Fluorescent proteins are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. We used molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variants, and its GFP homolog citrine. We employed implicit ligand sampling and locally enhanced sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. The pathway contains several oxygen hosting pockets, which were identified by the amino acid residues that form the pocket. We calculated the free-energy of an oxygen molecule at points along the path. We also investigated an RFP variant known to be significantly less photostable than mCherry and find much easier oxygen access in this variant. We showed that oxygen pathways can be blocked or altered, and barrel fluctuations can be reduced by strategic amino acid substitutions. The results provide a better understanding of the mechanism of molecular oxygen access into the fully folded mCherry protein barrel and provide insight into the photobleaching process in these proteins.

This thesis is focused for manufacturing of a gun barrels and his issues. In thesis is involved introducing of the company Česká zbrojovka a.s. and definition of hand fired guns. Written analysis present the state of a technological process and describe an individual phase of manufacturing of the semi-finished product of gun barrel and the following conclusion of a complex manufacturing of a gun barrel from the semi-finished product to the stand, when the gun barrel installed into the gun is. Subsequently are suggested possibly solutions of manufacturing rationalization and possibly purchasing of a new modern CNC machines. In the end is accomplished a technical-economic evaluation, which compares existing and suggesting of the technology.

This thesis deals with design of equipment for the production of PET termoplastic fibre to be used in 3D printing by means of FDM method. Concise survey of plastics processing methods is presented herein, furthermore, structural design of equipment with necessary engineering calculations is included as well. Drawing documents with the total economic evaluation is a part of this thesis too.

Im Rahmen eines Seminars über „Möbel als historische Quelle“ am Institut für Kunstgeschichte der Universität Leipzig im WS 2005/06 unter der Leitung des Verfassers wurde Sebastian Jung, Student der Kunstgeschichte, angeregt, eine Hausarbeit anzufertigen mit dem Thema ,,Gibt es geböttcherte Möbel? Eine Spurensuche in der Niederländischen Malerei“. Einige der folgenden Überlegungen entstammen dieser Arbeit. Die Gewerke der Tischler, Schnitzer, Drechsler und Zimmerleute sind holzverarbeitende Berufe, die immer auch Möbel angefertigt haben. Vom Böttcherhandwerk ist dies nicht bekannt, sieht man einmal von modernem Kneipeninterieur „im Böttcherstil“ ab. Das Besondere der Böttcherei ist, dass die einzelnen Holzteile – Dauben genannt – ohne Holzverbindungen nur mittels Reifen zusammengehalten werden. Dies benötigt eine regelmäßige Pflege durch den Nutzer, zum Beispiel durch das Nachschlagen der Reifen. Ist dies nicht gegeben, schwindet das Holz, die Reifen werden locker und es besteht so die Gefahr, dass alles auseinander fällt. Es handelt sich also bei geböttcherten Dingen um relativ empfindliche und pflegeintensive Gegenstände.

Prenatal alcohol exposure (PAE) alters limb development that may lead to structural and functional abnormalities of the limb reported in children diagnosed with Fetal Alcohol Spectrum Disorder. To determine whether PAE alters the central representation of the forelimb we used the rodent barrel cortex as our model system where it was possible to visualize and quantitatively measure the size of the forepaw representation in the forepaw barrel subfield (FBS) in first somatosensory cortex. In the present study, we examined the effects of PAE on pattern and size of the forepaw and forepaw representation in FBS in neonatal rats at gestational day 32 that corresponds to postnatal day 9. Pregnant Sprague-Dawley rats were chronically intubated with binge doses of ethanol (6 g/kg) from gestational day 1 through gestational day 20. The offspring of the ethanol treated dams comprised the ethanol (EtOH) group. The effect of PAE on the EtOH group was compared with a nutritional-controlled pairfed (PF) group and a normal chowfed (CF) group. The ventral (glabrous) surface area of the forepaw digits, length of digit 2 through digit 5, and the corresponding glabrous forepaw digit representations in the FBS were measured and compared between treatment groups. In rats exposed to in utero alcohol, the sizes of the overall glabrous forepaw and forepaw digits were significantly reduced in EtOH pups compared to CF and PF pups; overall glabrous forepaw area was 11% smaller than CF controls. Glabrous digit lengths were also smaller in EtOH rats compared to CF controls and significantly smaller in digit 2 through digit 4. The glabrous digit representation in FBS was 18% smaller in the EtOH group when compared to the CF treatment. However, PAE did not produce malformations in the forepaw or alter the pattern of the forepaw representation in FBS; instead, PAE significantly reduced both body and brain weights compared to controls. Unexpectedly, little or no correlation was observed between the size of the glabrous forepaw compared to the size of the glabrous forepaw representation in the FBS for any of the treatment groups. The present findings of PAE-related alterations in sensory periphery and the central cortical representation may underlie deficits in sensorimotor integration reported among children with Fetal Alcohol Spectrum Disorder.

High-throughput genomics projects have resulted in a rapid accumulation of protein sequences. Therefore, computational methods that can predict protein functions and functional sites efficiently and accurately are in high demand. In addition, prediction methods utilizing only sequence information are of particular interest because for most proteins, 3-dimensional structures are not available. However, there are several key challenges in developing methods for predicting protein function and functional sites. These challenges include the following: the construction of representative datasets to train and evaluate the method, the collection of features related to the protein functions, the selection of the most useful features, and the integration of selected features into suitable computational models. In this proposed study, we tackle these challenges by developing procedures for benchmark dataset construction and protein feature extraction, implementing efficient feature selection strategies, and developing effective machine learning algorithms for protein function and functional site predictions. We investigate these challenges in three bioinformatics tasks: the discovery of transmembrane beta-barrel (TMB) proteins in gram-negative bacterial proteomes, the identification of deleterious non-synonymous single nucleotide polymorphisms (nsSNPs), and the identification of helix-turn-helix (HTH) motifs from protein sequence.

A gun barrels performance can only be considered satisfactory provided there is sufficient heat-dissipation, thus not hindering the tactical use of the unit and the weapon itself. The added energy to the barrel can potentially permanently change the material structure of the steel and create a hazard.The thesis evaluates air cooling concepts relative to a barrel blank to assess its ability to handle heat flow and heat flux. The concepts have been designed in such a way that the ergonomic and practical weight limitations of a soldier have been taken into account. The results will provide a basis for future product development.The scientific work was limited to the section of the barrel nearest to the bore where the majority of the heat will be concentrated, hence that section with the biggest need for heat dissipation. For this to be done an assumption was made that the heat can only dissipate radially. The metal in this short section of the barrel is assumed to be heated equally along the barrel axis relative to the surrounding sections. The thesis was divided in two parts, a literature study and one with a deductive approach, where the research resulted in calculations in an iterative process.The calculations determined the cooling and heating time for each concept. Finally, the heat flow in and out was calculated.Based on the information that the calculations provided, a comparison could be done and an assessment for each concept to be a potential solution if it were to be applied in a sustained fire role. The conclusion was that an air cooled concept could never provide the heat flux needed to sufficiently cool a barrel in a sustained fire role due to all the thermal energy added for each shot fired.The composite concept that was evaluated has great potential in a lightweight weapon system. It provides quadruple cooling capability with no additional weight. It also has good potential for future development of the concept.

Introdução e objetivos: β-glicosidases da família GH1 das glicosil-hidrolases possuem um dobramento do tipo barril (β/α)8. Propõe-se que proteínas com este dobramento, que usualmente classificam-se como tendo um único domínio, na verdade são compostas por dois domínios, cada um deles correspondendo a um \"meio barril\" (β/α)4. Assim, as proteínas com dobramento barril (β/α)8 seriam provenientes de uma duplicação e fusão gênica de um ancestral \"meio barril\" (β/α)4. O objetivo geral deste projeto é investigar a existência de dois domínios (β/α)4, as metades N- e C-terminal, na estrutura (β/α)8 barril da β-glicosidase A de Thermotoga maritima (bglTm) e β-glicosidase B de Paenibacillus polymyxa (bglB) por meio de análise da desnaturação térmica e química dessas enzimas. Resultados: Para atingir esse objetivo foram introduzidas mutações que rompem contatos não covalentes entre os supostos domínios destas β-glicosidases. Os segmentos de DNA que codificam as enzimas selvagem e mutantes foram clonados em plasmídeo de expressão pLATE51 e as enzimas recombinantes expressas em Escherichia coli BL21(DE3). Foram purificadas com sucesso as enzimas selvagens e duas mutantes de bglTm, denominadas T1 e T2, que possuem 2 e 4 mutações respectivamente em resíduos na interface inter-metades. Para confirmar o enovelamento destas proteínas recombinantes foi empregada a análise de estruturas secundárias por dicroísmo circular, também o espectro de fluorescência intrínseca de triptofano e sua supressão por acrilamida e finalmente foram determinados parâmetros cinéticos. Observou-se que não houve mudanças significativas na exposição dos triptofanos das proteínas recombinantes, sugerindo que se encontram enoveladas, o que está de acordo com a observação de que as proteínas recombinantes mantêm sua atividade catalítica. Já pela análise de dicroísmo circular concluiu-se que a mutante T1 possui dobramento semelhante à selvagem bglTm e que T2 apresenta diferenças significativas, sendo as porcentagens de α-hélice de 21%, 22% e 10% para bglTm, T1 e T2, respectivamente. Em seguida demonstrou-se que T1 mantém a termo estabilidade semelhante à selvagem, enquanto que T2 tem termo estabilidade reduzida, apresentando kobs de 0,3 min-1 em 80°C e de 0,06 min-1 em 75 °C.. O cálculo de Tm através de Differential Scanning Fluorimetry foi feito para bglB e T2 (42 e 81.7 °C respectivamente), enquanto que bglTm e T1 mantiveram-se estáveis na faixa de temperatura analisada (até 95°C). Na análise do efeito da temperatura sobre a estrutura das mutantes T1 e T2 não se observou nenhuma evidência da presença dos dois supostos domínios (β/α)4. A análise da desnaturação por cloreto de guanidina mostrou que o c50 diminuiu para T2 (2,4 M), mas não mostrou alteração para T1 (4,5 M) em comparação à bglTm (4,3 M). Coerentemente, a estabilidade da enzima selvagem e de T1 na ausência de desnaturante é a mesma (ΔGH2O = 5,2 kcal/mol), mas se reduziu para a T2 (ΔGH2O = 3,5 kcal/mol). Os cálculos do parâmetro m mostraram uma cooperatividade semelhante na desnaturação de bglTm, T1 e T2, não evidenciando independência entre os dois supostos domínios (β/α)4. Em conclusão, a análise estabilidade da β-glicosidase bglTm frente à temperatura e ao cloreto de guanidina não revelaram a presença dos domínios (β/α)4 que correspondem às metades N- e C-terminal desta β-glicosidase. / Introduction and Aims: β-glucosidases from the family GH1 of the glycosil-hidrolases presents a (β/α)8 barrel folding. These proteins are usually classified as single domain, however it has been alternatively proposed that they actually are formed by two \"half barrel\" (β/α)4. Thus, (β/α)8 barrel proteins had evolved from an \"half barrel\" ancestor that underwent a duplication-fusion event. The general goal of this project is the search for the two putative (β/α)4 domains, which form the N- and C-terminal ends of the β-glucosidase A from Thermotoga maritima (bglTm) and β-glucosidase B from Paenibacillus polymyxa (bglB), detecting their presence through the thermal and chemical stability of these (β/α)8 barrel proteins. Results: Site-directed mutagenesis was employed to replace residues forming non-covalent interaction between the putative (β/α)4 domains. DNA segments coding for bglB, bglTm and mutant bglTm were cloned into the pLATE51 expression vector and produced as recombinant proteins in E. coli BL21(DE3). The bglB, bglTm and two mutant bglTm, hereafter called T1 and T2, with 2 and 4 mutations respectively on residues in the interface between the protein halves, were purified. They were stable folded as shown by detecting their catalytic activity upon two different substrates and also by circular dichroism (CD) and tryptophan fluorescence analysis. Nevertheless, T2 showed a decrease in the α-helix content (10 %) in the CD analysis, whereas bglTm and T1 are similar (22 %). The wild-type bglTm and T1 are thermostable, whereas T2 was inactivated after pre-incubation at high temperature (kobs = 0,3 min-1 at 80 °C and 0,06 min-1 at 75 °C). The Differential Scanning Fluorimetry experiments revealed Tm of 42 e 81.7 °C for bglB and T2, respectively, whereas wild-type bglTm and mutant T1 did not showed any thermal transition up to 95 °C. Indeed, the analysis of the thermal stability of T1 and T2 did not reveal any evidence of the putative (β/α)4 domains. Following that, the analysis of the protein denaturation by guanidine hydrochloride showed that the c50 for T2 was reduced (2.4 M), whereas no modification was observed for bglTm and T1 (4,3 and 4,5 M, respectively). In agreement the stability of bglTm and T1 (ΔGH2O = 5,2 kcal/mol) is similar, but it was reduced for T2 (ΔGH2O = 3,5 kcal/mol). The m parameter showed a similar cooperative denaturation for wild-type bglTm and mutants T1 and T2, but no evidence of the independent unfolding of the putative (β/α)4 domains was found. Conclusion: In conclusion, the analysis of the thermal and chemical stability of the bglTm did not reveal the presence of the putative (β/α)4 domains that form the N- and C-terminal end of these β-glucosidase.

Otto Scheeloch's U.S. Patent No. 134,442 of 1872 describes a unique firearm that uses triangular bullets. The current research effort evaluates the ballistic performance of Otto's disclosure for the very first time. To achieve this goal it was necessary to seek out surviving artifacts and scour the historical record in search of all the parameters needed to meticulously recreate the curious triangular cartridges and the corresponding gun barrel, with its matching twisted triangular bore. Every aspect of the resulting reproduction ammunition was made to be as authentic as possible, including the use of vintage civil war era bullet lead, bullet grease of period recipe, and the correct type of black powder propellant. 3D CAD (SolidWorksTM) was employed in designing the components, while advanced rapid prototyping (FDM & DMLS) techniques and investment casting were used in the physical construction of the ammunition and barrel. The ballistics testing was performed from a shooting rest over a range of 10-feet. Data was obtained for five rounds using a chronograph, paper targets and ballistic gel. The triangular bullets proved to be surprisingly accurate, consistent, and stable in flight. Data was recorded for sectional density, ballistic coefficient, muzzle velocity and energy, group size and penetration.

Das Ende der 90er Jahre neu entstandene Crystal Barrel-Experiment am Bonner Elektronenbeschleuniger ELSA, kurz CB-ELSA-Experiment, dient hauptsächlich dem Studium der Photoproduktion am Proton bei Strahlimpulsen zwischen 0.8 GeV/c und 3.2 GeV/c. Ziel dieses Experimentes ist es, neue Erkenntnisse über das Spektrum der leichten Baryonen zu gewinnen und auch einige wichtige Beiträge für ein besseres Verständnis des Spektrums der leichten Mesonen zu liefern. Die Besonderheit dieses Experimentes liegt in der Topologie der Detektoren. Das Herzstück bildet hierbei das Crystal Barrel-Kalorimeter, das unter Zuhilfenahme weiterer Detektoren die vollständige Erfassung von Ereignissen mit neutralen Mesonen über einen Raumwinkel von nahezu 4[pi] erlaubt. Die vorliegende Arbeit befasst sich zum einen mit dem Aufbau des Flüssig-Wasserstoff-Targets, für dessen Anpassung an die hohen Ansprüche des Experimentes eine umfangreiche Entwicklungsarbeit erforderlich war. Zum anderen bildet den Hauptteil der Arbeit die Untersuchung der photoinduzierten Reaktionen [gamma]proton nach proton[pi]0[pi]0 und [gamma]proton nach proton[pi]0[eta] bei einem Strahlimpuls unpolarisierter Elektronen von 2.6 GeV/c. Beim Studium dieser Reaktionen zeigen sich Hinweise auf missing resonances, die sequentiell über verschiedene Baryonenresonanzen zerfallen. Darüber hinaus gibt es deutliche Evidenzen für die f<sub>0</sub>(980)- und a<sub>0</sub>(980)-Photoproduktion, deren weitergehende Untersuchungen wichtige Beiträge zur Klärung des bis heute noch nicht verstandenen skalaren Mesonennonetts liefern könnten.