Abstract In conservation biology, informed and sound management decisions depend on target specific information about the life history and demography of the endangered populations. I used long-term, individual-based life history data (breeding and capture-recapture data) to examine life-history and demography in order to assess population viability and management. My study systems consist of metapopulations of two endangered, long-distance migratory wader species breeding on Baltic coastal meadows, the Temminck’s stint (Calidris temminckii) and the Southern Dunlin (Calidris alpina schinzii), whose breeding habitats are mainly managed by livestock grazing. Demographic models indicated that both study populations were sinks, which persist through immigration due to low local recruitment insufficient to compensate for adult mortality or emigration. Philopatric Temminck’s stints had higher nest and apparent adult survival compared to immigrants. Consideration of dispersal status in population models resulted in the most realistic estimates of population growth and revealed a low demographic impact of immigrants. Immigrants had low return rates after reproductive failure, suggesting that the difference in apparent survival was partly caused by different breeding dispersal strategies. Thus, management actions improving reproduction, and thus site fidelity, should also influence viability. An artificial nest experiment indicated high trampling rates under recommended stocking rates on managed meadows. This was attributed to similar space use of nesting Dunlin and cattle. Cattle presence did not affect nest predation. Young Dunlin preferred intensively grazed low sward habitat over high sward habitat. Reproductive success was also higher in low sward habitat. However, nests in the preferred habitat had the highest risk of being trampled if not artificially protected. Thus, low sward habitat was found to work as an ecological trap if grazing was started too early in the breeding season. Due to the sink nature of the Dunlin population even small reductions in reproductive success caused by trampling were detrimental to long term viability. The results encourage continuation of the use of cattle grazing as a management tool, but also highlight the need for more detailed consideration of local grazing practices, especially when sketching management plans for endangered species. / Tiivistelmä Uhanalaisten lajien suojelussa tehokkaiden suojelutoimien suunnittelu edellyttää tietoa hoitokohteen elinkierron eri vaiheista ja niiden demografisesta merkityksestä. Käytän tutkimuksessani pitkäaikaisaineistoja (pesintä ja merkintä-takaisinpyynti) kuvatakseni tutkimuslajien demografiaa ja arvioidessani populaatioiden elinkykyä sekä hoitotoimia. Tutkimuskohteinani ovat lapinsirrin ja etelänsuosirrin, kahden uhanalaisen kahlaajan, Perämeren rantaniityillä pesivät metapopulaatiot. Näiden lajien elinympäristöjä hoidetaan pääosin laiduntamalla. Demografinen mallinnus osoitti molempien populaatioiden olevan nielupopulaatioita, joiden säilyminen on tulomuuton varassa sillä poikastuotto ei kompensoi aikuisten kuolleisuutta ja poismuuttoa. Tutkimusalueella syntyneiden ja sinne rekrytoituneiden lapinsirrien pesä- ja aikuissäilyvyydet olivat paremmat kuin tulomuuttajilla. Tämän huomioiminen populaatiomalleissa tuotti realistisimman kasvukertoimen arvion ja osoitti paikallisten rekryyttien olevan tulomuuttajia merkittävämpiä populaation kasvun kannalta. Pesinnässään onnistuneet tulomuuttajat palasivat seuraavina pesimäkausina epäonnistuneita todennäköisemmin. Paikallisilla rekryyteillä vastaavaa eroa ei havaittu. Ilmiö heijastellee tulomuuttajien muuttoalttiutta myöhemminkin ja voi kertoa erilaisista pesimädispersaalistrategioista. Lisääntymismenestyksen parantaminen voisi parantaa elinkykyä myös pienentämällä poismuuttoa. Tekopesäkokeen perusteella pesien tallausriski oli laidunnetuissa ympäristöissä korkea vaikka laidunnuspaine noudatteli hoitosuosituksia. Tämä johtunee ainakin osin laskennallista korkeammasta todellisesta laidunpaineesta, mikä puolestaan johtui lintujen ja karjan yhtäläisestä tilankäytöstä. Rekrytoituvat suosirrit suosivat voimakkaasti laidunnettuja matalakasvuisia niittyjä, joissa poikastuotto oli parempi kuin, laiduntamattomilla niityillä. Toisaalta, tallausriski on voimakkaasti laidunnetuilla niityillä suuri ja jo pienetkin pesätappiot uhkaavat suosirripopulaation elinkykyä. Täten laitumet voivat toimia ns. ekologisina loukkuina, jos laidunnus aloitetaan liian aikaisin suhteessa pesintään. Laidunnus on kuitenkin suositeltava hoitomuoto, sillä se näyttää tuottavan parhaita pesimäympäristöjä etelänsuosirrille. Tulosten mukaan laidunnuksen ajoitusta, laajuutta ja laidunnuspainetta on muokattava kohdekohtaisesti, jotta saavutetaan paras tulos uhanalaisten lajien suojelun kannalta.

Modification of land use/land cover in South Florida has posed a major challenge in the region’s eco-hydrology by shifting the surface-atmosphere water and energy balance. Although drainage and development in South Florida took place extensively between the mid- and late- 20th century, converting half of the original Everglades into agricultural and urban areas, urban expansion still accounts for a dominant mode of surface cover change in South Florida. Changes in surface cover directly affect the radiative, thermophysical and aerodynamic parameters which determine the absorption and partitioning of radiation into different components at the Earth surface. The alteration is responsible for changing the thermal structure of the surface and surface layer atmosphere, eventually modifying surface-induced convection. This dissertation is aimed at analyzing the extent and pattern of land cover change in South Florida and delineating the associated development of urban heat island (UHI), energy flux alteration, and convective rainfall modification using observed data, remotely sensed estimates, and modeled results. Urban land covers in South Florida are found to have increased by 10% from 1974 to 2011. Higher Landsat-derived land surface temperatures (LST) are observed in urban areas (LSTu-r =2.8°C) with satisfactory validation statistics for eastern stations (Nash-Sutcliffe coefficient =0.70 and R2 =0.79). Time series trends, significantly negative for diurnal temperature range (DTR= -1°C, p=0.005) and positive for lifting condensation level (LCL > 20m) reveal temporal and conspicuous urban-rural differences in nocturnal temperature (ΔTu-r = 4°C) shows spatial signatures of UHI. Spatially higher (urban: 3, forest: 0.14) and temporally increasing (urban: 1.67 to 3) Bowen’s ratios, and sensible heat fluxes exceeding net radiation in medium and high-intensity developed areas in 2010 reflect the effect of urbanization on surface energy balance. Radar reflectivity-derived surface-induced convective rainfall reveals significantly positive mean differences (thunderstorm cell density: 6/1000 km2and rain rate: 0.24 mm/hr/summer, p < 0.005) between urban and entire South Florida indicating convective enhancement by urban covers. The research fulfils its two-fold purposes: advancing the understanding of post-development hydrometeorology in South Florida and investigating the spatial and temporal impacts of land cover change on the microclimate of a subtropical city.

The use of recycled concrete aggregates (RCA) has gained increased attention in the past few decades as an alternative to decrease the carbon footprint of concrete construction. Yet, most of the research performed so far demonstrates that RCA concrete displays inferior performance in the fresh and hardened states when compared to conventional concrete (CC). The latter is believed due to the fact that very often the different microstructure of RCA is not accounted for while the mix-proportioning of RCA concrete. Recently, a number of mix-design procedures accounting for RCA microstructure have been proposed. Amongst them, the Equivalent Volume (EV) method seems to be quite promising. The EV method may proportion RCA concrete made of coarse (CRCA) or fine (FRCA) RCA and is based on a companion CC. Previous research has demonstrated that the fresh and hardened properties of EV mix-designed CRCA are suitable for structural applications. Yet, very few research, analysis and quantification have been conducted on the fresh behaviour of EV mix- proportioned FRCA concrete. This work presents a comprehensive study on the rheological behaviour of EV mix-designed CRCA and FRCA concrete presenting distinct features (i.e. inner qualities, mineralogy, fabrication process, etc.) through the use of a planetary rheometer (IBB). Results show that the EV is capable of proportioning low embodied energy CRCA and FRCA concrete with shear thinning profiles. The latter suggests that these mixtures are suitable for applications under high torque regimes such as vibrated or pumped concrete.

High speed in waves, necessary in for instance rescue or military operations, often result in severe loading on both the craft and the crew. To maximize the performance of the high-speed craft (HSC) system that the craft and crew constitute, balance between these loads is essential. There should be no overload or underuse of crew, craft or equipment. For small high-speed craft systems, man is often the weakest link. The human exposure to vibration and shock results in injuries and other adverse health effects, which increase the risks for non-safe operations and performance degradation of the crew and craft system. To achieve a system in balance, the human acceleration exposure must be considered early in ship design. It must also be considered in duty planning and in design and selection of vibration mitigation systems. The thesis presents a simulation-based method for prediction and evaluation of the acceleration exposure of the crew on small HSC. A numerical seat model, validated with experimental full-scale data, is used to determine the crew's acceleration exposure. The input to the model is the boat acceleration expressed in the time domain (simulated or measured), the total mass of the seated human, and seat specific parameters such as mass, spring stiffness and damping coefficients and the seat's longitudinal position in the craft. The model generates seat response time series that are evaluated using available methods for evaluation of whole-body vibration (ISO 2631-1 \&amp; ISO 2631-5) and statistical methods for calculation of extreme values. The presented simulation scheme enables evaluation of human exposure to vibration and shock at an early stage in the design process. It can also be used as a tool in duty planning, requirements specification or for design of appropriate vibration mitigation systems. Further studies is proposed within three areas: investigation of the actual operational profiles of HSC, further development of seat models and investigation of the prevailing injuries and health problems among the crew of HSC. / <p>QC 20150126</p>

This thesis explores the spherically symmetric gravitational collapse of a massless scalar field in a minimally modified gravity theory denoted VCDM (V replaces $\Lambda$ in the $\Lambda$CDM abbreviation), a class of theories propagating the same degrees of freedom as general relativity at the expense of broken 4D diffeomorphism invariance. Numerical evolution of the equations of motion reveals that for small initial scalar profile amplitudes, no black hole forms from the collapse. However, for larger amplitudes, collapse leads to an apparent horizon's formation in finite time. Outside the horizon, the solution resembles the Schwarzschild geometry, while inside, the lapse function continues to decrease toward zero, implying the formation of a singularity/foliation breakdown. This suggests a need for a UV completion for the theory inside the horizon. Despite this, VCDM can describe the entire time evolution of the universe outside the black hole horizon without requiring knowledge of such a UV completion. / Denna uppsats undersöker den sfäriskt symmetriska gravitationskollapsen av ett masslöst skalärfält inom en minimalt modifierad gravitationsteori betecknad VCDM (där V ersätter $\Lambda$ i $\Lambda$CDM-förkortningen), en klass av teorier som propagerar samma frihetsgrader som den allmäna relativitetsteorin på bekostnad av bruten fyrdimensionell diffeomorfiinvarians. Numerisk utveckling av rörelseekvationerna visar att ett svart hål inte kan bildas om begynnelseamplituden hos den initiala skalärfältsprofilen är liten. För större amplituder bildas en uppenbar horisont på en ändlig tid. Utanför horisonten sammanfaller lösningen med Schwarzschildgeometrin, medan inuti horisonten fortsätter lapsefunktionen att falla mot noll, vilket implicerar formationen av en singularitet/sönderfall av rumtidsfolieringen. Detta tyder på att teorin är i behov av en UV-komplettering innanför horisonten. Trots detta kan VCDM beskriva hela universums tidsutveckling utanför det svarta hålet utan vetskap om en sådan UV-komplettering.

No description available.

Knochen und Muskel sind die wichtigsten Gewebe im muskuloskelettalen System welche dem Körper die Bewegungen möglich machen. Beide Gewebetypen sind hochgradig strukturierter Extrazellulärmatrix zugrundegelegt, welche die mechanischen und biologischen Funktionen bestimmen. In dieser Studie wurden die räumliche Verteilung der anisotropen elastischen Eigenschaften und der Gewebemineralisation im humanen kortikalen Femur untersucht mit akustischer Mikroskopie und Synchrotron-µCT. Die homogenisierten elastischen Eigenschaften wurden aus einer Kombination der Porosität und der Gewebeelastizitätsmatrix mit Hilfe eines asymptotischen Homogenisierungsmodells ermittelt. Der Einfluss der Gewebemineralisierung und der Strukturparameter auf die mikroskopischen und mesoskopischen elastischen Koeffizienten wurde unter Berücksichtigung der anatomischen Position des Femurschaftes untersucht. Es wurde ein Modell entwickelt, mit welchem der intramuskuläre Fettgehalt des porcinen musculus longissimus nichtinvasiv mittels quantitativem Ultraschall und dessen spektraler Analyze des Echosignals bestimmt werden kann. Muskelspezifische Parameter wie Dämpfung, spectral slope, midband fit, apparent integrated backscatter und cepstrale Paramter wurden aus den RF-Signalen extrahiert. Die Einflüsse der Muskelkomposition und Strukturparameter auf die spektralen Ultraschallparameter wurden untersucht. Die akustischer Parameter werden durch die Muskelfaserorientierung beeinflusst und weisen höhere Werte parallel zur Faserlängsrichtung als senkrecht zur Faserorientierung auf. Die in dieser Studie gewonnenen detaillierten und lokal bestimmten Knochendaten können möglicherweise als Eingabeparameter für numerische 3D FE-Simulationen. Darüber hinaus kann die Untersuchung von Veränderungen der lokalen Gewebeanisotropie neue Einsichten in Studien über Knochenumbildung geben. Diese auf Gewebeebene bestimmten Daten von Muskelgewebe können in numerischen Simulationen von akustischer Rückstreuung genutzt werden um diagnostische Methoden und Geräte zu verbessern. / Bone and muscle are the most important tissues in the musculoskeletal system that gives the ability to move the body. Both tissues have the highly oriented underlying extracellular matrix structure for performing mechanical and biological functions. In this study, the spatial distribution of anisotropic elastic properties and tissue mineralization within a human femoral cortical bone shaft were investigated using scanning acoustic microscopy and synchrotron radiation µCT. The homogenized meoscopic elastic properties were determined by a combination of porosity and tissue elastic matrix using a asymptotic homogenization model. The impact on tissue mineralization and structural parameters of the microscopic and mesocopic elastic coefficients was analyzed with respect to the anatomical location of the femoral shaft. A model was developed to estimate intramuscular fat of porcine musculus longissimus non-invasively using a quantitative ultrasonic device by spectral analysis of ultrasonic echo signals. Muscle specific acoustic parameters, i.e. attenuation, spectral slope, midband fit, apparent integrated backscatter, and cepstral parameters were extracted from the measured RF echoes. The impact of muscle composition and structural properties on ultrasonic spectral parameters was analyzed. The ultrasound propagating parameters were affected by the muscle fiber orientation. The most dominant direction dependency was found for the attenuation. The detailed locally assessed bone data in this study may serve as a real-life input for numerical 3D FE simulation models. Moreover, the assessment of changes of local tissue anisotropy may provide new insights into the bone remodelling studies. The data provided at tissue level and investigated ultrasound backscattering from muscle tissue, can be used in numerical simulation FE models for acoustical backscattering from muscle for the further improvement of diagnostic methods and equipment.

No description available.

Made available in DSpace on 2014-12-17T15:42:26Z (GMT). No. of bitstreams: 1 ZilvamMS_TESE_reduzido.pdf: 9108618 bytes, checksum: 613dad3fd1a359dce84e2af73b067934 (MD5) Previous issue date: 2013-02-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Surfactant-polymer interactions are widely used when required rheological properties for specific applications, such as the production of fluids for oil exploration. Studies of the interactions of chitosan with cationic surfactants has attracted attention by being able to cause changes in rheological parameters of the systems making room for new applications. The commercial chitosan represents an interesting alternative to these systems, since it is obtained from partial deacetylation of chitin: the residues sites acetylated can then be used for the polymer-surfactant interactions. Alkyl ethoxylated surfactants can be used in this system, since these non-ionic surfactants can interact with hydrophobic sites of chitosan, modifying the rheology of solutions or emulsions resultants, which depends on the relaxation phenomenon occurring in these systems. In this work, first, inverse emulsions were prepared from chitosan solution as the dispersed phase and cyclohexane as the continuous phase were, using CTAB as a surfactant. The rheological analysis of these emulsions showed pronounced pseudoplastic behavior. This behavior was attributed to interaction of "loops" of chitosan chains. Creep tests were also performed and gave further support to these discussions. Subsequently, in order to obtain more information about the interaction of chitosan with non-ionic surfactants, solutions of chitosan were mixed with C12E8 and and carried out rheological analysis and dynamic light scattering. The systems showed marked pseudoplastic behavior, which became less evident when the concentration of surfactant was increased. Arrhenius and KWW equations were used to obtain parameters of the apparent activation energy and relaxation rate distribution, respectively, to which were connected to the content of surfactant and temperature used in this work / As intera??es tensoativo-pol?mero s?o amplamente usadas quando s?o necess?rias propriedades reol?gicas para aplica??es espec?ficas, como a produ??o de fluidos para explora??o do petr?leo. Estudos das intera??es de quitosana com tensoativos cati?nicos tem chamado aten??o por serem capazes de causar mudan?as nos par?metros reol?gicos dos sistemas abrindo espa?o para novas aplica??es. A quitosana comercial representa uma alternativa interessante para estes sistemas, uma vez que ela ? obtida a partir da desacetila??o parcial da quitina: os s?tos acetilados residuais podem, ent?o, ser usados para as intera??es pol?mero-tensoativo. Tensoativos alquil etoxilados podem ser utilizados neste sistema, pois estes tensoativos n?o i?nicos podem interagir com s?tios hidrof?bicos da quitosana, modificando a reologia de solu??es ou emuls?es resultantes, os quais dependem do fen?meno de relaxa??o ocorrendo nestes sistemas. Neste trabalho, primeiramente, foram preparadas emuls?es inversas de solu??o de quitosana como fase dispersa e cicloexano como fase cont?nua usando CTAB como tensoativo. A an?lise reol?gica destas emuls?es mostrou pronunciado comportamento pseudopl?stico. Esta pseudoplasticidade foi atribu?da ? intera??o por la?os loops de cadeias de quitosana. Ensaios de flu?ncia tamb?m foram executados e deram maior suporte a estas discuss?es. Em seguida, a fim de se obter maiores informa??es sobre as intera??es da quitosana com tensoativos n?o i?nicos, solu??es de quitosana foram misturadas com C12E8 e levadas ?s an?lises reol?gica e de espalhamento din?mico de luz. Os sistemas tiveram elevado comportamento pseudopl?stico, o qual se tornava menos evidente, quando o teor de tensoativo foi aumentado. Equa??es de Arrhenius e de KWW foram usadas para obter par?metros de energia de ativa??o aparente e de distribui??o da taxa de relaxa??o, respectivamente, aos quais foram relacionados em fun??o do teor de tensoativo e da temperatura, usados neste trabalho

Die Pektat-Lyasen gehören zu einer Proteinfamilie, die meistens von pflanzenpathogenen Mikroorganismen sekretiert werden. Die Enzyme katalysieren den Abbau von Polygalakturonsäure, einem Hauptbestandteil in <br /> pflanzlichen Mittellamellen und Primärzellwänden. Der Abbau der alpha-1,4-verbrückten Galakturonsäurereste erfogt durch eine beta-Eliminierungsreaktion, dabei entsteht ein Produkt mit einer ungesättigten C4-C5 Bindung am nicht reduzierenden Ende, das durch spektroskopische Messungen beobachtet werden kann. Für die enzymatische Reaktion der Pektat-Lyasen ist Calcium nötig und das pH-Optimum der Reaktion liegt bei pH 8.5. Alle bis jetzt bekannten Strukturen der Pektat- und Pektin-Lyasen haben das gleiche Strukturmotiv - eine rechtsgängige parallele beta-Helix. Die Struktur der Pektat-Lyase aus Bacillus subtilis (BsPel) ist im Komplex mit Calcium gelöst worden. BsPel ist ein monomeres Protein mit einer ungefähren Molekularmasse von 43 kDa, das keine Disulfidbrücken enthält. Dies erlaubte sowohl eine effiziente rekombinante Expression des Wildtypproteins, als auch von destabilisierten Mutanten im Cytoplasma von E. coli. Parallele beta-Helices sind relativ große, jedoch verhältnismäßig einfach aufgebaute Proteine. Um detailliertere Informationen über die kritischen Schritte bei der in vitro-Faltung von parallelen beta-Helices zu erhalten, sollte in der vorliegenden Arbeit versucht werden, den Faltungsmechanismus dieses Proteins näher zu charakterisieren. Dabei sollte vor allem die Frage geklärt werden, welche Wechselwirkungen für die Stabilität dieses Proteins einerseits und für die Stabilität von essentiellen Faltungsintermediaten andererseits besonders wichtig sind.<BR><br>Rückfaltung von BsPel, ausgehend vom guanidiniumchlorid-denaturierten Zustand, war bei kleinen Proteinkonzentrationen und niedrigen Temperaturen vollständig möglich. GdmCl-induzierte Faltungsübergänge waren aber nicht reversibel und zeigten eine apparente Hysterese. Kinetische Messungen des Fluoreszenz- und CD-Signals im fernen UV ergaben eine extreme Denaturierungsmittelabhängigkeit der Rückfaltungsrate im Bereich des Übergangmittelpunktes. Der extreme Abfall der Rückfaltungsraten mit steigender Denaturierungsmittelkonzentration kann als kooperative <br /> Entfaltung eines essentiellen Faltungsintermediats verstanden werden. Dieses Faltungsintermediat ist temperaturlabil und kann durch den Zusatz Glycerin im Renaturierungspuffer stabilisiert werden, wobei sich die Hysterese verringert, jedoch nicht vollständig aufgehoben wird. Durch reverse Doppelsprungexperimente konnten zwei transiente Faltungsintermediate nachgewiesen werden, die auf zwei parallelen Faltungswegen liegen und beide zum nativen Zustand weiterreagieren können. Fluoreszenzemissionsspektren der beiden Intermediate zeigten, daß beide schon nativähnliche Struktur aufweisen. Kinetische Daten von Prolin-Doppelsprungexperimenten zeigten, daß Prolinisomerisierung den geschwindigkeitsbestimmenden Schritt in der Reaktivierung des denaturierten Enzyms darstellt. Desweiteren konnte durch Prolin-Doppelsprungexperimenten an Mutanten mit Substitutionen im Prolinrest 281 gezeigt werden, daß die langsame Renaturierung von BsPel nicht durch die Isomerisierung der einzigen cis-Peptidbindung an Prolin 281 verursacht wird, sondern durch die Isomerisierung mehrerer trans-Proline. Die beiden beobachteten transienten Faltungsintermediate sind somit wahrscheinlich zwei Populationen von Faltungsintermediaten mit nicht-nativen X-Pro-Peptidbindungen, wobei sich die Populationen durch mindestens eine nicht-native X-Pro-Peptidbindung unterscheiden.<BR><br>Der Austausch des Prolinrestes 281 gegen verschiedene Aminosäuren (Ala, Ile, Leu, Phe, Gly) führte zu einer starken Destabilisierung des nativen Proteins und daneben auch zu einer Reduktion in der Aktivität, da die Mutationsstelle in der Nähe der putativen Substratbindetasche liegt. Die Rückfaltungskinetiken der Prolinmutanten war bei 10&#176;C annähernd gleich zum Wildtyp und die geschwindigkeitsbestimmenden Schritte der Faltung waren durch die Mutation nicht verändert. Die durch die Mutation verursachte drastische Destabilisierung des nativen Zustands führte zu einem reversiblen Entfaltungsgleichgewicht bei pH 7 und 10&#176;C. GdmCl-induzierte Faltungsübergänge der Mutante P281A zeigten bei Messungen der Tryptophanfluoreszenzemission und der Aktivität einen kooperativen Phasenübergang mit einem Übergangsmittelpunkt bei 1.1 M GdmCl. Durch die Übereinstimmung der Faltungsübergänge bei beiden Messparametern konnten die Faltungsübergänge nach dem Zwei-Zustandsmodell ausgewertet werden. Dabei wurde eine freie Sabilisierungsenthalpie der Faltung für die Mutante von <nobr>-&nbsp;64.2&nbsp;&#177;&nbsp;0.4&nbsp;kJ/mol</nobr> und eine Kooperativität des Übergangs <br /> von <nobr>-&nbsp;58.2&nbsp;&#177;&nbsp;0.3&nbsp;kJ/(mol&#183;M)</nobr> bestimmt.<BR> <br /> <br>BsPel enthält, wie die meisten monomeren rechtsgängigen parallelen beta-Helix-Proteine, einen internen Stapel wasserstoffverbrückter Asparagin-Seitenketten. Die Mehrheit der erzeugten Mutanten mit Substitutionen im Zentrum der Asn-Leiter (N271X) waren als enzymatisch aktives Protein zugänglich. Die Auswirkung der Mutation auf die Stabilität und Rückfaltung wurde an den Proteinen BsPel-N271T und BsPel-N271A näher analysiert. Dabei führte die Unterbrechung des Asparaginstapels im Inneren der beta-Helix zu keiner drastischen Destabilisierung des nativen Proteins. Allerdings führten diese Mutationen zu einem temperatur-sensitiven Faltungsphänotyp und die Hysterese im Denaturierungsübergang wurde verstärkt. Offenbar wird durch die Unterbrechung des Asparaginstapel ein essentielles, thermolabiles Faltungsintermediat destabilisiert. Der Asparaginstapel wird somit bei der Faltung sehr früh ausgebildet und ist wahrscheinlich schon im Übergangszustand vorhanden. / Pectate lyases belong to a family of proteins secreted by plant pathogenic microbes. The enzymes cleave alpha-1,4 linked galacturonic acid by a beta-elimination that results in an unsaturated product, which can be quantified spectrophotometrically. Calcium is essential for the activity and the pH-optimum is near 8.5. All known structures of pectate and pectin lyases have the same structural motif - a right handed parallel beta-helix. The structure of pectate lyase from Bacillus subtilis (BsPel) has been solved in complex with calcium. It is a monomeric protein, with a molecular mass of about 43 kDa and without disulfide bonds. This allows its high-yield recombinant expression in the cytoplasm of Escherichia coli. Parallel beta-helices are relative large proteins, however with a simple folding topology. The objective of this work was to characterize the folding mechanism of BsPel. In particular we investigated the role of the interactions of certain residues in the parallel beta-helix for the stability of the native protein and the stability of essential folding intermediates.<br /> <br /> Refolding of BsPel was possible at low protein concentrations and low temperature. However, denaturation of BsPel was not freely reversible. De- and renaturation curves showed a large apparent hysteresis. Furthermore, the folding rate constant deduced from fluorescence and circulardichroism measurements showed a very strong dependence on denaturant concentrations near the midpoint of the renaturation transition. This can be explained with a cooperative unfolding of an essential folding intermediate. Upon stabilisation of the temperature-sensitive intermediate by addition of glycerol in the renaturation buffer, the hysteresis is reduced, but does not disappear. Reverse double mixing kinetic experiments have shown that two transient folding intermediates are on the folding pathway. These intermediates are on parallel pathways and both can fold to the native state. Fluorescence emission spectra have shown the native-like structure of both intermediates. Furthermore, data from proline double mixing kinetic experiments revealed that isomerization of peptidyl-prolyl bonds was responsible for the slow kinetics in the reactivation of the enzyme. However, the isomerization of the single cis-peptidyl-prolyl bond at Pro281 was not responsible for the slowest folding phase observed, but rather the isomerization of other trans-peptidyl-prolyl bonds. Thus, both transient folding intermediates observed probably represent two populations of folding intermediates with non-native X-Pro-peptide bonds. The difference of the two populations is at least one non-native X-Pro-peptide bond.<br /> <br /> Mutations of the proline 281 against various residues (Ala, Ile, Leu, Phe, Gly) resulted in a strong destabilization of the native protein. Also, the activity of the mutant proteins was strong reduced due to the position of the mutation site near the putative active center of the protein. At 10&#176;C the kinetic folding behavior of the proline mutants was not significant changed. However, the strong destabilization of the native state in the proline mutants resulted in a reversible folding equilibrium at pH 7 and 10&#176;C. The unfolding of the P281A mutant was reversible as determined by fluorescence emission and enzyme activity measurements. The coincidence of these detected transitions is consistent with a two-state equilibrium transition. At pH 7 and 10&#176;C the delta G&#176;(H₂O) for folding of P281A was <nobr>-&nbsp;64.2&nbsp;&#177;&nbsp;0.4&nbsp;kJ/mol,</nobr> with a midpoint of the transition at 1.1 M GdmCl and a cooperativity of <nobr>-&nbsp;58.2&nbsp;&#177;&nbsp;0.3&nbsp;kJ/(mol&#183;M).</nobr><br /> <br /> BsPel has an asparagine ladder in turn 2 of the parallel beta-helix with extensive network of side-chain hydrogen bonds between the Asn residues. Such an Asn-ladder is a conserved feature of many monomeric beta-helices crystallized so far. The middle Asn residue (271) was selected and exchanged for various residues. Most of the mutants were expressed at 25&#176;C as soluble and active proteins but with a significant reduction in yield. Mutants N271T and N271A were selected to study the stability and refolding of these proteins in comparison with the wild-type protein. The substitution in the Asn-ladder did not drastically destabilize the native protein, but caused a temperature-sensitive-folding (tsf) phenotype with an increased hysteresis in the de- and renaturation transition curves. In addition, the disruption of the Asn-ladder resulted in destabilization of an essential, thermosensitive folding intermediate. Thus, the Asn-ladder is formed very early during the folding, probably well before the transition state of folding.