modC is part of a set of genes, modABC, which encode the bacterial molybdate ABC transporter. ModC comprises the ATPase. Of the three structural Mod proteins, ModC has received the least attention, believed to be little different from ATPases of other ABC complexes. However, findings of multiple copies of modC in A. vinelandii and identification of a potential molybdate-binding domain in the C-terminal of ModC may point to a more complex role. ORF10 is one of three copies of modC in A. vinelandii; atypically, ORF10 is not found with genes encoding the other ABC transporter components but is instead found as part of the nif gene cluster, encoding molybdenum nitrogenase. The role of ORF10 is investigated here via sequence analysis and comparison of growth of a ORF10- A. vinelandii strain with wild type growth. Findings imply ORF10 optimizes growth under conditions requiring nitrogenixation, specifically under those conditions where molybdate-availability is limited.

Many multicellular cyanobacteria produce specialized nitrogenfixing heterocysts. During diazotrophic growth of Anabaena (Nostoc) sp. strain PCC 7120, a regulated developmental pattern of single heterocysts separated by about 10 to 20 photosynthetic vegetative cells is maintained along filaments. Heterocyst structure and metabolic activity function together to accommodate oxygensensitive nitrogen fixation, catalyzed by nitrogenase. In this work, we show that the promoter of the nifHDK genes that encode nitrogenase, lies upstream from the intergenic region between nifH and nifU. Excision of the fdxN element is required for transcription of the nifHDK genes. Fluorescence microscopy of reporter strain PnifHDgfp, in the chromosomal nif locus indicated that expression of nifHDK is blocked in mutants that are unable to excise the fdxN element after nitrogen deprivation. We proposed that a promoter upstream of the element, likely PnifB, is required for transcription of the nifHDK genes. Indeed, the PnifHDgfp reporter at an ectopic site did not show GFP fluorescence. A PnifBgfp reporter was expressed specifically in heterocysts indicating that a promoter for the nifB gene lies in the intergenic region upstream of nifB. A stem loop structure located in the intergenic region between nifH and nifU may act as a processing site for production of nifHDK transcripts. We also provide evidence that DevH, a transcriptional regulator, is involved in regulating the nifBfdxNnifSUHDK genes. DevH is a protein belonging to the cAMP receptor protein (CRP) family of proteins that are widespread in bacteria and regulate genes in response to a gamut of physiological conditions. We show that DevH binds specifically to the nifB upstream region but not to the immediate upstream region of nifH. We predict that DevH binds to an NtcAlike binding site upstream of nifB and functions as an activator of the nifBfdxNnifSUHDK genes. Finally, we show that sigE, which is expressed at 16 hours after nitrogen deprivation, is required for normal expression of some heterocyst specific genes, including nifHDK. A sigE mutant shows delayed and reduced expression of nifHDK and some middle and late genes. We hypothesize that DevH in concert with SigE upregulates the expression of nifHDK in heterocysts after nitrogen deprivation.

I am interested in discovering the role of field potential oscillations in producing synchrony within the song system of the male zebra finch brain. An important function attributed to neural synchrony is sensorimotor integration. In the production of birdsong, sensorimotor integration is crucial, as auditory feedback is necessary for the maintenance of the song. A cortical-thalamic-cortical feedback loop is thought to play a role in the integration of auditory and motor information for the purpose of producing song. Synchronous activity has been observed between at least two nuclei in this feedback loop, MMAN and HVC. Since low frequency field potential oscillations have been shown to play a role in the synchronization of nuclei within the brain of other model animals, I hypothesized that this may be the case in the zebra finch song system. In order to investigate whether oscillatory activity is a mechanism behind the synchronous activity observed between HVC and MMAN, I performed dual extracellular recordings of neural activity within the zebra finch song system. Results suggest that oscillations are likely not involved in the synchrony observed in these nuclei. Future study may reveal that the structure of the feedback loop is necessary, and possibly even sufficient, for the synchronous activity in the zebra finch song system.

Techn. Hochsch., Diss., 2002--Aachen.

The onset of solvent production by the clostridia involves regulation at the transcriptional level. The signal triggering the onset has not been identified, but redox and energetic states have been suggested as possible factors. Because several solvent-producing clostridia, including Clostridium acetobutylicum and Clostridium beijerinckii, are nitrogen-fixing organisms and both nitrogen-fixation and alcohol production (n-butanol, isopropanol and ethanol) are reductant-dependent processes, the effect of nitrogen fixation on the onset and progression of solvent production in C. beijerinckii NRRL B593 and vice versa was investigated. For this purpose, a defined growth medium containing three amino acids was developed for C. beijerinckii NRRL B593, and this medium was used for growing solvent-producing and nitrogen-fixing cultures. The nitrogen-fixing cultures produced solvents with a solventogenic shift, which appeared to coincide with a decrease in nitrogen-fixing activity. Measurement of specific activities of acetoacetate decarboxylase and aldehyde dehydrogenase and Northern blot analysis of the mRNA of the solvent-producing genes in samples harvested periodically from a nitrogen-fixing culture of C. beijerinckii showed the presence of both enzyme activities and the mRNA carrying the solvent-production genes throughout incubation. A 2.5-fold increase in the specific activity of acetoacetate decarboxylase and a 4.5-fold increase in the specific activity of aldehyde dehydrogenase were observed when the activities in the latest cell-free extract was compared with the activities in the earliest cell-free extract. When C. beijerinckii was grown in the medium containing 4 mM ammonium acetate, the onset of nitrogen fixation coincided with the onset of solvent production and prevented accumulation of solvents to high levels, which suggested competition between alcohol-producing enzymes and nitrogenase for the reductant. Recently, a 20-kb region of the genomic DNA of C. beijerinckii NRRL B593 that contained the nif genes and ORFs with other putative functions was sequenced in our laboratory. An examination of the nif clusters of C. beijerinckii, C. acetobutylicum and C. pasteurianum revealed apparent differences in the intervening ORFs which suggested differences in the regulation of nitrogen fixation in these organisms. Transcriptional analysis of genes in the nif cluster of C. beijerinckii by Northern blotting revealed four different transcripts. The absence of mRNAs of the nif-associated ORFs in RNA samples isolated from non-nitrogen-fixing cells indicated that the nif-associated ORFs are regulated in parallel to the nif genes. By studying the effect of ammonia addition on nitrogen-fixing activities of C. beijerinckii and C. pasteurianum, significant differences in the regulation of nitrogen-fixation in the two species were observed. C. beijerinckii NRRL B593, but not C. pasteurianum, showed a rapid decrease in nitrogen-fixing activity in vivo upon ammonium acetate addition. However, measurement of nitrogen-fixing activities in vitro before and after ammonium acetate addition showed the presence of active nitrogenase throughout growth in both organisms. The results suggest that the nitrogenase activity in C. beijerinckii NRRL B593 is inhibited when ammonia is available. A second nifH-hybridizing mRNA was detected in Northern blots during studies of the expression of nifH1 in C. pasteurianum. The mRNA was identified as that from either the nifH2 or nifH6 gene after sequencing the cDNA strands, which were generated by RT (Reverse Transcriptase)-PCR. In addition, Western blot analysis of the cell-free extracts of nitrogen-fixing cells of C. pasteurianum indicated the presence of a second NifH-related polypeptide. The two NifH-related polypeptides were separated by preparative gel electrophoresis and characterized by MALDI-TOF (Matrix-assisted Laser Desorption Ionization Time-Of-Flight) mass spectrometry. The results suggested the expression of NifH2/H6 protein in nitrogen-fixing cells of C. pasteurianum. The physiological significance of the expression of the nifH2 or nifH6 gene or both is yet to be determined. / Ph. D.

A system was developed for the controlled expression of genes in Azotobacter vinelandii by using genomic fusions to the sucrose catabolic regulon. This system was used for the functional analysis of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this analysis the scrX gene, contained within the sucrose catabolic regulon, was replaced by the A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, iscX gene cluster, resulting in duplicate genomic copies of these genes, one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional analysis of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This experimental strategy was used to show that IscS, IscU, HscBA and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in null growth only when cells are cultured under conditions of elevated oxygen, marking the first null phenotype associated with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted for HscBA could be partially reversed by culturing cells under conditions of low oxygen. These results are interpreted to indicate that HscBA and IscA could have functions related to the protection or repair of the primary IscS/IscU machinery when grown under aerobic conditions. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their respective functions and/or display a partial or complete dominant-negative growth phenotype were also identified using this system. Inactivation of the IscR repressor protein resulted in a slow growth phenotype that could be specifically attributed to the elevated expression of an intact [Fe-S] cluster biosynthetic system. This system was also used to investigate the extent to which the two [Fe-S] biosynthetic systems in A. vinelandii, Nif and Isc, can perform overlapping functions. Under normal laboratory growth conditions, no cross-talk between the two systems could be detected. However, elevated expression of Isc components as a consequence of inactivation of the IscR repressor protein results in a modest ability of the Isc [Fe-S] protein maturation components to replace the function of Nif-specific [Fe-S] protein maturation components. Similarly, when expressed at very high levels the Nif-specific [Fe-S] protein maturation components could functionally replace the Isc components. Oxygen levels were also found to affect the ability of the Nif and Isc systems to perform common functions. Nevertheless, the lack of significant reciprocal cross-talk between the Nif and Isc systems when they are produced only at levels necessary to satisfy their respective physiological functions, indicates a high level of target specificity with respect to [Fe-S] protein maturation. / Ph. D.

Hydrogen has an extensive potential as a clean and renewable energy source. Photosynthetic, non-sulphur, purple bacteria, Rhodobacter sphaeroides O.U.001 produces molecular hydrogen by nitrogenase enzyme. Nitrogenase enzyme is encoded by nifHDK genes and expression of the structural genes, nifHDK, is controlled by NifA which is encoded by nifA gene. The transcription of nifA is under the control of Ntr system and product of prrA gene. Relationship between the genes that have roles in nitrogenase synthesis should be understood well to increase biological hydrogen production. In this work, expression levels of nitrogenase encoding nifH and control genes nifA, prrA were examined at different physiological conditions. In addition to modifications in expression levels, changes in hydrogen production and growth capacity were also investigated in response to different concentrations of ammonium source, oxygen and different light intensities. In this study, it was found that increasing concentrations of ammonium chloride caused decrease in hydrogen production. Glutamate containing medium had the capacity for higher hydrogen production. The expression levels of nifH and nifA genes decreased with the increase in concentrations of ammonium chloride. There was a negative correlation between the expression levels of prrA gene and its target, nifA gene. Hydrogen production was observed even in aerobic conditions of the same media compositions. It was observed that different culture media had changing growth and hydrogen production capabilities at different light intensities. There was no direct proportion between the expression levels of nifH gene and amount of hydrogen at different light intensities.

The theoretical part of the thesis deals with economic theories relating to the unprofitable sector, definition and segmentation of public sector, and description and classification of unprofitable associations. Next there is detailed information about foundation -- legislative definition of terms, laws and obligations, as well as information about foundational mutual fund and foundation activities in practice -- a description of granting procedure. The practical part deals with analysis of tender procedure to foundation subsidy allocation at five chosen foundations. The way of evaluation of received applications is examined in detail, and then comparison of process is accomplished. The ending contains proposed procedure presenting optimal concept of open and transparent tender.

No description available.

Experimental results and simulations of imploding fusion concepts have identified the Rayleigh-Taylor (RT) instability as one of the largest inhibitors to achieving fusion. Understanding the origin and development of the RT instability will allow for the development of mitigating measures to dampen the instability growth, thus improving the chance that fusion concepts such as inertial confinement fusion (ICF) are successful. A study of 1D and 2D simulations are presented for investigating RT instability growth in deceleration stage of imploding geometries. Two cases of laser-driven implosion geometry, Cartesian and cylindrical, are used to study late stage deceleration-phase RT instability development on the interior surface of imploding targets. FLASH's hydrodynamic (HD) and magnetohydrodynamic (MHD) modeling capabilities are used for different laser and target parameters in order to study the RT instability and the impact of externally applied magnetic fields on their evolution. Several simulation regimes have been identified that provide novel insight into the impact that a seeded magnetic field can have on RT instability growth and the conditions under which magnetic field stabilization of the RT instability is observable. Finally, future work and recommendations are made. / Master of Science / The direction for the future of renewable energy is uncertain at this time; however, it is known that the future of human energy consumption must be green in order to be sustainable. Fusion energy presents an opportunity for an unlimited clean renewable energy source that has yet to be realized. Fusion is achieved only by overcoming the earthly limitations presented by trying to replicate conditions at the interior of stellar structures. The pressures, temperature, and densities seen in the interior of stars are not easily reproduced, and thus human technology must be developed to reach these difficult stellar conditions in order to harvest fusion energy. There are two main branches of developmental technology geared towards achieving the difficult conditions controlled nuclear fusion presents, magnetic confinement fusion (MCF) and inertial confinement fusion (ICF)[17]. Yet in both approaches barriers exist which have thwarted the efforts toward reaching fusion ignition which must be addressed through scientific discovery. Successfully reaching ignition is only the first step in the ultimate pursuit of a self sustaining fusion reactor. This work will focus on the experimental ICF configuration, and on one such inhibitor toward achieving ignition, the Rayleigh-Taylor (RT) instability. The RT instability develops on the surfaces of the fusion fuel capsules, targets, and causes nonuniform compression of the target. This nonuniform compression of the target leads to lower pressures and densities through the material mixing of fusion fuel and the capsule shell, which ultimately leads to challenges with reaching fusion ignition. The work presented here was performed utilizing the University of Chicago's FLASH code, which is a state-of-the-art open source radiation magneto-hydrodynamic (MHD) code used for plasma and astrophysics computational modeling [11]. Simulations of the RT instability are performed using FLASH in planar and cylindrical geometries to explore fundamental Rayleigh-Taylor instability evolution for these two different geometries. These geometries provide easier access for experimental diagnostics to probe RT dynamics. Additionally, the impact of externally applied magnetic fields are explored in an effort to examine if and how the detrimental instability can be controlled.