Periodontal disease (PD) is a polymicrobial infection characterized by inflammation of the gingiva, alveolar bone resorption, and tooth loss (edentulism). Treponema denticola along with Porphyromonas gingivalis and Tannerella forsythia are among the “Red Complex” and are main etiological agents in PD. Treponemes are a member of the Spirochaeta phylum and are obligate anaerobes, that express pyruvate ferredoxin oxidoreductase (PFOR). The enzyme catalyzes the oxidation of pyruvate to acetyl-CoA and reduced ferredoxin. Amixicile is a novel bacteriostatic derivative of nitazoxanide and an inhibitor of PFOR. In light of the fact that Treponemes express PFOR, this study was conducted to investigate the susceptibility of oral Treponemes to AMX. All oral Treponemes tested were susceptible to AMX and the MIC values were determined ranging of 1.5-4.5 μg mL-1 for an initial starting cell concentration of 1.9x106 cells mL-1. Other potentially therapeutic effects for AMX for T. denticola were investigated: motility, hydrogen sulfide production, and serum sensitivity. AMX reduced overall spirochete motility by 50% at sub-MIC concentrations. There was a dose dependent decrease in H2S production in T. denticola at sub-MIC and MIC values. Furthermore, prior exposure of AMX led to increases in serum sensitivity. Taking into account the fact that other periodontal red complex bacteria express PFOR, AMX could serve as a new selective adjunctive treatment for periodontal disease.

The objective of this study was to transform sweet pepper ferredoxin-like protein (PFLP) gene, which has antimicrobial properties, to tobacco and investigate the disease resistance abilities of transgenic tobacco. This protein interacts with another protein, harpin that is produced by the bacteria which is invading the plant tissues, and stimulates hypersensitivity response in plants, thus the spreading of disease is limited. Gene transfer was achieved to tobacco by Agrobacterium- mediated method and with indirect organogenesis / the explants were grown on selective media and then transferred to jars and pots respectively. Molecular and genetic analyses such as PCR, RT-PCR, Sequence Analysis and Northern Blot, were performed with plants which their seeds survived and grew on selective medium and also gave positive reactions for GUS histochemical assay. Finally, with putative transgenic plants, some hypersensitive response assays were carried out with Pseudomonas syringae and it was observed that the recovered plants showed hypersensitive response (HR) in the preliminary tests. These results indicated that putative transgenic tobacco plants which carry pflp transgene, can be used in disease resistance studies.

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Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation. / Master of Science

Toxoplasmose ist weltweit eine der am häufigsten auftretenden parasitären Zoonosen mit einer geschätzten Infektionsrate von über 30%. Toxoplasma gondii (Phylum: Apicomplexa) besitzt ein Plastid ähnliches Organell, den Apicoplasten. In diesem befindet sich das einzig bekannte Redoxsystem, welches aus der Ferredoxin-NADP+-Reduktase und Ferredoxin (Fd) besteht. Fd als Elektonendonator liefert Elektronen an verschiedene essentielle Stoffwechselwege, wie der Isoprenoidvorstufen- und Liponsäuresynthese. Um die bei einem Elektronentransfer benötigte direkte Protein-Protein-Interaktion eingehend zu analysieren, wurde ein bakterielles Reverse Two Hybrid System verwendet, womit die Interaktion von TgFd und TgLipA gezeigt werden konnte. Da angenommen wird, dass Fd eine zentrale Rolle in verschiedenen Stoffwechselwegen übernimmt, ist für einen Fd Knockout ein komplexer biochemischer Phänotyp zu erwarten, der möglicherweise zum Absterben der Parasiten führt. Zur Untersuchung dessen wurden zwei komplementäre Wege verfolgt. Eine der Strategien basierte auf dem grundsätzlichen Nachweis, dass Fd unerlässlich für das Überleben von T. gondii ist. Mit Hilfe des DiCre Systems sollte ein definierter genetischer Fd Knockout hergestellt werden, welcher jedoch nicht zweifelsfrei generiert werden konnte. Bei der zweiten Strategie kam ein konditionales Knockdown System zur Anwendung, bei welchem die Expression des Fd Gens nach Induktion herabreguliert wird. Mit Hilfe dessen konnten weitreichende Auswirkungen der Fd Defizienz auf T. gondii gezeigt werden: die Fettsäuresynthese der im Apicoplasten synthetisierten Fettsäuren ist reduziert sowie die Motilität durch eine beeinträchtigte Isoprenoidsynthese verringert, wodurch insgesamt drastische Auswirkungen auf das Parasitenwachstum gezeigt werden konnten. Beide Stoffwechsel sind vom Elektronendonator Fd abhängig und durch die Fd Herabregulation betroffen. Die Ergebnisse unterstreichen die essentielle Rolle des Fd-Redoxsystems von T. gondii. / Toxoplasmosis is one of the most common parasitic zoonoses world-wide, around 30% of human beings are infected. Toxoplasma gondii (phylum: Apicomplexa) contains a unique intracellular organelle derived from plastids, called apicoplast. The only known redox system in the apicoplast consists of the ferredoxin NADP+-reductase and its redox partner, ferredoxin (Fd). The latter donates electrons to different essential metabolic pathways in the apicoplast like the last two enzymes of the isoprenoid precursor biosynthesis and the lipoic acid synthesis. To dissect protein protein interactions for an electron transfer a bacterial reverse two hybrid system was used. The physical interaction of both proteins TgFd and TgLipA could be shown. Fd is supposed to play an important role in diverse metabolic pathways, hence a knock-out of the Fd gene is expected to generate a complex biochemical phenotype and be lethal to the parasite. Therefore two complementary approaches were used to analyze the role of TgFd in this context. The first strategy shall verify the essentiality of TgFd for the survival of T. gondii. It is based on the DiCre system whereby a defined genetic knock out of TgFd is produced. Respectives parasites have been generated, but at the end no genetic Fd knock out could be produced. In the second approach a conditional knock-down was generated, where the expression of the TgFd gene is repressed after induction. The Fd deficiency has wide ranging effects on T. gondii: The fatty acid synthesis of the apicoplast-synthesized fatty acids is reduced as well as the motility is decreased due to an affected isoprenoid synthesis. In total this leads to a dramatic inhibition of parasite growth. Both metabolic pathways depend upon the electron carrier Fd and thus are affected by Fd deficiency. The results underline the essential role of the ferredoxin redoxsystem of T. gondii.

Investigations of soft matter using ultrashort high intensity pulses have been made possible through the advent of X-ray free-electrons lasers. The last decade has seen the development of a new type of protein crystallography where femtosecond dynamics can be studied, and single particle imaging with atomic resolution is on the horizon. The pulses are so intense that any sample quickly turns into a plasma. This thesis studies the ultrafast transition from soft matter to warm dense matter, and the implications for structural determination of proteins.                    We use non-thermal plasma simulations to predict ultrafast structural and electron dynamics. Changes in atomic form factors due to the electronic state, and displacement as a function of temperature, are used to predict Bragg signal intensity in protein nanocrystals. The damage processes started by the pulse will gate the diffracted signal within the pulse duration, suggesting that long pulses are useful to study protein structure. This illustrates diffraction-before-destruction in crystallography. The effect from a varying temporal photon distribution within a pulse is also investigated. A well-defined initial front determines the quality of the diffracted signal. At lower intensities, the temporal shape of the X-ray pulse will affect the overall signal strength; at high intensities the signal level will be strongly dependent on the resolution. Water is routinely used to deliver biological samples into the X-ray beam. Structural dynamics in water exposed to intense X-rays were investigated with simulations and experiments. Using pulses of different duration, we found that non-thermal heating will affect the water structure on a time scale longer than 25 fs but shorter than 75 fs. Modeling suggests that a loss of long-range coordination of the solvation shells accounts for the observed decrease in scattering signal. The feasibility of using X-ray emission from plasma as an indicator for hits in serial diffraction experiments is studied. Specific line emission from sulfur at high X-ray energies is suitable for distinguishing spectral features from proteins, compared to emission from delivery liquids. We find that plasma emission continues long after the femtosecond pulse has ended, suggesting that spectrum-during-destruction could reveal information complementary to diffraction.