Role of PheE15 gate in ligand entry and nitric oxide detoxification function of Mycobacterium tuberculosis truncated hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O(2) and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O(2)/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

Development of IT Service Management in a Large International Company

The purpose of this Master’s thesis is to examine the IT Service Management (ITSM) capabilities of a large international company and develop a design for the IT Service Management of the company. The thesis consists of two parts: a literature review and a case study. The first part is the literature review. It examines different aspects of IT Service Management. The purpose of the literature review is to increase the credibility of the case study. In the case study, the IT Service Management of a large industrial company is assessed, and an improved design for IT Service Management is created. The services are limited to common IT services, and management is limited to operative management. To assess and develop the case company’s IT Service Management, the IT organization and its ITSM capabilities are analyzed, and on the basis of the analyses, an improved IT Service Management model is created. As a result of the analyses, it was found out that the management of the IT organization is function-oriented. Therefore, the organization needs a management model that breaks the functional borders and brings services to the center of management. The designed model aspires to achieve this by defining IT services and modeling management processes from the service perspective.

Vascular Function in Chronic Kidney Disease and in Renovascular Disease

Cardiovascular mortality is 15 to 30 times higher in patients with chronic kidney disease than in the age-adjusted general population. Even minor renal dysfunction predicts cardiovascular events and death in the general population. In patients with atherosclerotic renovascular disease the annual cardiovascular event and death rate is even higher. The abnormalities in coronary and peripheral artery function in the different stages of chronic kidney disease and in renovascular disease are still poorly understood, nor have the cardiac effects of renal artery revascularization been well characterized, although considered to be beneficial. This study was conducted to characterize myocardial perfusion and peripheral endothelial function in patients with chronic kidney disease and in patients with atherosclerotic renovascular disease. Myocardial perfusion was measured with positron emission tomography (PET) and peripheral endothelial function with brachial artery flow-mediated dilatation. It has been suggested that the poor renal outcomes after the renal artery revascularization could be due to damage in the stenotic kidney parenchyma; especially the reduction in the microvascular density, changes mainly evident at the cortical level which controls almost 80% of the total renal blood flow. This study was also performed to measure the effect of renal artery stenosis revascularization on renal perfusion in patients with renovascular disease. In order to do that a PET-based method for quantification of renal perfusion was developed. The coronary flow reserve of patients with chronic kidney disease was similar to the coronary flow reserve of healthy controls. In renovascular disease the coronary flow reserve was, however, markedly reduced. Flow-mediated dilatation of brachial artery was decreased in patients with chronic kidney disease compared to healthy controls, and even more so in patients with renovascular disease. After renal artery stenosis revascularization, coronary vascular function and renal perfusion did not improve in patients with atherosclerotic renovascular disease, but in patients with bilateral renal artery stenosis, flow-mediated dilatation improved. Chronic kidney disease does not significantly affect coronary vascular function. On the contrary, coronary vascular function was severely deteriorated in patients with atherosclerotic renovascular disease, possibly because of diffuse coronary artery disease and/or diffuse microvascular disease. The peripheral endothelial function was disturbed in patients with chronic kidney disease and even more so in patient with atherosclerotic renovascular disease. Renal artery stenosis dilatation does not seem to offer any benefits over medical treatment in patients with renovascular disease, since revascularization does not improve coronary vascular function or renal perfusion.

Seeder-fertilizer machine: energetic demand as a function of vegetal covering handling and manure deposition shank depth in no-tillage system

The purpose of this work was evaluating the energetic demand of a seeder-fertilizer machine as a function of the type and handling of vegetal covering culture and of the fertilizer deposition shank depth. A Valtra BM100 tractor was used implemented to pull a high precision seeder-fertilizer machine with four ranks of seeding, spaced 0.9 m for maize culture. Experiment was conducted with design in randomized blocks in factorial plots, in the Laboratory of Machines and Agricultural Mechanization experimental area (LAMMA) of UNESP-Jaboticabal, using two covering cultures (black-mucuna and crotalaria), three handlings of this covering, two mechanical (straw crusher and roller knife) and one chemical (pulverization of herbicide), performed 120 days after seeding of covering cultures and three depths of fertilizer deposition shank, completing 18 treatments, with four repetitions, totaling 72 observations. Parameters of displacement speed, gliding, force on traction bar, peak force, power on pulling bar and fuel consumption were evaluated. It was possible to conclude that force on traction bar was less for depths of 0.11 and 0.14 m of fertilizer plough shank, the same occurring for peak force, power on traction bar and volumetric consumption. The specific consumption was lower at a depth of 0.17 m of fertilizer plough shank. Covering cultures and their handlings did not interfere in the performance of machines under inquiry.

The regulation and function of collagenase-3 (MMP-13) in cutaneous wound healing and squamous cell carcinoma

Matrix metalloproteinase-13 (MMP-13) is a potent proteolytic enzyme, whose expression has been previously associated with fetal bone development and postnatal bone remodeling and with adult gingival wound healing. MMP-13 is also known to be involved in the growth and invasion of various cancers including squamous cell carcinoma (SCC) of the skin. The aim of this study was to further elucidate the function and regulation of MMP-13 in wound repair and cancer. In this study, it was shown that fetal skin fibroblasts express MMP-13 in response to transforming growth factor-β in a p38 MAP kinase dependent manner. In addition, MMP-13 was found to be expressed in vivo by wound fibroblasts in human fetal skin grafted on SCID mice. Adenovirally delivered expression of MMP-13 enhanced collagen matrix contraction by fibroblasts in vitro in association with altered cytoskeletal structure, enhanced proliferation and survival. These results indicate that MMP-13 is involved in cell-mediated collagen matrix remodeling and suggest a role for MMP-13 in superior matrix remodeling and scarless healing of fetal skin wounds. Using an MMP-13 deficient mouse strain, it was shown that MMP-13 is essential for the normal development of experimental granulation tissue in mice. MMP-13 was implicated in the regulation of myofibroblast function and angiogenesis and the expression of genes involved in cellular proliferation and movement, immune response, angiogenesis and proteolysis. Finally, epidermal mitogen, keratinocyte growth factor (KGF) was shown to suppress the malignant properties of skin SCC cells by downregulating the expression of several target genes with potential cancer promoting properties, including MMP-13, and by reducing SCC cell invasion. These results provide evidence that MMP-13 potently regulates cell viability, myofibroblast function and angiogenesis associated with wound healing and cancer. In addition, fibroblasts expressing MMP-13 show high collagen reorganization capacity. Moreover, the results suggest that KGF mediates the anti-cancer effects on skin SCC

Fuel consumption of a tractor in function of wear, of ballasting and tire inflation pressure

When using appropriate inflation pressures and load capacity (ballast), it may obtain a higher yield and prolongation of the life of the tire, besides it may minimize the problems of loss of traction, increased slippage and fuel consumption. This study aimed to evaluate the fuel consumption of a tractor operating with new and worn tires in three conditions of ballasting and three inflation pressures, when driving on compacted soil with vegetation cover. The experiment was conducted at the experimental unit from the Department of Animal Science, Federal University of Lavras, state of Minas Gerais, Brazil, in an agricultural soil compacted by cattle trampling and with vegetation cover. It was used a tractor 4x2 with front wheel assist, of a 65.62 kW engine power. The tires were of R1 type, diagonal (front: 12.4 to 24; and rear: 18.4 to 30), the average height of the clutches of the new tires were 0.3 and 0.35 m for front and rear tires, respectively, and for the worn tires were 0.018 and 0.0045 m, for the front and the rear tires, respectively. The results showed advantages for the tractor equipped with new tires.

Energetic efficiency of an agricultural tractor in function of tire inflation pressure

The tire inflation pressure, among other factors, determines the efficiency in which a tractor can exert traction. It was studied the effect of using two tire inflation pressures, 110.4 kPa in the front and rear wheels, 124.2 kPa in the front wheel and 138 kPa in the rear wheels, the energetic efficiency of an agricultural tractor of 147 kW of engine power, in the displacement speed of 6.0 km.h-1, on track with firm surface, with the tractor engine speed of 2000 rpm. For each condition of the tire pressure, the tested tractor was subjected to constant forces in the drawbar of 45 kN and 50 kN, covering 30 meters. It was used a randomized complete block with a 2x2 factorial arrangement (tire pressure and drawbar power) with four replications, totaling 16 experimental units. Data were subjected to analysis of variance, using the Tukey test at 5% probability for comparison averages. The lowest hourly and specific fuel consumption, the lowest slippage of the wheelsets and the highest efficiency in the drawbar was obtained with the tire inflation pressure of 110.4 kPa in the front and rear tires of the tractor, highlighting that lower pressures improve energetic and operational performance of the tractor.

Quality of peanut mechanical sowing in function of seeding density and size of the seeds

ABSTRACTPeanut crop (Arachis hypogaeaL.) mechanization has been improved over the years; however there are drawbacks that affect the quality of operations. Thus, this article’s objectives were to evaluate the operational performance of the mechanized sowing of peanut crop according to seeding densities (10, 14, and 18 seeds m-1) and seed sizes (21 and 23 mm). It was observed that the seeds of 23 mm had shorter average number of days to emergence and a higher percentage of emergences, occurring the opposite to the seeding density of 18 seeds m-1. The higher the seeding density, the largest was the plant stand, whereas the 23 mm seed obtained the best results and the same with the seeding density of 14 seeds m-1 that had a higher percentage of normal spacing. The densities of 14 and 18 seeds m-1 reflected in higher yields, being always superior to the 23 mm seeds.

Ankle-brachial index, high-sensitivity C-reactive protein and endothelial function in a cardiovascular risk population

Atherosclerotic vascular disease is the leading cause of death in the Western world. Its main three manifestations are coronary heart disease, cerebrovascular disease, and peripheral arterial disease. Asymptomatic peripheral arterial disease is usually diagnosed using the ankle brachial index, and values ≤ 0.90 are used to determine the diagnosis. The classical risk factors of peripheral arterial disease, such as smoking and diabetes, are well known and early interventions are mandatory to improve the prognosis. What is not well known is the role of inflammation as a risk factor. Yet, a novel approach to cardiovascular diseases is the measurement of endothelial function. In this thesis, we studied the ankle-brachial index, C-reactive protein and endothelial function in a cardiovascular risk population. A total of 2856 subjects were invited to the study and 2085 (73%) responded. From these subjects, a cohort of 1756 risk persons was screened. We excluded the subjects with previously known cardiovascular disease or diabetes, because they were already under systematic follow-up. Out of the study subjects, 983 (56%) were women and 773 (44%) men. The ankle brachial index and high-sensitivity C-reactive protein were measured from 1047 subjects. Endothelial function was assessed by measuring reactive hyperemia pulse amplitude tonometry from 66 subjects with borderline peripheral arterial disease. In this study, smoking was a crucial risk factor for peripheral arterial disease. Subclinical peripheral arterial disease seems to be more common in hypertensive patients even without comorbidities. The measurement of the ankle brachial index is an efficient method to identify patients at an increased cardiovascular risk. High-sensitivity C-reactive protein did not correlate with the ankle brachial index or peripheral arterial disease. Instead, it correlated with measures of obesity. In a cardiovascular risk population with borderline peripheral arterial disease, nearly every fourth subject had endothelial dysfunction. This might point out a subgroup of individuals in need of more intensive treatment for their risk factors.

The Structure and Function of αI Domains in Collagen Receptor and Leukocyte Integrins

Integrins are heterodimeric, signaling transmembrane adhesion receptors that connect the intracellular actin microfilaments to the extracellular matrix composed of collagens and other matrix molecules. Bidirectional signaling is mediated via drastic conformational changes in integrins. These changes also occur in the integrin αI domains, which are responsible for ligand binding by collagen receptor and leukocyte specific integrins. Like intact integrins, soluble αI domains exist in the closed, low affinity form and in the open, high affinity form, and so it is possible to use isolated αI domains to study the factors and mechanisms involved in integrin activation/deactivation. Integrins are found in all mammalian tissues and cells, where they play crucial roles in growth, migration, defense mechanisms and apoptosis. Integrins are involved in many human diseases, such as inflammatory, cardiovascular and metastatic diseases, and so plenty of effort has been invested into developing integrin specific drugs. Humans have 24 different integrins, four of which are collagen receptor (α1β1, α2β1, α10β1, α11β1) and five leukocyte specific integrins (αLβ2, αMβ2, αXβ2, αDβ2, αEβ7). These two integrin groups are quite unselective having both primary and secondary ligands. This work presents the first systematic studies performed on these integrin groups to find out how integrin activation affects ligand binding and selectivity. These kinds of studies are important not only for understanding the partially overlapping functions of integrins, but also for drug development. In general, our results indicated that selectivity in ligand recognition is greatly reduced upon integrin activation. Interestingly, in some cases the ligand binding properties of integrins have been shown to be cell type specific. The reason for this is not known, but our observations suggest that cell types with a higher integrin activation state have lower ligand selectivity, and vice versa. Furthermore, we solved the three-dimensional structure for the activated form of the collagen receptor α1I domain. This structure revealed a novel intermediate conformation not previously seen with any other integrin αI domain. This is the first 3D structure for an activated collagen receptor αI domain without ligand. Based on the differences between the open and closed conformation of the αI domain we set structural criteria for a search for effective collagen receptor drugs. By docking a large number of molecules into the closed conformation of the α2I domain we discovered two polyketides, which best fulfilled the set structural criteria, and by cell adhesion studies we showed them to be specific inhibitors of the collagen receptor integrins.

Role of the canonical transient receptor potential channel 2 (TRPC2) in thyroid cell function

Kalciumjonen reglerar flera processer i celler såsom transkribering av gener, celldelning, cellernas rörlighet och celldöd. Därför har cellerna utvecklat många mekanismer för att reglera den intracellulära kalciumkoncentrationen. Kalciumkanaler spelar en viktig roll i denna regleringsprocess. TRPC-kanalerna (eng. canonical transient receptor potential) är en familj av jonkanaler med sju medlemmar (TRPC1-7) vars regleringsmekanismer och fysiologiska roller är varierande. TRPC2-kanalens fysiologiska signifikans, samt hur kanalen regleras, är dåligt karakteriserad. För första gången, rapporterar vi närvaron av TRPC2 kanalen i råttans sköldkörtelceller samt primära sköldkörtelceller från råtta. Hos gnagare har TRPC2 antagits vara exklusivt uttryckt i det vomeronasala organet. För att undersöka den fysiologiska betydelsen av kanalen, har vi utvecklat stabila celler med nedreglerat TRPC2 (shTRPC2) m.h.a. shRNA-teknik. Nedreglering av TRPC2 resulterade i stora skillnader i flera viktiga cellulära funktioner och i regleringen av sköldkörtelcellernas cellsignalering. Nedreglering av TRPC2 orsakade minskad agonist-beroende frigivning av kalcium från det endoplasmatiska nätverket, samt minskat agonist-beroende inflöde av extracellulärt kalcium, men ökade det basala kalciuminflödet. Uttrycket av PKCβ1 och PKCδ, SERCA-aktiviteten och kalciumhalten i det endoplasmatiska nätverket minskade i shTRPC2 celler. Kommunikation mellan kalcium- och cAMP-signalering påvisades vara TRPC2-beroende, vilket visades reglera uttrycket av TSH-receptorn. Vi undersökte också betydelsen av TRPC2 kanalen i reglering av sköldkörtelcellers proliferation, migration, vidhäftning och invasion; processer som alla var dämpade i shTRPC2 celler. Samamnfattningsvis påvisade dessa resultat en ny och viktig fysiologisk betydelse för TRPC2 kanalerna.

Glycolipid and ceramide transfer proteins : important determinants for their function

Lipider är viktiga biomolekyler, eftersom de bygger upp alla cellulära membran. Glykolipider, dvs. lipider som innehåller socker, är dessutom betydelsefulla som signaleringsmolekyler vid olika processer. Det är essentiellt att regleringen av syntesen, nedbrytningen samt transporten av lipider i cellen är noggrant koordinerade, och faktorer som kan påverka lipidmetabolismen är därför viktiga att undersöka. Denna avhandling har undersökt två olika lipidbindande proteiner, glykolipidtransportprotein (GLTP) och ceramidtransportprotein (CERT). GLTPs biologiska funktion är ännu oklar, dock vet man att GLTP har förmåga att binda olika glykolipider samt överföra dessa lipider mellan olika lipidmembraner. CERT har däremot visats kunna transportera ceramid från det endoplastiska retiklet (ER) till Golgi-apparaten, för produktion av sfingomyelin. I detta avhandlingsarbete undersöktes lokaliseringen av GLTP i celler med olika metoder, bl.a. konfokalmikroskopi, samt olika centrifugeringsmetoder. Genom att överuttrycka GLTP i celler och därefter analysera halten nysyntetiserade glykolipider, kunde även sambandet mellan GLTP-uttrycket och dessa lipider undersökas. I avhandlingen identifierades ytterligare en specifik aminosyrasekvens hos GLTP. Denna sekvens visades kunna binda till VAP-A, ett integralt ER protein, med en tidigare fastställd viktig funktion vid regleringen av lipidtransporten. I avhandlingen analyserades även hur ceramidtransporten mellan två olika membraner, medierad av CERT, påverkas av egenskaper i ceramidens omgivning. För att undersöka detta användes artificiella modellmembraner samt fluorimetriska metoder. Sammansättningen och packningen hos lipidmembranerna visades ha en stor betydelse för den CERT-katalyserade ceramidtransporten. Sammanfattningsvis antyder resultaten från avhandlingen att det existerar flera faktorer som kan påverka aktiviteten av GLTP och CERT, vilka i sin tur har förmåga att reglera lipidmetabolismen.

Photosynthetic characteristics of hybrid and conventional rice plants as a function of plant competition

The objective of this work was to evaluate the characteristics related to the photosynthetic ability of hybrid and inbred rice varieties, as a way to assess which of the two presented higher potential to stand out under conditions of competition. The trial was set in a greenhouse in completely randomized block design and 2 x 6 factorial scheme with four replications. Factor A consisted of rice varieties (hybrid or inbred) and factor B by competition levels. Treatments consisted in maintaining one plant of either BRS Pelota (inbred) or Inov (hybrid) variety at the center of the plot, under competition with 0, 1, 2, 3, 4 or 5 plants of the variety BRS Pelota at the periphery of the experimental unit, according to the treatment. Fifty days after emergence (DAE), sub-stomatal CO2 concentration (Ci - mmol mol-1), photosynthetic rate (A - mmol m-2 s-1) and CO2 consumed (DC - mmol mol-1) were quantified, as well as shoot dry mass(SDM).Hybrid plants present higher photosynthesis capacity than inbred plants, when competing with up to 3 times its own density. When under the same competitive intensity, hybrid plants surpass the inbred. However, it should be emphasized that, when in farm condition, the lower competitive capacity with weeds often attributed to the hybrid varieties, probably is due to their lower planting density, but if weed competition is kept at low levels, hybrid rice plants may perform in the same way or usually better than inbred plants.

How can we agree on some product agnostic repository function fundamentals?

Presentation at Open Repositories 2014, Helsinki, Finland, June 9-13, 2014

Regulation of HSF2 and its function in mitosis

The cell is continuously subjected to various forms of external and intrinsic proteindamaging stresses, including hyperthermia, pathophysiological states, as well as cell differentiation and proliferation. Proteindamaging stresses result in denaturation and improper folding of proteins, leading to the formation of toxic aggregates that are detrimental for various pathological conditions, including Alzheimer’s and Huntington’s diseases. In order to maintain protein homeostasis, cells have developed different cytoprotective mechanisms, one of which is the evolutionary well-conserved heat shock response. The heat shock response results in the expression of heat shock proteins (Hsps), which act as molecular chaperones that bind to misfolded proteins, facilitate their refolding and prevent the formation of protein aggregates. Stress-induced expression of Hsps is mediated by a family of transcription factors, the heat shock factors, HSFs. Of the four HSFs found in vertebrates, HSF1-4, HSF1 is the major stress-responsive factor that is required for the induction of the heat shock response. HSF2 cannot alone induce Hsps, but modulates the heat shock response by forming heterotrimers with HSF1. HSFs are not only involved in the heat shock response, but they have also been found to have a function in development, neurodegenerative disorders, cancer, and longevity. Therefore, insight into how HSFs are regulated is important for the understanding of both normal physiological and disease processes. The activity of HSF1 is mainly regulated by intricate post-translational modifications, whereas the activity of HSF2 is concentrationdependent. However, there is only limited understanding of how the abundance of HSF2 is regulated. This study describes two different means of how HSF2 levels are regulated. In the first study it was shown that microRNA miR-18, a member of the miR-17~92 cluster, directly regulates Hsf2 mRNA stability and thus protein levels. HSF2 has earlier been shown to play a profound role in the regulation of male germ cell maturation during the spermatogenesis. The effect on miR-18 on HSF2 was examined in vivo by transfecting intact seminiferous tubules, and it was found that inhibition of miR-18 resulted in increased HSF2 levels and modified expression of the HSF2 targets Ssty2 and Speer4a. HSF2 has earlier been reported to modulate the heat shock response by forming heterotrimers with HSF1. In the second study, it was shown that HSF2 is cleared off the Hsp70 promoter and degraded by the ubiquitinproteasome pathway upon acute stress. By silencing components of the anaphase promoting complex/cyclosome (APC/C), including the co-activators Cdc20 and Cdh1, it was shown that APC/C mediates the heatinduced ubiquitylation of HSF2. Furthermore, down-regulation of Cdc20 was shown to alter the expression of heat shock-responsive genes. Next, we studied if APC/C-Cdc20, which controls cell cycle progression, also regulates HSF2 during the cell cycle. We found that both HSF2 mRNA and protein levels decreased during mitosis in several but not all human cell lines, indicating that HSF2 has a function in mitotic cells. Interestingly, although transcription is globally repressed during mitosis, mainly due to the displacement of RNA polymerase II and transcription factors, including HSF1, from the mitotic chromatin, HSF2 is capable of binding DNA during mitosis. Thus, during mitosis the heat shock response is impaired, leaving mitotic cells vulnerable to proteotoxic stress. However, in HSF2-deficient mitotic cells the Hsp70 promoter is accessible to both HSF1 and RNA polymerase II, allowing for stress-inducible Hsp expression to occur. As a consequence HSF2-deficient mitotic cells have a survival advantage upon acute heat stress. The results, presented in this thesis contribute to the understanding of the regulatory mechanisms of HSF2 and its function in the heat shock response in both interphase and mitotic cells.