Dissertations / Theses on the topic 'Motility behaviour'

Motility and chemotaxis have been implicated in the process of biofilm formation in a wide range of species. Using a combination of microscopy and image analysis, genetics, microbiology and biochemistry, the initial approach of Rhodobacter sphaeroides cells to a solid surface has been characterised. Interestingly, these data suggest that for R. sphaeroides alterations in motility and swimming behaviour may result in differences in biofilm formation simply by changing the number of cells which reach the surface. This is in contrast to a few other well-studied species where the motility apparatus, the flagellum, has been shown to play an active role in surface sensing and the transition to biofilm growth. Tracking swimming cells and measuring surface attachment revealed that changes in motility affect the ability of cells to attach to a surface, with non-motile cells attaching least and mutants with frequent stops attaching less than smooth swimming cells with few stops. Tracking attaching cells and classifying their method of attachment revealed that flagellar tethering is not essential for R. sphaeroides attachment. Competition assays with fluorescently labelled strains showed that the initial imbalance between motile and non-motile cells remains as microcolonies develop over 48 hours,and the proportion of non-motile cells remains fairly constant. Development on a surface over 48 hours was similar for motile and non-motile strains, including aflagellate strains, once attached. Using parameters calculated by tracking swimming cells to calculate the effective diffusion coefficient in a simple model of cell movement suggested that motion alone could explain the differences in attachment without assuming different cell properties. In particular, aflagellate strains might be hindered from surface attachment by their reduced motility alone. This is interesting since some other bacterial species use the flagellum as a surface sensor.

Microorganisms have evolved in a low Reynolds number environment and have adapted their behaviour to its viscosity. Here, we consider some features of behaviour observed in microorganisms and use hydrodynamic models to show that these behaviours emerge from physical interactions, including hydrodynamic friction, hydrodynamic interactions and mechanical constraints. Swimming behaviour is affected by surfaces and observations of Vibrio cholerae show that it swims near a surface with two distinct motility modes. We develop a model which shows that friction between pili and the surface gives the two motility modes. The model is extended to study the behaviour of bacteria which are partially attached to a surface. Observations of Shewanella constrained by a surface show several different behaviours. The model shows that different degrees of surface constraint lead to different types of behaviour; the flexibility of the flagellar hook and the torque exerted by the flagellar motor also cause different behaviours. Near surface behaviour is important for understanding the initial stages of biofilm formation. Chlamydomonas swims using synchronous beating of its two flagella. A simple model of Chlamydomonas is developed to study motility and synchronisation. This model shows that the stability of synchronisation is sensitive to the beat pattern. Run-and-tumble behaviour emerges when we include intrinsic noise, without the need for biochemical signalling. The model is also used to show how observed responses of the flagella to light stimuli produce phototaxis. Finally we study hydrodynamic synchronisation of many cilia and consider the stability of metachronal waves in arrays of hydrodynamically coupled cilia. This thesis shows that physical interactions are responsible for many behavioural features and that physical models provide a useful technique for exploring open questions in biology.

This thesis investigates bacterial motility from the mechanism permitting individual selfpropulsion to the complex collective flocking motility in Escherichia coli and Bacillus subtilis cells. Understanding bacterial swimming has intrigued scientists for decades and recently there has been a growing interest in collective swimming behaviour. The first part of this thesis reviews the characteristics of E. coli and B. subtilis cells subsequently describing the governing physics and constraints of self-propulsion in the low Reynolds regime. The second part of this thesis presents three self-contained experimental sections, examining individual swimming in non-conventional body shaped cells and subsequently focusing on concentrated bacterial swimming in normal cells. We first investigated motility in mutant spherical E. coli cells KJB24 motivated by simulations, which often model bacteria as self-propelled spheres. Somewhat unexpectedly these spherical cells do not exhibit runs and tumbles but diffuse slower than expected. As an introduction to working with microbiology and to familiarise with microbiology techniques we investigated why these spherical cells do not swim. Secondly we investigated how cellular motility varies as a function of body length by inhibiting cell division in wild-type E. coli with cephalexin; which remained motile despite body elongation. Fluorescent flagella visualization provided evidence of multiple bundle formations along the lateral walls as a mechanism to sustain motility. The average swimming velocity, body and flagella rotation rates, the number of flagella and number of flagella bundles were extracted experimentally as a function of length. The extracted experimental parameters for normal sized cells were consistent with Purcell’s model. We explored simple adaptations and scaling of this model to describe motility for filamentous cells, which agrees with experimental values. The main focus is on collective behaviour of B. subtilis by examining the onset from individual swimming to collective motility using time-lapse microscopy. Results demonstrated a smooth transition where cells self-organize into domains expanding rapidly by recruiting cells. We present advancements in B. subtilis fluorescent flagella staining which revealed unexpected multiple flagella bundle arrangements during runs, contradictory to general conjectures. Novel visualisation of flagella filaments during reversal events is presented in both E. coli and B. subtilis cells, providing experimental evidence for complex flagella ‘flipping’. Cellular reversal is hypothesized as a mechanism for quorum polarity facilitating collective swimming. We present novel flagella imaging in the setting of collective behaviour showing evidence to support quorum polarity. Subsequently we extracted the run length distributions of cells as a function of concentration, yielding a decreasing trend with increasing concentration. Using particle tracking we quantitatively extracted the mean squared displacement of swimming cells versus passive tracers at different concentrations during collective swimming, these novel results are discussed in respect to recent simulations. These presented experiments provide new insights into collective behaviour improving current understanding of this phenomenon.

Most eukaryotic cell motility relies on plasma membrane protrusions, which depend on the actin cytoskeleton and its tight regulation. The SCAR/WAVE complex, a pentameric assembly comprising SCAR/WAVE, Nap1, CYFIP/Pir121, Abi and HSPC300, is a key driver of actin-based protrusions such as pseudopods. SCAR/WAVE is thought to activate the Arp2/3 complex, a crucial actin nucleator, after being itself activated by upstream signals such as active Rac1. Despite recent progress on the study of the SCAR/WAVE complex, its regulation is still incompletely understood, with Nap1’s role being particularly enigmatic. Upon screening for potential Nap1 binding partners in the social amoeba Dictyostelium discoideum – a well established model organism in the study of the actin cytoskeleton and cell motility – we found FAM49, a ~36 kDa protein of unknown function which is highly conserved in Metazoa (animals) and evolutionarily closer species such as D. discoideum. Interestingly, D. discoideum’s FAM49 and its homologs contain a DUF1394 domain, which is also predicted in CYFIP/Pir121 proteins and most likely involved in their direct binding to active Rac1, which in turn contributes to SCAR/WAVE’s activation. FAM49’s unknown role, apparent high degree of conservation and potential connections to SCAR/WAVE and Rac1 persuaded us to start investigating its function and biological relevance in D. discoideum, leading to the work presented in this thesis. Several pieces of our data collectively support a function for FAM49 in modulating the protrusive behaviour, and ultimately motility, of D. discoideum cells, as well as a regulatory link between FAM49 and Rac1. FAM49’s involvement in protrusion regulation was first hinted at by our observation that GFP-tagged FAM49 is enriched in pseudopods. The possibility of a link with Rac1 was then strengthened by two additional observations: first, pseudopodial GFP-FAM49 is substantially co-enriched with active Rac, both showing fairly comparable spatio-temporal accumulation dynamics; second, when dominant-active (G12V) Rac1 is expressed in cells, it triggers the recruitment and persistent accumulation of GFP-FAM49 at the plasma membrane, where both become highly co-enriched. We subsequently determined that fam49 KO cells differ from wild-type cells in the way they protrude and move, as assessed in under-agarose chemotaxis assays. In particular, our data indicate that fam49 KO cells tend to display a lower degree of global protrusive activity, their protrusions extend more slowly and are less discrete, and the cells end up moving at lower speeds and with higher directional persistence. This phenotype was substantially rescued by FAM49 re-expression. While re-expressing FAM49 in fam49 KO cells we generated putative FAM49 overexpressor cells; compared to wild-type cells, they displayed atypically thin pseudopods and what seemed to be an excessively dynamic, and perhaps less coordinated, protrusive behaviour. Additional data in our study suggest that pseudopods made by fam49 KO cells are still driven by SCAR/WAVE, which is clearly not being replaced by WASP (as is now known to be the case in D. discoideum cells lacking a functional SCAR/WAVE complex). Nonetheless, the peculiar dynamics of those pseudopods imply that SCAR/WAVE’s activity is regulated differently when FAM49 is lost, though it remains to be determined how. This thesis is the first report of a dedicated study on FAM49 and lays the foundation for future research on it.

This work explores bacterial motility from the mechanisms of propulsion of an individual cell to the complex behavior of collective motility. The shear modulus of bacterial flagella was measured by stretching isolated flagella with an optical trap and by measuring force extension curves of the stretched flagella shedding light onto the me-chanics involved in the motility of single micro-organisms. Experiments in concentrated suspensions of bacteria show collective behavior with large scale mixing on a time and length scale greater than can be understood from the standard model of "run and tumble" motility of a single organism are reported. To further understand the transition from individual to collective motility a novel form of motility where an individual bacterium can reverse direction without changing cell orientation is reported here. These experiments further the understanding of bacterial motility.

The effect of intracerebroventricular (ICV) injection of corticotrophin releasing hormone (CRH) and related compounds on appetite, behavior, and gastric motility in lines of chickens, one selected for low body weight (LWS) and the other high body weight (HWS), was determined. Nucleotide sequence and expression patterns of the CRHr2 receptor, involved in appetite regulation, were also determined. Some individuals of the LWS line are anorexics and many die simply from not eating while some individuals in the HWS line are compulsive eaters and exhibit obesity. CRH is a 41 residue peptide that initiates an organismâ s stress response and is a potent inhibitor of appetite. An ICV injection of CRH dose-dependently decreased feed intake in both lines but did not effect water intake. When CRH receptor antagonists were ICV injected an increase in feed intake in the LWS line but not in the HWS line was observed, however the appetite reducing effect of CRH was attenuated in the HWS line but not in the LWS line. The LWS line has higher concentration of corticosterone than does the HWS line. In both lines at all times treatment with CRH caused an increase in locomotion and no CRH-treated chicks from either line slept post injection. Chicks from the LWS line that were treated with CRH exhibited other anxiety related behaviors sooner than the HWS line. The LWS line showed a liner increase in crop emptying time as the dose of ICV CRH increased. The HWS line responded with a quadratic dose response to CRH treatment. Polymorphisms in the CRHr2 receptor were found in both lines in the same positions, thus we concluded these differences do not significantly contribute to body weight differences. However, differences detected in expression patterns between lines for the CRHr2 receptor may contribute to their different body weights. We conclude that differences in the CRH system, its concentrations and differential receptor action, of these two lines may be partly responsible for their altered body weight phenotype.
Ph. D.

Transitioning cattle from a high-forage to a high-concentrate diet increases the risk for ruminal acidosis and is often related to decreased feed intake, which compromises animal health and performance. Since control of feed intake and rumen motility are closely related, we hypothesized that a reduction in rumen motility may be associated with a reduction in feed intake during this transition. Computer programs were created to analyze feed disappearance and rumen pressure data for feeding behavior as well as identification and characterization of rumen contractions, respectively. This method enabled timely analysis of large datasets and removed subjectivity associated with manual analysis. In the second part of this series, cattle were moderately transitioned from a 70% to a 90% concentrate diet, and SARA was induced. Although, reductions in feed intake were modest, on day 2 of high-grain feeding, animals slowed feed consumption rate and displayed a reduction in rumen contraction frequency, amplitude, and duration. Next, an abrupt transition from 50% to 90% concentrate was used to induce ruminal acidosis and cause some animals to stop eating. The abrupt increase in dietary concentrate was also associated with reductions in rumen motility. Patterns of ruminal pH, viscosity, and motility changes were related to when cattle reduced feed intake. Endotoxin quantification in blood samples from the ruminal vein, portal vein, and mesenteric artery suggested the point of endotoxin translocation into blood was across the ruminal epithelium. Additionally, the greater the concentration of endotoxin in the plasma, the more likely animals were to go “off-feed.” By understanding the physiological and behavioral mechanisms by which cattle adapt to high-grain diets, we can improve animal health and performance through these diet transitions.

Cell-substrate interactions influence various cellular processes such as morphology, motility, proliferation and differentiation. Actin dynamics within cells have been shown to be influenced by substrate stiffness, as NIH 3T3 fibroblasts grown on stiffer substrates tend to exhibit more prominent actin stress fiber formation. Circular dorsal ruffles (CDRs) are transient actin-rich ring-like structures within cells, induced by various growth factors, such as the platelet-derived growth factor (PDGF). CDRs grow and shrink in size after cells are stimulated with PDGF, eventually disappearing ten of minutes after stimulation. As substrate stiffness affect actin structures and cell motility, and CDRs are actin structures which have been previously linked to cell motility and macropinocytosis, the role of substrate stiffness on the properties of CDRs in NIH 3T3 fibroblasts and how they proceed to affect cell behavior is investigated. Cells were seeded on Poly-dimethylsiloxane (PDMS) substrates of various stiffnesses and stimulated with PDGF to induce CDR formation. It was found that an increase in substrate stiffness increases the lifetime of CDRs, but did not affect their size. A mathematical model of the signaling pathways involved in CDR formation is developed to provide insight into this lifetime and size dependence, and is linked to substrate stiffness via Rac-Rho antagonism. CDR formation did not affect the motility of cells seeded on 10 kPa stiff substrates, but is shown to increase localized lamellipodia formation in the cell via the diffusion of actin from the CDRs to the lamellipodia. To further probe the influence of cell-substrate interactions on cell behavior and actin dynamics, a two dimensional system which introduces a dynamically changing, reversible and localized substrate stiffness environment is constructed. Cells are seeded on top of thin PDMS nano-membranes, and are capable of feeling through the thin layer, experiencing the stiffness of the polyacrylamide substrates below the nano-membrane. The membranes are carefully re-transplanted on top of other polyacrylamide substrates with differing stiffnesses. This reversible dynamic stiffness system is a novel approach which would help in the investigation of the influence of reversible dynamic stiffness environments on cell morphology, motility, proliferation and differentiation in various cells types.

Suspensions of the aerobic bacteria {\it Bacilus subtilis} develop patterns and flows from the interplay of motility, chemotaxis and buoyancy.In sessile drops, such bioconvectively driven flows carry plumes down the slanted meniscus and concentrate cells at the drop edge, while in pendant drops such self-concentration occurs at the bottom.These dynamics are explained quantitatively by a mathematical model consisting of oxygen diffusion and consumption, chemotaxis, and viscous fluid dynamics.Concentrated regions in both geometries comprise nearly close-packed populations, forming the collective ``Zooming BioNematic'' (ZBN) phase.This state exhibits large-scale orientational coherence, analogous to the molecular alignment of nematic liquid crystals, coupled with remarkable spatial and temporal correlations of velocity and vorticity, as measured by both novel and standard applications of particle imaging velocimetry.To probe mechanisms leading to this phase, response of individual cells to steric stress was explored, finding that they can reverse swimming direction at spatial constrictions without turning the cell body.The consequences of this propensity to flip the flagella are quantified, showing that "forwards" and "backwards" motion are dynamically and morphologically indistinguishable.Finally, experiments and mathematical modeling show that complex flows driven by previously unknown bipolar flagellar arrangements are induced when {\it B. subtilis} are confined in a thin layer of fluid, between asymmetric boundaries.The resulting driven flow circulates around the cell body ranging over several cell diameters, in contrast to the more localized flows surrounding free swimmers.This discovery extends our knowledge of the dynamic geometry of bacteria and their flagella, and reveals new mechanisms for motility-associated molecular transport and inter-cellular communication.

Protection of potable water supplies demands a better understanding of the factors controlling migration of disease causing bacteria in subsurface environments. In this study, the migration behaviour of the waterborne pathogenic microorganisms Escherichia coli O157:H7 and Yersinia enterocolitica was investigated in water saturated granular systems. Both facultative bacteria were grown under aerobic and anaerobic conditions and further acclimatized to a microaerophilic or fully aerated environment for 21 h. Experiments were conducted using laboratory-scale packed columns over controlled extreme dissolved oxygen (DO) concentrations. The observed differences in the transport potential of these pathogens were found to depend strongly on the antecedent growth conditions under the tested environmental settings as well with the environmental DO in certain conditions. Further microbial characterization using cell titrations and FTIR spectroscopy gave a greater insight on the source of the surface charge that was found to dominate the attachment phenomena in sand packed columns. Techniques also revealed a probable role of other cell surface macromolecules (LPS) that could account for non-DLVO behaviour. The results illustrate the importance of considering physicochemical conditions relevant to the natural subsurface environment when designing laboratory transport experiments as evidenced by variations in microbe migration as a function of the DO under growth and acclimation.
Keywords: bacterial adhesion, bacterial transport, DLVO, physicochemical characterization, dissolved oxygen, porous media.

BACKGROUND:Colony stimulating factor-1 (CSF-1) plays an important role in ovarian cancer biology and as a prognostic factor in ovarian cancer. Elevated levels of CSF-1 promote progression of ovarian cancer, by binding to CSF-1R (the tyrosine kinase receptor encoded by c-fms proto-oncogene).Post-transcriptional regulation of CSF-1 mRNA by its 3' untranslated region (3'UTR) has been studied previously. Several cis-acting elements in 3'UTR are involved in post-transcriptional regulation of CSF-1 mRNA. These include conserved protein-binding motifs as well as miRNA targets. miRNAs are 21-23nt single strand RNA which bind the complementary sequences in mRNAs, suppressing translation and enhancing mRNA degradation.RESULTS:In this report, we investigate the effect of miRNAs on post-transcriptional regulation of CSF-1 mRNA in human ovarian cancer. Bioinformatics analysis predicts at least 14 miRNAs targeting CSF-1 mRNA 3'UTR. By mutations in putative miRNA targets in CSF-1 mRNA 3'UTR, we identified a common target for both miR-128 and miR-152. We have also found that both miR-128 and miR-152 down-regulate CSF-1 mRNA and protein expression in ovarian cancer cells leading to decreased cell motility and adhesion in vitro, two major aspects of the metastatic potential of cancer cells.CONCLUSION:The major CSF-1 mRNA 3'UTR contains a common miRNA target which is involved in post-transcriptional regulation of CSF-1. Our results provide the evidence for a mechanism by which miR-128 and miR-152 down-regulate CSF-1, an important regulator of ovarian cancer.

L’homéostasie sodique joue un rôle prépondérant lors de la carcinogénèse prostatique. Cependant, le rôle du canal de fuite sodique NALCN lors de la carcinogénèse prostatique était totalement inconnu. L’objectif principal de cette étude était d’étudier le rôle de NALCN dans la dérégulation de l’homéostasie sodique lors de la tumorigenèse prostatique. Tout d’abord, nous avons montré sur des coupes de tissus prostatiques que l’expression de NALCN est augmentée dans les cancers. De plus, NALCN est surexprimé dans les lignées cancéreuses prostatiques les plus agressives. Nous avons vérifié la fonctionnalité du canal NALCN et de ses protéines associées via des expériences d’imagerie sodique dans ces lignées. Grâce à notre étude, nous montrons que NALCN n’est pas impliqué dans le cycle cellulaire, la viabilité cellulaire, l’apoptose ni la prolifération. En revanche, nous avons démontré que ce canal affecte grandement la motilité, la migration et l’invasion de nos lignées cellulaires cancéreuses prostatiques. Nous avons montré que NALCN et le proto-oncogène Src sont co-localisées dans le cancer de la prostate, notamment au niveau de structures appelées invadopodes. Enfin, nous avons prouvé par des études in vivo que la croissance tumorale et la formation de métastases sont inhibées lorsque l’expression du canal NALCN est diminuée. En conclusion, nos données mettent en évidence que le canal NALCN est un acteur important dans l’augmentation du potentiel métastatique des cellules cancéreuses de prostate à la fois in vitro et in vivo. NALCN peut donc être considéré comme une nouvelle cible thérapeutique permettant de diminuer l’agressivité des cancers de la prostate
Importantly, altered Na+ homeostasis was implemented in prostate carcinogenesis. However, until now nothing was known about a newly discovered Na+ leak channel, NALCN, and its role in prostate malignancy. Therefore, the main objective of this study was to investigate the involvement of NALCN as a potential candidate of the deregulated Na+-dependent signalling mechanisms in prostate cancer. Interestingly, NALCN represented distinctly different localization patterns and levels of expression between human healthy and cancer prostate tissues. Indeed, NALCN was expressed preferentially in highly aggressive prostate cancer cell lines. Na+ imaging results verified on functionality of NALCN channelosome in these cells. Our study also revealed that NALCN was not involved in cell cycle, viability, apoptosis and proliferation, but significantly affected motility, migration and invasiveness of the prostate cancer cells. Interestingly, it was already reported that protooncogene Src family kinase is recruited to the NALCN complex. In this study, we confirmed that NALCN and Src kinase are co-localized in human prostate cancer cells, particularly in the structures that represent invadopodia formation sites. Furthermore, in vivo studies confirmed that NALCN downregulation inhibits tumour growth and metastasis formation. Overall, these data provide evidence on NALCN contribution to the increased metastatic potential of human prostate cancer cells in vitro and in vivo. Therefore, NALCN could provide new perspective molecular target for the disease suppression, in particular at its advanced stages

Der ubiquitär exprimierte, multifunktionale Glucosetransporter GLUT8 gehört zur Klasse III der Familie der passiven Glucosetransporter, die aus insgesamt 14 Proteinen besteht. Die fünf Mitglieder der Klasse IIII unterscheiden sich strukturell leicht von den Mitgliedern der Klasse I und II (Joost und Thorens, 2001). GLUT8 besitzt ein N-terminales Dileucin-Motiv, das Teil eines [DE]XXXL[LI] Motivs ist, welches für die Sortierung des Transporters in späte Endosomen und Lysosomen verantwortlich ist (Augustin et al., 2005). Da bis heute kein Signal identifiziert wurde, das eine Translokation des Transporters zur Plasmamembran auslöst, wird eine intrazelluläre Funktion von GLUT8 vermutet (Widmer et al., 2005). Im Rahmen der vorliegenden Arbeit wurde die intrazelluläre Funktion des Transporters in der Regulation der Glucosehomöostase des Körpers durch Analyse einer Slc2a8-knockout-Maus untersucht. Die homozygote Deletion des Transporters erbrachte lebensfähige Nachkommen, die sich augenscheinlich nicht von ihren Wildtyp-Geschwistern unterschieden. Allerdings wurde bei Verpaarungen heterozygoter Mäuse eine verminderte Anzahl an Slc2a8-/--Nachkommen beobachtet, die signifikant von der erwarteten Mendel’schen Verteilung abwich. Da Slc2a8 die höchste mRNA-Expression in den Testes aufwies und die Überprüfung der Fertilität mittels verschiedener homozygoter Verpaarungen eine Störung der weiblichen Fortpflanzungsfähigkeit ausschloss, wurden die Spermatozoen der Slc2a8-/--Mäuse eingehender untersucht. Als Ursache für die verringerte Anzahl von Slc2a8-/--Geburten wurde eine verminderte Prozentzahl motiler Slc2a8-/--Spermien ermittelt, die durch eine unzureichende mitochondriale Kondensation in den Spermien bedingt war. Diese Veränderung war mit einem reduzierten mitochondrialen Membranpotential assoziiert, was eine verminderte ATP-Produktion nach sich zog. Somit scheint GLUT8 in den Spermien an einem intrazellulären Transportprozess beteiligt zu sein, der einen Einfluss auf die oxidative Phosphorylierung der Mitochondrien ausübt. Im Gehirn wurde Slc2a8 besonders stark im Hippocampus exprimiert, der in der Regulation von körperlicher Aktivität, Explorationsverhalten, Erinnerungs- und Lernprozessen sowie Angst- und Stressreaktionen eine Rolle spielt. Außerdem wurde GLUT8 im Hypothalamus nachgewiesen, der unter anderem an der Regulation der Nahrungsaufnahme beteiligt ist. Die Slc2a8-/--Mäuse zeigten im Vergleich zu ihren Slc2a8+/+-Geschwistern eine signifikant gesteigerte körperliche Aktivität, die zusammen mit der von Membrez et al. (2006) publizierten erhöhten Zellproliferation im Hippocampus auf eine Nährstoffunterversorgung dieses Areals hindeutet. Die Nahrungsaufnahme war in Abwesenheit von GLUT8 nicht verändert, was zusammen mit dem nur geringfügig niedrigeren Körpergewicht der Slc2a8-/--Mäuse eine Funktion von GLUT8 im Glucose-sensing der Glucose-sensitiven Neurone des Gehirns ausschließt. Das leicht reduzierte Körpergewicht der Slc2a8-/--Mäuse ließ sich keinem bestimmten Organ- oder Gewebetyp zuordnen, sondern schien durch eine marginale Gewichtsreduktion aller untersuchten Gewebe bedingt zu sein. Zusammen mit den erniedrigten Blutglucosespiegeln und der anscheinend gesteigerten Lebenserwartung zeigten die Slc2a8-/--Mäuse Symptome einer leichten Nährstoffunterversorgung. GLUT8 scheint daher am Transport von Zuckerderivaten, die während des lysosomalen/endosomalen Abbaus von Glykoproteinen anfallen, beteiligt zu sein. Die so wiederaufbereiteten Zucker dienen dem Körper offenbar als zusätzliche Energiequelle.
The family of facilitative glucose transporters consists of 14 different members in human, which are divided into three classes (Joost and Thorens, 2001). The class III family member GLUT8 contains an amino-terminal dileucine sorting signal, which is part of the highly conserved [DE]XXXL[LI] motif responsible for the localization of GLUT8 in lysosomes and late endosomes (Augustin et al., 2005). To date there is no stimulus known, which translocates the transporter to the plasma membrane, therefore an intracellular function rather than at the cell surface is considered (Widmer et al., 2005). The aim of the present dissertation was to analyze the intracellular role of GLUT8 in the regulation of whole body glucose homeostasis, by the characterization of the corresponding knockout mice (Slc2a8-/-). Slc2a8-/- mice were viable and showed no obvious disparity to their wild-type littermates. However, analysis of the offspring distribution of heterozygous mating provided a reduced number of born Slc2a8-/- offspring which differed significantly from the expected Mendelian distribution. Because Slc2a8 mRNA is expressed at highest levels in the testis and the female Slc2a8-/- mice showed no alterations in fertility, we further investigated the function of Slc2a8-/- spermatozoa. An impaired mitochondrial condensation in the Slc2a8-/- spermatozoa, which was associated with decreased ATP levels resulted in a reduced number of motile Slc2a8-/- sperm, which appeared to be responsible for the reduced number of born Slc2a8-/- offspring. Therefore in sperm cells GLUT8 seems to be important for an intracellular transport process, which exerts an influence on the oxidative phosphorylation in the mitochondria. In the brain Slc2a8 is expressed at highest levels in the hippocampus, which is important for the regulation of physical activity, exploration behaviour, memory and learning as well as anxiety related behaviour. Additionally, GLUT8 was detected in the hypothalamus, which is amongst others involved in the regulation of food intake. The Slc2a8-/- mice showed a significant increase in locomotor activity, which indicates a moderate undersupply of the hippocampus area. According to this finding the group of Membrez et al. (2006) observed a raised cell proliferation in the hippocampus of Slc2a8-/- mice. The fact that no alterations in food intake and only a moderate reduction in body weight was detected in Slc2a8-/- mice, indicates that GLUT8 is not important for the hypothalamic glucose sensing. The marginal decreased body weight of the Slc2a8-/- mice appeared to be associated with a slightly reduced weight of different tissues. Together with the lowered blood glucose concentrations and the apparently enhanced lifespan, the Slc2a8-/- mice showed symptoms of a moderate undersupply compareable to caloric restriction. Thus, we hypothesize that GLUT8 is important for the transport of sugar derivatives which arise during lysosomal/endosomal degradation of glycoproteins. These recycled sugars may serve as an additional energy source in the cell.

Die vorliegende Arbeit untersucht die tatsächliche und potenzielle Mobilität von Kindern aus einer ‎informellen Siedlungsstruktur in einer Megacity des globalen Südens am Fall Ezbet El-Hagganas im ‎Großraum Kairos; die Entwicklung von Mobilitätsgewohnheiten in der Kindheitsphase ‎und bezieht sich dabei nicht nur auf Kinder als Nutzer, sondern berücksichtigt zudem deren ‎unmittelbares soziales Umfeld und den gesamtgesellschaftlichen Kontext. Der theoretische Rahmen für die ‎Untersuchung der Mobilität von Kindern basiert auf Icek Ajzens Theorie des geplanten Verhaltens ‎sowie auf Vincent Kaufmanns Konzeptualisierung des Motilitätsbegriffes. Primärdaten wurden in ‎einer Feldstudie und in Fokusgruppen erhoben. Die Ergebnisse deuteten auf eine hohe Prävalenz der ‎unabhängigen Mobilität von Kindern (child independent mobility, CIM) auf deren am häufigsten zu ‎bewältigenden Strecke (zur Schule) hin, zunächst durch nicht motorisierte Formen der ‎Verkehrsteilnahme, aber weitgehend auch mit Hilfe von verschiedenen formellen wie informellen ‎Nahverkehrsmitteln. Es wurden sozio-kulturelle und sozialpsychologische Einflussfaktoren seitens ‎sowohl Kindern als auch ihren Eltern identifiziert, die die Verkehrsteilnahme von Kindern hemmen ‎oder befördern können. Gleichermaßen wurden hemmende ‎Faktoren untersucht. Die Akzeptanz des Radfahrens von Kindern wurde als Fallbeispiel für ‎unerschlossenes Mobilitätspotenzial untersucht und deutet auf die Prävalenz sozialpsychologischer ‎Faktoren hin, die Entscheidungsfaktoren rationaler und praktischer Natur entgegenstehen und die die ‎Verkehrsmittelnutzung von Kindern mindern; hier benannt als Nutzungslücke (appropriation gap). Die ‎Ergebnisse wurden schließlich in Zusammenhang mit dem Konzept der Verkehrsteilnahme gestellt ‎und erlauben die Diskussion von Implikationen für die politische Ebene und für den Diskurs von ‎Mobilität in benachteiligten Gruppen der Gesellschaft.
This study investigates the nature of children's actual and potential mobility in a case study of an ‎‎informal settlement in a megacity of a developing country; namely Ezbet El-Haggana in Greater Cairo ‎and explores the nature of the childhood ‎phase of developing mobility practices and habits, not only ‎as enabled by children themselves as ‎commuters, but also as enabled by parents and the surrounding ‎community and society. A theoretical framework was constructed through which ‎children's mobility is ‎investigated. It is based on Icek Ajzen's Theory of Planned Behavior and Vincent ‎Kaufmann's ‎conceptualization of Motility. The data was primarily collected through a field survey and ‎focus groups. ‎Results indicated high prevalence of child independent mobility (CIM) in the most ‎frequent trip (to ‎school), not only through active transport but also largely through different formal ‎and informal ‎transport services available. There are socio-cultural and socio-psychological factors ‎among both the ‎parents and the children that constitute the appropriation of mobility options that ‎cater to children, ‎either enabling or inhibiting their mobility. Through the experiences of children's ‎mobility, the acquired ‎skills and adaptive attitudes by both children and parents enhance children's ‎potential mobility ‎compared to children in wealthier communities that may be granted less mobility ‎rights or have less ‎competences among other factors. Inhibiting factors were also ‎investigated; the specific case of ‎acceptance of cycling for children. This exemplified prevalence of socio-psychological factors that ‎overshadow ‎practical and rational aspects of choice; it is articulated here as an appropriation gap. ‎Results were ‎finally associated with the conceptualization of appropriation to discuss implications for ‎policy and for ‎the discourses of mobility in disadvantaged communities.‎

La première partie de ma thèse est consacrée à l’étude de l’auto-organisation de souches génétiquement modifiées de bactéries Escherichia coli. Ce projet, réalisé en collaboration avec des biologistes synthétiques de l’Université de Hong Kong, a pour objectif l’exploration et le décryptage d’un nouveau mécanisme d’auto-organisation dans des colonies bactériennes multi-espèces. Cela a été inspiré par la question fascinante de comment les écosystèmes bactériens comprenant plusieurs espèces de bactéries peuvent s’auto-organiser dans l’espace. En considérant des systèmes dans lesquels deux souches de bactéries régulent mutuellement leurs motilités, j’ai pu montrer que le contrôle de densité réciproque est une voie générique de formation de motifs: si deux souches tendent à faire augmenter mutuellement leur motilité (la souche A se déplace plus vite quand la souche B est présent, et vice versa), ils subissent un processus de formation de motifs conduisant à la démixtion entre les deux souches. Inversement, l’inhibition mutuelle de la motilité conduit à la formation de motifs avec colocalisation. Ces résultats ont étévalidés expérimentalement par nos collaborateurs biologistes. Par la suite, j’ai étendu mon étude à des systèmes composés de plus de deux espèces en interaction, trouvant des règles simples permettant de prédire l’auto-organisation spatiale d’un nombre arbitraire d’espèces dont la motilité est sous contrôle mutuel. Cette partie de ma thèse ouvre une nouvelle voie pour comprendre l’auto-organisation des colonies bactériennes avec des souches concurrentes, ce qui est une question importante pour comprendre la dynamique des biofilms ou des écosystèmes bactériens dans les sols. Le deuxième problème traité dans ma thèse est inspiré par le comportement collectif des moteurs moléculaires se déplaçant le long des microtubules dans le cytoplasme des cellules eucaryotes. Un modèle pertinent pour le mouvement des moteurs moléculaires est donné par un système paradigmatique de non-équilibre appelé Processus Asymmetrique d’Exclusion Simple, en anglais Asymmetric Simple Exclusion Process (ASEP). Dans ce modèle sur réseau unidimensionnel, les particules se déplacent dans les sites voisins vides à des taux constants, avec un biais gauche-droite qui déséquilibre le système.Lorsqu’il est connecté à ses extrémités à des réservoirs de particules, l’ASEP est un exemple prototypique de transitions de phase unidimensionnelles guidées par les conditions aux limites. Les exemples réalistes, cependant, impliquent rarement une seule voie:les microtubules sont constitués de plusieurs pistes de tubuline auxquelles les moteurs peuvent s’attacher. Dans ma thèse, j’explique comment on peut théoriquement prédire le comportement de phase de systèmes à plusieurs voies complexes, dans lesquels les particules peuvent également sauter entre des voies parallèles. En particulier, je montre que la transition de phase unidimensionnelle vue dans l’ASEP survit cette complexité supplémentaire mais implique de nouvelles caractéristiques telles que des courants transversaux stables non-nulles et une localisation de cisaillement
The first part of my thesis is devoted to studying the self-organization of engineered strains of run-and-tumble bacteria Escherichia coli. This project, carried out in collaboration with synthetic biologists at Hong Kong University, has as its objective the exploration and decipherment of a novel self-organization mechanism in multi-species bacterial colonies. This was inspired by the fascinating question of how bacterial ecosystems comprising several species of bacteria can self-organize in space. By considering systems in which two strains of bacteria mutually regulate their motilities, I was able to show that reciprocal density control is a generic pattern-formation pathway: if two strains tend tomutually enhance their motility (strain A moves faster when strain B is present, and conversely),they undergo a pattern formation process leading to demixing between the two strains. Conversely, mutual inhibition of motility leads to pattern formation with colocalization. These results were validated experimentally by our biologist collaborators. Subsequently, I extended my study to systems composed of more than two interacting species, finding simple rules that can predict the spatial self-organization of an arbitrary number of species whose motility is under mutual control. This part of my thesis opens up a new route to understand the self-organization of bacterial colonies with competing strains, which is an important question to understand the dynamics of biofilms or bacterial ecosystems in soils.The second problem treated in my thesis is inspired by the collective behaviour ofmolecular motorsmoving along microtubules in the cytoplasm of eukaryotic cells. A relevant model for the molecularmotors’ motion is given by a paradigmatic non-equilibrium system called Asymmetric Simple Exclusion Process (ASEP). In this one-dimensional lattice- based model, particles hop on empty neighboring sites at constant rates, with a leftright bias that drives the systemout of equilibrium. When connected at its ends to particle reservoirs, the ASEP is a prototypical example of one-dimensional boundary driven phase transitions. Realistic examples, however, seldom involve only one lane: microtubules are made of several tubulin tracks to which the motors can attach. In my thesis, I explained how one can theoretically predict the phase behaviour of complex multilane systems, in which particles can also hop between parallel lanes. In particular, I showed that the onedimensional phase transition seen in the ASEP survives this additional complexity but involves new features such as non-zero steady transverse currents and shear localization

University of Technology Sydney. Faculty of Science.
Microorganisms live in tight associations with corals, but the ecological interactions and microbial functions and behaviours underpinning these relationships are not yet fully understood. The goal of this thesis is to define coral-microbe interactions by exploring how the composition, behaviour and function of microbial communities vary throughout a coral reef and how increasing sea water temperatures can affect coral-microbial relationships. As a first step to achieving this aim, In Chapter 1 we used metagenomics to characterise patterns in microbial composition and metabolic capacity across different niches, including coral-associated and non-coral associated microenvironments, on Heron Island, the Great Barrier Reef (GBR). We found that the composition and metabolic potential of coral reef bacteria is highly heterogeneous across a coral reef ecosystem, with a shift from an oligotrophy-dominated community (e.g. SAR11, Prochlorococcus, Synechococcus) in the open water and sandy substrate niches, to a community characterised by an increased frequency of copiotrophic bacteria (e.g. Vibrio, Pseudoalteromonas, Alteromonas) in the coral seawater niches. Among the major functional patterns observed were significant increases in genes associated with bacterial motility and chemotaxis in samples associated with the surfaces of coral colonies. The observation of increased motility and chemotaxis near to coral surfaces is notable given previous evidence that these phenotypes may be involved in coral disease processes. The research presented in this chapter was published in Microbial Ecology (2014 67 (3): 540-552) To investigate these patterns in chemotaxis further we next (Chapter 2) directly examined the potential ecological role of chemotaxis among coral-associated bacteria, by using laboratory based and in situ chemotaxis assays to test levels of chemotaxis among natural communities of coral reef microbes. We examined the behavioural responses towards several chemoattractants known to be released by corals and their symbiotic dinoflagelletes including amino acids, carbohydrates, ammonium chloride, and dimethylsulfonopropionate (DMSP). Using these approaches we found that bacteria associated with the surfaces of the corals exhibited high levels of chemotaxis, particularly towards DMSP and several amino acids. Levels of chemotaxis by coral-associated bacteria were consistently higher than those demonstrated by non-coral associated bacteria. This work was published in the ISME Journal (doi: 10.1038/ismej.2014.261) We next extended the in situ chemotaxis assays to examine the chemotactic behaviour of bacteria associated with other important coral reef organisms, sponges. These results redefine the sponge-symbiont acquisition paradigm whereby we show for that bacteria use chemotaxis to locate their sponge host on a coral reef. This work is in preparation for submission to the ISME Journal. After defining some of the functions and behaviours involved in coral reef microbiology, we next examined how these processes may shift under changing environmental conditions, associated with climate change. To determine how environmental variability, specifically thermal stress, influences bacterial community composition, behaviour and metabolic capacity, manipulation experiments were conducted using the coral Pocillopora damicornis. To investigate the dynamics of coral-associated vibrios under heat stress, in Chapter 4 we used Vibrio-specific amplicon sequencing approaches and qPCR to quantify shifts in the abundance and composition of natural populations of Vibrio, with a specific focus on the putative coral pathogen V. coralliilyticus. These experiments revealed that increasing seawater temperatures can favour the proliferation of potential coral pathogens among a natural mixed microbial community. This work has been published in Frontiers in Microbiology (6:432.doi: 10.3389/fmicb.2015.00432). In Chapter 5, we decided to explore the entire coral-associated community by using metagenomics and metatranscriptomics to investigate how the phylogeny and function of coral associated microbes shift resulting from increasing seawater temperatures. We found a dramatic shift in the community from Endozoicomonaceae being dominant in the control corals, while there was an appearance of the vibrios under increasing sea water temperatures in line with our findings from chapter 4. We also observed functional shifts that involved an upregulation of chemotaxis and motility genes at higher temperatures and were shown to be affiliated with vibrios, a genus which contains several putative coral pathogens. Taken together our data demonstrate that coral reef bacterial communities are highly dynamic and that key groups of copiotrophic bacteria have the capacity to use behaviours such as chemotaxis to use nutrient gradients to potentially locate and colonize benthic host animals including corals and sponges. Increasing seawater temperatures causes dramatic changes in the coral-associated bacterial community, allowing for the proliferation of potential coral pathogens and increased expression of behavioural phenotypes that may promote successful infection of corals.

博士
國立陽明大學
臨床醫學研究所
94
Ghrelin, a novel 28-amino acid peptide, was recently identified in the stomach. Acylated ghrelin has two unique biologic characteristics: (1) the only known orexigenic gut-brain peptide to increase meal size; (2) the unique post-translational modification of O-n-octanoylation at serine 3. Acylated ghrelin is involved in the regulation of gastrointestinal motility, cardiac performance, anxiety, and energy balance. On the other hand, des-acyl ghrelin has been reported to be devoid of any endocrine activities. However, plasma des-acyl ghrelin concentration accounts for more than 90% of total circulating ghrelin, and the ratio of des-acylated to acylated ghrelin in rat hypothalamus is 2: 1. The concept of des-acyl ghrelin as a traditionally-thought non-functional peptide has been particularly challenged. Recently, des-acyl ghrelin has been reported to inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT pathways in vitro, inhibit the proliferation of breast canrcinoma cell line, reduce glycerol release from rat epididymal adipocyte, directly bind to the membranes of PC-3 prostate tumor cells. Des-acyl ghrelin exhibits more potent cardiotropic action than acylated ghrelin on guinea pig papillary muscle ex vivo. More recently, des-acyl ghrelin has been shown to promote bone marrow adipogenesis in vivo by a direct peripheral action. Converging evidence indicate that des-acyl ghrelin has its unique biologic effects and roles. However, the effects of des-acyl ghrelin on ingestive behavior and gastrointestinal motility remain uninvestigated. Therefore, an in vivo whole animal model: rats, was used to conduct the prospective studies. Intraperitoneal injection of des-acyl ghrelin inhibited food intake in food-deprived rats, but not free-feeding rats. Intraperitoneal injection of acylated ghrelin stimulated food intake in both food-deprived and free-feeding rats. Capsaicin treatment did not alter intraperitoneal-injected des-acyl ghrelin induced anorectic effect, but blocked the anorexigenic effect induced by intraperitoneal-injected acylated ghrelin. Similarly, intrcisternal injection of des-acyl ghrelin inhibited food intake in food-deprived rats, but not free-feeding rats. Intracisternal injection of acylated ghrelin stimulated food intake in both food-deprived and free-feeding rats. For the studies of gastrointestinal motility, intravenous and intracerebroventricular injection of des-acyl ghrelin disrupted fasted antral motility, but not duodenal motility, in conscious rats. Capsaicin treatment did not alter the disruption of fasted antral motility induced by des-acyl ghrelin, but truncal vagotomy blocked it. Pretreatment with intracerebroventricular injection of the non-selective CRF and selective CRF2 receptor antagonists prevented the disruption of fasted antral motility induced by intravenous and intracerebroventricular injection of des-acyl ghrelin. However, pretreatment with intracerebroventricular injection of the selective CRF1 receptor antagonist did not prevent the disruption of fasted antral motility induced by intravenous and intracerebroventricular injection of des-acyl ghrelin. Another experiment indicated that intracisternal injection of des-acyl ghrelin dose-dependently inhibited the non-nutrient semiliquid gastric emptying, without altering the small intestinal transit. On the contrary, intracisternal injection of acylated ghrelin accelerated the non-nutrient semiliquid gastric emptying and increased the geometric center of the small intestinal transit, but did not alter the running percentage of small intestinal transit. For immunohistochemical studies, intraperitoneal injection of des-acyl and acylated ghrelin both increased c-fos expression in arcuate nucleus and paraventricular nucleus. Intraperitoneal injection of des-acyl ghrelin did not stimulated c-fos expression in the nucleus of the solitary tract, but, however, intraperitoneal injection of acylated ghrelin activated c-fos expression in the nucleus of the solitary tract. Taken together, the inhibitory effect of des-acyl ghrelin on food intake is short-lived and discrete in food-deprived rats. The effects of des-acyl ghrelin on food intake and fasted antral motility are not vagally or the nucleus of the solitary tract-mediated. The disruption of des-acyl ghrelin on fasted antral motility in conscious rats is probably through brain CRF2 receptor and vagal efferent dependent. The results from these studies opened up a lot of interesting questions about acylated and des-acyl ghrelin research dogmas. Des-acyl ghrelin regulated ingestive behavior, antral motility and gastric emptying. To investigate the target which des-acyl ghrelin acts on, especially the unique speficic receptor, as well as the physiologic and neuroanatomic interactions, might help understand the roles of des-acyl ghrelin in modulating ingestive behavior, gastrointestinal motility and energy balance. To develop reliable RIA or ELISA kits, for separate measurement of acylated and des-acyl ghrelin, also help translate basic science results into clinical research, efficiently solving the growing health and economic problems caused by obesity and anorexia-cacheixa in this modern society.

The individual behaviour of prokaryotic organisms such as bacteria often gives rise to complexity that is commonly associated with multicellular behaviour. The transition from unicellular to multicellular behaviour occurs in response to chemical signals, called autoinducers, which bacteria generate and receive internally within a given population. These autoinducers control the gene expression necessary for the emergence of group-behaviour-phenotype. This phenomenon is called quorum sensing (QS). An example of the quorum sensing control of gene regulation has been the luminescence (lux) operon in Vibrio fischeri. The luxI and ainS quorum signalling systems work in conjunction to regulate luminescence in V. fischeri. LuxI and AinS are acyl-synthases that catalyse the production of the autoinducers C6-HSL and C8-HSL respectively. These autoinducers bind to LuxR, a transcriptional activator of the lux operon, which activates expression of the lux genes causing an increase in luminescence. It was shown that quorum signalling also affects motility and biofilm formation in bacteria. However, the evidence with respect to these phenotypes is conflicting and inconclusive, the reason being the state of quorum is ambiguously defined. It is not properly known whether the observed collective behaviour is purely a result of physical crowding of bacteria, or that both chemical signalling and crowding contribute to this phenomenon. This work attempts to address these issues by studying luminescence, motility, and biofilm, a diverse set of behaviours, yet closely linked to each other in V. fischeri-squid symbiosis. We studied the luminescence response of V. fischeri to both endogenous and externally added signals at per-cell and population level. Experiments with ES114, a wild-type strain of V. fischeri, and ainS mutant showed that (i) luminescence per cell does not mutually correlate with the cell-density, indicating that bacteria do not show greater response to the signal at higher densities; (ii) the activity of the lux signalling circuit shows a strong dependence on the growth stage, (iii) the cells do not show enhanced growth, i.e., they do not derive fitness benefits at higher densities in response to the signal. We anticipated that the culture with a higher cell-density should exhibit greater per-cell-luminescence. However, we found that the luminescence curve of the culture with lower density crosses that of the cultures with higher densities during the exponential phase. Kinetic modelling of the luxI mRNA expression showed that the expression profile qualitatively agrees with the luminescence trend observed in the cultures, supporting the observation that growth-phase plays a major role in regulating the luminescence gene expression. We also studied the effect of autoinducers on motility of V. fischeri. V. fischeri uses flagella to move into the inner crypts of the light organ of the squid. The bacterium secretes autoinducers, encounters secretions of the light organ, and slows down during the final stage of colonization process. Studies have shown that flagellar elaboration is repressed as a consequence of ainS signalling. However, those studies were soft-agar migration assays and carried out with the mutant strain of ainS. We measured real-time planktonic motility of ES114 and the signalling mutant strains of V. fischeri in response to autoinducers added exogenously at different concentrations. We found that the autoinducers do not affect the motility of the strains. We also showed that reduction in motility is purely a consequence of physical crowding of bacteria, and chemical signalling may not be involved in the process. It was shown that reduction in motility leads to biofilm formation. Motile bacteria must lose flagella in order to form biofilm, and signalling controls biofilm formation in many species. Our study on motility showed that reduction in motility occurs because of physical crowding in V. fischeri. Hence, we explored the possibility that physical crowding might lead to formation of biofilm rather than signalling in this species. We quantified exopolysaccharide production by crystal violet assay, which revealed that planktonic cells produce exopolysaccharides, in addition to biofilm cells. The study revealed that V. fischeri cells always produce exopolysaccharides irrespective of their physiological state. We examined the effect of signalling on biofilm in ES114 and the mutant strains using gene-expression analysis. We quantified the expression of various genes involved in biofilm formation and found that both ES114 and the mutants expressed rscS and sypP indicating that exopolysaccharide production is not under the control of autoinducers. Therefore, we hypothesized that biofilm formation in V. fischeri may be a result of physical agglomeration of cells. Our observations indicate that the state of quorum is inadequately defined and there is no direct measure of the underlying process. Multicellular behaviour in V. fischeri is regulated by a complex interplay of cell-density, signalling, and other factors such as the growth phase of the culture, indicating that the state of quorum employs different mechanisms to regulate various phenotypes. Our study reveals that QS is an intricate process, and the accepted mechanisms for QS are incomplete at best.