Agent Based Traffic Signals Regulating Flow On a Basic Grid

A simulation study on traffic light optimisation with agent-based behaviour of the traffic signals

Photoactivatable siRNAs and molecular beacons

Die Verfügbarkeit synthetischer Oligonukleotide hat der Entwicklung einer Vielzahl molekularbiologischer, biochemischer und medizinischer Anwendungen den Weg geebnet. Und sind viele diese Anwendungen für sich genommen schon hochinteressant, so eröffnet die Kombination mehrerer Methoden oft noch ganz neue Möglichkeiten. In der vorliegenden Doktorarbeit ist es gelungen, die Technik der photolabilen Schützung auf die Anwendungen von siRNAs und molecular beacons zu übertragen und diesen damit die Option der orts- und zeitaufgelösten Aktivierung zu ermöglichen. Durch die Einführung eines Nukleotids mit 2-(2-nitrophenyl)propyl-geschützter Nukleobase in eine siRNA, konnte der katalytische Schritt der RNA-Interferenz, die mRNA-Spaltung, unterbunden werden. Hierzu wurde das photolabil modifizierte Nukleotid so in der siRNA positioniert, dass es gegenüber der mRNA-Schnittstelle bzw. in unmittelbarerer Nachbarschaft zu dieser lag. Dabei war das modifizierte Nukleotid selbst kein Ribonukleotid sondern ein Desoxynukleotid. Zuvor konnte gezeigt werden, dass die Einführung einzelner Desoxynukleotide in eine siRNA keinerlei Einfluss auf deren Aktivität hat. Als Modellsystem diente der RFP/eGFP-Reportergenassay, wobei die Plasmide mit der siRNA in die verwendeten HeLa-Zellen kotransfiziert wurden. Die verwendete siRNA regulierte dabei die eGFP-mRNA, die gemessene Fluoreszenz wurde auf die RFP-Fluoreszenz normiert. In der Studie gelang es, ein sauberes „An/Aus-Verhalten“ zu erzielen, das heißt, die modifizierte siRNA zeigte zunächst keinerlei Einfluss auf die eGFP-mRNA. Bestrahlte man diese siRNA jedoch für drei Minuten bei 366 nm, erzielte man eine Unterdrückung der eGFP-Expression, die der einer unmodifizierten siRNA entsprach. Dies funktionierte für vor der Transfektion bestrahlte siRNAs ebenso, wie für solche, die erst nach der Transfektion in der Zelle entschützt wurden. Vereinfachte Darstellung der lichtaktivierbaren RNA Interferenz. Links: solange die Photoschutzgruppe (rot) auf dem Führungsstrang sitzt wird das Substrat des RISC nicht geschnitten. Rechts: Bestrahlung mit UV-Licht entfernt die Photoschutzgruppe und aktiviert die RNAi-Maschinerie. Ein bis jetzt ungeklärtes und noch näher zu untersuchendes Phänomen ist die Stabilität der Modifikationen in der Zelle. Aus bisher nicht eindeutig zu benennender Ursache fand nach einer definierten Zeit eine Aktivierung der ausgeschalteten siRNA statt, ohne dass diese bestrahlt wurde. Versuche mit photolabil modifizierten Nukleotiden an anderen Positionen innerhalb der siRNA, sowie eine Fluoreszenz-Korrelationsspektroskopie-Studie mit fluoreszenzmarkierter siRNA und fluoreszenzmarkiertem RISC erlaubten es Rückschlüsse auf den Schritt der RNAi zu ziehen, der durch die Einführung der Basenmodifikation blockiert wird. Offenbar handelt es sich tatsächlich um den katalytischen Schritt der mRNA-Spaltung, ein Einbau der modifizierten siRNA in den RISC findet statt. Zudem zeigte die erfolgreiche Inaktivierung der für die FCS-Studie genutzten anti-TK siRNA, dass der Ansatz, die Modifikation im Bereich der Schnittstelle einzubauen, von der anti-eGFP siRNA auf andere siRNAs übertragbar ist. Im zweiten erfolgreichen Projekt gelang es, molecular beacons durch Einführung zahlreicher photolabiler Basenmodifikationen lichtaktivierbar zu machen. Hierzu wurde ein bereits beschriebener GAPDH-molecular beacon verwendet. Modifiziert man die Schleife dieses molecular beacon mit sieben photolabilen Basenschutzgruppen (NPP und NPE) so gelingt es die Bindung desselben an seine komplementäre Ziel-RNA komplett zu unterbinden, während ein GAPDH-beacon mit drei oder fünf Modifikationen noch in verringertem Maße bindungsfähig ist. Dieses Verhalten wurde sowohl mittels einfachen Auslesens der Fluoreszenzintensität, als auch anhand eines PA-Geles belegt. Eine große Herausforderung bei diesem Projekt stellte die Aufreinigung des hochmodifizierten molecular beacon dar, der neben Fluorophor und Quencher auch zahlreiche Photoschutzgruppen trägt. Diese gelang schließlich durch den Einsatz einer extra densely bond-RP-HPLC-Säule und wiederholter HPL-Chromatographie. Ebenfalls konnte der große Vorteil eines lichtaktivierbaren molecular beacon, die Möglichkeit der präzisen Ortsauflösung der Aktivierung, dargestellt werden. Hierzu wurde modellhaft die Ziel-RNA auf einer Objektträgeroberfläche immobilisiert. Die dann aufgetragene molecular beacon-Lösung zeigte zunächst keine Fluoreszenz. Diese trat erst nach Bestrahlung und auch nur begrenzt auf den wenige Quadratmikrometer großen Bestrahlungsbereich auf.The availability of synthetic oligonucleotides paved the way for the development of a multitude of molecular biological, biochemical and medical applications. And as many of these applications might already be exciting separately, combining some of these methods establishes even further prospects. Within the PhD thesis at hand the technique of photolabile protection could successfully be transferred to the applications of siRNAs and molecular beacons. Therewith these methods additionally gained the feature of spatiotemporal control. By introducing a nucleotide bearing a 2-(2-nitrophenyl)propyl-protected nucleobase into an siRNA, the catalytic step of RNA-interference – the mRNA cleavage - could be prevented. To achieve this the photolabilely modified nucleotide was placed within the siRNA opponent to the mRNA cleavage site or adjacent to this position. The modified nucleotide itself was no ribonucleotide but a deoxynucleotide. Before, it could be shown that introducing single deoxynucleotides into an siRNA had no effect on siRNA activity. The RFP/eGFP-reportergene-assay was chosen as a model system. Both plasmides were cotransfected with the siRNA. The siRNA which was used targeted eGFP-mRNA, the fluorescence detected was normalized to RFP-fluorescence. In the present study a clean „on/off-behaviour“ was obtained. Initially, the modified siRNA did not show any influence on the eGFP-mRNA. However, irradiation of the siRNA with UV-light (366 nm) lead to a suppression of eGFP-expression which was equal to the extent of suppression triggered by a non-modified siRNA. Re-activation worked for siRNAs which were exposed to UV-light prior to transfection as well as for siRNAs irradiated post transfection inside living cells. Schematic of light-activatable RNA-interference. Left: as long as the protection group (red) is located on the guide-strand the mRNA-substrate of RISC is not cleaved. Right: Irradition with UV-light removes the photolabile protecting group and RNA-interference is activated. A phenomenon yet to be investigated is stability of the modification within the cytoplasm. After a certain time, a small amount of inactivated siRNAs were re-activated without irradiation and till recently no reasonable explanation could be conceived. Additionally experiments with modifications at different positions within the siRNA as well as a fluorescence-cross-correlation-spectroscopy-study using a labelled anti-TK siRNA and a labelled RISC were performed. This allowed for drawing conclusions concerning the very step of RNAi, which is blocked by introduction of the photolabile protecting group. Apparently blockage really is a matter of the catalytic step of mRNA-cleavage as the modified siRNA is still introduced into RISC. Furthermore the successful inactivation of the anti-TK siRNA clearly showed, that the approach of introducing the modification in close proximity of the cleavage-site can be transferred from the anti-eGFP siRNA to other siRNAs. During the second successful project molecular beacons could be altered light-activatable by introducing several photolabile nucleobase-modifications. For this study an already published GAPDH-molecular beacon was used. By modifying the loop region of this molecular beacon with seven photolabile protecting groups (NPE and/or NPP) hybridization of the beacon to its complementary target-RNA was completely abolished whereas the same molecular beacon bearing only three or five such modifications showed a residual target-binding capacity. The characteristics of light-activatable molecular beacons were followed both via fluorescence read-out and polyacrylamide gel electrophoresis (PAGE). A major challenge of this project was constituted by the purification of this highly modified system. The light-activatable molecular beacon not only carries a fluorophor and a quencher but additionally several photolabile protection groups. In the end sufficient purification could be achieved by using an xtra densely bond-RP-HPLC-column an performing several rounds of HPL-chromatography. Moreover the major advantage of a light-activatable molecular beacon could be demonstrated: the capability of precise spatio-temporal addressing. As a model system, target-RNA was immobilised to the surface of a glass-slide. Adding molecular-beacon solution did not result in any fluorescence. Only after irradiation fluorescence could be detected and still the area lighting up was restricted to the very region, which was exposed to UV-light

Body Image Perceptions and Eating Patterns Among Preadolescent Children

Preadolescent children are showing patterns of body distortion and unhealthy dieting practices to attempt weight manipulation. The present study examined body image perceptions and interest in weight manipulation among 215 preadolescent children (girls, n =101) (mean age = 10) in third, fourth, and fifth grades. To measure body image perceptions, children were asked to answer two multiple choice questions: I think I am: fat, skinny, in-between; and I would like to: lose weight, gain weight, stay the same. Interest in weight manipulation was measured using the Children’s Eating Attitude Test (ChEAT). Results indicated that children show a fairly accurate perception of their body shape and size and this remained consistent for both genders and across all age groups. ANOVA results showed children who felt they were “fat” had a significantly higher level of disturbed eating patterns (ChEAT mean = 14.00) compared to the children who felt they were “in-between” (ChEAT mean = 6.95) (p = .009). Children who indicated they would like to “lose weight” had significantly higher ChEAT scores (mean = 9.19) than those who wanted to “stay the same” (mean = 6.56) (p = .033). ChEAT scores for girls showed a significant difference between the “fat” group (mean = 29.00) and the “in-between” group (mean = 6.81) (p < .001) and between the “fat” group and the “skinny” group (mean = 10.78) (p = .002). Twenty-five percent of this sample of children indicated a desire to “lose weight” (mean body mass index = 21.35) and 67% desired to “stay the same” (mean body mass index = 17.45). Preadolescent children are showing a desire for a thinner body type and are already developing a motivation to avoid weight gain or obesity through dietary manipulation. Early identification of body distortion and disturbed eating patterns could help with education and prevention strategies

Bildungspolitik als Gesellschaftspolitik

[Abstract fehlt

Validity and reliability of the session RPE method for monitoring exercise training intensity

Objective. The Session Rating of Perceived Extertion (RPE) is a method of measuring exercise intensity that may be useful for the quantitative assessment of exercise training programmes. However, there are inadequate data regarding the validity and reliability of the Session RPE method. This study was designed to evaluate both the validity and reliability of the Session RPE method in comparison to objective measures (%HRpeak, %HRreserve and %VO2peak) of exercise intensity. Methods. Fourteen healthy volunteers (7 male, 7 female) performed 6 randomly ordered 30-minute constant-load exercise bouts at 3 different intensities, with each intensity being repeated. Oxygen consumption (VO2) and heart rate (HR) were measured throughout each exercise bout and normalised to maximal values obtained during a preliminary maximal exercise test. Thirty minutes following the conclusion of each exercise bout, the subject rated the global intensity of the bout using a modification of the Category Ratio (CR) (0 - 10) RPE scale. This rating was compared to the mean value of objectively measured exercise intensity across the duration of the bout. Results. There were significant non-linear relationships between Session RPE and %VO2peak (R2 = 0.76), %HRpeak (R2 = 0.74) and %HRreserve (R2 = 0.71). There were no significant differences between test and retest values of %VO2peak, %HRpeak, %HRreserve and Session RPE during the easy (47 v. 47%, 65 v. 66%, 47 v. 48% and 2.0 v. 1.9), moderate (69 v. 70%, 83 v. 84%, 74 v. 75%, and 4.2 v. 4.3) and hard (81 v. 81%, 94 v. 94%, 91 v. 91% and 7.3 v. 7.4) exercise bouts. Correlations between repeated bouts for %VO2peak (r = 0.98), %HRpeak (r = 0.98), %HRreserve (r = 0.98) and Session RPE (r = 0.88) were significant and strong. Conclusions. The results support the validity and reliability of the Session RPE method of monitoring exercise intensity, although as might be predicted for a subjective method the Session RPE was less precise than the objective measures of exercise training intensity. South African Journal of Sports Medicine Vol. 18 (1) 2006: pp. 14-1

Body Image Dissatisfaction Among Third, Fourth, and Fifth Grade Children

Body image dissatisfaction has become increasingly more prevalent among the preadolescent population over recent years. This study examines the level of body image dissatisfaction among 261 third, fourth, and fifth grade girls and boys. A pictorial scale was used to assess how the participants viewed their current body shape, their ideal body shape, and what they believed to be the ideal body shape of the opposite gender. Overall results indicated that 50.6% of the children surveyed were dissatisfied with their current body shape, 41.8% wanted to be thinner, while 8.8% wanted to be larger. Fifty percent of the girls were dissatisfied with their current body shape, with 45.1% wishing to be thinner. Boys showed similar trends, with 48.9% dissatisfied and 38.9% wishing to be thinner. A greater percentage of boys wanted to be larger than their current body shape (12.3%) compared to girls (4.9%). A significant difference was found between genders regarding the difference between scores of their current self and ideal self, where girls selected a smaller ideal body shape than the boys. An encouraging finding was that the level of body dissatisfaction decreased from third grade to fifth grade among both genders. Girls, however, still wished to be thinner over time. Boys, on the other hand, indicated a preference for a somewhat larger body shape over time. These results indicate that body image dissatisfaction exists prior to adolescence among this sample of children. Prevention strategies and education are encouraged among this age group

CAN THE TALK TEST BE USED TO PREDICT TRAINING INDUCED CHANGES IN VENTILATORY THRESHOLD?

This study was designed to assess the ability of the Talk Test (TT) to track training-related changes in ventilatory threshold (VT). Thirteen recreational athletes (mean±SD; age, 20.5±1.91 years, (Males=7, Females=6) completed two incremental exercise tests (one with respiratory gas exchange and one with the TT) before and after 6-weeks of self-directed increases in training load. The TT was used to predict VT by assessing the ability to speak comfortably after 3-minute exercise stages based on speech comfort during a 100-word passage. Training load was documented using exercise logs based on sRPE and training duration. Repeated measures ANOVA, with Tukey’s post-hoc analysis, was used to detect differences between the changes in power output at the equivocal stage of the Talk Test and VT measured by gas exchange (p&lt;.05). Significant mean differences were found between pre vs post training power output at the equivocal stage of the Talk Test (125+40.8 vs 135+29.8 Watts) and measured VT (116+32.4 vs 134+32.4 Watts) (p&gt;.05). However, the increase in power output at VT (+15.5%) was significantly underestimated by the change in power output at equivocal stage of the TT (+8.0%).&nbsp; The correlation between changes in power output at VT and PO at the equivocal stage of the TT was r=0.66. However, about 50% of subjects did not change their power output at the equivocal stage of the TT, so the individual correspondence between Talk Test and measured VT was only moderately strong.</p

Online-Marketing Basics für B2B

Das vorliegende Paper fasst die Erkenntnisse zum Einsatz von Online-Marketing für B2B Unternehmen zusammen. Es basiert auf der Bachelorarbeit von Frau Lena-Marie Mikat, die von September 2017 bis Januar 2018 von Prof. Dr. Monika Engelen betreut wurde. Neben einer Recherche der grundlegenden Theorie wurden die Online-Präsenzen der 25 größten B2B-Unternehmen des Oberbergischen Kreises analysiert. Der Kerninhalt dieses Papers ist wie B2B Unternehmen von Online-Marketing anwen-den können. Dabei liegt der Fokus auf die Websitegestaltung für B2B-Unternehmen mit Tipps zur strategischen Planung und operativen Gestaltung