Standard hydrothermal and gas-buffering techniques have been used to synthesize and investigate phase relations of amphiboles on the join Mg₇Si₈O₂₂(OH)₂-Fe₇Si₈O₂₂(OH)₂. Synthesized amphiboles from Mg₆Fe₁Si₈O₂₂(OH)₂ to Mg₁Fe₆Si₈O₂₂(OH)₂ are optically orthorhombic. Variation of unit cell parameters with composition suggests that they are members of a single, continuous solid solution. (a(Å) = 18.577(12) + 0.001284(190)X_Fe, b(Å) = 17.942(11) + 0.004862(170)X_Fe, c(Å) = 5.285(3) + 0.000617(50)X_Fe, V(ų) = 1760.8(1.7) + 0.8226(280)X_Fe ; X_Fe = mole % Fe end-member) Electron diffraction patterns of composition Mg₅Fe₂ are consistent with that of an orthorhombic amphibole with an a unit cell repeat of ~18.6 Å, but are unlike any known amphibole structure type. Even though the structure type is unknown, measured shifts in peak locations on the powder X-ray diffraction patterns allow compositions to be measured to within ±3 mole % Fe end-member. Combining these results with those of Forbes (1971) and Greenwood (1963), it is now clear that the entire range of amphiboles across the join can be synthesized. No change in unit cell parameters as a function of f_O₂ was observed, and thus there is no evidence for solution of oxy-amphibole component. At 2 kbar and f_O₂ defined by the MH buffer the maximum extent of 2 solution of Fe end-member in amphibole is 12 and 22 mole% at 725° and 630°C respectively; amphibole is unstable below 630°C, being replaced by the assemblage + talc + quartz + magnetite + hematite. At f_O₂ defined by the NNO buffer the extent of solid solution expands to 54, 62, and 65 mole % Fe end-member at 725°, 625°, and 600°C, respectively. Results obtained in this study have been combined with previously published data to produce a T-X section of the upper thermal stability of amphibole at 2 kbar and f_O₂ defined by the FMQ buffer. Temperatures for 2 the reaction: amphibole -> pyroxene + quartz + vapor decrease from ~765°C for the pure Mg end-member to ~710°C for 62 mole % Fe end-member. The breakdown reaction: amphibole -> olivine + quartz + vapor, was observed for the more iron-rich amphiboles, and takes place at ~675°C for amphibole of 73 mole % Fe end-member. Reversed tie lines have been determined between Fe-Mg amphiboles and pyrrhotites in the presence of excess magnetite and quartz at 2 kbar, and 650°, 675°, 700°, and 725°C. This assemblage represents the simultaneous equilibria: amphibole + O₂ -> magnetite + quartz + H₂O (1) amphibole + S₂ -> pyrrhotite + quartz + H₂O + O₂. (2) At 700°C, amphiboles of 29, 41, 49, and 57 mole % Fe end-member coexist with pyrrhotites of N = 0.928, 0.934, 0.943, and 0.950, respectively. Compositions of coexisting amphibole-pyrrhotite pairs apparently are not seriously affected by temperature over the range investigated although scatter of the amphibole data does not allow a rigorous analysis. Sulfur fugacity for runs was determined from pyrrhotite compositions while f_O₂ was known from an experimentally determined magnetite-pyrrhotite curve. Knowledge of these two fugacities allowed calculation of fugacities of all species, including H2o, assuming an H-0-S vapor, and thus reactions (1) and (2) were located in terms of f_O₂and f_S₂. Several models for the amphibole solid solution were used to explain the variation in composition of coexisting amphibole-pyrrhotite pairs at 700°C. The precision of measurement of both the amphibole compositions and the fugacities of volatile species does not justify other than an ideal solution model. A standard state enthalpy of formation (H°_{298°;1 atm}) of Fe₇Si₈O₂₂(OH)₂ amphibole from the elements of -2262 kcal/mole was calculated from a log K_eq vs 1/T plot for reaction (1). The results in the S-free system have been used to estimate temperatures of formation of amphibole-bearing metamorphic and extrusive igneous rocks. Application of the results in the S-containing system is limited by the scarcity of data on natural amphibole-sulfide assemblages. / Ph. D.

In order to assess the potential of increased mechanization of shortwood harvesting in the man-made forests of Sao Paulo, Brazil, surveys and field studies of present systems were made to provide a basis for evaluation. From this information typical domestic systems with field debarking and without field debarking were compared to mechanized systems used in other countries. The analysis of twenty-two systems were made using a deterministic mathematical model to predict total direct cost per stere, man hour per stere, and capital requirements per stere of annual production. In addition, labor and capital requirements to supply a 500 ton per day pulpmill were calculated. The analysis indicated that: (1) field debarking almost doubles direct cost, dramatically increases labor requirements, and significantly increases capital requirements when portable debarkers are used: (2) mechanical loading appears to be both cost effective and an efficient use of capital; (3) additional mechanization results in increases cost, however, this situation is due largely to the prevailing low labor rates which could change in the future; and (4) if shortages of wood labor develops, the transition from domestic systems with field debarking to mechanical systems without field debarking could be made without a major infusion of capital. This study has shown that there is a great need for the standardization of terminology and improved methods of data collection and analysis. The use of computer simulation for systems analysis should be implemented as soon as feasible to provide a sound basis for research and planning. / M.S.

Borrelia burgdorferi is an elongated and helically shaped bacterium that is the causal agent of the tick-borne illness Lyme disease. The disease manifests with initial flu-like symptoms and, in many cases, the appearance of a skin rash called erythema migrans at the site of the tick bite. If left untreated the disease might cause impairment of various organs such as the skin, heart, joints and the nervous system. The bacteria have a parasitic lifestyle and are always present within a host. Hosts are usually ticks or different animals and birds that serve as reservoirs for infection. B. burgdorferi are unable to synthesize building blocks for many vital cellular processes and are therefore highly dependent on their surroundings to obtain nutrients. Because of this, porins situated in the outer membrane, involved in nutrient uptake, are believed to be very important for B. burgdorferi. Except for a role in nutrient acquisition, porins can also have a function in binding extracellular matrix proteins, such as integrins, and have also been implicated in bacterial adaptation to new environments with variations in osmotic pressure. P13 and P66 are two integral outer membrane proteins in B. burgdorferi previously shown to have porin activities. In addition to its porin function, P66 also has integrin binding activity. In this thesis, oligomeric structures formed by the P13 and P66 protein complexes were studied using the Black lipid bilayer technique in combination with nonelectrolytes. Initial attempts were also made to study the structure of P13 in Nanodiscs, whereby membrane proteins can insert into artificial lipid bilayers in their native state and the structure can be analyzed by electron microscopy. In addition, the role of P13 and P66 in B. burgdorferi osmotic stress adaptation was examined and also the importance and role of the integrin-binding activity of P66 in B. burgdorferi infections in mice. Using Black lipid bilayer studies, the pore forming activity of P13 was shown to be much smaller than previously thought, exhibiting activity at 0.6 nS. The complex formed by P13 was approximately 300 kDa and solely composed of P13 monomers. The channel size was calculated to be roughly 1.4 nm. Initial Nanodisc experiments showed a pore size of 1.3 nm, confirming the pore size determined by Black lipid bilayer experiments. P66 form pores with a single channel conductance of 11 nS and a channel size of 1.9 nm. The porin assembles in the outer membrane into a large protein complex of 420 kDa, containing exclusively P66 monomers. The integrin-binding function of P66 was found to be important for efficient bacterial dissemination in the murine host but was not essential for B. burgdorferi infectivity. Neither P13 nor P66 had an active role in osmotic stress adaptation. Instead, two p13 paralogs were up-regulated at the transcript level in B. burgdorferi cultured under glycerol-induced osmotic stress. / Borrelia burgdorferi är en bakterie med många unika egenskaper som orsakar sjukdomen Lyme borrelios. Borrelia kan idag lätt behandlas med antibiotika om sjukdomen upptäcks i ett tidigt stadium. Det är först om sjukdomen tillåts fortgå som symptom som nervsmärta och ansiktsförlamning kan uppstå och dessutom vara svåra att koppla till en Borrelia-infektion. Multiresistenta bakterier har blivit en stor del av vår vardag och även om Borrelia-bakterierna idag inte är resistenta mot flertalet antibiotika är det kanske speciellt viktigt, innan det är för sent, med forskning som kan leda till upptäckter av unika angreppsställen för nya läkemedel. Målet med denna avhandling var att studera hur två Borrelia proteiner, P13 och P66, ser ut, är uppbyggda och även vilken funktion de har. Dessa proteiner är tänkbara vaccinkandidater eftersom de sitter i yttre membranet hos bakterierna och sticker ut på ytan mot våra värdceller, vilket gör att vi reagerar mot dem vid en infektion. P13 och P66 är också viktiga kanaler för bakterierna vid upptag av näringsämnen och byggstenar från omgivningen. Ämnen som bakterierna inte kan producera själva. Pga. denna funktion är P13 och P66 tänkbara proteiner för blockering med ett läkemedel som skulle förhindra bakterien från att föröka sig i och med att de förlorar möjligheten att tillgodogöra sig näring. Detta i sin tur skulle leda till att vårt eget immunförsvar hinner rensa undan bakterierna innan infektionen blivit för stor och vi blivit sjuka. P66 har förutom porin funktionen även en adhesions funktion när proteinet kan binda integriner som sitter på olika typer av celler i vår kropp, bl. a. immunceller och epitelceller i våra blodkärl och vävnader. Den integrin bindande funktionen är viktig för bakterierna vid en infektion eftersom det gör det möjligt för bakterierna att binda till våra celler. Ett steg som är viktigt för att de senare ska kunna ta sig ut från blodkärlen till våra vävnader. P13 och P66 visade sig kunna bilda stora proteinkomplex i ytter membranet hos bakterierna med en storlek på 300 kDa respektive 420 kDa. De är inga specifika poriner som bara kan transportera en viss typ av molekyl med t.ex. en viss laddning, utan kan ombesörja upptaget av många olika typer av ämnen. Eliminering av p66 orsakade att ett annat adhesionsprotein, uppreglerades. En omplacering av ett normalt cytoplasmatiskt lokaliserat chaperon-protein till ytter-membranet hos bakterierna kunde också ses i frånvaro av P66. Chaperonet GroEL har i andra bakterier, bl. a. Helicobacter pylori, bakterien som orsakar magsår, beskrivits som ett protein som kan förflytta sig till ytan av bakterierna och där ha en liknande funktion som P66, dvs. att binda extracellulära matrisprotein. Förändringen i uttryck av adhesionsproteinet och förflyttningen av chaperonet till membranet var en följd av p66-eliminering och mest troligt ett sätt för bakterierna att komplettera den förlorade integrinbindande funktionen av P66. Det har tidigare visats att poriner är involverade i skyddet mot osmotisk stress i andra bakterier. Denna funktion hos P13 och P66 i Borrelia kunde inte ses när bakterier utsattes för osmotisk stress med glycerol, som orsakar en form av membranstress. Däremot kunde vi med hjälp av transkriptomanalys se att Borrelia-bakterier uppreglerade transkriptionen av två paraloger till P13 vid hyper-osmotisk stress. Borrelia bakteriens användning av dessa paraloga proteiner har tidigare trotts ske enbart i frånvaro av ett funktionellt P13 protein. Nu visade det sig att P13-paraloger har en egen funktion även i närvaro av P13, nämligen att vara involverade i regleringen av hyperosmotisk stress och därmed skydda bakterierna i denna stressituation. Andra gener som påverkades av osmotisk stress med glycerol var gener för stressfaktorer och pumpar i inre membranet hos bakterien.

No description available.

Borrelia burgdorferi is a pathogenic spirochete which cycles between its arthropod vector and vertebrate host. If transmitted to humans, B. burgdorferi causes Lyme disease, an infection which can impair different organs, such as the skin, joints, nervous system and heart. Alterations in protein expression due to the different environments Borrelia encounters during its complicated life cycle require advanced adaptation mechanisms. The outer surface-exposed proteins play a critical role in survival and pathogenesis of Borrelia in different hosts and tissues, being involved in avoiding the host immune response, adhesion to different tissues and nutrient acquisition. This thesis aimed to characterize integral outer membrane proteins which play a role in solute and nutrient uptake, and provides support for their role in the environmental adaptation of Borrelia. In this thesis, three B. burgdorferi proteins, P13, BBA01 and P66, were shown to be porins, and characterized structurally and functionally using a combination of biochemical, biophysical and genetic methods. The channel-forming function of the 13 kDa protein, P13, was elucidated by a lipid bilayer assay. Post-translational processing of P13 occurred at the C-terminus by C-terminal processing protease (CtpA)-dependent cleavage. The membrane-spanning architecture of P13 was determined by epitope mapping and computer-based structural predictions which revealed that P13 is an unusual porin, not possessing the structural properties of conventional porins: rather than forming β-barrels, it is predicted to span the membrane with hydrophobic α-helices. p13 belongs to a paralogous gene family. The transcription of p13 and other gene family members during in vitro growth and in a mouse infection model was therefore investigated. The paralog BBA01, which has the highest sequence homology to P13, is expressed during in vitro growth in all three Lyme disease causing species, although at very low levels. Like P13, BBA01 is also processed by CtpA and exhibits very similar channel-forming activity. Furthermore, in the absence of P13, a proportion of total BBA01 protein is relocated to the bacterial surface with strong indications that BBA01 and P13 are functionally interchangeable. P66, an integrin binding protein, was also determined to be a porin. The oligomeric state of native P66, elucidated by chemical cross-linking, indicated that P66 forms trimers, as do the majority of conventional porins. Electron crystallography and a projection map of P66 crystals at 2.2 nm resolution revealed tetragonal unit cell symmetry with the area intercalated between the assembled protein structures consistent with the approximate expected size of the channel formed by P66. Finally, the biological relevance of two porins, P13 and P66, was demonstrated in a double mutant displaying a stress response as revealed by increased sensitivity to high osmolarity and elevated expression of the B. burgdorferi heat-shock protein HtrA homolog.

Borrelia is a Gram-negative, corkscrew-shaped bacterium transmitted by infected ticks or lice. Borreliae are subdivided into pathogens of two diseases: Lyme disease, caused mainly by B. burgdorferi, B. afzelii and B. garinii; and relapsing fever caused primarily by B. duttonii, B. hermsii, B. recurrentis or B. crocidurae. Both diseases differ in their manifestations, duration times and dissemination patterns. Antibiotics are the major therapeutics, although unfortunately antibiotic treatment is not always beneficial. To date, drug resistance mechanisms in B. burgdorferi are unknown. Transporters of the resistance-nodulation-division (RND) family appear to be involved in drug resistance, especially in Gram-negative bacteria. They consist of three components: a cytoplasmic membrane export system, a membrane fusion protein (MFP), and an outer membrane factor (OMP). The major antibiotic efflux activity of this type in Escherichia coli is mediated by the tripartite multidrug resistance pump AcrAB-TolC. Based on the sequence homology we conclude that the besA (bb0140), besB (bb0141) and besC (bb0142) genes code for a similar efflux system in B. burgdorferi. We created a deletion mutant of besC. The minimal inhibitory concentration (MIC) values of B. burgdorferi carrying an inactive besC gene were 4- to 8-fold lower than in the wild type strain. Animal experiments showed that the besC mutant was unable to infect mice. Black lipid bilayer experiments were carried out to determine the biophysical properties of purified BesC. This study showed the importance of BesC protein for B. burgdorferi pathogenicity and resistance to antibiotics, although its importance in clinical isolates is not known. Due to its small genome, Borrelia is metabolically and biosynthetically deficient, thereby making it highly dependent on nutrients provided by their hosts. The uptake of nutrients by Borrelia is not yet completely understood. We describe the purification and characterization of a 36-kDa protein that functions as a putative dicarboxylate-specific porin in the outer membrane of Borrelia. The protein was designated as DipA, for dicarboxylate-specific porin A. DipA was biophysically characterized using the black lipid bilayer assay. The permeation of KCl through the channel could be partly blocked by titrating the DipA-mediated membrane conductance with increasing concentrations of different organic dicarboxylic anions. The obtained results imply that DipA does not form a general diffusion pore, but a porin with a binding site specific for dicarboxylates which play important key roles in the deficient metabolic and biosynthetic pathways of Borrelia species. The presence of porin P66 has been shown in both Lyme disease and relapsing fever spirochetes. In our study, purified P66 homologues from Lyme disease species B. burgdorferi, B. afzelii and B. garinii and relapsing fever species B. duttonii, B. recurrentis and B. hermsii were compared and their biophysical properties were further characterized in black lipid bilayer assay. Subsequently, the channel diameter of B. burgdorferi P66 was investigated in more detail. For this study, different nonelectrolytes with known hydrodynamic radii were used. This allowed us to determine the effective diameter of the P66 channel lumen. Furthermore, the blockage of the channel after addition of nonelectrolytes revealed seven subconducting states and indicated a heptameric structure of the P66 channel. These results may give more insight into the functional properties of this important porin.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 5 uppsatser.