Synthesis and characterization of sugar containing hydrophilic and hydrophobic polymers with potential application in medicine

Dissertation toobtaina Master of Science degree in Bioorganics

Electrochemically prepared polypyrrole-2-carboxylic acid films: synthesis protocols and studies on biosensors

The process for obtaining polypyrrole-2-carboxylic acid (PPY-2-COOH) films in acetonitrile was investigated using cyclic voltammetry, electrochemical quartz crystal microgravimetry (EQCM), and infrared spectroscopy (FTIR). Different potential ranges were applied during cyclic voltammetry experiments with the aim of obtaining films without and with the presence of controlled amounts of water added in acetonitrile. The FTIR spectra of the films have evidenced that cations and anions from the electrolyte solution were incorporated into the PPY-2-COOH structure, with a preferential adsorption of cations. After chemically immobilizing polyphenoloxidase (tyrosinase, PPO), PPY-2-COOH/PPO films were build for amperometric detection of catechol, establishing a linear limit of concentrations ranging from 5.0 x 10-4 to 2.5 x 10-2 mol L-1.

Dielectric behaviour of Hf-doped CaCu3Ti4O12 ceramics obtained by conventional synthesis and reactive sintering

CaCu3(Ti4xHfx)O12 ceramics (JC = 0.04, 0.1 and 0.2) were prepared by conventional synthesis (CS) and through reactive sintering (RS), in which synthesis and sintering of the material take place in one single step. The microstructure and the dielectric properties of Hf-doped CCTO (CCTOHf) have been studied by XRD, FE-SEM, AFM, Raman and impedance spectroscopy (IS) in order to correlate the structure, microstructure and the electrical properties. Samples prepared by reactive sintering show slightly higher dielectric constant than those prepared by conventional synthesis in the same way than the pure CCTO. Dielectric constant and dielectric losses decrease slightly increasing Hf content. For CCTOHf ceramics with x> 0.04 for CS and x> 0.1 for RS, a secondary phase HfTi04 appears. As expected, the reactive sintering processing method allows a higher incorporation of Hf in the CCTO lattice than the conventional synthesis one.

Effect of the Synthesis Route on the Microstructure and the Dielectric Behavior of CaCu3Ti4O12 Ceramics

CaCu3Ti4O12 (CCTO) was prepared by a conventional synthesis (CS) and through reaction sintering, in which synthesis and sintering of the material take place in one single step. The microstructure and the dielectric properties of CCTO have been studied by XRD, FE-SEM, EDS, AFM, and impedance spectroscopy to correlate structure, microstructure, and electrical properties. Samples prepared by reactive sintering show very similar dielectric behavior to those prepared by CS. Therefore, it is possible to prepare CCTO by means of a single-step processing method.

Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters

Treatment of human cancers with an inherent antigen-processing defect due to a loss of peptide transporters (TAP-1 and TAP-2) and/or MHC class I antigen expression remains a considerable challenge. There is now an increasing realization that tumor cells with down-regulated expression of TAP and/or MHC class I antigens display strong resistance to cytotoxic T lymphocyte (CTL)mediated immune control, and often fail to respond to the conventional immunotherapeutic protocols based on active immunization with tumor-associated epitopes (TAE) or adoptive transfer of tumor-specific T cells, In the present study, we describe a novel approach based on immunization with either genetically modified tumor cells or naked DNA vectors encoding TAE fused to an endoplasmic reticulum (ER) signal sequence (ER-TAE) which affords protection against challenge by melanoma cells with down-regulated expression of TAP-1/2 and MHC class I antigens. In contrast, animals immunized with a vaccine based on TAE alone showed no protection against tumor challenge. Although MHC-peptide tetramer analysis showed a similar frequency of antigen-specific CTL in both ER-TAE- and TAE-immunized mice, functional analysis revealed that CTL activated following immunization with ER-TAE displayed significantly higher avidity for TAE when compared to animals immunized with the TAE alone, These observations provide a new strategy in anti-cancer vaccine design that allows activation of a highly effective and well-defined CTL response against tumors with down-regulated expression of TAP and MHC class I antigens.

Development of 2-oxazoline-based hydrogels and porous polyester microparticles in scCO2

Thesis submitted to Faculdade de Ciências e Tecnologia from Universidade Nova de Lisboa in partial fulfillment of the requirements for the obtention of the degree of Master of Science in Biotechnology

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

Jejum pré-operatório de 8 horas ou de 2 horas: o que revela a evidência?

Insulin resistance is a transitory phenomenon of the metabolic response to trauma. In uncomplicated operations it lasts for 2-4 weeks postoperatively, and is directly related to the magnitude of the injury. The fasting status caused by conventional fasting protocols aggravates this resistance and may induce hyperglycemia. Conventional preoperative fasting time may aggravate this resistance and increment the elevation of glycemia especially because it is frequently longer than the expected 6-8h and may reach 10-16 hs. Additionally, overnight fasting may cause variable degrees of dehydration depending on the extension of the fasting period. Recently, various societies of anesthesia and nutrition have changed their guidelines to propose a reduction of preoperative fasting to 2h with clear fluids containing carbohydrates. These new protocols (ACERTO, ERAS) are based on the safety of this routine as consistently demonstrated by various randomized trials and a meta-analysis.

Réparation par excision de nucléotides des dommages induits par rayons ultraviolets dans les mélanomes humains

Les mélanomes malins (MM) constituent le deuxième type de cancer le plus fréquent chez les jeunes adultes canadiens (entre 20 et 44 ans) ainsi qu’un des rares cancers dont l’incidence augmente annuellement. À moins que les MM ne soient excisés à temps par chirurgie, les chances de survie des patients sont pratiquement nulles puisque ce type de tumeur est très réfractaire aux traitements conventionnels. Il est bien connu que l’exposition aux rayons ultraviolets (UV), induisant des photoproduits génotoxiques, est une déterminante majeure dans l’acquisition de MM. À cet effet, la réparation par excision de nucléotides (NER) est la ligne de défense principale contre le développement des mélanomes puisqu’elle est la voie de réparation prépondérante en ce qui a trait aux dits photoproduits. Malgré cela, la contribution potentielle de défauts de la NER au développement des MM dans la population normale n’est toujours pas bien établie. Notre laboratoire a précédemment développé une méthode basée sur la cytométrie de flux qui permet de mesurer la NER en fonction du cycle cellulaire. Cette méthode a déjà mise en évidence qu’une déficience de l’activité de la protéine ATR peut mener à une déficience de la NER exclusive à la phase S dans des fibroblastes humains. Pareillement, nous avons démontré que plusieurs lignées cellulaires cancéreuses modèles comportent une déficience en NER en phase S, suggérant qu’une telle déficience puisse caractériser certains types de cancers. Nous avons voulu savoir si une déficience en NER en phase S pouvait être associée à une proportion significative de mélanomes et si le tout pouvait être attribuable à une diminution de l’activité d’ATR. Nos objectifs ont donc été de : (i) mesurer l’efficacité de la NER en fonction du cycle cellulaire dans les MM en comparaison avec les mélanocytes primaires, (ii) vérifier si le niveau d’activité d’ATR corrèle avec l’efficacité de la NER en phase S dans les lignées de MM et (iii) voir si un gène fréquemment muté dans les mélanomes (tels PTEN et BRAF) pouvait coopérer avec ATR pour réguler la NER en phase S dans les mélanomes. Nous avons démontré que 13 lignées de MM sur 16 ont une capacité grandement diminuée à réparer les photoproduits induits par UV spécifiquement en phase S. De plus, cette déficience corrèle fortement avec une réduction de l’activation d’ATR et, dans plusieurs lignées de MM, avec une phosphorylation d’Akt plus importante. L’utilisation d’ARN interférent ou d’un inhibiteur du suppresseur de tumeurs PTEN, a permis, en plus d’augmenter la phosphorylation d’Akt, de réduire la réparation des photoproduits et l’activation d’ATR dans les cellules en phase S. En addition, (i) l’expression ectopique de la protéine PTEN sauvage dans des lignées déficientes en PTEN (mais pas d’une protéine PTEN sans activité phosphatase) ou (ii) l’inhibition pharmacologique d’Akt a permis d’augmenter la réparation en phase S ainsi que l’activation d’ATR. En somme, cette étude démontre qu’une signalisation d’ATR dépendante de PTEN/Akt amenant à une réparation déficiente des photoproduits génomiques causés par les UV en phase S peut être déterminante dans le développement des mélanomes induits par UV.

Use of knowledge regarding LH receptors to improve superstimulatory treatments in cattle

Embryo transfer is a biotechnology that has been used worldwide to increase the production of offspring from female bovines. Treatments to induce multiple ovulations (superovulation) have evolved from superstimulatory protocols that depended upon detection of oestrus to treatments that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-timed AI (FTAI). The protocols associated with FTAI facilitate animal handling and produce at least as many viably embryos as conventional treatment protocols that required detection of oestrus. Recent knowledge regarding LH receptors (LHR) and follicular development can be applied to improve embryo transfer protocols. In fact, improvements in the superstimulatory treatment called the 'P-36 protocol', which include hormones that stimulate LHR, indicate that adjustments related to LHR availability may increase bovine embryo yield compared with conventional protocols based on the detection of oestrus.

Expressão dos genes MDR-1, TP53, BCL-2 e BAX em tumor venéreo transmissível canino e sua relação com a agressividade e resposta à terapia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência de elementos terras raras nas propriedades catalíticas de óxidos derivados de hidrotalcitas para produção de biodiesel etílico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Rod-shaped plasmonic sensors

Plasmonic nanoparticles exhibit strong light scattering efficiency due to the oscillations of their conductive electrons (plasmon), which are excited by light. For rod-shaped nanoparticles, the resonance position is highly tunable by the aspect ratio (length/width) and the sensitivity to changes in the refractive index in the local environment depends on their diameter, hence, their volume. Therefore, rod-shaped nanoparticles are highly suitable as plasmonic sensors.rnWithin this thesis, I study the formation of gold nanorods and nanorods from a gold-copper alloy using a combination of small-angle X-ray scattering and optical extinction spectroscopy. The latter represents one of the first metal alloy nanoparticle synthesis protocols for producing rod-shaped single crystalline gold-copper (AuxCu(1-x)) alloyed nanoparticles. I find that both length and width independently follow an exponential growth behavior with different time-constants, which intrinsically leads to a switch between positive and negative aspect ratio growth during the course of the synthesis. In a parameter study, I find linear relations for the rate constants as a function of [HAuCl4]/[CTAB] ratio and [HAuCl4]/[seed] ratio. Furthermore, I find a correlation of final aspect ratio and ratio of rate constants for length and width growth rate for different [AgNO3]/[HAuCl4] ratios. I identify ascorbic acid as the yield limiting species in the reaction by the use of spectroscopic monitoring and TEM. Finally, I present the use of plasmonic nanorods that absorb light at 1064nm as contrast agents for photoacoustic imaging (BMBF project Polysound). rnIn the physics part, I present my automated dark-field microscope that is capable of collecting spectra in the range of 450nm to 1750 nm. I show the characteristics of that setup for the spectra acquisition in the UV-VIS range and how I use this information to simulate measurements. I show the major noise sources of the measurements and ways to reduce the noise and how the combination of setup charactersitics and simulations of sensitivity and sensing volume can be used to select appropriate gold rods for single unlabeled protein detection. Using my setup, I show how to estimate the size of gold nano-rods directly from the plasmon linewidth measured from optical single particle spectra. Then, I use this information to reduce the distribution (between particles) of the measured plasmonic sensitivity S by 30% by correcting for the systematic error introduced from the variation in particle size. I investigate the single particle scattering of bowtie structures — structures consisting of two (mostly) equilateral triangles pointing one tip at each other. I simulate the spectra of the structures considering the oblique illumination angle in my setup, which leads to additional plasmon modes in the spectra. The simulations agree well with the measurements form a qualitative point of view.rn

Fine tuning for the tropics: application of eDNA technology for invasive fish detection in tropical freshwater ecosystems

Invasive species pose a major threat to aquatic ecosystems. Their impact can be particularly severe in tropical regions, like those in northern Australia, where >20 invasive fish species are recorded. In temperate regions, environmental DNA (eDNA) technology is gaining momentum as a tool to detect aquatic pests, but the technology's effectiveness has not been fully explored in tropical systems with their unique climatic challenges (i.e. high turbidity, temperatures and ultraviolet light). In this study, we modified conventional eDNA protocols for use in tropical environments using the invasive fish, Mozambique tilapia (Oreochromis mossambicus) as a detection model. We evaluated the effects of high water temperatures and fish density on the detection of tilapia eDNA, using filters with larger pores to facilitate filtration. Large-pore filters (20 μm) were effective in filtering turbid waters and retaining sufficient eDNA, whilst achieving filtration times of 2-3 min per 2-L sample. High water temperatures, often experienced in the tropics (23, 29, 35 °C), did not affect eDNA degradation rates, although high temperatures (35 °C) did significantly increase fish eDNA shedding rates. We established a minimum detection limit for tilapia (1 fish/0.4 megalitres/after 4 days) and found that low water flow (3.17 L/s) into ponds with high fish density (>16 fish/0.4 megalitres) did not affect eDNA detection. These results demonstrate that eDNA technology can be effectively used in tropical ecosystems to detect invasive fish species. © 2016 John Wiley & Sons Ltd.

Supported metal nanoparticles for sustainable green catalytic processes

Preformed Au nanoparticles supported on activated carbon and TiO2 were synthesised by sol-immobilisation. Polyethylene glycol, polyvinyl pyrrolidone and polyvinyl alcohol were used as stabilisers for the gold nanoparticles at different polymer/Au wt/wt ratios for each polymer. The effect of polymer/Au wt/wt ratios was investigated on (i) the average nanoparticle size, (ii) catalytic activity for two reactions, 4-nitrophenol reduction and glucose oxidation to glucaric acid. 4-nitrophenol reduction is recognised as a model reaction for nanomaterial catalytic activity tests; glucose oxidation to glucaric acid is a reaction that is traditionally carried out with concentrated nitric acid, for which alternative reaction pathways are looked for in an effort to reduce its environmental impact. The catalysts were characterised from the nanoparticle synthesis by colloidal method by means of UV-vis spectroscopy and DLS analysis, to the immobilisation step by XRD and TEM. The effect of the polymer:Au wt/wt ratio on nanoparticle size depends on the polymer nature, and point out the need to optimise supported nanoparticle synthesis protocols in the future depending on the type of stabiliser. The catalytic tests revealed that the polymers interact with Au nanoparticles through different active sites. Activated carbon (AC) and TiO2 were compared as supports for Au nanoparticles stabilised by PVA at PVA/Au 0,65 wt/wt. AC-supported Au NPs were the most active for glucose oxidation while TiO2-stabilised Au NPs were five times more active in 4-nitrophenol reduction that AC-supported NPs. Hence support and stabiliser are important parameters that should be optimised in order to achieve high catalytic activity for a given reaction.