123 research outputs found

    Zebrafish Models for Development and Disease 2.0

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    The special issue (Zebrafish Models for Development and Disease 2.0) is a collection of articles highlighting research using the zebrafish (Danio rerio) experimental organism. Research described in this special issue addresses various developmental biology, genetic, biomedical and neuroscience topics that should be of general interest to the biomedical research community

    Microfuidic Devices and Open Access Tool for Localized Microinjection and Heart Monitoring of Drosophila Melanogaster

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    This thesis aims to address the current research gaps associated with the use of Drosophila larvae as an in-vivo model for cardiac toxicity and cardiac gene screening. In objective 1, we have developed a hybrid multi-tasking microfluidic platform that enables desired orientation, reversible immobilization, and localized microinjection of intact Drosophila larvae for recording heart activities upon injection of controlled dosages of different chemicals. In objective 2. we have developed software for in-vivo quantification of essential heartbeat parameters on intact Drosophila larvae. Several image segmentation and signal processing algorithms were developed to detect the heart, extract the heartbeat signal, and quantify heart rate and arrhythmicity index automatically, while other heartbeat parameters were quantified semi-automatically using the M-mode. In objective 3a, we demonstrated the application of our microfluidic device and heartbeat quantification software for investigating the effect of different chemicals (e.g., serotonin and heavy metals) on Drosophila larval heart function. Also, we applied our technology to genetically modified Drosophila larvae to investigate the effect of metal responsive transcription factor (MTF-1) against heavy metals cardiac toxicity (objective 3b)

    Microfuidic Devices and Open Access Tool for Localized Microinjection and Heart Monitoring of Drosophila Melanogaster

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    This thesis aims to address the current research gaps associated with the use of Drosophila larvae as an in-vivo model for cardiac toxicity and cardiac gene screening. In objective 1, we have developed a hybrid multi-tasking microfluidic platform that enables desired orientation, reversible immobilization, and localized microinjection of intact Drosophila larvae for recording heart activities upon injection of controlled dosages of different chemicals. In objective 2. we have developed software for in-vivo quantification of essential heartbeat parameters on intact Drosophila larvae. Several image segmentation and signal processing algorithms were developed to detect the heart, extract the heartbeat signal, and quantify heart rate and arrhythmicity index automatically, while other heartbeat parameters were quantified semi-automatically using the M-mode. In objective 3a, we demonstrated the application of our microfluidic device and heartbeat quantification software for investigating the effect of different chemicals (e.g., serotonin and heavy metals) on Drosophila larval heart function. Also, we applied our technology to genetically modified Drosophila larvae to investigate the effect of metal responsive transcription factor (MTF-1) against heavy metals cardiac toxicity (objective 3b)

    Effects of petroleum-based and biopolymer-based nanoplastics on aquatic organisms: A case study with mechanically degraded pristine polymers

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    Mismanaged plastic litter submitted to environmental conditions may breakdown into smaller fragments, eventually reaching nano-scale particles (nanoplastics, NPLs). In this study, pristine beads of four different types of polymers, three oil-based (polypropylene, PP; polystyrene, PS; and low-density polyethylene, LDPE) and one bio-based (polylactic acid, PLA) were mechanically broken down to obtain more environmentally realistic NPLs and its toxicity to two freshwater secondary consumers was assessed. Thus, effects on the cnidarian Hydra viridissima (mortality, morphology, regeneration ability, and feeding behavior) and the fish Danio rerio (mortality, morphological alterations, and swimming behavior) were tested at NPLs concentrations in the 0.001 to 100 mg/L range. Mortality and several morphological alterations were observed on hydras exposed to 10 and 100 mg/L PP and 100 mg/L LDPE, whilst regeneration capacity was overall accelerated. The locomotory activity of D. rerio larvae was affected by NPLs (decreased swimming time, distance or turning frequency) at environmentally realistic concentrations (as low as 0.001 mg/L). Overall, petroleum- and bio-based NPLs elicited pernicious effects on tested model organisms, especially PP, LDPE and PLA. Data allowed the estimation of NPLs effective concentrations and showed that biopolymers may also induce relevant toxic effectsThe authors acknowledge the financial support provided by the Spanish Government (Ministerio de Ciencia e Innovación): PID2020-113769RBC21/22, PLEC2021-007693 and the Thematic Network of Micro- and Nanoplastics in the Environment (RED2018-102345-T, EnviroPlaNet Network) and by the Portuguese Government: Project NanoPlanet (2022.02340.PTDC) and financial support to CESAM (UIDP/ 50017/ 2020+UIDB/50017/2020+LA/P/0094/2020), through FCT/MCTES. MTB is the recipient of a FPU (FPU17/01789) pre-doctoral contract by the Spanish Ministerio de Ciencia, Innovación y Universidade

    3D orbital tracking microscopy: from cells to organisms

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    Development and characterisation of a zebrafish larval model to investigate mechanisms for pathophysiology of intracranial hypertension in cryptococcal meningitis

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    Cryptococcosis is a fungal infection caused by members of the genus Cryptococcus. Worldwide, the most prevalent pathogen of this genus is the encapsulated saprophyte species Cryptococcus neoformans. Cryptococcosis most commonly occurs as an opportunistic airborne lung infection which can disseminate to most organ systems. The central nervous system appears particularly susceptible to developing a pathology from the infection, with more than half of cryptococcosis patients diagnosed with cryptococcal meningitis despite infection in multiple organs. Cryptococcal meningitis (CM) is a meningoencephalitis (infection of the brain parenchyma and meninges) which globally accounts for 19% (13-24) of AIDS-related mortality (Rajansingham et al., 2022). In 2020, reports show annual incidence of 152 000 cases of cryptococcal meningitis, resulting in 112 000 cryptococcal-related deaths, almost half of which are in eastern and southern Africa (Rajansingham et al., 2022). 50-70% of CM cases present with a pathologically elevated intracranial pressure (intracranial hypertension) (Graybill et al., 2000; Jarvis et al., 2014; Kagimu et al., 2022;). This thesis aims to improve our understanding of intracranial hypertension in CM to help identify potential targets for treatment, by developing and testing new models of intracranial hypertension in CM. Three different approaches were used – theoretical, in vitro rheology and in vivo in zebrafish. Zebrafish was chosen as the core experimental system in which to develop new models due to its physiology, tractability for live imaging and susceptibility to cryptococcosis. In zebrafish larvae, the dynamic nature of cranial vasculature compartments and the CSF during infection was examined using wide field and light sheet microscopy techniques. The physical properties of tissues and fluids when interacting with cryptococcal yeast cells was modelled with theoretical and in vitro rheological measurements. In vitro it was found that viscosity of fluids may increase in the presence of heat killed C. neoformans, but whether this change is pathologically significant requires further investigation. Using a model of cryptococcal infection in zebrafish larvae, a “pulsation” phenomenon was identified, consisting of vasodilation and constriction in the cranial vasculature with an impact on vessel wall permeability. The findings in this work, are reflective of the CM pathology as seen in human patients and suggest impaired CSF and blood flow homeostasis may contribute to intracranial hypertension in CM

    Fast Objective Coupled Planar Illumination Microscopy

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    Among optical imaging techniques light sheet fluorescence microscopy stands out as one of the most attractive for capturing high-speed biological dynamics unfolding in three dimensions. The technique is potentially millions of times faster than point-scanning techniques such as two-photon microscopy. This potential is especially poignant for neuroscience applications due to the fact that interactions between neurons transpire over mere milliseconds within tissue volumes spanning hundreds of cubic microns. However current-generation light sheet microscopes are limited by volume scanning rate and/or camera frame rate. We begin by reviewing the optical principles underlying light sheet fluorescence microscopy and the origin of these rate bottlenecks. We present an analysis leading us to the conclusion that Objective Coupled Planar Illumination (OCPI) microscopy is a particularly promising technique for recording the activity of large populations of neurons at high sampling rate. We then present speed-optimized OCPI microscopy, the first fast light sheet technique to avoid compromising image quality or photon efficiency. We enact two strategies to develop the fast OCPI microscope. First, we devise a set of optimizations that increase the rate of the volume scanning system to 40 Hz for volumes up to 700 microns thick. Second, we introduce Multi-Camera Image Sharing (MCIS), a technique to scale imaging rate by incorporating additional cameras. MCIS can be applied not only to OCPI but to any widefield imaging technique, circumventing the limitations imposed by the camera. Detailed design drawings are included to aid in dissemination to other research groups. We also demonstrate fast calcium imaging of the larval zebrafish brain and find a heartbeat-induced motion artifact. We recommend a new preprocessing step to remove the artifact through filtering. This step requires a minimal sampling rate of 15 Hz, and we expect it to become a standard procedure in zebrafish imaging pipelines. In the last chapter we describe essential computational considerations for controlling a fast OCPI microscope and processing the data that it generates. We introduce a new image processing pipeline developed to maximize computational efficiency when analyzing these multi-terabyte datasets, including a novel calcium imaging deconvolution algorithm. Finally we provide a demonstration of how combined innovations in microscope hardware and software enable inference of predictive relationships between neurons, a promising complement to more conventional correlation-based analyses

    3D orbital tracking microscopy: from cells to organisms

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    Surface functionalized iron oxide nanoparticles for applications in biomedical sciences

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    This thesis focuses on the functionalization of iron oxide nanoparticles (Fe3O4) and their applications in biomedical sciences. Each chapter represents an independent research project that has been conducted within a different collaboration. For each project, magnetic Fe3O4 iron oxide nanoparticles have been functionalized or modified to suit its requirements. This thesis aims to show how versatile and most promising Fe3O4 iron oxide nanoparticles are, and how their unique properties in size, shape, and magnetism can be utilized for a broad range of applications in biomedicines. Chapter 2 focuses on magnetic resonance (MR), computed tomography (CT), and intravascular ultrasound (IVUS) as essential diagnostic imaging techniques and how iron oxide nanoparticles can potentially be used as contrast agents across all three imaging modalities. Contrast agents are commonly used to enhance the imaging quality and thus provide more detail for assessment. However, previous studies using nanoparticles for MR and CT were prepared with surface coating stabilizers, which in turn can compromise the use in clinical studies. In this chapter, gold-iron oxide nanoparticles (Au*MNP) are presented as a multi-modal contrast agent. Using a chemically grafting method without stabilizers, presenting nanoparticles with pristine surfaces that allow for further functionalization in molecularly targeted theragnostic applications. In Chapter 3, the response of HepaRG liver cells to nanoparticles is examined in two methods, 2D and 3D cell culturing. By analyzing the cell response in 2D and 3D cultures an accurate estimation of the toxicity of nanoparticles can be made. The toxicity of iron oxide was assessed using commercially available cell assays (CellTiter-Glo and PrestoBlue), however, the experiments suggested some restrictions that could alter the data and therefore resemble inaccurate results. For this purpose, non-invasive imaging techniques based on impedance (xCELLigence system) and Coherent Anti-Stokes Raman Scattering (CARS) were used to analyze the cell toxicity. The results showed that those methods provided a much deeper insight into the cell viability and proliferation of HepaRG cells. Furthermore, valuable data on the immediate effects in real-time and long-term exposures can be captured of the same culture. Chapter 4 presents the cell internalization process of iron oxide nanoparticles, captured using the unique holographic cell imaging technique of a HoloMonitor M4 microscope. In most cases where the cell internalization process is monitored, only one or two cells can be visualized and tracked at the same time. This is not the case with this technique, where hundreds of cells can be simultaneously visualized, analyzed, and monitored over time. Unlike single-cell observation, the system takes pictures of the cell culture at a high capture rate, allowing to observe and interpret the cell dynamics, cell morphologies, and cell reactions to nanoparticles (e.g., toxicity and apoptosis). Measuring the kurtosis and skewness of MCF-7 cancer cells for 72 hours after nanoparticle exposure, showed that cell splitting and proliferation took place, and no extraordinary damages or cell death was caused by the internalization of the particles. Furthermore, every step of the internalization process was monitored and captured in visible data for the first time. Chapter 5 demonstrates magnetic molecularly imprinted polymer networks and spheres (MMIPs) for the selective binding of antibiotics. MIPs are polymers that can be synthesized with highly selective and reusable binding sites. Their combination with magnetic iron oxide can be used as a useful tool to monitor and remove antibiotic pollutants from freshwater sources and food products. MMIPs are prepared using a microemulsion technique containing vinyl-functionalized iron oxide to selectively bind the model antibiotics erythromycin (ERY) and ciprofloxacin (CPX). The results show that MMIPs prepared using this technique are highly selective towards their respective template molecule in methanol/water and milk matrix, are recyclable, and most importantly open to modification. In Chapter 6, zebrafish larvae are exposed to polyethylene glycol coated iron oxide supported gold nanoparticles for further toxicity assessment. In general, toxicological data is gathered in vivo, however, the translational values from in vitro to in vivo are sometimes questionable due to the complexity of the organism. Zebrafish larvae are used as an intermediate method for in vivo experiments, as they can be used 96 hours after hatching, unlike larger animals such as mice, rats, or rabbits which take a much longer time to reach adulthood, sometimes up to 3 months. Also, a single female zebrafish can spawn about 200-300 eggs per week, which can generate an extensive data set from a small-scale experimental setup. The results presented in this chapter showed a 100% survival rate of all exposed zebrafish larvae between a range of concentrations from 0 - 2mM (0 - 463.1 mg/L). Chapter 7 summarizes the key findings and developments presented in this thesis, suggestions for future work within each research project, and proposes future applications. Chapter 8 includes a list of journal publications produced from this thesis.financial support provided from Heriot-Watt University (FOS
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