; Followed by 25.5% cases with level V; Clark II in 23.5%; Clark III in 20.6%; And Clark I in only 1 case (1.0%). There were metastases in 47% of the cases and the main localization sites were: lymph nodes, brain, skin and lung. Regarding the clinical evolution of the patients, there were 26 cases of deaths due to melanoma (25.5%). The survival curve calculated at the 60-month follow-up was 73.0%. Univariate analysis revealed significant associations between nuclear overexpression of OCT4 and the following variables: Breslow index with thickness > 2.1 mm (p = 0.021, OR: 2.64, 95% CI: 1.15-6.05 ); Levels of Clark, IV and V (p = 0.001, OR: 4.11, 95% CI: 1.79-9.46); ulceration present (p≤0.0001; OR: 459.0; 95% CI: 51.67-4077.27); Presence of metastases (p <0.0001, OR: 40.25, 95% CI: 12.90- 125.62), and death from cutaneous melanoma (p <0.0001). The significant associations between cytoplasmic hyperexpression of OCT4 and the following variables were: present ulceration (p = 0.015, OR: 2.73, 95% CI: 1.21-6.16); Presence of metastases (p = 0.004, OR: 3.34, 95% CI: 1.47 - 7.62) and death from cutaneous melanoma (p = 0.039, OR 2.67, 95% CI 1.05 - 6,77). And the significant associations between the cytoplasmic hyperexpression of NANOG and the following variables were: present ulceration (p≤ 0.0001); Presence of metastases (p ≤ 0.0001) and death due to cutaneous melanoma (p = 0.030). Our study demonstrated a strong association of the OCT4
O melanoma cutâneo é um tipo de câncer de pele que tem origem nos melanócitos, afetando predominantemente adultos jovens e de meia-idade. Diversas evidências sugerem a existência células-tronco tumorais para o melanoma. Os genes POU5F1/OCT4, NANOG vem sendo estudados como marcadores células-tronco tumorais. O OCT4 e o NANOG estão envolvidos na manutenção da pluripotência e autorrenovação das células-tronco embrionárias indiferenciadas. Este estudo tem por objetivo avaliar os fatores epidemiológicos, clínico-patológicos e expressão da proteína OCT4 e NANOG em amostras de melanoma cutâneo. Foram selecionados 102 casos para o estudo epidemiológico, clínico e patológico, diagnosticados entre os anos de 2004 a 2008. Para estudo de sobrevida foram selecionadas pacientes com seguimento de até 60 meses, com óbito registrado. Dos 102 casos avaliados foram observados 62,7% do gênero feminino e 37,3% masculino; com média de idade de 57,2 anos e 63,1 respectivamente (p= 0,0026). A idade de maior prevalência ao diagnóstico do melanoma foi entre 51 a 70 anos (44,1% p= 0,023). Nesse estudo, houve predomínio das lesões localizadas no tronco (32,3%). Ao exame histopatológico quanto ao tipo de crescimento do tumor, houve predomínio do tipo extensivo superficial em 52,9% dos casos. Segundo o índice de Breslow predominaram as lesões com ≤1,0mm em 39,2% dos indivíduos, seguidas pelas lesões >4,0mm em 23,5% dos casos. De acordo com o nível de Clark 29,4% dos casos foram classificados no nível IV; seguidas por 25,5% casos com nível V; Clark II em 23,5%; Clark III em 20,6%; e Clark I em apenas 1 caso (1,0%). Houve metástases em 47% dos casos e os principais sítios de localização foram: linfonodos, cérebro, pele e pulmão. Em relação à evolução clínica dos pacientes ocorreram 26 casos de óbitos por melanoma (25,5%). A curva de sobrevida calculada no seguimento de 60 meses foi de 73,0%. A análise univariada revelou associações significativas entre a hiperexpressão nuclear de OCT4 e as seguintes variáveis: índice de Breslow com espessura de > 2,1 mm (p= 0,021; OR: 2,64; IC 95%: 1,15 - 6,05); níveis de Clark, IV e V (p= 0,001; OR: 4,11; IC 95%: 1,79 - 9,46); ulceração presente (p≤ 0,0001; OR: 459,0; IC 95%: 51,67 - 4077,27); presença de metástases (p≤ 0,0001; OR: 40,25; IC 95%: 12,90 - 125,62) e óbito por melanoma cutâneo (p≤ 0,0001). As associações significativas entre a hiperexpressão citoplasmática de OCT4 e as seguintes variáveis foram: ulceração presente (p= 0,015; OR: 2,73; IC 95%: 1,21 - 6,16); presença de metástases (p= 0,004; OR: 3,34; IC 95%: 1,47 - 7,62) e óbito por melanoma cutâneo (p= 0,039; OR: 2,67; IC 95%: 1,05 - 6,77). E as associações significativas entre a hiperexpressão citoplasmática de NANOG e as seguintes variáveis foram: ulceração presente (p≤ 0,0001); presença de metástases (p≤ 0,0001) e óbito por melanoma cutâneo (p= 0,030). Nosso estudo demostraram uma forte associação dos genes OCT4 e NANOG como os piores fatores prognósticos do melanoma cutâneo.

Block copolymer nanoparticles (NPs) have gained great attention among researcher for various medical application mainly due to their extraordinary optical, chemical, and biological properties. The current thesis presents design of multifunctional polymeric NPs for imaging and drug delivery system (DDS) with an in-vitro study of their participation in drug release and cell viability. The NPs were synthesized using reversible addition chain fragmentation transfer (RAFT)-mediated emulsion polymerization via polymerization induce self-assembly (PISA) approach. The environment-friendly emulsion polymerization process of n-buytl acrylate (n-BA) in water is highly efficient. The process produced uniform NPs which would have control over the particle size and molecular weight of the compound. Herein we report a novel simultaneous encapsulation of camptothecin (CPT) and Nile red (NR) into poly(ethylene glycol) methyl ether methacrylate-co-N-hydroxyethyl acrylamide-b-poly n-buytlacrylate (PEGA-co-HEAA)-b-P(n-BA) during the particles formation with a small particle size of 66 nm, high conversion ~80% and encapsulation efficiency of ~50%. The In vitro drug release of the CPT from the NPs exhibited an initial burst (70-80%) within 6h. cell viability was evaluated for the NPs against RAW 264.7 cell line, which indicated the designed NPs are biocompatible and not toxic.

The following project presents in silico investigation of axonal damage in Diffuse Axonal Injury (DAI). When axons face a shear force, orientation of the lipids in the axonal membrane gets disrupted. Depending on the value of the force, a tensile strain causes the axons to get partially or fully deformed and in some cases a pore forms in the membrane. Using Molecular Dynamic (MD) simulation and a coarse grain model, a series of bilayers with various bilayer structure (single bilayer, parallel bilayer and cylindrical bilayer) and similar composition to biological axonal membrane were simulated. This was initially done to investigate the strain rate dependency of the bilayers, and their viscoelastic ability on returning to their original shape from their deformed forms. To achieve this, various deformation velocities were applied to the bilayers reaching 20% strain and relaxing the bilayer after. Additionally, the bilayers were deformed further until they reached a pore. It was found that the bilayers can almost recover from their deformed forms to their original length when they were deformed at 20% strain level. In conjunction, no correlation between the deformation velocity and lipid deformation was observed. Further, it was found that bilayers with different lipid percentage to axonal bilayer has different strain values for water penetration and for pore formation. The strain value for cylindrical bilayer was found very high compared to the strain values found in vitro. The strain for pore formation of parallel and single bilayer was found to be around 80% to 90% and for water penetration was found to be 70% for single bilayer and 50% for parallel bilayer. A slight difference in strain for pore formation between single and parallel bilayer was found which showed the bilayer structure can play a role in simulation results. The effect of the length in the simulations results was also observed where shorter bilayers showed lower strain for pore formation compared to longer bilayers.

Publicación a texto completo no autorizada por el autor
Determina el efecto de un suplemento sobre los marcadores bioquímicos musculares y el rendimiento físico en ratones sometidos a nado forzado. Desarrolla un estudio analítico, experimental, transversal y prospectivo. Utilza 35 ratones Mus musculus machos adultos de 8 semanas de edad, con un peso promedio de 35,43 g, los cuales fueron distribuidos de manera aleatoria en 5 grupos (n=7) para recibir el siguiente tratamiento vía peroral: grupo I y II: suero fisiológico 10 mL/kg; y los grupos III, IV y V recibieron el suplemento deportivo a dosis de 150, 300 y 800 mg/kg de peso. Posteriormente se sometió a los grupos II, III, IV y V al test de nado forzado hasta el agotamiento. Para determinar el rendimiento físico se tomó el tiempo (minutos) de nado forzado hasta el agotamiento, con respecto a los marcadores bioquímicos se tomó la actividad enzimática del Lactato deshidrogenasa (LDH) y lipoperoxidación a nivel muscular. Encuentra que el suplemento presenta un efecto de incremento en la resistencia física de los grupos IV y V en comparación del grupo II, también se observó una reducción en la actividad la lactato deshidrogenasa (LDH) en los grupos III y IV en comparación del grupo número II, además con respecto a la lipoperoxidación se encontró una reducción en los grupos III, IV y V en comparación del grupo II. Concluye que el suplemento presenta un efecto favorable en la resistencia física y sobre los marcadores bioquímicos musculares.
Tesis

The objective of this study was to determine the effectiveness of a 6 week afterschool nutrition and physical activity intervention administered by a registered dietitian with the help of a humanoid robot targeting elementary school aged children aged 6-12 years. The study was conducted across four Young Men’s Christian’s Association (YMCA) sites in Miami-Dade County, Florida (N= 114, Mean age: 8.16 ±1.57 years) using a pretest-posttest quasi-experimental design via randomly assigned intervention (two sites; n=63) and comparison groups (two sites; n=51). The validated Coordinated Approach to Child Health (CATCH) kids club questionnaire and the validated Previous Day Physical Activity Recall (PDPAR) were used to assess nutrition and physical activity knowledge, attitudes/beliefs and behavior change. The Inbody 230 instrument (Biospace, California) was used to calculate body composition and weight. Body Mass Index (BMI) percentiles and associated BMI z-scores for age and gender were calculated based on the Center for Disease Control and Prevention (CDC) growth charts. Data measures were collected at baseline (week 0) and one-week post intervention (week 7). Statistical analysis included independent t-test, paired t-test, chi-squared test, Wilcoxon signed ranks test and logistic regression. Results indicated that nutrition knowledge score significantly increased from 67.43% ±21.03 to 81.31% ±18.47 in the intervention group (p

Les sutures crâniennes ont été régulièrement étudiées au fil des siècles puisque déjà Vésale établissait une relation entre l’âge et la synostose suturale.

Leur imprécision a quelque peu confiné l’observation de ces sutures dans un certain oubli, justifié en partie.

Il est clair que leur fiabilité quant à l’estimation de l’âge au décès reste discutable et ce, pour diverses raisons.

Leur observation est difficile et dès lors sujette à subjectivité dans l’appréciation de leurs stades de fusion.

De plus, leur apparence sur le crâne sec peut être altérée par divers artéfacts de conservation (cire, vernis…).

Outre l’observation des classiques sutures ectocrâniennes de voûte et l’utilisation des méthodes habituelles en la matière (méthode de Acsádi et Nemeskéri, méthode de Masset), nous avons orienté notre étude vers des sutures peu voire pas exploitées, à savoir les sutures palatines, les sutures fronto-naso-maxillaires et les sutures de l’os zygomatique.

Ces trois groupes suturaux ont la particularité d’évoluer de manière très lente vers la fusion, à tel point que peu d’individus en présentent une oblitération complète.

Face au vieillissement de la population et de par notre expérience médico-légale et anthropologique d’étude de pièces osseuses de personnes âgées, nous avons estimé qu’il était utile de se pencher sur des collections particulières de sujets d’âge avancé, afin d’apprécier l’évolution morphologique de ces sutures faciales.

Si certaines personnes fort âgées gardent malgré tout des caractéristiques suturales peu évoluées, il existe dans l’ensemble, une progression suturale quasiment constante en fonction de l’âge.

Nous avons tenté de la cerner, en attribuant à ces sutures, des degrés bien définis de cette progressive fusion et par là, l’aboutissement à un coefficient moyen d’oblitération suturale, se traduisant aisément en pourcentage d’oblitération ou pouvant être introduit dans des équations de régression.

Tout en connaissant les limites de cette approche, nous pouvons estimer qu’elle peut rendre des services lors de l’étude de restes humains squelettisés, notamment s’ils appartiennent à des personnes fort âgées, dès lors qu’à ces stades de vieillissement, peu de méthodes restent encore applicables. D’autre part, l’approche en pourcentage d’oblitération suturale rend également des services lorsque les crânes étudiés sont fragmentés. C’est la situation que nous rencontrons actuellement lors de l’étude d’une très importante collection anthropologique à l’Institut royal des Sciences naturelles de Belgique.

Cette observation suturale pourra également – à l’avenir – trouver un terrain d’approche fort utile par l’étude des sutures en CT-Scan ou en micro-CT.

Enfin, grâce à ces techniques modernes d’imagerie médicale, les sutures trouvent un regain d’intérêt dans une application toute particulière, qui est celle de l’identification comparative, puisqu’il apparaît que le « dessin sutural » s’avère tout à fait propre à chaque individu.

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Cranial sutures were regularly studied during centuries since Vésale already established a relationship between age at death and sutural fusion.

Their inaccuracy somewhat confined the observation of these joints in a certain lapse of memory, partly justified.

Various reasons clearly indicate that their reliability for age at death estimation remains debatable.

Their observation is difficult and consequently prone to subjectivity in the appreciation of their stages of fusion.

Moreover, their appearance on dry cranium can be modified by various artefacts from preserving methods (wax, varnished…).

In addition to the traditional observation of ectocranial sutures (with the usual methods such as Acsádi and Nemeskéri method and Masset method), we have directed our study towards not much exploited sutures, namely the palatine sutures, the fronto-naso-zygomatic sutures and the sutures of the zygomatic bone.

These three sutural groups are characteristic by evolving very slowly to fusion, and than, only few individuals present a complete obliteration of the latter.

Because of general population ageing and our medicolegal and anthropological experience of skeletal remains in elderly, we estimated the usefulness in studying particular collections of old people, in order to appreciate the morphological evolution of these facial joints.

Despite some very old people who present little evolved sutural characteristics, we consider as a whole, an almost constant sutural progression according to age.

We tried to define degrees of this progressive fusion and by the way, the result with a sutural obliteration average coefficient, can be easily translate as a percentage obliteration or able to be introduced into regression equations.

The limits of this approach are well known but we estimate that it is possible to help the examination of human skeletal remains in elderly, although at these stages of ageing, some methods remain still applicable. In addition, the approach expressed as a percentage of sutural obliteration is also very helpful when craniums are fragmented. This situation is currently observed on the anthropological collections at the Royal Belgian Institute of Natural Sciences.

Furthemore in the future, the sutural observation will find an interesting and useful approach by the study of the joints in CT-Scan or micro-CT.

At least, thanks to these modern techniques of medical imagery, the sutures find a renewed interest in a very particular application, such as comparative identification. It appears that the "sutural drawing" proves completely specific to each individual.

Doctorat en sciences médicales
info:eu-repo/semantics/nonPublished

To increase the survival rate from infectious- and noncommunicable diseases, reliable diagnostic during the preliminary stages of a disease onset is of vital importance. This is not trivial to achieve, a highly sensitive and selective detection system is needed for measuring the low concentrations of biomarkers available. One possible route to achieve this is through biosensing based on plasmonic nanostructures, which during the last decade have demonstrated impressive diagnostic capabilities. These nanoplasmonic surfaces have the ability to significantly enhance fluorescence- and Raman signals through localized hotspots, where a stronger then normal electric field is present. By further utilizing a periodic sub-wavelength nanohole array the extraordinary optical transmission phenomena is supported, which open up new ways for miniaturization. In this study a nanoplasmonic chip (NPC) composed of a nanohole array —with lateral size on the order of hundreds of nanometer— covered in a thin layer of gold is created. The nanohole array is fabricated using soft nanoimprint lithography on two resists, hydroxypropyl cellulose (HPC) and polymethyl methacrylate (PMMA). An in depth analysis of the effect of thickness is done, where the transmittance and Raman scattering (using rhodamine 6G) are measured for varying gold layers from 5 to 21 nm. The thickness was proved to be of great importance for optimizing the Raman enhancement, where a maximum was found at 13 nm. The nanohole array were also in general found beneficial for additionally enhancing the Raman signal. A transmittance minima and maxima were found in the region 200-1000 nm for the NPCs, where the minima redshifted as the thickness increased. The extraordinary transmission phenomena was however not observed at these thin gold layers. Oxygen plasma treatment further proved an effective treatment method to reduce the hydrophobic properties of the NPCs. Care needs be taken when using thin layers of gold with a PMMA base, as the PMMA structure could get severely damaged by the plasma. HPC also proved inadequate for this projects purpose, as water-based fluids easily damaged the surface despite a deposited gold layer on top.

碩士
國立清華大學
材料科學工程學系
100
With the property of sol-gel transition in physiological condition and numerous advantages from peptide-based material, self assembling peptide (SAP) has been regarded as a promising 3D hydrogel scaffold or cell/gene (drug) carrier for the applications in tissue engineering and drug delivery system. The additional functional motifs with various modification ratios and multi-extended diversity can be conjugated and extended at the terminal residue sequence of specific-designed SAPs to enrich SAP possessing therapeutic functionality for different biopharmaceutical applications and serve as medical disease treatment modality. In this study, we specifically enriched RADA16, one of the widely used SAPs, with different functional motifs and evaluated the medical applications of the functionalized SAPs in hemostasis, liver tissue regeneration and brain neural tissue repair in the central nervous system. In the first part of this study, the physiochemical properties and gelation mechanism of SAPs were in detail investigated. The pKa1, pKa2, and isoelectric point (pI) was obtained by pH titration curve. The peptide showed net-charged at neutral pH value. The protein secondary structure of β-sheet arrangement was confirmed by circular dichroism (CD) spectroscopy. The morphology of nanofibers about 25nm diameter was observed by atomic force microscopy (AFM). The gelation behavior in terms of mechanical property of hydrogel was determined by oscillatory rheology test. As for in vitro experiments, the relative low cytotoxicity of peptide hydrogel was examined by Live/Dead assay and the high cell proliferation ability was shown in 3D hydrogel culture by MTS assay. The results demonstrated that the encapsulated neural stem cells well survived with 3D culture in SAP hydrogel. We also found that even with extended motifs, the gelation behavior, physical and chemical properties of functionalized SAPs were still preserved. It is believed that the functionalized SAPs can be a promising material for follow-up tissue engineering applications. In hemostasis and liver tissue regeneration study, specifically extended fragments of functional motifs derived from fibronectin and laminin, GRGDS and YIGSR, was designed to evaluate the capability of these functional SAPs in the effect of immediate hemostasis and accelerative liver tissue regeneration. The results showed that hemostasis can be achieved within 10 seconds and the histological analyses demonstrated by H/E stain and immunohistochemistry revealed that SAPs with these extended functional motifs significantly enhanced liver tissue regeneration in rat liver wound model. It is reported that SAPs enriched with the extended functional motifs not only maintained their spontaneous self-assembly property but also extensively advanced the potential therapeutic effect in hemostasis and liver tissue regeneration. In the study of brain neural tissue repair in the central nervous system, the functionalized RADA16-IKVAV peptide hydrogel was used to encapsulate neural stem cells (NSCs) for investigation of the effectiveness in brain tissue regeneration. The results showed that after 6 weeks transplantation, the regenerative extracellular matrix (ECM) of brain neural tissue was noticed. The specific neuronal protein markers, such as βIII tubulin, MAP2, NF-H were positively stained in the region of wound defect area, suggesting regenerative process of neuronal cells in the brain cerebral cortex. In summary, we have successfully developed functionalized SAPs with multiple extended motifs, including RADA16-GRGDS, RADA16-YIGSR and RADA16-IKVAV, and their physiochemical properties were also carefully evaluated for the comparison with RADA16 nanopeptides. These functionalized SAPs can not only be used alone as 3D nanoscale scaffold for immediate hemostasis and accelerative liver tissue wound healing, but also be incorporated with neural stem cells for the improvement of brain tissue repair in the central nervous system.

碩士
中臺科技大學
生物科技暨醫學工程研究所
105
This study proposes composite masks with avirulence, humectancy, and whitening properties. Polymeric materials, including polyvinyl pyrrolidone (PVP), gelatin (GEL), and Morus alba (MA), are combined with spunlace nonwoven fabrics in order to form cosmetic care products. This study can be divided into three stages. In the 1st stage： natural Chinese herbal medicine are extracted and evaluated for whitening efficacy. In the 2nd stage, PVP/GEL/MA nanofiber membranes are prepared. In the last stage, membranes are then combined with nonwoven fabrics. Natural Chinese herbal medicine, including Sophora japonica, Spatholobus suberectus, Morus alba, and Polygonatum odoratum are respectively immersed in pure water and 95% ethanol solution in order to have the Chinese herbal medicine extracts. The extracts are tested in terms of extractability, biocompatibility, and MTT assay, thereby characterizing the properties of the four Chinese herbal medicine. Tyrosinase inhibition ratio is used to examine the water-extraction Morus alba (MA) extract to be the optimal whitening extract. Moreover, the PVP/GEL/MA solution is electrospun into spunlance nonwoven, forming PVP/GEL/MA composite masks. The test results show that MA herbal extract at a concentration of 0.5 mg/ml has good inhibition ratio against tyrosinase, and the MTT assay result is 88 %, indicating a good biocompatibility with L929 fibroblasts. The electrospinning film has a smooth fiber formation when a 20 wt% PVA/GEL mixture is used. The addition of GEL and MA increases both the viscosity and electrical conductivity of the mixtures. The electrospun nanofibers are slim without bead-shaped and have a diameter of 210 nm with the settings of a voltage of being 20kV, a collect distance of being 15 cm, and an electrospinning rate being 0.5 mg/hr. Based on the contact angle meter restuls, the combination of PVP/GEL/MA film and spunlance nonwoven results in a water contact angle of 29.2°, suggesting a good hydrophility. The water vapor transmission rate of PVP/GEL/MA mask is 600-800 g/m2day, which is close to that of the human skin (350 g/m2day), which means the masks have good air-permeable property and suitable for the skin. Moreover, according to the tyrosinase inhibition measurement, the masks have a drug delivery period of 25 minutes at a release rate of 75 %, and an inhibition ratio of 81.42±1.6%. Namely, the whitening effect of the proposed composite masks is slightly higher than that of ready-made masks. In addition, the proposed composite masks have an MTT assay result at 88.79%, which shows that PPVP/GEL-MA composite masks does not obstruct the cell growth and have good biocompatibility. All test results confirm that the PVP/GEL/MA composite membranes have good whitening efficacy and biocompatibility.

Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
Introdução: A abordagem de utilizar nano medicina para tratar dentre outras doenças, o cancro vem sendo utilizada e estudada cada vez mais para ultrapassar as barreiras encontradas na terapia convencional. A utilização do polímero anfifílico não tóxico como o Pluronic F127 para a formação de nano partículas traz grandes vantagens por formar micelas de substâncias biologicamente ativas com tamanhos de 10 a 100 nm que tem como objetivo entregar fármacos pouco solúveis as células, além de terem a habilidade de entrar passivamente via aumento de permeabilidade e retenção das células tumorais aumentando assim a biodisponibilidade e acúmulo do fármaco nas células de interesse, diminuindo efeitos adversos causados por quimioterápicos. A Salinomicina (SAL) é um antibiótico isolado de Streptomyces albus que recentemente mostrou ser um novo candidato ao tratamento de cancro por inibir seletivamente células estaminais presentes no cancro responsáveis pelo ressurgimento da doença e resistência aos fármacos quimioterápicos.Objetivos: O objetivo principal deste estudo é o design de micelas poliméricas (PM) para distribuição de SAL para células cancerígenas e sua caracterização em termos de diâmetro médio (MD), carga de superfície (ZP) e índice de poli dispersão (PDI). A capacidade de encapsulaçao de MP SAL é determinada e sua eficácia in vitro em relação ao modelo NSCLC foi avaliada e comparada com a atividade livre do fármaco.Métodos: A formação de micelas foi feita pela técnica de reidratação de filme, onde o solvente orgânico contendo uma mistura de fármaco e polímero é evaporado a fim de formar um filme polimérico. Esse filme então é reidratado com água e agitado e há a formação de micelas. Ensaios in vitro como MTT, wound healing e imunocitoquímica foram realizados em triplicata com cultura celular em placas de 96 e 24 poços seguidos de tratamentos com fármaco livre (SAL), fármaco encapsulado (PM SAL) em micelas bem como micelas vazias (Plain PM) em diferentes tempos de incubação. Para a imunocitoquímica, anticorpos anti Pg-p e Vimentina foram utilizados a fim de observar no microscópio de fluorescência os níveis de expressão destas proteínas.Resultados: A caracterização das micelas confirmou que o polímero utilizado foi capaz de formar micelas com tamanhos e índice de poli dispersão esperados (<100nm e < 0,4 respectivamente) já o potencial zeta foi diretamente alterado a medida que a concentração de polímero aumentava, ou seja, quanto maior a concentração de polímero mais próximo da neutralidade a carga de superfície. Para os resultados de MTT, SAL em sua forma não encapsulada mostrou ter efeito dose e tempo dependente em células de cancro de pulmão após 48 horas e além disso, mostrou ser mais eficaz do que em sua forma encapsulada.Nos ensaios de invasão celular (wound healing) SAL e PM SAL mostraram efeitos semelhantes na retenção de migração celular após 24 horas. Nos resultados de imunocitoquímica, SAL e PM SAL mostraram resultados muito semelhantes em inibir a expressão de Pg-p. Por fim, quanto a expressão de vimentina, pode-se observar que quando a concentração de fármaco foi aumentada para 20 uM, houve uma diminuição mais acentuada da expressão de vimentina nas células tratadas com PM SAL. Conclusão: As micelas poliméricas apresentaram as características propostas para a entrega de SAL. Nos ensaios de viabilidade celular, não demonstraram ter efeito citotóxico mais acentuados do que em sua forma livre, porém as PM SAL resistiram ao processo biológico e sua eficácia foi demonstrada na inibição da migração celular, na evasão da Pg-p e inibição da vimentina. Isto indica que as PM SAL podem ser um eficiente coadjuvante no tratamento de NSCLC. Estudos posteriores serão necessários mais para entender melhor a internalização das micelas assim como se faz necessária a funcionalização com anticorpos para que as micelas sejam direcionadas para as células de interesse.
Introduction: The approach of using nano medicine to treat several diseases, for cancer has been increasingly used and studied to overcome the barriers found in conventional therapy. The use of the non-toxic amphiphilic polymer such as Pluronic F127 for the formation of nano particles brings great advantages by forming micelles of biologically active substances with sizes from 10 to 100 nm. This technique aims to deliver poorly soluble drugs to the cells, in addition to achieve the ability to passively enter via enhanced permeability and retention (EPR) of tumour cells thereby increasing the bioavailability and accumulation of the drug in the cells of interest, decreasing adverse effects caused by chemotherapeutics. Salinomycin (SAL) is an isolated Streptomyces albus antibiotic that has recently been shown to be a new candidate for cancer treatment by selectively inhibit cancer stem cells responsible for disease resurgence and resistance to chemotherapeutic drugs.Objectives: The main goal of this study is the design of polymeric micelles (PM) for SAL delivery to cancer cells and their characterization in terms of medium diameter (MD), surface charge (ZP) and poly dispersion index (PDI). MP SAL loading capacity is determined and it’s in vitro in effect against NSCLC model was assessed and compared with the free drug activity.Methods: The preparation of micelles was done by the film rehydration technique, where the organic solvent containing a mixture of drug and polymer is evaporated to form a polymer film. This film is then rehydrated with water and vortex to form micelles. In vitro assays such as MTT, wound healing and Immunocytochemistry were performed in triplicate with cell culture in 96 and 24 plates followed by treatments with free drug (SAL), encapsulated drug (PM SAL) in micelles and empty micelles (Plain PM) in different incubation time points. For immunocytochemistry, anti-Pg-p and vimentin antibodies were used to observe the levels of expression of these proteins in the fluorescence microscope.Results: The micelles characterization confirmed that the polymer was able to form micelles with expected MD and PDI (<100nm and <0.4 respectively), ZP was directly altered as a function on the polymer concentration. For the MTT results, SAL in its non encapsulated form has shown to have dose and time dependent effect on lung cancer cells after 48 hours and furthermore proved to be more effective than in its encapsulated form. The wound healing results SAL and PM SAL had similar effects on retention of cell migration after 24 hours. In the results of immunocytochemistry, SAL and PM SAL seems to have very similar results in inhibiting Pg-p expression. Finally, as for Vimentin expression, it can be seen that, when the drug concentration was increased to 20 uM, there was more marked decrease in Vimentin expression in the cells treated with PM SAL.Conclusion: The polymeric micelles has presented the characteristics proposed for SAL delivery. In the cell viability assays, PM SAL were not shown to have more marked cytotoxic effect than in their free form, but PM SALs resisted the biological process and their efficacy was demonstrated in inhibition of cell migration, Pg-p evasion and inhibition of vimentin expression. This indicates that PM SAL can be an efficient adjunct in the treatment of NSCLC. Further studies will be needed for better understanding of the micelles internalization process, also the functionalization with antibodies is necessary to directing the micelles to the cells of interest.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
No âmbito da Unidade Curricular de Estágio Curricular do Mestrado Integrado em Ciências Farmacêuticas da Faculdade de Farmácia da Universidade de Coimbra, o presente documento apresenta, sob a forma de análise SWOT (Strengths, Weaknesses, Opportunities and Threats), o relatório de estágio em Indústria Farmacêutica, relativo ao estágio realizado na Farmalabor, em Condeixa-a-Nova, com início a 8 de janeiro e término a 29 de março de 2018, bem como o relatório de estágio em Farmácia Comunitária, realizado na Farmácia de Celas, em Coimbra, entre os dias 2 de abril e 15 de julho de 2018. O presente documento inclui ainda a monografia intitulada “Medicamentos de base nanotecnológica na terapia anti-tumoral, no mercado e em ensaios clínicos”.O cancro é hoje uma das principais causas de mortalidade a nível global. A quimioterapia convencional apresenta várias limitações, como a entrega não específica dos agentes terapêuticos, levando muitas vezes a problemas de toxicidade. Nos últimos anos, o crescimento rápido da nano-medicina na área da oncologia tem demonstrado inúmeros avanços através do desenvolvimento de várias nano-terapias. Estas terapias recorrem a nano-transportadores com o objetivo de proporcionar um tratamento direcionado, alterando o perfil de farmacocinética das terapias convencionais e entregando o fármaco no local-alvo do tumor, de forma a diminuir os efeitos adversos e aumentar a eficácia terapêutica. Baseados em estratégias de passive targeting, active targeting e libertação por resposta a estímulos, estes nano-medicamentos têm alcançado resultados extraordinários em ensaios clínicos, tendo sido alguns já aprovados pelas entidades reguladoras do medicamento, ambicionando alcançar um enorme peso no futuro da terapia anti-tumoral.
Within the scope of the course unit of Curricular Internship of the Integrated Master’s degree in Pharmaceutical Sciences of the Faculty of Pharmacy of the University of Coimbra, the present document exhibits, in the form of a SWOT analysis (Strengths, Weaknesses, Opportunities and Threats), the report of the Pharmaceutical Industry Internship, performed at Farmalabor, in Condeixa-a-Nova, from 8th of January to 29th of March 2018, and the report of the Community Pharmacy Internship, performed at Farmácia de Celas, in Coimbra, from 2nd of April to 15th of July 2018. The present document also includes the monograph entitled “Nanotechnology-based drugs in anticancer therapy, on the market and in clinical trials”.Cancer is now a leading cause of global mortality. Conventional chemotherapy has several limitations, such as non-specific delivery of therapeutic agents, often leading to toxicity problems. In the last few years, the rapid growth of nanomedicine in the field of oncology has demonstrated numerous advances through the development of various nanotherapies. These therapies rely on nanocarriers to provide targeted treatment by altering the pharmacokinetic profile of conventional therapies and delivering the drug to the target site of the tumor to decrease adverse effects and increase therapeutic efficacy. Based on strategies of passive targeting, active targeting and stimuli-responsive and triggered release, these nanomedicines have achieved extraordinary results in clinical trials, and some have already been approved by regulatory authorities, aiming to achieve a huge weight in the future of anticancer therapy.

With 50,310 related deaths this year, colorectal cancer (CRC) has emerged as the second largest cause of cancer related deaths among Americans. While 70 million Americans are considered at-risk of developing CRC, it is highly curable if detected early. Cohesin proteins, which hold sister chromatids together during replication, have emerged as a potential biomarker in multiple cancer lines. Because of their probable role in DNA replication, DNA repair, chromatin nano-architecture, and gene expression, this paper assessed whether cohesion proteins could be used as a potential biomarker for colorectal cancer risk stratification. While cohesin protein mutations have been reported in different cancers and involved in chromosomal instability, its role in early cancer formation has yet to be observed. Using immunohistochemical and Quantitative Real Time PCR analysis, this thesis assessed the protein and RNA expression levels of cohesin proteins SA-1, NIPBL, and SMC3 from human biopsies at different stages and locations of colorectal cancer development. The results showed that SA-1, a structural cohesion subunit, was significantly (p<0.01) down regulated in cancerous compared to normal tissue. The SA-1 protein was also down regulated in the involved mucosa adjacent to CRC polyps. The cohesion loading protein, NIPBL, was also significantly (p<0.01) under expressed in cancerous versus normal tissue. The RNA expression analysis of rectal mucosa showed that SMC3 and SA-1 was over expressed two fold in patients harboring hyperplastic and adenomous polyps, giving evidence that cohesin proteins are differentially expressed throughout the field of carcinogenesis. Our results demonstrate for the first time that cohesion dysregulation is an early event in human colorectal cancer development and may serve as an important biomarker of field carcinogenesis.

The thesis begins with a brief introduction and relevant literature references. The novelty of synthesis, methodology and results of the work reported in the thesis and highlighted subsequently. The thesis consist of three parts, Part A of the thesis consist of five chapters describing new methods of synthesis of orthovanadates, mainly dealing with the structure and photocatalytic properties of synthesized materials. Part B of the thesis consist of two chapters dealing with an unique crystallization methodology for subliming and low melting organic compounds and the crystal structure determination via single crystal X-ray diffraction. Part C consists of one chapter presenting the ab-initio approach of structure determination via powder X-ray diffraction methods. Chapter 1 of the thesis presents the synthesis of three zircon type lanthanide orthovanadates, LnVO4 (Ln = Ce, Pr and Nd) via a new solution based approach at room temperature and photodegradation of a variety of water pollutants have been investigated. Chapter 2 describes the importance of microwave synthesis to produce nano particles of the zircon type lanthanide orthovanadates LnVO4. The importance of the surface area in photocatalysis is evaluated. Chapter 3 consist of the synthesis of a series of new compounds, Ln0.95φ0.05Mo0.15V0.85O4 (Ln = Ce, Pr and Nd) via solid state method. The photocatalytic activities of these compounds are investigated both under UV exposure and sunlight. Chapter 4 presents the synthesis of MxCe1-xVO4+ (M = Li, Ca and Fe) with x = 0.1, 0.25 and 0.05 respectively. Different kinds of dyes and organics are degraded under UV radiation and the specificity towards the same are evaluated. Chapter 5 describes a comparative photocatalytic conversion of cyclohexane and benzene to cyclohexanol, cyclohexanone and phenol respectively by LnVO4, LnMo0.15V0.85O4 and MxCe1-xVO4+ (Ln = Ce, Pr and Nd, M= Li, Ca and Fe with x = 0.1, 0.25 and 0.05 respectively). Kinetics of above photoconversions are established by proposing a mechanism and determining the rate constants. Chapter 6 describes the development of a novel apparatus for the crystallization of anhydrous adenine, whose structure has not been solved over the last few decades. The crystal structure is solved via single crystal X-ray diffraction. Chapter 7 presents a modified design for crystallization of low melting organic compounds and co-crystals. A new polymorph of anhydrous thymine has been grown with this apparatus and its structure has been analyzed and compared with the known form of thymine. Chapter 8 presents the ab-initio approach of structure determination via powder X-ray diffraction methods. The methodology of using the direct space approach for the determination of the crystal structure of N-(2-fluorophenyl) benzamide (a drug intermediate), which could not yield good quality single crystals, is outlined.

Tese de doutoramento, Ciências Biomédicas (Ciências Biopatológicas), Universidade de Lisboa, Faculdade de Medicina, 2017
Rheumatoid arthritis (RA) is a chronic, systemic and immune-mediated inflammatory disease that mainly affects the synovial membrane of multiple small joints. As a consequence, RA results in cartilage and bone damage, leading to functional impairment and an increase in morbidity and mortality. Early diagnosis and adequate treatment are critical to prevent RA progression, as joint destruction can occur immediately after its onset. The most characteristic feature of RA is synovial hyperplasia, which is mediated by several immune cells, such as T-cells, B-cells, neutrophils, macrophages and by a complex cytokine network, especially interleukin (IL)-1β, tumor necrosis factor (TNF) and IL-6. RA inflammatory environment induces osteoclastogenesis, promoting disturbances in skeletal bone remodeling, which ultimately leads to the development of secondary osteoporosis and consequent bone fragility. An opportunity for a more effective treatment intervention was identified in early RA, when permanent damage can be prevented and a higher number of patients can achieve remission. Early treatment intervention might also interfere systemically with bone biology preventing bone micro and nano architectural damage. The development of therapeutic strategies able to control both inflammation and bone degradation, with a high rate of disease remission, low incidence of side effects and low production costs is still an unmet medical need in RA. Our hypothesis is that the impact of inflammation on bone micro and nano properties (intrinsic bone tissue properties, independent of the overall bone architecture and directly dependent on the way bone cells, collagen and calcium crystals interact) occurs almost immediately, upon first symptoms, and that these effects can be prevented by early intervention with drugs able to control inflammation and capable of interfering also with bone metabolism. This thesis characterizes the early events of bone damage in RA and explores the effect of novel treatment interventions in this context. Accordingly, in the first part of this thesis, we used an adjuvant induced arthritis (AIA) rat model and observed a synovial sublining layer infiltration, increased lining layer cells, bone erosions and cartilage surface damage present since the early stages of arthritis, as well as increased levels of IL-6. This inflammatory environment promotes osteoclastogenesis, which is related to the observed local bone erosion and may interfere systemically with bone skeletal remodeling. Indeed, AIA animals showed an increased bone turnover, as depicted by increased CTX-I (Carboxy-terminal telopeptide of type I collagen) and P1NP (amino terminal propeptides of type I collagen) levels since the early stages of arthritis. Bone histology was consistent with this early onset spur of bone remodeling. Arthritic animals showed concentric lamellas in secondary osteons (SO), which are the consequence of intense bone remodeling. On the contrary, healthy animals presented more parallel-lamellae (PL) structures than SO structures and these PL structures are 10% harder than SO structures, representing the mature bone structure (normal bone remodeling). Thus, arthritic bone tissue was composed of a larger number of younger, less mineralized and less hard structures, explaining the reduced hardness that we have observed by nanoindentation. Moreover, an increased area occupied by osteocyte lacunae was detected early on in the arthritis process. This apparent change of osteocyte morphology might be related to bone necrosis, leading to mineral loss, decreased hardness and possibly mechanical weakness. In addition, we have also demonstrated that arthritis induces mineral and collagen loss in trabecular bone since the early phase of arthritis development. At a higher organizational level data, micro computed tomography (micro-CT) revealed in arthritic animals a lower fraction of cortical and trabecular bone volume with reduced trabecular thickness together with a higher trabecular separation, in comparison with controls. Results also demonstrated cortical differences in polar moment of inertia, suggesting mechanical weakness in arthritic groups since the early phase of arthritis. Furthermore, cortical and trabecular porosity were increased in the arthritic groups compared to healthy controls. We also confirmed these observations by classic histomorphometry, which demonstrated a decreased structural integrity in arthritic animals. Coherent with these structural defects, our results also showed that in very early arthritis bone has low mechanical competence. Altogether, these results revealed that inflammation promotes bone nano and micro structural disturbances, leading to bone fragility since the early stages of arthritis. In addition, we also provided the basis for using the AIA animal model of arthritis as an adequate model for studying the impact of inflammation on bone and for assessing candidate compounds for the control of arthritis and its associated bone damage. The quest for new RA treatments, more effective at inflammation and bone damage control, safer and less expensive is still a major need. Previously, we had demonstrated that celastrol, acts by downregulating IL1β and TNF production, was a promising RA therapeutic candidate. Herein we have demonstrated that celastrol was able to reduce the number of synovial B and T-cells as well as fibroblasts and CD68 macrophages. Accordingly, we showed that celastrol protects cartilage and bone from inflammation-induced focal damage. At a systemic level, we observed a reduction in bone turnover together with preservation of bone structural and mechanical properties. Moreover, celastrol therapy showed superior effects if administrated in an early phase of arthritis development, which highlights the importance of an early treatment to limit inflammation-induced bone damage. Tofacitinib was also tested in order to assess the effects on micro and nano structural and mechanical properties of bone in an AIA rat model of arthritis. Tofacitinib is a selective inhibitor of janus kinase 1 (JAK1) and janus kinase 3 (JAK 3). Results showed significant reduced arthritis manifestations, synovial tissue inflammation and bone erosions, accompanied by a reduced bone turnover rate and a predominance of parallel structures on bone tissue. At tissue level, measurements performed by nanoindentation showed that tofacitinib increased bone cortical and trabecular hardness. However, micro-CT and 3-point bending tests revealed that tofacitinib did not revert the effects of arthritis on cortical and trabecular bone structure and mechanical properties. This effect on bone might be related to the mechanism of action of tofacitinib which has complex and conflictual molecular interactions with bone. We suggest that these interactions have an overall negative effect not totally compensated by the benefits resulting from the control of inflammation. On the other hand, tofacitinib may require more exposure time to have an impact on bone quality. Overall, the results of the present thesis support the hypothesis that the impact of inflammation on bone micro and nano properties occurs almost immediately, upon the appearance of first symptoms. Moreover, these observed effects can be prevented by very early intervention with drugs able to control inflammation and capable of interfering with bone metabolism.
Pfizer – ASPIRE 2013 Prize; ECTS/AMGEN Bone Biology 2014 Prize

Abstract Aim: The purpose of the study is to show the influence of Proton Pump Inhibitors (PPI in form of esomeprazole) on the bioactivation of dietary nitrate (sodium-nitrate solution) in submaximal exercise, through affecting the gastric pH. Method: Randomized, doubled-blinded, placebo-controlled and crossover study with six subjects (mean ± SD, age 29 ± 5years, height 170 ± 5 centimeters, weight 70 ± 5 Kg, BMI 24,36 ± 1,75 Kg/m2 blood pressure 119/ 77 ± 6 mmHg, 3 male and 3 female). They were tested in two different trials. Every trial consisted of two parts. One part was cycling on 4 different submaximal stages (80W/60RPM, 80W/90RPM, 120W/60RPM, 120W/90RPM) for 5 minutes each, with 90 minutes rest in between. The same protocol was repeated. In the beginning of the resting time a sodium nitrate solution (NaNO3-, 10mg/kg body weight) was ingested. VO2, VCO2, RER, VE, Lactate, Glucose, heart rate and blood pressure were recorded. Venous blood samples were taken. Whether esomeprazole (10mg) or a placebo were taken 24h, 12h and directly before being tested in both trials. Subjects were pleased to have a nitrate poor diet starting when taking the pills. An information sheet was provided. Results: No significant differences were found between the post values and the treatment. Tendencies of a higher oxygen consumption when taking esomeprazole (2,62%) in comparison to placebo (0,11%) were observed. Systolic BP decreased by 3,91% with the placebo while it decreased just 2,04% with esomeprazole after intake. Sex-specific differences occurred in the metabolism of esomeprazole and dietary nitrate. RER showed a significant post nitrate difference between the female and male participants with t=.006 and a significance in predietary nitrate intake. VE in female (40,79 ± 7,20 L/min) and (50,03 ± 10,09 L/min) in male were as well significant (t=.017). Conclusion: Tendencies of effects of PPI are seen in the post-values of VO2 and BP after intake of dietary nitrate. Gender-differences are shown in RER and VE. More research is needed to see the impact of dietary nitrate on the human body under submaximal load.