RdoA is a eukaryotic-like serine/threonine protein kinase found in Salmonella typhimurium. It is a downstream effector of the Cpx stress response pathway and has been phenotypically characterized to have a functional role in flagellin phase variation and long-term bacterial survivability. Structurally, RdoA is homologous to, choline kinase and aminoglycoside (3’) phosphotransferase IIIa (APH[3’]IIIa). These kinases all belong to a protein kinase superfamily and share highly conserved residues/motifs in their catalytic domain. In RdoA seven of these conserved amino acids were proposed to have functional roles in the phosphotransfer mechanism. Mutation of these proposed catalytic domain residues resulted in a loss of in vitro kinase activity and in vivo RdoA function for a majority of the mutants. Four of the mutants also exhibited decreased levels of stable RdoA compared to wildtype. Many protein kinases regulate activity through phosphorylation of an activation loop. Although RdoA does not contain a canonical activation loop, its carboxyl terminus is proposed to play a similar regulatory function. Mutations of a putative autophosphorylation target in the carboxyl terminus resulted in loss of in vitro kinase activity. Truncations of this region also resulted in loss of kinase activity, as well as decreasing RdoA stability. The length of the carboxyl terminus in the kinase was shown to be an important determinant in the overall structural stability of RdoA. Mutational analyses of conserved amino acid residues surrounding the putative substrate-binding cleft of RdoA revealed site specific mutants with diminished in vitro phosphorylation activity and/or RdoA levels. A subset of these mutants for which no in vitro kinase activity was detected were still able to complement RdoA function in vivo. Taken together these results indicate that this region of the protein is important for RdoA function. In summary, this work has generated a panel of RdoA mutants with several unique phenotypes that will facilitate characterization of RdoA function and of regions of the protein / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-29 21:35:42.815

Bakgrund: Det finns flertalet modeller av pulsklockor på marknaden med funktioner som skall kunna uppskatta individens maximala syreupptagningsförmåga. En av dessa modeller är Polar Vantage V, som enligt tillverkaren kan uppskatta individens VO2max genom funktionen”konditionstest”. Testet utförs genom ett fem minuters vilotest. Testvärdet beräknas sedan från hjärtfrekvens samt personliga faktorer. Syfte: Studien jämförde Polar Vantage V uppskattade testvärde med en direkt mätning av VO2max, med frågeställningen om detta test var pålitligt och användbart i praktiken. Metod: En experimentell studiedesign användes som inkluderade 11 män samt 5 kvinnor som deltagare (N=16). Alla deltagare utförde Polar Vantage V’s konditionstest i vila, för att sedan utföra en maximal prestation utfört på löpband. För mätning av VO2max användes ergospirometri-systemet Jaeger®Vyntus CPX med halvmask. Resultat:Kendall Tau-b test visade signifikant starkt positivt samband mellan Vantage V’s testvärde och VO2max från Vyntus CPX. Wilcoxon Signed Rank test visade ingen signifikant skillnad mellan testvärdet (grupp medelvärde) från Vantage V och Vyntus CPX. Slutsats: På grund av stora individuella skillnader i testvärdet mätt med ”Konditionstestet” från Vantage V, bör individen tolka resultatet med aktsamhet. Vantage V’s ”Konditionstest” verkar emellertid kunna ge en bra uppskattning av VO2max på gruppnivå för friska vuxna i 20-36 års ålder. / Background: There are several models of fitness devices on the market with functions that claims to have the capability to estimate the capacity of a individuals maximum oxygen uptake. One of these models is the Polar Vantage V fitness watch, which according to the manufacturer can estimate the VO2max through the function "Fitness test". The test is performed through a five-minute rest test. The test result is then calculated thru various variables such as the individuals resting heart rate and personal factors. Purpose: To assess the estimated test value of the Polar Vantage V “Fitness test” with a direct measurement of VO2max, with the research question whether this test was valid and useful in practice. Method: This study used an experimental design and included 11 men and 5 women as participants (N = 16). All participantsfirst performed the Polar Vantage V's “Fitness test”, then proceeded to perform a maximum performance using a treadmill. For measurement of VO2max, the cardiopulmonary exercisetesting system Jaeger®Vyntus CPX with half mask was used. Results: Kendall Tau-b test presented a significant strong positive relationship between Vantage V’s “Fitness test” and VO2max -value from the Vyntus CPX. The Wilcoxon Signed Rank test presented a nonsignificant difference between the test value (group mean value) from Vantage V “Fitness test”and VO2max from Vyntus CPX. Conclusion: Due to large individual differences in the test value measured by the "Fitness test" from Vantage V, one should interpret the test-result with care. However, the Vantage V "Fitness test" seems to be able to provide a good estimate of theVO2max at group level in healthy adults aged between 20-36.

In this work we present the first characterization of a Cu2+-transporting ATPase. The thermophilic bacteria Archaeoglobus fulgidus contains two genes, CopA and CopB, encoding for CPx-ATPases. CopB belongs to the subgroup IB-4 of the CPX-ATPases. These enzymes are characterized by a CPH motif in the 6th transmembrane domain and a His-rich N-terminus metal binding domain (MBD). CopB was heterologously expressed in E. coli. Membranes were prepared and used to measure activity. CopB was active at high temperature (75º C), high ionic strength and pH 5.7. The enzyme was activated by Cu2+, and in to a lesser extent by Ag+ and Cu+. CopB showed a Vmax = 5 µmol/mg/h and a high apparent affinity (K1/2 = 0.28 ± 0.09 μM) for Cu2+. Uptake of 64Cu2+ into everted vesicles was also measured in order to show that Cu2+ is not only activating the enzyme but being transported. Compared with CopB-WT, CopB-T (lacking the N-terminus MBD) did not show any difference in its activation by the different metal ions, demonstrating that the cytoplasmic MBD has no role in the metal selectivity. CopB-T also showed a 40 % decrease in the ATPase activity. CopB-WT and CopB-T presented similar levels of phosphorylation. However, CopB-T exhibited a reduced rate of dephosphorylation (slower transition from the E2P to the E2 conformation). These observations suggest a regulatory role for the cytoplasmic MBD.

The thesis deals with verifying the acoustic properties of road markings in real conditions the vehicle travels. It contains basic information about the acoustics of the sources of traffic noise and methods of measuring noise. Below are the main information about the horizontal signposts and its impact on safety. The practical part contains descriptions of results, estimation of measurement and subsequently implemented economic assessment.

No description available.

Noise is a growing health concern as urban residents increases rapidly and more reports of noise causing sleep disturbances and increasing the risk of cardiovascular health problems are published. Noise has a negative influence on life quality. This life quality deficiency also shows in housing and office pricing in noisy environments. Housing and office prices are often higher in quiet areas than in noisy areas. Therefore noise is both a health issue and has big economic consequences. The biggest contributor to the road traffic noise is the tire/road noise at speeds above 50km/h. Therefore this is an important aspect to monitor and the problem has to be alleviated. In this thesis the client Ramböll has gotten many contracts concerning the state of the roads from governmental institutes. As a part of the evaluation of the roads they want to implement the noise emission as a factor. This gives the government institutions another parameter that helps in the decision of which part of the road network to refurbish first. In the effort to decide what is best way for Ramböll to measure noise a new setup for measuring noise has been developed. The most used ways of measuring tire/road-noise is the close proximity (CPX) -method and the statistical pass-by (SPB) -method. These methods both give accurate results but they have drawbacks. The SPB measurements are time consuming and only give noise levels for a small patch of a road. CPX measurement on the other hand require costly and time consuming development of a measurement trailer. Certifying the trailer and maintenance work of it is expensive. So this report shows a first step in how to build a measurement setup and what aspects were taken into consideration when it was designed. A close proximity measurement setup in the form of a tube with a microphone placed inside it was built and installed underneath the measurement vehicle. The  measurement setup designed and built was named the Tube-CPX measurement setup in this report. The measurements performed with this Tube-CPX measurement setup show promising results. Similarities between CPX measurement setup and the Tube-CPX setup have been found both in the frequency spectrum as well as in the relation with pass-by measurement noise levels. The repeatability of the Tube-CPX measurements is even better than the compared CPX measurements. Although results are promising more work is required before the Tube-CPX setup can be seen in operation at Ramböll. In particular the question which source causes which sound pressure is required to understand the measured levels. And also more controlled pass-by measurements have to be performed to determine the relation of the absolute values measured at the tire/road impact spot to the values that are most interesting, namely the sound pressure levels that the human at the side of the road depicts.

No description available.

This Thesis concerns the development of fullerene-like (FL) carbon nitride (CNx) thin films and the discovery of phosphorus-carbide (CPx) compounds. The work dedicated to CPx include first-principles theoretical simulations of the growth and properties of FL-CPx structures. I have employed DC magnetron sputtering methods to synthesize both CNx and CPx thin films. The deposition conditions for CPx films were chosen on the basis of the theoretical results as well as from the experience from the deposition of FL-CNx thin films. The characterization of the CPx films is divided into three main directions: structural characterization by transmission electron microscopyand scanning electron microscopy, analysis of the amount of elements and chemical bonds presentin the structure by X-ray photoelectron spectroscopy and Auger spectroscopy, and mechanicalproperty analysis by nanoindentation. The CPx films exhibit a short range orderedstructure with FL characteristics for substrate temperature of 300 °C and for a phosphorus content of 10-15 at.%, which isconsistent with the theoretical findings. These films also displayed the best mechanical properties in terms of hardness and resiliency, which are better than those of the corresponding FL-CNx films. For the FL-CNx thin film material, I find that the surface water adsorption is lower compared to commercial computer hard disk top coatings. Following that line the dangling bonds in FL-CNx coatings have been investigated  by electron spin resonance (ESR). The absence of ESR signal for FL-CNx indicates very low density of dangling bonds in the material, which explains the low water adsorption tendency. The potential for using highly elastic FL-CNx coatings in an automotive valve-train environment has also been investigated. CNx coatings of different nitrogen content were investigated using microscopy, wear testing, nanoindentation testing, and in an experimental cam-tappet testing rig. The FL-CNx coating with the higher value of hardness/elastic modulus showed greater durability in cam-tappet wear testing.

All cells maintain essential metal nutrients at optimal levels by metal homeostasis. P-type ATPases, a crucial superfamily of integral membrane proteins, are involved in the active transport of metal ions across biological membranes driven by the motive force of ATP- hydrolysis. The PIB-type ATPase subfamily, also called CPx-ATPases, fulfills a key role in heavy metal homoeostasis among the most widespread species from bacteria to human. In humans, the defect in copper transporters is the direct cause of severe neurological and hepatic disorders such as Wilson and Menkes diseases, therefore, understanding the molecular function of these pumps is of paramount importance in human health. Cu+-ATPases have two transmembrane metal binding sites (TM-MBS) and three cytosolic domains, namely the actuator (A-domain) and phosphorylation and nucleotide-binding domain (PN), and regulatory N-terminal heavy metal binding domain (HMBD). Here, we have studied the Legionella pneumophila CopA (LpCopA) and its isolated cytosolic domains to improve our understanding of the functional interaction of the protein domains during metal transport relate this to the known structure of this ATPase. To elucidate how cytosolic ligands (Cu+ and nucleotide) stimulate the interactions among the cytosolic domains and may transmit conformational changes to the TM-MBS, the interactions among recombinant isolated cytosolic domains were first examined biochemically by co-purification and spectroscopically by circular dichroism, time-resolved fluorescence and site-directed fluorescent labeling assays. The Cu+-dependent interaction between the A-domain and HMBD has been postulated as a mechanism for activating the ATPase cycle. This question was addressed here by studying copper-dependent interactions between the isolated expressed domains. Spectroscopic evidence is provided that an HMBD-A complex is formed in the presence of Cu+ which binds with 100-200 nM affinity to the recombinant HMBD. In contrast, the A-domain interacts with the PN domain in a nucleotide-dependent fashion. This molecular recognition is required for the dephosphorylation step in the catalytic cycle. The interaction was investigated in more detail by the use of a decameric peptide derived from the PN-binding interface of the A-domain and carrying the conserved TGE-motif involved in dephosphorylation. Its binding to the isolated PN domain in a weakly nucleotide-dependent manner, is demonstrated here by stopped-flow fluorescence spectroscopy. Several ATPase assays were modified to assess the functionality of the PN-domain and full length LpCopA. The peptide was found to reduce the catalytic turnover of full length LpCopA. This agrees with the expected slowing down of the reformation of the PN-A-domain interaction since the peptide occupies their binding interface. Thus, the synthetic peptide provides a means to study specifically the influence of PN-A-domain interactions on the structure and function of LpCopA. This was done by time-correlated single photon counting (TCSPC) method. The time-dependent Stokes shift of the environmentally sensitive fluorophore BADAN which was covalently attached to the conserved CPC-motif in the TM-MBS was measured. The data indicate that the interior of the ATPase is hydrated and the mobility of the intra-protein water varies from high to low at C382 at the “luminal side” and C384 at the “cytosolic side” of the TM-MBS, respectively. This finding is consistent with the recent MD simulation of LpCopA, bringing the first experimental evidence on a luminal-open conformation of E2~P state. The A-domain-derived decapeptide, although binding to the cytosolic head piece, induces structural changes also at the TM-MBS. The peptide-stabilized state (with a disrupted PN-A interface) renders the C384 environment more hydrophobic as evidenced by TCSPC. Taken together, the data from cytosolic domain interactions, ATPase assays and of time-dependent Stoke shift analyses of BADAN-labeled LpCopA reveal the presence of hydrated intramembraneous sites whose degree of hydration is regulated by the rearrangement of cytosolic domains, particularly during the association and dissociation of the PN-A domains. Copper affects this arrangement by inducing the linkage of the A-domain to the HMBD. The latter appears to play not only an autoinhibitory but also a chaperone-like role in transferring Cu+ to the TM-MBS during catalytic turnover.

The task of the master's thesis is introduction with the problems of traffic noise, focusing on noise from road traffic. There is a description what is the noise, how it is formed, its resources and what are the methods of measuring. The following are the various noise reduction measures such as noise barriers and low noise pavements. The main attention is devoted to the noise generated at the tire / road that is reduced by these pavements. The practical part describes the implemented measurements on individual sections at various locations in our country. The measurement was carried by slightly modified method CPX with reference tire directly at the vehicle. The main aim was to evaluate the measurement data, make comparison of different low-noise surfaces with each other and with the commonly used surfaces. Then quantification of the rate reduction of the noise emission for a given section and verification of input data for noise modeling. In conclusion, the obtained results are summarized and based on them were confirmed very good acoustic properties with the recommendation to continue with measurements in the future.