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Microfluidics is the science and technology of manipulating fluids at small scales ranging from microlitres (10$^{-6}$) to picolitres (10$^{-12}$). The fundamental physics is distinct from fluid behaviour on bulk scales and laminar flow is the key characteristic on this scale. Microfluidic systems have a wide range of applications in many disciplines from engineering to physics, chemistry and biotechnology. In this thesis, I explore different strategies exploiting the capabilities of microfluidic devices for manipulating and analysing biomolecules. A particular focus of the work is on the study of amyloid fibrils. These species are protein aggregates related to a wide range of human diseases and functional materials. In chapter 3 I demonstrate an efficient way to separate particles in different sizes based on a microfluidic diffusion method. This method enables us to explore the properties of amyloid fibrils, such as their growth kinetics and interaction with small molecules. Rapid binding information could be obtained with only microlitres of sample in tens of seconds time scale. A further manipulation method for charged particles is introduced in chapter 4, based on the integration of microfluidics and free flow electrophoresis. I present a very effective and simple way to overcome one of the most critical problem in this situation. High electric filed can be applied through two streams of conductive solutions, with all the electrolysis by products, e.g., gas bubbles and other deposits, removed simultaneously without interfering with the system. In addition to microfluidic devices made by soft lithography in PDMS, I also set up a hot embossing fabrication process with the Teflon material (chapter 5). Teflon has many advantages compared with PDMS, such as lower protein adsorption, higher mechanical strength and better chemical compatibility. With different materials and structures, microfluidic devices can be expanded to more applications.

Curcumin is the active ingredient present in the roots and rhizomes of the tropical plant known as Curcuma Longa (Turmeric). Scientific research has identified curcumin to possess various pharmacological properties including, anti-inflammatory and anticancer activities. This novel research focused on investigating the advantageous properties of curcumin in combination with current therapeutic options for the treatment of conditions of an inflammatory nature such as rheumatoid arthritis (RA) and cancer, specifically, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM). Previous microarray results from our laboratory was able to identify a set of 74 genes which were differentially expressed in untreated human fibroblast-like synoviocytes isolated from RA patients (HFLS-RA), methotrexate (MTX) and curcumin treated cells, individually and in combination. From this list, 13 genes were selected for further qRT-PCR analysis due to the established role they play in the process of inflammation. The change in expression of these 13 candidate genes were monitored in order to investigate the efficacy of curcumin as a therapeutic agent whilst identifying potential therapeutic biomarkers for the treatment of these inflammatory conditions. HFLS-RA cells were treated with curcumin and MTX, individually and in combination. The novel finding from this study highlighted that individual treatment with curcumin was the most effective since it lowered the expression of 11 out of the 13 candidate genes. The levels of CD248, HSPA6, MMP1, MMP13 and TNFSF10 were sharply downregulated across all treatment conditions, allowing the identification of potential therapeutic targets for intervention in RA. As normal synoviocytes (HFLS) were also studied alongside HFLS-RA cells, it was possible to also identify potential diagnostic biomarkers for RA. The expression of CD248, HSPA6, MMP1, MMP13 and TNFSF10 was significantly higher (p < 0.001) in HFLS-RA cells compared to normal synoviocytes (HFLS). This highlighted the use of these genes as potential diagnostic biomarkers as they were suggested to play a role in the pathogenesis of RA. A549 lung carcinoma cells were treated with curcumin and cisplatin, individually and in combination. Curcumin treatment alone was the most effective and downregulated the expression of 11 inflammatory response genes studied, ANGPTL7, CD248, CH25H, COL14A1, CXCL12, HSPA6, IFITM1, IL-7, MMP1, MMP13 and TNFSF10. Cisplatin was only able to decrease the expression of 7 genes, however, in combination with curcumin, a total of 9 genes were downregulated. Curcumin was able to work in synergy with cisplatin in order to potentiate the effects of this cytotoxic agent and help to overcome cisplatin-resistance mechanisms. U87-MG glioblastoma cells were treated with curcumin and TMZ, individually and in combination. Curcumin lowered the expression of 10 candidate genes compared to TMZ which downregulated 8. Combination treatment was the most effective as it decreased the expression of ANGPTL7, CD248, CH25H, COL14A1, CXCL12, CYTL1, IFITM1, IL-6, IL-7, MMP1 and TNFSF10. The use of curcumin in combination with TMZ sensitises glioma cells to the activity of TMZ, thereby increasing the therapeutic potential of the drug. Curcumin was able to specifically target mediators involved in the inflammatory pathway, and so the use of this natural compound for RA, NSCLC and GBM can improve the efficacy of current therapeutic options, limit undesirable side effects and help to overcome resistance mechanisms deployed by the cells.

Living cells are dependent upon protein synthesis for virtually all cellular functions. The cellular machine responsible for protein synthesis, called the ribosome, is formed through the association of two unequally sized subunits, each composed of RNA and proteins. Proper assembly of each subunit is essential to ribosome function and therefore essential to the cellular life cycle. Previous studies focused on dissecting the assembly of the small ribosomal subunit (30S subunit) from E. coli have shown that 21 proteins sequentially assemble on the 16S rRNA at multiple nucleation sites. For the first time, we are able to monitor changes in the secondary and tertiary structure of the 16S rRNA upon the addition of single proteins during assembly by using time-dependent chemical probing. Results from these studies suggest that protein S17 induces multiple structural changes in 16S rRNA by first binding to helix 11 and then helix 7. S20 also induces changes in the rRNA by interacting with helix 9, 11, 44 and 13 in that order. These structural formations and rearrangements then prepare the binding sites for additional proteins (S12 and S16, respectively). This study demonstrates that time-dependent chemical probing is able to monitor the assembly of the 30S subunit at a level of detail never before seen. These studies also suggest that many motifs in the 16S rRNA structure are formed as a result of the proteins binding, lending evidence to the hypothesis that the function of ribosomal proteins is to shape and/or hold the RNA structure in place.

Um biossensor de afinidade química baseado no fenômeno da reflexão total interna é apresentado. Nesse, as propriedades particulares apresentadas pelas componentes ortogonais de polarização do campo elétrico da luz permitem que ambas se cancelem na vizinhança do ângulo crítico da reflexão interna, proporcionando um meio para sua determinação unívoca, permitindo monitorar o mesmo como função do índice de refração de uma amostra de interesse. Um estudo acerca dos principais aspectos teóricos envolvidos no fenômeno da reflexão total interna foi realizado, a fim de explorar e justificar os fenômenos observados. A montagem experimental utilizada nos experimentos consiste de um laser de HeNe emitindo em 633 nm, um polarizador, uma lente semicilíndrica de alto índice de refração, produzida com um vidro denso do tipo flint, um filme produzido com um anticorpo, que é depositado sobre essa lente, um analisador paralelamente orientado com relação ao eixo de transmissão do polarizador, um dispositivo transdutor, que converte um sinal luminoso em sinais elétricos, uma solução aquosa do antígeno específico e de um computador, onde as informações coletadas pelo transdutor são tratadas por meio de um programa de aquisição de dados desenvolvido em uma plataforma gráfica de programação. O programa empregado permite o acompanhamento das variações do perfil luminoso, devido à interação do filme de anticorpos com a solução de antígenos, coletado pelo transdutor em tempo real, ou seja, é possível acompanhar toda evolução da reação química entre um par de espécies químicas em tempo real. Para confirmação da efetividade da técnica, foram comparadas medidas obtidas com um refratômetro comercial, onde foi constatado que o sistema possui sensibilidade suficiente para acompanhar mudanças da ordem de 10-5 unidades do índice de refração. / A chemical affinity biosensor based on the phenomenon of total internal reflection is presented. In it, the particular properties exhibited by the orthogonal polarization components of the electric field of light allow them to cancel in the vicinity of the critical angle of the internal reflection, providing means for its univocal determination, allowing its monitoring as a function of the refractive index of a sample of interest. A study about the theoretical aspects involved in the total internal reflection phenomenon was carried out in order to explore and justify the observed phenomena. The experimental set up consists of a HeNe laser emitting at 633 nm; a polarizer; a high refractive index semi-cylindrical lens, produced with a dense flint type glass; a film produced with an antibody, which is deposited over the aforementioned lens; an analyzer oriented parallel to the transmission axis of the polarizer; a transducer device, which converts a light signal into electric signals; an aqueous solution of the specific antigen and a computer, where the information collected by the transducer is treated with a data acquisition software developed in a graphical programming platform. The software allows one to monitor changes in the light profile due to interaction of the antibody with the antigen solution, collected by the transducer in real time, i.e. it&rsquo;s possible to follow the evolution of the chemical reaction between a couple of chemical species in real time. To confirm the effectiveness of the technique a comparison with measurements obtained with a commercial refractometer was carried out, where it was verified enough sensitivity to follow changes of the order of 10-5 refractive index units.

It was once thought either impossible or inefficient to photopolymerize a thickness greater than a thin film because of the optical attenuation of light into the depth of the sample. However, if several considerations are allowed, it is indeed possible. Three particular modifications are essential to enhance light penetration into the depth of the system. An initiator that absorbs in a region of the spectrum where no other components absorb maximizes the incident light intensity for photolysis of the initiator. Concentration and/or molar absorptivity of the initiator lower than typically used in thin films enhance light penetration. Finally, photobleaching initiators exhibit decreased absorbance upon photolysis and thus allow light to penetrate more deeply into the system with time. A need to model these systems is born out of the desirability to use light to initiate polymerizations of all sorts, including thicker systems. In this project, a set of differential equations describing the spatial and temporal evolution of the light intensity gradient, photoinitiator concentration gradient, and the photoinitiation rate profile are developed for a thick polymer system. The generalized model accounts for the consumption of initiator, evolution of the products of photolysis, diffusion of the initiator and photolysis products, and absorbance by all system components. The purpose of these studies was to characterize further these systems so that results accurately capture the photoinitiation process. Several key objectives have been accomplished, including the effects of illumination with polychromatic incident light, various illumination schemes, and verification of the predicative ability of the model. The ultimate goal of this project was two fold; first, to build a tool that models photopolymerization systems well, and second, to develop a means for choosing reaction components for photopolymerization applications. To understand and predict how these systems work contributes significantly to the photopolymerization field because it allows the user to predict system behavior accurately and to choose system components appropriate for a particular application.

The most prevalent polymerization methods are those which fall under the category of thermal polymerizations. The ease of initiation and the abundant knowledge present for these long time standards make implementation straightforward. However in some applications, the drawbacks may be numerous and the use of light induced polymerizations may be advantageous. Because of distinct advantages obtained using photopolymerizations as opposed to the more conventional thermally induced polymerizations science continues to further the knowledge and applications of photopolymers. Structured illumination is one such expansion of photopolymer knowledge and is a method by which variations in light intensity set up differential reaction rates evolving in the migration of monomer. The method can be tailored to produce cured systems with enhanced properties such as the reduction of stress or the control of gloss. Polymerization shrinkage is important for many applications since it leads to residual stresses which can deform a system and undermine its optical or mechanical properties. Also, while photopolymerized coatings are generally high in gloss, it is a characteristic of a polymer system that can have great impact on its function and appearance. Utilizing the simple method of structured illumination, and thereby controlling the coating system for both reductions of stress and gloss, can lead to great advantages for the finished product. This contribution looks at not only producing coatings using the method of structured illumination but also characterizes their properties by standard and unconventional means, alike. Mathematical modeling of the shrinkage, stress and monomer migration is also present in this work.

This thesis examines the use of capillary electrophoresis for the study of several biomolecules and their interactions and viruses. The first two experimental chapters focus on its utility for thermodynamic and kinetic analysis of molecules. Chapter one focuses on the use of non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), to calculate the dissociation constant for the interaction between double stranded microRNA-122 and protein p19. NECEEM was used to calculate the rate constants (koff = 0.059 ± 0.013 s-1, kon = 0.0022 ± 0.0008 s-1M-1) and the dissociation constant between miR-122 and wild type p19 (Kd = 27 ± 9 nM). A new method was developed to calculate the rate constant koff, by using multiple electric fields; which resulted in a koff value of 0.072 ± 0.022 s-1. In chapter two, the dissociation constant, Kd, was determined between HIV trans-activation response element and nuclear protein TOE1. It was demonstrated that TOE1, more specifically peptides ER19 and ED35, were binding to TAR with Kd values of 4.08 ± 0.19 µM for ER19 and 7.43 ± 1.60 µM for ED35. The discovery of the peptides’ inhibitory action of viral replication at the transcription level is a significant step towards further elucidating mechanisms for host response to HIV-1 infection. The third chapter focuses on the use of capillary electrophoresis for studying vesicular stomatitis virus (VSV) and vaccinia virus (VV). A new method was developed for quantification of VSV, using dithiothreitol. Furthermore, CE was used to study the preservation of VSV by a previously selected aptamer construct (quadramer) during freeze-thaw cycles. It was found that the infectivity of quadramer and aptamer pool-protected virus was higher than pure virus after 60 freeze−thaw cycles. It was also found that adding quadramers to the virus without freezing (cycle 0) increased the virus infectivity by 30%. We also investigated the potency of a carbohydrate-based ice recrystallization inhibitor, N-octyl-D-gluconamide (NOGlc) for its ability to eliminate the cold chain and stabilize the potency of VV. Viral potency after storage at room temperature demonstrated that NOGlc conserved the infectivity of VV, during 40 days.