Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-10-18T12:24:30Z No. of bitstreams: 1 DISS_2014_Laila Natasha Santos Brandão.pdf: 1739750 bytes, checksum: 16eb165dd6c3a1476424ddf9bd18fe73 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-11-07T15:29:45Z (GMT) No. of bitstreams: 1 DISS_2014_Laila Natasha Santos Brandão.pdf: 1739750 bytes, checksum: 16eb165dd6c3a1476424ddf9bd18fe73 (MD5) / Made available in DSpace on 2017-11-07T15:29:45Z (GMT). No. of bitstreams: 1 DISS_2014_Laila Natasha Santos Brandão.pdf: 1739750 bytes, checksum: 16eb165dd6c3a1476424ddf9bd18fe73 (MD5) Previous issue date: 2014-03-05 / CAPES / A possibilidade de detecção de agentes patogênicos representou um grande passo para a ciência. O desenvolvimento e melhorias nas técnicas de diagnóstico como a técnica de Reação em Cadeia da Polimerase (PCR), ao longo dos anos ainda requer infraestrutura laboratorial, apesar de sua sensibilidade e custo decrescente, investimentos em equipamentos e cuidados com fontes de contaminação externa. Técnicas que possam ser executas com facilidade e pouca mão de obra ou exigência de pessoas capacitadas, têm grande possibilidades de aplicabilidade. Neste estudo desenvolveu-se uma técnica de rápida execução e baixo custo, para detecção de um dos principais agentes causadores de pneumonias em granjas de suínos o Actinobaccilus pleuropneumoniae, com sensibilidade 93,8% e especificidade de 84,6% em amostras de pulmões com e sem lesão. O teste Kappa entre a PCR e nanopartícula de ouro foi 0,684 representando boa concordância. A técnica pode ser utilizada como alternativa aos testes convencionais, já que é de fácil e rápida execução e não exige infraestrututra e mão de obra especializada. / The possibility of detection of pathogens was a major step for science as a whole, developing and improving these techniques over the years to increase visibly. The development of the technique of Polymerase Chain Reaction ( PCR ) , although its sensitivity and decreasing cost over the years, it's still a handy little technique that requires laboratory infrastructure, high investments in equipment and care with sources of external contamination. Techniques that can be performed through the ease and little manpower or requirement of skilled people, always have great scope of applicability. This study develops a technique for quick and low- cost detection of a major causative agent of pneumonia in swine herds the Actinobaccilus pleuropneumoniae , with 93.8 % sensitivity and 84.6% specificity in samples of lungs with and without injury, the Kappa test between PCR and gold nanoparticle was 0,684 representing good agreement . The technique can be used as an alternative to conventional tests, since it is easy and quick to implement and does not require infrastructure and skilled labor.

When exposed to light, metal nanoparticles exhibit a phenomenon known as LSPR, Localized Surface Plasmon Resonance. The wavelengths at which LSPR occurs is very dependent on the refractive index of the surrounding medium. Binding of biomolecules to the surface of gold nanoparticles result in a change in the refractive index that can be detected spectrophotometrically by monitoring the LSPR peak shift. When functionalized with the corresponding ligand(s), gold nanoparticles can be utilized in biosensors to detect the presence and concentration of a predetermined analyte. However, the system must exhibit high specificity and give rise to a detectable shift for analytes in the desired concentration range to be of commercial interest. The aim of the diploma project was to investigate and optimize the biofunctionalization and performance of a fiber optics based LSPR biosensor.  Three ligand systems were investigated for detection of antibodies (IgG), insulin and avidin. Binding of the analyte to the ligand caused a shift of a few nanometers when using spherical gold nanoparticles. The shifts were significantly larger when using gold nanorods. When using the IgG and insulin ligands, only minor unspecific binding was observed. The setup thus shows great potential for use in a wide range of sensing applications.

A microfluidic lab-on-a-chip (LOC) device for in-situ synthesis of gold nano-particles was developed. The long term goal is to develop a portable hand-held diagnostic platform for monitoring water quality (e.g., detecting metal ion pollutants). The LOC consists of micro-chambers housing different reagents and samples that feed to a common reaction chamber. The reaction products are delivered to several waste chambers in a pre-defined sequence to enable reagents/ samples to flow into and out of the reaction chamber. Passive flow actuation is obtained by capillary driven flow (wicking) and dissolvable microstructures called ‘salt pillars’. The LOC does not require any external power source for actuation and the passive microvalves enable flow actuation at predefined intervals. The LOC and the dissolvable microstructures are fabricated using a combination of photolithography and soft lithography techniques. Experiments were conducted to demonstrate the variation in the valve actuation time with respect to valve position and geometric parameters. Subsequently, analytical models were developed using one dimensional linear diffusion theory. The analytical models were in good agreement with the experimental data. The microvalves were developed using various salts: polyethylene glycol, sodium chloride and sodium acetate. Synthesized in-situ in our experiments, gold nano-particles exhibit specific colorimetric and optical properties due to the surface plasmon resonance effect. These stabilized mono-disperse gold nano-particles can be coated with bio-molecular recognition motifs on their surfaces. A colorimetric peptide assay was thus developed using the intrinsic property of noble metal nano-particles. The LOC device was further developed on a paper microfluidics platform. This platform was tested successfully for synthesis of gold nano-particles using a peptide assay and using passive salt-bridge microvalves. This study proves the feasibility of a LOC device that utilizes peptide assay for synthesis of gold nano-particles in-situ. It could be highly significant in a simple portable water quality monitoring platform.

Cancer is the second leading cause of death in the United States (more than half a million people each year), and even with billions of dollars in medical effort patients are rarely cured. This dissertation research is devoted to meeting this medical need by providing new cancer therapeutics that are more potent and safer than current chemotherapies. This is achieved by using two state of the art anticancer “warheads”: 1) gold nanoparticle (AuNP) technology and 2) a new class of epigenetic anticancer small molecules, histone deacetylase inhibitors (HDACi). These warheads are then selectively delivered to cancer cells via “homing devices” targeted to receptors that are overexpressed in the cancers. This work primarily focuses on the androgen receptor (AR) to target prostate cancer. The 1st chapter sets the stage, providing scientific rationale and background for the central hypothesis: small molecules that engage the AR can, upon conjugation to a therapeutic agent, enable selective delivery of that agent to prostate cancer cells. Chapter 2 delves into the structural molecular biology of the androgen receptor. There is a survey of the crystallographic data for all nuclear receptors, providing structural information which is used to build AR homology models for antagonist and inverse agonist modes of ligand binding. These models are used to design AR targeting ligands (Chapters 3, 5, 6 and 7). The application of the targeting technology is illustrated by attaching them to AuNPs for selective delivery to prostate cancer cells (Chapter 3). Next, in order to appreciate the importance of using targeting agents in HDACi cancer therapeutics, we reviewed this recently emerged field in Chapter 4. In this chapter we argue that the failure of HDACi in solid tumors, despite more than 500 clinical trials in the last decade, is primarily due to an inability of these small molecules to accumulate at effective concentrations in the cancer. We provide an analysis of the paradigms being pursued to overcome this barrier, including HDAC isoform selectivity, localized administration, and targeting cap groups to achieve selective tissue and cell type distribution. In Chapter 5, this last approach (targeting cap groups, or a “homing device”) is illustrated with HDACi targeted to prostate cancer via antiandrogens that bind the AR. The second generation of improved “homing devices” is disclosed in Chapter 6 (for both AuNPs and HDACi), in addition to preliminary ADMET data and safety studies in mice. Excitingly, our three dimensional understanding of binding to the AR allowed design and structure-activity-relationship studies that lead to the first reported examples of AR inverse agonists (Chapter 7) Several points of significance: • AuNP targeted to AR ∙ have the strongest binding affinity ever reported (IC50 ~14 picomolar) ∙ are actively recruited to prostate cancer cells ∙ overcome treatment resistance in advanced prostate cancer cells ∙ exhibit nanomolar anticancer potency ∙ resolved the identity of the “membrane AR” as the GPRC6A • HDACi targeted to AR ∙ have HDACi activity and AR binding affinity superior to their clinical precursors ∙ exhibit potent AR antagonist activity ∙ induce AR translocation to the nucleus in a HDACi dependent fashion ∙ selectively and potently kill prostate cancer cells that express AR ∙ are safer than Tylenol®, as tested in small animals • Pure AR binding ligand studies ∙ resulted in the discovery of the first examples of AR inverse agonists, which are vastly more potent that clinically available antiandrogens for prostate cancer ∙ work via a never-before-seen mechanism of action, by localizing to the nucleus and recruiting corepressors to actively shut off AR genes

Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis, with an incidence in a quarter of the world population. Despite the scientific and technological advances, an effective diagnostic method has not yet been found that allows an early diagnosis and, also, to detect the strain present in the patient. The combination of nanotechnology with molecular diagnostics has shown promising advances offering new possibilities, such as the development of nanoflares.  Nanoflares represent a new class of molecular probes, composed of gold nanoparticles functionalized with a recognition sequence that can be amplified by rolling circle amplification (RCA) technique, producing a fluorescence signal.  This thesis focuses in the synthesis of gold nanoparticles, with different coatings and sizes, as well as their subsequent application in the preparation and optimization of nanoflares for the genotyping of synthetic M. tuberculosis targets using RCA technique. The different preparations of nanoflares have an impact in the assay sensitivity, showing two times increase in sensitivity for citrate-coated nanoparticles with respect to those coated with PEG. Furthermore, it was observed that the sensitivity is directly related to the synthesized particle size.  Sensitivity is also affected by the application of a purification post-treatment of the synthesis product. This post-treatment reduces the sensitivity of nanoflares by up to 37% but, by contrast, extends its useful life.  The results obtained are shown as a proof of concept for a future cost-effective, rapid and robust in situ diagnostic method that identifies the strain of tuberculosis present in the patient.

<p>Differences in the wavelengths of the surface plasmon band of gold nanoparticles (AuNP) – before and after particle aggregation – are widely used in bioanalytical assays. However, the gold surfaces in such bioassays can suffer from exchange and desorption of non-covalently bound ligands and from non-specific adsorption of bio-molecules. Silica shells on the surfaces of the gold can extend the available surface chemistries for bioconjugation and potentially avoid these issues. Therefore, silica was grown on gold surfaces primed with polyvinylpyrrolidone (PVP) using either hydrolysis/condensation of tetraethyl orthosilicate under basic conditions or diglycerylsilane at neutral pH. The former precursor permitted slow, controlled growth of shells from about 1.7 to 4.3 nm thickness. By contrast, silica shells formed within an hour using diglyceroxysilane; the thickness was insensitive to changes in silane concentration and incubation time and could be tuned using different molecular weight PVP to prime the particles. The control over shell thickness is discussed with respect to the PVP interface, the electrical double layer, and interpenetrating organic-inorganic hybrid structures. Within the range of shell thicknesses synthesized, the presence of a silica shell on the gold nanoparticles did not significantly affect the absorbance maximum (~ 5 nm) of unaggregated particles. However, the change in absorbance wavelength upon aggregation of the particles was highly dependent on the thickness of the shell. With silica shells coating the AuNP, there was a significant decrease in the absorbance maximum of the aggregated particles, from ~578 to ~536 nm, as the shell thicknesses increased from ~1.7 to ~4.3 nm, due to increased distance between adjacent gold cores. These studies provide guidance for the iv development of colorimetric assays using silica coated AuNP. Such particles also show potential for application in 2D and 3D nanostructured assemblies.</p> / Master of Science (MSc)

Electron Backscatter Diffraction (EBSD) is a well-developed technique used to perform quantitative microstructure analysis in the Scanning Electron Microscope (SEM); however, it has not been widely applied towards studying nanostructures. This work focuses on the use and limitations of EBSD in the characterization of Au nanoparticles on an MgAl2O4 substrate. Samples under investigation are prepared by depositing a thin film of Au on an MgAl2O4 substrate, and then finally heated in a furnace to induce dewetting and cluster formation. The challenges of obtaining crystallographic information from nanoparticles using EBSD are qualitatively and quantitatively described through an evaluation of the quality of the diffraction pattern at various locations of the primary electron beam on the nanoparticle. It is determined that for a high quality Electron Backscatter Diffraction Pattern (EBSP), the production of diffracted backscattered electrons travelling towards the detector must be high and the depth of the source point must be low. The top of the nanoparticle, where the local geometry of the system is similar to the geometry of a macroscopically flat sample, is found to produce diffraction patterns of the highest quality. On the other hand, reversed-contrast EBSPs are observed when the beam is positioned near the bottom of the nanoparticle. In addition, crystallographic information for each individual nanoparticle is gathered using EBSD. Each individual AuNP is observed to be single crystalline. Finally, the complete ensemble of crystalline orientations for individual nanoparticles is then compared to the global averaged crystallinity of the sample, as measured by X-ray diffraction. These results show that EBSD promises to be a powerful and robust technique in the characterization of nanoparticles. / Thesis / Master of Applied Science (MASc)