Rhodobacter sphaeroides O.U.001 is a purple non-sulphur bacterium producing hydrogen under photoheterotrophic, nitrogen limited conditions. Hydrogen is produced by Mo-nitrogenase but substantial amount of H2 is reoxidized by a membrane bound uptake hydrogenase. In this study, hydrogen production and the expression of structural nitrogenase genes were investigated by varying molybdenum and iron ion concentrations. These two elements are found in the structure of Mo-nitrogenase and they are important for functioning of the enzyme. The results showed that hydrogen production and nifD gene expression increased upon increase in molybdenum concentration. Increasing iron concentration had also positive effect on hydrogen production and nifK gene expression. To improve the hydrogen producing capacity of R. sphaeroides O.U.001, hupSL genes encoding uptake hydrogenase were disrupted in two different methods. In the first method, hup genes were disrupted by gentamicin resistance gene insertion. In the second method, part of the hup gene was deleted without using antibiotic resistance gene. The wild type and the hup- mutant cells showed similar growth patterns but substantially more hydrogen was produced by the mutant cells. The genes coding for hox1 hydrogenase of Thiocapsa roseopersicina was aimed to be expressed in R. sphaeroides O.U.001 to produce H2 under nitrogenase repressed and mixotrophic conditions. The hox1 hydrogenase genes of T. roseopersicina were cloned and transferred to R. sphaeroides. Although the cloning was successful, the expression of hydrogenase was not achieved by using either the native promoter of hox1 hydrogenase or the crtD promoter of T. roseopersicina.

Rhodobacter capsulatus is a photosynthetic, purple non-sulfur (PNS) bacterium that produces biohydrogen via photofermentation. Nitrogenase enzyme is responsible for hydrogen production / during fixation of molecular nitrogen into ammonium, hydrogen is produced. Since this process is an energetically expensive process for the cell, hydrogen production is strictly controlled at different levels. When ammonium is present in the environment, hydrogen production completely ceases. The key proteins in the regulation of nitrogenase by ammonium are two PII proteins / GlnB and GlnK. &lsquo / Hyvolution&rsquo / , 6th framework EU project, aims to achieve maximum hydrogen production by combining two hydrogen production processes / dark fermentation and photofermentation. In the first stage of the overall process, biomass is used for hydrogen production in dark fermentation process. Then, the effluent of dark fermentation is further utilized by photosynthetic bacteria to produce more hydrogen. However, the effluent of dark fermentation contains high amount of ammonium, which inhibits photofermentative hydrogen production. In order to achieve maximum hydrogen production, ammonium regulation of nitrogenase enzyme in R.capsulatus has to be released. For this purpose, all PII signal transduction proteins of R.capsulatus (GlnB and GlnK) were targeted to be inactivated by site-directed mutagenesis. The internal parts of glnB and glnK genes were deleted individually without using antibiotic cassette insertion. The successful glnB mutant was obtained at the end of mutagenesis studies. In the case of glnK mutation, the suicide vector was constructed and delivered into the cells. However, glnK mutant could not be obtained. The effect of ammonium on glnB mutant R.capsulatus was investigated and compared with wild type. Biomass of the bacterial cultures, pH of the medium and amount of produced hydrogen were periodically determined. Moreover, the concentrations of acetic, lactic, formic and propionic acids in the medium were periodically measured. Both wild type and glnB mutant grew on acetate and effectively utilized acetate. Ammonium negatively affected hydrogen production of glnB mutant and wild type. The ammonium inhibition of hydrogen production did not release in glnB mutant due to the presence of active GlnK protein in the cell / hence, inactivation of one of PII proteins was not enough to disrupt ammonium regulation of the cell. Moreover, kinetic analysis of bacterial growth and hydrogen production were done. Growth data fitted to the Logistic Model and hydrogen production data fitted to the Modified Gompertz Model.

The planning and scheduling requirement of industry can not be satisfied by traditional scheduling systems. Companies need to put extra human resource to fix the result made by these systems. The main reason is an improper assumption of infinite capacity adopted by these scheduling systems. In order to improve the scheduling result, this research refers plastic injection industry¡¦s characters to implement a decision support system. The decision support system integrates Forward Finite Loading and Constraint Directed into our algorithm in order to minimize the increase in total cost, and raise the capacity balance between machines .According to the result of the practical research, we can prove this decision support system is more effective and efficiency than the traditional scheduling method.

The purpose of this study was to investigate the post-conflict reconciliation, consolation, solicited affiliation, stress and redirection in the captive Formosan macaques¡]Macaca cyclopis¡^in the Taipei Zoo. I used the post-conflict and matched-control (PC-MC) method to calculate the conciliatory tendency (CCT) and the triadic contact tendency (TCT) in adult macaques. The mean CCT for kin (83.33%) was significantly higher than that for non-kin (0.36%), and victims initiated reconciliation toward aggressors in higher rank classes significant more than both of them were in the same rank. The mean TCTs of aggressors and the victims were similar toward different triadic contact opponents (opponent¡¦s kin, own kin, unrelated individual). The ratio of the attracted pairs of victims who reconciled with aggressors by sociosexual behavior (15.23%) was significant higher than dispersed pairs (non-exist). In addition, the ratio of attracted pairs of victims who reconciled with unrelated third party by affiliation (46.72%) was significant higher than the dispersed pairs (21.76%). The similar situation also occurred in sociosexual behavior (28.68% verse 1.75%). However, aggressors and victims had similar chance to take the initiative affiliation after conflict (P > 0.05) The frequency of self-directed behavior (SDB) of Formosan macaques was slightly higher in the first 4 minutes in PC. The frequency of SDB after reconciliation (14.6 bouts/100 min) was not significantly lower than that before reconciliation (23.2 bouts/100 min) or when affiliation behavior did not occur (22.3 bouts/100 min). When the conflict opponents were kin, the SDB frequency (16.1 bouts/100 min) was not significantly lower than non-kin (24.1 bouts/100 min). The targets of redirect aggression were mostly unrelated individuals (82.61%). The mean CCT of the victims (16.50%) did not significant differ from the mean consolation TCT of the victims (48.81%), which indicated that reconciliation and consolation played similar critical roles after conflict. The sociosexual behavior performed by victims only occurred in PC (34.85%), which indicated the purpose of sociosexual behavior in reconciliation was to prevent further attack from aggressors. The chance of victims did not involve reconciliation and consolation, but solicited affiliation with a third party in PC was 23.32%. This indicated that the solicited affiliation might function to exchange the aggressive supports from the third party in the following conflicts.

The RNA World hypothesis suggests that RNA, or a proto-RNA, existed in an early form of life that had not yet developed the ability to synthesize protein enzymes. This hypothesis, by some interpretations, implies that nucleic acid polymers were the first polymers of life, and must have therefore spontaneously formed from simple molecular building blocks in the "prebiotic soup." Although prebiotic chemists have searched for decades for a process by which RNA can be made from plausible prebiotic reactions, numerous problems persist that stand in the way of a chemically-sound model for the spontaneous generation of an RNA World (e.g., strand-cyclization, heterogeneous backbones, non-selective ligation of activated nucleotides). The Molecular Midwife hypothesis, proposed by Hud and Anet in 2000, provides a possible solution to several problems associated with the assembly of the first nucleic acids. In this hypothesis, nucleic acid base pairs are assembled by small, planar molecules that resemble molecules which are known today to intercalate the base pairs of nucleic acid duplexes. Thus, the validity and merits of the Molecular Midwife hypothesis can be, to some extent, explored by studying the effects of intercalation on the non-covalent assembly of nucleic acids. In this thesis, I explore the role of the sugar-phosphate backbone in dictating the structure and thermodynamics of nucleic acid intercalation by using 2′,5′-linked RNA intercalation as a model system of non-natural nucleic acid intercalation. The solution structure of an intercalator-bound 2′,5′ RNA duplex reveals structural and thermodynamic aspects of intercalation that provide insight into the origin of the nearest-neighbor exclusion principle, a principle that is uniformly obeyed upon the intercalation of natural (i.e. 3′,5′-linked) RNA and DNA. I also demonstrate the ability of intercalator-mediated assembly to circumvent the strand-cyclization problem, a problem that otherwise greatly limits the polymerization of short oligonucleotides into long polymers. Together, the data presented in this thesis illustrate the important role that the nucleic acid backbone plays in governing the thermodynamics of intercalation, and provide support for the proposed role of intercalator-mediated assembly in the prebiotic formation of nucleic acids.

<p>Recombinant Candida antarctica lipase B was successfullyproduced in the methylotropic yeast Pichia pastoris. Thespecific activities of Candida antarctica lipase B produced inPichia pastoris and commercial Candida antarctica lipase B fromNovozymes were the same. In shake-flask cultivations theexpression levels were about 25 mg L-1. Production levels couldbe increased to 1.5 g L-1, using a fermentor. A model tosimulate growth and oxygen consumption was described. The highcell density growth could be explained by the low maintenancecoefficient of Pichia pastoris. Enrichment of the aeration withoxygen increased the recombinant protein production. The lipasewas also produced as a fusion to a cellulose binding module.The cellulose binding module did not interfere with thespecific activity of the lipase. With this fusion proteincatalytic reactions can be performed in close proximity to acellulose surface. The binding module can also function as anaffinity tag for purification. Establishment of the Candidaantarctica lipase B production system allowed the engineeringof Candida antarctica lipase B variants. Four differentvariants were produced in order to investigate if electrostaticinteractions contributed to enantioselectivity. Theenantioselectivity of two halogenated secondary alcohols wasdoubled for the Ser47Ala variant. Thisimplied thatelectrostatic interactions are important forenantioselectivity. The Trp104His variant showed a decrease inenantioselectivity for all tested substrates. This was causedby an increase in the size of the stereoselectivity pocket.Symmetrical secondary alcohols of different size were used tomap the stereoselectivity pocket. A substituent as large as apropyl or isopropyl could be accommodated in the pocket of theTrp104His variant. In the wild-type lipase thestereoselectivity pocket was estimated to fit an ethyl group.The enzyme variants were subjected to a thermodynamic study, toelucidate changes in the enthalpic and entropic contributionsto enantioselectivity. The enthalpic and entropic contributionschanged for the different lipase variants and werecompensatory. The compensation was not perfect, allowing forchanges in enantioselectivity.</p><p>In general one can conclude that rational design of newenzyme properties, in order to change the substrateselectivity, is feasible if based on a thorough model ofsubstrate enzyme interactions.</p><p><b>Key words:</b>Protein expression, Candida antarctica lipaseB, Pichia pastoris, sitedirected mutagenesis, fermentation,selectivity</p>

Involving patients in medical decisions by acknowledging patients personal values and individual preferences has become an important goal of providing ethical medical care. Despite a general movement towards a model of shared decision-making, many patients do not fully meet their preferred role in practice. The decision whether or not to accept a kidney once it is offered to a patient awaiting transplant has historically been made predominantly by the transplant surgeon with little involvement from the patient. Because dialysis can provide long-term renal replacement, declining a kidney is a viable option. Patient changes over time and inherent heterogeneity of donor kidneys make this an authentic decision requiring careful analysis of costs and benefits from the patient perspective. The purpose of this study is to improve our understanding of how patients and transplant surgeons prioritize different factors when deciding whether or not to accept a kidney that has become available, in order to empower patients to become more involved in the decision-making process. Phase I: We developed a comprehensive list of factors that patients might consider important through qualitative interviews with patients, and deliberation with a transplant surgeon (SK) and a transplant nephrologists (RF). Phase II: We quantified the relative importance of each factor for patients on the transplant list and for transplant surgeons with a computerized survey using Maximum Differences Scaling. We developed relative importance scores using Heirarchical Bayes analysis, and tested for associations between patient characteristics and relative importance scores using Spearmans correlation coefficient and the Mann Whitney U test for continuous and categorical variables respectively. Of the factors evaluated, patients placed the greatest value on Kidney quality, How closely matched you are to the kidney, and How strongly your surgeon feels you should accept the kidney. Relative importance of different factors did not change based on patient demographic characteristics. Patients who are on the waiting list longer give less importance to kidney quality (standard beta estimate -0.23, p value 0.03) and more importance to How difficult it is for you to be matched to a donor (ie whether or not you are sensitized) (standard beta estimate 0.28, p value 0.01). Surgeons placed the greatest value on Kidney quality, How difficult it is for the patient to be matched to a kidney (ie whether or not the patient is sensitized), and The age of the donor. This pilot study suggests a role for standardized education tools to help empower patients to be involved in this difficult decision. Development of decision aids can be guided by the results of this project.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 114-126.

Thesis (M.S. in Applied Physics)--Naval Postgraduate School, December 2003. / Thesis advisor(s): William Colson, Robert Armstead. Includes bibliographical references (p. 67-68). Also available online.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 169 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.