<p><i>In vitro</i> production of embryos allows efficient management of herd genetics, reduction of disease impact, and if used in combination with other reproductive technologies it could aid in preserving the threatened genetic diversity of swine. <i>In vitro</i> maturation (IVM) is identified as a deficient step in porcine in vitro production (IVP) of embryo systems, which decreases the overall success of IVP. There are problems encountered in each step of IVP; chromosomal abnormalities and decreased cell numbers in blastocysts during <i>in vitro</i> culturing (IVC), and low monospermic fertilization rates during <i>in vitro</i> fertilization (IVF) may be a result of insufficient IVM. As an addition to maturation media, porcine follicular fluid (pFF) can affect IVM. Estrogen can be found in high concentrations in pFF; possibly contributing to the effects seen when pFF is added to IVM. The objective of this thesis was to investigate the effects of estrogen supplementation during IVM on IVP of porcine embryos.</p> <p>The first objective was to evaluate the <i>in vitro</i> maturation rates of porcine oocytes in two maturation media: protein-free and 10% pFF supplemented. Nuclear maturation of oocytes was evaluated using Lamin/Dapi staining of oocytes matured in protein-free and 10% pFF maturation media to ensure the efficiency of the protein-free media. Protein-free and 10% pFF media mature oocytes at similar rates (91% and 89% respectively).</p> <p>The transcripts within the oocyte can be altered based on the <i>in vitro</i> maturation environment, so the second objective was to observe the expression of four chosen maternal effect genes: Basonuclin-1 (<i>BNC1</i>), Nucleoplasmin 2 (<i>NPM2</i>), Zygote arrest 1 (<i>ZAR1</i>), and Tripartite-motif protein-24 (<i>TRIM24</i>), using oocytes matured in 50 ng/ml, 100 ng/ml, or 1000 ng/ml of estradiol 17-&beta; (E₂), 10% pFF, or protein-free maturation media. Expression of maternal effect genes, was shown by the &Delta;Ct (cycle threshold) values, obtained from the difference between the Ct values of the normalizing gene (<i>GAPDH</i>) and the genes of interest evaluated through QRT-PCR. Values of &Delta;Ct were analyzed in place of fold change to avoid data manipulation. The &Delta;Ct expression of <i>TRIM24</i> in 0 ng/ml E₂ maturation medium and the 10% pFF maturation medium were significantly different (p<0.05) from the non-matured control, the other maternal determinant genes did not differ in their expression under any treatment.</p> <p>We hypothesized that estradiol's effects on IVM would be evident when analyzing cleavage and blastocyst formation rates. Cleavage and blastocyst formation rates were examined following <i>in vitro</i> fertilization of oocytes matured in 100 ng/ml E₂, 10% pFF, or a protein-free maturation medium to investigate the effect of estradiol on IVP embryos. Cleavage rates for the E₂ (n= 252; 60.2%) or 10% pFF (n= 256; 55.7%) additions to the maturation media did not differ (p>0.05) when compared to the protein-free maturation media (n=264; 54.9%). Both 10% pFF and E<syb>2</sub> groups had significantly higher blastocyst formation rates (p&le;0.05) than the protein-free maturation media (n=264; 3.5%), although no statistical difference was observed between the blastocyst formation rates of the 10% pFF (n=256; 12.4%) and E2 (n=252; 14.6%) groups.</o> <p>As a final study, the global gene expression of oocytes matured in a control protein-free media and the protein-free media supplemented with 100 ng/ml E₂ or 10% pFF was investigated using microarray analysis. Genes were not differentially expressed among the matured groups with the outlined threshold values of -2 &ge; log2(fold change) &ge; 2, and adjusted p-value &le;0.05. A total of 16 differentially expressed genes between the non-matured and all matured groups exceeded this threshold. Of these genes, 6 are novel transcribed regions with evidence of being an embryonic EST, and 1 is a novel protein-coding gene. The other genes are FBJ murine osteosarcoma viral oncogene homolog (<i>FOS</i>), Vimentin (<i>VIM</i>), Capthesin C (<i>CTSC</i>), Selenium binding protein 1 (<i>SELENBP1</i>), Poly(A) binding protein cytoplasmic 1 (<i>PABPC1</i>), Tissue factor pathway inhibitor 2 (<i>TFPI2</i>), Cysteine-rich, angiogenic inducer 61 (<i>CYR61</i>), Acyl-CoA synthetase long-chain family member 6 (<i>ACSL6</i>), and Phospholipase A2 group VII (<i>PLA2G7</i>).</p> <p>In conclusion, successful nuclear maturation of oocytes derived of prepubertal gilt abbatoire derived ovaries can be achieved without pFF or hormone supplementation. The expression of maternal determinant genes is not affected in a dose dependant manner, and removal of E₂ or supplementation of pFF during maturation may alter the expression of <i>TRIM24</i> from the non-matured control; where no other maternal effect gene changes through maturation. Estradiol has a similar effect as pFF during <i>in vitro</i> maturation of porcine oocytes as seen by cleavage and blastocyst formation rates. And media does not affect the global gene expression of porcine oocytes, though there is a temporal control of gene expression through maturation.</p>

Microbial metal reduction contributes to biogeochemical cycling, and reductive precipitation provides the basis for bioremediation strategies designed to immobilize radionuclide contaminants present in the subsurface. Facultatively anaerobic ×-proteobacteria of the genus Shewanella are present in many aquatic and terrestrial environments and are capable of respiration on a wide range of compounds as terminal electron acceptor including transition metals, uranium and transuranics. S. putrefaciens is readily cultivated in the laboratory and a genetic system was recently developed to study U(VI) reduction in this organism. U(VI) reduction-deficient S. putrefaciens point mutant Urr14 (hereafter referred to as CCMB1) was found to retain the ability to respire several alternate electron acceptors. In the present study, CCMB1 was tested on a suite of electron acceptors and found to retain growth on electron acceptors with high reduction potential (E¡¬0) [O2, Fe(III)-citrate, Mn(IV), Mn(III)-pyrophosphate, NO3-] but was impaired for anaerobic growth on electron acceptors with low E¡¬0 [NO2-, U(VI), dimethyl sulfoxide, trimethylamine N-oxide, fumarate, ×-FeOOH, SO32-, S2O32-]. Genetic complementation and sequencing analysis revealed that CCMB1 contained a point mutation (H108Y) in a CcmB homolog, an ABC transporter permease subunit required for c-type cytochrome maturation in E. coli. The periplasmic space of CCMB1 contained low levels of cytochrome c and elevated levels of free thiol equivalents (-SH), an indication that redox homeostasis was disrupted. Anaerobic growth ability, but not cytochrome c maturation activity, was restored to CCMB1 by adding exogenous disulfide bond-containing compounds (e.g., cystine) to the growth medium. To test the possibility that CcmB transports heme from the cytoplasm to the periplasm in S. putrefaciens, H108 was replaced with alanine, leucine, methionine and lysine residues via site-directed mutagenesis. Anaerobic growth, cytochrome c biosynthesis or redox homeostasis was disrupted in each of the site-directed mutants except H108M. The results of this study demonstrate, for the first time, that S. putrefaciens requires CcmB to produce c-type cytochromes under U(VI)-reducing conditions and maintain redox homeostasis during growth on electron acceptors with low E¡¬0. The present study is the first to examine CcmB activity during growth on electron acceptors with widely-ranging E¡¬0, and the results suggest that cytochrome c or free heme maintains periplasmic redox poise during growth on electron acceptors with E¡¬0 < 0.36V such as in the subsurface engineered for rapid U(VI) reduction or anoxic environments dominated by sulfate-reducing bacteria. A mechanism for CcmB heme translocation across the S. putrefaciens cytoplasmic membrane via heme coordination by H108 is proposed.

ABSTRACT MATURATION OF SHAVIAN WOMEN: A STUDY OF THE MATURATION PROCESSES OF FEMALE PRATOGONISTS IN PYGMALION AND MRS. WARREN&#039 / S PROFESSION M.A., Department of English Literature May, 2009, 112 pages George Bernard Shaw is a celebrated playwright for his depiction of emancipated women. His women, regardless of the conditions they are in at the beginning of the play, experience a maturation process in the flow of the events and especially discussions which direct the change in his characters. In this thesis, the maturation processes Vivie Warren and Eliza Doolittle experience are analyzed in the plays Mrs. Warren&#039 / s Profession and Pygmalion, respectively. Vivie is a typical Shavian heroine who is educated and free-spirited even at the beginning of the play. At the end, she chooses to start a professional life breaking with the domestic and social boundaries by rejecting to see her mother or marry Frank. Likewise, Eliza, who is a simple flower girl at the beginning of the play, seems to bear the free spirit Vivie has because she earns her living and makes her own decision of taking phonetics courses, which causes the events in the play to take place. At the end, she rejects marrying to support her life and chooses to pursue phonetics as a profession to earn her living. As a result, her free-spirited personality leads her to her maturation process. In this study, it is concluded that no matter what their starting point is, both Shavian women bear the characteristics of New Woman at the beginning of the play which facilitates their progress into New Women at the end of the plays.

We identified a synthetic hexapeptide that blocks Magnaporthe grisea appressorium formation, in artificial hydrophobic surface. The results suggest that peptides interfere with surface recognition. M. grisea non pathogenic pth1 mutants were complemented by N. crassa orthologous gene suggesting that the biochemical function of pth1 has not evolved specifically to play a role in appressorium development.

The effects of unilateral eyestalk ablation, diets and sex ratios were evaluated on two wild populations of Farfantepenaeus aztecus in a closed recirculating maturation system. Ovarian development and spawning frequencies of ablated females in both studies were higher than the non-ablated females. Replacement of bloodworms in maturation diet with enriched adult Artemia sp. had no negative effect on the number of eggs spawned and resulted in increased hatch and survival rates from Nauplius I to Zoea I. Life span of ablated females fed enriched Artemia sp. was longer than ablated females fed bloodworms. Replacement of the expensive bloodworm diet component with adult enriched Artemia sp. is possible without negative impact on female reproductive performance. Reducing male to female ratio from 2:1 to 1:1 resulted in a 1.25% decrease in spawning activities of ablated females. The life cycle of pond-raised F1 generation F. aztecus also was completed in the closed recirculating system using unilateral eyestalk ablation as previously described. This study found diets that contained an enriched adult Artemia sp. component performed superior (i.e. hatch rate, nauplii and zoea production) to a diet containing bloodworms. Six consecutive larval rearing trials evaluated changes in select water quality indicators and their effect on growth, survival, and stress tolerance of F. aztecus postlarvae cultured in artificial seawater under closed recirculating and flow-through conditions. The closed recirculating larval rearing system successfully produced five-day-old postlarvae (PL) from Zoea I (Z1) with similar dry weights, lengths and stress resistance to PL produced under standard water exchange practices. The trickling biofilters were found to be a limiting component of this system. A submerged coral biofilter was added to the system and effectively processed culture water for re-use. Addition of the submerged biofilter resulted in improved survival rates in Trials 4, 5 and 6. These studies demonstrate maturation and larval rearing of F. aztecus is feasible in closed recirculating systems. Implementation of these systems in hatcheries bolsters biosecurity while reducing the environmental impact of hatchery effluent. Recirculating and re-use systems are therefore essential in the further development of sustainable hatchery programs for endemic species.

La microcine J25 (MccJ25) est un peptide antibactérien inhibiteur de l'ARN polymérase, produit par Escherichia coli AY25 selon la voie ribosomique. Sa structure tridimensionnelle en forme de lasso résulte du clivage d'un précurseur, McjA, et de la formation sur le peptide C-terminal résultant, d'une liaison amide Gly1-Glu8. Nous avons d'abord étudié le mode d'import de MccJ25 dans les bactéries et avons montré in vivo et in vitro qu'elle parasite un transporteur du complexe fer/ferrichrome, FhuA. Ainsi, MccJ25 se lie à FhuA avec un Kd de 1.2 µM, pour former un complexe de stœchiométrie 2:1. La boucle en épingle à cheveu béta définie par les résidus Val11-Pro16 de MccJ25 est nécessaire à cette étape de reconnaissance. Dans un second temps, nous avons étudié la biosynthèse de MccJ25. Nous avons montré par inactivation et complémentation de gènes que les deux protéines, McjB et McjC codées par le cluster génétique de la microcine, sont nécessaires au processus de maturation. McjA, McjB et McjC produites en système recombinant ont permis de montrer que McjB et McjC sont capables de catalyser la conversion de McjA (précurseur linéaire inactif) en MccJ25 dotée d'activité antibactérienne et structurée en lasso. L'étude des similarités de séquences de McjB et McjC indique que McjB serait responsable du clivage protéolytique de McjA et McjC de la formation de la liaison Gly1-Glu8.

Les précurseurs neuraux adultes possèdent une plasticité cellulaire suggérant un rôle dans l'apparition de pathologies mais aussi un potentiel curatif inespéré. Cependant, l'emploi clinique de ces cellules nécessite une connaissance des mécanismes biologiques contrôlant leur prolifération, maturation ou spécification cellulaire. Dans cette thèse, nous avons étudié l'expression des protéines S100 A1 et B dans les cellules progénitrices d'oligodendrocytes (OPC) et les cellules souches astrocytaires. Nous avons démontré que <br />1) toutes les cellules gliales expriment précocement la S100A1 alors que la S100B est liée à leur maturation <br />2) la S100B régule la maturation des OPC <br />3) les cellules souches astrocytaires adultes sont maintenues dans un stade de développement immature (S100B-) grâce à l'EGF, afin de conserver leurs propriétés germinales.<br />Ces résultats démontrent un lien entre les protéines S100A1/B, la maturation des cellules gliales et leurs propriétés de différenciation cellulaire.

Thesis (D. Min.)--Bethel Seminary, St. Paul, MN, 2008. / Abstract. Includes bibliographical references (leaves 153-158).

This thesis explores engagement in organizational politics among managers. There is increasing recognition that organizational politics are ubiquitous in organizational life and critically important in managerial roles. Drawing on micro perspectives in extant literature on organizational politics, this research attempts to better understand managerial engagement in politics by focusing not only on managers’ ability to engage in politics, but also on their willingness to do so. As such, the research examines what managerial political will and political skill entail, as well as how political will and skill develop. In doing so, special consideration is paid to gender, an aspect largely ignored in extant research on organizational politics. Adopting a qualitative exploratory approach, the empirical study consisted of semi-structured interviews with 38 managers (20 women and 18 men) in two global companies. The thesis makes four key theoretical contributions. First, it conceptualizes and identifies three dimensions political will, a previously neglected factor pertaining to managerial political engagement. Second, the study reconciles and refines the dimensionality of political skill, as related to existing models in field. Third, the thesis introduces a novel developmental perspective on political will and skill, proposing an initial model of political maturation. This model outlines three stages of political maturation by mapping out developmental patterns in managers’ political will and skill. The model also identifies triggers of political maturation. Finally, the thesis unpacks the role of gender in managers’ political will, skill and their maturation, demonstrating the importance of making gender visible and voiced when investigating managers’ engagement in organizational politics. In articulating these contributions, the study thoroughly accounts for the impact of organizational context on the political will, skill and maturation journey of male and female managers.

The conflict between HIV-1 and the host immune system plays out over a time-scale of months and years, and on a grander scale in the co-evolution of lentiviruses and the immune systems of their host species. Directed evolution of HIV-1 entry inhibitors using controlled randomization together with a display system offers a means of recapitulating one side of this conflict in vitro on an accelerated time-scale. To address limitations in existing display systems, we constructed a vector (pDQ1) integrating phage-display and mammalian-expression systems. This vector displays on phage when expressed in bacteria, and as an Fc-fusion when expressed in tissue culture, thus accelerating the iterative process of randomization, display, and characterization. We demonstrated the utility of this vector in the evolution of a CD4-mimetic peptide.