Alcohol use and abuse among college/university students continues as a major public health concern.  One potential source of alcohol-related harm is the inability of students to estimate their current level of intoxication (gBAC).  The current four field studies use breathalyzers to investigate student gBAC and the efficacy of a variety of BAC-feedback tools at promoting greater awareness of driving risks while under the influence of alcohol.     The research was conducted across 89 nights spanning seven academic semesters from Fall 2009 to Fall 2012.  Research tables were setup between the hours of 6:00pm and 2:00am at three locations near downtown bar establishments and one on-campus location near a late-night dining facility.  Unique subject codes were created to track participants across multiple nights of participation.  In total, 12,432 blood alcohol concentration (BAC) readings were collected from 10,225 unique individuals. Study 1 examined general epidemiology across all nights.  The average BAC of drinking participants was .100 mL/L.  Results revealed significant differences in BAC as a function of demographic and environmental factors.  Additionally, it was found the average student was incorrect in estimating his or her BAC by .034 mL/L. Studies 2 and 3 examined the accuracy of BAC-estimation tools (i.e., nomograms, sobriety tests, and phone applications) and the efficacy of these tools to increase awareness of driving-related risks.  On average, both nomograms and BAC-estimation phone applications were incorrect in estimating BAC by over .05 mL/L.  Sobriety tests performed slightly better than chance at discriminating BACs of .08 mL/L.   Participants receiving BAC-feedback had increased awareness of driving risk across levels of intoxication. Nomogram and breathalyzer feedback tended to promote healthier perceptions of external risk.  Sobriety testing shifted the internal perception of feeling less safe to drive.  No effect was observed for BAC-estimation phone applications. Study 4 found individuals who received breathalyzer feedback across multiple nights of the research were significantly more accurate at estimating their BAC.  Specifically, individuals on the fifth night of participation were .017 mL/L more accurate at estimating their BAC as compared to the first night.  Future research areas and policy implications are discussed. / Ph. D.

The adrenal gland is an important endocrine organ, protecting the body against acute and chronic stress. The adrenal cortex consists of three morphologically and functionally distinct zones: the outer zona glomerulosa (zG), the zona fasciculata (zF), and the innermost zona reticularis (zR). In rodents, zG cells produce mineralocorticoids (mainly aldosterone), while zF cells secrete glucocorticoids (mainly corticosterone). The functions of zG and zF are defined by the mutually exclusive expression of Cyp11b2 and Cyp11b1 that encode the enzymes aldosterone synthase and 11β-hydroxylase, which catalyze the terminal reactions in the production of aldosterone and corticosterone, respectively. This thesis aims to investigate the maintenance of the definitive mouse adrenal cortex. This involves studies to identify the location of adrenal stem/progenitor cells, and the mechanisms by which differentiated adrenocortical cells are replenished in the adult mice. BrdU pulse-chase studies provided valuable information about cell division and cell fate under physiological or pathophysiological stimulations. The distribution of adrenocortical cells with nuclei stained positively for BrdU and/or Ki67 was identified. Ki67 labelling marked actively dividing cells and showed that adrenocortical cells originate at or around the zG/zF interface. BrdU labelling indicated that, following cell division, cells are displaced inwards and outwards. Acute angiotensin II treatment was shown to have no significant effects on the cell proliferation or turnover in any of the adrenocortical zones. The pathophysiological effects of long-term ACTH treatment were analyzed in a mouse model of congenital adrenal hyperplasia caused by a null mutation of Cyp11b1. Cell hypertrophy was evident in all regions of the adrenal cortex due to the impaired negative-feedback of the HPA axis. Adrenocortical cell proliferation was also increased particularly in the outer zona fasciculata at the border between zG and zF where adrenocortical stem/progenitor cells might be located. The intervening steps between cell proliferation and the final differentiation into steroidogenic zG and zF cells have yet to be discovered. A visual method of monitoring levels of Cyp11b2 and Cyp11b1would offer a convenient approach to track the stages of adult stem cell differentiation that lead to normal adrenal maintenance in vivo and in vitro. In the present study an AS-mCherry-11B-EGFP BAC construct was successfully engineered, in which Cyp11b2 and Cyp11b1 were substituted by mCherry and EGFP, respectively. This BAC construct was characterized in mouse adrenocortical Y1 cells. It was determined that EGFP faithfully recapitulated the expression of Cyp11b1. Forskolin or cAMP treatment induced a rapid cell rounding effect and caused the increased expression of EGFP transgene and endogenous Cyp11b1. An attempt was made to establish a transgenic mouse model, in which zG and zF cells would be marked with mCherry and EGFP respectively, allowing the differentiation of an adrenocortical stem cell to be traced. Following microinjection of the BAC into mouse zygotes, twoAS-mCherry-11B-EGFP transgenic founder mice were identified. Unfortunately, neither of them was able to transmit the transgene through germline, suggesting the mosaicism of transgene integration. Indeed, mosaicism of the transgenic adrenals was demonstrated by RT-PCR and immunostaining, which also revealed that the exogenous EGFP expression faithfully recapitulated the endogenous Cyp11b1 in adrenals. Although it is assumed that expression of Cyp11b2 and Cyp11b1 are mutually exclusive, zG and zF cells may have the plasticity to allow the transition from one cell type into another. The AS-mCherry-11B-EGFP BAC construct is a useful tool for studying in vitro ES cell differentiation towards the adrenocortical lineage. Transgenic AS-mCherry-11B-EGFP ES cells were successfully differentiated into mesenchymal stem cells, as identified by the expression of molecular markers for the mesenchymal lineage. It has been reported that steroidogenic factor (Sf1) can promote the differentiation of MSCs into steroidogenic cells, and Shh plays an important role in Sf1 expression and the consequent adrenal development. However, Shh treatment failed to achieve transformation of mesenchymal cells into adrenocortical cells. It is thought there might be a requirement for additional factors to combine with Shh in promoting the transdifferentiation of ESC-derived mesenchymal cells. Future studies will focus on the genetic control of Cyp11b1 and Cyp11b2 in transgenic AS-mCherry-11B-EGFP ES cells. In conclusion, the location and fate of the adrenocortical progenitor cells were demonstrated by the BrdU pulse-chase studies in different mouse models. An AS-mCherry-11B-EGFP BAC construct was generated, and used to study the mutual and differential controls of Cyp11b1 and Cyp11b2 expression in adrenocortical cells in vitro and in transgenic mice in vivo.

In Lymphozyten wird nach Antigenaktivierung die Expression des Nfatc1-Gens durch Aktivierung des P1-Promoters stark induziert. Dagegen ist die, durch den Promoter P2 vermittelte Expression ebenso wie die der anderen NFAT Faktoren c2 und c3 konstitutiv. Die Akkumulation der dabei gebildeten Isoform NFATc1/αA ist sowohl für Effektorfunktionen wie die Zytokinproduktion sowie die Proliferation und das Überleben der aktivierten Zellen wichtig (Chuvpilo et al., 2002). Um die Expression des Nfatc1-Gens auf Einzelzellebene messen zu können, wurden BAC (bacterial artificial chromosom) transgene Mauslinien generiert, die einen 210kb großen Bereich des Nfatc1-Gens der Maus enthalten. In diesen Lokus wurde ein eGFP-Reportergen innerhalb des allen Isoformen gemeinsamen, dritten Exons integriert. In dieser Arbeit wird durch semiquantitative RT-PCR-Experimente von Gesamt-Milzzellen und TLymphozyten gezeigt, dass in den B6/NFATc1-eGFP-BAC-Reportermäusen die Expression der eGFP-cDNA analog zum endogenen Nfatc1-Lokus der Kontrolle der beiden Promotoren P1 und P2 unterliegt. In Western Blot Experimenten wird in diesen Zellen mittels eines NFATc1α-spezifischen Antikörpers eine induzierbare und CsA-sensitive α-GFP-Isoform - vergleichbar mit der endogenen NFATc1α-Isoform - nachgewiesen. Gleichzeitig zeigen NFATc1-Antikörper das konstitutiv exprimierte GFPβ-Protein. Die Korrelation der Expression von NFATc1 und GFP auf mRNA- und Proteinebene machen in B6/NFATc1-eGFP-BAC-Reportermäusen das GFP-Protein somit zu einem sensitiven und spezifischen Marker der NFATc1-Aktivität. In FACS-Analysen gibt der Anstieg der GFP-Fluoreszenzintensität bei Stimulation von Gesamt- Milzzellen bzw. T-Lymphozyten um bis auf das Dreifache die Induktion von NFATc1 wider. Unter dem Einfluss von CsA verbleibt die GFPFluoreszenzintensität auf dem Niveau unstimulierter Zellen. Die GFPFluoreszenz korreliert darüber hinaus bei Primärstimulation mit der Expression des IL-2-Gens, dessen Promotor mit 5 NFAT-Bindestellen den Prototyp eines NFATc1-Targets darstellt (Serfling et al., 1989). Die Analyse der Koexpression von NFATc1 und GFP mittels Fluoreszenzmikroskopie zeigt in allen stimulierten, GFP-positiven CD4+-Lymphozyten die nukleäre Lokalisation von 75 NFATc1, vor allem von NFATc1α. Die Analyse des GFP-Phänotyps in alloreaktiven T-Zellen zeigt bei Abstoßungsreaktionen in vitro („Mixed Lymphocyte Reactions“) eine selektive Zunahme der Fluoreszenz dieser Zellen um bis auf das Vierfache, was die Rolle von NFATc1 für die Effektorfunktion aktivierter T-Lymphozyten verdeutlicht. GFP und das endogene NFATc1 werden bei Stimulation konventioneller T-Zellen (Tcons, CD4+CD25-FoxP3-) stark exprimiert, während natürliche regulatorische T-Zellen (nTregs, CD4+CD25+FoxP3+) konstant geringe NFATc1- und GFP-Konzentrationen zeigen. In induzierten regulatorischen T-Lymphozyten (iTregs) supprimiert TGF- β konzentrationsabhängig die GFP-Fluoreszenz bis auf das Niveau unstimulierter Lymphozyten. Während in nTregs die Suppression des Nfatc1- Gens im wesentlichen durch FoxP3 erfolgt (Torgerson et al., 2009), scheint dies in iTregs vor allem über den TGF-β Signalweg vermittelt zu werden. Die Analyse der GFP-Expression in den verschiedenen Stadien der TZellentwicklung zeigt weiterhin deutliche Unterschiede in der Aktivität des Nfatc1-Gens. Dies wird durch die starke Aktivität des BAC-Genlokus in CD4- CD8- DN Thymozyten, welche eine sechsfach höhere GFP-Expression aufweisen als CD4+CD8+ DP Zellen, deutlich. / Activation of lymphocytes causes a high level of Nfatc1 due to P1-promoter induction, whereas the P2-promoter leads to an constitutive expression of NFATc2/c3. NFATc1/αA is essential for effector-function, proliferation and survival of activated cells (Chuvpilo et al., 2002). Here we show that the eGFP-cDNA integrated in a NFATc1-eGFP-BAC-construct is under control of both promoters (P1, P2) and correlates with the NFATc1-Expression on mRNA and protein-levels. In FACS-analysis there is an increase in eGFP-fluorescence intensity, which is highly CsA-sensitive. In natural and induced Tregs we observed a low level of eGFP-expression correlating with concentration of TGF-β. CD4-CD8- DN cells show the highest eGFP-Expression in thymocytes.

Made available in DSpace on 2014-06-12T15:04:33Z (GMT). No. of bitstreams: 2 arquivo4858_1.pdf: 1670085 bytes, checksum: e9633b164026a38f15e739944c5a046d (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A definição das espécies dentro dos gêneros pode ser problemática e as espécies de Citrus configuram um exemplo clássico de classificação conflitante. O presente estudo visou auxiliar na compreensão da sistemática deste grupo, ampliando a análise citogenética dos gêneros Citrus e Poncirus. Inicialmente foi analisada a variabilidade cariotípica dos pomelos (toranja × laranjadoce), indicando diferentes cariótipos entre as cultivares. Provavelmente, os diferentes cariótipos são resultantes de eventos de hibridação independentes, envolvendo diversos acessos de toranja. No entanto, as toranjas analisadas mostraram cariótipos idênticos e homozigotos, corroborando sua classificação como espécie pura. O segundo estudo dedicou-se à análise de 13 acessos de tangerinas. Em todos os acessos, o cromossomo D/5S-45S foi observado em homozigose e considerado um bom marcador para este grupo. A tangerina é classificada com uma das espécies puras, porém os dados obtidos sugerem que compreendam três espécies básicas: C. sunki, C. reshni e C. deliciosa, todas com cariótipos homomórficos, sendo que as duas primeiras foram idênticas. O terceiro trabalho visou refinar a análise cromossômica a partir da FISH de seqüências de cópia única. A espécie utilizada foi P. trifoliata, importante fonte de genes de resistência para Citrus. A análise da hibridização foi realizada comparativamente ao padrão de bandeamento CMA/DAPI e localização de sítio de DNAr 45S, permitindo reconhecer os nove pares cromossômicos, além de mapear a região de resistência ao VTC

Quinoa (Chenopodium quinoa) is an Andean crop adapted to harsh environmental conditions and containing a high and well-balanced protein composition. Two seed storage proteins, the 2S albumin and 11S globulin, are the major amino acid reservoirs for the developing seedling. An in-depth study of the genes encoding these proteins is necessary to understand the source of quinoa's protein quality. Our objectives include identification and sequencing of the 11S genomic and cDNA clones, analysis of 11S expression profiles in different quinoa accessions and evaluation of evolutionary relationships between the sequence of the 11S gene in quinoa and homologous genes in other species. Clones containing 11S cDNA and genomic DNA were identified and sequenced. Two copies of the gene were found to be present at two different loci in the quinoa genome. The amino acid composition of the 11S gene was also analyzed. Results show that the 11S gene contains a well-balanced assortment of essential amino acids with relatively high levels of glutamate/glutamine, aspartate/asparagine, arginine, serine, leucine and glycine, typical of other 11S seed storage proteins. Total RNA and globulin was extracted from seed collected at different developmental intervals from nine quinoa accessions. Expression profiles were determined by evaluating 11S transcript levels using relative quantification real time RT-PCR and comparing relative 11S globulin accumulation using sodium SDS-PAGE. The 11S gene was found to be expressed during late-maturation regardless of differences in maturation rate. A portion of the amino acid sequence of the 11S and homologous genes was chosen for phylogenetic analysis. Fifty five such sequences from 50 different plant species were assembled and aligned. Two phylogenetic reconstructions, one using the parsimony method and the other using Bayesian analysis, were generated in order to analyze evolutionary relationships between the 11S gene in quinoa and homologous genes in other species. Relationships shown by both reconstructions for sequences from closely-related species were consistent with taxonomic clustering. Both reconstructions showed less resolution involving relationships between distantly-related angiosperm taxa indicating a wide divergence of sequence at the angiosperm level and a need for additional angiosperm sequence data for finer resolution.

Nous présentons dans cette thèse un nouveau modèle de contrôle d'accès dénommé Or-BAC, Organization Based Access Control. Il vise à pallier les limites des modèles de sécurité existants tout en simplifiant la spécification d'une politique de sécurité. Nous proposons un modèle plus riche et plus modulaire qui permet de distinguer la rédaction de la politique de sécurité de son implantation. Ceci est rendu possible par l'abstraction des entités traditionnelles du contrôle d'accès: les sujets sont employés dans des rôles, les objets sont utilisés dans des vues et les actions implémentent des activités. De plus l'organisation dans laquelle un règlement de sécurité est défini prend une place centrale dans ce nouveau modèle. On peut ainsi analyser l'interopérabilité d'organisations ayant chacune leur politique de sécurité et par ailleurs modéliser la structure des organisations. Trois autres aspects sont détaillés dans ce mémoire. Premièrement, afin d'obtenir un règlement de sécurité dynamique, nous intégrons une large variété de contextes. De tels contextes permettent d'activer ou de désactiver des autorisations. Deuxièmement, nous offrons la possibilité d'exprimer des autorisations négatives et définissons une méthode de gestion des conflits entre autorisations positives et négatives qui a la particularité d'être paramétrable et de permettre de détecter et surtout de prévenir les conflits. Enfin, nous associons à notre modèle un modèle d'administration, AdOr-BAC, qui permet de gérer l'ensemble d'une politique de sécurité Or-BAC de façon flexible et décentralisée. Nous présentons également deux travaux de mise en œuvre: l'adaptation de notre modèle dans un environnement réseau et le développement d'OToKit, une maquette de saisie et de validation d'une politique de sécurité Or-BAC.

The codling moth, Cydia pomonella (Lepidoptera; Tortricoidea) is a major pest of pome fruitand walnut orchards in the world. Due to the intensive chemical control C. pomonella has developed a high resistance to various insecticides. One of the mechanisms of the resistance is acetylcholinesterase insensitivity to carbamates and organophosphates. The insensitivity is based on mutations in one of two genes for acetylcholinesterase. This study deals with testing a hypothesis suggesting that one gene coding for acetylcholinesterase in the codling moth was translocated to the Z sex chromosome. The hypothesis has been latersupported by sex-linked inheritance of insecticide resistance in a related species, Grapholita molesta, and also by a large size of sex hromosomes in the codling moth.

BACKGROUND:Sugarcane breeding has significantly progressed in the last 30 years, but achieving additional yield gains has been difficult because of the constraints imposed by the complex ploidy of this crop. Sugarcane cultivars are interspecific hybrids between Saccharum officinarum and Saccharum spontaneum. S. officinarum is an octoploid with 2n=80 chromosomes while S. spontaneum has 2n=40 to 128 chromosomes and ploidy varying from 5 to 16. The hybrid genome is composed of 70-80%S. officinaram and 5-20%S. spontaneum chromosomes and a small proportion of recombinants. Sequencing the genome of this complex crop may help identify useful genes, either per se or through comparative genomics using closely related grasses. The construction and sequencing of a bacterial artificial chromosome (BAC) library of an elite commercial variety of sugarcane could help assembly the sugarcane genome.RESULTS:A BAC library designated SS_SBa was constructed with DNA isolated from the commercial sugarcane variety SP80-3280. The library contains 36,864 clones with an average insert size of 125 Kb, 88% of which has inserts larger than 90 Kb. Based on the estimated genome size of 760-930 Mb, the library exhibits 5-6 times coverage the monoploid sugarcane genome. Bidirectional BAC end sequencing (BESs) from a random sample of 192 BAC clones sampled genes and repetitive elements of the sugarcane genome. Forty-five per cent of the total BES nucleotides represents repetitive elements, 83% of which belonging to LTR retrotransposons. Alignment of BESs corresponding to 42 BACs to the genome sequence of the 10 sorghum chromosomes revealed regions of microsynteny, with expansions and contractions of sorghum genome regions relative to the sugarcane BAC clones. In general, the sampled sorghum genome regions presented an average 29% expansion in relation to the sugarcane syntenic BACs.CONCLUSION:The SS_SBa BAC library represents a new resource for sugarcane genome sequencing. An analysis of insert size, genome coverage and orthologous alignment with the sorghum genome revealed that the library presents whole genome coverage. The comparison of syntenic regions of the sorghum genome to 42 SS_SBa BES pairs revealed that the sorghum genome is expanded in relation to the sugarcane genome.

The purposes of this study are¡G(1) comparing the treatment efficiency with advanced and traditional drinking water treatment plants in southern Taiwan¡F(2) assessing the treatment efficiency and formation of disinfection by-products in advanced water treatment processes¡F(3) assessing the feasibility of wastewater recycling and treatment efficiency of wastewater treatment units¡F(4) evaluating corrosion of drinking water transportation pipelines and reproducing of chlorination by-products. This study found that the removal efficiency of turbidity, iron, manganese, coliform group and total bacterial count were approximately 99% by advanced and traditional purification processes. The concentrations of ammonia-N (NH3-N), nitrite nitrogen and nitrate nitrogen were lower drinking water quality standard. Pellet softening process was designed following coagulation/sedimentation unit to increase 8~14% and 6~20% removal efficiency of alkalinity and total hardness (TH) concentrations. The removal efficiency of total dissolved solids (TDS) was approximately 3~15% by advanced water treatment processes better than traditional water treatment processes. In the formation of disinfection by-products (DBPs), the trihalomethanes (THMS) and haloacetic acid (HAA5) were efficiently decreased by advanced purification processes. Bromate concentrations which lower detection limit were treated by ozonation process during the study periods. Advanced treatment processes should control the dosage of ozone and post-chlorine to avoid production of DBPs. In wastewater reuse, the treatment efficiency of suspended solids (SS) was 48¡ã99%, respectively, showing the significant removal efficiency of the wastewater process. However, the removal efficiencies of NH3-N, total organic carbon (TOC) and chemical oxygen demand (COD) are limited by wastewater treatment processes. Because NH3-N, TOC and COD of the mixing supernatant and raw water are regulated raw water quality standards, supernatant reuse is feasible and workable during wastewater processes at this plant. Overall, analytical results indicated that supernatant reuse is feasible. The Chengcing Lake water treatment plant significantly reduced alkalinity, Ca2+ concentration and TH concentration via pellet softening treatment: however, reducing the Langelier saturation index (LSI) value of water could cause some adverse effects on distribution systems. Operational conditions by Pingding water treatment plant was added base to water can be tried to adjust pH to maintain a slightly positive LSI value, whereas for water with low hardness and alkalinity.

Master's thesis. / Format: PDF. Bibl.