<p> Bacterial cell division is an essential process, which is initiated by forming the Z-ring as a cytoskeletal scaffold at the midcell site, followed by the recruitment of a series of divisome proteins. In <i>Escherichia coli</i> (Ec), at least 15 divisome proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsB, FtsL, FtsI, FtsW, FtsN, FtsE, FtsX, ZapA, AmiC, EnvC) have been implicated in this process. The components of the cell division machinery proteins in <i>Neisseria gonorrhoeae</i> (Ng) differs from <i>E. coli. N. gonorrhoeae</i> possesses FtsA, but lacks FtsB. ZipA and FtsL in <i>N. gonorrhoeae</i> have low identity to ZipA and FtsL from <i>E. coli</i>. Our laboratory has studied the central division protein FtsZ in <i>N. gonorrhoeae</i>. Thus, my research investigated the role of <i>N. gonorrhoeae</i> FtsA in cell division and investigated the interactions between divisome proteins from <i>N. gonorrhoeae</i> to understand divisome assembly.</p> <p>This study determined the association of FtsA_Ng with FtsZ</sub>Ng and other divisome proteins in <i>N. gonorrhoeae</i> and identified the functional domains of FtsA<sun>Ng</sub> involved in these interactions using a bacterial two-hybrid (B2H) assay. FtsA_Ng interacted with FtsZ_Ng, FtsK_Ng, FtsW_Ng, FtsQ_Ng, and FtsN_Ng. Self-interactions of FtsA_Ng and FtsZ_Ng were also detected. FtsI_Ng, FtsE_Ng and FtsX_Ng did not interact with FtsA_Ng. The 2A₁, 2A₂ and 2B domains of FtsA_Ng were sufficient to interact with FtsZ_Ng independently. Domain 2A₁ interacted with FtsK_Ng and FtsN_Ng. Domain 2B of FtsA_Ng interacted with FtsK_Ng, FtsQ_Ng, and FtsN_Ng. Domain 2A₂ of FtsA_Ng interacted with FtsQ_Ng, FtsW_Ng, and FtsN_Ng. These data suggest that FtsA in <i>N. gonorrhoeae</i> plays a key role in interactions with FtsZ and other divisome proteins.</p> <p>The potential interactions between divisome proteins in <i>N. gonorrhoeae</i> were examined using B2H assays. The comparisons between the <i>N. gonorrhoeae</i> divisome protein interaction network and those of <i>E. coli</i> and <i>S. pneumoniae</i> indicates that the divisome protein interactome of <i>N. gonorrhoeae</i> is more similar to that of <i>S. pneumoniae</i> and differs from that of <i>E. coli</i>. The comparisons revealed that compared to the interactions in <i>E. coli</i> and <i>S. pneumoniae</i>, more interactions between divisome proteins upstream of FtsA_Ng (including FtsA_Ng) and downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i> while fewer interactions between divisome proteins downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i>. Possible reasons for this include the inability of ZipA_Ng to interact with other divisome proteins and the absence of FtsL and FtsB in <i>N. gonorrhoeae</i>, resulting in the lack of an FtsQ-FtsB-FtsL complex in <i>N. gonorrhoeae</i>. These results indicate a possibly different divisome assembly in <i>N. gonorrhoeae</i> from that proposed models for <i>E. coli</i>.</p> A model for FtsA_Ng structure was predicted based on structural homology modeling with the resolved crystal structure of <i>Thermotoga maritima</i> FtsA. Four domains on the molecule were identified, designated 1A, 1C, 2B and 2A (including 2A₁ and 2A₂). Domains 2A and 2B of FtsA were highly conserved based on multi-sequence alignments of FtsAs from 30 bacteria. FtsA_Ng located to the division site in <i>N. gonorrhoeae</i> cells and the ratio of FtsA to FtsZ ranged from 1:24 to 1: 33 in three <i>N. gonorrhoeae</i> strains, which gave a lower cellular concentration of FtsA compared to other organisms.</p> <p>I also determined that overexpression of FtsA_Ng in <i>E. coli</i> led to cell filamentous in rod-shaped <i>E. coli</i> and cell enlargement and aggregation in mutant, round <i>E. coli</i>. FtsA_Ng failed to complement an <i>ftsA</i>_Ec-deletion <i>E. coli</i> strain although the overexperssion of FtsA_Ng disrupted <i>E. coli</i> cell division. In addition, overexpression of FtsA_Ng only affected cell division in some cells and its localization in <i>E. coli</i> was independent of interaction with <i>E. coli</i> FtsA or FtsZ. These results indicate that FtsA_Ng exhibits a species-specific functionality and <i>E. coli</i> is not a suitable model for studying FtsA_Ng functionality.</p> <p>This is the first study to characterize FtsA from <i>N. gonorrhoeae</i> in cell division. I identified novel functional domains of FtsA_Ng involved in interactions with other divisome proteins. The <i>N. gonorrhoeae</i> divisome protein interaction network determined by B2H assays provides insight into divisome assembly in <i>N. gonorrhoeae</i></p>.

Eukaryotic translation initiation factor 5A (eIF-5A) is the only known cellular protein that contains the post-translationally derived amino acid, hypusine. Initially, eIF-5A was named as a translation initiation factor because of its capability to stimulate the formation of methionyl-puromycin, which mimics the first peptide bond formation during protein synthesis, under <em>in vitro</em> conditions. Subsequently, however, this proposed function of eIF-5A has been questioned because a similar effect on translation was not observed <em>in situ</em>. Moreover, eIF-5A appears not to be required for general protein synthesis. Rather, there is evidence that it facilitates the translation of specific subsets of mRNAs required for cell proliferation as well as apoptosis. <br /><br /> There are two isoforms of eIF-5A in the human genome which have designated eIF-5A1 and eIF-5A2. The objective of the present study was to gain an increased understanding of the roles of eIF-5A1 and eIF-5A2 during apoptosis and cell proliferation using human cell lines as a model system. Apoptosis was induced by treating the cells with Actinomycin D or sodium nitroprusside (SNP), which initiate programmed cell death by different mechanisms. It was observed for both normal and cancer cells that eIF-5A1 protein is up-regulated during apoptosis induced by Actinomycin D or SNP, whereas eIF-5A1 mRNA is constitutively expressed and does not change in abundance during this treatment. The up regulation of eIF-5A1 protein levels in the absence of a corresponding up-regulation in eIF-5A1 mRNA suggests that eIF-5A1 may be post-transcriptionally regulated. Moreover, eIF-5A1 protein up-regulation was stronger in normal cells than in cancer cells. By contrast, eIF-5A2 protein was below detection levels during apoptosis in both normal and cancer cells, although the corresponding transcript was detectable by semi-quantitative RT-PCR. This is attributable to inefficient translation of eIF-5A2 mRNA. <br /><br /> The effects of eIF-5A1 and eIF-5A2 on cell proliferation were examined by modulating the levels of serum in cultures of UACC-1598 cells, which are ovarian cancer cells that express high levels of both isoforms of eIF-5A. Serum starvation, which induces cell cycle arrest and ensuing apoptosis, followed by the re-addition of serum had no effect on the transcript levels of either eIF-5A1 or eIF-5A2. However, eIF-5A1 and eIF-5A2 proteins were both up-regulated within 24 hours of the initiation of serum starvation, and this coincided temporally with the onset of apoptosis as measured by TUNEL and a subsequent decline in viable cells. <br /><br /> The data indicate that eIF-5A1 and eIF-5A2 are both post-transcriptionally regulated and that they have functionally redundant roles in apoptosis.

This thesis presents consolidated research results of a low-cost, high efficiency, high throughput detection system for fluorescence-based bio-assays. Such high throughput screening process is an invaluable tool for the multifaceted field of Systems Biology, where it is widely used in genomics and proteomics for drug and gene discovery applications. The thesis is divided into three parts: addressing the feasibility of using hydrogenated amorphous silicon photodiodes as the sensor, the development of an associated compact model suitable for circuit-level simulations, and integration of the sensors and switches to realize the array. Requirements of fluorescent bio-assays demand low sensor dark current densities in the order of 10¯¹¹A/cm² at room temperature. Fabrication of high quality segmented a–Si:H n–i–p photodiodes with such specification is achieved by tailoring defects at photodiode junction sidewalls, where both the dry etching and passivation conditions play important roles. Measurements of the fabricated photodiodes at different temperatures allowed the extraction of reverse current components, which are necessary in modeling such sensors in Verilog-A. Two prototype array designs are fabricated with pixel dimensions matching ANSI standard microwell plates. The functionalities of the small arrays are demonstrated with green LEDs to simulate fluorescent dyes that are commonly used in the high throughput bio-assay processes.

Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria that colonize the rhizosphere and, when applied to crops, enhance the growth of plants. Plant growth-promoting rhizobacteria may enhance plant growth either by direct or indirect mechanisms. The direct mechanisms of action include nitrogen fixation,production of phytohormones and lowering of ethylene concentrations. The objective of this study was to determine whether Pseudomonas putida strain 6-8 isolated from the rhizosphere of legume crops grown in Saskatchewan fields was able to promote the growth of canola cv. Smart and lentil cv. Milestone plants by direct mechanisms. Initial studies determined the effect of strain 6-8 and other known phytohormoneproducing PGPR strains on the growth of canola and lentil plants both in gnotobiotic and growth chamber conditions. Variations in the results were observed, as there were significant differences among trials. Strain 6-8 enhanced the growth of canola cv. Smart in growth pouches but not in pots in growth chamber studies. In the case of lentil cv.Milestone, strain 6-8 had no significant effect in growth pouches, but it significantly increased root dry weight, shoot dry weight and root surface area in pots in growth chamber studies. A similar effect was observed with wild-type strains GR12-2 and G20- 18. Strain GR12-2 was consistent in promoting the growth of lentil cv. Milestone both in growth pouches and in pots in growth chambers when compared to other strains and the control. The ability of the PGPR strains to produce auxin and cytokinin phytohomones in pure culture and in the canola rhizosphere was tested using the enzyme linked immunosorbent assay (ELISA). All the PGPR strains produced indole compounds and the concentration of the indoles produced increased with increasing concentrations of the precursor tryptophan. There were no significant differences among PGPR strains in production of indole-3-acetic acid (IAA) when assayed using ELISA. The concentrations of IAA secreted by PGPR strains were extremely low (0.19 µg/ml 9.80 µg/ml). Strain 6-8 produced the cytokinins, isopentenyl adenosine (IPA), zeatin riboside (ZR) and dihydroxyzeatin riboside (DHZR) in pure culture. Indole-3-acetic acid was detected in supernatants obtained from canola growth pouches inoculated with PGPR strains, but there were no significant differences in the concentrations of IAA secreted among PGPR strains. Significantly higher concentrations of IPA and ZR were observed in the rhizosphere of canola inoculated with strain 6-8 than in the non-inoculated control. Strain 6-8 produced siderophores, solubilized inorganic phosphate and used 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, as sole nitrogen source. These traits are considered to be alternative mechanisms for direct plant growth promotion. A qualitative and quantitative study of root colonization by strain 6-8 was conducted by tagging the strain with green fluorescent protein in conjunction with confocal laser scanning microscopy and by conventional plating. The populations of strain 6-8 were higher on canola roots than on lentil roots by conventional plating. Similar results were also observed in confocal laser scanning microscopy (CLSM) studies after 5, 7 and 9 days for canola and 3, 6 and 9 days for lentil. Pseudomonas putida strain 6-8 produced cytokinins and also possessed other direct growth promoting characteristics. The ability of strain 6-8 to promote the growth of canola cv. Smart in growth pouches and lentil cv. Milestone in growth chamber studies may be related to these direct growth promoting characteristics. Strain 6-8 may have potential for development as a plant growth-promoting rhizobacterial inoculant.

none

Electrospinning is a technique to achieve nanometer scale fibers. Similar to the conventional spin methods of making fabric, the viscous polymer solution is ejected from a spinneret; stretched and solidified in the air, the solution forms the fibers. The different part of electrospinning among others is that the fibers are driven by the electrostatic force, which induces the repulsion inside the liquid and further reduces the diameter. The resultant product is a non-woven membrane, which is porous; and the pore size is around several nanometers to a micrometer wide. In this work, the relationship between the diameter of electrospun silica fibers, experimental parameters such as concentration and voltage, and between pore size of the fiber membrane and experimental time were studied. Materials used in the process are Polyvinylpyrrolidone (PVP), butanol and spin-on-glass coating solution, which act as polymer carrier, solvent, and silica-precursor, respectively. Polymer/silica precursor composite fibers were ejected from the needle of a plastic syringe when an electrical field, as high as several kV/cm, was applied. Then silica fibers were achieved by baking the composite ones at 773 oK for 12 h. Electrospun silica nanofibers were characterized as a function of polymer solution parameters. The calcined fibers were examined by using a field emission scanning electron microscope. The results showed that the fiber diameters decrease with decreasing proportion of polymer and silica precursor, and increase with a higher electric field. Pore sizes, defined as the grid areas enclosed by fibers on nearby layers, were also examined and showed no time-dependent tendency when the electrospin time was between 1-5 min. Fiber membranes were then used as the platform for protein detection. The results were compared with the control, which used glass slides as the platform. The results make it possible to make a more sensitive biosensing device.

Some pathogenic bacteria including Haemophilus influenzae and Neisseria meningitides produce a protease called IgA1 protease to impair a major antibody, immunoglobulin A1 (IgA1), on human mucosal surfaces. The iga mRNA is initially translated into a precursor containing four distinct domains: a 31-amino acids signal peptide which leads the precursor to the periplasmic space, an 105-kDa protease domain which cleaves host IgA1 molecule, a £]-domain responsible for autotransportation of the protease domain, and a short linker between the protease and the £]-domains. The autotransporter £]-domain can be further divided into three subdomains in Neisseria protease: an extracellular linking region £\-protein and a membrane-embedded £]-core, between which there is a distinguished sequence called surface region. The hydrolytic function of the protease and the transporter role of £]-core had been studied extensively, but the £\-protein and the surface regions were less defined, or had their role characterized. Thus this study is designed to reveal the possible pathogenic functions of the £\-protein and the surface region in bacterial adherence to human cell surfaces. To complete this project, recombinant £\-protein and the surface region were expressed in IgA1 protease-negative E. coli strain (UT5600) respectively and purified to homogeneity. These recombinant proteins were used in cellular assays for bacterial adhesion on human lung cancer cell (A549). Four different invasive strains of pathogenic bacteria (IgA1 protease-positive or negative), were recruited in adherence assays to determine the effect of the purified £\-protein and the surface region on bacterial adherence to A549 cells. Results showed that the both £\-protein and the surface region played a role in bacterial adherence in a species-dependent manner.

Abstract At present, diagnostic reagent is the most fruitful and important realm in internal biotech product commercialization. Compared to other biotech medicine with the industrial characteristics of complications such as: high investment, high risk, and long-term research and development; diagnostic reagent, relatively, acquires a lower threshold for technique, a short-term research and development, less investment and a short time for feedback. In addition, people gradually acquire the notions of prophylaxis and health care, as a result, the potentiality of diagnostic reagent comes to the market¡¦s notice. The process of diagnostic reagent development can be divided by the mainstream techniques into four phases including: the technology of chemical test, the technology of enzyme test, the technology of immunoassay test, the technology of nuclease molecule test and biochip. From this, we know that the technology of immunity test is still the mainstream in the market, but there is a trend toward the development of the technology of nuclease molecule immunoassay. General Biologicals Corp. is the first built diagnostic reagent manufacture in our country, and, so far, it is the only diagnostic reagent manufacture that meets with the Department of Health¡¦s CGMP standard. The General Biologicals Corp. is the only internal manufacture that produces ELISA and EIA microplate. However, the General Biologicals Corps has less than 1% of six hundred million, and foreign manufactures have it all. The General Biologicals Corps went public in recent years, and the company has a microplate immunoassay diagnostic reagent factory for over twenty years. Those doctors, examiners and sales clerks who used the immunoassay reagent produced by the General Biologicals Corps are all in high positions in this industry. They know this company and also their reagent kits which have a low sensitivity. More important is that the reagent kits are not improved for all these years. The bad impression of the company makes the customers and collaborators have doubts about the quality, the attitude of the personnel and the sincerity of the company. It results in a trust crisis. In light of the five competitiveness analyses in the executive strategies: dynamic competition of the company in enterprise; S.W.O.T competitiveness analysis; smiling curve and bitter smiling curve; red ocean and blue ocean strategy and, finally, the administration of registering examined medical instrument, to see the integral competitiveness of the company in the industrial competitive environment. Can the General Biologicals Corp. seek league and collaboration in the competitors to seize the internal market, and then the overseas market. By these analyses and advises, to reengeneering the General Biologicals Corp. has a new management on the visible foundation. Key Word¡GImmunity Test, Nuclear Molecular Immuno assay. Five Force, Reengeneering

No description available.

The D-optimal design problems in polynomial regression models with a one-dimensional control variable and k-dimensional response variable Y=(Y_1,...,Y_k) where there are some common unknown parameters are discussed. The approximate D-optimal designs are shown to be independent of the covariance structure between the k responses when the degrees of the k responses are of the same order. Then, the exact n-point D-optimal designs are also discussed. Krafft and Schaefer (1992) and Imhof (2000) are useful in obtaining our results. We extend the proof of symmetric cases for k>= 2.