Raman spectroscopy for point of care urinary tract infection diagnosis

Urinary tract infections (UTIs) are one of the most common bacterial infections experience by humans, with 150 million people suffering one or more UTIs each year. The massive scale at which UTIs occurs translates to a tremendous health burden comprising of patient morbidity and mortality, massive societal costs and a recognised contribution to expanding antimicrobial resistance. The considerable disease burden caused by UTIs is severely exacerbated by an outdated diagnostic paradigm characterised by inaccuracy and delay. Poor accuracy of screening tests, such as urinalysis, lead to misdiagnosis which in turn result in delayed recognition or overtreatment. Additionally, these screening tests fail to identify the causative pathogen, causing an overreliance on broad-spectrum antimicrobials which exacerbate burgeoning antimicrobial resistance. While diagnosis may be accurately confirmed though culture and sensitivity testing, the prolonged delay incurred negates the value of the information provided doing so. A novel diagnostic paradigm is required that that targets rapid and accurate diagnosis of UTIs, while providing real-time identification of the causative pathogen. Achieving this precision management is contingent on the development of novel diagnostic technologies that bring accurate diagnosis and pathogen classification to the point of care. The purpose of this thesis is to develop a technology that may form the core of a point-of-care diagnostic capable of delivering rapid and accurate pathogen identification direct from urine sample. Raman spectroscopy is identified as a technology with the potential to fulfil this role, primarily mediated though its ability to provide rapid biochemical phenotyping without requiring prior biomass expansion. Raman spectroscopy has demonstrated an ability to achieve pathogen classification through the analysis of inelastically scattered light arising from pathogens. The central challenge to developing a Raman-based diagnostic for UTIs is enhancing the weak bacterial Raman signal while limiting the substantial background noise. Developing a technology using Raman spectroscopy able to provide UTI diagnosis with uropathogen classification is contingent on developing a robust experimental methodology that harnesses the multitude of experimental and analytical parameters. The refined methodology is applied in a series of experimental works that demonstrate the unique Raman spectra of pathogens has the potential for accurate classification. Achieving this at a clinically relevant pathogen load and in a clinically relevant timeframe is, however, dependent on overcoming weak bacterial signal to improve signal-to-noise ratio. Surface-enhanced Raman spectroscopy (SERS) provides massive Raman signal enhancement of pathogens held in close apposition to noble metal nanostructures. Additionally, vacuum filtration is identified as a means of rapidly capturing pathogens directly from urine. SERS-active filters are developed by applying a gold nanolayer to commercially available membrane filters through physical vapour deposition. These SERS-active membrane filter perform multiple roles of capturing pathogens, separating them from urine, while providing Raman signal enhancement through SERS. The diagnostic and classification performance of SERS-active filters for UTIs is demonstrated to achieve rapid and accurate diagnosis of infected samples, with real-time uropathogen classification, using phantom urine samples, before piloting the technology using clinical urine samples. The Raman technology developed in this thesis will be further developed toward a clinically implementable technology capable of ameliorating the substantial burden of disease caused by UTIs.Open Acces

Electromagnetic Waves

This book is dedicated to various aspects of electromagnetic wave theory and its applications in science and technology. The covered topics include the fundamental physics of electromagnetic waves, theory of electromagnetic wave propagation and scattering, methods of computational analysis, material characterization, electromagnetic properties of plasma, analysis and applications of periodic structures and waveguide components, and finally, the biological effects and medical applications of electromagnetic fields

Fragilidade : prevalência, fatores associados e tratamento através do treinamento de força

Orientadores: Marco Carlos Uchida, Bruno RodriguesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Educação FísicaResumo: A fragilidade é um estado potencialmente reversível de maior vulnerabilidade à desfechos de saúde negativos, o qual ocorre como resultado do comprometimento biológico multissistêmico e aspectos socioambientais. A prevalência de fragilidade na Europa e na Ásia já foi estabelecida e, mais recentemente, pesquisadores sugeriram que a incidência de fragilidade em três anos ao rendo do mundo é de 13,6%. Embora os países da América do Sul sejam uma economia emergente, ainda sofrem com a pobreza, desnutrição, moradia precária, falta de informação e baixa qualidade de vida, todas variáveis associadas ao desenvolvimento da fragilidade. No entanto, a prevalência de fragilidade na América do Sul ainda é pouco explorada. Portanto, o presente projeto de doutorado investigou a prevalência de fragilidade na América do Sul. A gênese e a progressão da fragilidade estão associadas à muitos desfechos negativos relacionados à saúde, como limitações da mobilidade e anormalidades cardiovasculares. Em contraste, o alto consumo proteico parece estar associado negativamente ao status de fragilidade. Notavelmente, a definição operacional de fragilidade ainda é difícil pela ausência de uma definição unívoca, e mais de 60 instrumentos diferentes para a sua avaliação estão atualmente disponíveis. Algumas investigações observaram que esses instrumentos não capturam necessariamente o mesmo construto, o que permite supor que a associação entre fragilidade e outros fatores possa ser dependente do instrumento utilizado. Com base nessas premissas, este projeto investigou a relação entre o status de fragilidade e a ingestão de proteínas, função física e parâmetros relacionados à hipertensão arterial sistêmica usando 4 instrumentos diferentes. O estabelecimento da fragilidade como um problema de saúde pública levou pesquisadores a examinar terapias para colaborar com o tratamento dessa condição. Muita atenção tem sido dada ao exercício físico, principalmente ao treinamento de força (TF), dados os inúmeros estudos que relataram melhorias nos parâmetros relacionados à fragilidade em resposta aos programas de TF. No entanto, mesmo que os programas tradicionais de TF, ou também chamados treinamento de resistência a baixa velocidade (LSRT), pareçam ser uma ferramenta poderosa para melhorar a força muscular, seus efeitos da mobilidade são menos pronunciados. Nesse contexto, evidências que a potência muscular, a capacidade de exercer força em um curto intervalo de tempo, diminui precocemente e está mais associada à tarefas de mobilidade do que a força muscular, levou à suposição de que os protocolos de RT de alta velocidade (HSRT) poderiam causar maiores melhorias na mobilidade do que o LSRT. Ensaios clínicos randomizados, revisões sistemáticas e 11 metanálises testaram essa hipótese e os resultados ainda são controversos. Além disso, as investigações são baseadas em idosos robustos, de modo que os efeitos dos programas de TF no status de fragilidade e no desempenho físico de idosos frágeis não foram descritos anteriormente. Assim, o presente projeto investigou os efeitos do HSRT e LSRT no status de fragilidade. Secundariamente, foi examinado os efeitos de ambos os programas de TF no desempenho físico, função cognitiva e pressão arterial, dada sua estreita associação com à fragilidadeAbstract:DoutoradoAtividade Fisica AdaptadaDoutor em Educação Física01P-1870/2016, 23P-6076/2018CAPE

The effects of DMT and associated psychedelics on the human mind and brain

This work presents seven investigations conducted with the aim to determine the effects of DMT (a compound which is able to cause remarkable effects in consciousness) and associated psychedelic drugs on the human brain and mind. Including a variety of neuroimaging (EEG, MEG and fMRI), phenomenological, psychometric and naturalistic research methods, these are the first controlled investigations of the impact of DMT in the human resting brain. Results revealed that DMT disrupted several brain mechanisms associated with top-down control (alpha power, integrity of high-level networks, modularity), increased measures related to entropy, or disorder (Lempel-Ziv complexity, novel pairwise connectivity) and immersive states of consciousness (delta/theta power), with some of these effects following the experiential trajectories of the DMT state. We also observed a significant temporal correlation between some of these effects (alpha power and default-mode network integrity fluctuations), which were supported by LSD effects of reduced feedback connectivity and neural adaption mechanisms. suggesting that the psychedelic brain state is one of reduced modularity, increased integration and functional plasticity. These findings were complemented by psychological studies showing that the DMT state is one of immersive visual imagery, intense somatic experiences and partial disconnection from the environment, which we found shared significant overlap with near- death experiences. DMT administration also resulted in positive mental health outcomes in healthy volunteers providing evidence for the first time that DMT may provide a useful alternative to currently- investigated psychedelic treatments. Finally, results from our last study performed in naturalistic environments revealed that psychedelics are able to have a transformative potential on core beliefs concerning the fundamental nature of reality and consciousness for up to 6 months, with important social and bioethical implications. Collectively these results attest to the strong impact that psychedelics have on varied human domains, which range experience, brain activity, mental health, intersubjectivity and beliefs.Open Acces

Neural representation of social, monetary and chocolate reinforcer processing

Little attention has been paid to social reinforcer processing compared with food and monetary reinforcers, in the reward-related functional magnetic resonance imaging (fMRI) literature. This is surprising as social reinforcers pervade our daily lives and are often experienced more frequently than food or monetary reinforcers. The question of whether social reinforcers are processed in the same or different brain regions as other reinforcer types remains poorly understood. In this thesis, three fMRI studies were employed to investigate this question, in healthy individuals. The experimental paradigms focused on two main aspects of reward processing: neural patterns of activation associated with different reward types and valance, and also correlations between neural activation to rewards and participants’ hedonic level. The studies reported in this thesis revealed that amygdala and a subregion of the OFC responded more sensitively to social reinforcers than monetary, or food reinforcers, indicating social reinforcers modulate the affective response more strongly in the brain reward network. The results also provide evidence for a medial-lateral functional dissociation in the OFC to rewards and punishment, so that medial OFC responded more strongly to rewards and lateral OFC to punishments. Moreover, fMRI study-1 revealed a crossover interaction between reinforcement valence and reward type in the lateral OFC, indicating this region may be involved in the functional integration of both reward type and valence. This is consistent with the theory of a common neural currency, for valuing different rewards in the OFC. As activation in the reward network may also be attributed to the hedonic experience of gaining rewards, fMRI study-2 and study-3 also explored the relationship between BOLD activity in response to rewards and participants’ hedonic scores. These two studies demonstrated highly significant correlations between BOLD activity in the OFC (positive correlation) and insula (negative correlation) and self-reported levels of hedonic response. The findings of the correlations between reward and hedonic level could have important implications for understanding how human hedonic levels affect responses to various reinforcements

Molecular mechanisms of PAH function in response to phenylalanine and tetrahydrobiopterin binding

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism (IEM) caused by mutations in the phenylalanine hydroxylase (PAH) gene. The molecular mechanism underlying deficiency of the PAH protein is, in most of the cases, loss of function due to protein misfolding. PAH mutations induce disturbed oligomerisation, decreased stability and accelerated degradation of hepatic PAH, a key enzyme in phenylalanine metabolism. Since the development of a phenylalanine-restricted diet in the 1950ies, PKU is a prototype for treatable inherited diseases. About 60 years later, the natural PAH cofactor tetrahydrobiopterin (BH4) was shown to act as a pharmacological chaperone stabilising the misfolded PAH protein. In consequence, BH4 (KUVAN®) was introduced to the pharmaceutical market as an alternative treatment for BH4-responsive PAH deficiency. Therefore, PKU is also regarded as a prototype for a pharmacologically treatable protein misfolding disease. Despite the progress in PKU therapy, knowledge on the molecular basis of PKU and the BH4 mode of action was still incomplete. Biochemical and biophysical characterisation of purified variant PAH proteins, which were derived from patient’s mutations, aimed at a better understanding of the molecular mechanisms of PAH loss of function. We showed that local side-chain replacements induce global conformational changes with negative impact on molecular motions that are essential for physiological enzyme function. The development of a continuous real-time fluorescence-based assay of PAH activity allowed for robust analysis of steady state kinetics and allosteric behaviour of recombinantly expressed PAH proteins. We identified positive cooperativity of the PAH enzyme towards BH4, where cooperativity does not rely on the presence of phenylalanine but is determined by activating conformational rearrangements. In vivo investigations on the mode-of-action of BH4 revealed differences in pharmacodynamics but not in pharmacokinetics between different strains of PAH-deficient mice (wild-type, Pahenu1/1 and Pahenu1/2). These observations pointed to a significant impact of the genotype on responsiveness to BH4. The available database information on PAH function associated with PAH mutations was based on non-standardised enzyme activity assays performed in different cellular systems and under different conditions usually focusing on single PAH mutations. These inconsistent data on PAH enzyme activity hindered robust prediction of the patient’s phenotype. Furthermore, assays on single PAH mutations do not reflect the high allelic and phenotypic heterogeneity of PKU with 89 % of patients being compound heterozygotes. In addition, the knowledge on enzyme function and regulation in the therapeutic and pathologic metabolic context was still scarce. In order to get more insight into the interplay of the PAH genotype, the phenylalanine concentration and BH4 treatment, we performed functional analyses of both, single, purified PAH variants as well as PAH full genotypes in the physiological, pathological and therapeutic context. The analysis of PAH activity as a function of phenylalanine and BH4 concentrations enabled determination of the optimal working ranges of the enzyme and visualisation of differences in the regulation of PAH activity by BH4 and phenylalanine depending on the underlying genotype. Moreover, these PAH activity landscapes allowed for setting rules for dietary regimens and pharmacological treatment based on the genotype of the patient. Taken together, precise knowledge on the mechanism of the misfolding-induced loss of function in PAH deficiency enabled a better understanding of the molecular mode of action of pharmacological rescue of enzyme function by BH4. We implemented the combination of genotype-specific functional analyses together with biochemical, clinical and therapeutic data of individual patients as a powerful tool for phenotype prediction and paved the way for personalised medicine strategies in phenylketonuria

A Nano-sized Dose of Toxicology: Elucidating the Disconnect Between Nanomaterial Dosimetry and Biological Effects

Nanotechnologies continue to permeate a multitude of industries, with diverse applications ranging from pesticides to fuel additives. The unusual behavior of nanomaterials that drives their innovation also complicates the job of toxicologists tasked with assessing their potential environmental and public health impacts. This dissertation investigates the underlying reasons for uncertainty associated with the biological effects of nanomaterials. Chapter 2 and Chapter 3 focus on nanoparticles present in commercially available pesticide formulations and assess how particle size influences the environmental risk of a pesticide’s active ingredient. These studies reveal a size-specific effect on the toxicity of the nanoparticles despite normalized concentrations of active ingredient and highlight the potential for nanoparticles to mask the hydrophobic behavior of some chemicals. In Chapters 4 and 5, the focus shifts to soybeans and their microbiome as they respond to soils amended with cerium oxide nanoparticles. Utilizing metagenomic software to analyze large data sets and predict changes in ecological functionality, these companion chapters correlate plant growth with bacterial community structure and emphasize the atypical dose-response relationship of nanoparticles in terrestrial systems. Further, they address importance of acknowledging the difference between pristine and aged nanoparticle exposures. This body of work demonstrates the capacity for nanomaterials to confound toxicological assessment if the disconnect between nanomaterial dosimetry and biological response is not accounted for