The sigma-1 receptor, although originally classified as an opioid receptor is now thought of as distinct receptor class, sharing no homology with any other known mammalian protein. The receptor has been implicated with a number of diseases including cancer and depression. Modulation of the receptors activity with agonists has potential antidepressant activity whereas antagonists lead to death of cancer cells. Using radioligand binding assays, utilizing the cancer cell line MDA-MB-468, which highly expresses the sigma-1 receptor, a series of novel specific, high affinity, sigma-1 receptor ligands have been characterised. These ligands differed from any previous sigma- 1 receptor ligand in that they are very simple ammonium salts, containing a single nitrogen atom and either straight or branched carbon chains. The binding studies revealed that the straight-chain ammonium salts gave nH values of 1 whereas the branched-chain ammonium salts had statistically significant lower nH values. The ammonium salts were tested for sigma-1 receptor activity in vitro using ratiometric Fura-2 calcium assays and the MTS cell proliferation assay. Branched-chain ammonium salts appeared to have sigma-1 receptor antagonist like effects on cytoplasmic calcium and cell proliferation, whereas the straight-chain ammonium salts behaved as sigma-1 receptor agonists. Three ammonium salts stood out as potential effective sigma-1 receptor drugs, the straight-chain ammonium salt dipentylammonium, and two branched-chain ammonium salts, bis(2-ethylhexyl)ammonium and triisopentylammonium. The ammonium salts were then tested in vivo. Dipentylammonium showed significant antidepressant properties when tested in behavioural models for depression and bis(2-ethylhexyl)ammonium and triisopentylammonium were able to significantly inhibit the growth of tumours implanted in mice. Finally I looked at the coupling of the sigma-1 receptor with G-proteins and show that sigma-1 receptor antagonists dose dependently reduce G-protein activity and inhibition of G-proteins enhanced the sigma-1 antagonists' effects of calcium signalling.

Cells have lesion bypass DNA polymerases (DNAPs), often in the Y-Family, which synthesize passed DNA damage. One class of Y-Family DNAPs includes hDNAP k, EcDNAP IV and SsDbh, which insert accurately opposite N2-dG adducts, including BP-N2-dG formed from benzo[a]pyrene (BP). Another class includes hDNAP h, EcDNAP V and SsDpo4, which insert accurately opposite UV-damage. For correct Watson-Crick pairing between BP-N2-dG and dCTP, the BP moiety must be in the minor groove. On the minor groove side of the active site, k/IV/Dbh-class DNAPs have large openings that accommodate the BP moiety. Primer extension assays with purified proteins show that DNAP IV correctly inserts dCTP opposite BP more than 10-fold faster than it mis-inserts dATP, dGTP, or dTTP. In contrast, h/V/Dpo4-class DNAPs have small active site openings, which cannot accommodate BP and lead to a distorted structure and increased mutagenesis; e.g., Dpo4 has dGTP and dATP insertion rates that are 10-fold greater than those of dCTP. The opening in Dpo4 is plugged and bulky, whereas DNAP IV has a relatively spacious cavity. Consistent with this model, mutants of Dpo4 with a larger opening insert up to 10-fold more accurately opposite BP-N2-dG. Near the active site, Dpo4 has a single non-covalent bridge (NCB) between the little finger domain and the thumb-palm-fingers domain. DNAP IV and Dbh have a second, distal NCB that is 8 angstroms away from the active site towards the 3' end of the template DNA. Dpo4 becomes nearly 5-fold more accurate when mutated to carry a distal NCB, suggesting that NCB's also help control mutagenesis. Lastly, the active site of Dpo4 has a cavity in the major groove side, which may allow base flipping and dGTP insertion opposite -BP, while k/IV/Dbh-type polymerases do not. When this cavity is plugged in Dpo4 by mutagenesis or the introduction of an N-clasp motif, dGTP rates increase by nearly 20-fold. In conclusion, this data suggests that three structural regions contribute to accurate dCTP insertion opposite BP-N2-dG by k/IV/Dbh-class DNAPs: a large opening on the minor groove side near the active site, a cavity on the major groove side, and the number of non-covalent bridges between the little finger domain and the thumb-palm-fingers domain.

Pureza é um dos principais atributos de qualidade de matérias-primas farmacêuticas, já que a identificação e determinação quantitativa de impurezas podem ajudar a controlar/minimizar o risco de efeitos adversos de medicamentos. O papel dos métodos de doseamento, tanto específicos como não específicos, em caracterizar a qualidade de matérias-primas farmacêuticas, tem sido questionado. Por outro lado, a abordagem do balanço de massas, que quantifica cada impureza orgânica bem como substâncias voláteis e cinzas, e então subtrai a soma destas impurezas de 100%, pode oferecer uma melhor maneira de determinar com exatidão se o fármaco atende aos critérios regulatórios de aceitação e de detectar com confiança mudanças não esperadas na qualidade do fármaco. O objetivo do estudo foi comparar métodos de doseamento compendiais com essa abordagem de balanço de massas na determinação do conteúdo de substância ativa e caracterização da qualidade de matérias-primas farmacêuticas. Os fármacos empregados neste estudo foram o nifedipino, o diazepam, a glibenclamida e a estavudina. A identificação e caracterização dos fármacos foram realizadas por meio de determinação do ponto de fusão e espectroscopia no infravermelho. Para a determinação quantitativa foram empregados os métodos presentes na Farmacopéia Brasileira, United States Pharmacopeia e British Pharmacopeia. O detector de aerossol carregado (CAD) foi acoplado ao sistema de cromatografia à líquido de alta eficiência para comparação das respostas com o detector ultravioleta. Os resultados demonstraram a viabilidade do método indireto, que oferece dados mais precisos que os do método direto, além de favorecer um controle de qualidade mais focado no perfil de impurezas. / Purity is one of the main attributes of quality of bulk drug materials, since the identification and quantitative determination of impurities can help to avoid or at least control/minimize the risk of their contribution to the side effects profile of drug materials. The role of assay methods, either specific or non-specific, in characterizing the quality of bulk drug materials, has been questioned. On the other hand, the mass balance approach, which quantifies every organic impurity as well as volatile substances and ashes, and then subtracts the sum of these impurities from 100%, can provide a better way to accurately determine if the drug substance meets the regulatory acceptance criteria and to reliably detect unexpected changes in the quality of the drug substance. The objective of this study was to compare compendial assay methods with a mass balance approach in the determination of the active ingredient content and characterization of the quality of bulk drug materials. The identification and characterization of the drugs were accomplished through determination of melting point and infrared spectroscopy. The methods from the Farmacopeia Brasileira, United States Pharmacopeia and British Pharmacopoeia were used for the quantitative determination of the drugs. The drug related impurities were determined by high performance liquid chromatography (HPLC) equipped with an ultraviolet (UV) detector and charged aerossol detector (CAD) in tandem. The results demonstrated the feasibility of the indirect method, which offers more precise data than those from the direct method, besides it enables the quality control to be focused on the impurity profile.

In the nature, lots of organism apply different kinds of lights such as flourscence or luminoscence for some purposes such as defence or hunting. Firefly luciferase and Bacterial luciferase are the most famous ones which have been used to design Biosensors or Bioreporters in recent decades. Their applications are so extensive from detecting pollutions in the environment to medical and treatment usages. To design Copper Bioreporter, copper resistance promoter from COP operon which plays an important role in Pseudomonas syringae and pGL3 plasmid which has luciferase gene were utilized. To achieve that target, sequences of promoter were synthesized and inserted to pCR2.1 vector, then suitable primers with considering restriction sites were designed to get high concentration of DNA. After digestion of pGL3 and interested gene by Nhe I and Sac I enzymes, ligation was performed, and then recombinat plasmids were transferred to E. coli BL-21 as a host cell. Finallay, luciferase assay of designed bioreporter was performed by Luminometer in presence of different concentration of CuSO4. The result was maginificant that confirmed design of Copper Bioreporter.

Molecular imaging of the human body is beginning to revolutionize drug development, drug delivery targeting, prognostics and diagnostics, and patient screening for clinical trials. The primary clinical tool of molecular imaging is Positron Emission Tomography (PET), which uses radioactively tagged probes (radioligands) for the in vivo quantification of blood flow, metabolism, protein distribution, gene expression and drug target occupancy. While many radioligands are used in human research, only a few have been adopted for clinical use. A major obstacle to translating these tools from bench-to-bedside is that PET images acquired using complex radioligands can not be properly interpreted or quantified without arterial blood sampling during the scan. Arterial blood sampling is an invasive, risky, costly, time consuming and uncomfortable procedure that deters subjects' participation and requires highly specialized medical staff presence and laboratories to run blood analysis. Many approaches have been developed over the years to reduce the number of blood samples for certain classes of radioligands, yet the ultimate goal of zero blood samples has remained illusive. In this dissertation we break this proverbial blood barrier and present for the first time a non-invasive PET quantification framework. To accomplish this, we introduce novel image processing, modeling, and tomographic reconstruction tools. First, we developed dedicated pharmacokinetic modeling, machine learning and optimization framework based on the fusion of Electronic Health Records (EHR) data with dynamic PET brain imaging information. EHR data is used to infer individualized metabolism and clearance rates of the radioligand from the body. This is combined with simultaneous estimation on multiple distinct regions of the PET image. A substantial part of this effort involved curating, and then mining, an extensive database of PET, EHR and arterial blood sampling data. Second, we outline a new tomographic reconstruction and resolution modeling approach that takes into account the scanner point spread function in order to improve the resolution of existing PET data-sets. This technique allows visualization and quantification of structures smaller than previously possible. Recovery of signal from blood vessels and integration with the non-invasive framework is demonstrated. We also show general applicability of this technique for visualization and signal recovery from the raphe, a sub-resolution cluster of nuclei in the brain that were previously not detectible with standard techniques. Our framework can be generalizable to all classes of radioligands, independent of their kinetics and distribution within body. Work presented in this thesis will allow the PET scientific and clinical community to advance towards the ultimate goal of making PET cost-effective and to enable new clinical use cases.

Os organismos vivos estão expostos à ação oxidativa de espécies reativas de oxigênio (ERO), causando uma série de doenças degenerativas como câncer, aterosclerose, diabetes, artrite e doenças do coração. Estudos têm demonstrado que o consumo de substâncias antioxidantes na dieta diária podem prevenir estes processos oxidativos que provocam o envelhecimento precoce do organismo. Nas últimas décadas tem se destacado o interesse em encontrar antioxidantes naturais para o emprego em produtos alimentícios ou farmacêuticos, com a finalidade de substituir antioxidantes sintéticos, os quais apresentam restrições devido ao seu potencial tóxico. Nesse trabalho, são comparados os resultados de medidas da capacidade antirradicalar de vários derivados fenólicos incluindo produtos naturais obtidos com os ensaios utilizando o radical estável DPPH• e o cátion radical ABTS•+, que apresentam vantagens em relação à simplicidade do método analítico e facilidade na coleta de dados, além da reprodutibilidade dos resultados. Além disso, desenvolveu-se um ensaio com DPPH• para avaliar a capacidade antirradicalar de compostos fenólicos em meio ácido, hidroalcoólico e tamponado, para possibilitar a obtenção de valores da capacidade antirradicalar de flavonoides e compostos análogos em meio aquoso em diferentes estados de ionização. Também, foi utilizado o ensaio quimiluminescente com luminol/hemina/H2O2, desenvolvido pelo nosso grupo de pesquisa, para a determinação da capacidade antirradicalar de extratos, fases e frações de Baccharis regnelli e proposto o novo parâmetro \"Porcentagem de Trolox\" para expressar adequadamente esta capacidade em misturas complexas. A sensibilidade do ensaio luminol comprovou ser maior que a de outros métodos e adequado para medir a capacidade antirradicalar de misturas complexas de produtos naturais, auxiliando no isolamento de novas substâncias com atividade antirradicalar. / Living organisms are exposed to the oxidative action of reactive oxygen species (ROS), causing a series of degenerative diseases such as cancer, atherosclerosis, diabetes, arthritis and heart disease. Studies have shown that consumption of antioxidant substances in the daily diet can prevent these oxidative processes that cause premature aging of the organism. Much attention has been paid in the last decades on the discovery of new natural antioxidants for its use in food or pharmaceutical industry, with the aim of replace synthetic antioxidants, which have restrictions due to their toxic potential. In this study, we compared the results from antiradical capacity determinations of several phenolic derivatives including natural products obtained by two different assays. One of them utilizes the stable radical DPPH• and the other the radical cation ABTS•+ as reagents and both have the advantage of a simple analytical method and ease in data collection as well as high data reproducibility. Furthermore, we have developed a method with DPPH• for the evaluation of the antirradicalar capacity of phenolic compounds in acid and buffered hydroalcoholic media, in order to facilitate the determination of the antiradical capacity of flavonoids and similar compounds in aqueous ambient and to allow differentiation between the capacity of different ionization states of these derivatives. Finally the chemiluminescent luminol/hemin/H2O2 assay, developed by our research group, has been utilized for the determination of the antiradical capacity of extracts, phases and fractions of Baccharis regnelli and the new parameter \"Trolox Percentage\" is being proposed to adequately express the antiradical capacity of complex mixtures. The sensibility of the luminol assay has been found to be higher than that of other methods and is shown to be suitable for the determination of antiradical activity parameters of complex natural product mixtures, contributing to the isolation of new substances with antiradical activity.

O objetivo deste estudo foi avaliar, através de ensaios de tração, os efeitos do desenvolvimento de miopatia metabólica secundária a administração de corticosteróide sobre propriedades biomecânicas dos músculos diafragma e gastrocnêmio medial de coelhos. Foram estudadas 30 coelhas albinas adultas da raça Nova Zelândia, divididas em dois grupos de 15: Grupo Experimental (GE), que recebeu injeções subcutâneas de succinato sódico de 21 metil-prednisolona (Solumedrol Ò; Pharmacia - Up John N.N. / S.A. – Puurs - Bélgica) nas doses de 2 mg/kg/dia, e Grupo Controle (GC) que recebeu soro fisiológico a 0,9% por via subcutânea em volumes proporcionais. Ambos os grupos foram tratados durante 21 dias consecutivos. Os ensaios de tração foram realizados utilizando uma Máquina Universal de Ensaios do Laboratório de Bioengenharia da Faculdade de Medicina de Ribeirão Preto - USP. Para o hemi-diafragma esquerdo foram feitos 24 ensaios, 12 para o GE e 12 para o GC e excluídos 3 animais de cada grupo devido a problemas técnicos. Para o gastrocnêmio medial esquerdo foram feitos 30 ensaios, sendo 15 no GE e 15 no GC. A análise histoenzimológica dos músculos hemi-diafragma e gastrocnêmio medial direitos foi feita em 3 coelhos do GE e 3 do GC. O peso médio final dos animais no GE foi 3,6 Kg e no GC 4,0 Kg. A variação média percentual de peso final no GE foi - 8,4% e no GC 3,1%. O valor médio de peso final do gastrocnêmio no GE foi 5,6g e no GC 7,0g. Os valores médios de área, largura e espessura do gastrocnêmio no GE foram 2,4 x 10-4 m 2, 21,7 mm e 5,4 mm, respectivamente e no GC 2,8 x 10-4 m 2, 24,1 mm e 6,7.mm Nos diafragmas os valores médios de tensão e deformação do limite máximo, tensão e deformação do limite de proporcionalidade e módulo de elasticidade no GE comparado ao GC utilizando o teste t de Student e teste de Mann Whitney, não mostraram diferenças estatísticamente significantes. Nos gastrocnêmios os valores médios de carga máxima, carga e deformação do limite de proporcionalidade e rigidez no GE comparado ao GC, pelo teste t de Student não mostraram diferença estatística significante. Porém, o valor médio de deformação máxima no GE foi 26,63 x10-3 m e no GC 32,33 x10-3 m, mostrando significância estatística entre os grupos através do teste t de Student. Quanto aos locais de ruptura, no GE, 9 foram no ventre muscular, 2 na porção miotendínea distal e 4 na origem e no GC 8 ocorreram no ventre muscular, 5 na porção miotendínea distal e 2 na origem. Do ponto de vista histopatológico observamos que: a miopatia metabólica apresentou-se mais evidente nos diafragmas do GE; houve alterações metabólicas leves nos gastrocnêmios do GE e ocorreu aumento da succinato de desidrogenase (SDH) e Tricrômicro de Gomori modificado nos diafragmas do GC. Diante disto, concluímos que: 1) Os diafragmas não mostraram alterações de suas propriedade biomecânicas, nas fases plástica e elástica, apresentando a mesma capacidade de alongamento em ambos os grupos, suportando cargas semelhantes; 2) O músculo gastrocnêmio medial manteve suas características e capacidade de alongamento na fase elástica. Entretanto, o tratamento com esteróide, na fase plástica, levou a uma significativa redução do seu limite máximo de deformação, apresentando uma menor capacidade de alongamento, embora com mesma carga máxima do controle. / The aim of this study was to assess, using traction assays, the effects of the metabolic myopathy secondary to corticosteroids on biomechanical features of the diaphragm and the medial gastrocnemius muscles of rabbits. The study was composed of 30 albino adult rabbits of the New Zealand breed, which were divided into 2 groups of 15: Experimental Group (EG), which received subcutaneous injections of sodium succinate of 21 methil-prednisolone with doses of 2mg/Kg/day, and the Control Group (CG) which received subcutaneous Saline in proportional volumes. Both groups were treated during 21 consecutive days. The traction assays were performed by applying the Universal Machine of Assays from the Bioengenering Laboratory at the Medical School of Ribeirão Preto - USP. For the left hemi-diaphragm, 24 assays were performed, 12 for EG and 12 CG. Three animals from each group were excluded due to technical problems. For the left medial gastrocnemius, 30 assays were done, 15 for the EG and 15 for CG. The histoenzymologic analysis of both the hemi-diaphragm muscles and the right mild gastrocnemius were performed in three rabbits of the EG, and 3 of the CG. The final average weight for the animals in EG was 3,6Kg, and in the CG was 4,0Kg. The average percentage variation from the initial to the final weight for the EG was -8,4% and for the CG 3,1%. The final average weight of gastrocnemius was 5,1g in the EG and 7,0g in the CG. The average value of the area width and thickness of the EG was 2,4 x 10 -4 m 2, 21,7 mm and 5,4 mm, respectively, and for the CG 2,8 x 10-4 m 2, 24,1 mm and 6,7mm. In the diaphragms, the average values of the tension and deformity in the maximum threshold, tension and deformity at the proportional threshold and elasticity module in the EG compared to the CG, through the Student t Test and the Mann Whitney test, presented no significant statistical differences. In the gastrocnemius, average values of the maximum load, load and deformity within the threshold of proportions and stiffness in the EG compared to the CG, according to the Student t test, did not present a significant statistical difference. However, the average values of maximum deformity were 26,63 x10 -3 m in the EG and 32,33 x10-3 m in the GC, showing statistical significant difference between groups. Concerning the locations of the rupture in the EG, 9 were in the muscle belly, 2 in the miotendinous distal junction, and 4 in the origin; in the CG the rupture ocurred 8 times in the muscle belly , 5 in miotendinous distal junction and 2 in the origin. From a histopathological view, we have observed that metabolic myophaty was presented clearly evident in the diaphragms of the EG; there were slight metabolic alterations in the gastrocnemius of the EG, and there was an increase of the succinic dehydrogenase (SDH) and modified Gomori Trichrome in the diaphragms of the CG. In this manner, we conclude that: 1) The diaphragms didn´t show alterations of their biomechanical properties, in elastic and plastic phases, showing the same elongation capacity in both groups, with equal loads; 2) The medium gastrocnemius muscles, kept their biomechanical features and elongation capacity in elastic phase. However, the steroid treatment lead to a significant decrease of the elongation capacity in the plastic phase, with maximal loads similar to the control group; 3) It was not found a relationship between histological evidences of metabolic miopathy and changes in the biomechanical properties of the studied muscles.

As proteínas inibidoras de poligalacturonases (PGIPs) presentes na parede celular são capazes de limitar o potencial destrutivo da poligalacturonase (PG) fúngica e, assim, constituem um tipo importante dentre os diversos sistemas de defesa do tecido vegetal frente à infecção fúngica. No mamão, o ataque fitopatogênico é o principal causador de danos pós-colheita, e sua alta susceptibilidade pode estar relacionada com a baixa eficácia ou pouca abundância dos meios de defesa anti-fitopatogênica. Uma vez que isso pode estar relacionado com as PGIPs e nada se conhece sobre o papel dessas proteínas nesse fruto, o objetivo do trabalho foi clonar os genes das PGIPs de mamoeiro e definir seu padrão de expressão em diferentes órgãos e tecidos e ao longo do amadurecimento. Para tanto, foram identificadas no genoma do mamoeiro, a partir de critérios que definem a identidade de uma PGIP, duas prováveis sequências dentre 13 candidatas iniciais. Ambas foram clonadas a partir das sequências genômicas e de cDNA, sequenciadas e sua identidade confirmada, sendo denominadas Cppgip4 e Cppgip6. As análises de expressão relativa em diversos tecidos e idades fisiológicas do mamoeiro demonstraram que os dois genes apresentaram diminuição da expressão com o desenvolvimento dos frutos, sendo que com a polpa apresentou redução dos níveis de expressão relativa de Cppgip4 em até 18 vezes dos 30 dias pós-antese (DPA) ao 9 dias pós-colheita (DPC). Na casca também houve redução significativa da expressão com o desenvolvimento. Para a expressão absoluta, nos frutos, sementes, caules, raízes e folhas, o número de cópias de ambos os transcritos decresceu com o desenvolvimento, sendo cerca de cem mil vezes mais abundante para Cppgip6 que para Cppgip4. As tentativas de expressão de proteínas recombinantes em Pichia pastoris não geraram resultado positivo, provavelmente em virtude das condições ideais de indução ainda não terem sido estabelecidas corretamente para o ensaio. A atividade de PGIPs extraídas diretamente do tecido foi medida por análise de difusão em ágar empregando pectinase de Aspergillus niger e revelou uma tendência à diminuição da porcentagem de inibição à medida que os frutos se desenvolveram, em concordância com os resultados da análise por qPCR. O conjunto de resultados sugere que a expressão varia com o estádio de desenvolvimento do fruto e é tecido-específica, possivelmente em resposta à diferente susceptibilidade dos tecidos ao ataque fitopatogênico, indicando que menores níveis de transcritos e atividade no amadurecimento, período de maior susceptibilidade, poderiam sinalizar para a regulação do processo degradativo marcando o início da senescência. / Polygalacturonase inhibiting proteins (PGIPs) present in plant cell walls are able to inhibit the destructive action of fungal polygalacturonase (PG). In this way, they constitute an important type of plant defense system against fungal infections. In papaya fruit, the pathogenic attack is the main cause of post harvesting loss, and its high susceptibility may be related to the low efficiency or low abundance of anti-phytopathogenic defense. Since this fact could be related to PGIPs expression and little is known about the response of these proteins in the fruit, the aim of the present work was to clone the genes of PGIPs papaya fruit and set their expression pattern in different organs and tissues throughout fruit ripening. Thus, two probable PGIP sequences among 13 initial candidates were identified in the papaya genome by using specific criteria. Both sequences were cloned from cDNA and genomic samples, sequenced and confirmed its identity, and then being named Cppgip4 and Cppgip6. Analysis of relative expression in various tissues at different physiological stages demonstrated that both genes were down regulated during fruit development. The relative expression levels of Cppgip4 in papaya pulp was reduced by 18 times from the 30 days post-anthesis (DPA) to the 9 days post-harvest (DPH). Similarly, gene expression in papaya peel was significant down regulated during fruit development. Absolute expression analysis revealed gene expressions in the fruit pulp, seed, stem, root and leaf were also down regulated within development. Moreover, Cppgip6 gene expression was a hundred thousand times more abundant than Cppgip4. The recombinant protein expression in Pichia pastoris did not result positive, probably because of the ideal conditions of induction have not been properly established the yet. The activity of PGIPs extracted directly from the tissue was measured by the agar diffusion assay using pectinase from Aspergillus niger and showed decrease of inhibition during fruit developed in accordance with the results of the qPCR analysis. Based on the results it is possible to suggest the expression of these genes varies temporally with the developmental stage of the fruit and is tissue-specific, possibly in response to the different susceptibility of tissues to pathogenic attack. In addition, the lowest levels of PGIP expression were achieved at the fruit ripening, when the susceptibility to fungal infection is high and could signal for regulating the degradation process characterized by the onset of senescence.

Protein engineering strategies, such as directed evolution, generate large libraries of enzyme variants, typically in the range of 106-108 variants. However, the availability of rapid, robust high-throughput screening methods has often limited the impact of directed evolution in discovering enzymes with enhanced catalyst performance. Raman spectroscopy is an established analytical technique, providing molecular specific information, permitting analysis in aqueous solutions and as such is an attractive, alternative screening method for biological systems. Although an inherently weak physical phenomenon, enhanced Raman scattering techniques, such as surface enhanced Raman scattering (SERS) and ultraviolet resonance Raman (UVRR) spectroscopy, can be used to overcome the associated sensitivity issues. Herein, we successfully monitored xanthine oxidase (XO) catalysed conversions of xanthine to uric acid, before extending to hypoxanthine, using two contrasting Raman scattering enhanced approaches. Firstly, a SERS-based assay was developed utilising silver nanoparticles to measure analytes directly and quantitatively on micromolar scale, in the absence of chromogenic substrates or lengthy chromatography. Secondly, a UVRR approach was developed enabling monitoring of the XO-mediated reaction in real-time and without the need to quench the system. Significantly, both methods demonstrated over &gt;30 fold reduction in acquisition times (when compared to conventional HPLC analysis), and offered excellent medium-term reproducibility and accuracy of results over significant time periods. Furthermore, investigations were made into developing this SERS-based assay into an enantiomeric screen using another vibrational spectroscopy approach, Raman optical activity (ROA), along with circular dichroism (CD). Successful chiral reduced nanoparticles were synthesised, with multiple characterisation techniques employed, affording enantiopure Au-cysteine and Ag-tyrosine colloids. However, it was not possible to generate consistent and reproducible SEROA responses, with these techniques ultimately being unsuccessful in analysing these chiral sensitive nanoprobes, and thus differentiating between the D- and L- forms. Finally, a novel SERS-based approach, in combination with the standard addition method (SAM), was developed for the routine analysis of uric acid (end product in XO catalysed reaction(s) and biomarker for various diseases), at clinically relevant levels in urine samples from patients. Results were highly comparable and in very good agreement with HPLC analyses, with an average < 9% difference in predictions between the two analytical approaches across all samples analysed, and a 60-fold reduction in acquisition time (when compared with HPLC). Together, the research presented in this thesis demonstrates the suitability of Raman enhanced techniques for quantitative analysis, measuring the analytes directly using a portable Raman instrument and, most importantly, offering significant reductions in acquisition times when compared to established analytical techniques.

Various recent studies have suggested that regular intake of some non-steroidal anti-inflammatory drugs (NSAIDs) have a preventative effect against several types of tumours including breast cancer. The term nanotechnology refers to technology in which one-billionth of a meter is used as a scale for chemical particle size. This work aims to study the effect of both ibuprofen and aspirin on DNA damage using peripheral blood lymphocytes from breast cancer patients and comparing the results with those from healthy females as a control using the Comet and micronucleus assays. Western blot analysis (WBA) was used to investigate the effect of these drugs on XRCC3 and p53 proteins, whereas QPCR was to evaluate this effect on p53, cox1 and cox2 genes. Two hundred fifty ng/ml of ibuprofen (NP and bulk) and 500 ng/ml of aspirin (NP and bulk) were used to treat the lymphocytes. Both aspirin and ibuprofen caused a reduction in DNA damage and micronucleus formation. Aspirin, both forms, showed a reduction in DNA damage in the Comet and micronucleus assays. Ibuprofen both forms, by contrast, showed a statistically significant reduction in micronucleus frequency in the micronucleus assay, while its preventative effect with the Comet assay was weak or insignificant. NPs of both agents were more effective than bulk sizes. Using the Comet repair assay, aspirin and ibuprofen nano form catalysed DNA repair to a greater extent than their bulk forms. Also, both sizes showed better repair with NSAIDs compared to samples repaired without NSAIDs. In WBA aspirin increased the expression of XRCC3 protein in healthy cells. However, both NSAIDs decreased that expression in cells from BC patients. Furthermore, aspirin increased p53 expression in BC patients lymphocytes. With the QPCR method, results of both aspirin forms increased the expression of the p53 gene in BC patient cells statistically significantly. Both drugs reduced cox1 expression in healthy volunteers and cancer patients lymphocytes. Moreover, cox2 reduction was only in lymphocytes from BC patients. The results of this work are consistent with the view that NSAIDs, particularly aspirin and ibuprofen, could have a promising role in cancer treatment including breast cancer.