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1.
  • Alvarez, Francisco J., et al. (author)
  • Diverse Nitrogen Sources in Seminal Fluid Act in Synergy To Induce Filamentous Growth of Candida albicans
  • 2015
  • In: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 81:8, s. 2770-2780
  • Journal article (peer-reviewed)abstract
    • The pathogenic fungus Candida albicans is the leading cause of vulvovaginal candidiasis (VVC). VVC represents a major quality- of-life issue for women during their reproductive years, a stage of life where the vaginal epithelium is subject to periodic hormonally induced changes associated with menstruation and concomitant exposure to serum as well as potential intermittent contact with seminal fluid. Seminal fluid potently triggers Candida albicans to switch from yeastlike to filamentous modes of growth, a developmental response tightly linked to virulence. Conversely, vaginal fluid inhibits filamentation. Here, we used artificial formulations of seminal and vaginal fluids that faithfully mimic genuine fluids to assess the contribution of individual components within these fluids to filamentation. The high levels of albumin, amino acids, and N-acetylglucosamine in seminal fluid act synergistically as potent inducers of filamentous growth, even at atmospheric levels of CO2 and reduced temperatures (30 degrees C). Using a simplified in vitro model that mimics the natural introduction of seminal fluid into the vulvovaginal environment, a pulse of artificial seminal fluid (ASF) was found to exert an enduring potential to overcome the inhibitory efficacy of artificial vaginal fluid (AVF) on filamentation. These findings suggest that a transient but substantial change in the nutrient levels within the vulvovaginal environment during unprotected coitus can induce resident C. albicans cells to engage developmental programs associated with virulent growth.
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2.
  • Boban, Mirta, et al. (author)
  • A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation
  • 2014
  • In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 127:16, s. 3603-3613
  • Journal article (peer-reviewed)abstract
    • The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of similar to 45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.
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3.
  • Boban, Mirta, et al. (author)
  • Atypical Ubiquitylation in Yeast Targets Lysine-less Asi2 for Proteasomal Degradation
  • 2015
  • In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:4, s. 2489-2495
  • Journal article (peer-reviewed)abstract
    • Proteins are typically targeted for proteasomal degradation by the attachment of a polyubiquitin chain to epsilon-amino groups of lysine residues. Non-lysine ubiquitylation of proteasomal substrates has been considered an atypical and rare event limited to complex eukaryotes. Here we report that a fully functional lysine-less mutant of an inner nuclear membrane protein in yeast, Asi2, is polyubiquitylated and targeted for proteasomal degradation. Efficient degradation of lysine-free Asi2 requires E3-ligase Doa10 and E2 enzymes Ubc6 and Ubc7, components of the endoplasmic reticulum-associated degradation pathway. Together, our data suggest that non-lysine ubiquitylation may be more prevalent than currently considered.
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5.
  • Chernomoretz, Ariel, et al. (author)
  • The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium inaugural meeting report
  • 2016
  • In: Microbiome. - : Springer Science and Business Media LLC. - 2049-2618. ; 4
  • Journal article (peer-reviewed)abstract
    • The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium is a novel, interdisciplinary initiative comprised of experts across many fields, including genomics, data analysis, engineering, public health, and architecture. The ultimate goal of the MetaSUB Consortium is to improve city utilization and planning through the detection, measurement, and design of metagenomics within urban environments. Although continual measures occur for temperature, air pressure, weather, and human activity, including longitudinal, cross-kingdom ecosystem dynamics can alter and improve the design of cities. The MetaSUB Consortium is aiding these efforts by developing and testing metagenomic methods and standards, including optimized methods for sample collection, DNA/RNA isolation, taxa characterization, and data visualization. The data produced by the consortium can aid city planners, public health officials, and architectural designers. In addition, the study will continue to lead to the discovery of new species, global maps of antimicrobial resistance (AMR) markers, and novel biosynthetic gene clusters (BGCs). Finally, we note that engineered metagenomic ecosystems can help enable more responsive, safer, and quantified cities.
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6.
  • Chng, Kern Rei, et al. (author)
  • Cartography of opportunistic pathogens and antibiotic resistance genes in a tertiary hospital environment
  • 2020
  • In: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 26, s. 941-951
  • Journal article (peer-reviewed)abstract
    • Although disinfection is key to infection control, the colonization patterns and resistomes of hospital-environment microbes remain underexplored. We report the first extensive genomic characterization of microbiomes, pathogens and antibiotic resistance cassettes in a tertiary-care hospital, from repeated sampling (up to 1.5 years apart) of 179 sites associated with 45 beds. Deep shotgun metagenomics unveiled distinct ecological niches of microbes and antibiotic resistance genes characterized by biofilm-forming and human-microbiome-influenced environments with corresponding patterns of spatiotemporal divergence. Quasi-metagenomics with nanopore sequencing provided thousands of high-contiguity genomes, phage and plasmid sequences (>60% novel), enabling characterization of resistome and mobilome diversity and dynamic architectures in hospital environments. Phylogenetics identified multidrug-resistant strains as being widely distributed and stably colonizing across sites. Comparisons with clinical isolates indicated that such microbes can persist in hospitals for extended periods (>8 years), to opportunistically infect patients. These findings highlight the importance of characterizing antibiotic resistance reservoirs in hospitals and establish the feasibility of systematic surveys to target resources for preventing infections. Spatiotemporal characterization of microbial diversity and antibiotic resistance in a tertiary-care hospital reveals broad distribution and persistence of antibiotic-resistant organisms that could cause opportunistic infections in a healthcare setting.
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7.
  • Danko, David, et al. (author)
  • A global metagenomic map of urban microbiomes and antimicrobial resistance
  • 2021
  • In: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 184:13, s. 3376-3393
  • Journal article (peer-reviewed)abstract
    • We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.
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8.
  • Darkahi, Bahman, et al. (author)
  • Effectiveness of antibiotic prophylaxis in cholecystectomy : a prospective population-based study of 1171 cholecystectomies
  • 2012
  • In: Scandinavian Journal of Gastroenterology. - : Informa UK Limited. - 0036-5521 .- 1502-7708. ; 47:10, s. 1242-1246
  • Journal article (peer-reviewed)abstract
    • Background.The aim of this study was to assess the benefit from antibiotic prophylaxis (AP) during cholecystectomy in a population-based cohort study.Methods.All cholecystectomies performed in Uppsala County, 2003-2005, were registered prospectively according to a standardized protocol. High-risk procedures (HP) were defined as operations for acute cholecystitis and procedures including exploration of the common bile duct. Infections requiring surgical or percutaneous drainage and non-surgical infections that prolonged hospital stay were defined as major infectious complications (IC).Results. Altogether 1171 patients underwent cholecystectomy. AP was given to 130 of 867 (15%) of the patients undergoing low-risk procedures (LP) and 205 of 304 (67%) of those undergoing H-R P. Major IC were seen in 6 of 205 (3%) of the patients undergoing H-R P with AP and 1 of 99 of the patients undergoing H-R P without AP. No major IC was seen after L-R P. Minor IC were seen after 5 of 205 (2%) HP with AP, 1 of 99 (1%) HP without AP, 0 of 130 (0%) LP with AP, and 2 of 737 (0.3%) LP without AP. In univariate logistic analysis, the overall risk for IC was found to be higher with AP (p < 0.05), but the increase did not remain significant if adjusting for age, gender, ASA class, H-R P/L-R P and surgical approach or limiting the analysis to major IC.Conclusion. There is no benefit from AP in uncomplicated procedures. The effectiveness of antibiotic prophylaxis in complicated cholecystectomy must be evaluated in randomized controlled trials.
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9.
  • Davis, Monica M., et al. (author)
  • Wild-Type Drosophila melanogaster as a Model Host to Analyze Nitrogen Source Dependent Virulence of Candida albicans
  • 2011
  • In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:11, s. e27434-
  • Journal article (peer-reviewed)abstract
    • The fungal pathogen Candida albicans is a common cause of opportunistic infections in humans. We report that wild-type Drosophila melanogaster (OrR) flies are susceptible to virulent C. albicans infections and have established experimental conditions that enable OrR flies to serve as model hosts for studying C. albicans virulence. After injection into the thorax, wild-type C. albicans cells disseminate and invade tissues throughout the fly, leading to lethality. Similar to results obtained monitoring systemic infections in mice, well-characterized cph1Δ efg1Δ and csh3Δ fungal mutants exhibit attenuated virulence in flies. Using the OrR fly host model, we assessed the virulence of C. albicans strains individually lacking functional components of the SPS sensing pathway. In response to extracellular amino acids, the plasma membrane localized SPS-sensor (Ssy1, Ptr3, and Ssy5) activates two transcription factors (Stp1 and Stp2) to differentially control two distinct modes of nitrogen acquisition (host protein catabolism and amino acid uptake, respectively). Our results indicate that a functional SPS-sensor and Stp1 controlled genes required for host protein catabolism and utilization, including the major secreted aspartyl protease SAP2, are required to establish virulent infections. By contrast, Stp2, which activates genes required for amino acid uptake, is dispensable for virulence. These results indicate that nutrient availability within infected hosts directly influences C. albicans virulence.
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10.
  • Gustafsson, Jan-Eric, 1949, et al. (author)
  • School, Learning and Mental Health : A systematic review
  • 2010
  • Reports (other academic/artistic)abstract
    • Rapporten presenterar resultaten från en systematisk översikt av forskning om skola, lärande och barns psykiska hälsa. Kungliga Vetenskapsakademiens Hälsoutskottet har givit uppdraget att genomföra en sådan översikt till en arbetsgrupp som har arbetat med uppdraget från hösten 2008 till mars 2009. Det första syftet med översikten är att genomföra en kartläggning av forskning inom det breda fält som behandlar frågor om skola, lärande och barns och ungdomars psykiska hälsa. Det andra syftet är att genomföra en narrativ syntes av forskning som undersökt orsaksförhållanden mellan psykisk hälsa å ena sidan och skolresultat och lärande å den andra sidan. Det tredje syftet är att redovisa resultat från forskning som har studerat svenska barns och ungdomars erfarenheter och upplevelser av skola och undervisningssituationer. För att uppnå de första två syftena genomfördes systematiska litteratursökningar i bibliografiska databaser av artiklar publicerade i vetenskapliga internationella tidskrifter inom olika discipliner. Det tredje syftet undersöktes med litteratursökningar av kvalitativa svenska studier i bibliografiska databaser. Slutsatser På grundval dels av kartläggningen av forskning om skola, lärande och psykisk hälsa, dels av de två fördjupade översikterna kan följande slutsatser dras: • Omfattningen av forskning som undersöker relationerna mellan olika aspekter av skola och psykisk hälsa är begränsad och i synnerhet gäller detta forskning som undersöker organisationsfaktorer och undervisnings-faktorer, aktiviteter, läroplaners utformning, resurser, specialpedagogiskt stöd, och olika former av betyg och bedömning. • Tidiga svårigheter i skolan och i synnerhet läs- och skrivsvårigheter kan orsaka internaliserande och externaliserande psykiska problem. • Svårigheter i skola och psykiska problem tenderar att vara stabila över tid. • Skolrelaterade hälsoproblem tenderar att minska när eleverna börjar på gymnasiet och får tillgång till nya områden av aktiviteter, roller och valmöjligheter. • Att genomföra stora ansträngningar utan att detta leder till resultat är relaterat till utveckling av depression. Problem i skolan med skolresultat och prestationer orsakar inter-naliserande symptom för flickor under tonåren. • Det finns samband mellan olika typer av psykiska problem och de är också relaterade till ett brett spektrum av somatiska och psykosomatiska symptom. • Internaliserande och externaliserande psykiska problem har negativa effekter på skolprestationer genom mekanismer som är delvis ålders- och genusspecifika. • Kompetenser och prestationer i skolan är relaterade till psykisk hälsa. • Goda resultat i skolan har en positiv effekt på självuppfattning. • En god självuppfattning bidrar inte direkt till bättre resultat, men andra faktorer som är relaterade till självuppfattning (motivation och upplevd inre/yttre kontroll) påverkar lärande och resultat • Relationer med klasskamrater och lärare bidrar till processer som kopplar skolmisslyckande till psykisk ohälsa. Relationer med kamrater och lärare kan också skydda mot utvecklingen av psykiska problem. • Jämförelser med klasskamrater påverkar självuppfattningen, med effekter som varierar beroende på gruppsammansättning och typ av skola.
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11.
  • Jenull, Sabrina, et al. (author)
  • The histone chaperone HIR maintains chromatin states to control nitrogen assimilation and fungal virulence
  • 2021
  • In: Cell Reports. - : Elsevier BV. - 2211-1247. ; 36:3
  • Journal article (peer-reviewed)abstract
    • Adaptation to changing environments and immune evasion is pivotal for fitness of pathogens. Yet, the underlying mechanisms remain largely unknown. Adaptation is governed by dynamic transcriptional re-programming, which is tightly connected to chromatin architecture. Here, we report a pivotal role for the HIR histone chaperone complex in modulating virulence of the human fungal pathogen Candida albicans. Genetic ablation of HIR function alters chromatin accessibility linked to aberrant transcriptional responses to protein as nitrogen source. This accelerates metabolic adaptation and increases the release of extracellular proteases, which enables scavenging of alternative nitrogen sources. Furthermore, HIR controls fungal virulence, as HIR1 deletion leads to differential recognition by immune cells and hypervirulence in a mouse model of systemic infection. This work provides mechanistic insights into chromatin-coupled regulatory mechanisms that fine-tune pathogen gene expression and virulence. Furthermore, the data point toward the requirement of refined screening approaches to exploit chromatin modifications as antifungal strategies.
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12.
  • Khmelinskii, Anton, et al. (author)
  • Protein quality control at the inner nuclear membrane
  • 2014
  • In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 516:7531, s. 410-
  • Journal article (peer-reviewed)abstract
    • The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression(1). The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asicomplex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer(5), we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.
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15.
  • Liu, Ning-Ning, et al. (author)
  • Intersection of phosphate transport, oxidative stress and TOR signalling in Candida albicans virulence
  • 2018
  • In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 14:7
  • Journal article (peer-reviewed)abstract
    • Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.
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16.
  • Ljungdahl, Jonas, 1979- (author)
  • Structure and properties of Vasa oak
  • 2006
  • Licentiate thesis (other academic/artistic)abstract
    • The Vasa ship is not adequately supported. Measurements of the hull show that the ship deforms and rotate towards the port side. In addition, damages on the hull at support areas have been observed. The damages are due to high compressive loads. At damaged zones the support has been removed and the loads are thus transferred to adjacent support stanchions. In order to design an improved support, knowledge of the mechanical behaviour of the material is needed. In particular, radial modulus, strength and deformation mechanisms are of interest. In the present study, the mechanical behaviour of recent oak and oak from Vasa is studied. Furthermore, effects of PEG content, degradation and moisture on the properties of Vasa oak are investigated. Oak is characterized by a very abrupt change from earlywood to latewood, where the latewood is much denser than earlywood. Also present in oak are large rays in the radial direction of the wood. Small specimens were tested in compression using Digital Speckle Photography (DSP) in order to obtain strain fields of the whole specimen surface. This technique also provided data on failure mechanisms. Dynamic mechanical thermal analysis (DMTA) was performed to establish differences in moisture softening. In radial compression, modulus and strength of Vasa oak are reduced by 50% compared with recent oak. A significant change of failure mechanism is observed for Vasa oak. In recent oak, failure in radial compression is by continuous folds of rays in the earlywood followed by continued plastic collapse of the earlywood layer. In Vasa oak rays show a more brittle fracture in each earlywood region. DMTA results indicate no effect on moisture softening of Vasa oak from presence of PEG although more work is needed to confirm this. Moisture adsorption for PEG-extracted Vasa oak is not significantly higher than for recent oak below 60% RH, suggesting that the extent of degradation of Vasa oak is limited. Vasa oak containing PEG is much more hygroscopic than PEG-extracted Vasa oak already at 50%. This difference is increasing with increasing relative humidity.
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18.
  • Ljungdahl, Per O (author)
  • Amino-acid-induced signalling via the SPS-sensing pathway in yeast.
  • 2009
  • In: Biochemical Society Transactions. - 0300-5127 .- 1470-8752. ; 37:Pt 1, s. 242-7
  • Journal article (peer-reviewed)abstract
    • Yeast cells rely on the SPS-sensing pathway to respond to extracellular amino acids. This nutrient-induced signal transduction pathway regulates gene expression by controlling the activity of two redundant transcription factors: Stp1 and Stp2. These factors are synthesized as latent cytoplasmic proteins with N-terminal regulatory domains. Upon induction by extracellular amino acids, the plasma membrane SPS-sensor catalyses an endoproteolytic processing event that cleaves away the regulatory N-terminal domains. The shorter forms of Stp1 and Stp2 efficiently target to the nucleus, where they bind and activate transcription of selected genes encoding a subset of amino acid permeases that function at the plasma membrane to catalyse the transport of amino acids into cells. In the present article, the current understanding of events in the SPS-sensing pathway that enable external amino acids to induce their own uptake are reviewed with a focus on two key issues: (i) the maintenance of Stp1 and Stp2 latency in the absence of amino acid induction; and (ii) the amino-acid-induced SPS-sensor-mediated proteolytic cleavage of Stp1 and Stp2.
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19.
  • Ljungdahl, Per O., et al. (author)
  • Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae
  • 2012
  • In: Genetics. - : Oxford University Press (OUP). - 0016-6731 .- 1943-2631. ; 190:3, s. 885-929
  • Journal article (peer-reviewed)abstract
    • Ever since the beginning of biochemical analysis, yeast has been a pioneering model for studying the regulation of eukaryotic metabolism. During the last three decades, the combination of powerful yeast genetics and genome-wide approaches has led to a more integrated view of metabolic regulation. Multiple layers of regulation, from suprapathway control to individual gene responses, have been discovered. Constitutive and dedicated systems that are critical in sensing of the intra-and extracellular environment have been identified, and there is a growing awareness of their involvement in the highly regulated intracellular compartmentalization of proteins and metabolites. This review focuses on recent developments in the field of amino acid, nucleotide, and phosphate metabolism and provides illustrative examples of how yeast cells combine a variety of mechanisms to achieve coordinated regulation of multiple metabolic pathways. Importantly, common schemes have emerged, which reveal mechanisms conserved among various pathways, such as those involved in metabolite sensing and transcriptional regulation by noncoding RNAs or by metabolic intermediates. Thanks to the remarkable sophistication offered by the yeast experimental system, a picture of the intimate connections between the metabolomic and the transcriptome is becoming clear.
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20.
  • Llopis-Torregrosa, Vicent, et al. (author)
  • Trk1-mediated potassium uptake contributes to cell-surface properties and virulence of Candida glabrata
  • 2019
  • In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
  • Journal article (peer-reviewed)abstract
    • The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1 Delta cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1 Delta mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.
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21.
  • Martins, António, 1976-, et al. (author)
  • Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor controlled Ssy5 signaling protease
  • Journal article (peer-reviewed)abstract
    • The Saccharomyces cerevisiae Ssy5 signaling protease is a core component of the plasma membrane (PM)-localized SPS (Ssy1-Ptr3-Ssy5)-sensor. In response to extracellular amino acids, the SPS-sensor orchestrates the proteasomal degradation of the inhibitory Ssy5 prodomain. The unfettered catalytic (Cat)-domain cleaves latent transcription factors Stp1 and Stp2, freeing them from negative N-terminal regulatory domains. By studying the spatial and temporal constraints affecting the unfettered Cat-domain, we found that it can cleave substrates not associated with the PM; the Cat-domain efficiently cleaves Stp1 even when fused to the carboxy-terminal of the endoplasmic reticulum (ER) membrane protein Shr3. The amino acid-induced cleavage of this synthetic membrane-anchored substrate occurs in a Δtether strain lacking ER-PM junctions. We report that the bulk of the Cat-domain is soluble, exhibits a disperse intracellular distribution and is subject to ubiquitylation. Cat-domain ubiquitylation is dependent on Ptr3 and the integral PM casein kinase I (Yck1/2). Time-course experiments reveal that the non- and ubiquitylated forms of the Cat-domain are stable in cells grown in the absence of inducing amino acids. By contrast, amino acid induction significantly accelerates Cat-domain degradation. These findings provide novel insights into the SPS-sensing pathway and suggest that Cat-domain degradation is a requisite for resetting SPS-sensor signaling.
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22.
  • Martins, António, et al. (author)
  • Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor-controlled Ssy5 signaling protease
  • 2019
  • In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 30:21, s. 2709-2720
  • Journal article (peer-reviewed)abstract
    • The Saccharomyces cerevisiae Ssy5 signaling protease is a core component of the plasma membrane (PM)-localized SPS (Ssy1-Ptr3-Ssy5) sensor. In response to extracellular amino acids, the SPS-sensor orchestrates the proteasomal degradation of the inhibitory Ssy5 prodomain. The unfettered catalytic (Cat)-domain cleaves latent transcription factors Stp1 and Stp2, freeing them from negative N-terminal regulatory domains. By studying the spatial and temporal constraints affecting the unfettered Cat-domain, we found that it can cleave substrates not associated with the PM; the Cat-domain efficiently cleaves Stp1 even when fused to the carboxy terminus of the endoplasmic reticulum (ER) membrane protein Shr3. The amino acid-induced cleavage of this synthetic membrane-anchored substrate occurs in a Delta tether strain lacking ER-PM junctions. We report that the bulk of the Cat-domain is soluble, exhibits a disperse intracellular distribution, and is subject to ubiquitylation. Cat-domain ubiquitylation is dependent on Ptr3 and the integral PM casein kinase I (Yck1/2). Time-course experiments reveal that the non-and ubiquitylated forms of the Cat-domain are stable in cells grown in the absence of inducing amino acids. By contrast, amino acid induction significantly accelerates Cat-domain degradation. These findings provide novel insights into the SPS-sensing pathway and suggest that Cat-domain degradation is a requisite for resetting SPS-sensor signaling.
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23.
  • Martins, António, et al. (author)
  • Ssy5 is a signaling serine protease that exhibits atypical biogenesis and marked S1 specificity
  • 2018
  • In: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 293:22, s. 8362-8378
  • Journal article (peer-reviewed)abstract
    • Ssy5 is a signaling endoprotease that plays a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival of yeast (Saccharomyces cerevisiae) cells. In response to extracellular amino acids, Ssy5 proteolytically activates the transcription factors Stp1 and Stp2, leading to enhanced Ssy1-Ptr3-Ssy5 (SPS) sensor-regulated gene expression. Ssy5 comprises a catalytic (Cat) domain and an extensive regulatory prodomain. Ssy5 is refractory to both broad-spectrum and serine protease-specific inhibitors, confounding its classification as a protease, and no information about Ssy5's cleavage-site preferences and its mechanism of substrate selection is available. Here, using mutational and inhibition experiments, we investigated the biogenesis and catalytic properties of Ssy5 and conclusively show that it is a serine protease. Atypical for the majority of serine proteases, Ssy5's prodomain was obligatorily required in cis during biogenesis for the maturation of the proteolytic activity of the Cat domain. Autolysis and Stp1 and Stp2 cleavage occurred between a cysteine (at the P1 site) and a serine or alanine (at the P1 site) and required residues with short side chains at the P1 site. Substitutions in the Cat domain affecting substrate specificity revealed that residues Phe-634, His-661, and Gly-671 in the S1-binding pocket of this domain are important for Ssy5 catalytic function. This study confirms that the signaling protease Ssy5 is a serine protease and provides a detailed understanding of the biogenesis and intrinsic properties of this key enzyme in yeast.
  •  
24.
  • Martins, António, 1976- (author)
  • The cell biology and catalytic properties of the nutrient-induced signaling endoprotease Ssy5
  • 2018
  • Doctoral thesis (other academic/artistic)abstract
    • Cells continuously sense and respond to changes in the presence, quality and quantity of external and internal nutrients. Specific signaling proteases have been identified based on their roles in processing or destruction of distinct sets of downstream effector proteins in response to environmental cues. The Saccharomyces cerevisiae Ssy5 signaling endoprotease has a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival. Ssy5 is a core component of the Ssy1–Ptr3-Ssy5 (SPS) sensor, which enables yeast cells to respond to extracellular amino acids and induce their uptake. Ssy5 cleaves transcription factors Stp1 and Stp2, permitting their translocation to the nucleus where they enhance the expression of amino acid permease genes. This thesis focuses on Ssy5, its biogenesis and catalytic properties (paper I), the spatial determinants underlying Ssy5 function in SPS-sensor context (paper II) and substrate cleavage (paper III).Ssy5 is comprised of pro- and catalytic-(Cat)-domains. The Cat-domain possesses characteristic hallmarks of a serine protease; however, serine protease-specific inhibitors have limited effect, confounding its classification. In paper I we unambiguously show that Ssy5 is a serine protease, define the precise sites of cleavage in Stp1 and Stp2, and describe the sequence specific requirements of their cleavage. The uniquely large prodomain (381 amino acids) has two essential functions. Initially, it is required in cis for the maturation of the Cat-domain, helping to overcome a folding barrier that is reflected in the high stability of the Cat-domain. Subsequent to attaining enzymatic competence, Ssy5 undergoes an autolytic cleavage event. The domains remain associated and the prodomain functions to fetter the proteolytic activity of the Cat-domain.The plasma membrane (PM) localization of Ssy1 has recently been questioned in a report that postulated that Ssy1 is a component of the endoplasmic reticulum (ER) and contributes to the formation of ER-PM junctions. In paper II, using mutational and subcellular fractionation experiments we critically examined this notion that is inconsistent with the current understanding of Ssy5 activation, i.e., the unfettering of the Cat-domain occurs in strict association with Ssy1 at the PM. The data show that Ssy1 is indeed a PM protein, and importantly, Ssy5-activation occurs independent of ER-PM junctions. A di-acidic ER exit motif was identified that is critical for proper PM localization and function of Ssy1. In paper III, we report that the Cat-domain is post-translationally modified in a manner dependent on Ptr3 and the PM casein kinase I (Yck1/2), consistent with Ssy5 activation occurring at the PM. Strikingly, the activated Cat-domain is capable of properly cleaving Stp1 fused to an ER membrane protein. The amino acid-induced cleavage of this artificial membrane-bound substrate occurs in a Δtether strain (ist2Δ scs2Δ scs22Δ tcb1Δ tcb2Δ tcb3Δ) lacking ER-PM junctions. These findings indicate that the activated Cat-domain can bind and functionally interact with substrates distant from the PM. Finally, we show that the Cat-domain is degraded faster in amino acid-induced cells. These findings provide novel insights into the SPS-sensing pathway and demonstrate for the first time that the resetting of the SPS-sensing system correlates with Cat-domain degradation.
  •  
25.
  • Myronidi, Ioanna, 1986-, et al. (author)
  • ER-localized Shr3 is a selective co-translational folding chaperone necessary for amino acid permease biogenesis
  • 2023
  • In: Journal of Cell Biology. - 0021-9525 .- 1540-8140. ; 222:9
  • Journal article (peer-reviewed)abstract
    • Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Shr3, an ER membrane–localized chaperone in Saccharomyces cerevisiae, is required for the functional expression of a family of 18 amino acid permeases (AAP) comprised of 12 MS. We have used comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with a modified split-ubiquitin approach to probe chaperone–substrate interactions in vivo. Shr3 selectively interacts with nested C-terminal AAP truncations in marked contrast to similar truncations of non-Shr3 substrate sugar transporters. Shr3–AAP interactions initiate with the first four MS of AAP and successively strengthen but weaken abruptly when all 12 MS are present. Shr3–AAP interactions are based on structural rather than sequence-specific interactions involving membrane and luminal domains of Shr3. The data align with Shr3 engaging nascent N-terminal chains of AAP, functioning as a scaffold to facilitate folding as translation completes.
  •  
26.
  • Omnus, Deike J., 1981-, et al. (author)
  • A phosphodegron controls nutrient-induced proteasomal activation of the signaling protease Ssy5
  • 2011
  • In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 22:15, s. 2754-2765
  • Journal article (peer-reviewed)abstract
    • Regulated proteolysis serves as a mechanism to control cellular processes. The SPS (Ssy1-Ptr3-Ssy5) sensor in yeast responds to extracellular amino acids by endoproteolytically activating transcription factors Stp1 and Stp2 (Stp1/2). The processing endoprotease Ssy5 is regulated via proteasomal degradation of its noncovalently associated N-terminal prodomain. We find that degradation of the prodomain requires a conserved phosphodegron comprising phosphoacceptor sites and ubiquitin-accepting lysine residues. Upon amino acid induction, the phosphodegron is modified in a series of linked events by a set of general regulatory factors involved in diverse signaling pathways. First, an amino acid-induced conformational change triggers phosphodegron phosphorylation by the constitutively active plasma membrane-localized casein kinase I (Yck1/2). Next the prodomain becomes a substrate for polyubiquitylation by the Skp1/Cullin/Grr1 E3 ubiquitin ligase complex (SCF(Grr1)). Finally, the modified prodomain is concomitantly degraded by the 26S proteasome. These integrated events are requisite for unfettering the Ssy5 endoprotease, and thus Stp1/2 processing. The Ssy5 phosphoacceptor motif resembles the Yck1/2- and Grr1-dependent degrons of regulators in the Snf3/Rgt2 glucose-sensing pathway. Our work defines a novel proteolytic activation cascade that regulates an intracellular signaling protease and illustrates how general signaling components are recruited to distinct pathways that achieve conditional and specific signaling outputs.
  •  
27.
  •  
28.
  • Omnus, Deike J., et al. (author)
  • Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron
  • 2014
  • In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 25:23, s. 3823-3833
  • Journal article (peer-reviewed)abstract
    • The Ssy1-Ptr3-Ssy5 (SPS)-sensing pathway enables yeast to respond to extracellular amino acids. Stp1, the effector transcription factor, is synthesized as a latent cytoplasmic precursor with an N-terminal regulatory domain that restricts its nuclear accumulation. The negative regulatory mechanisms impinging on the N-terminal domain are poorly understood. However, Stp1 latency depends on three inner nuclear membrane proteins, Asi1, Asi2, and Asi3. We report that the N-terminal domain of Stp1 contains a small motif, designated RI, that fully accounts for latency. RI is modular, mediates interactions with the plasma membrane, and can retain histone Htb2 in the cytoplasm. A novel class of STP1 mutations affecting RI were isolated that are less efficiently retained in the cytoplasm but remain under tight negative control by the Asi proteins. Intriguingly, these mutant proteins exhibit enhanced stability in strains lacking ASI1. Our results indicate that RI mediates latency by two distinct activities: it functions as a cytoplasmic retention determinant and an Asi-dependent degron. These findings provide novel insights into the SPS-sensing pathway and demonstrate for the first time that the inner nuclear membrane Asi proteins function in a degradation pathway in the nucleus.
  •  
29.
  •  
30.
  • Omnus, Deike J., 1981- (author)
  • Regulatory mechanisms of amino acid-induced signaling in Saccharomyces cerevisiae
  • 2014
  • Doctoral thesis (other academic/artistic)abstract
    • This thesis describes studies aimed at elucidating the molecular mechanisms that regulate the SPS (Ssy1-Ptr3-Ssy5) signal transduction pathway in the yeast Saccharomyces cerevisiae. This pathway is induced by extracellular amino acids and facilitates their uptake. The most downstream effectors of the SPS pathway, the homologous transcription factors Stp1 and Stp2 (Stp1/2), are synthesized as latent precursors with N-terminal regulatory domains that restrict their nuclear accumulation. Amino acid-induced signaling, initiated by the plasma membrane localized receptor Ssy1, leads via Ptr3 to the activation of the endoprotease Ssy5. Active Ssy5 cleaves the regulatory domains in Stp1/2. As a consequence, the processed transcription factors lacking their N-terminal domains accumulate in the nucleus and activate the transcription of amino acid permease genes to enhance the uptake capacity of cells.Ssy5 is synthesized as a zymogen precursor that processes itself into a prodomain and catalytic (Cat) domain that remain non-covalently associated. We found that the prodomain functions as an inhibitor of the Cat domain. Signaling triggers the degradation of the prodomain by the proteasome, thereby releasing Cat domain activity (paper I). We identified a motif in the prodomain that functions as inducible phosphodegron. Upon signaling, this motif is phosphorylated which triggers prodomain polyubiquitylation, and as a consequence, its proteasomal degradation (paper II). Also, we found that Ptr3 functions to mediate prodomain phosphorylation upon signaling and that protein phosphatase 2A constitutively mutes phosphorylation-dependent activation of Ssy5 (paper III).Finally, in addition to the regulation of the processing protease Ssy5, the control of transcriptional activity of Stp1 depends on a motif within its N-terminal regulatory domain, designated Region I. We found that Region I mediates latency by functioning as cytoplasmic retention determinant and nuclear degron (paper IV).
  •  
31.
  • Omnus, Deike J., 1981-, et al. (author)
  • Rts1-protein phosphatase 2A antagonizes Ptr3-mediated activation of the signaling protease Ssy5 by casein kinase I
  • 2013
  • In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 24:9, s. 1480-1492
  • Journal article (peer-reviewed)abstract
    • Ligand-induced conformational changes of plasma membrane receptors initiate signals that enable cells to respond to discrete extracellular cues. In response to extracellular amino acids, the yeast Ssy1-Ptr3-Ssy5 sensor triggers the endoproteolytic processing of transcription factors Stp1 and Stp2 to induce amino acid uptake. Activation of the processing protease Ssy5 depends on the signal-induced phosphorylation of its prodomain by casein kinase I (Yck1/2). Phosphorylation is required for subsequent Skp1/Cullin/Grr1 E3 ubiquitin ligase-dependent polyubiquitylation and proteasomal degradation of the inhibitory prodomain. Here we show that Rts1, a regulatory subunit of the general protein phosphatase 2A, and Ptr3 have opposing roles in controlling Ssy5 prodomain phosphorylation. Rts1 constitutively directs protein phosphatase 2A activity toward the prodomain, effectively setting a signaling threshold required to mute Ssy5 activation in the absence of amino acid induction. Ptr3 functions as an adaptor that transduces conformational signals initiated by the Ssy1 receptor to dynamically induce prodomain phosphorylation by mediating the proximity of the Ssy5 prodomain and Yck1/2. Our results demonstrate how pathway-specific and general signaling components function synergistically to convert an extracellular stimulus into a highly specific, tuned, and switch-like transcriptional response that is critical for cells to adapt to changes in nutrient availability.
  •  
32.
  • Pantazopoulou, Marina, et al. (author)
  • Cdc48 and Ubx1 participate in a pathway associated with the inner nuclear membrane that governs Asi1 degradation
  • 2016
  • In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 129:20, s. 3770-3780
  • Journal article (peer-reviewed)abstract
    • The nuclear envelope is a barrier comprising outer and inner membranes that separate the cytoplasm from the nucleoplasm. The two membranes have different physical characteristics and protein compositions. The processes governing the stability of inner nuclear membrane (INM) proteins are not well characterized. In Saccharomyces cerevisiae, the INM Asi1-Asi3 complex, principally composed of integral membrane proteins Asi1 and Asi3, is an E3 ubiquitin ligase. In addition to its well-documented function in endoplasmic reticulum (ER)-associated degradation, the Doa10 E3 ubiquitin ligase complex partially localizes to the INM. The Asi1-Asi3 and Doa10 complexes define independent INM-associated degradation (INMAD) pathways that target discrete sets of nuclear substrates for proteasomal degradation. Here, we report that Asi1 is rapidly turned over (t(1/2)<= 30 min). Its turnover depends on ubiquitin-mediated degradation by nucleus-localized proteasomes, exhibiting a clear requirement for the E2 ubiquitin-conjugating enzyme Ubc7, Cue1 and the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs largely independently of the Asi1-Asi3 or Doa10 complexes, indicating that it is subject to quality control at the INM in a manner distinct from that of the characterized INMAD pathways.
  •  
33.
  •  
34.
  • Pantazopoulou, Marina, 1983- (author)
  • Protein Quality Control at the Inner Nuclear Membrane – The Asi complex in Saccharomyces cerevisiae
  • 2016
  • Doctoral thesis (other academic/artistic)abstract
    • The nuclear envelope is a barrier comprised of outer and inner membranes that separate the cytoplasm from the nucleoplasm. The outer (ONM) and inner (INM) membranes have different physical characteristics and protein compositions. In contrast to the extensive data available on the protein quality control processes operating in the cytoplasm, endoplasmic reticulum and the nucleoplasm, the mechanisms controlling protein turnover at the INM are poorly documented. The work presented in this thesis focuses on Asi1, Asi2 and Asi3, three bona-fide integral INM proteins of the yeast Saccharomyces cerevisiae. By contrast to mammalian cells, yeast progress through the cell cycle with a closed mitosis, that is cells divide in the absence of the cyclical fragmentation/reassembly of the nuclear membrane. Consequently, examining the processes affecting the stability of the Asi proteins in yeast may provide useful paradigms for understanding the turnover of INM components in non-dividing, terminally differentiated and post-mitotic cells of metazoan origin.The results have contributed to the elucidation of the biological function of Asi1 and Asi3, which are homologous proteins with C-terminal RING domains. Asi1 and Asi3 function together as a dimeric E3 ubiquitin ligase complex that operates with ubiquitin conjugating enzymes Ubc6 and Ubc7. The Asi1/3 complex ubiquitylates transcription factors Stp1 and Stp2 when they gain inappropriate access to the nucleus in the absence of SPS-sensor activation. Intriguingly, the Asi1/3 complex also mediates the turnover of multiple membrane proteins that primarily localize to other cell membranes. This latter finding indicates that the barrier function of nuclear pore complexes is not as tight as previously thought. Consistently, asi1 null mutations are synthetic lethal when introduced into hrd1Δ ire2Δ cells with compromised ER-associated degradation (ERAD) and unfolded protein response (UPR) pathways. Together the results define Asi1/3 as components of a novel quality control pathway operating in association with the INM that acts to safeguard the identity and maintain the function of the nuclear compartment. Asi1 and Asi2 exhibit rapid turnover and their turnover is ubiquitin-dependent, exhibiting a clear requirement for Ubc7. The ubiquitylated forms of Asi1 and Asi2 are degraded by nuclear-localized proteasomes; the ubiquitylated forms exhibit enhanced stability in sts1-2 mutants. Asi1 turnover requires Cue1, the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs unimpeded in cells lacking a functional Asi1/3 complex and in cells lacking Doa10, an E3 ligase complex also known to function at the INM. Consequently, Asi1 is subject to a quality control pathway associated with INM but that is distinct from the Asi1/3 and Doa10 INM- associated degradative (INMAD) pathways. This thesis documents work that clearly demonstrates that the INM is a highly dynamic structure that possesses multiple and active quality control pathways.
  •  
35.
  • Pfirrmann, Thorsten, et al. (author)
  • SOMA : A Single Oligonucleotide Mutagenesis and Cloning Approach
  • 2013
  • In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:6
  • Journal article (peer-reviewed)abstract
    • Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.
  •  
36.
  • Pfirrmann, Thorsten, et al. (author)
  • The prodomain of Ssy5 protease controls receptor-activated proteolysis of transcription factor Stp1
  • 2010
  • In: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 30:13, s. 3299-309
  • Journal article (peer-reviewed)abstract
    • Extracellular amino acids induce the yeast SPS sensor to endoproteolytically cleave transcription factors Stp1 and Stp2 in a process termed receptor-activated proteolysis (RAP). Ssy5, the activating endoprotease, is synthesized with a large N-terminal prodomain and a C-terminal chymotrypsin-like catalytic (Cat) domain. During biogenesis, Ssy5 cleaves itself and the prodomain and Cat domain remain associated, forming an inactive primed protease. Here we show that the prodomain is a potent inhibitor of Cat domain activity and that its inactivation is a requisite for RAP. Accordingly, amino acid-induced signals trigger proteasome-dependent degradation of the prodomain. A mutation that stabilizes the prodomain prevents Stp1 processing, whereas destabilizing mutations lead to constitutive RAP-independent Stp1 processing. We fused a conditional degron to the prodomain to synthetically reprogram the amino acid-responsive SPS signaling pathway, placing it under temperature control. Our results define a regulatory mechanism that is novel for eukaryotic proteases functioning within cells
  •  
37.
  • Ring, Andreas, 1983- (author)
  • In vivo analysis of amino acid permease folding in yeast
  • 2019
  • Doctoral thesis (other academic/artistic)abstract
    • Plasma membrane (PM) proteins are critical for cells to respond to environmental cues, such as the availability of nutrients. The yeast Saccharomyces cerevisiae is able to sense extracellular amino acids using the SPS sensing system. Activation of the multimeric PM-localized SPS(Ssy1-Ptr3-Ssy5)-sensor complex occurs upon binding of external amino acids to Ssy1, inducing a conformational change. In a Ptr3-mediated event, the catalytic activity of the Ssy5 endoprotease is unfettered, leading to the proteolytic processing of two latent transcription factors, Stp1 and Stp2. Ssy1, the primary sensor component, is a non-transporting member of the amino acid permease (AAP) family of transport proteins, a family of eighteen complex integral membrane proteins comprised of 12 transmembrane segments (TMS). The AAPs exhibit a common requirement for the endoplasmic reticulum (ER)-localized membrane chaperone Shr3 to fold and to be transported to the PM. The absence of Shr3 leads to the accumulation of misfolded AAP species that are targeted for ER-associated degradation. Thus, proper Shr3 function is required as the most upstream and most downstream component of the SPS sensing system. In paper I, we investigate the chaperone function of Shr3. We report a surprisingly low level of sequence specificity underlies Shr3-AAP interactions. We used a split-ubiquitin approach to probe Shr3-AAP interactions in vivo. The Shr3-AAP interactions initiate early after the first two-to-four TMS of AAPs insert into the ER membrane, successively strengthening and then diminishing after all 12 TMS partition into the membrane. In paper II, we clarified the localization and trafficking determinants of Ssy1. A study by Kralt et al. 2015 reported that Ssy1 primarily localizes to the ER and is sorted to ER-PM tethers. These reported findings are clearly incompatible with the accepted model of amino acid sensing by the SPS-sensor. We critically re-examined the localization of Ssy1 and found that it indeed localizes to the PM, and importantly does so independent of ER-PM tethers. We also identified a novel ER exit motif in the carboxy-terminal tail of Ssy1 required for proper PM localization and SPS-sensor function. In paper III, we report that Ssy5 is able to cleave substrates in unusual contexts, i.e., an engineered substrate carrying rearranged recognition and cleavage determinants placed ectopically at the carboxy terminus of Stp1, and an ER-anchored substrate with Stp1 fused to the carboxy terminus of Shr3. Strikingly, Ssy5 catalyzed cleavage of Shr3-Stp1 in cells lacking ER-PM tethers, indicating that once activated, Ssy5 can find and cleave substrates distant from the PM. Consequently, cells must be able to rein in the activity of the Ssy5 protease to prevent spurious and improper proteolysis. Consistent with this notion, we report that the catalytic domain of Ssy5 is ubiquitylated in a Ptr3 and Yck1/2 dependent manner, and under amino acid-inducing conditions is subject to degradation. We propose a model that degradation of the Ssy5 catalytic domain is essential for resetting the SPS sensing system and a requisite for cells to regain the ability to correctly sense extracellular amino acids.
  •  
38.
  • Ring, Andreas, et al. (author)
  • Ssy1 functions at the plasma membrane as a receptor of extracellular amino acids independent of plasma membrane‐endoplasmic reticulum junctions
  • 2019
  • In: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 20:10, s. 775-784
  • Journal article (peer-reviewed)abstract
    • Evidence from multiple laboratories have implicated Ssy1, a non‐transporting amino acid permease, as the receptor component of the yeast plasma membrane (PM)‐localized SPS (Ssy1‐Ptr3‐Ssy5)‐sensor. Upon binding external amino acids, Ssy1 is thought to initiate signaling events leading to the induction of amino acid permease gene expression. In striking contrast, Kralt et al. 2015 (Traffic 16:135‐147) have questioned the role of Ssy1 in amino acid sensing and reported that Ssy1 is a component of the endoplasmic reticulum (ER), where it reportedly participates in the formation of ER‐PM junctions. Here, we have re‐examined the intracellular location of Ssy1 and tested the role of ER‐PM junctions in SPS sensor signaling. We show that the C‐terminal of Ssy1 carries a functional ER‐exit motif required for proper localization of Ssy1 to the PM. Furthermore, ER‐PM junctions are dispensable for PM‐localization and function of Ssy1; Ssy1 localizes to the PM in a Δtether strain lacking ER‐PM junctions (ist2Δ scs2Δ scs22Δ tcb1Δ tcb2Δ tcb3Δ), and this strain retains the ability to initiate signals induced by extracellular amino acids. The data demonstrate that Ssy1 functions as the primary amino acid receptor and that it carries out this function at the PM.
  •  
39.
  • Ring, Andreas, et al. (author)
  • The ER membrane chaperone Shr3 co-translationally assists biogenesis of related polytopic membrane protein substrates
  • Other publication (other academic/artistic)abstract
    • Polytopic membrane proteins with multiple transmembrane segments (TMS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Discrete sets of polytopic membrane proteins in Saccharomyces cerevisiae require ER membrane-localized chaperones (MLC) to prevent aggregation and fold properly. Shr3, the best characterized MLC, is specifically required for the functional expression of amino acid permeases (AAP), a family of transport proteins comprised of twelve TMS. We performed comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with split-ubiquitin approaches to probe chaperone-substrate (Shr3-AAP) interactions in vivo. We report a surprisingly low level of sequence specificity underlies Shr3-AAP interactions, which initiate after the first 2 to 4 TMS of AAP partition into the membrane. The Shr3-AAP interactions successively strengthen and then weaken as all 12 TMS are inserted. Thus, Shr3 acts transiently in a co-translationally manner to prevent TMS of translation intermediates from engaging in non-productive interactions, thereby preventing AAP misfolding during biogenesis.
  •  
40.
  • Sandblom, Gabriel, et al. (author)
  • Validation of Gastrointestinal Quality of Life Index in Swedish for assessing the impact of gallstones on health-related quality of life
  • 2009
  • In: Value in Health. - : Elsevier BV. - 1098-3015 .- 1524-4733. ; 12:1, s. 181-184
  • Journal article (peer-reviewed)abstract
    • OBJECTIVE: The aim of the present study was to validate a Swedish translation of the Gastrointestinal Quality of Life Index (GIQLI) questionnaire in patients with gallstone disease. METHODS: Sensitivity to change, internal consistency, and test-retest stability were tested in 187 consecutive patients who underwent planned cholecystectomy. Construct validity was assessed by comparing the GIQLI score with the bodily pain scale of SF-36 and four single-item questions in a separate group of 104 patients. RESULTS: A significant increase in all five domains as well as in the overall GIQLI score 6 months after surgery (all P < 0.05) was seen. All five domains correlated significantly with other measures of gallstone-related symptoms except one single-item question. Intraclass correlations ranged from 0.62 to 0.87. Cronbach's alpha ranged from 0.77 to 0.89. CONCLUSION: The Swedish translation of GIQLI has a high validity and reliability for assessing the impact of gallstones on quality of life.
  •  
41.
  • Silao, Fitz Gerald S., 1985-, et al. (author)
  • Amino Acid Sensing and Assimilation by the Fungal Pathogen Candida albicans in the Human Host
  • 2022
  • In: Pathogens. - : MDPI AG. - 2076-0817. ; 11:1
  • Journal article (peer-reviewed)abstract
    • Nutrient uptake is essential for cellular life and the capacity to perceive extracellular nutrients is critical for coordinating their uptake and metabolism. Commensal fungal pathogens, e.g., Candida albicans, have evolved in close association with human hosts and are well-adapted to using diverse nutrients found in discrete host niches. Human cells that cannot synthesize all amino acids require the uptake of the “essential amino acids” to remain viable. Consistently, high levels of amino acids circulate in the blood. Host proteins are rich sources of amino acids but their use depends on proteases to cleave them into smaller peptides and free amino acids. C. albicans responds to extracellular amino acids by pleiotropically enhancing their uptake and derive energy from their catabolism to power opportunistic virulent growth. Studies using Saccharomyces cerevisiae have established paradigms to understand metabolic processes in C. albicans; however, fundamental differences exist. The advent of CRISPR/Cas9-based methods facilitate genetic analysis in C. albicans, and state-of-the-art molecular biological techniques are being applied to directly examine growth requirements in vivo and in situ in infected hosts. The combination of divergent approaches can illuminate the biological roles of individual cellular components. Here we discuss recent findings regarding nutrient sensing with a focus on amino acid uptake and metabolism, processes that underlie the virulence of C. albicans. 
  •  
42.
  • Silao, Fitz-Gerald S., 1985-, et al. (author)
  • Diverse mechanisms control amino acid-dependent environmental alkalization by Candida albicans
  • 2024
  • In: Molecular Microbiology. - 0950-382X .- 1365-2958.
  • Journal article (peer-reviewed)abstract
    • Candida albicans has the capacity to neutralize acidic growth environments by releasing ammonia derived from the catabolism of amino acids. The molecular components underlying alkalization and its physiological significance remain poorly understood. Here, we present an integrative model with the cytosolic NAD+-dependent glutamate dehydrogenase (Gdh2) as the principal ammonia-generating component. We show that alkalization is dependent on the SPS-sensor-regulated transcription factor STP2 and the proline-responsive activator Put3. These factors function in parallel to derepress GDH2 and the two proline catabolic enzymes PUT1 and PUT2. Consistently, a double mutant lacking STP2 and PUT3 exhibits a severe alkalization defect that nearly phenocopies that of a gdh2-/- strain. Alkalization is dependent on mitochondrial activity and in wild-type cells occurs as long as the conditions permit respiratory growth. Strikingly, Gdh2 levels decrease and cells transiently extrude glutamate as the environment becomes more alkaline. Together, these processes constitute a rudimentary regulatory system that counters and limits the negative effects associated with ammonia generation. These findings align with Gdh2 being dispensable for virulence, and based on a whole human blood virulence assay, the same is true for C. glabrata and C. auris. Using a transwell co-culture system, we observed that the growth and proliferation of Lactobacillus crispatus, a common component of the acidic vaginal microenvironment and a potent antagonist of C. albicans, is unaffected by fungal-induced alkalization. Consequently, although Candida spp. can alkalinize their growth environments, other fungal-associated processes are more critical in promoting dysbiosis and virulent fungal growth. 
  •  
43.
  • Silao, Fitz Gerald S., et al. (author)
  • Glutamate dehydrogenase (Gdh2)-dependent alkalization is dispensable for escape from macrophages and virulence of Candida albicans
  • 2020
  • In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 16:9
  • Journal article (peer-reviewed)abstract
    • Candida albicans cells depend on the energy derived from amino acid catabolism to induce and sustain hyphal growth inside phagosomes of engulfing macrophages. The concomitant deamination of amino acids is thought to neutralize the acidic microenvironment of phagosomes, a presumed requisite for survival and initiation of hyphal growth. Here, in contrast to an existing model, we show that mitochondrial-localized NAD+-dependent glutamate dehydrogenase (GDH2) catalyzing the deamination of glutamate to α-ketoglutarate, and not the cytosolic urea amidolyase (DUR1,2), accounts for the observed alkalization of media when amino acids are the sole sources of carbon and nitrogen. C. albicans strains lacking GDH2 (gdh2-/-) are viable and do not extrude ammonia on amino acid-based media. Environmental alkalization does not occur under conditions of high glucose (2%), a finding attributable to glucose-repression of GDH2 expression and mitochondrial function. Consistently, inhibition of oxidative phosphorylation or mitochondrial translation by antimycin A or chloramphenicol, respectively, prevents alkalization. GDH2 expression and mitochondrial function are derepressed as glucose levels are lowered from 2% (~110 mM) to 0.2% (~11 mM), or when glycerol is used as primary carbon source. Using time-lapse microscopy, we document that gdh2-/- cells survive, filament and escape from primary murine macrophages at rates indistinguishable from wildtype. In intact hosts, such as in fly and murine models of systemic candidiasis, gdh2-/- mutants are as virulent as wildtype. Thus, although Gdh2 has a critical role in central nitrogen metabolism, Gdh2-catalyzed deamination of glutamate is surprisingly dispensable for escape from macrophages and virulence. Consistently, using the pH-sensitive dye (pHrodo), we observed no significant difference between wildtype and gdh2-/- mutants in phagosomal pH modulation. Following engulfment of fungal cells, the phagosomal compartment is rapidly acidified and hyphal growth initiates and sustained under consistently acidic conditions within phagosomes. Together, our results demonstrate that amino acid-dependent alkalization is not essential for hyphal growth, survival in macrophages and hosts. An accurate understanding of the microenvironment within macrophage phagosomes and the metabolic events underlying the survival of phagocytized C. albicans cells and their escape are critical to understanding the host-pathogen interactions that ultimately determine the pathogenic outcome.
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44.
  • Silao, Fitz Gerald S., et al. (author)
  • Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans
  • 2019
  • In: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 15:2
  • Journal article (peer-reviewed)abstract
    • Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence. Author summary Candida albicans is an opportunistic fungal pathogen that exists as a benign member of the human microbiome. Immunosuppression, or microbial dysbiosis, can predispose an individual to infection, enabling this fungus to evade innate immune cells and initiate a spectrum of pathologies, including superficial mucocutaneous or even life-threatening invasive infections. Infectious growth is attributed to an array of virulence characteristics, a major one being the ability to switch morphologies from round yeast-like to elongated hyphal cells. Here we report that mitochondrial proline catabolism is required to induce hyphal growth of C. albicans cells in phagosomes of engulfing macrophages, which is key to evade killing by macrophages. The finding that proline catabolism, also required for the utilization of arginine and ornithine, is required to sustain the energy demands of hyphal growth underscores the central role of mitochondria in fungal virulence. In contrast to existing dogma, we show that in C. albicans, mitochondrial function is subject to glucose repression, amino acid-induced signals are strictly dependent on Ras1 and the SPS-sensor is the primary sensor of extracellular amino acids. The results provide a clear example of how C. albicans cells sense and respond to host nutrients to ensure proper nutrient uptake and survival.
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45.
  • Silao, Fitz-Gerald S., 1985-, et al. (author)
  • Proline catabolism is a key factor facilitating Candida albicans pathogenicity
  • 2023
  • In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 19:11 NOVEMBER
  • Journal article (peer-reviewed)abstract
    • Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.
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46.
  • Silao, Fitz Gerald S., 1985- (author)
  • The Role of Proline Catabolism in Candida albicans Pathogenesis
  • 2019
  • Doctoral thesis (other academic/artistic)abstract
    • Candida albicans is an opportunistic fungal pathogen that has evolved in close association with human hosts. Pathogenicity is linked to an array of virulence characteristics expressed in response to environmental cues and that reflect the requirement to take up and metabolize nutrients available in the host. Metabolism generates the energy to support the bioenergetic demands of infectious growth, including the ability to reversibly switch morphologies from yeast to filamentous hyphal forms. Amino acids are among the most versatile nutrients available in the hosts as they can serve as both carbon and nitrogen sources, be transformed to key metabolic intermediates, or utilized to modulate extracellular pH via deamination forming ammonia. Of the proteinogenic amino acids, proline is unique in having a secondary amine covalently locked within an imine ring. Accumulating evidence implicates proline catabolism as being critical in the pathogenesis of many human diseases, ranging from bacterial and parasitic infections to cancer progression. This work focuses on the role of proline catabolism on C. albicans  pathogenesis.Paper I describes how proline induces filamentous growth in C. albicans. Hyphal growth is induced by an increase in intracellular ATP, a positive regulator of the Ras1/cAMP/PKA pathway. Proline is a direct substrate for ATP production, its catabolism in the mitochondria by proline oxidase (Put1) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (Put2) leads to the generation of FADH2 and NADH, respectively. Arginine and ornithine induce filamentous growth due to being catabolized to proline. Strikingly, mitochondrial proline catabolism is essential for hyphal growth and escape from macrophages.Paper II documents that proline catabolism is an important regulator of reactive oxygen species (ROS) homeostasis in C. albicans. When cells depend on proline as an energy source, the activities of the two catabolic enzymes Put1 and Put2 must operate in synchrony; perturbation of these highly regulated catabolic steps exerts deleterious effects on growth. Cells lacking PUT2 exhibit increased sensitivity to exogenous proline. This sensitivity is linked to ROS generation, likely due to the accumulation of the toxic intermediate P5C. Consistently, a put2-/- mutant is avirulent in Drosophila and in a 3D skin infection model, and hypovirulent in neutrophils and a systemic murine infection model.Paper III shows that the enzymatic step directly downstream of Put2, the deamination of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase (Gdh2), releases the ammonia responsible for the alkalization of the extracellular environment when C. albicans  cells grow in the presence of amino acids. Cells lacking GDH2 do not alkalinize the medium. Alkalization is thought to induce hyphal growth in cells engulfed by macrophages. Surprisingly, filamentous growth of gdh2-/- cells is not impaired in filament-inducing media, or importantly, in situ in the phagosome of primary murine macrophages. Thus, alkalization is not a requisite for filamentous growth within macrophages.The results demonstrate that under physiologically relevant host conditions, proline catabolism is important for C. albicans pathogenesis. Further studies are warranted to determine the applicability of this pathway as a potential target for therapeutic approaches aimed at combating this major fungal pathogen.
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47.
  • Sollerbrant, Kerstin, et al. (author)
  • The Coxsackievirus and adenovirus receptor (CAR) forms a complex with the PDZ domain-containing protein ligand-of-numb protein-X (LNX)
  • 2003
  • In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:9, s. 7439-7444
  • Journal article (peer-reviewed)abstract
    • The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown. A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR. LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind to and regulate the expression level of the cell-fate determinant Numb. CAR was able to bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream sequences. The CAR binding region in LNX was mapped to the second PDZ domain. CAR and LNX were also shown to colocalize in vivo in mammalian cells. We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.
  •  
48.
  • Vedin, Per, et al. (author)
  • Standardbroar : en aktiv beställaråtgärd för en ökad kostnadsstyrning och produktivitet, för alla!
  • 2024
  • In: Sammanställning av referat från Transportforum 2024. - Linköping : Statens väg- och transportforskningsinstitut. ; , s. 407-407
  • Conference paper (other academic/artistic)abstract
    • Trafikverket har tagit steget att börja standardisera komplexa byggdelar av infrastrukturen. Först ut är väg- och järnvägsbroar i stora infrastrukturprojekt. Det här är mer än en standardiserad ritning. Det är ett genomtänkt arbetssätt från ax till limpa. Här har alla aktörers möjligheter och utmaningar beaktas från tidig planering till förvaltning av anläggningen. Det är ett nytt unikt arbetssätt och en aktiv beställaråtgärd. För att demonstrera nyttan har arbetet skett i ett pågående projekt, Norrbotniabanan, en ny 27 mil lång järnväg mellan Umeå och Luleå med ca 250 broar. Norrbotniabanan är inte ett unikt projekt när det gäller antal broar. Andra stora järnvägsprojekt planeras i andra delar av Sverige med liknande förutsättningar. Gemensamt för alla dessa projekt är att tillgången på resurser med rätt kompetens är begränsad. Det gäller inte bara hos beställare utan även leverantörer, projektörer och entreprenörer.  Idén om att bygga enklare och effektivare är inte unik eller ny och har under decennier angripits på olika sätt från Trafikverket som beställare. Trafikverket har på olika sätt försökt ge byggbranschen förutsättningar att bli effektivare och utvecklas. Ett exempel har varit att öka andelen totalentreprenader. Entreprenader där en entreprenör både ansvarar för projekteringen och av byggandet. Ett annat exempel har varit att i större utsträckning beställa en funktion istället för att tillhandhålla detaljerade lösningar. I januari 2022 påbörjades ett arbete med att ta fram ett första förslag på arbetssätt och metodik. Resultatet är tre broar, två järnvägsbroar och en vägbro, som projekterats så långt som möjligt utan att ta hänsyn till platsspecifika förhållanden. Arbetet inkluderar alla delar i byggprocessen och alla inblandade, från projektledare i tidiga skeden till hantverkaren som river den sista formen och förvaltaren som kontinuerligt kontrollerar funktionen många år efter bron tagits i bruk.  Förutsättningarna att leda den här typen av större förändringar har stora projekt som Norrbotniabanan med långa genomförandetider och stora mängder. Det är en förutsättning för en utvecklad kostnadsstyrning och kostnadskontroll vilket här stavas Standardbroar! En aktiv beställaråtgärd som bedöms ge 70 % lägre projekteringskostnad, 20 % lägre anläggningskostnad och en halverad produktionstid. 
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49.
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