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1
Gormley, Padhhraig J. "An investigation of advanced data-driven identification methods for systems biology." Thesis, Queen's University Belfast, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.534735.
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Gray, Kurtis N. "A Molecular Phylogeny of the Echeneoidea (Perciformes: Carangoidei) and an Investigation of Population Structuring Within the Echeneidae." W&M ScholarWorks, 2005. https://scholarworks.wm.edu/etd/1539617836.
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Wang, Benjamin X. Ph D. Massachusetts Institute of Technology. "Investigation of two-component signaling systems in Pseudomonas aeruginosa and their roles in the mucus barrier." Thesis, Massachusetts Institute of Technology, 2021. https://hdl.handle.net/1721.1/130821.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February, 2021Cataloged from the official PDF of thesis.
Includes bibliographical references.
All living things must be able to sense and respond to signals in their environment in order to grow and survive. For bacteria, a common means through which this occurs is via two-component signaling systems, which are typically comprised of a membrane-bound histidine kinase that senses external cues signals, and a cognate cytoplasmic response regulator that triggers changes in gene expression. The importance of these systems is highlighted by the fact that they have been identified in the vast majority of sequenced bacteria. However, despite their prevalence, much remains to be discovered about these sensory systems. In particular, both the actual processes that these systems control, along with the signals that they sense and respond to, remain poorly characterized in many cases.
In this work, I further characterize two-component signaling systems in the opportunistic pathogen Pseudomonas aeruginosa, which is most well-known for chronically infecting the lungs of patients with cystic fibrosis. I begin by screening a collection of in-frame deletion mutants of each histidine kinase in the PA14 strain against a dozen virulence-associated phenotypes, including different types of motility, biofilm formation, virulence factor production, and antibiotic resistance. Through this approach, I identify nearly two dozen of these proteins as important regulators of virulence. As P. aeruginosa is a human-associated mucosal pathogen, I next search for host-derived signals in mucus that act through these sensory systems in P. aeruginosa. I identify mucins and their associated glycans as signals that act through the RetS histidine kinase and the GacS-GacA two-component signaling system.
One major output of this signaling is the downregulation of the type VI secretion system, which suggests that mucin glycans may serve as host-derived "safety signals" that suppress microbial competition under non-dysbiotic conditions. Finally, I characterize the molecular mechanisms by which RetS inhibits GacS activity to better understand the unusual interactions between these two histidine kinases. Overall, this work underscores the diverse and important roles that two-component signaling systems play in bacteria, and begins to shed light on how microbes like P. aeruginosa utilize these systems to sense and respond to signals in the host.
by Benjamin X. Wang.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
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Dubaj, Vladimir, and n/a. "Novel optical fluorescence imaging probe for the investigation of biological function at the microscopic level." Swinburne University of Technology, 2005. http://adt.lib.swin.edu.au./public/adt-VSWT20060905.084615.
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Existing optic fibre-bundle based imaging probes have been successfully used to image
biological signals from tissue in direct contact with the probe tip (Hirano et al. 1996).
These fibre-bundle probe systems employed conventional fluorescence microscopy and
thus lacked spatial filtering or a scanned light source, two features used by laser
scanning confocal microscopes (LSCMs) to improve signal quality. Improving the
methods of imaging tissue in its natural state, deep in-vivo and at cellular resolution is
an ever-present goal in biological research. Within this study, a novel (580 μm
diameter) optic fibre-bundle direct-contact imaging probe, employing a LSCM, was
developed to allow for improved imaging of deep biological tissue in-vivo. The new
LSCM/probe system possessed a spatial resolution of 10 μm, and a temporal resolution
of 1 msec. The LSCM/probe system was compared to a previously used direct-contact
probe system that employed a conventional fluorescence microscope. Quantitative and
qualitative data indicated that the LSCM/probe system possessed superior image
contrast and quality. Furthermore, the LSCM/probe system was approximately 16 times
more effective at filtering unwanted contaminating light from regions below the
imaging plane (z-axis). The unique LSCM/probe system was applied to an exploratory
investigation of calcium activity of both glial and neuronal cells within the whisker
portion of the rat primary somatosensory cortex in-vivo. Fluorescence signals of 106
cells were recorded from 12 female Sprague Dawley rats aged between 7-8 weeks.
Fluo-3(AM) fluorophore based calcium fluctuations that coincided with 10 - 14 Hz
sinusoidal stimulation of rat whiskers for 0.5-1 second were observed in 8.5% of cells (9
of 106). Both increases and decreases in calcium levels that coincided with whisker
stimulation were observed. Of the 8.5 % of cells, 2.8% (3 cells) were categorized as
glial and 5.7% (6 cells) as neuronal, based on temporal characteristics of the observed
activity. The remaining cells (97 of 106) displayed sufficient calcium-based intensity
but no fluctuations that coincided with an applied stimulus. This was partially attributed
to electronic noise inherent in the prototype system obscuring potential very weak cell
signals. The results indicate that the novel LSCM/probe system is an advancement over
previously used systems that employed direct-contact imaging probes. The miniature
nature of the probe allows for insertion into soft tissue, like a hypodermic needle, and
provides access to a range of depths with minimal invasiveness. Furthermore, when
combined with selected dyes, the system allows for imaging of numerous forms of
activity at cellular resolution.
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Price, Heather Leigh. "Investigation of larval sensory systems in the marine bryozoan, Bugula neritina." DigitalCommons@CalPoly, 2015. https://digitalcommons.calpoly.edu/theses/1410.
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Bugula neritina is a sessile marine bryozoan with a pelagic larval stage. Larvae frequently settle on boat hulls, facilitating the introduction of B. neritina to bays and estuaries worldwide. Adrenergic agonists, such as the vertebrate hormone noradrenaline, inhibit larval settlement in a variety of marine invertebrate species, including B. neritina. Light also inhibits B. neritina larval settlement, yet the underlying mechanisms by which light and adrenergic compounds exert their effects on larvae are not well understood. Octopamine is considered the invertebrate analog of noradrenaline, and may be an endogenous hormone involved in larval settlement pathways. I observed the effects of the adrenergic agonist noradrenaline and the adrenergic antagonist phentolamine on larval settlement, and found that high concentrations of noradrenaline increased larval mortality, inhibited larval attachment, and increased larval swimming behavior. High concentrations of phentolamine also increased larval mortality, but increased larval attachment and decreased larval swimming behavior. I used fluorescent labeling and microscopy to localize sensory system components, and found that larvae possess adrenergic-like receptors, as well as tyrosine hydroxylase-like and octopamine-like immunoreactivity. I also exposed larvae to phentolamine in both dark and light conditions, and found that light significantly inhibited larval attachment, but phentolamine blocked those inhibitory effects. These results suggest that B. neritina larvae possess adrenergic-like receptors, which serve as the binding sites for noradrenaline and phentolamine. These are likely octopamine receptors, and octopamine may be one endogenous compound involved in controlling larval phototaxis and settlement behavior. Light may increase octopamine production, thereby stimulating cilial activity, extending swimming behavior, and preventing larvae from attaching to a substrate. This research sheds light on previously unknown sensory mechanisms in B. neritina larvae, and may aid in the development of new biofouling control strategies.
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Hyder, Jennifer A. "An Investigation of the Effects of Increased Tidal Inundation, Competition, and Facilitation on Salt Marsh Systems." Thesis, University of South Florida, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=3700275.
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The low-lying topographic nature of salt marshes makes plants in these communities particularly vulnerable to increased salinity and inundation exposure associated with sea level rise. Both increased salinity and inundation have been cited as major causes of reduced plant performance and survival in marsh and areas fringing marsh. In addition to limitations imposed by physical stress, interspecific interactions have also been shown to mediate the performance and survival of salt marsh and salt marsh fringing species. The Stress Gradient Hypothesis (SGH) postulates that species interactions shift from competitive to facilitative as stress levels increase and predicts that (a) the frequency and intensity of facilitative interactions increase as conditions become more stressful for plants and (b) the strength of competitive interactions increases as abiotic stress levels diminish. The SGH has been rigorously tested to examine how both the frequency and intensity of species interactions change under varying physical stress levels. Studies conducted in salt marsh systems have shown facilitation to be as strong of a driving force as competition in influencing plant performance and survival and have shown that while competition appears to be the pervasive force in the less physically stressful terrestrial zones fringing salt marshes, facilitation influences the performance and survival of species in harsher marsh areas. Under conditions of sea level rise, it remains unclear if the nature of interspecific interactions would shift as stress levels change. This research endeavors to examine the interplay between abiotic stresses and biotic interactions under conditions of increased salinity and inundation exposure.
The first study presented here investigated the effects of increased inundation and soil salinity associated with sea level rise on four salt marsh fringing species, and assesses how competition and facilitation impact survival of salt marsh fringing plant survival under these changing conditions. All plant species experienced reduced growth and photosynthetic inhibition below their current distributional positions, both in the presence and absence of neighboring above ground vegetation. The findings also signal a potential shift in the nature of interspecific interactions from competition to facilitation to neutral as plants begin to experience increased salt and inundation exposure.
The second study aimed to disentangle the effects of increased soil salinity and increased soil moisture on four salt marsh fringing species, and to examine the effects of plant neighbors. The results showed that fringe plants exposed to increased inundation experienced a two-fold reduction in performance and survival over 750 g pure salt addition, suggesting that inundation may be a more important limiting factor than salinity with rising sea levels. Landward transplants at the forest-fringe margin exposed to lower soil salinity and decreased inundation exhibited a three-fold increase in performance and survival when compared to controls. Neighbor manipulation studies, which consisted of trimming neighboring vegetation to ground level, again suggested that interspecific interactions in salt marsh fringing species may shift from competitive to facilitative with climate-induced sea level rise. Overall, our findings suggest that salt marsh fringing species may not be able to tolerate changing conditions associated with sea level rise and their survival may hinge on their ability to migrate towards higher elevations.
The final experiment tested the Stress Gradient Hypothesis and investigated the relative importance of facilitation and competition in a salt marsh system under varying stress levels. This study also ascertained whether salt or inundation exposure is the primary influence on salt marsh plant performance and survival. As in previous studies, our findings suggest that many salt marsh plants don't require, but merely tolerate harsher abiotic conditions. The results showed that plants at higher elevations were depressed by strong competitive pressure from neighboring fringe species while plants at lower elevations benefited from the presence of neighbors. Collectively, the results of these studies indicate that species interactions are an integral driver of plant distribution in salt marsh communities. Furthermore, our findings indicate that changing stress levels may not always result in a shift in the nature of interspecific interactions. These studies have endeavored to show that the interplay between competition and facilitation interacts with physical processes to determine the growth and performance of both fringe and marsh plant species. The paucity of studies examining the roles of species interactions and changing abiotic stress levels on multiple salt marsh and salt marsh fringing species warrants the need for additional research. The responses of salt marsh and salt marsh fringing species to sea level rise can not only serve as very valuable and sensitive indictors of climate change, but will also aid in predicting the future location of the marsh-fringe-forest ecotone, which is predicted to shift inland as sea levels continue to rise.
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Britton, Oliver Jonathan. "Combined experimental and computational investigation into inter-subject variability in cardiac electrophysiology." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:6299240d-0528-4662-8e1f-5025f39e730f.
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The underlying causes of variability in the electrical activity of hearts from individuals of the same species are not well understood. Understanding this variability is important to enable prediction of the response of individual hearts to diseases and therapies. Current experimental and computational methods for investigating the behaviour of the heart do not incorporate biological variation between individuals. In experimental studies, experimental results are averaged together to control errors and determine the average behaviour of the studied organism. In computational studies, averaged experimental data is usually used to develop models, and these models therefore represent a 'typical' organism, with all information on variability within the species having been lost. In this thesis we develop a methodology for modelling variability between individuals of the same species in cardiac cellular electrophysiology, motivated by the inability of traditional computational modelling approaches to capture experimental variability. A first study is conducted using traditional modelling approaches to investigate potentially pro-arrhythmic abnormalities in rabbit Purkinje fibres. A comparison with experimental recordings highlights their wide variability and the inability of existing computer modelling approaches to capture it. This leads to the development of a novel methodology that integrates the variability observed in experimental data with computational modelling and simulation, by building experimentally-calibrated populations of computational models, that collectively span the variability seen in experimental data. We apply this methodology to construct a population of rabbit Purkinje cell models. We show that our population of models can quantitatively predict the range of responses, not just the average response, to application of the potassium channel blocking drug dofetilide. This demonstrates an important potential application of our methodology, for predicting pro-arrhythmic drug effects in safety pharmacology. We then analyse a data set of experimental recordings from human ventricular tissue preparations, and use this data to develop a population of human ventricular cell models. We apply this population to study how variability between individuals alters the susceptibility of cardiac cells to developing drug-induced repolarisation abnormalities. These abnormalities can increase the chance of fatal arrhythmias, but the mechanisms that determine individual susceptibility are not well-understood.
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Parikh, Jaimit B. "Theoretical Investigation of Intra- and Inter-cellular Spatiotemporal Calcium Patterns in Microcirculation." FIU Digital Commons, 2015. http://digitalcommons.fiu.edu/etd/1927.
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Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined.
Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC.
From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.
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Chee, Meng May. "Investigations into telomere biology in systemic sclerosis." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/3615/.
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Systemic sclerosis (SSc) is a complex multisystem autoimmune connective tissue disease of yet unknown aetiology. It is characterised by vascular damage, autoimmunity and progressive fibrosis in the skin and internal organs, with 2 main subsets of disease: diffuse cutaneous SSc (dcSSc) and limited cutaneous SSc (lcSSc), classified mainly according to skin involvement. Although there has been tremendous progress in the understanding and treatment of autoimmune diseases in the last 20 years, SSc remains a disease with high morbidity and mortality, with no proven treatments that improve long-term outcome. There is therefore a need to improve the understanding of such a devastating disease to facilitate the development of future treatments and improve the prognosis for patients with SSc. Telomeres are nucleo-protein complexes, comprising tandem TTAGGG repeats at the ends of eukaryotic chromosomes, which protect the chromosomes from end-to-end fusion, recombination and degradation. There are a variety of telomere binding proteins which maintain telomere stability and function in sensing, signalling and repairing DNA damage. Telomere lengths have been used as a marker of biological aging for over a decade, and the technologies for measuring telomere lengths have also advanced in recent years. Quantitative real time PCR (QPCR) has now superseded Southern Blotting (SB) for telomere length measurements. Telomere erosion has been implicated in a wide range of diseases and its impact on autoimmune diseases remains unclear. As SSc is associated with particular autoantibodies which have been linked to telomeres, this thesis sought to explore telomere biology in SSc. Initial work measuring telomere lengths in SSc patients using the SB method revealed surprising results, with a lack of age-related telomere erosion in patients with lcSSc, which was in contrast to published literature at the time. The aim of this research was to measure telomere lengths of peripheral blood mononuclear cells (PBMC) in patients with SSc and related connective tissue diseases using QPCR, with the hypothesis that telomere lengths would be shorter in disease, and to explore telomere binding protein genes, with a view to gaining insight into any mechanistic differences in telomere biology in the different subsets of disease. Measurement of telomere lengths of PBMC from healthy controls and patients using QPCR showed that telomere lengths were significantly shorter in disease compared to controls, but when adjusted for age, there was no statistically significant difference in telomere lengths of patients with lcSSc compared to controls. Taking into account the complexity of telomere biology, it is possible that mediators of the inflammatory reaction at the systemic level or autoantibodies against nucleoprotein complexes directly or indirectly interfere with the homeostasis of telomere length in blood. This could occur through disrupting the proteins of the shelterin complex or associated factors relating to DNA repair. Hence, the gene expression of telomere binding proteins and other genes associated with inflammation and DNA repair were explored in patients with SSc using Taqman Low Density Arrays. The majority of these genes were under expressed in disease compared to controls, but when samples were age-matched, only 4 genes were under-expressed in patients with dcSSc: BCL2, POT1, SIRT6 and WRN, and 4 genes were under-expressed in lcSSc: ATM, BCL2, STAU1 and WRN. There was no correlation between telomere lengths and gene expression. These observations are intriguing, and the role of BCL2 and WRN merit further investigation in patients with SSc. This work has confirmed that telomeres shorten with age and disease, in keeping with the original hypothesis and published literature. However, there was no significant difference in age-related telomere erosion in patients with lcSSc. Gene expression analysis of telomere associated genes in SSc revealed lower expression in disease compared with controls. Whether this observation is a cause or effect of disease remains to be proven, but suggests that telomeres may be implicated in the pathophysiology of SSc and there are mechanistic differences between the subsets of disease.
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Wilcock, Paul. "A systems biology approach for investigating oral squamous cell carcinoma (OSCC)." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/a-systems-biology-approach-for-investigating-oral-squamous-cell-carcinoma-oscc(8ec3728b-1928-450f-b467-76996fa970fb).html.
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A systems biology approach was adopted in order to assess various aspects of the disease oral squamous cell carcinoma. Three main aims were addressed; assess the ability of CoCl2 to mimic the hypoxic response in a eukaryotic cell line, assess the role of PDE4D in oral squamous cell carcinoma (OSCC) and the construction of a normoxic/hypoxic mathematical model to identify therapeutic targets.Cancer cells often acquire a revised metabolism which aids in initiation, survival and progression of the tumour. This is predominantly due to the transcription factor HIF-1 which is activated under hypoxic conditions. Certain compounds such as cobalt chloride (CoCl2) have been used extensively to inhibit the degradation of HIF-1α and simulate hypoxia. CoCl2 is likely to have off-target effects on metabolism; these effects were examined when exposing human telomerase reverse transcriptase (hTERT) cells to 100μM CoCl2. Gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) based metabolomics were utilised in combination with ELISA assays for HIF-1α and ATP. Central metabolism was accurately mimicked when hTERT cells were subjected to 100μM CoCl2, however; it was apparent that this concentration of CoCl2 does not induce an equal extent of hypoxia as 1% oxygen. A number of off-target effects of CoCl2 were observed in secondary metabolism, specifically in lipids and fatty acids. In conclusion, CoCl2 should be used with caution as a hypoxic mimicker with the caveat that interpretation of results should be restricted to its effects on central metabolism.The transcription factor CREB has the ability to regulate approximately 4000 genes, a number of which are associated with cancer initiation and progression. Cyclic adenosine monophosphate (cAMP) is required to activate CREB and is partially regulated through its degradation via the enzyme phosphodiesterase type 4D (PDE4D). A homozygous deletion of PDE4D has been associated with OSCC; however; the exact consequence of this deletion has not been fully elucidated. PDE4D was knocked down in the OSCC cell line BicR16 and cellular proliferation, migration, resistance to ionising radiation and central metabolism was investigated using MTT, scratch, clonogenic and GC-MS, respectively. The knockdown resulted in an increase in proliferation, migration and radiation resistance suggesting the role of PDE4D as a TSG. Amino acids, cholesterol, fatty acids, carbohydrates and TCA intermediates were found to be altered in concentration.A mathematical model of glycolysis, TCA and glutaminolysis under normoxia and hypoxia was constructed through the amalgamation of two established models from the literature. New reactions, parameters and metabolite concentrations were added and unnecessary entities were deleted. COmplex PAthway SImulator (COPASI) was utilised to construct the model before validating the model using experimental data from the literature and steady state and flux analyses. Sensitivity analysis and a reduction in external glucose and glutamine were mimicked and the alterations in hypoxic and normoxic metabolism analysed. The reactions vCSII, vGS, vPGK and vGII were identified as potential therapeutic targets which may affect metabolism in hypoxia only. However, certain validation methods proved unsuccessful and hence the model requires further work before attempting the analyses again.
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Yen, Jennifer Lee. "Investigating the zebrafish system for modelling cancer genomics and biology." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648122.
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Xie, Zhi. "Modelling genetic regulatory networks: a new model for circadian rhythms in Drosophila and investigation of genetic noise in a viral infection process." Phd thesis, Lincoln University. Agriculture and Life Sciences Division, 2007. http://theses.lincoln.ac.nz/public/adt-NZLIU20070712.144258/.
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In spite of remarkable progress in molecular biology, our understanding of the dynamics and functions of intra- and inter-cellular biological networks has been hampered by their complexity. Kinetics modelling, an important type of mathematical modelling, provides a rigorous and reliable way to reveal the complexity of biological networks. In this thesis, two genetic regulatory networks have been investigated via kinetic models.
In the first part of the study, a model is developed to represent the transcriptional regulatory network essential for the circadian rhythms in Drosophila. The model incorporates the transcriptional feedback loops revealed so far in the network of the circadian clock (PER/TIM and VRI/PDP1 loops). Conventional Hill functions are not used to describe the regulation of genes, instead the explicit reactions of binding and unbinding processes of transcription factors to promoters are modelled. The model is described by a set of ordinary differential equations and the parameters are estimated from the in vitro experimental data of the clocks components. The simulation results show that the model reproduces sustained circadian oscillations in mRNA and protein concentrations that are in agreement with experimental observations. It also simulates the entrainment by light-dark cycles, the disappearance of the rhythmicity in constant light and the shape of phase response curves resembling that of experimental results. The model is robust over a wide range of parameter variations. In addition, the simulated E-box mutation, perS and perL mutants are similar to that observed in the experiments. The deficiency between the simulated mRNA levels and experimental observations in per01, tim01 and clkJrk mutants suggests some differences in the model from reality. Finally, a possible function of VRI/PDP1 loops is proposed to increase the robustness of the clock.
In the second part of the study, the sources of intrinsic noise and the influence of extrinsic noise are investigated on an intracellular viral infection system. The contribution of the intrinsic noise from each reaction is measured by means of a special form of stochastic differential equation, the chemical Langevin equation. The intrinsic noise of the system is the linear sum of the noise in each of the reactions. The intrinsic noise arises mainly from the degradation of mRNA and the transcription processes. Then, the effects of extrinsic noise are studied by means of a general form of stochastic differential equation. It is found that the noise of the viral components grows logarithmically with increasing noise intensities. The system is most susceptible to noise in the virus assembly process. A high level of noise in this process can even inhibit the replication of the viruses.
In summary, the success of this thesis demonstrates the usefulness of models for interpreting experimental data, developing hypotheses, as well as for understanding the design principles of genetic regulatory networks.
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Miu, Peter. "An electrophysiological investigation of monosialoganglioside in the mammalian central nervous system." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41186.
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This thesis focuses on the functional role of monosialoganglioside (GM1) in neuronal activity and synaptic transmission of rat hippocampal slices. The slices were placed in an interface recording chamber and constantly superfused with oxygenated saline at 33$ sp circ$. Both extracellular and intracellular (current- and voltage-clamp) recording techniques were used to measure the field potentials and postsynaptic responses respectively. Our findings indicated that GM1 induces a small inward current in CA1 pyramidal neurons, depresses their high voltage activated (HVA) Ca$ sp{2+}$ currents, and selectively facilitates the excitatory synaptic inputs while reducing the inhibitory ones.The most probable mechanism underlying the selective enhancement of excitatory inputs by GM1 is an increase in glutamate release, since the amplitudes and frequency of spontaneous miniature postsynaptic responses, recorded either in the presence or absence of presynaptic cell firing, were cosistently increased. When L-glutamate was applied iontophoretically in the dendritic region, GM1 transiently potentiated the postsynaptic glutamate currents, thus further indicating a GM1-induced enhancement of glutamatergic transmission. In contrast, both spontaneous and evoked inhibitory postsynaptic responses were suppressed by GM1. This effect is dependent on changes in excitatory inputs to inhibitory interneurons because in the presence of tetrodotoxin and/or kynurenic acid, GM1 did not alter the amplitude of the monosynaptic IPSPs or the frequency of spontaneous miniature IPSCs.
Both the GM1-induced inward current and the reduction of postsynaptic HVA Ca$ sp{2+}$ currents were antagonised by kynurenic acid, suggesting that these effects might be caused by glutamate receptor activation. By raising intraneuronal Ca$ sp{2+}$ concentration, the potentiated glutamate release would trigger Ca$ sp{2+}$-dependent Ca$ sp{2+}$ inactivation, and thus explain the reduction in HVA Ca$ sp{2+}$ currents.
In conclusion, most of the GM1 actions observed in this project can be explained on the basis of a GM1-induced facilitation of excitatory transmission, mediated especially via enhanced glutamate release.
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Cizmeci, Deniz. "A systems biology approach to investigating host-pathogen interactions in infection with Burkholderia pseudomallei." Thesis, University of Exeter, 2017. http://hdl.handle.net/10871/29963.
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This thesis applies systems approaches in order better to understand host-pathogen interactions in infectious diseases; it focuses on the intracellular bacterium Burkholderia pseudomallei, the causative agent of the human disease melioidosis. Little is known about the epigenetic changes in host cells during infection. This study assesses genome-wide patterns of the epigenetic marker DNA methylation in host cells following infection with B. pseudomallei. The studies of this thesis concern the infection of human macrophage-like U937 cells with B. pseudomallei and the DNA methylation levels were measured during the early stages of infection. Analyses reveal significant changes in infected cells (compared to uninfected controls) at multiple locations in the host DNA. Most of the methylation changes in infected cells are losses rather than gains in methylation. Five different differential methylation patterns (constant, early, late, transient, and oscillatory) are identified. Differentially methylated sites mapped to genes that may affect virulence, e.g. genes involved in actin regulation, immune response, inflammatory response, and nitric oxide generation. The thesis also measures whole blood DNA methylation profiles of patients diagnosed with melioidosis in order to test the potential role of host DNA methylation in melioidosis. The results demonstrate that patients with melioidosis are separated from healthy subjects by their distinct methylation profiles. The differentially methylated regions reported here can potentially be used as biomarkers for classification and prognostication of infectious diseases. In addition to exploring the changes to the host, a comprehensive understanding of the pathogen interference and the search for countermeasures requires a framework that assesses how the host changes the pathogen metabolically. In this thesis, to understand the role of trehalose pathway in virulence, computational models were constructed by integrating kinetic information, genomics data and literature surveys. Existing kinetic models of the trehalose pathway were implemented and extended allowing for the in silico investigation of the trehalose mutant. Further, metabolic networks of B. pseudomallei were analysed at the genome scale to identify molecular links between trehalose and metabolic pathways such as glycolysis. The genome- scale reconstruction of the B. pseudomallei metabolic network was used to simulate growth under different conditions and predict the effects of gene knockouts. This thesis not only expands the existing knowledge about B. pseudomallei infection, the novel approaches employed here will stimulate a wider understanding of the applications of systems biology to host-pathogen research and defence needs.
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Finneran, James Joseph. "An experimental and theoretical investigation of the mechanics of the goldfish peripheral auditory system /." Connect to resource, 1997. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1230661897.
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Sherwood, Amanda R. "INVESTIGATING THERAPEUTIC OPTIONS FOR LAFORA DISEASE USING STRUCTURAL BIOLOGY AND TRANSLATIONAL METHODS." UKnowledge, 2013. http://uknowledge.uky.edu/biochem_etds/13.
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Lafora disease (LD) is a rare yet invariably fatal form of epilepsy characterized by progressive degeneration of the central nervous and motor systems and accumulation of insoluble glucans within cells. LD results from mutation of either the phosphatase laforin, an enzyme that dephosphorylates cellular glycogen, or the E3 ubiquitin ligase malin, the binding partner of laforin. Currently, there are no therapeutic options for LD, or reported methods by which the specific activity of glucan phosphatases such as laforin can be easily measured. To facilitate our translational studies, we developed an assay with which the glucan phosphatase activity of laforin as well as emerging members of the glucan phosphatase family can be characterized. We then adapted this assay for the detection of endogenous laforin activity from human and mouse tissue. This laforin bioassay will prove useful in the detection of functional laforin in LD patient tissue following the application of therapies to LD patients. We subsequently developed an in vitro readthrough reporter system in order to assess the efficacy of aminoglycosides in the readthrough of laforin and malin nonsense mutations. We found that although several laforin and malin nonsense mutations exhibited significant drug-induced readthrough, the location of the epitope tag used to detect readthrough products dramatically affected our readthrough results. Cell lines established from LD patients with nonsense mutations are thus required to accurately assess the efficacy of aminoglycosides as a therapeutic option for LD. Using hydrogen-deuterium exchange mass spectrometry (DXMS), we then gained insight into the molecular etiology of several point mutations in laforin that cause LD. We identified a novel motif in the phosphatase domain of laforin that shares homology with glycosyl hydrolases (GH) and appears to play a role in the interaction of laforin with glucans. We studied the impact of the Y294N and P301L LD mutations within this GH motif on glucan binding. Surprisingly, these mutations did not reduce glucan binding as expected, rather enhancing the binding of laforin to glucans. These findings elucidate the mechanism by which laforin interacts with and acts upon glucan substrates, providing a target for the development of therapeutic compounds.
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Shi, Huilin. "Investigation of Common Bases of Sympathetic Nervous System and Neuroblastoma Development." University of Toledo Health Science Campus / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=mco1244058100.
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Frawley, Laura E. (Laura Elizabeth). "Investigation of the role of polyploidization in glial cells during the development of the drosophila nervous system." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/117882.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.Cataloged from PDF version of thesis.
Includes bibliographical references.
Organogenesis is a complex process encompassing cell determination, cell differentiation, cell proliferation, and cell size regulation. The proper orchestration of these events ensures that each organ is scaled correctly and can function properly. Polyploidization is a process by which cells increase their DNA content and is used across species to generate large cells. Our lab had previously determined that subperineurial glia (SPG) of the Drosophila melanogaster nervous system become polyploid by both the endocycle and endomitosis. These are two cell cycle variants employed to produce polyploid cells that differ in the latter undergoing some aspects of mitosis but not cytokinesis. Polyploidization of the SPG is critical for blood-brain barrier (BBB) function. Here, we determined that the developmental switch from endocycling to endomitotic SPG occurs in about 70% of SPG in the brain lobes by the second larval instar. The SPG in the ventral nerve cord and peripheral nervous system solely endocycle. We demonstrated that both the Notch signaling pathway and the String Cdc25 phosphatase are critical in determining whether SPG endocycle or endomitose. Experiments manipulating the percentage of cells that are endocycling versus endomitotic highlight key differences between endocycling and endomitotic SPG. We find that endomitotic SPG cells are capable of achieving higher ploidy and cell area values than endocycling cells and are essential to the integrity of the BBB. Strikingly, we find that endocycling SPG within the ventral nerve cord retain the ability to undergo endomitosis when the Notch signaling pathway or the String Cdc25 phosphatase are altered. Further, we showed that a second glial cell type in the peripheral nervous system, wrapping glia (WG), is polyploid and determined that total WG ploidy correlates with nerve length. Interestingly, when WG ploidy was reduced, we found that axonal ensheathment is defective. We also established that the three WG per peripheral nerve differentially contribute to overall ploidy. Axonal ensheathment throughout the entire nerve seems to be dependent on position along the anterior-posterior larval body axis. Finally, we find that reduction of DNA replication components causes reduced WG ploidy only in longer peripheral nerves.
by Laura E. Frawley.
Ph. D.
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Paton, R. C. "Some investigations into the use and meaning of system in biological education." Thesis, University of Leeds, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233889.
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Beaulieu, Angela Denise. "Investigations of milk protein and lipid synthesis utilizing an in vitro bovine mammary cell culture system /." The Ohio State University, 1994. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487858417984017.
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Robinson, David. "An investigation into novel molecules involved in the development of the nervous system of Drosophila melanogaster." Thesis, University of Greenwich, 2015. http://gala.gre.ac.uk/18054/.
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A major facet of nervous system development entails the projection of axons from neuronal cell bodies towards synaptic partners, such as other neurons or muscles. This process, referred to as “axon guidance,” relies upon receptors on axons detecting secreted or membrane-associated guidance cues within the developing organism, which attract or repel axons. While many cues and receptors have been identified over the last few decades, many within studies of the relatively simple fly embryonic nervous system, bioinformatic analyses of the Drosophila proteome suggest numerous uncharacterised proteins might be implicated in directing axonal growth. Such proteins are predicted to be expressed at the cell membrane, to harbour domains common to established axon guidance proteins, and to be expressed in the nervous system while axons are extending. The current study focuses on three genes predicted to encode proteins with the above characteristics: CG7565, CG31814, and otk2. An examination of a line with a P-element in the coding sequence of CG7565 revealed aberrations throughout the embryonic motor neurons. Abnormalities in these nerves were also observed in a line with a deficiency spanning CG7565, as well as in embryos misexpressing CG7565 in motor neurons or somatic muscle. Motor neuron projections were absent in embryos harbouring a P-element in the 5’ UTR of CG31814, which were rescued by the precise excision of the transposon. In null otk2 mutant embryos, various motor axons were absent or abnormal, and aberrations in the same branches were apparent when misexpressing otk2 in motor neurons or somatic muscle. Moreover, analyses of transheterozygous embryos provide evidence that otk2 genetically interacts with the established axon guidance genes, otk, sema-1a, and fz2, and that otk interacts with fz2. Analysis of a line with a deficiency that removes otk and otk2 revealed more severe phenotypes than were observed in single mutants, providing further support for cooperation between the off-tracks. Thus, taken together these observations implicate three largely uncharacterised genes in axon guidance and reveal novel insights into the signalling pathways in which these and established axon guidance genes participate.
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Davis, Jon Franklin. "A Functional, Anatomical, and Molecular Investigation of Natural Reward: Sexual Plasticity and Limbic System." Cincinnati, Ohio : University of Cincinnati, 2005. http://www.ohiolink.edu/etd/view.cgi?acc%5Fnum=ucin1123831570.
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Thesis (Ph. D.)--University of Cincinnati, 2005.Title from electronic thesis title page (viewed Mar. 23, 2006). Includes abstract. Keywords: limbic, reward, plasticity. Includes bibliographical references.
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Adamska-Venkatesh, Agnieszka [Verfasser]. "Spectroscopic investigations of [FeFe] hydrogenases and related model systems / Agnieszka Adamska-Venkatesh." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2015. http://d-nb.info/1072224569/34.
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Rossmanith, Eva. "Breeding biology, mating system and population dynamics of the Lesser Spotted Woodepcker (Picoides minor) : combining empirical and model investigations." Phd thesis, Universität Potsdam, 2005. http://opus.kobv.de/ubp/volltexte/2005/532/.
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The protection of species is one major focus in conservation biology. The basis for any management concept is the knowledge of the species autecology. In my thesis, I studied the life-history traits and population dynamics of the endangered Lesser Spotted Woodpecker (Picoides minor) in Central Europe. Here, I combine a range of approaches, from empirical investigations of a Lesser Spotted Woodpecker population in the Taunus low mountain range in Germany, the analysis of empirical data and the development of an individual-based stochastic model simulating the population dynamics.In the field studies I collected basic demographic data of reproductive success and mortality. Moreover, breeding biology and behaviour were investigated in detail. My results showed a significant decrease of the reproductive success with later timing of breeding, caused by deterioration in food supply. Moreover, mate fidelity was of benefit, since pairs composed of individuals that bred together the previous year started earlier with egg laying and obtained a higher reproductive success. Both sexes were involved in parental care, but the care was only shared equally during incubation and the early nestling stage. In the late nestling stage, parental care strategies differed between sexes: Females considerably decreased feeding rate with number of nestlings and even completely deserted small broods. Males fed their nestlings irrespective of brood size and compensated for the females absence. The organisation of parental care in the Lesser Spotted Woodpecker is discussed to provide the possibility for females to mate with two males with separate nests and indeed, polyandry was confirmed.
To investigate the influence of the observed flexibility in the social mating system on the population persistence, a stochastic individual-based model simulating the population dynamics of the Lesser Spotted Woodpecker was developed, based on empirical results. However, pre-breeding survival rates could not be obtained empirically and I present in this thesis a pattern-oriented modelling approach to estimate pre-breeding survival rates by comparing simulation results with empirical pattern of population structure and reproductive success on population level. Here, I estimated the pre-breeding survival for two Lesser Spotted Woodpecker populations on different latitudes to test the reliability of the results.
Finally, I used the same simulation model to investigate the effect of flexibility in the mating system on the persistence of the population. With increasing rate of polyandry in the population, the persistence increased and even low rates of polyandry had a strong influence. Even when presuming only a low polyandry rate and costs of polyandry in terms of higher mortality and lower reproductive success for the secondary male, the positive effect of polyandry on the persistence of the population was still strong.
This thesis greatly helped to increase the knowledge of the autecology of an endangered woodpecker species. Beyond the relevance for the species, I could demonstrate here that in general flexibility in mating systems are buffer mechanisms and reduce the impact of environmental and demographic noise.
Der Schutz von Arten ist eine der Hauptaufgaben des Naturschutzes. Für die Erstellung von Schutzkonzepten sind Informationen zur Autökologie der Zielart notwendige Voraussetzung. Der Kleinspecht (Picoides minor) ist in vielen Teilen seines Verbreitungsgebietes bestandsbedroht, das Wissen zur Biologie und Verhalten der Art ist jedoch lückenhaft. Ziel meiner Arbeit war es daher, demographische Parameter der Populationsdynamik des Kleinspechts zu erfassen, die als Grundlage für Populationsgefährdungsanalysen benötigt werden. Da Untersuchungen in Schweden eine gewisse Flexibilität im Paarungssystem des Kleinspechts zeigten, sollte darüber hinaus das Paarungssystem und sein Einfluss auf die Persistenz der Population untersucht werden.
Die Arbeit umfasste eine Reihe von methodischen Ansätzen, von empirischen Untersuchungen an einer Kleinspechtpopulation im hessischen Vordertaunus über die Aufbereitung von empirischen Daten bis hin zur Entwicklung und Auswertung eines stochastischen individuenbasierten Modells zur Simulation der Populationsdynamik.
Die Ergebnisse der empirischen Untersuchung zeigten eine Abnahme des Reproduktionserfolgs mit fortschreitendem Legebeginn. Die Zusammensetzung der Nestlingsnahrung ließ vermuten, dass dies durch eine Verschlechterung der Nahrungsversorgung begründet war. Paartreue war bei der Reproduktion von Vorteil, da Individuen, die schon im vorherigen Jahr zusammen gebrütet hatten, einen früheren Legebeginn und damit einen höheren Fortpflanzungserfolg aufwiesen als neu formierte Paare. Beide Geschlechter investierten in die Brutpflege, jedoch war die Aufteilung nur während der Bebrütung der Eier und in der ersten Hälfte der Nestlingsperiode gleichmäßig. In der späten Nestlingsperiode konnten geschlechtsspezifische Strategien im elterlichen Investment identifiziert werden: die Weibchen verringerten die Versorgungsrate in Abhängigkeit des Wertes der Brut - gemessen in der Zahl der Nestlinge - und gaben die Versorgung kleiner Bruten ganz auf. Die Männchen dagegen kompensierten dieses Verhalten, so dass auch von den Weibchen verlassene Bruten erfolgreich waren. Interessanterweise konnte mehrmals die Verpaarung von einem Weibchen mit zwei Männchen beobachtet werden. Das Auftreten dieses polyandrischen Paarungssystems wird in der Arbeit als Resultat der Aufteilung der Brutpflege diskutiert.
Die bestätigte Flexibilität im Paarungssystem könnte Einfluss auf die Persistenz der Population haben. Die Persistenz von Populationen kann jedoch nicht empirisch gemessen werden. Daher entwickelte ich ein individuen-basiertes stochastisches Modell zur Simulation der Populationsdynamik des Kleinspechts, dass auf den empirischen Daten basiert. Allerdings fehlten Überlebensraten der ausgeflogenen Jungvögel, die im Feld nicht ermittelt werden kann. Daher testete ich hier eine Methode, die durch den Vergleich von Simulationsergebnissen mit eigenen empirischen Daten zur Populationsstruktur und zum Reproduktionserfolg auf der Ebene der Gesamtpopulation die Überlebensrate der Jungvögel abschätzt. Die Überlebensraten wurde zusätzlich für eine Population des Kleinspechtes ermittelt, deren Datengrundlage aus Freilandstudien in Schweden stammten. Durch den Vergleich der Raten für die beiden Populationen konnte die Aussagefähigkeit des Modells und die Güte der Abschätzungen untersucht werden. Im letzten Teil meiner Arbeit nutzte ich das Modell schließlich, um die Auswirkungen des Paarungssystems auf die Überlebensfähigkeit der Population zu untersuchen. Im Modell konnte ein Weibchen polyandrisch sein, wenn es gute Brutbedingungen hatte und das Geschlechterverhältnis zum Männchen hin verschoben war. Zusätzlich variierte ich die Wahrscheinlichkeit, dass unter diesen Umständen Polyandrie auftritt. Im Model wurden 3 Szenarien getestet: (i) strenge Monogamie, (ii) gelegentliche Polyandrie und (iii) gelegentliche Polyandrie unter der Annahme von Kosten für das sekundäre Männchen in Form von höherer Mortalität und geringerem Reproduktionserfolg. Es zeigte sich, dass selbst sehr geringe Polyandrieraten und die Annahme von Kosten noch einen deutlichen positiven Einfluss auf die Persistenz der Population ausüben. Die Flexibilität im Paarungssystem dient damit als Puffermechanismus gegen demographisches Rauschen und Umweltrauschen.
Diese Arbeit trät dazu bei, die Autökologie des Kleinspechts besser zu verstehen und ist damit wichtige Grundlage für Schutzkonzepte in Mitteleuropa. Über die artspezifische Bedeutung hinaus, leistet die Arbeit einen Beitrag zur Untersuchung von Methoden zur Abschätzung fehlender demographischer Parameter sowie zur Identifizierung von Puffermechanismen. Eine wichtige Schlussfolgerung meiner Arbeit ist es, dass die Flexibilität artspezifischen Verhaltens in zukünftigen Populationsgefährdungsanalysen integriert werden sollte, um die Qualität von Prognosen zur Persistenz von Populationen zu verbessern.
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Nyrén, Karl. "Phylogenetic analysis of secretion systems in Francisellaceae and Legionellales : Investigating events of intracellularization." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-448062.
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Host-adapted bacteria are pathogens that, through evolutionary time and host-adaptive events, acquired the ability to manipulate hosts into assisting their own reproduction and spread. Through these host-adaptive events, free-living pathogens may be rendered unable to reproduce without their host, which is an irreversible step in evolution. Francisellaceae and Legionellales, two orders of Gammaproteobacteria, are cases where host-adaptation has lead to an intracellular lifestyle. Both orders use secretion systems, in combination with effector proteins, to invade and control their hosts. A current view is that Francisellaceae and Legionellales went through host-adaptive events at two separate time points. However, F. hongkongensis, a member of Francisellaceae shares the same secretion system as the order of Legionellales. Additionally, two host-adapted Gammaproteobacteria, Piscirickettsia spp. and Berkiella spp., swaps phylogenetic positions between Legionellales and Francisellaceae depending on methods applied - indicating shared features of Francisellaceae and Legionellales. In this study, we set up a workflow to screen public metagenomic data for candidate host-adaptive bacteria. Using this data, we attempted to assert the phylogenetic position and possibly resolve evolutionary events that occurred in Legionellales, F. hongkongensis, Francisellaceae, Piscirickettsia spp. and Berkiella spp. We successfully acquired 23 candidate host-adapted MAGs by (i) scanning for genes, among reads before assembly, using PhyloMagnet, and (ii) screening for complete secretion systems with MacSyFinder. The phylogenetic results turned out indecisive in the placement ofBerkiella spp. and Piscirickettsia. However, results found in this study indicate that, contrary to previous beliefs, it is possible that it was one intracellularization event of a common ancestor that gave rise to the intracellular lifestyle of Francisellaceae and Legionellales.
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Kingston, Demelza. "Investigating central nervous system trypanosomosis in working equids in The Gambia." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/30682/.
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Working equids, vital to many of the world’s most economically vulnerable people, face many challenges to their health, welfare and productivity. In The Gambia, West Africa, appropriate nutrition, husbandry and veterinary assistance are limited, while infectious disease is a constant threat, particularly the parasitic disease trypanosomosis. The prevalence of generalised trypanosomosis in working equids attending the Gambia Horse and Donkey Trust show in 2013 using PCR was 55.4%. Trypanosoma congolense was most prevalent (47.0%), followed by T. vivax (15.7%) and T. brucei s.l. (2.4%). Mixed infections were common (9.4%) and T. congolense/ T. vivax coinfection appeared to have the greatest clinical effect. Spread of T. brucei parasites to the central nervous system (CNS), confirmed using immunohistochemistry and PCR, causes severe CNS dysfunction. Horses showed spastic paraparesis that rapidly progressed to recumbency, while donkeys more often displayed somnolence and cranial nerve dysfunction with a slower deterioration. The disease was fatal in all cases. Histopathology revealed diffuse lymphocytic-plasmacytic meningoencephalo-myelitis with marked perivascular cuffing, particularly in the white matter. T cells were prominent in this first study of lymphocyte distribution in equine CNS trypanosomosis. Extensive reactive astrocytosis was also demonstrated. Currently, a reliable diagnosis of equine CNS requires post mortem samples. The loop-mediated isothermal amplification (LAMP) assay was assessed for the diagnosis of equine T. brucei infection for the first time in both blood and cerebrospinal fluid (CSF). An entomological survey showed that Glossina morsitans submorsitans was common in dry woodland areas while G. palpalis gambiensis was found in riverine habitats. The prevalence of T. brucei in the midguts of Glossina specimens was 1.7% and equine DNA was found in tsetse bloodmeals, providing evidence for ongoing interaction between host, parasite and vector. Atylotus agrestis, vector of T. vivax and T. congolense, was present in large numbers in village areas. Equine DNA was detected in one A. agrestis specimen, however, no evidence of T. brucei in association with these flies was found. Finally, microsatellite genotyping was used for the first time to investigate T. brucei populations in equine trypanosomosis in The Gambia. The results revealed a heterogenous population, providing further evidence for a tsetse-transmitted mode of transmission. No evidence of population clustering by disease type or host species was detected, suggesting that host factors determine pathogenesis. Initial evidence for the involvement of the tsetse vector supports evaluation of vector control methods although further analysis of T. brucei populations in insect vectors and their relationships with those infecting equids is recommended. The clinicopathological descriptions will be of use in further study of equine CNS trypanosomosis and the development of new therapeutics and LAMP has the potential to facilitate research, especially in the study of CNS infection which has, up to now, relied on post mortem confirmation.
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Goschorska, Maja. "Investigating the mechanisms of cell competition in mammals using in vitro systems." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/290214.
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Cell competition leads to elimination of a viable cell population, by fitter cells. Despite over forty years of research, the molecular mechanisms of competition in mammals are poorly understood. During my PhD I have investigated the mechanisms of competition by exploring an established mammalian cell culture system, in which wild-type MDCK cells eliminate scribble-deficient cells, and I have also developed a novel cell culture system to model mammalian competition. My work contributed to the discovery that scribble-deficient cells are eliminated not by biochemical exchange among cells, but by mechanical compaction. We termed this phenomenon mechanical competition. I employed transcriptional profiling to determine the molecular signature of mechanical losers, and identified activation of p53 signalling as their hallmark. My colleagues and I then demonstrated that elevation of p53 is both necessary and sufficient to trigger mechanical competition. In further investigating the mechanisms of mechanical competition, I found that compaction activates ROCK in scribble-deficient cells, and that this is required for their elimination. Inhibition of Src signalling in mechanical losers also protected them form out-competition, and integrin signalling is another pathway likely involved in mechanical competition. While investigating p53 competition, we observed that p53-high and p53-low cells engage in directional migration, with p53-high cells always at the migrating front. As a side-project, I investigated the role of p53 in directional migration, by exploring an established model with a single leader cell and multiple followers. We established a method to generate multinucleated leaders on demand. By creating leaders from p53-deficient cells, I established that p53 signalling is required for some, but not all multinucleated cells to trigger collective migration, thus implicating p53 signalling in a type of migration involved in wound healing. Finally, I successfully modelled p53-driven mechanical competition in a differentiated primary tracheal epithelial cell culture, thereby establishing a novel system to study mammalian competition, and also proving that p53 competition is conserved between different mammalian epithelia. Considering the involvement of p53, mechanical competition may play a major role in cancer.
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Hille, Frank [Verfasser]. "Investigation of Spacer Acquisition Mechanisms in Type V-A CRISPR-Cas Systems / Frank Hille." Berlin : Humboldt-Universität zu Berlin, 2020. http://d-nb.info/1215570341/34.
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Bates, Nicholas Robert. "Investigation of the physical and biological controls of the oceanic CO2 system in the Sargasso Sea." Thesis, University of Southampton, 1995. https://eprints.soton.ac.uk/358400/.
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Charles, Sarah Lucy. "Investigations into the physiological actions of nitric oxide in the nervous systems of the ring dove and the rat." Thesis, University of Central Lancashire, 1996. http://clok.uclan.ac.uk/18873/.
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A wealth of physiological and pathophysiological roles have been ascribed to nitric oxide (NO) in the nervous system. Many of these seem diverse and unrelated. However, the widespread role NO has to play in modulation of the nervous system suggests an underlying common and fundamental role. The ongoing popularity of NO research has lead to a mass of literature on the subject yet great controversy still prevails as to the true physiological function of NO in the nervous system. In this thesis, the existing literature has been critically evaluated. Histological, autoradiographic and electrophysiological research approaches have been used to investigate further the physiological role for NO in the nervous system in the hope of explaining some of the controversy that still prevails. Due to an unforseen move, two different models have been used during the investigations, the brain and the pituitary gland of the ring dove (Streptopelia risoria) and the superior cervical ganglion (SCG) of the rat. The histological NADPH-diaphorase (NDP) technique was used to show the distribution of the NO synthesising enzyme nitric oxide synthase (NOS) for the first time in the ring dove brain. Previously, the NDP distribution has been mapped in the brain of other species and found to be distinct from all other known neurotransmitters. NADPH-diaphorase is a known marker of the mammalian pontine cholinergic reticular formation. However, findings presented here strongly suggest that the distribution of NOS is far more widespread in structures comprising the avian reticular formation. The distribution of the NO synthesising enzyme and the freely diffusible property of NO make it a highly attractive messenger molecule that might coordinate the spatially distinct structures of the reticular formation enabling them to act as one functional unit. Nitric oxide is released following NMDA receptor activation. Both the presence and the distribution of this component of the NO-cGMP pathway have been shown here for the first time in the ring dove brain. This has been achieved using receptor autoradiography of the [3H]-MK-801, a ligand that binds to the NMDA receptor channel. The ring dove is a popular model for the study of behaviour changes and the hormonal control of the avian breeding cycle. Here, a study was set up to compare regional [3H]-MK-801 ligand binding in the brains of non breeding birds (naive) and first time breeders. Quantitative receptor autoradiographic analysis of the ligand binding has shown that significant regional binding changes occur during the breeding cycle of the ring dove. This result implies that the NO-cGMP pathway might have a physiological role in the breeding cycle of the dove and that further investigation of this matter would be worth undertaking. Here, histological and electrophysiological approaches using the rat SCG model have been employed to investigate the role of NO-cGMP pathway in modulation of synaptic transmission. Histological mapping of the pathway components has reconfirmed that NOS is present in the presynaptic fibres and terminals, but it also suggests that it is restricted to a subpopulation of the principal neurones in the ganglion. Immunocytochemistry of the NO-donor induced cGMP accumulations shows that the satellite cells surrounding the principal neurones are major target sites for NO. This finding is consistent with that seen in the dorsal root ganglion (DRG), and supports a glial target for NO in this tissue. Although, NO-donors depolarise the ganglion and enhance synaptic transmission when extracellularly recording the compound action potential (CAP), intracellular recording did not reveal such clear responses. This difference was attributed to the fact that a subpopulation of the principal neurones receives a NO input. These findings suggest that the SCG is not a particularly efficient model for intracellular investigation of the NOcGMP pathway. Both intracellular and extracellular investigations into involvement of NO-cGMP pathway in ganglionic L TP suggest that it does not have a role to play in long-term potentiation (L TP) of synaptic efficacy in this tissue. Here, intracellular and extracellular approaches agreed. Further to this, extracellular recording ofLTP of the CAP supported the involvement of the pathway in a short term enhanced synaptic efficacy by a mechanism distinct from L TP. Moreover, it has been shown here that L TP of the epsp can be reliably produced in the SCG. It is therefore suggested that the SCG is a suitable preparation for both intracellular and extracellular investigation of LTP at the peripheral synapse.
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Muhammad, Nefertiti. "INVESTIGATING THE PORE COMPOSITION OF THE CHLOROPLAST TWIN ARGININE TRANSPORT SYSTEM." Miami University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=miami1543609222190572.
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Kis, Zoltan. "Development of novel synthetic and systems biology tools for investigating and obviating the effect of atherogenic blood flow on vascular cells." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/33249.
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The high mortality from cardiovascular diseases is caused by atherosclerosis. Atherosclerosis develops due to multiple factors, including biomechanical factors, such as shear stress generated by the flow of blood on the inner lining of blood vessels. In order to tackle this serious life-threatening condition, we aimed to develop synthetic and systems biology tools for studying the effect of atherogenic shear stress regimes on vascular cells. Our tools could potentially also lead to the identification of drug targets and of drug candidates against cardiovascular disease. The first tool that we developed is a network of genes consisting of a shear stress sensor module, a reporter module and a linker module which signals from the sensor to the reporter module. This circuit is capable of processing the shear stress or ligand activation signal into a fluorescent readout, allowing screening for drug candidate compounds that modify the activity of the shear stress sensor. Instead of the reporter module, the gene network could be coupled to therapeutic genes in order to express these genes under atherogenic shear stress conditions. Our second developed tool is a flow chamber which facilitates exposure of vascular cells to linearly increasing shear stress along the length of the channel floor for in vitro cellular biomechanical studies. This device outperforms currently available linear shear stress inducing devices in terms of the magnitude of shear stress range, linearity of shear stress along the channel length, and the large sampling area granted by the uniformity of shear stress across the channel width. The third tool that we developed is an electroporation and flow device capable of inserting genetic material into primary vascular cells in their adherent state, exposing these cells to fluid flow. This device allows investigations into cardiovascular mechanotransduction pathways under relevant physiological flow conditions.
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Satler, Jordan. "Do ecological communities co-diversify? An investigation into the Sarracenia alata pitcher plant system." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1467855198.
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Dunn, Karen. "Investigation into the Nrf2 signalling system dynamics and its crosstalk with the NF-κB signalling pathway at the single cell level." Thesis, University of Liverpool, 2013. http://livrepository.liverpool.ac.uk/16373/.
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The Nrf2 system plays an important role in the regulation of the redox state of the cell. Protection from oxidative and genotoxic damage is achieved by Nrf2-mediated expression of antioxidant and phase-2 detoxification enzymes. The Nrf2 network topology possesses several feedback mechanisms that could play a role in the dynamics of Nrf2 protein. To address this issue, a range of molecular reagents were generated, including plasmid and Bacterial Artificial Chromosome (BAC) expression systems. These allowed the localization and dynamics of Nrf2 to be analysed at near endogenous expression levels. Specifically, we generated and characterised an Nrf2-venus BAC stable SK-N-AS cell line using state-of-the-art molecular biology and microscopy techniques including Fluorescence In Situ Hybridization (FISH) and Fluorescence Correlation Spectroscopy (FCS). Single cell imaging showed oscillations in Nrf2 levels that were heterogeneous amongst the cell population. These novel tools were applied to investigate the reciprocal effects of crosstalk between the Nrf2 and NF-κB signalling networks. These interactions have direct implications for a range of physiological and pathological processes including cancer, ageing and inflammation. While systems biology approaches have made important contributions to our understanding of the NF-κB dynamics, we still have a relatively poor understanding of how this system integrates with other regulatory networks in different cell types. Single live-cell imaging of the dynamics of these pathways suggested reciprocal dynamics, where stimulation of Nrf2 delays rather than inhibits NF-κB dynamics and Nrf2 oscillates out-of-phase to robust p65 oscillations. The tools developed during this study will be an important resource for further investigations of the Nrf2 system and its crosstalk with other signalling pathways. The BAC could also be used to generate novel reporter transgenic mice. Such data could be used to inform predictive mathematical models, which have direct implications for rational drug evaluation in either normal or diseased cells and tissues.
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Lalovic, Aleksandra. "Genetic studies of suicidal behaviour : investigation of genes involved in the serotonergic system and cholesterol metabolism." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79022.
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Substantial evidence has accumulated indicating that a genetic predisposition underlies suicidal behaviour, and that the mediating mechanism may involve decreased serotonergic activity and/or low serum cholesterol level. Most association studies have focused on genes involved in the serotonergic tophan hydroxylase (TPH) gene has been extensively examined and conflicting findings have been reported. The meta-analysis presented here was conducted to clarify the role of the TPH gene in suicidal behaviour. No overall association between the TPH gene and suicidal behaviour was found. A shift in focus to genes related to regulation of cholesterol level may provide useful clues. Thus, five genes encoding proteins involved in cholesterol biosynthesis and metabolism were investigated for a role in suicidal behaviour. No association was detected between any of the genes examined and suicide, suggesting that none of the genes investigated plays a major role in the etiology of suicide. Further studies in a larger sample are necessary to exclude possible small genetic effects.
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Gärdes, Astrid [Verfasser]. "A bilateral model system for the molecular investigation of diatom-bacteria interactions / Astrid Gärdes." Bremen : IRC-Library, Information Resource Center der Jacobs University Bremen, 2010. http://d-nb.info/1035032279/34.
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Boopathy, Sivakumar. "Investigating Structural and Functional Defects in ALS-causing Profilin 1 Variants." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/923.
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Mutations in profilin 1 (PFN1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that targets motor neurons. PFN1 is a 15 kDa protein that is best known for its role in actin dynamics. However, little is known about the pathological mechanisms of PFN1 in ALS. In this dissertation, it is demonstrated that certain familial ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in neuronal cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported functional defects in cell-based assays. The source of this destabilization is illuminated by the crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of one ALS variant and predicting a non-surface exposed cavity in another. Functional biochemical experiments point to abnormalities in actin filament nucleation and elongation caused by PFN1 mutants. In HeLa cells, PFN1 is essential for the generation of actin-rich filopodia and expression of mutant PFN1 alters filopodia density further supporting a pathogenesis mechanism involving actin cytoskeleton. Taken together, this dissertation infers that the pathogenesis of ALS due to mutations in PFN1 can be mediated at least by two possibly related mechanisms, a destabilization of the native PFN1 structure and an impact on the actin assembly processes.
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Boopathy, Sivakumar. "Investigating Structural and Functional Defects in ALS-causing Profilin 1 Variants." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/923.
Full textAbstract:
Mutations in profilin 1 (PFN1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that targets motor neurons. PFN1 is a 15 kDa protein that is best known for its role in actin dynamics. However, little is known about the pathological mechanisms of PFN1 in ALS. In this dissertation, it is demonstrated that certain familial ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in neuronal cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported functional defects in cell-based assays. The source of this destabilization is illuminated by the crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of one ALS variant and predicting a non-surface exposed cavity in another. Functional biochemical experiments point to abnormalities in actin filament nucleation and elongation caused by PFN1 mutants. In HeLa cells, PFN1 is essential for the generation of actin-rich filopodia and expression of mutant PFN1 alters filopodia density further supporting a pathogenesis mechanism involving actin cytoskeleton. Taken together, this dissertation infers that the pathogenesis of ALS due to mutations in PFN1 can be mediated at least by two possibly related mechanisms, a destabilization of the native PFN1 structure and an impact on the actin assembly processes.
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Müller, Britta Katrin [Verfasser]. "Systems biological investigation of aerobic and anaerobic aromatic catabolism in the bacterium Aromatoleum aromaticum EbN1 / Britta Katrin Müller." Braunschweig : Technische Universität Braunschweig, 2016. http://d-nb.info/1175818380/34.
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Bakhti, Mostafa. "Investigation of myelin membrane adhesion and compaction in the central nervous system." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2012. http://hdl.handle.net/11858/00-1735-0000-000D-FB5C-B.
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Myelin ist eine mehrschichtige Membran, die die Axone in peripheren (PNS) und
Zentrale Nervensystem (ZNS) umhüllt. Die Bildung und Anordnung dieser Struktur ist ein mehrstufiger Prozess, der durch eine Vielzahl extrazellulärer Faktoren reguliert wird. Im ZNS wird Myelin von Oligodendrozyten gebildet. Während der Entwicklung differenzieren die Vorläufer dieser Zellen zu reifen Oligodendrozyten aus. Nachdem sie das geeignete Signal aus ihrer Umgebung erhalten haben, beginnen die Oligodendrozyten die Axone mit Myelinmembranen einzuhüllen. Allerdings sind die Signale, die diesen Prozess initiieren unbekannt. Mit dieser Arbeit zeigen wir, dass Oligodendrozyten kleine Mikrovesikel - so genannte Exosomen - in den extrazellulären Raum freisetzen, welche die terminale Differenzierung von Oligodendrozyten und die anschließende Myelinbildung verhindern. Es konnte gezeigt werden, dass diese inhibitorische Wirkung durch die Aktivität der RhoA-ROCK-Signalkaskade vermittelt wird. Bemerkenswerterweise war die Exosomenfreisetzumg durch Oligodendrozyten signifikant reduziert, wenn die Zellen mit konditioniertem Medium von Neuronen inkubiert wurden. Unsere Ergebnisse legen nahe, dass Exosomen, die von Oligodendrozyten produziert werden, Zellen in einem pre-myelinisierten Stadium halten, während die Sekretion von Exosomen in Gegenwart neuronaler Signale reduziert wird und autoinhibitorische Signale aufgehoben werden. Somit können Neuronen die Bildung und Freisetzung von Exosomen regulieren, welche von Oligodendrozyten freigesetzt werden, um die Biogenese und Assemblierung der Myelinmembran zu koordinieren.
Im zweiten Teil der Arbeit wurde die Frage, wie die Kompaktierung des Myelins vermittelt wird, erörtert. Während bekannt ist, dass MBP die Interaktion zwischen Myelinmembranen von cytoplasmatischer Seite aus organisiert, ist der zugrundeliegende molekulare Mechanismus der Interaktion zwischen den äußeren Membranen nach wie vor unklar. Im Allgemeinen erfordert die Interaktion zwischen zwei gegenüberliegenden Membranen die Expression von Adhäsionsmolekülen und die Entfernung von repulsiven Komponenten.
Daher untersuchten wir die Rolle des Proteolipid-Proteins (PLP), als mutmaßliches Adhäsionsmolekül, und die Glykocalix, als repulsive Struktur während der Myelinkompaktierung im ZNS. Wir analysierten die Adhäsion von aufgereinigten Myelinpartikeln mit den primären Oligodendrozyten, um die Wechselwirkung zwischen den Myelinschichten zu imitieren. Mit diesem System haben wir gezeigt, dass PLP die Adhäsionsfähigkeit der Myelinmembran erhöht. Mittels Single Particle Force-Spektroskopie fanden wir außerdem heraus, dass PLP die physikalische Stabilität von Myelin verbessert. Zusätzlich beobachteten wir eine signifikante Reduzierung in der Glykokalix während der Oligodendrozytenreifung, die mit einer Zunahme in ihrer Oberflächenaffinität gegenüber den Myelinpartikeln korreliert. Weitere Analysen zeigten, dass die negative Ladung der Zuckeranteile, hauptsächlich der Sialinsäure, für die Verringerung der Myelinadhäsion verantwortlich ist. Daher schlagen wir vor, dass die Adhäsionseigenschaften von PLP zusammen mit der Reduzierung der Glykokalyx, die Adhäsion der Myelinmembran und die Kompaktierung im ZNS organisieren.
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Mussard, Chase W. "In Vitro Investigation of the Effect of Exogenous Ubiquitin on Processes Associated with Atherosclerosis." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/honors/327.
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Atherosclerosis, characterized by the build-up of cholesterol, immune cells and cellular debris within arterial walls, is accelerated following myocardial infarction by poorly understood mechanisms. Ubiquitin, a small, well-studied intracellular protein involved in protein turnover via the proteasome pathway, has recently been shown to exert extracellular effects on cardiac myocytes, in vitro, and in mice undergoing myocardial remodeling. This study investigates the potential role of extracellular ubiquitin in atherosclerosis by determining its effects on two critical atherosclerotic processes: the migration of vascular smooth muscles cells and the uptake of modified LDL by monocyte/macrophages in foam cell formation. In the presence of ubiquitin, smooth muscle cell migration was accelerated and foam cell formation was enhanced, suggesting that ubiquitin has an active role in atherosclerosis.
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Robinson, Jeffrey G. "An investigation of the seasonal and spatial occurrence of coliform bacteria in a distribution system." Virtual Press, 1992. http://liblink.bsu.edu/uhtbin/catkey/845953.
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Bacteriological data from 1980 to 1991 were reviewed to determine whether coliform bacteria occurred seasonally and spatially within a midwestern city's distribution system. Coliform bacteria are used as microbiological indicator organisms to determine if a public water supply is safe for consumption. The public water_ distribution system examined had at least a twelve year history of the presence of coliform bacteria. Previous investigations have described the occurrence of the coliform bacteria as sporadic because there were no apparent patterns to their presence. An analysis of bacteriological data has not previously been performed to specifically detect seasonal and spatial occurrences of coliform bacteria.This study attempted to determine if seasonal or spatial patterns of coliform occurrences exist within the in the dominant coliform species. Data indicate that the highest percentage of coliform positive samples occurred in the summer, followed by fall, then winter, with spring having the lowest percentage of coliform positive samples. While Enterobacter cloacae was the dominant coliform species during the spring, summer and fall, Klebsiella oxytoca was the dominant coliform during the winter. Coliform occurrence throughout the distribution system was variable among the 43 sample sites. The percentage of positive samples from the various sites ranged from 0% to 10.5%. The five sites with the highest percentage of coliform positive samples were at the extremities of the distribution system. E. cloacae was dominant at 88% of the sites. K. oxytoca was dominant at 9% sites, which typically had a low percentage of coliform positive samples.Department of Biology
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Steyn, Natassja Lise. "Investigating the localisation of the ESX-3 secretion system in Mycobacterium smegmatis." Thesis, Stellenbosch : Stellenbosch University, 2012. http://hdl.handle.net/10019.1/71959.
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Thesis (MScMedSc)--Stellenbosch University, 2012.ENGLISH ABSTRACT: Mycobacterium tuberculosis is a pathogenic organism that infects a third of the world’s population and causes approximately 2 million deaths per year. Extensive research has been done on this pathogen, however our knowledge of the mechanisms of pathogenicity remain limited. The M. tuberculosis genome contains five ESAT-6 gene cluster regions, ESX-1 to 5, which encode specialized type VII secretion systems. These secretion systems are known to secrete members of the ESAT-6/CFP-10 and PE/PPE protein families, some of which contribute to the pathogenicity and phagosomal escape of the pathogen. ESX-3 has been shown to be essential for in vitro growth and survival of M. tuberculosis. The expression of ESX-3 in M. tuberculosis is regulated by IdeR and Zur, in response to intracellular iron and zinc concentrations, respectively. Interestingly, ESX-3 is not essential for the growth and survival of the saprophytic organism M. smegmatis. In this study, we aimed to identify the subcellular localisation of the individual components of the ESX-3 secretion system in the non-pathogenic, fast-growing organism M. smegmatis. The esx conserved component (ecc) genes from ESX-3 were expressed from the episomal expression vector pDMNI as fusion proteins with green fluorescent protein (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3), MSMEG_0623 (eccD3) and MSMEG_0626 (eccE3) were successfully cloned into pDMNI and expression of fusion proteins was confirmed by Western blotting for MSMEG_0615-GFP, MSMEG_0616-GFP and MSMEG_0626-GFP in M. smegmatis. In the M. smegmatis ESX-3 knock-out (with MSMEG_0615 to MSMEG_0626 deleted) expression was confirmed for MSMEG_0615-GFP and MSMEG0626-GFP. Fluorescent microscopy determined that MSMEG_0615-GFP localised to a single mycobacterial pole in both strains. MSMEG_0616-GFP and MSMEG_0626-GFP were found to be membrane associated in M. smegmatis, while MSMEG_0626-GFP was found to be membrane associated in the M. smegmatis ESX-3 knock-out. The unipolar localisation of MSMEG_0615-GFP suggests that the assembled ESX-3 secretion system apparatus is situated at a single pole in M. smegmatis. Therefore, we hypothesize that MSMEG_0615 might act as a recruiter protein that is involved in the assembly of ESX-3 at the mycobacterial pole.
AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is ‘n patogene organisme wat ‘n derde van die wêreld se bevolking infekteer en eis jaarliks 2 miljoen lewens deur tuberkulose. Ten spyte van uitgebreide navorsing, is daar min kennis oor die meganismes van patogenisiteit van hierdie organisme. Die M. tuberculosis genoom bevat vyf duplikasies van die ESAT-6 geen groep gebiede, ESX-1 tot 5, wat kodeer vir gespesialiseerde Tipe VII sekresie sisteme. Hierdie sekresie sisteme is bekend vir die sekresie van lede van die ESAT-6/CFP-10 en PE/PPE proteïen families, waarvan sommige bydra tot die patogenisieit en fagosomale ontsnapping van hierdie organisme. ESX-3 is noodsaaklik vir die in vitro groei en oorlewing van M. tuberculosis. Die uitdrukking van ESX-3 in M. tuberculosis word gereguleer deur IdeR en Zur in reaksie op intrasellulêre yster en sink konsentrasies, onderskeidelik. ESX-3 word nie benodig vir die groei en oorlewing van die saprofitiese organisme M. smegmatis nie. Hierdie studie was gemik om die sub-sellulêre lokalisering van ESX-3 te identifiseer in die niepatogeniese en vinnig-groeiende organisme, M. smegmatis. Die “esx conserved component” (ecc) gene van ESX-3 is uitgedruk vanaf die episomale uitdrukkingsvektor pDMNI as gekombineerde proteïene met die groen fluoreserende proteïen (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3), MSMEG_0623 (eccD3) en MSMEG_0626 (eccE3) is suksesvol gekloneer en die uitdrukking van die gekombineerde proteïene is bevestig deur Western oordrag vir MSMEG_0615-GFP, MSMEG_0616-GFP en MSMEG_0626-GFP in M. smegmatis. In die M. smegmatis ESX-3 uitklopmutant (met MSMEG_0615 tot MSMEG_0626 uitgeslaan) is uitdrukking bevestig vir MSMEG_0615-GFP en MSMEG0626-GFP. Fluoresensie mikroskopie het bepaal dat MSMEG_0615-GFP gelokaliseer is by ‘n enkele mikobakteriese pool in beide stamme. MSMEG_0616-GFP en MSMEG_0626-GFP was membraan-geassosieerd in M. smegmatis, terwyl en MSMEG_0626-GFP geassosieer het met die membraan in die M. smegmatis uitklopmutant. MSMEG_0615 het gelokaliseer by ‘n enkele pool in M. smegmatis en dit dui aan dat die saamgestelde ESX-3 sekresie sisteem apparaat slegs by ‘n enkele pool voorkom in M. smegmatis. Ons hipotiseer dat MSMEG_0615 dalk mag optree as ‘n werwer proteïen wat betrokke is by die samestelling van die ESX-3 sekresie sisteem by die mikrobakteriese pool.
Stellenbosch University
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Ternes, Svenja [Verfasser]. "Investigation of the endocannabinod system using in vivo and in vitro models / Svenja Ternes." Bonn : Universitäts- und Landesbibliothek Bonn, 2013. http://d-nb.info/1054691975/34.
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Whelan, Jillian Nicole. "Investigation of Respiratory Syncytial Virus Structural Determinants and Exploitation of the Host Ubiquitin System." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6431.
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Respiratory syncytial virus (RSV) is a globally circulating, non-segmented, negative sense (NNS) RNA virus that causes severe lower respiratory infections. This study explored several avenues to ultimately expand upon our understanding of RSV pathogenesis at the protein level. Evaluation of RSV intrinsic protein disorder increased the relatively limited description of the RSV structure-function relationship. Global proteomics analysis provided direction for further hypothesis-driven investigation of host pathways altered by RSV infection, specifically the interaction between the RSV NS2 protein and the host ubiquitin system. NS2 primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. The goal was to identify NS2 residues important for interaction with the host ubiquitin system, as well as describe the mechanism by which NS2 induces host protein ubiquitination. Bioinformatics analysis provided a platform for development of loss-of-ubiquitin-function NS2 mutants. Combining critical mutations as double or triple NS2 ubiquitin mutants displayed an additive effect on reducing NS2-induced ubiquitination. Recombinant RSV (rRSV) containing NS2 ubiquitin mutations maintained their effect on ubiquitin expression during infection in addition to limiting STAT2 degradation activity. NS2 ubiquitin mutants decreased rRSV growth and increased levels of innate immune responses, indicating a correlation between NS2’s ubiquitin function and antagonism of type I IFN to enhance viral replication. Finally, several proteomics strategies were employed to identify specific cellular proteins ubiquitinated by NS2 to further define host-pathogen interactions during RSV infection. This study demonstrates an effective approach for limiting viral protein function to enhance immune responses during infection.
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Park, Minwoo. "2',3'-Cyclic nucleotide 3'-phosphodiesterase : investigation of interaction with Fyn tyrosine kinase during the development of nervous system, and mitochondrial import of CNP2 isoform." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81423.
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2',3'-Cyclic nucleotide 3'-phosphodiesterase is a protein highly expressed in oligodendrocytes in the central nervous system, and it is believed to be an important regulator of myelination. In this report, two aspects of CNP are investigated, each introduced in their own chapters.First, a possible interaction of CNP with Fyn tyrosine kinase during myelination is investigated. Fyn is an important factor known to be active during the process of myelination, and CNP contains a number of possible tyrosine phosphorylation sites. Furthermore, their presence in an isolated domain of cell membrane called lipid rafts, further strengthened the possibility of interaction. However, no evidence was found which could support the possibility, neither in new born mouse brain nor in in-vitro experiment using transfected cells expressing Fyn and CNP.
Secondly, the role of CNP2 isoform in mitochondria is investigated. CNP2 is found to be associated with mitochondria, and its 20 amino acid segment at the N-terminus possesses characteristics of a mitochondrial import signal. Furthermore, two known sites of serine phosphorylation in the N terminus of CNP2 show influence on mitochondrial localization of the protein. However, results collectively suggests that CNP2 is not imported into mitochondria, as pulse chase did not show the typical processing of what was suspected to be the N-terminal import signal sequence. Furthermore, CNP was degraded when partially purified mitochondria was subjected to protease action, showing that CNP is not enclosed by mitochondrial membranes. Two serine residues at positions 9 and 22 in the N-terminus of CNP2 are likely phosphorylated by both PKA and PKC, and is responsible for the decrease in mitochondrial localization of CNP2.
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Kwok, Chiu Wai [Verfasser], and Uwe [Akademischer Betreuer] Strähle. "In vitro cell culture systems for the investigation of the morphogen Sonic hedgehog (Shh) / Chiu Wai Kwok ; Betreuer: Uwe Strähle." Heidelberg : Universitätsbibliothek Heidelberg, 2011. http://d-nb.info/1179782674/34.
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Choi, Sungwook. "Investigation of LIN-28 Function in Somatic Gonadal Development and Fertility, and Characterization of the LIN-28 Isoforms in C. elegans Hermaphrodites." eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/991.
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lin-28 was first characterized as a developmental timing regulator in Caenorhabditis elegans. Loss of lin-28 function (lin-28(lf)) mutants skip the hypodermal cell fates specific to the 2nd larval stage. Here, we studied two aspects of lin-28 which had not yet been investigated.
First, we show that lin-28(lf) mutants exhibit reduced fertility associated with abnormal somatic gonadal morphology. In particular, the abnormal spermatheca-uterine valve morphology of lin-28(lf) hermaphrodites traps embryos in the spermatheca, which disrupts ovulation and causes embryonic lethality. The same genes downstream of lin-28 in the regulation of hypodermal developmental timing also act downstream of lin-28 in somatic gonadal morphogenesis and fertility. Importantly, we find that hypodermal expression, but not somatic gonadal expression, of lin-28 is sufficient for restoring normal somatic gonadal morphology in lin-28(lf) mutants. We propose that the abnormal somatic gonadal morphogenesis of lin-28(lf) hermaphrodites results from temporal discoordination between the accelerated hypodermal development and normally timed somatic gonadal development. Thus, our findings exemplify how a cell-intrinsic developmental timing program can also control proper development of other interacting tissues, cell non-autonomously.
We also investigated the expression patterns and functions of two lin-28 isoforms in C. elegans. Our analysis of spatial expression patterns suggests that lin-28a and lin-28b are co-expressed in diverse tissues. Consistently, neither of isoform specific knock-out mutant, lin-28a(lf) or lin-28b(lf), exhibits defects in hypodermal development, somatic gonad, or fertility, indicating functional redundancy of two isoforms. Our study will contribute to further investigation of lin-28 isoforms by providing the mutants of each isoform as well as the primary analysis of their phenotypes.
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Stephan, Till [Verfasser]. "Investigation of the mitochondrial contact site and cristae organizing system and its role in cristae formation / Till Stephan." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://d-nb.info/1220504513/34.
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Nagel, Maximilian [Verfasser], Marc [Akademischer Betreuer] Spehr, and Ivan [Akademischer Betreuer] Manzini. "Physiological investigation of sensory signaling mechanisms in the mouse accessory olfactory system / Maximilian Nagel ; Marc Spehr, Ivan Manzini." Aachen : Universitätsbibliothek der RWTH Aachen, 2019. http://d-nb.info/1211487830/34.
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