The accurate and safe diagnosis of breast cancer is a significant societal issue, with annual disease incidence of 48,000 women and around 370 men in the UK. Early diagnosis of the disease allows more conservative treatments and better patient outcomes. Microcalcifications in breast tissue are an important indicator for breast cancers, and often the only sign of their presence. Several studies have suggested that the type of calcification formed may act as a marker for malignancy and its presence may be of biological significance. In this work, breast calcifications are studied with FTIR, synchrotron FTIR, ATR FTIR, and Raman mapping to explore their disease specific composition. From a comparison between vibrational spectroscopy and routine staining procedures it becomes clear that calcium builds up prior to calcification formation. Raman and FTIR indicate the same size for calcifications and are in agreement with routine staining techniques. From the synchrotron FTIR measurements it can be proven that amide is present in the centre of the calcifications and the intensity of the bands depends on the pathology. Special attention is paid to the type of carbonate substitution in the calcifications relating to different pathology grades. In contrast to mammography, Raman spectroscopy has the capability to distinguish calcifications based on their chemical composition. The ultimate goal is to turn the acquired knowledge from the mapping studies into a clinical tool based on deep Raman spectroscopy. Deep Raman techniques have a considerable potential to reduce large numbers of normal biopsies, reduce the time delay between screening and diagnosis and therefore diminish patient anxiety. In order to achieve this, a deep Raman system is designed and after evaluation of its performance tested on buried calcification standards in porcine soft tissue and human mammary tissue. It is shown that, when the calcification is probed through tissue, the strong 960 cm-1 phosphate band can be used as a pseudo marker for carbonate substitution which is related to the pathology of the surrounding tissue. Furthermore, the first study in which human breast calcifications are measured in bulk tissue with a thickness of several millimetres to centimetres is presented. To date, measurements have been performed at 41 specimens with a thickness up to 25 mm. Measurements could be performed through skin and blue dye. The proposed deep Raman technique is promising for probing of calcifications through tissue but will need refinement before being adopted in hospitals.

A novel lidar ceilometer prototype based on divided lens optics has been designed, built, characterised, and tested. The primary applications for this manufacturable ground-based sensor are the determination of cloud base height and the measurement of vertical visibility. First, the design, which was developed in order to achieve superior performance at a low cost, is described in detail, along with the process used to develop it. The primary design considerations of optical signal to noise ratio, range-dependent overlap of the transmitter and receiver channels, and manufacturability, were balanced to develop an instrument with good signal to noise ratio, fast turn-on of overlap for detection of close range returns, and a minimised number of optical components and simplicity of assembly for cost control purposes. Second, a novel imaging method for characterisation of transmitter-receiver overlap as a function of range is described and applied to the instrument. The method is validated by an alternative experimental method and a geometric calculation that is specific to the unique geometry of the instrument. These techniques allow the calibration of close range detection sensitivity in order to acquire information prior to full overlap. Finally, signal processing methods used to automate the detection process are described. A novel two-part cloud base detection algorithm has been developed which combines extinction-derived visibility thresholds in the inverted cloud return signal with feature detection on the raw signal. In addition, standard approaches for determination of visibility based on an iterative far boundary inversion method, and calibration of attenuated backscatter profile using returns from a fully-attenuating water cloud, have been applied to the prototype. The prototype design, characterisation, and signal processing have been shown to be appropriate for implementation into a commercial instrument. The work that has been carried out provides a platform upon which a wide range of further work can be built.

Nitrocellulose is the main component in many types of ammunition, propellants and explosives. The principles of production for nitrocellulose have not changed much since the 19th century when it started being industrially produced for this purpose. The character of the nitrocellulose has a large effect on the end products abilities. The aim of this study was to develop a method that would be able to characterise and distinguish between nitrocellulose from different manufacturers to be able to relate the character of the nitrocellulose to the properties of ammunition, propellants and explosives. Samples were dissolved in acetone and analysed by GC/MS and data were then analysed by multivariable statistics. FTIR was also used to characterise the nitrocellulose. Results from both methods showed very small differences when chromatograms and spectra were analysed. This study shows that GC/MS and FTIR are not suitable for this type of characterisation. The differences between the data were not sufficient to be able to separate the samples from each other. / Nitrocellulosa är den viktigaste komponenten i många typer av ammunition, drivmedel och sprängämnen. Principerna för produktionen av nitrocellulosa har inte förändrats mycket sedan det börjades produceras industriellt för detta ändamål på 1800 talet. Karaktären av nitrocellulosa har en stor inverkan på slutproduktens egenskaper. Syftet med denna studie var att utveckla en metod som skulle kunna karaktärisera och skilja mellan nitrocellulosa från olika tillverkare för att kunna relatera karaktären av nitrocellulosa till egenskaperna hos ammunition, drivmedel och sprängämnen. Proverna löstes i aceton och analyserades med GC/MS och data analyserades med multivariabel statistik. FTIR användes också för att karakterisera nitrocellulosan. Resultaten för båda proverna visade mycket små skillnader när kromatogram och spektra analyserades. Denna studie visar att GC/MS och FTIR inte är lämpliga för denna typ av karaktärisering. Skillnaderna i data var inte tillräckliga för att kunna skilja proverna från varandra.

Flagelina, a subunidade estrutural dos flagelos bacterianos, pode ser empregada em estratégias vacinais como carregadora de epitopos antigênicos fusionados ou como adjuvante. Neste trabalho avaliamos a imunogenicidade e o potencial vacinal das flagelinas Salmonella enterica sorovar Typhimurium em camundongos BALB/c após administração pelas vias oral ou intraperitoneal. Os resultados indicam que animais imunizados pela via oral com linhagens flageladas de S. Typhimurium não desenvolvem respostas de anticorpos, sistêmicos ou secretados, anti-flagelina, por outro lado, ativam respostas celulares específicas para as flagelinas em função da via de administração utilizada. Observamos ainda que as diferentes flagelinas testadas apresentam efeito adjuvante quando co-administradas pela via nasal com a fímbria CFA/I de ETEC. Em suma, os resultados apresentados contribuem para um melhor conhecimento sobre as propriedades imunológicas e adjuvantes das flagelinas de S. Typhimurium e agregam informações para o uso racional dessas proteínas em formulações vacinais. / Flagellin, the structural subunit of bacterial flagella, can be used in vaccine development as a fused antigenic epitope carrier or as an adjuvant. In this work we evaluated the immunogenicity and vaccine approach of S. Typhimurium flagellin in BALB/c mice after administration by oral or intraperitoneal route. Our results indicate that mice immunized by oral route with flagellated S. Typhimurium strains do not develop any anti-flagellin antibody response, be it serum or secreted. On the other hand, specific anti-flagellin cellular response was observed depending on the chosen route of immunization with S. Typhimurium attenuated strain. Furthermore, all flagellins tested proved to be efficient adjuvants when co-administrated with CFA/I fimbriae by nasal route. Together, these results contribute to a better understanding of the immunological and adjuvant properties of S. Typhimurium flagellins and also provide information on the rational use of these proteins in vaccine development.

A Esquistossomose é uma das mais importantes doenças endêmicas do mundo, com mais de 200 milhões de pessoas infectadas em 76 países. É estimado que mais de 600 milhões de pessoas estejam em áreas endêmicas. Em 2003 dados do transcriptoma do Schistosoma mansoni foram disponibilizados. As informações de proteínas codificadas permitiram a análise de suas funções, e auxiliaram na procura de novos candidatos vacinais. A análise do transcriptoma permitiu a identificação de três famílias de proteínas homólogas à dineína de cadeia leve (DLC) de mamíferos. Uma delas é a família L8, com ao menos 18 membros, com proteínas em torno de 10 kDa. Essas proteínas são expressas em diferentes estágios do ciclo de vida do Schistosoma mansoni. Duas DLCs foram reconhecidas no tegumento de S. japonicum, sugerindo que elas são expostas ao sistema imune do hospedeiro. Salmonelas atenuadas como vacinas vivas têm sido descritas como bons veículos para apresentação de antígenos heterólogos. No nosso laboratório uma importante ferramenta tem sido desenvolvida para auxiliar o uso de salmonelas como vacinas vivas recombinantes, foi desenvolvido um vetor plasmididal baseado no regulon soxRS para controlar a expressão de genes heterólogos in vivo. Este sistema de expressão promove a expressão de proteínas recombinantes sob condições de estresse oxidativo, como aquele imposto ao microorganismo dentro do macrófago. Para investigar o potencial vacinal das DLCs, três genes foram selecionados para serem clonados e expressos em E. coli e em salmonelas atenuadas. A antigenicidade e imunogenicidade desses parálogos foram testadas em camundongos depois de serem imunizados com proteínas purificadas ou com salmonelas recombinantes. As DLCs mostraram ser bastante imunogênicas, aumentando os título de IgG. A proteína DLC1 também aumentou os níveis de IgE no soro dos animais, fato que poderia estar relacionado com reações alérgicas observadas em populações infetadas. Altos níveis de IgE também podem ser relacionados com uma marca de resistência existente em pessoas que vivem em áreas endêmicas. Depois de imunizados os animais foram desafiados com cercárias para investigar possível proteção. Foi observado que a imunização com proteínas purificadas resultou em aproximadamente 40% de diminuição da carga parasitária. A análise dos granulomas hepáticos com 45 dias depois da infecção indicou uma significativa redução, maior que 70% das áreas dos granulomas, sgerindo que a imunização com as DLCs promove uma importante interferência na formação dos granulomas hepáticos. Por outro lado, nossos estudos com salmonelas atenuadas recombinantes, carregando as DLCs, mostraram que foram ineficientes na apresentação dos antígenos ao sistema imune. Relacionando os resultados de diminuição de carga parasitária e das áreas dos granulomas depois da imunização com as DLCs purificadas sugere-se que essas proteínas podem ser consideradas como interessantes candidatos vacinais, uma vez que elas afetam as mais importantes causas da patologia da esquistossomose. / Schistosomiasis is one of the most important endemic diseases in the world, with more than 200 million people infected in 76 countries. It is estimated that more than 600 million people live in endemic areas. In 2003 extensive data from the transcriptome of Schsitosoma mansoni was made available. The information on the encoded proteins allowed the analysis of protein function and improved the search for vaccine candidates. The analysis of the transcriptome allowed the identification of three families of proteins homologs to the mammalian dynein light chain (DLC). One of these was the L8 family, with at least 18 members, all proteins with around 10 kDa. These proteins were found to be expressed in the different stages of the S. mansoni life cycle. Two DLCs were recognized in the tegument of S. japonicum, suggesting that they are exposed to the host immune system. Attenuated salmonellas, as live vaccines, have been described as good vehicles for presentation of heterologous antigens. At our laboratory an important tool has been developed to improve the use of salmonellas as live recombinant vaccines, a plasmidial vector based on the soxRS regulon to control the expression of heterologous genes in vivo. This expression system promotes the expression of recombinant proteins under conditions of oxidative stress, such as that imposed to the microorganism in the macrophage environment. To investigate the vaccine potential of DLCs, three genes were selected to be cloned and expressed in E. coli and in attenuated salmonellas. Antigenicity and immunogenicity of these paralogous were tested in mice after immunization with purified proteins or with the recombinant salmonellas. The DLCs were proven to be very immunogenic, increasing the IgG titers. DLC1 also increased the IgE levels in the sera of animals, what could be related to allergenic reactions observed in infected population. High level of IgE can also be related to the resistance mark of people living in endemic areas. After immunization, the animals were challenged with cercarias to investigate the protective profile. It was observed that immunization with purified proteins resulted in approximately 40% decreasing in the worm burden. The analysis of the hepatic granulomas 45 days after infection indicated a significant, up to 70 %, reduction of the granuloma areas, suggesting that immunization with DLCs promotes important interference in the hepatic granuloma formation. On the other hand, our studies with the attenuated recombinant salmonellas, carrying DLCs, showed no effective antigen presentation to the mice immune system. Taking together, the results of decreasing the worm burden and the granuloma size after immunization with purified DLCs suggest that these proteins could be considered as interesting vaccine candidates, affecting the main causes of the pathology of schistosomiasis.

Seasonal influenza outbreaks are associated with morbidity and mortality in the United States. Though children are the most susceptible to influenza infection and are most likely to transmit the illness to others, many children are not vaccinated. The purpose of this study was to examine the relationship between seasonal influenza vaccination Advisory Committee on Immunization Practices (ACIP) recommendations, demographic characteristics, socioeconomic factors, and vaccine type among children over 3 consecutive flu seasons. This quantitative cross-sectional study was guided by the social ecology of health model. Secondary data from 3 consecutive flu seasons (2014-2015, 2015-2016, and 2016-2017) provided by the National Health Interview Survey was used for this study. Binary logistic regression and chi-square were used to analyze the data. A relationship between socioeconomic status, demographics (age, race, and family income) and vaccine type (live-attenuated influenza vaccine [LAIV]/inactivated influenza vaccine) was established among U.S. children; those who received LAIV were most likely to be White elementary school age children with a higher family income. Demographic and socioeconomic status was not considered influential in LAIV uptake for race, health insurance status, or family income. ACIP recommendations by age and year had the greatest impact on flu vaccine choice for this sample population. The results of this study can lead to social change by providing information for policy that can increase vaccine uptake, which can result in lower health cost and reduced illness and death rates associated with the flu, especially for those most at risk.