The complexity of Drosophila innate immunity

Metazoans rely on efficient mechanisms to oppose infections caused by pathogens. The immediate and first-line defense mechanism(s) in metazoans, referred to as the innate immune system, is initiated upon recognition of microbial intruders by germline encoded receptors and is executed by a set of rapid effector mechanisms. Adaptive immunity is restricted to vertebrate species and it is controlled and assisted by the innate immune system. Interestingly, most of the basic signaling cascades that regulate the primeval innate defense mechanism(s) have been well conserved during evolution, for instance between humans and the fruit fly, Drosophila melanogaster. Being devoid of adaptive signaling and effector systems, Drosophila has become an established model system for studying pristine innate immune cascades and reactions. In general, an immune response is evoked when microorganisms pass the fruit fly’s physical barriers (e.g. cuticle, epithelial lining of gut and trachea), and it is mainly executed in the hemolymph, the equivalent of the mammalian blood. Innate immunity in the fruit fly consists of a phenoloxidase (PO) response, a cellular response (hemocytes), an antiviral response, and the NF-κB dependent production of antimicrobial peptides referred to as the humoral response. The JAK/STAT and Jun kinase signaling cascades are also implicated in the defence against pathogens.

The ecology and evolution of invasive Potamopyrgus antipodarum in Australia

The spread of invasive organisms is one of the greatest threats to ecosystems and biodiversity worldwide. Understanding the evolutionary and ecological factors responsible for the transport, introduction, establishment and spread of invasive species will assist the development of control strategies. The New Zealand mudsnail, Potamopyrgus antipodarum (Gray 1843) (Gastropoda: Hydrobiidae), is a global freshwater invader, with populations established in Europe, Asia, the Americas and Australia. While sexual and asexual P. antipodarum coexist in the native range, invasive populations reproduce by parthenogenesis, producing dense populations that compete for resources with native species. Potamopyrgus antipodarum is a natural model system for the study of evolutionary and ecological processes underlying invasion. This thesis assesses the invasion history, genetic diversity and ecology of P. antipodarum in Australia, with particular focus on: a) potential source populations, b) distribution and structure of populations, and c) species traits related to the establishment, persistence and spread of invasive P. antipodarum. Genetic analyses were carried out on specimens collected for this study from New Zealand and Australia, along with existing museum samples. In combination with published data, the analyses revealed low genetic diversity among and within invasive populations in south-eastern Australia, relative to New Zealand populations. Phylogenetic relationships inferred from mitochondrial sequences indicated that the Australian populations belong to clades dominated by parthenogenetic haplotypes that are known to be present in Europe and the US. These ‘invasive clades’ are likely to originate from the North Island of New Zealand, and suggest a role for selection in determining genetic composition of invasive populations. The genotypic diversity of Australian P. antipodarum was low, with few, closely related clones distributed across south-eastern Australia. The pattern of clone distribution was not consistent with any assessed geographical or abiotic factors; instead a few, widely-distributed clones were present in high frequencies at most sites. Differences in clone frequencies were found, which may indicate differential success of clonal lineages. A range of traits have been proposed as facilitators of invasion success, and within-species variation in these traits can promote differential success of genotypes. Using laboratory-based experiments, the performance of the three most common Australian clones was tested across a suite of invasion-relevant traits. Ecologically-relevant variation in traits was found among the clones. These differences may have determined the spatial distribution of clones, and may continue to do so into the future. This thesis found that the P. antipodarum invasion of Australia is the result of few introductions of a small number of globally-invasive genotypes that vary in ecologically-relevant traits. From a source of considerable genetic diversity in the native range, very few genotypes have become invasive. Those that are invasive appear to be very successful at continental scales. These findings highlight a capacity in asexual invaders to successfully invade, and potentially adapt to, a broad range of ecosystems. The P. antipodarum invasion system is amenable to research using combinations of field-based studies, molecular and laboratory approaches, and is likely to yield significant, broadly-applicable insights into invasion.

Scanning Laser Optical Tomography Resolves Structural Plasticity during Regeneration in an Insect Brain

Background: Optical Projection Tomography (OPT) is a microscopic technique that generates three dimensional images from whole mount samples the size of which exceeds the maximum focal depth of confocal laser scanning microscopes. As an advancement of conventional emission-OPT, Scanning Laser Optical Tomography (SLOTy) allows simultaneous detection of fluorescence and absorbance with high sensitivity. In the present study, we employ SLOTy in a paradigm of brain plasticity in an insect model system. Methodology: We visualize and quantify volumetric changes in sensory information procession centers in the adult locust, Locusta migratoria. Olfactory receptor neurons, which project from the antenna into the brain, are axotomized by crushing the antennal nerve or ablating the entire antenna. We follow the resulting degeneration and regeneration in the olfactory centers (antennal lobes and mushroom bodies) by measuring their size in reconstructed SLOTy images with respect to the untreated control side. Within three weeks post treatment antennal lobes with ablated antennae lose as much as 60% of their initial volume. In contrast, antennal lobes with crushed antennal nerves initially shrink as well, but regain size back to normal within three weeks. The combined application of transmission-and fluorescence projections of Neurobiotin labeled axotomized fibers confirms that recovery of normal size is restored by regenerated afferents. Remarkably, SLOTy images reveal that degeneration of olfactory receptor axons has a trans-synaptic effect on second order brain centers and leads to size reduction of the mushroom body calyx. Conclusions: This study demonstrates that SLOTy is a suitable method for rapid screening of volumetric plasticity in insect brains and suggests its application also to vertebrate preparations.

Telomeres in aging and disease: lessons from zebrafish

Age is the highest risk factor for some of the most prevalent human diseases, including cancer. Telomere shortening is thought to play a central role in the aging process in humans. The link between telomeres and aging is highlighted by the fact that genetic diseases causing telomerase deficiency are associated with premature aging and increased risk of cancer. For the last two decades, this link has been mostly investigated using mice that have long telomeres. However, zebrafish has recently emerged as a powerful and complementary model system to study telomere biology. Zebrafish possess human-like short telomeres that progressively decline with age, reaching lengths in old age that are observed when telomerase is mutated. The extensive characterization of its well-conserved molecular and cellular physiology makes this vertebrate an excellent model to unravel the underlying relationship between telomere shortening, tissue regeneration, aging and disease. In this Review, we explore the advantages of using zebrafish in telomere research and discuss the primary discoveries made in this model that have contributed to expanding our knowledge of how telomere attrition contributes to cellular senescence, organ dysfunction and disease.

Understanding immobilised enzymes by NMR spectroscopy

Enzyme immobilisation is the conversion of a soluble enzyme molecule into a solid particle form. This allows the recovery of the enzyme catalyst for its re-use and avoids protein contamination of the product streams. A better understanding of immobilised enzymes is necessary for their rational development. A more rational design can help enormously in the applicability of these systems in different areas, from biosensors to chemical industry. Immobilised enzymes are challenging systems to study and very little information is given by conventional biochemical analysis such as catalytic activity and amount of protein. Here, solid-state NMR has been applied as the main technique to study these systems and evaluate them more precisely. Various approaches are presented for a better understanding of immobilised enzymes, which is the aim of this thesis. Firstly, the requirements of a model system of study will be discussed. The selected systems will be comprehensibly characterised by a variety of techniques but mainly by solid-state NMR. The chosen system will essentially be the enzyme α-chymotrypsin covalently immobilised on two functionalised inorganic supports – epoxide silica and epoxide alumina – and an organic support – Eupergit®. The study of interactions of immobilised enzymes with other species is vital for understanding the macromolecular function and for predicting and engineering protein behaviour. The study of water, ions and inhibitors interacting with various immobilised enzyme systems is covered here. The interactions of water and sodium ions were studied by 17O and 23Na multiple-quantum techniques, respectively. Various pore sizes of the supports were studied for the immobilised enzyme in the presence of labelled water and sodium cations. Finally, interactions between two fluorinated inhibitors and the active site of the enzyme will be explored using 19F NMR, offering a unique approach to evaluate catalytic behaviour. These interactions will be explored by solution-state NMR firstly, then by solid-state NMR. NMR has the potential to give information about the state of the protein in the solid support, but the precise molecular interpretation is a difficult task.

The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.

Ecology, physiology and performance in high-rate anaerobic digestion

The design demands on water and sanitation engineers are rapidly changing. The global population is set to rise from 7 billion to 10 billion by 2083. Urbanisation in developing regions is increasing at such a rate that a predicted 56% of the global population will live in an urban setting by 2025. Compounding these problems, the global water and energy crises are impacting the Global North and South alike. High-rate anaerobic digestion offers a low-cost, low-energy treatment alternative to the energy intensive aerobic technologies used today. Widespread implementation however is hindered by the lack of capacity to engineer high-rate anaerobic digestion for the treatment of complex wastes such as sewage. This thesis utilises the Expanded Granular Sludge Bed bioreactor (EGSB) as a model system in which to study the ecology, physiology and performance of high-rate anaerobic digestion of complex wastes. The impacts of a range of engineered parameters including reactor geometry, wastewater type, operating temperature and organic loading rate are systematically investigated using lab-scale EGSB bioreactors. Next generation sequencing of 16S amplicons is utilised as a means of monitoring microbial ecology. Microbial community physiology is monitored by means of specific methanogenic activity testing and a range of physical and chemical methods are applied to assess reactor performance. Finally, the limit state approach is trialled as a method for testing the EGSB and is proposed as a standard method for biotechnology testing enabling improved process control at full-scale. The arising data is assessed both qualitatively and quantitatively. Lab-scale reactor design is demonstrated to significantly influence the spatial distribution of the underlying ecology and community physiology in lab-scale reactors, a vital finding for both researchers and full-scale plant operators responsible for monitoring EGSB reactors. Recurrent trends in the data indicate that hydrogenotrophic methanogenesis dominates in high-rate anaerobic digestion at both full- and lab-scale when subject to engineered or operational stresses including low-temperature and variable feeding regimes. This is of relevance for those seeking to define new directions in fundamental understanding of syntrophic and competitive relations in methanogenic communities and also to design engineers in determining operating parameters for full-scale digesters. The adoption of the limit state approach enabled identification of biological indicators providing early warning of failure under high-solids loading, a vital insight for those currently working empirically towards the development of new biotechnologies at lab-scale.

Cell-cell communication in three dimensional microenvironments

Cellular behavior is dependent on a variety of extracellular cues required for normal tissue function, wound healing, and activation of the immune system. Removed from their in vivo microenvironment and cultured in vitro, cells lose many environmental cues and that may result in abberant behavior, making it difficult to study cellular processes. In order to mimic native tissue environments, optical tweezer and microfluidic technologies were used to place cells within defined areas of the culture environment. To provide three dimensional supports found in natural tissues, hydrogel scaffolds of poly (ethylene glycol) diacrylate and the basement membrane matrix Matrigel were used. Optical tweezer technology allowed precision placement and formation of homotypic and heterotypic arrays of human U937, HEK 293, and porcine mesenchymal stem cells. Alternatively, two microfluidic devices were designed to pattern Matrigel scaffolds. The first microfluidic device utilized laminar flow to spatially pattern multiple cell types within the device. Gradients of soluble molecules were then be formed and manipulated across the Matrigel scaffolds. Patterning Matrigel using laminar flow techniques require microfluidic expertise and do not produce consistent patterning conditions, limiting their use difficult in most cell culture laboratories. Thus, a buried Matrigel polydimethylsiloxane (PDMS) device was developed for spatial patterning of biological scaffolds. Matrigel is injected into micron sized channels of PDMS fabricated by soft lithography and allowed to thermally cure. Following curing, a second PDMS device was placed on top of the buried Matrigel channels to support media flow. In order to validate these systems, a cell-cell communication model system was developed utilizing LPS and TNFα signaling with fluorescent reporter systems to monitor communication in real time. We demonstrated the utility of microfluidic devices to support the cell-cell communication model system by co culturing three cell types within Matrigel scaffolds and monitoring signaling activity via fluorescent reporters.

Decision tools for bacterial blight resistance gene deployment in rice-based agricultural ecosystems

Attempting to achieve long-lasting and stable resistance using uniformly deployed rice varieties is not a sustainable approach. The real situation appears to be much more complex and dynamic, one in which pathogens quickly adapt to resistant varieties. To prevent disease epidemics, deployment should be customized and this decision will require interdisciplinary actions. This perspective article aims to highlight the current progress on disease resistance deployment to control bacterial blight in rice. Although the model system rice-Xanthomonas oryzae pv. oryzae has distinctive features that underpin the need for a case-by-case analysis, strategies to integrate those elements into a unique decision tool could be easily extended to other crops.

Differences in grain growth of calcite: a field-based modeling approach

Normal grain growth of calcite was investigated by combining grain size analysis of calcite across the contact aureole of the Adamello pluton, and grain growth modeling based on a thermal model of the surroundings of the pluton. In an unbiased model system, i.e., location dependent variations in temperature-time path, 2/3 and 1/3 of grain growth occurs during pro- and retrograde metamorphism at all locations, respectively. In contrast to this idealized situation, in the field example three groups can be distinguished, which are characterized by variations in their grain size versus temperature relationships: Group I occurs at low temperatures and the grain size remains constant because nano-scale second phase particles of organic origin inhibit grain growth in the calcite aggregates under these conditions. In the presence of an aqueous fluid, these second phases decay at a temperature of about 350 °C enabling the onset of grain growth in calcite. In the following growth period, fluid-enhanced group II and slower group III growth occurs. For group II a continuous and intense grain size increase with T is typical while the grain growth decreases with T for group III. None of the observed trends correlate with experimentally based grain growth kinetics, probably due to differences between nature and experiment which have not yet been investigated (e.g., porosity, second phases). Therefore, grain growth modeling was used to iteratively improve the correlation between measured and modeled grain sizes by optimizing activation energy (Q), pre-exponential factor (k0) and grain size exponent (n). For n=2, Q of 350 kJ/mol, k0 of 1.7×1021 μmns−1 and Q of 35 kJ/mol, k0 of 2.5×10-5 μmns−1 were obtained for group II and III, respectively. With respect to future work, field-data based grain growth modeling might be a promising tool for investigating the influences of secondary effects like porosity and second phases on grain growth in nature, and to unravel differences between nature and experiment.

A contribution towards the performance and structural understanding of copper and nickel salts as ammonia stores

The ongoing depletion of fossil fuels and the severe consequences of the greenhouse effect make the development of alternative energy systems crucially important. While hydrogen is, in principle, a promising alternative, releasing nothing but energy and pure water. Hydrogen storage is complicated and no completely viable technique has been proposed so far. This work is concerned with the study of one potential alternative to pure hydrogen: ammonia, and more specifically its storage in solids. Ammonia, NH3, can be regarded as a chemical hydrogen carrier with the advantages of strongly reduced flammability and explosiveness as compared to hydrogen. Furthermore, ammine metal salts presented here as promising ammonia stores easily store up to 50 wt.-% ammonia, giving them a volumetric energy density comparable to natural gas. The model system NiX2–NH3 ( X = Cl, Br, I) is studied thoroughly with respect to ammine salt formation, thermal decomposition, air stability and structural effects. The system CuX2–NH3 ( X = Cl, Br) has an adverse thermal decomposition behaviour, making it impractical for use as an ammonia store. This system is, however, most interesting from a structural point of view and some work concerning the study of the structural behaviour of this system is presented. Finally, close chemical relatives to the metal ammine halides, the metal ammine nitrates are studied. They exhibit interesting anion arrangements, which is an impressive showcase for the combination of diffraction and spectroscopic information. The characterisation techniques in this thesis range from powder diffraction over single crystal diffraction, spectroscopy, computational modelling, thermal analyses to gravimetric uptake experiments. Further highlights are the structure solutions and refinements from powder data of (NH4)2[NiCl4(H2O)(NH3)] and Ni(NH3)2(NO3)2, the combination of crystallographic and chemical information for the elucidation of the (NH4)2[NiCl4(H2O)(NH3)] formation reaction and the growth of single crystals under ammonia flow, a technique allowing the first documented successful growth and single crystal diffraction measurement for [Cu(NH3)6]Cl2.

A new, fast and flexible radiative transfer method for Venus general circulation models

We present a new radiation scheme for the Oxford Planetary Unified Model System for Venus, suitable for the solar and thermal bands. This new and fast radiative parameterization uses a different approach in the two main radiative wavelength bands: solar radiation (0.1-5.5 mu m) and thermal radiation (1.7-260 mu m). The solar radiation calculation is based on the delta-Eddington approximation (two-stream-type) with an adding layer method. For the thermal radiation case, a code based on an absorptivity/emissivity formulation is used. The new radiative transfer formulation implemented is intended to be computationally light, to allow its incorporation in 3D global circulation models, but still allowing for the calculation of the effect of atmospheric conditions on radiative fluxes. This will allow us to investigate the dynamical-radiative-microphysical feedbacks. The model flexibility can be also used to explore the uncertainties in the Venus atmosphere such as the optical properties in the deep atmosphere or cloud amount. The results of radiative cooling and heating rates and the global-mean radiative-convective equilibrium temperature profiles for different atmospheric conditions are presented and discussed. This new scheme works in an atmospheric column and can be easily implemented in 3D Venus global circulation models. (C) 2014 Elsevier Ltd. All rights reserved.

A perfect day for zebrafish : neuromodulation of sleep in a diurnal vertebrate

Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish (Danio rerio), a diurnal vertebrate, has emerged as a promising model system for sleep and arousal research.

In this thesis, I describe two studies on sleep/arousal pathways that I conducted using zebrafish, and I discuss how the findings can be combined in future projects to advance our understanding of vertebrate sleep/arousal pathways. In the first study, I discovered a neuropeptide that regulates zebrafish sleep and arousal as a result of a large-scale effort to identify molecules that regulate behavioral states. Taking advantage of facile zebrafish genetics, I constructed mutants for the three known receptors of this peptide and identified the one receptor that exclusively mediates the observed behavioral effects. I further show that the peptide exerts its behavioral effects independently of signaling at a key module of a neuroendocrine signaling pathway. This finding contradicts the hypothesis put forth in mammalian systems that the peptide acts through the classical neuroendocrine pathway; our data further generate new testable hypotheses for determining the central nervous system or alternative neuroendocrine pathways involved.

Second, I will present the development of a chemigenetic method to non-invasively manipulate neurons in the behaving zebrafish. I validated this technique by expressing and inducing the chemigenetic tool in a restricted population of sleep-regulating neurons in the zebrafish. As predicted by established models of this vertebrate sleep regulator, chemigenetic activation of these neurons induced hyperactivity, whereas chemigenetic ablation of these neurons induced increased sleep behavior. Given that light is a potent modulator of behavior in zebrafish, our proof-of-principle data provide a springboard for future studies of sleep/arousal and other light-dependent behaviors to interrogate genetically-defined populations of neurons independently of optogenetic tools.

Purification, biochemical characterization and localization at single cell resolution of Matrix Gla Protein (MGP) and Bone Gla Protein (BGP) in the teleost fish Argyrosomus regius

Tese de dout. em Química, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2002

Estudo da aplicação de molas magnéticas na recolha da energia de fontes renováveis

Dissertação de Mestrado, Engenharia Elétrica e Eletrónica, Especialização em Sistemas de Energia e Controlo, Instituto Superior de Engenharia, Universidade do Algarve, 2016