Invasive fungal infections are a significant clinical challenge especially for hospitalized patients as traditional antifungal therapy often fails to resolve these infections. The ability of Candida to undergo yeast-to-hyphae morphological transition is central to this invasive behavior. Morphogenesis is also important for the formation of biofilms which are highly structured communities of microorganisms attached to one another or substratum and embedded within a protective extracellular matrix material. The refractory nature of cells within a biofilm to current antifungal therapies has created a need for alternative antifungal agents for the management of Candida biofilm-related infections. The novel antifungal occidiofungin is a natural product produced by the soil bacteria Burkholderia contaminans shown to be effective against a broad range of fungi including Candida spp. Prior studies have demonstrated that occidiofungin inhibits yeast-to-hyphae morphogenesis in the dimorphic yeast, C. albicans, likely through its impact on disrupting F-actin organization. To extend these findings, the efficacy of occidiofungin on morphogenesis of C. albicans and C. tropicalis strains under different inducing conditions was evaluated. Further, given the role of biofilm on pathogenicity, the anti-biofilm properties of occidiofungin against Candida species was examined using an in vitro static biofilm model developed on a silicon elastomer disk. The accumulated data indicate that occidiofungin inhibits hyphal transformation regardless of the inducing conditions used and prevents hyphal extension when added to cells post switching. Moreover, morphologically switching cells were more sensitive to occidiofungin than their yeast counterpart. In addition, occidiofungin effectively blocks biofilm formation at all stages of development and reduces dispersed cells from the biofilm for both C. albicans and C. tropicalis. Confocal data revealed alterations in actin organization with occidiofungin treatment for both morphologically switching and biofilm cells. These findings correlate with prior observations for occidiofungin activity on yeast form cells indicating the broad activity of occidiofungin against fungi at various stages of pathogenic growth and supports efforts to pursue occidiofungin as a potential therapeutic against Candida based infections.

Blutplättchen werden von Megakaryozyten im Knochenmark in einem Prozess produziert, an dem Aktin beteiligt ist. Aktin-Depolymerisierungsfaktor (ADF) und Cofilin sind Aktin-bindende Proteine, die als entscheidende Regulatoren im Aktinumsatz agieren, indem sie das Schneiden und Depolymerisieren von Filamenten unterstützen. Die Bedeutung von ADF/Cofilin und des Aktinumsatzes in der Bildung von Blutplättchen ist gegenwärtig nicht bekannt. In der vorliegenden Arbeit wurden Mäuse untersucht, die eine konstitutive ADF-Defizienz und/oder die eine konditionale n-Cofilin Defizienz (Cre/loxP) aufweisen. Um Cofilin nur in Megakaryozyten und Blutplättchen auszuschalten, wurden Cofilinfl/fl Mäuse mit PF4-Cre Mäusen verpaart. ADF- oder n-Cofilin-defiziente Mäuse hatten keinen oder nur einen geringen Phänotyp in Blutplättchen. Eine Defizienz von ADF und n-Cofilin führte hingegen zu einem beinahe kompletten Verlust der Blutplättchen, was mit Defekten in der Bildung von Plättchenzonen in Knochenmark-Megakaryozyten einherging. Weitere Untersuchungen an in vitro und ex vivo kultivierten Megakaryozyten zeigten eine Reduzierung der Bildung von Proplättchen und das Fehlen der typischen Verdickungen der Proplättchen. Diese Daten zeigen redundante aber essentielle Funktionen von ADF und n-Cofilin im terminalen Schritt der Plättchenbildung in vitro und in vivo, und belegen erstmals eine wichtige Rolle des Aktinumsatzes in diesem Prozess. Im zweiten Teil dieser Dissertation wurden die Mechanismen untersucht, die für die zelluläre Regulierung des Hauptkollagenrezeptors auf Blutplättchen, Glykoprotein VI (GPVI), verantwortlich sind. Nach einer Gefäßwandverletzung wird subendotheliales Kollagen freigelegt, wodurch GPVI die Aktivierung von Blutplättchen vermittelt, und damit zur Blutstillung (Hämostase), aber auch zum Verschluss eines verletzten Gefäßes beitragen kann, was letztendlich zu einem Myokardinfarkt oder einem Schlaganfall führen kann. Deshalb ist GPVI ein attraktives Zielprotein für eine anti-thrombotische Therapie, insbesondere weil frühere Studien gezeigt haben, dass anti-GPVI Antikörper eine irreversible Herunterregulierung des Rezeptors auf zirkulierenden Blutplättchen mittels Internalisierung und Abspaltung induzieren. Es wird vermutet, dass Metalloproteinasen der ADAM (a disintegrin and metalloproteinase domain) - Familie das Abspalten vermitteln, jedoch fehlt in vivo der Beweis dafür. Um die Mechanismen des Abspaltungsprozesses des GPVI Rezeptors in vivo besser verstehen zu können, wurden zwei Mauslinien, GPVI- und konditionale ADAM10-defiziente Mäuse, generiert und zusätzlich sogenannte „low TACE (TNFalpha converting enzyme)“ Mäuse analysiert. Es konnte gezeigt werden, dass GPVI in vitro von ADAM10 oder TACE in Abhängigkeit der Signalwege, die zum Abspalten des Rezeptors führen, geschnitten werden kann. Darüberhinaus wurde GPVI in vivo nach Antikörperverabreichung in ADAM10-defizienten Mäusen und „low TACE“ Mäusen herunterreguliert, was vermuten lässt, dass entweder beide Metalloproteinasen an diesem Prozess beteiligt sind oder noch eine zusätzliche Metalloproteinase für die GPVI Regulation in vivo verantwortlich ist. / Platelets are produced by bone marrow megakaryocytes in a process involving actin dynamics. Actin-depolymerizing factor (ADF) and cofilin are actin-binding proteins that act as key regulators in actin turnover by promoting filament severing and depolymerization. The overall significance of ADF/cofilin function and actin turnover in platelet formation is presently unclear. In the first part of this thesis, platelet formation and function were studied in mice constitutively lacking ADF and/or mice with a conditional deficiency (Cre/loxP) in n-cofilin. To delete cofilin exclusively in megakaryocytes and platelets, cofilinfl/fl mice were crossed with PF4 (platelet factor 4)-Cre mice. While a single-deficiency in ADF or n-cofilin resulted in no or only a minor platelet formation defect, respectively, a double-deficiency in ADF and n-cofilin led to an almost complete loss of platelets. Bone marrow megakaryocytes of ADF/n-cofilin-deficient mice showed defective platelet zone formation. Interestingly, in vitro and ex vivo megakaryocyte differentiation revealed reduced proplatelet formation and absence of platelet-forming swellings. These data establish that ADF and n-cofilin have redundant but essential roles in the terminal step of platelet formation in vitro and in vivo. In the second part of the thesis, mechanisms underlying cellular regulation of the major platelet collagen receptor, glycoprotein VI (GPVI), were studied. GPVI mediates platelet activation on exposed subendothelial collagens at sites of vascular injury, and thereby contributes to normal hemostasis but also to occlusion of diseased vessels in the setting of myocardial infarction or stroke. Thus, GPVI is an attractive target for anti-thrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and ectodomain shedding. Metalloproteinases of the ADAM (a disintegrin and metalloproteinase domain) family are suspected to mediate this ectodomain shedding, but in vivo evidence for this is lacking. To study the mechanism of GPVI regulation in vivo, two mouse lines, Gp6 knock-out and Adam10fl/fl, PF4-Cre mice, were generated and in addition low TACE (TNFalpha converting enzyme) mice were analyzed. It was shown that GPVI can be cleaved in vitro by ADAM10 or TACE depending on the shedding-inducing signaling pathway. Moreover, GPVI was down-regulated in vivo upon antibody injection in ADAM10-deficient and low TACE mice suggesting that either both or an additional metalloproteinase is involved in GPVI regulation in vivo.

Actin is a highly abundant protein in most eukaryotic cells. The assembly of actin monomers to double helical filaments is crucial for many cellular functions, including cell movement and cell division. Actin filament assembly in cells occurs in a crowded intracellular environment consisting of various molecules, including cations and organic osmolytes. Recent studies show that cation binding stiffens actin filaments, and a small organic osmolyte trimethylamine-N-oxide (TMAO) modulates filament assembly. However, how cations and TMAO combined affect actin filament mechanics is not understood. We hypothesize that depending on the concentrations of cations and osmolytes, there will be different effects on the stiffness and assembly of actin filaments. In this study, using TIRF we evaluate actin filament mechanics and assembly. Our findings indicate that when TMAO is present alone, it can increase the elongation rate and stiffness of actin filaments, however the inclusion of potassium levels alongside TMAO reduces the persistence length of actin filaments, suggesting a decrease in filament stiffness compared to the influence of TMAO alone. Furthermore, the elongation rate of actin filaments decreases when both TMAO and potassium ions are present. This study will help us better understand how cations and osmolytes together can affect actin filament mechanics in the living cells.

This research examines the role of actin cytoskeleton filaments in chondroinduction by cyclic hydrostatic pressurization. A chondroinductive hydrostatic pressurization system was developed and characterized. A pressure of 5 MPa at 1 Hz frequency, applied for 7200 cycles (4 hours intermittent) per day, induced chondrogenic differentiation in C3H10T1/2 cells while 1800 cycles (1 hour intermittent) did not induce chondrogenesis. Quantitative analysis of chondrogenesis was determined as sulfated glycosaminoglycan synthesis and rate of collagen synthesis while qualitative analysis was obtained as Alcian Blue staining and collagen type II immunostaining. Actin disruption using 2 uM Cytochalasin D inhibited the enhanced sGAG synthesis in the chondroinductive hydrostatic pressurization environment and significantly inhibited rate of collagen synthesis to the mean level lower than that of the non-pressurized group. These results suggest an involvement of actin cytoskeleton filaments in mechanotransduction of cyclic hydrostatic pressure.

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