Einleitung: Fibroblasten bilden essentielle Komponenten des Bindegewebes und reagieren auf Bestrahlung mit langwelligem Licht. In der Zellkultur lässt sich durch Lichtsignale eine gerichtete Migration (= Phototaxis) induzieren. Da die Migration von Fibroblasten eine zentrale Rolle für die Wundheilung und Regeneration des Bindegewebes spielt, hat die Phototherapie mit langwelligem, nicht-thermischem Licht besondere klinische Bedeutung erlangt. Eindeutige Vorgaben für phototherapeutische Anwendungsparameter existieren bisher jedoch nicht. Fragestellung: Frühere Versuchsanordnungen zur vitalmikroskopischen Untersuchung der Phototaxis von 3T3-Fibroblasten arbeiteten in geschlossenen Kulturflaschen mit Latex- Kügelchen als Lichtquellen, die von externen Strahlungsquellen aktiviert wurden. Die vorliegende Arbeit beschreibt die Entwicklung einer faseroptischen Anordnung mit freier Positionierbarkeit in der offenen Kulturschale. Die primäre Fragestellung war die Eignung der Methode zur Erstellung eines Aktionsspektrums für Phototaxis photoresponsiver, adhärent wachsender Zellen. Material und Methoden: Die Versuche wurden mit NIH-3T3-Zellen der Schweizer Maus durchgeführt. Um die Ergebnisse nicht durch Fremd-Chromophore zu verfälschen, wurden native Zellen ohne Färbemethoden vitalmikroskopisch untersucht. Eine speziell entwickelte Faseroptik und LED-Lichtquellen mit verschiedenen Wellenlängen ermöglichten die direkte Einbringung und Positionierung der Lichtsignale auf dem Boden der Kulturschale. Ein wissenschaftliches Bildbearbeitungsprogramm wurde durch ein Plug-In so modifiziert, dass die Bewegungs- und Zeitparameter halbautomatisch erfasst werden konnten. Ergebnisse: Bei 18 von 44 Langzeitversuchen konnte eindeutiges phototaktisches Verhalten mit Zellkontakt zur Lichtquelle induziert werden. In 16 Fällen kam es zur migratorischen Annäherung und in 10 Fällen zu keiner Reaktion bzw. einer diskret negativen Phototaxis. Das positive phototaktische Verhalten der beiden reagierenden Gruppen war hochsignifikant, der zweiseitige p-Wert des Binomialtests beträgt 0,000388. Bei 77,3 % der Versuche konnte positive Phototaxis demonstriert werden. Schlussfolgerungen: Das neu entwickelte Verfahren eignet sich zur Untersuchung einer Phototaxis und kann damit prinzipiell zur Erstellung von phototaktischen Aktionsspektren eingesetzt werden. Im Vergleich zu bisherigen Methoden können die Bestrahlungsstärken direkt am Lichtwellenleiter gemessen und unerwünschte Fremdkörper-induzierte Wechselwirkungen eliminiert werden. Durch die freie Positionierbarkeit der Lichtquelle in der Kulturschale können die zu beobachtenden Zellen beliebig ausgewählt werden.:INHALTSVERZEICHNIS IV ABBILDUNGSVERZEICHNIS VII TABELLENVERZEICHNIS IX ABKÜRZUNGSVERZEICHNIS X 1. EINLEITUNG 1 2. DEFINITIONEN UND STAND DER FORSCHUNG 5 2.1 LICHT 5 2.1.1 Sonnenlicht 5 2.1.2 Licht in der Physik 5 2.1.3 Licht in der Biologie 6 2.1.3.1 Taxis 7 2.1.4 Licht in der Medizin 8 2.1.4.1 Historischer Überblick 8 2.2 PHOTOBIOLOGIE VON ROT UND NAHINFRAROT 10 2.2.1 Photochemische Grundlagen 10 2.2.2 Haut 11 2.2.2.1 Aufbau und Funktion 11 2.2.2.2 Optische Eigenschaften 12 2.2.3 Fibroblasten 13 2.2.4 Zellmigration 15 2.2.5 Mitochondrien 16 2.3 PHOTOTAXIS BEI 3T3-FIBROBLASTEN 17 3. FRAGESTELLUNG 23 4. MATERIAL UND METHODEN 25 4.1 GERÄTE, LABORMATERIALIEN UND SOFTWARE 25 4.2 ZELLMATERIAL UND KULTURBEDINGUNGEN 25 4.3 ZELLPRÄPARATION 27 4.4 VERSUCHSAUFBAU 27 4.5 SHUTTER-SIMULATION DER OBJEKTBELEUCHTUNG 32 4.6 ZUFUHR VON DESTILLIERTEM WASSER 33 4.7 VERSUCHSABLAUF 33 4.8 VERMESSUNG DES BEWEGUNGSVERHALTENS 35 4.9 STATISTISCHE BERECHNUNG 36 5. ERGEBNISSE 37 5.1 AUSWAHLKRITERIEN FÜR GÜLTIGE VERSUCHE 37 5.2 GRUPPENEINTEILUNG DER GÜLTIGEN VERSUCHE 38 5.2.1 Die Gruppe "Touchdown" (TD) 38 5.2.2 Die Gruppe "Annäherung/Closer" (CL) 39 5.2.3 Die Gruppe "still/entf./no reaction" (NO) 39 5.2.4 Zusammenfassung des Gruppenverhaltens 40 5.3 GRAFISCHE DARSTELLUNGEN DER AUSGEWERTETEN VERSUCHE 41 5.4 PRIMÄRE FRAGESTELLUNG: PHOTOTAKTISCHE REAKTION 49 5.5 FRAGESTELLUNG 2: EINFLUSS DER MIGRATIONSMODALITÄT 49 5.6 EINFLUSS VON WELLENLÄNGE UND LICHTMODULATION 51 5.6.1 Fragestellung 3: Einfluss der Wellenlänge 51 5.6.2 Fragestellung 4: Einfluss der Pulsationsfrequenz 52 5.7 FRAGESTELLUNG 5: ROLLE DER AUSGANGSPOSITION 53 5.8 LOKOMOTORISCHE PARAMETER 54 5.8.1 Fragestellung 6: Zurückgelegte Wegstrecke 54 5.8.2 Fragestellung 7: Migrationsgeschwindigkeit 56 5.9 FRAGESTELLUNG 8: EINFLUSS DER VITALITÄT 57 5.9.1 Definition der mittleren Vitalität vitmean 57 5.9.2 Definition der relativen Vitalität vitrel 57 5.9.3 Die relative Vitalität vitrel der Zellen FBc, FB1 und FB2 58 5.9.4 Die mittlere Vitalität vitmean in den Versuchsgruppen 59 5.9.5 Die relative Vitalität vitrel des zentralen Fibroblasten 60 5.10 ATYPISCHES ZELLVERHALTEN 61 5.10.1 Atypisches Verhalten in Versuchen mit normalen 3T3-Zellen 61 5.10.1.1 Hohe Geschwindigkeit und große Wegstrecke 61 5.10.1.3 "Überfall" eines Fibroblasten 63 5.10.1.2 Zentraler Fibroblast migriert unter den Lichtwellenleiter 64 5.10.2 Atypisches Verhalten bei frischen humanen Monozyten 66 6. DISKUSSION 68 6.1 ERGEBNISSE 68 6.1.1 Primäre Fragestellung: Phototaktische Reaktion 68 6.1.2 Fragestellung 2: Einfluss der Migrationsmodalität 70 6.1.3 Fragestellung 3: Einfluss der Wellenlänge 71 6.1.4 Fragestellung 4: Einfluss der Pulsationsfrequenz 74 6.1.5 Fragestellung 5: Rolle der Ausgangsposition 78 6.1.6 Fragestellung 6: Zurückgelegte Wegstrecke 79 6.1.6.1 Differenz Startposition - Minimalposition zum LWL 79 6.1.6.2 Insgesamt zurückgelegte Wegstrecke 80 6.1.7 Fragestellung 7: Migrationsgeschwindigkeit 80 6.1.8 Fragestellung 8: Einfluss der Vitalität 81 6.1.9 Atypisches Zellverhalten 83 6.1.9.1 Versuche mit 3T3-Zellen 83 6.1.9.1.1 Hohe Geschwindigkeit und große Wegstrecke 83 6.1.9.1.2 "Überfall" eines Fibroblasten 84 6.1.9.1.3 Phototaxis bei hyperosmotischem Stress 85 6.1.9.2 Atypisches Verhalten bei frischen humanen Monozyten 86 6.1.10 Zusätzliche Fragestellungen 87 6.1.10.1 Wie lange ist die maximale Versuchsdauer im offenen System 88 6.1.10.2 Spielt der Aspekt der potentiellen Verkeimung eine relevante Rolle? 88 6.1.10.3 Spielt die Dimension der Lichtquelle eine objektivierbare Rolle 88 6.1.10.4 Kann die Bestrahlungsstärke gemessen anstatt geschätzt werden 89 6.1.10.5 Können Rückschlüsse auf das "Auge der Zelle" gezogen werden? 90 6.2 SCHLUSSFOLGERUNG 93 6.3 AUSBLICK 94 7. ZUSAMMENFASSUNG 96 7.1 SUMMARY 97 8. LITERATURVERZEICHNIS 98 9. ANLAGEN 117 9.1 LABORGERÄTE UND MATERIALIEN 117 9.1.1 Mikroskope 117 9.1.2 Software: 117 9.1.3 Geräte: 118 9.1.4 Verbrauchsmaterialien 118 9.1.5 Medien, Puffer, Lösungen 119 9.2 EIGENE GERÄTSCHAFTEN 120 9.2.1 Frequenzgenerator 120 9.2.2 Halbleiter-Lichtquellen 121 9.2.2.1 Modulierbare Lichtquelle 121 9.2.2.2 Kohärente Lichtquelle 122 9.2.2.3 Photon Micro-Light LED-Lichtquellen mit Pulsmodus f = 1Hz 124 9.2.2.4 Bestrahlungsstärken der verwendeten Lichtquellen 124 9.2.3 Systemträger 125 9.2.4 LWL und Optokoppler 126 9.2.5 Modifizierte Microsoft-Maus 126 9.2.6 Eigene Software 127 10. DANKSAGUNG 128 11. EIDESSTATTLICHE ERKLÄRUNG 130 / Background: Fibroblasts are essential constituents of connective tissue and react upon irradiation with long-wavelength light. In cell culture, directed migration (= phototaxis) can be induced by light stimuli. Fibroblast migration and activity play a central role for wound healing and connective tissue regeneration. In consequence, phototherapy with non-thermal, long wavelength light obtained increasing clinical importance. However, accepted guidelines for phototherapeutical treatment parameters are still lacking. Objective: Existing assays for microscopic observation of phototactic reaction of 3T3- fibroblasts in a live cell chamber use closed cell culture flasks with small light-scattering latex particles attached to the surface of the flask bottom prior to cell seeding, which can be illuminated by an external light source. The present work describes the development of a fiberoptic assembly providing free positioning of the light source in an open culture dish. The primary objective is the eligibility of this method for determination of a phototactic action spectrum for photoresponsive adherent cell species. Materials and Methods: Experiments were conducted using Swiss mouse NIH-3T3-cells. Native cells without staining were used for elimination of potential dye-induced cell-light interferences. A light microscope with field illumination shutter simulation and live cell chamber was used in combination with a specially devised LED-light-fed fiberoptic assembly providing different wavelengths, which could be directly positioned on the inner bottom of the culture dish. A scientific image processing application was modified by a special plug-in for semi-automatic acquisition of cellular locomotion parameters and temporal data token. Results: A total of 44 experiments were conducted. 18 essays resulted in full phototactic reaction characterized by pseudopodium contact with the fiberoptic aperture, another 16 essays showed migratory approximation without direct contact with the light source. 10 essays displayed no reaction or discrete negative phototaxis. The positive phototaxis of the two responding groups was highly significant in the two-sided binomial test (p = 0.000388). For 77.3 % of the experiments positive phototaxis could be demonstrated. Conclusions: The newly developed assay is appropriate for the induction of phototactic behaviour and can be utilized for the determination of phototactic action spectra. In contrast to existing methods the irradiances can be measured directly at the aperture of the optical fiber and biasing foreign object-induced effects can be eliminated. Due to arbitrary positioning of the light source the cells can be chosen freely for examination.:INHALTSVERZEICHNIS IV ABBILDUNGSVERZEICHNIS VII TABELLENVERZEICHNIS IX ABKÜRZUNGSVERZEICHNIS X 1. EINLEITUNG 1 2. DEFINITIONEN UND STAND DER FORSCHUNG 5 2.1 LICHT 5 2.1.1 Sonnenlicht 5 2.1.2 Licht in der Physik 5 2.1.3 Licht in der Biologie 6 2.1.3.1 Taxis 7 2.1.4 Licht in der Medizin 8 2.1.4.1 Historischer Überblick 8 2.2 PHOTOBIOLOGIE VON ROT UND NAHINFRAROT 10 2.2.1 Photochemische Grundlagen 10 2.2.2 Haut 11 2.2.2.1 Aufbau und Funktion 11 2.2.2.2 Optische Eigenschaften 12 2.2.3 Fibroblasten 13 2.2.4 Zellmigration 15 2.2.5 Mitochondrien 16 2.3 PHOTOTAXIS BEI 3T3-FIBROBLASTEN 17 3. FRAGESTELLUNG 23 4. MATERIAL UND METHODEN 25 4.1 GERÄTE, LABORMATERIALIEN UND SOFTWARE 25 4.2 ZELLMATERIAL UND KULTURBEDINGUNGEN 25 4.3 ZELLPRÄPARATION 27 4.4 VERSUCHSAUFBAU 27 4.5 SHUTTER-SIMULATION DER OBJEKTBELEUCHTUNG 32 4.6 ZUFUHR VON DESTILLIERTEM WASSER 33 4.7 VERSUCHSABLAUF 33 4.8 VERMESSUNG DES BEWEGUNGSVERHALTENS 35 4.9 STATISTISCHE BERECHNUNG 36 5. ERGEBNISSE 37 5.1 AUSWAHLKRITERIEN FÜR GÜLTIGE VERSUCHE 37 5.2 GRUPPENEINTEILUNG DER GÜLTIGEN VERSUCHE 38 5.2.1 Die Gruppe "Touchdown" (TD) 38 5.2.2 Die Gruppe "Annäherung/Closer" (CL) 39 5.2.3 Die Gruppe "still/entf./no reaction" (NO) 39 5.2.4 Zusammenfassung des Gruppenverhaltens 40 5.3 GRAFISCHE DARSTELLUNGEN DER AUSGEWERTETEN VERSUCHE 41 5.4 PRIMÄRE FRAGESTELLUNG: PHOTOTAKTISCHE REAKTION 49 5.5 FRAGESTELLUNG 2: EINFLUSS DER MIGRATIONSMODALITÄT 49 5.6 EINFLUSS VON WELLENLÄNGE UND LICHTMODULATION 51 5.6.1 Fragestellung 3: Einfluss der Wellenlänge 51 5.6.2 Fragestellung 4: Einfluss der Pulsationsfrequenz 52 5.7 FRAGESTELLUNG 5: ROLLE DER AUSGANGSPOSITION 53 5.8 LOKOMOTORISCHE PARAMETER 54 5.8.1 Fragestellung 6: Zurückgelegte Wegstrecke 54 5.8.2 Fragestellung 7: Migrationsgeschwindigkeit 56 5.9 FRAGESTELLUNG 8: EINFLUSS DER VITALITÄT 57 5.9.1 Definition der mittleren Vitalität vitmean 57 5.9.2 Definition der relativen Vitalität vitrel 57 5.9.3 Die relative Vitalität vitrel der Zellen FBc, FB1 und FB2 58 5.9.4 Die mittlere Vitalität vitmean in den Versuchsgruppen 59 5.9.5 Die relative Vitalität vitrel des zentralen Fibroblasten 60 5.10 ATYPISCHES ZELLVERHALTEN 61 5.10.1 Atypisches Verhalten in Versuchen mit normalen 3T3-Zellen 61 5.10.1.1 Hohe Geschwindigkeit und große Wegstrecke 61 5.10.1.3 "Überfall" eines Fibroblasten 63 5.10.1.2 Zentraler Fibroblast migriert unter den Lichtwellenleiter 64 5.10.2 Atypisches Verhalten bei frischen humanen Monozyten 66 6. DISKUSSION 68 6.1 ERGEBNISSE 68 6.1.1 Primäre Fragestellung: Phototaktische Reaktion 68 6.1.2 Fragestellung 2: Einfluss der Migrationsmodalität 70 6.1.3 Fragestellung 3: Einfluss der Wellenlänge 71 6.1.4 Fragestellung 4: Einfluss der Pulsationsfrequenz 74 6.1.5 Fragestellung 5: Rolle der Ausgangsposition 78 6.1.6 Fragestellung 6: Zurückgelegte Wegstrecke 79 6.1.6.1 Differenz Startposition - Minimalposition zum LWL 79 6.1.6.2 Insgesamt zurückgelegte Wegstrecke 80 6.1.7 Fragestellung 7: Migrationsgeschwindigkeit 80 6.1.8 Fragestellung 8: Einfluss der Vitalität 81 6.1.9 Atypisches Zellverhalten 83 6.1.9.1 Versuche mit 3T3-Zellen 83 6.1.9.1.1 Hohe Geschwindigkeit und große Wegstrecke 83 6.1.9.1.2 "Überfall" eines Fibroblasten 84 6.1.9.1.3 Phototaxis bei hyperosmotischem Stress 85 6.1.9.2 Atypisches Verhalten bei frischen humanen Monozyten 86 6.1.10 Zusätzliche Fragestellungen 87 6.1.10.1 Wie lange ist die maximale Versuchsdauer im offenen System 88 6.1.10.2 Spielt der Aspekt der potentiellen Verkeimung eine relevante Rolle? 88 6.1.10.3 Spielt die Dimension der Lichtquelle eine objektivierbare Rolle 88 6.1.10.4 Kann die Bestrahlungsstärke gemessen anstatt geschätzt werden 89 6.1.10.5 Können Rückschlüsse auf das "Auge der Zelle" gezogen werden? 90 6.2 SCHLUSSFOLGERUNG 93 6.3 AUSBLICK 94 7. ZUSAMMENFASSUNG 96 7.1 SUMMARY 97 8. LITERATURVERZEICHNIS 98 9. ANLAGEN 117 9.1 LABORGERÄTE UND MATERIALIEN 117 9.1.1 Mikroskope 117 9.1.2 Software: 117 9.1.3 Geräte: 118 9.1.4 Verbrauchsmaterialien 118 9.1.5 Medien, Puffer, Lösungen 119 9.2 EIGENE GERÄTSCHAFTEN 120 9.2.1 Frequenzgenerator 120 9.2.2 Halbleiter-Lichtquellen 121 9.2.2.1 Modulierbare Lichtquelle 121 9.2.2.2 Kohärente Lichtquelle 122 9.2.2.3 Photon Micro-Light LED-Lichtquellen mit Pulsmodus f = 1Hz 124 9.2.2.4 Bestrahlungsstärken der verwendeten Lichtquellen 124 9.2.3 Systemträger 125 9.2.4 LWL und Optokoppler 126 9.2.5 Modifizierte Microsoft-Maus 126 9.2.6 Eigene Software 127 10. DANKSAGUNG 128 11. EIDESSTATTLICHE ERKLÄRUNG 130

The circadian clock is an endogenous timer that oscillates with a period of approximately 24 hours and is reset upon environmental time cues such as the daily light/ dark or temperature cycles. Chlamydomonas reinhardtii is an ideal model organism for research on the circadian clock, because it shows several well-characterized behaviors that exhibit a circadian rhythm. Its circadian rhythm of phototaxis (swimming toward light) has been automated. Former action spectrum studies using the circadian phototaxis rhythm as an indicator surprisingly found that pulses of blue light were not effective in resetting the circadian clock of dark-adapted cells. This may have been because of the particular strain used in the study (the cell wall-deficient strain CW15). It may also have been due to the additional phase shift caused by the act of placing the cultures into the monitoring machine at particular times during their circadian cycle. This additional phase shift was most likely the result of the white background light present when monitoring the rhythm of phototaxis. The phototaxis monitoring process was improved by using narrow-wavelength LEDs specific for phototaxis as test lights and by omitting the background light between test light cycles. This study demonstrates that the modifications prevent any phase shifts due to the cultures being placed into the monitoring machine. Using a further improved experimental set-up and the wild-type strain CC124, this study unambiguously shows that blue light of 440nm is effective in resetting the circadian clock in Chlamydomonas reinhardtii. Because of this difference in blue light response to the earlier study, the action spectrum of the entire visible light range was also evaluated. Effective wavelengths for resetting the circadian clock in wild-type C. reinhardtii were found to occur at 400nm, 440nm, 540nm, and 640-660nm, corresponding to near UV-A, blue, green, and red light, respectively. With the exception of 440nm, these findings are congruent with previous action spectrum studies for the cell wall-deficient strain CW15.

Dans cette thèse, nous nous intéressons aux mécanismes précurseurs de la formation d'un biofilm dans le cas de la cyanobactérie Synechocystis sp. PCC 6803, en tant que micro-organisme modèle. Nous avons en premier lieu observé la motilité de cellules qui sédimentent puis diffusent librement sur une surface en verre. De plus, nous avons relevé les détails de la motilité des bactéries, qui est intermittente : des périodes de mouvements directionnels (les ``runs'') alternent avec des périodes de mouvements localisés (les ``tumbles''). Le coefficient de diffusion résultant décroît avec le temps, avant de se stabiliser; en revanche des souches mutantes relarguant moins de substance extracellulaire gardent un coefficient de diffusion constant au cours de l'expérience. Nous proposons un modèle de ralentissement basé sur une marche aléatoire à "temps continu" influencée par le recouvrement progressif de la surface par les exopolysaccharides secrétés par les bactéries. Les bactéries pourraient ainsi "reconnaître" le type de surface sur lequel elles évoluent et adapter leur motilité en conséquence, ce qui constitue une étape préliminaire dans la formation d'un biofilm. Nous avons vérifié ce point avec une étude de la diffusion sur des surfaces de différentes rigidités. L'expérience est cette fois prolongée pour étudier l'effet de la dureté du substrat sur la morphologie du biofilm. La proportion de bactéries non motiles est plus importante sur les surfaces molles. Cet effet à l'échelle de la particule conditionne en grande partie la morphologie des micro-colonies émergentes après plusieurs jours de culture, avec davantage de micro-colonies et une densité cellulaire plus hétérogène sur les surfaces molles. Nous construisons un modèle qui prend en compte la division cellulaire et le ralentissement de la dynamique individuelle, et permettant de conclure sur le lien entre rigidité de surface, dynamique cellulaire, et formation de micro-colonies. Le troisième chapitre expérimental aborde la réponse du système à des changements de conditions lumineuses, d'abord de manière isotrope puis en introduisant une lumière directionnelle. En conditions isotropes, les échelons d'intensité lumineuse perturbent les temps caractéristiques de ``run'' et de ``tumble''. Nous analysons ces variations dans le cadre de la théorie de la réponse linéaire. Nos données suggèrent qu'il est possible de décrire la réponse à une perturbation d'intensité lumineuse de manière similaire à des "stimuli" chimiques, avec la même fonction réponse. Sous un flux lumineux directionnel, nous observons une phototaxie complexe pour laquelle une fraction des cellules a un mouvement aléatoire pendant qu'une autre est sensible à l'anisotropie de l'éclairage. L'orientation des déplacements s'effectue dès l'introduction du flux lumineux, mais les temps de ``run'' et de ``tumble'' continuent d'évoluer pendant la période du flux lumineux suivant une tendance proche de celle observée en conditions isotropes. Ces résultats mettent en évidence le couplage entre l'intensité lumineuse et l'anisotropie de l'éclairage dans les mécanismes responsables de la phototaxie. / In this work, we focus on the early stages of biofilm formation, using the cyanobacterium Synechocystis sp. PCC 6803 as a model micro-organism. First, we observe the motility of cells that have just reached the surface after sedimentation, and are then let free to diffuse on a glass surface. The resulting diffusion coefficient decreases with time, until it reaches a plateau value; whereas mutant strains secreting less extracellular substance do not exhibit such a slowdown. To explore the mechanism at work, we investigate the details of bacterial motility, which is intermittent: periods of directional movements (``runs'') alternate with periods of localized movements (``tumbles''). We propose a slowdown model based on a continuous-time random walk, influenced by the progressive surface coverage with the exopolysaccharides secreted by the bacteria. They could then ``recognize'' the surface onto which they are diffusing and adapt consequently their motility, which establishes a preliminary step for the biofilm formation. We have adressed this issue by studying diffusion onto surfaces with different stiffnesses. The experimentation is extended in order to analyze the effect of surface toughness on the biofilm morphology. The proportion of non-motile bacteria is higher on softer surfaces. This effect on the individual particle affects the shape of emergent microcolonies after several days of growth, with more microcolonies and a more heterogeneous surface density on soft surfaces. We build a model that takes into account cellular division and individual dynamics slowdown that enables us to point out the relation between surface rigidity, cellular dynamics, and microcolonies formation. The third experimental chapter tackles the response of the system to changes in light conditions, first in an isotropic way, then by introducing a directional light. For isotropic conditions, steps of light intensity disrupt the characteristic ``run'' and ``tumble'' times. We analyze these variations in the framework of the linear response theory. Our data suggest that it is possible to describe the response to light disruptions in the same way as what has been done for chemical ``stimuli'', with a similar response function. With a directional luminous flux, we observe complex phototaxis, in which some fraction of the cellular population displays random movements, whereas another one is sensitive to the lighting anisotropy. The displacements are oriented as soon as the luminous flux is switched on, and the ``run'' and ``tumble'' times also respond this change. Our results show that light intensity triggers bacterial motility, whether it is oriented or not.

This thesis introduces a design combining an emerging area in robotics with a well established mathematical research topic: swarm intelligence and Voronoi tessellations, respectively. The main objective for this research is to design an economical and robust swarm system to achieve distributed control. This research combines swarm intelligence with Voronoi tessellations to localize a source and create formations. Extensive software coding must be implemented for this design, such as the development of a discrete centroidal Voronoi tessellation (CVT) algorithm. The ultimate purpose of this research is to advance the existing Mobile Actuator and Sensor Network (MASnet) platform to eventually develop a cooperative robot team that can sense, predict, and nally neutralize a diusion process. Previous work on the MASnet platform has served as a foundation for this research. While growing closer to the MASnet goal, results also provide stimulating discoveries for mathematical and swarm research areas.

In der vorliegenden Arbeit wurde die Funktion der beiden cyanobakteriellen Phytochrome Cph1 und Cph2 untersucht. Dafür wurde zunächst das Wachstum von Mutanten mit einem inaktivierten cph1- bzw. cph2-Gen unter verschiedenen Lichtbedingungen analysiert. Das Wachstum aller Phytochrommutanten war unter Starklicht beeinträchtigt. Dahingegen wuchs die cph1-Mutante im FRL schlechter als der Wildtyp, während das Wachstum der cph2-Mutante im RL vermindert war. Eine cph1/cph2-Doppelmutante zeigte unter allen Lichtbedingungen eine Wachstumsreduktion, die der jeweiligen Einzelmutante ähnlich war. Die genaue Ursache für die Beeinträchtigung des Wachstums der Phytochrommutanten konnte nicht ermittelt werden. Die verschiedenen Aspekte der Photosynthese, wie Pigmentzusammensetzung, maximale Netto-Sauerstofffreisetzungsrate und 77K-Fluoreszenzemission, waren in den Phytochrommutanten nicht signifikant verändert. Bei Synechocystis sp. PCC 6803 konnte eine lichtgerichtete Bewegung beobachtet werden, wobei man aufgrund von Aktionsspektren der Motilität eine Funktion der Phytochrome oder phytochromähnlichen Proteine bei der Steuerung der phototaktischen Bewegung vermutet. Dem Cph1-Protein konnte hierfür keine Rolle zugeordnet werden. Dahingegen scheint der Photorezeptor Cph2 die lichtgerichtete Bewegung der Zellen in Richtung einer Blaulichtquelle zu inhibieren. Eine Interaktion mit einem klassischen Blaulicht-Rezeptor konnte ausgeschlossen werden. / The function of the cyanobacterial phytochromes Cph1 and Cph2 was investigated. At first, the growth of mutants with an inactivated cph1 or cph2 gene was analysed under different light conditions. The growth of all phytochrome mutants was affected under high-light conditions. However, the cph1 mutant grew slower than the wild type in far-red light, whilst the cph2 mutant revealed a reduced growth under red light conditions. A decreased growth of the cph1/cph2 double mutant was observed under all light conditions with a growth rate similar to the corresponding single mutant. The exact reason for the growth impairment of the phytochrome mutants could not be ascertained. Different aspects of photosynthesis (pigment composition, maximal net-oxygen evolution and 77K fluorescence emission) were not changed significantly in the phytochrome mutants. Synechocystis sp. PCC 6803 shows a movement towards a light source. Based on action spectra of motility phytochromes and phytochrome-like proteins are supposed to have a function in regulating the phototactic movement. An influence of the Cph1 protein in the phototactic movement was not demonstrated. Whereas, the Cph2 protein seems to be involved in the inhibition of the cell movement towards blue light. An interaction with a typical blue-light receptor was excluded.

O segundo mensageiro cGMP está envolvido em diversas funções celulares incluindo a visão em mamíferos. Embora trabalhos anteriores mostrassem variações nos níveis de cGMP durante o ciclo de vida de Blastocladiela emersonii e evidências da existência de enzimas específicas envolvidas na sua síntese (guanilato ciclase) e degradação (cGMP fosfodiesterase), nenhum genoma de fungo publicado até o momento mostrou a existência de genes codificando estas enzimas. Este fato é atribuído por evolucionistas à completa perda de motilidade dos fungos em geral, já que cGMP está primordialmente associado a células com cílios. Blastocladiomicetos, como Blastocladiella, apresentam células móveis em pelo menos um estágio do seu ciclo de vida, o que poderia explicar a existência dessa via nesses fungos. Uma investigação no banco de ESTs de B. emersonii revelou a existência de cDNAs codificando parte de prováveis guanilato ciclases (BeGC1, BeGC2 e BeGC3) e uma possível cGMP fosfodiesterase (BePDE). Assim, este trabalho buscou confirmar a existência destas enzimas e caracterizar a sinalização por cGMP em B. emersonii. A proteína recombinante selvagem correspondente ao domínio catalítico de BePDE mostrou atividade de degradação sobre cGMP e a mutação E389A foi capaz de alterar a especificidade por cGMP. Com o sequênciamento do genoma de B. emersonii obteve-se as sequências completas das guanilato ciclases. Em BeGC2 não foi possível identificar o ligante responsável por sua ativação. Em BeGC3, a presença de um domínio Heme-Pas sugeriu sua ativação por óxido nítrico. A presença de um domínio rodopsina em BeGC1 sugeriu sua ativação por luz. Experimentos de microscopia por imunofluorescência localizaram BeGC1 no \"eyespot\", BeGC2 no capacete nuclear e BeGC3 no citoplasma de zoósporos de B. emersonii. Verificamos também que zoósporos realizam fototaxia em direção à luz verde e que a adição de hidroxilamina, inibidor de rodopsina, ou do inibidor de guanilato ciclase LY83583 tem efeito negativo na fototaxia, bem como impede o aumento dos níveis de cGMP observado em zoósporos expostos à luz verde. O bloqueio da síntese de retinal por Norflurazon também inibiu a fototaxia sendo esta restaurada quando adicionamos retinalA1. Estes dados, juntamente com o fato de o domínio rodopsina de BeGC1 ser a única rodopsina presente no genoma, indicam que BeGC1 é responsável pela fototaxia nos zoósporos de B. emersonii. O genoma do fungo apresenta ainda um possível canal de potássio ativado por cGMP (BeCNG1) localizado na membrana plasmática de zoósporos, similar ao canal regulado por cGMP envolvido na visão em humanos. Ensaios de microfluorimetria também evidenciaram a presença de um canal ativado por cGMP relacionado com o influxo de potássio e a motilidade dos zoósporos. Um modelo para a via de sinalização da fototaxia em B.emersonii foi proposto e comparado com a sinalização presente na visão de mamíferos, destacando a existência de cGMP e rodopsina em ambos os processos e sugerindo uma possível origem comum. Portanto, os resultados obtidos suportam a existência da sinalização por cGMP em B. emersonii, além de indicar o papel dessa sinalização na fototaxia dos zoósporos, sendo esta a primeira via de sinalização por cGMP caracterizada em fungos. / The second messenger cyclic GMP is involved in a wide array of cellular processes including vision in mammals. Although previous studies demonstrated changes in cGMP levels during the life cycle of Blastocladiela emersonii and evidences of specific enzymes involved in its synthesis (guanylyl cyclase) and hydrolysis (cGMP-phosphodiesterase), no fungal genome published so far shows the presence of genes encoding these enzymes. Evolutionists attribute the absence of cGMP signaling pathways in higher fungi to the sedentary life style of these organisms, since cGMP is primarily associated with ciliated cells. However, blastocladiomycetes like Blastocladiella, have motile cells in at least one stage of their life cycle, which could explain the existence of this pathway in these primitive fungi. Inspection of B. emersonii EST data bank, revealed cDNAs encoding part of three putative guanylyl cyclases (BeGC1, BeGC2 e BeGC3) and one possible cGMP phosphodiesterase (BePDE). Thus, the purpose of this study was to confirm the existence of these enzymes and characterize the cGMP signaling pathway in this model. The recombinant protein containing the wild type catalytic domain of BePDE presented activity towards hydrolysis of cGMP and the E389A mutation of this domain changed the cGMP specificity of this enzyme. The complete nucleotide sequence of the guanylyl cyclases were obtained by sequencing of B. emersonii genome. In BeGC2 we were unable identify the ligand responsible for its activation, but in BeGC3, the presence of a Heme-Pas domain suggested its activation by nitric oxide. The presence of a rhodopsin domain in BeGC1 suggested its activation by light. Immunofluorescence microscopy localized BeGC1 in the \"eyespot\" structure, BeGC2 in the nuclear cap and BeGC3 in the cytoplasm of zoospores of B. emersonii. We found that Blastocladiella zoospores performed phototaxis toward green light and photobleaching of rhodopsin function using hydroxylamine prevented both phototaxis and the increased cGMP levels observed when zoospores were exposed to green light. The same effect was observed using the guanylyl cyclase inhibitor LY83583. Inhibition of retinal synthesis using Norflurazon prevented the phototaxis response, which could be restored by zoospore complementation with retinalA1. The BeGC1 gene is the only rhodopsin found in the draft assembly of B. emersonii genome, which indicates that BeGC1 is responsible for phototaxis observed in zoospores. We also found in the genome a possible cGMP-activated potassium channel (BeCNG1), localized in the plasma membrane of the zoospores, which is similar to the cGMP-activated channel involved in human vision. In addition, microfluorimetry assays revealed the presence of a cGMP-activated potassium channel involved in potassium influx and zoospore motility. The signaling model of B. emersonii phototaxis was proposed and compared with the mammalian vision system, with cGMP and rhodopsin acting in both signaling pathways, suggesting a common origin. Altogether our data indicate that Blastocladiella emersonii has a cGMP signaling system involved in phototaxis, being the first cGMP signaling pathway characterized in fungi.

Thesis (M.S.)--Oregon State University, 2011. / Printout. Includes bibliographical references (leaves 71-79). Also available on the World Wide Web.

O segundo mensageiro cGMP está envolvido em diversas funções celulares incluindo a visão em mamíferos. Embora trabalhos anteriores mostrassem variações nos níveis de cGMP durante o ciclo de vida de Blastocladiela emersonii e evidências da existência de enzimas específicas envolvidas na sua síntese (guanilato ciclase) e degradação (cGMP fosfodiesterase), nenhum genoma de fungo publicado até o momento mostrou a existência de genes codificando estas enzimas. Este fato é atribuído por evolucionistas à completa perda de motilidade dos fungos em geral, já que cGMP está primordialmente associado a células com cílios. Blastocladiomicetos, como Blastocladiella, apresentam células móveis em pelo menos um estágio do seu ciclo de vida, o que poderia explicar a existência dessa via nesses fungos. Uma investigação no banco de ESTs de B. emersonii revelou a existência de cDNAs codificando parte de prováveis guanilato ciclases (BeGC1, BeGC2 e BeGC3) e uma possível cGMP fosfodiesterase (BePDE). Assim, este trabalho buscou confirmar a existência destas enzimas e caracterizar a sinalização por cGMP em B. emersonii. A proteína recombinante selvagem correspondente ao domínio catalítico de BePDE mostrou atividade de degradação sobre cGMP e a mutação E389A foi capaz de alterar a especificidade por cGMP. Com o sequênciamento do genoma de B. emersonii obteve-se as sequências completas das guanilato ciclases. Em BeGC2 não foi possível identificar o ligante responsável por sua ativação. Em BeGC3, a presença de um domínio Heme-Pas sugeriu sua ativação por óxido nítrico. A presença de um domínio rodopsina em BeGC1 sugeriu sua ativação por luz. Experimentos de microscopia por imunofluorescência localizaram BeGC1 no \"eyespot\", BeGC2 no capacete nuclear e BeGC3 no citoplasma de zoósporos de B. emersonii. Verificamos também que zoósporos realizam fototaxia em direção à luz verde e que a adição de hidroxilamina, inibidor de rodopsina, ou do inibidor de guanilato ciclase LY83583 tem efeito negativo na fototaxia, bem como impede o aumento dos níveis de cGMP observado em zoósporos expostos à luz verde. O bloqueio da síntese de retinal por Norflurazon também inibiu a fototaxia sendo esta restaurada quando adicionamos retinalA1. Estes dados, juntamente com o fato de o domínio rodopsina de BeGC1 ser a única rodopsina presente no genoma, indicam que BeGC1 é responsável pela fototaxia nos zoósporos de B. emersonii. O genoma do fungo apresenta ainda um possível canal de potássio ativado por cGMP (BeCNG1) localizado na membrana plasmática de zoósporos, similar ao canal regulado por cGMP envolvido na visão em humanos. Ensaios de microfluorimetria também evidenciaram a presença de um canal ativado por cGMP relacionado com o influxo de potássio e a motilidade dos zoósporos. Um modelo para a via de sinalização da fototaxia em B.emersonii foi proposto e comparado com a sinalização presente na visão de mamíferos, destacando a existência de cGMP e rodopsina em ambos os processos e sugerindo uma possível origem comum. Portanto, os resultados obtidos suportam a existência da sinalização por cGMP em B. emersonii, além de indicar o papel dessa sinalização na fototaxia dos zoósporos, sendo esta a primeira via de sinalização por cGMP caracterizada em fungos. / The second messenger cyclic GMP is involved in a wide array of cellular processes including vision in mammals. Although previous studies demonstrated changes in cGMP levels during the life cycle of Blastocladiela emersonii and evidences of specific enzymes involved in its synthesis (guanylyl cyclase) and hydrolysis (cGMP-phosphodiesterase), no fungal genome published so far shows the presence of genes encoding these enzymes. Evolutionists attribute the absence of cGMP signaling pathways in higher fungi to the sedentary life style of these organisms, since cGMP is primarily associated with ciliated cells. However, blastocladiomycetes like Blastocladiella, have motile cells in at least one stage of their life cycle, which could explain the existence of this pathway in these primitive fungi. Inspection of B. emersonii EST data bank, revealed cDNAs encoding part of three putative guanylyl cyclases (BeGC1, BeGC2 e BeGC3) and one possible cGMP phosphodiesterase (BePDE). Thus, the purpose of this study was to confirm the existence of these enzymes and characterize the cGMP signaling pathway in this model. The recombinant protein containing the wild type catalytic domain of BePDE presented activity towards hydrolysis of cGMP and the E389A mutation of this domain changed the cGMP specificity of this enzyme. The complete nucleotide sequence of the guanylyl cyclases were obtained by sequencing of B. emersonii genome. In BeGC2 we were unable identify the ligand responsible for its activation, but in BeGC3, the presence of a Heme-Pas domain suggested its activation by nitric oxide. The presence of a rhodopsin domain in BeGC1 suggested its activation by light. Immunofluorescence microscopy localized BeGC1 in the \"eyespot\" structure, BeGC2 in the nuclear cap and BeGC3 in the cytoplasm of zoospores of B. emersonii. We found that Blastocladiella zoospores performed phototaxis toward green light and photobleaching of rhodopsin function using hydroxylamine prevented both phototaxis and the increased cGMP levels observed when zoospores were exposed to green light. The same effect was observed using the guanylyl cyclase inhibitor LY83583. Inhibition of retinal synthesis using Norflurazon prevented the phototaxis response, which could be restored by zoospore complementation with retinalA1. The BeGC1 gene is the only rhodopsin found in the draft assembly of B. emersonii genome, which indicates that BeGC1 is responsible for phototaxis observed in zoospores. We also found in the genome a possible cGMP-activated potassium channel (BeCNG1), localized in the plasma membrane of the zoospores, which is similar to the cGMP-activated channel involved in human vision. In addition, microfluorimetry assays revealed the presence of a cGMP-activated potassium channel involved in potassium influx and zoospore motility. The signaling model of B. emersonii phototaxis was proposed and compared with the mammalian vision system, with cGMP and rhodopsin acting in both signaling pathways, suggesting a common origin. Altogether our data indicate that Blastocladiella emersonii has a cGMP signaling system involved in phototaxis, being the first cGMP signaling pathway characterized in fungi.

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.

The brown marmorated stink bug (Halyomorpha halys) (Stål) is known for overwintering in human homes. Studies were conducted to understand the behavior of adult bugs in response to characteristics of potential overwintering structures including gap sizes, light, and presence of dead conspecifics. In a test where bugs were placed in increasingly tight boxes, most bugs settled in gaps 4.5 - 5.5 mm high. None settled in a space less than 3.5 mm high. In boxes with constant tightness, bugs tended to move to the back, and primarily settled along walls and in corners. In both box types, bugs tended to turn and face the cavity entrance during settling. In tests of responses of disturbed overwintering bugs to common household light bulbs over the course of a year, bugs were taken from shelters and exposed to lit bulbs. Bug responses were seasonal, with attraction to light bulbs in spring and summer, but little or no consistent response in fall or winter. The lack of response occurred more often at night than in afternoons. Because not all bugs survive the winter, corpses can accumulate. Single shelter-seeking H. halys were found not to respond to the presence of dead aggregations from the previous years unless touch was possible, in which case aggregations were joined. Dead aggregations from the same year had a repellent effect. Presence of a single dead bug from the same year did not provide any survivorship advantage to overwintering bugs. Results suggest possible improvements to trap shelters. / Master of Science in Life Sciences / The brown marmorated stink bug (Halyomorpha halys) is known for overwintering in human homes. This research investigated the responses of these bugs to some characteristics of overwintering structures, including crevice tightness preference, light sources, and dead bugs leftover from previous years. Tightness experiments indicated that bugs in crevices with hard walls prefer to settle in spaces between 4.5 and 5.5 mm high. Bugs also tended to stay on the floor, go back as far as they could, and face the entrance of the crevice. These findings will help improve overwintering box designs for collection and research, and will help pest control professionals focus their efforts. When bugs were disturbed and exposed to common household light bulbs, they tended to move toward the bulbs in late summer and in spring, but responded less in fall and winter, and responded more often in the afternoon than at night. Bugs were also exposed to other dead bugs in several ways. When lone bugs were exposed to groups of dead bugs from previous years, they went to them only if they could touch them. Single dead bugs had less of an impact. Exposure to groups of dead bugs from the same year drove live bugs away. Overwintering brown marmorated stink bugs do not appear to eat other bugs that have recently died. These results suggest several possible improvements to the designs of trap shelters used for research and control.