1,203 research outputs found

    Multi-Isotope Multi-Pinhole SPECT Bildgebung in kleinen Labortieren: Experimentelle Messungen und Monte Carlo Simulationen

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    Single photon emission computed tomography (SPECT) in small laboratory animals has become an integral part of translational medicine. It enables non-invasive validation of drug targeting, safety and efficacy in living organisms, which is progressively gaining importance in pharmaceutical industry. The increasing demand for efficiency in pharmaceutical research could be addressed by novel multitracer study designs. Multi-isotope multi-pinhole sampling allows validation of multiple tracers in a single experiment and consolidation of consecutive research trials. Due to physical and technical limitations, however, image quality and quantification can be substantially reduced. Advanced corrective procedures are required to establish multi-isotope multi-pinhole SPECT as a reliable and quantitative imaging technique for widespread use. For this purpose, the present work aimed to investigate the technical capabilities and physical limitations of multi-isotope multi-pinhole SPECT imaging in small laboratory animals. Based on experimental measurements and Monte Carlo simulations, specific error sources have been identified and procedures for quantitative image correction have been developed. A Monte Carlo simulation model of a state-of-the art SPECT/CT system has been established to provide a generalized framework for in-silico optimization of imaging hardware, acquisition protocols and reconstruction algorithms. The findings of this work can be used to improve image quality and quantification of SPECT in-vivo data for multi-isotope applications. They guide through the laborious process of multi-isotope protocol optimization and support the 3R welfare initiative that aims to replace, reduce and refine animal experimentation.Die Einzelphotonen-Emissionscomputertomographie (SPECT) in kleinen Labortieren hat sich als wichtiger Bestandteil der translationalen Medizin etabliert. Sie ermöglicht die nicht-invasive Validierung der Zielgenauigkeit, Wirksamkeit und Sicherheit von Wirkstoffen in lebenden Organismen und gewinnt zunehmend an Bedeutung in der pharmazeutischen Industrie. Die Forderung nach mehr Effizienz in der pharmazeutischen Forschung könnte durch neuartige Multitracer-Studien adressiert werden. Die Multi-Isotopen Akquisition mit Multi-Pinhole Kollimatoren ermöglicht die Validierung mehrerer Tracer in einem einzelnen Experiment und die Konsolidierung konsekutiver Bildgebungsstudien. Aufgrund physikalischer und technischer Limitationen ist die Bildqualität und Quantifizierbarkeit bei diesem Verfahren jedoch häufig reduziert. Um die Multi-Isotopen SPECT als zuverlässige und quantitative Bildgebungsmethode für den breiten Einsatz zu etablieren sind komplexe Korrekturverfahren erforderlich. Ziel der vorliegenden Arbeit war daher, die technischen Möglichkeiten und physikalischen Limitationen der Multi-Isotopen SPECT-Bildgebung in kleinen Labortieren systematisch zu untersuchen. Mithilfe von experimentellen Messungen und Monte Carlo Simulationen wurden spezifische Fehlerquellen identifiziert und Verfahren zur quantitativen Bildkorrektur entwickelt. Zudem wurde das Monte-Carlo Modell eines neuartigen SPECT/CT-Systems etabliert, um eine Plattform für die in-silico Optimierung von Bildgebungshardware, Aufnahmeprotokollen und Rekonstruktionsalgorithmen zu schaffen. Die Ergebnisse dieser Arbeit können die Bildqualität und Quantifizierbarkeit von SPECT in-vivo Daten für Multi-Isotopen Anwendungen verbessern. Sie führen beispielhaft durch den Prozess der Multi-Isotopen Protokolloptimierung und unterstützen die 3R-Initiative mit dem Ziel, experimentelle Tierversuche zu vermeiden (Replace), zu vermindern (Reduce) und zu verbessern (Refine)

    Spatial frequency domain imaging towards improved detection of gastrointestinal cancers

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    Early detection and treatment of gastrointestinal cancers has been shown to drastically improve patients survival rates. However, wide population based screening for gastrointestinal cancers is not feasible due to its high cost, risk of potential complications, and time consuming nature. This thesis forms the proposal for the development of a cost-effective, minimally invasive device to return quantitative tissue information for gastrointestinal cancer detection in-vivo using spatial frequency domain imaging (SFDI). SFDI is a non-invasive imaging technique which can return close to real time maps of absorption and reduced scattering coefficients by projecting a 2D sinusoidal pattern onto a sample of interest. First a low-cost, conventional bench top system was constructed to characterise tissue mimicking phantoms. Phantoms were fabricated with specific absorption and reduced scattering coefficients, mimicking the variation in optical properties typically seen in healthy, cancerous, and pre-cancerous oesophageal tissue. The system shows accurate retrieval of absorption and reduced scattering coefficients of 19% and 11% error respectively. However, this bench top system consists of a bulky projector and is therefore not feasible for in-vivo imaging. For SFDI systems to be feasible for in-vivo imaging, they are required to be miniaturised. Many conditions must be considered when doing this such as various illumination conditions, lighting conditions and system geometries. Therefore to aid in the miniaturisation of the bench top system, an SFDI system was simulated in the open-source ray tracing software Blender, where the capability to simulate these conditions is possible. A material of tunable absorption and scattering properties was characterised such that the specific absorption and reduced scattering coefficients of the material were known. The simulated system shows capability in detecting optical properties of typical gastrointestinal conditions in an up-close, planar geometry, as well in a non-planar geometry of a tube simulating a lumen. Optical property imaging in the non-planar, tubular geometry was done with the use of a novel illumination pattern, developed for this work. Finally, using the knowledge gained from the simulation model, the bench top system was miniaturised to a 3 mm diameter prototype. The novel use of a fiber array producing the necessary interfering fringe patterns replaced the bulky projector. The system showed capability to image phantoms simulating typical gastrointestinal conditions at two wavelengths (515 and 660 nm), measuring absorption and reduced scattering coefficients with 15% and 6% accuracy in comparison to the bench top system for the fabricated phantoms. It is proposed that this system may be used for cost-effective, minimally invasive, quantitative imaging of the gastrointestinal tract in-vivo, providing enhanced contrast for difficult to detect cancers

    Light microscopy applied to study defective macrophage phagocytosis in chronic obstructive pulmonary disease

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    Chronic obstructive pulmonary disease (COPD) is a progressive lung condition characterised by airflow limitation, primarily caused by cigarette smoking and other air pollutants, which results in irreversible damage to the lungs. This leads to alveolar destruction, reduced lung elasticity, narrowing of the small airways and fibrosis, and is associated with an increased number of phagocytic immune cells, including macrophages and neutrophils. Despite increased number of phagocytes, approximately half of COPD patients exhibit bacterial colonisation that are associated with increased numbers of exacerbations that often lead to hospitalisation. Bacterial colonisation may be explained, in part, by defective phagocytosis of pathogens by macrophages, although the mechanism involved is unclear. Light microscopy is widely used to study cell biology, although the ability to image cellular processes within cells and microorganisms is conventionally limited by the diffraction of light, as well as additional considerations of signal to noise that impacts the imaging speed and contrast. However, advances in light microscopy, including computational and physical techniques, to improve spatial resolution and image contrast prompted the investigation of their potential use in exploring phagocytosis. This project initially explored the potential of super-resolved microscopy and computational techniques, to allow image acquisition at a resolution of less than 20nm. These techniques can be used to identify and image sub-cellular proteins and cellular processes previously unobtainable, including phagocytosis. This thesis aimed to utilise live, cell imaging and super-resolved microscopy techniques to identify differences in morphology, cytoskeletal proteins, and mitochondria in macrophages. The most practically useful technique was found to be wide-field epifluorescence imaging with computational deconvolution, which was applied to study macrophages from COPD subjects, smokers and non-smoking control subjects with single cell analysis. This work confirmed that COPD macrophages were poorer at phagocytosing bacteria, and this was associated with structural changes in COPD macrophages. Using high-resolution microscopy, COPD macrophages were shown to be larger compared to non-smoker cells, with significantly longer microtubules observed in both lung tissue-derived and blood monocyte-derived macrophages (MDM), together with a reduction in actin-containing podosomes. Mitochondria function can be a key factor that affects cytoskeletal restructuring in cells, with the mitochondria membrane potential being important in the function of ATP generation, with mitochondria under normal conditions displaying a polarised phenotype, whereby they are highly negatively charged, due to the influx of electrons required for the electron transport chain. However, a significantly depolarised mitochondria is the result of a more positively charged membrane potential, which can result in impaired ATP production and cause cell death, which is associated with several pathologies. Work performed in this thesis using microscopy explored mitochondrial differences in COPD MDM, with a significantly depolarised mitochondrial membrane potential (ΔΨm) compared to non-smoker controls, but no difference in mitochondria reactive oxygen species (mROS). Mitochondrial antioxidants, SkQ1 and PQQ, were able to improve bacterial phagocytosis in COPD MDM by up to 60% but had no effect on ΔΨm or mROS. Work in this thesis has shown the use of microscopy and single cell image analysis can detect differences in key structural proteins and the mitochondria, whilst capable of being used as a screening tool for phagocytic correctors such as mitochondrial antioxidants, which were shown to increase phagocytosis with no alterations in mitochondrial function.Open Acces

    The study of renal function and toxicity using zebrafish (Danio rerio) larvae as a vertebrate model

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    Zebrafish (Danio rerio) is a powerful model in biomedical and pharmaceutical sciences. The zebrafish model was introduced to toxicological sciences in 1960, followed by its use in biomedical sciences to investigate vertebrate gene functions. As a consequence of many research projects in this field, the study of human genetic diseases became instantly feasible. Consequently, zebrafish have been intensively used in developmental biology and associated disciplines. Due to the simple administration of medicines and the high number of offspring, zebrafish larvae became widely more popular in pharmacological studies in the following years. In the past decade, zebrafish larvae were further established as a vertebrate model in the field of pharmacokinetics and nanomedicines. In this PhD thesis, zebrafish larvae were investigated as an earlystage in vivo vertebrate model to study renal function, toxicity, and were applied in drug-targeting projects using nanomedicines. The first part focused on the characterization of the renal function of three-to four-dayold zebrafish larvae. Non-renal elimination processes were additionally described. Moreover, injection techniques, imaging parameters, and post-image processing scripts were established to serve as a toolbox for follow-up projects. The second part analyzed the impact of gentamicin (a nephrotoxin) on the morphology of the pronephros of zebrafish larvae. Imaging methodologies such as fluorescent-based laser scanning microscopy and X-ray-based microtomography were applied. A profound comparison study of specimens acquired with different laboratory X-ray-based microtomography devices and a radiation facility was done to promote the use of X-ray-based microtomography for broader biomedical applications. In the third part, the toxicity of nephrotoxins on mitochondria in renal epithelial cells of proximal tubules was assessed using the zebrafish larva model. Findings were compared with other teleost models such as isolated renal tubules of killifish (Fundulus heteroclitus). In view of the usefulness and high predictability of the zebrafish model, it was applied to study the pharmacokinetics of novel nanoparticles in the fourth part. Various in vivo pharmacokinetic parameters such as drug release, transfection of mRNA/pDNA plasmids, macrophage clearance, and the characterization of novel drug carriers that were manipulated with ultrasound were assessed in multiple collaborative projects. Altogether, the presented zebrafish model showed to be a reliable in vivo vertebrate model to assess renal function, toxicity, and pharmacokinetics of nanoparticles. The application of the presented model will hopefully encourage others to reduce animal experiments in preliminary studies by fostering the use of zebrafish larvae

    Hemodynamic Quantifications By Contrast-Enhanced Ultrasound:From In-Vitro Modelling To Clinical Validation

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    Development and Application of Suspect and Nontarget Screening to Characterize Organic Micropollutants in Aquatic Environments of New York State

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    Organic micropollutants (OMPs) have presented a global challenge to water resources management due to concerns over their adverse impacts on aquatic biota and human health at low exposure concentrations (e.g., at ng/L to ÎĽg/L levels in aquatic systems). OMPs encompass an extensive array of synthetic organic compounds (e.g., pharmaceuticals, pesticides, personal care products, household chemicals, industrial additives) and their transformation products. My research has been centered around establishing analytical methods based on liquid chromatography-high-resolution mass spectrometry (LC-HRMS), with a focus on the development and application of suspect and nontarget screening workflows for the identification and prioritization of OMPs in inland lakes, streams, and urban wastewater in New York State. In Chapter 1, I collaborated with volunteers from the Citizens Statewide Lake Assessment Program and scientists at the Upstate Freshwater Institute to conduct the first statewide investigation of OMP occurrence in New York inland lakes. Through this project, I developed a suspect screening method based on LC-HRMS to identify and quantify 65 OMPs in 314 lake water samples collected by volunteers from 111 lakes, ponds, and reservoirs across the state. I also performed partial least squares regression and multiple linear regression analyses to prioritize total dissolved nitrogen, specific conductance, and a wastewater-derived fluorescent organic matter component as the best combination of explanatory predictors for the inter-lake variability in OMP occurrence patterns. I further applied the exposure-activity ratio approach to estimate the potential for biological effects associated with OMPs. My work demonstrated that engaging an established network of citizen volunteers offers a viable approach to increasing the spatiotemporal coverage of OMP monitoring while raising public awareness of their prevalence. In Chapter 2, I collaborated with Drs. Christa Kelleher and Rebecca Schewe to investigate the occurrence patterns of OMPs in streams draining mixed-use watersheds in central New York. I combined the use of polar organic chemical integrative samplers (POCIS) with suspect screening and nontarget screening based on LC-HRMS to identify and quantify 133 OMPs in samples collected from 20 stream sites over two sampling seasons. I also performed hierarchical clustering to establish the co-occurrence profiles of OMPs in connection with watershed attributes indicative of anthropogenic influences. I further evaluated the feasibility of deploying POCIS for estimating daily average loads of OMPs and their potential for biological effects in streams via screening-level risk assessments. My work supported the prospect of combining passive sampling with high-resolution accurate mass screening for the multi-watershed characterization of OMP contamination status in streams. In Chapter 3, I collaborated with colleagues from the School of Public Health to pursue one of the earliest wastewater-based epidemiology studies on population-level substance use during the COVID-19 pandemic. I developed and validated an online solid-phase extraction method for sample preconcentration before LC-HRMS analyses to achieve rapid screening of health and lifestyle-related substances in urban wastewater. I applied this method to quantify the levels of 26 pharmaceuticals and lifestyle chemicals in wastewater influent samples collected from six sewersheds in central New York over a period spanning the rising and falling of COVID-19 prevalence. I back-calculated the population-level consumption rates of antidepressants, antiepileptics, antihistamines, antihypertensives, and central nervous system stimulants and further identified their co-variation with disparities in household income, marital status, and/or age of the contributing populations as well as the detection frequency of SARS-CoV-2 RNA in wastewater and the COVID-19 test positivity within the sewersheds. My work highlighted the utility of high-throughput wastewater analysis for assessing substance use patterns during a public health crisis such as COVID-19

    Surface-Mounted Metal-Organic Frameworks as the Platform for Surface Science: Photoreactivity, Electroreactivity, and Thermal Reactivity

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    Bisher haben Forscher Modellsysteme wie Einkristallmetalle oder Metalloxide entwickelt, um reale Pulversysteme besser zu verstehen. Es bestehen jedoch immer noch Fragen hinsichtlich der Oberflächenstruktur und Reaktivität von MOFs (Metall-organische Gerüstverbindungen). Glücklicherweise bieten oberflächenorientierte SURMOFs (surface-oriented SURMOFs) einen alternativen Ansatz für den Aufbau von Modellplattformen zur Untersuchung dieser grundlegenden Aspekte von MOFs. Diese Arbeit konzentriert sich auf die organische Photochemie, Elektrokatalyse und thermische Pyrolyse von MOFs aus einer physikalisch-chemischen Perspektive unter Verwendung von Oberflächenwissenschaftstechniken und SURMOF-Plattformen. Das Ziel dieser Arbeit besteht nicht nur darin, das Wissen über MOFs und SURMOFs zu erweitern, sondern auch die Leistungsfähigkeit von Oberflächenwissenschaftstechniken und -methoden im Bereich chemischer Reaktionen zu demonstrieren. Zu diesem Zweck verwendet die Arbeit eine hochmoderne UHV-IRRAS-Apparatur (Ultra-High-Vacuum Infrared Reflection Absorption Spectroscopy). Ein auf der Oberfläche montiertes MOF (SURMOF) Modellsystem mit Azid-Seitenketten wurde erfolgreich hergestellt und genau überwacht, um chemische Veränderungen während des Betriebs zu erfassen. Die umfassenden Ergebnisse, die durch die Kombination von IRRAS mit in situ XRD, MS und XPS erzielt wurden, zeigen, dass die Photoreaktion von Azid durch die Bildung von hochaktiven Nitren-Gruppen initiiert wird, die anschließend mit benachbarten C=C-Bindungen des Gerüsts reagieren und Pyrrol-Derivate durch intramolekulare Aminierung erzeugen. Ein hochwertiges ZIF-67-SURMOF wurde in einem Flüssigphasen-Schicht-für-Schicht-Verfahren hergestellt und erstmals in der Sauerstoffentwicklungskatalyse (OER) eingesetzt. Die katalytisch aktiven Spezies, CoOOH, in den SURMOF-Derivaten wurden identifiziert, was Einblicke in die Mechanismen der strukturellen Transformation und die Struktur-Leistung-Beziehungen bietet. Durch Zugabe von Ni und B wurde die Überspannung auf 375 mV bei 10 mA/cm2 reduziert. Zusätzlich wurden in situ IRRAS und XPS verwendet, um die strukturellen Übergänge von ZIF-67 zu kohlenstoffhaltigen Materialien mit Stickstoffelementen zu enthüllen. NEXAFS-Daten zeigen eine abschließende graphitische Struktur der kohlenstoffhaltigen Materialien nach Pyrolyse bei 900 K. Hoffentlich kann diese Arbeit das grundlegende Verständnis und die Anwendungsfelder von auf MOF und SURMOF basierenden Materialien erweitern

    On Centrality and Population Size Effects in Urban Pollution: A Meta-Analysis of NO2 and Heat Islands and Spatial Analysis of NO2

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    Theoretical and experimental investigation of the plasmonic probe

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    The diffraction limit does not permit us to reveal information from dimensions smaller than roughly one-half of the wavelength. Hence, it was traditionally impossible to optically interact selectively with nanoscale features. However, with the increasing trend towards nanoscience and nanotechnology, nano-optics science emerged. A central goal of nano-optics is to extend optical techniques to length scales beyond the diffraction limit. In recent years, several new approaches have been developed to overcome this limitation. Tip-enhanced near-field microscopy techniques, such as TERS and PiFM, are among the innovations built around an AFM system. In this work, first, an algorithm for batch processing of the measured AFM data is introduced and utilized to analyze the height distribution of the inactivated SARS-CoV-2 samples. In the next chapter, plasmonic probes, as the crucial components of any near-field optical microscopy techniques, are modeled and investigated. Here, complex and realistic particle shapes are used to analyze particle-based probes’ near- and far-field behavior. It has been shown that only the frontmost particle is decisive for the near-field signal. On the other hand, the rest of nanoparticles enhance the scattering intensity. Apart from the optical responses, the mechanical properties of tips are also modeled because higher harmonics of the tip’s oscillations are the foundation of new patents such as torsional force microscopy and photo-induced force microscopy (PiFM). In the last chapter, the PiFM is introduced and employed in various applications, i.e., plasmonic probe’s quality, field mapping, and optical response of the plasmonic NPs
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