Note:

Trier, Universiẗat, Diss., 2004.

Saarbrücken, Universiẗat, Diss., 2003. / Erscheinungsjahr an der Haupttitelstelle: 2002.

Despite the importance of molecular recognition in nearly all aspects of protein function, the determinants of specificity for enzyme-substrate and protein-protein interactions are poorly understood. The majority of these complexes involving bacterial luciferase from V. harveyi have yet to be fully characterized. Luciferase catalyzes the reaction of molecular oxygen, FMNH2 and a long-chain aliphatic aldehyde yielding FMN, the corresponding carboxylic acid and blue-green light. In vivo, luciferase is thought to obtain FMNH2 following transfer from a transiently associated oxidoreductase. To identify the oxidoreductase responsible for providing FMNH2 in E. coli, bioluminescence was compared using single gene deletion strains deficient in either a homolog to the endogenous V. harveyi oxidoreductase (Frp) or an oxidoreductase distantly related to luxG from V. fischeri (Fre). Fre is responsible for reducing flavin in vivo but does not physically interact with luciferase. The association between luciferase and the flavin product is also described. Luciferase was crystallized and subjected to soaking with high concentrations of FMN. A model was obtained for luciferase bound to FMN. Using molecular dynamics, models for the enzyme:aldehyde, enzyme:FMNH2, and luciferase bound to several reaction intermediates are presented. Finally, a conserved loop region adjacent to the active center was investigated for the ability to facilitate protein:protein interaction between luciferase and the endogenous Frp oxidoreductase. Following alanine mutagenesis of the charged residues throughout this loop, it appears that the residues targeted by this study are not components of a docking platform but facilitate a lid-gating mechanism of paramount importance for catalytic function.

?rea de concentra??o: Produ??o animal / Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-12-22T12:13:05Z No. of bitstreams: 2 vilmar_rodrigues_sousa_junior.pdf: 525685 bytes, checksum: 6b5cf8f68fbb09851f53a993f9d39d69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-12-22T12:37:57Z (GMT) No. of bitstreams: 2 vilmar_rodrigues_sousa_junior.pdf: 525685 bytes, checksum: 6b5cf8f68fbb09851f53a993f9d39d69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2015-12-22T12:37:57Z (GMT). No. of bitstreams: 2 vilmar_rodrigues_sousa_junior.pdf: 525685 bytes, checksum: 6b5cf8f68fbb09851f53a993f9d39d69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2010 / Objetivou-se com esta pesquisa avaliar a influ?ncia da ilumina??o artificial, na creche, sobre o desempenho produtivo e estado sanit?rio dos leit?es e caracterizar as condi??es ambientais dentro das salas dos programas de ilumina??o artificial. O experimento foi realizado no Centro Nacional de Pesquisa em Su?nos e Aves (CNPSA-Embrapa), no Sistema de Produ??o de Su?nos, localizado no munic?pio de Conc?rdia, regi?o Oeste do Estado de Santa Catarina. O experimento foi realizado em dois per?odos, de 19 de junho de 2008 a 24 de julho de 2008 e de 10 de junho de 2009 a 15 de julho de 2009, com clima t?pico de inverno. Foram alocados seis animais em cada baia e utilizadas seis baias (blocos) centrais por sala. Os animais foram resultantes de cruzamento f?meas (Landrace x Large-White) e machos (MS 115), desmamados com 28,3 ? 2,1 dias de idade e peso de 9 ? 1,2 kg, de acordo com o manejo da granja. Os tratamentos foram associados ? ilumina??o da sala, sendo em cada uma aplicado um tratamento diferente: Programa LN - Ilumina??o natural (controle); Programa 16L:8E - Ilumina??o artificial de 16 horas di?rias de luz e 8 hora de escuro (16L:8E); Programa 23L:1E - Ilumina??o artificial de 23 horas di?rias de luz e 8 horas de escuro (23L:1E). As vari?veis analisadas foram: desempenho (ganho de peso di?rio, ganho de peso acumulado, consumo de ra??o di?rio, consumo de ra??o acumulado, consumo de ?gua di?rio); ocorr?ncia de diarr?ia nos leit?es; vari?veis meteorol?gicas (temperatura de bulbo seco, umidade e velocidade do ar) e ilumin?ncia. O planejamento experimental foi em blocos completos casualizados, com tr?s tratamentos, duas repeti??es e 35 dias de medi??o e as m?dias comparadas pelo teste t. N?o houve influ?ncia da ilumina??o sobre as vari?veis de desempenho. A frequ?ncia de diarr?ia n?o foi influenciada pelos programas de ilumina??o. As diferen?as encontradas nas vari?veis meteorol?gicas n?o influenciaram o desempenho dos su?nos. Os programas de ilumina??o avaliados n?o apresentaram melhorias no desempenho dos leit?es em fase de creche que justificassem a sua utiliza??o. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Zootecnia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, [2010]. / ABSTRACT The objective of this research was to evaluate the influence of artificial lighting in the nursery, on performance and sanitary conditions of piglets and characterize the environmental conditions inside the rooms of the programs of artificial lighting. The experiment was held at the National Center for research on pigs and poultries (CNPSA-EMBRAPA), the system of pig production in the city of Concordia, west of the state of Santa Catarina. The experiment was conducted in two periods, from June, 19 to July, 24 of 2008 and June, 10 to July, 15 of 2009, with typical winter weather. Six animals were allocated in each stall and used six stalls (blocks) in central room. The animals resulted from a cross between females (Landrace x Large White) and males (MS 115), weaned at 28.3 ? 2.1 days of age and weight of 9 ? 1.2 kg, according to the management of the poultry farm. The treatments were associated with the lighting of the room, being assigned to each one a different treatment: LN Program - natural lighting (control); Program 16L: 8E - Artificial lighting with 16 hours of light and 8 hours of dark (16L: 8E). Program 23L: 1E - Artificial lighting with 23 hours of light and 8 hours of dark (23L: 1D). The analyzed variables were: performance (daily weight gain, total weight gain, daily feed intake, total feed intake, daily water consumption), diarrhea in piglets; meteorological variables (dry bulb temperature, humidity and speed air) and illuminance. The experimental planning was in randomized complete blocks with three treatments, two repetitions and 35 days of measurement and the averages compared by t test. There was no influence of illumination on the performance variables. The frequency of diarrhea was not influenced by lighting programs. The differences in meteorological variables did not influence the performance of pigs. The evaluated lighting programs did

<I>Vibrio fischeri</I>, a symbiotic bioluminescent bacterium, serves as one of the best understood model systems for a mechanism of cell-density dependent bacterial gene regulation known as quorum sensing. During quorum sensing in <I>V. fischeri</I>, an acylated homoserine chemical signal (autoinducer) is synthesized by the bacteria and used to sense their own species in a given environment. As the autoinducer levels rise, complexes form between the autoinducer and the N-terminal domain of a regulatory protein, LuxR. In response to autoinducer binding, LuxR is believed to undergo a conformational change that allows the C-terminal domain to activate transcription of the luminescence or <I>lux</I> operon. To further understand the mechanism of LuxR-dependent transcriptional activation of the <I>lux</I> operon, PCR-based site-directed mutagenesis procedures have been used to generate alanine-substitution mutants in the C-terminal forty-one amino acid residues of LuxR, a region that has been hypothesized to play a critical role in the activation process. An <I>in vivo</I> luminescence assay was first used to test the effects of the mutations on LuxR-dependent activation of the <I>lux</I> operon in recombinant <I>Escherichia coli</I>. Luciferase levels present in cell extracts obtained from these strains were also quantified and found to correlate with the luminescence results. Eight strains encoding altered forms of LuxR exhibited a "dark" phenotype with luminescence output less than 50% and luciferase levels less than 50% of the wildtype control strain. Western immunoblotting analysis with cell extracts from the luminescence and luciferase assays verified that the altered forms of LuxR were expressed at levels approximately equal to wildtype. Therefor, Low luminescence and luciferase levels could be the result of a mutation that either affects the ability of LuxR to recognize and bind its DNA target (the <I>lux</I> box) or to establish associations with RNA polymerase (RNAP) at the <I>lux</I> operon promoter necessary for transcriptional initiation. A third <I>in vivo </I>assay was used to test the ability of the altered forms of LuxR to bind to the <I>lux</I> box (DNA binding assay/repression). All of the LuxR variants exhibiting the "dark" phenotype in the luminescence and luciferase assay were also found to be unable to bind to the <I>lux</I> box in the<I> </I>DNA binding assay. Therefore, it can be concluded that the alanine substitutions made at these positions affect the ability of LuxR to bind to the <I>lux</I> box in the presence and absence of RNA polymerase. Another class of mutants exhibited wildtype phenotypes in the luminescence and luciferase assays but were unable to bind to the <I>lux</I> box in the DNA binding assay. The alanine substitutions made at these amino acid residues may be making contacts with RNAP that are important for maintaining the stability of the DNA binding region of LuxR. Alanine substitutions made at these positions have a defect in DNA binding at the promoter of the <I>lux</I> operon only in the absence of RNAP. None of the alanine substitutions made in the C-terminal forty-one amino acids of LuxR were found to affect activation of transcription of the <I>lux</I> operon without also affecting DNA binding. Taken together, these results support the conclusion that the C-terminal forty-one amino acids of LuxR are important for DNA recognition and binding of the <I>lux</I> box rather than positive control of the process of transcription initiation. / Master of Science

This thesis is based around the use of energy harvesting in systems, specifically for a Bluetooth Low Energy (BLE) mesh testbed. This BLE mesh is located in a well lit lab and is currently powered by mains electricity. Systems such as the BLE mesh are considered Internet of Things (IoT). The market for these systems is rapidly expanding and in turn so is the energy use. Many systems are powered by battery, and the need to replace a battery with an energy harvesting system has arisen. This thesis will explore the possibilities to power a node in this mesh and introduce a level of intelligence to allow the system to better predict available energy to harvest. The lab the BLE mesh is in is characterised for potential energy sources. Light is chosen to be an exceptional power source, from here a lux metre is created from a photovoltaic (PV) cell. This PV cell would function as both the power for the system and provide a method to measure the current light intensity. This would help add a layer of intelligence to the system to allow future systems to better understand how much energy is available. This idea can be implemented in other harvesters as well. / Denna avhandling är baserad på användningen av energiskördning i ett system, specifikt i en Bluetooth Low Energy (BLE)-mesh. Denna BLE-mesh befinner sig i ett väl upplyst labb och är för närvarande strömdrivet genom huvudström. System som BLE-mesh anses vara en del av Internet of Things (IoT). Marknaden för dessa system expanderar snabbt och därmed ökar också energiförbrukningen. Många system är batteridrivna och det har uppstått ett växande behov av att ersätta batteriet med ett energiskördsystem. Denna avhandling kommer att utforska möjligheterna att strömförsörja en nod i denna mesh och införa en intelligensnivå för att förbättra systemets möjlighet att förutspå energi som är tillgänglig för att skördas. Labbet där BLE-meshen befinner sig karakteriseras för potentiella energikällor. Ljus valdes som en exceptionell kraftkälla, och därifrån skapas en luxmeter från en fotovoltaisk (PV) cell. Denna PV-cell ska fungera både som strömkälla för systemet och utgöra en metod för att mäta nuvarande ljusintensitet. Detta skulle bidra till att lägga till ett skikt av intelligens i systemet för att göra det möjligt för framtida system att bättre förutspå tillgänglig skördbar energi. Denna idé kan även implementeras i andra energiskördsystem.

abstract: Lux Aeterna is scored for Flute (Piccolo), Bb Clarinet (Bass Clarinet), Oboe, Bassoon (Contra-Bassoon), French Horn, Trumpet in C, Tenor Trombone, Soprano Voice, Alto Voice, Tenor Voice, Bass Voice, 2 Violins, Viola, Cello and Double Bass. The piece lasts 17-18 minutes. The text, in its original Latin, is from the Requiem Mass. The pitch material for the four voices is directly derived from the original Gregorian Chant (B, C#, D, E, F#, G, A). The primary pitch materials for the instrumental ensemble are drawn from the overtone series, with the fundamental of B natural. As found in the natural overtone series, the chords produced in this composition include microtones found both naturally from the series along with microtones added by the composer to create harmonic friction. This treatment of microtonal materials is juxtaposed with the “pure” nature of the B minor modal scale sung by the 4 vocalists. Lux Aeterna uses the performance space so that the sixteen performers and conductor surround the audience in an oval shape. Entrances of instruments are determined by their position around the audience and the way in which those sounds interact with one another across the space of the hall. The instruments are strategically placed so that timbres can be balanced in a specific way and the listener can hear the pitches blend in the acoustics of the hall. The goal is to create an immersive listening experience for the audience. Although there are some spectral techniques involved in this composition, no computers or software programs were used to analyze harmonic materials. The pitch material is either from the overtone series or the B minor scale. Essentially, the music is instinctually composed. / Dissertation/Thesis / Doctoral Dissertation Music 2019

La température ambiante joue un rôle direct dans le fonctionnement et le développement de la plante. L'augmentation des températures ambiantes mondiales pose un défi important aux espèces de plantes sauvages et cultivées. La plupart des espèces de plantes ajustent leur cycle de reproduction et leur développement pour optimiser leur survie et leur forme par temps ambiant élevé (Barnabás et al., 2008; Fitter et Fitter, 2002; Willis et al., 2008). Ces adaptations conduisent généralement à des hypocotyles allongés, à une réduction du nombre de feuilles au moment de la floraison, à une transition accélérée des phases de croissance végétative à reproductive, à une réduction du nombre de graines, à des gousses plus petites et à une diminution de la surface foliaire. Face au changement climatique rapide, en particulier à l'augmentation de la température permissive à la croissance ambiante, il est urgent de régler la thermoréponse des usines pour adapter les installations aux changements climatiques et garantir la production alimentaire future.L'expression de PIF4 est contrôlée par le complexe du soir (EC), un complexe à 3 protéines comprenant ELF3, ELF4 et LUX, en fonction de la température. La CE est capable de réprimer l'expression de PIF4 en se liant à des motifs spécifiques sur le promoteur de PIF4 à une température de croissance ambiante inférieure. Cependant, cette répression de l'expression de PIF4 est éliminée à une température ambiante de croissance plus élevée, ce qui entraîne un vieillissement prématuré des plantes.RésultatsLes trois protéines de la CE ont été produites en utilisant une stratégie d’expression différente, ELF4 et LUX dans E. coli, et ELF3 dans des cellules d’insecte. Les méthodes de purification des protéines et les tampons ont été optimisés pour obtenir des ELF3 et LUX stables. Une chromatographie d'exclusion de taille a été réalisée pour vérifier les états oligomères des protéines. LUX étant la seule protéine de la CE à posséder un domaine de liaison à l'ADN connu, elle a été utilisée pour effectuer des tests de déplacement de la mobilité afin de comprendre l'affinité de liaison de LUX pour ses motifs ADN cibles obtenus à partir de tests de puces de liaison de protéines. D'après les tests de décalage de mobilité, il a été observé que le domaine de liaison à l'ADN (DBD) seul pouvait se lier aux motifs d'ADN de son substrat à des concentrations molaires nano alors qu'une quantité de protéine excédentaire de 10 fois était nécessaire pour que la longueur totale de LUX obtienne la même quantité de liaison. Des pistes de cristallisation à haut débit ont été réalisées pour LUX et LUX-DBD pleine longueur avec deux motifs de liaison différents. Aucun cristal n'a été obtenu pour le LUX complet, alors que des cristaux ont été obtenus pour le LUX DBD avec les deux motifs d'ADN. La structure de LUX DBD en complexe avec ses motifs cibles a été résolue par diffraction aux rayons X. À partir de la structure Crystal, il a été constaté que LUX DBD avait adopté une structure à 3 hélices, la seconde hélice étant responsable de la lecture de la base. Fait intéressant, il a été observé qu'un résidu d'arginine présent au niveau de l'hélice n-terminale servait de pince pour interagir avec le motif ADN cible.En utilisant les tests de mutagenèse dirigée et de décalage de mobilité, il a été confirmé que cette arginine présente à la position 146 de la protéine est essentielle pour déterminer l'affinité. Il a été constaté que l'affinité de liaison était réduite d'un facteur 5 lorsque cet acide aminé était modifié. En outre pour comprendre son effet in planta. Les lignes de lux mutantes ont été complétées par une longueur totale de type sauvage et une version substituée par Arg14Ala de la longueur totale de LUX. Grâce à ces expériences, nous avons pu montrer que la complémentation complète du mutant R146A n’était pas observée, alors que la version de type sauvage était capable de compléter complètement le phénotype du mutant / Ambient temperature plays a direct role in plant functioning and development. Increase in global ambient temperatures poses a significant challenge to wild and cultivated plant species. Most plant species adjust reproductive timing and development to optimize survival and fitness in higher ambient temperatures (Barnabás et al., 2008; Fitter and Fitter, 2002; Willis et al., 2008). These adaptations generally lead to elongated hypocotyls, fewer leaves at time of flowering, accelerated transition from vegetative to reproductive growth phases, fewer seeds, smaller seed pods and decreased leaf area. In the face of rapid climate change, specifically increased ambient growth permissive temperatures, tuning plant thermoresponse is urgently needed to engineer plants for adaptation to climate change and for securing future food production.PIF4 expression is controlled by evening complex (EC), a 3 protein complex comprising of ELF3, ELF4 and LUX, in a temperature dependent manner. The EC is able to repress PIF4 expression by binding to specific motifs at the promoter of PIF4 at lower ambient growth temperature. However this repression of PIF4 expression is removed at higher ambient growth temperature leading to premature ageing of plantsResultsAll three proteins of EC were produced using different expression strategy, ELF4 and LUX in E. coli while ELF3 in insect cells. Protein purification methods and buffers were optimized to obtain stable ELF3 ELF4 and LUX. Size exclusion chromatography was done to verify oligomeric states of the proteins. Since LUX is the only protein in the EC which has known DNA binding domain, it was used for doing mobility shift assays to understand the binding affinity of LUX for its target DNA Motifs which were obtained from protein binding microarrays assays. From the mobility shift assays, it was observed that the DNA binding domain (DBD) alone could bind to its substrate DNA motifs at Nano molar concentrations while 10 fold excesses amount of protein was required for the full length LUX to obtain same amount of binding. High throughput crystallization trails were carried out for full length LUX and LUX- DBD with two different binding motifs. No crystals were obtained for full length LUX while Crystals were obtained for LUX DBD with both the DNA Motifs. Structure for LUX DBD in complex with its target motifs were solved through X-Ray diffraction. Using site directed mutagenesis and Mobility shift assays it was confirmed that this Arginine present at the 146 position of the protein is critical for determining affinity. It was found that the binding affinity was reduced by a factor of 5 when this amino acid was changed. Further to understand its effect in planta.. With this experiments we were able to show that with the R146A mutant full complementation wasn’t observed while the wildtype version was able to completely complement the mutant phenotype.To understand temperature based dynamics of the complex, ELF3, which is the most intrinsically disordered protein of the three proteins that constitute the complex, was studied for structural variation through CD spectroscopy and DLS experiments. From these experiments it was found that ELF3 attains a β-sheet like confirmation at higher temperature while a more globular confirmation at lower temperatures. It was found that this activity of ELF is reversible allowing for flexibility of the whole complex. We found that there were prion like domains in ELF3 protein which were primarily responsible for transition to β-sheet structure at higher temperature.In Order to engineer plants that could survive at higher ambient temperature, we decided to mutate promoter elements of PIF4 through CRISPR/Cas9 to obtain plants that can survive higher temperature.

No description available.