Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation

Studying gene function in the post-genome era requires methods to localize and inactivate proteins in a standardized fashion in model organisms. While genome-wide gene disruption and over-expression efforts are well on their way to vastly expand the repertoire of Drosophila tools, a complementary method to efficiently and quickly tag proteins expressed under endogenous control does not exist for fruit flies. Here, we describe the development of an efficient procedure to generate protein fusions at either terminus in an endogenous genomic context using recombineering. We demonstrate that the fluorescent protein tagged constructs, expressed under the proper control of regulatory elements, can rescue the respective mutations and enable the detection of proteins in vivo. Furthermore, we also adapted our method for use of the tetracysteine tag that tightly binds the fluorescent membrane-permeable FlAsH ligand. This technology allows us to acutely inactivate any tagged protein expressed under native control using fluorescein-assisted light inactivation and we provide proof of concept by demonstrating that acute loss of clathrin heavy chain function in the fly eye leads to synaptic transmission defects in photoreceptors. Our tagging technology is efficient and versatile, adaptable to any tag desired and paves the way to genome-wide gene tagging in Drosophila

A study of the temperature influence on biological nitrogen reduction

Sverige och dess avloppsreningsverk har, tillsammans med andra baltiska länder, problem med att uppfylla de reningskrav som finns angående utsläppen av kväve i de sjöar och hav. Att höja temperaturen i den biologiska kvävereningen är ett tänkbart alternativ för att förbättra reningen även under den årstiden då inkommande vatten är som lägst. Hammarby Sjöstadsverk har tillsammans med Oskarshamns Kommun tagit fram ett projekt kallat ITEST, Increased Technology and Efficiency in Sewage Treatment, där projektets huvudsakliga uppgift är att studera hur temperaturen påverkar den biologiska kvävereningsprocessen. Tillsammans med projektet har syftet med detta examensarbete varit att studera hur temperaturen, samt andra viktiga faktorer, påverkar den biologiska kvävereningen, och då framförallt nitrifikationen. Målet har varit att genomföra tester i en försöksanläggning i pilot-skala och att genomföra en litteraturstudie om temperaturens påverkan på kvävereningen samt kvävets påverkan på miljön i samhället. Projektet utfördes på Sjöstadsverket där en pilotanläggning med två opererande linjer hade satts upp. I den ena linjen reglerades temperaturen och den andra linjen agerade referenslinje. Prover och analyser har gjort från de båda linjerna, där kvävefraktionerna ammonium, nitrat och totalkväve har studerats. Temperaturens inverkan på denitrifikationen samt nitrifikationen har därefter studerats och diskuterats. Resultatet av litteraturstudiearbete visar att kvävereduktionen är beroende av temperaturen och att det bör vara möjligt att öka kvävereduktionen genom att höja temperaturen för den biologiska kvävereningsprocessen. Dock kunde ingen tydlig slutsats dras från de försök i testanläggningen som gjordes. Kvävereningen i de båda linjerna visade ett relativt liknande resultat trots temperaturskillnaden. Troligtvis var denna temperaturskillnad för liten för att ett tydligt resultat skulle kunna urskiljas.Sweden and its municipal wastewater treatments plants, has as other Baltic countries, problem meeting the requirement on nitrogen compounds discharged via municipal wastewater. Raising the temperature of the wastewater, in the biological nitrogen removing process, is a possible alternative in order to achiev a higher reduction efficiency during the cold season when the wastewater has a bad cleaning outcome because of the low temperature. Hammarby Sjöstadsverk has, together with Oskarshamns municipality, developed a project named ITEST,-( Increased Technology and Efficiency in Sewage Treatment), where the project is based on studying the temperature influence on the biological nitrogen reduction process. The aim with this thesis, has been to study how the temperature, but also other important factors, influence the biological nitrogen reduction process, and then mainly the nitrification process. The main objective has been to run tests in a pilot plant and to do a literature review about the temperature influence on the nitrogen reduction process and the influence on the environment. The performance of the project was at Sjöstadsverket where two testing line had been set up. The testing plant concisted of one line with adjusting temperature and another line acted as a reference line. Measurements have been performed from both lines, where the outgoing concentrations of ammonia, nitrate and totalnitrogen has been studied. From the studies a discussion of the temperature influence of the denitrification and the nitrification process has been fulfilled. The result of the literature study part of thesis thesis shows that the nitrogen reduction rate is dependent on the temperature of the wastewater and it thought to be possible to get a better treatment in the biological nitrogen removal step if the temperature can be higher. The test in the pilot plant didn´t show a clear result. The nitrogen reduction rate showeds from both lines quite similar results despite the differences of both lines temperature. The difference of temperature in the two lines was probably too small to show a clear result

A study of the temperature influence on biological nitrogen reduction

Sverige och dess avloppsreningsverk har, tillsammans med andra baltiska länder, problem med att uppfylla de reningskrav som finns angående utsläppen av kväve i de sjöar och hav. Att höja temperaturen i den biologiska kvävereningen är ett tänkbart alternativ för att förbättra reningen även under den årstiden då inkommande vatten är som lägst. Hammarby Sjöstadsverk har tillsammans med Oskarshamns Kommun tagit fram ett projekt kallat ITEST, Increased Technology and Efficiency in Sewage Treatment, där projektets huvudsakliga uppgift är att studera hur temperaturen påverkar den biologiska kvävereningsprocessen. Tillsammans med projektet har syftet med detta examensarbete varit att studera hur temperaturen, samt andra viktiga faktorer, påverkar den biologiska kvävereningen, och då framförallt nitrifikationen. Målet har varit att genomföra tester i en försöksanläggning i pilot-skala och att genomföra en litteraturstudie om temperaturens påverkan på kvävereningen samt kvävets påverkan på miljön i samhället. Projektet utfördes på Sjöstadsverket där en pilotanläggning med två opererande linjer hade satts upp. I den ena linjen reglerades temperaturen och den andra linjen agerade referenslinje. Prover och analyser har gjort från de båda linjerna, där kvävefraktionerna ammonium, nitrat och totalkväve har studerats. Temperaturens inverkan på denitrifikationen samt nitrifikationen har därefter studerats och diskuterats. Resultatet av litteraturstudiearbete visar att kvävereduktionen är beroende av temperaturen och att det bör vara möjligt att öka kvävereduktionen genom att höja temperaturen för den biologiska kvävereningsprocessen. Dock kunde ingen tydlig slutsats dras från de försök i testanläggningen som gjordes. Kvävereningen i de båda linjerna visade ett relativt liknande resultat trots temperaturskillnaden. Troligtvis var denna temperaturskillnad för liten för att ett tydligt resultat skulle kunna urskiljas.Sweden and its municipal wastewater treatments plants, has as other Baltic countries, problem meeting the requirement on nitrogen compounds discharged via municipal wastewater. Raising the temperature of the wastewater, in the biological nitrogen removing process, is a possible alternative in order to achiev a higher reduction efficiency during the cold season when the wastewater has a bad cleaning outcome because of the low temperature. Hammarby Sjöstadsverk has, together with Oskarshamns municipality, developed a project named ITEST,-( Increased Technology and Efficiency in Sewage Treatment), where the project is based on studying the temperature influence on the biological nitrogen reduction process. The aim with this thesis, has been to study how the temperature, but also other important factors, influence the biological nitrogen reduction process, and then mainly the nitrification process. The main objective has been to run tests in a pilot plant and to do a literature review about the temperature influence on the nitrogen reduction process and the influence on the environment. The performance of the project was at Sjöstadsverket where two testing line had been set up. The testing plant concisted of one line with adjusting temperature and another line acted as a reference line. Measurements have been performed from both lines, where the outgoing concentrations of ammonia, nitrate and totalnitrogen has been studied. From the studies a discussion of the temperature influence of the denitrification and the nitrification process has been fulfilled. The result of the literature study part of thesis thesis shows that the nitrogen reduction rate is dependent on the temperature of the wastewater and it thought to be possible to get a better treatment in the biological nitrogen removal step if the temperature can be higher. The test in the pilot plant didn´t show a clear result. The nitrogen reduction rate showeds from both lines quite similar results despite the differences of both lines temperature. The difference of temperature in the two lines was probably too small to show a clear result

Fibre reinforced concrete : fracture mechanics methods applied on self-compacting concrete and energetically modified binders

Fibre reinforced concrete (FRC) is a type of building material that is increasing in use. As new types on concrete develops the need to update the knowledge on the use of fibre reinforcement increases. This thesis covers test methods for FRC that ranges from standard beam tests to sophisticated uniaxial tension tests. The consequence of applying fibre reinforcement in concrete made using various modified cementitious binders has also been studied. The binders used in this study are energetically modified cements (EMC). They consist of a mix of ordinary cement and different fillers, which has been processed through a vibrating mill. The influence of fibre reinforcement in self-compacting concrete (SCC) has been studied with regard to its feasibility in full-scale production. The fibre reinforced SCC has been studied with regard to its ability to retain its workability after addition of fibres and on its toughness properties. Finally, early age concrete and its fracture mechanical properties have been modelled. FRC is often used in structures where its task is to distribute cracks originating from stresses caused by volume changes in combination with restraint. In these cases, the appearance of cracks is a time-dependent phenomenon that occurs mainly in early ages. In this study a finite element model for localisation of strains in discreete cracks, the Inner Softening Band model, has been adopted and implemented with a model for the visco-elastic behaviour of early age concrete.Godkänd; 2000; 20061117 (haneit

Temperatursprickor i betongkonstruktioner : beräkningsmetoder för hydrationsspänningar och diagram för några vanliga typfall. Del A, B och C

Godkänd; 1997; 20091027 (ysko

Design of high performance concrete structures : a Swedish design handbook

Godkänd; 1999; 20061112 (ysko)</p

Synaptic PI(3,4,5)P3 Is Required for Syntaxin1A Clustering and Neurotransmitter Release

Contains fulltext : 117715.pdf (publisher's version ) (Closed access)PI(3,4,5)P3 is a low-abundance lipid thought to play a role in the regulation of synaptic activity; however, the mechanism remains obscure. We have constructed novel split Venus-based probes and used superresolution imaging to localize PI(3,4,5)P3 at Drosophila larval neuromuscular synapses. We find the lipid in membrane domains at neurotransmitter release sites, where it concentrates with Syntaxin1A, a protein essential for vesicle fusion. Reducing PI(3,4,5)P3 availability disperses Syntaxin1A clusters and increasing PI(3,4,5)P3 levels rescues this defect. In artificial giant unilamellar vesicles, PI(3,4,5)P3 also induces Syntaxin1A domain formation and this clustering, in vitro and in vivo, is dependent on positively charged residues in the Syntaxin1A-juxtamembrane domain. Functionally, reduced PI(3,4,5)P3 causes temperature-sensitive paralysis and reduced neurotransmitter release, a phenotype also seen in animals expressing a Syntaxin1A with a mutated juxtamembrane domain. Thus, our data indicate that PI(3,4,5)P3, based on electrostatic interactions, clusters Syntaxin1A at release sites to regulate neurotransmitter release

Sports and exercise-related tendinopathies:a review of selected topical issues by participants of the second International Scientific Tendinopathy Symposium (ISTS) Vancouver 2012

<p>In September 2010, the first International Scientific Tendinopathy Symposium (ISTS) was held in Umea, Sweden, to establish a forum for original scientific and clinical insights in this growing field of clinical research and practice. The second ISTS was organised by the same group and held in Vancouver, Canada, in September 2012. This symposium was preceded by a round-table meeting in which the participants engaged in focused discussions, resulting in the following overview of tendinopathy clinical and research issues. This paper is a narrative review and summary developed during and after the second ISTS. The document is designed to highlight some key issues raised at ISTS 2012, and to integrate them into a shared conceptual framework. It should be considered an update and a signposting document rather than a comprehensive review. The document is developed for use by physiotherapists, physicians, athletic trainers, massage therapists and other health professionals as well as team coaches and strength/conditioning managers involved in care of sportspeople or workers with tendinopathy.</p>

Overview of the JET results

Since the last IAEA Conference JET has been in operation for one year with a programmatic focus on the qualification of ITER operating scenarios, the consolidation of ITER design choices and preparation for plasma operation with the ITER-like wall presently being installed in JET. Good progress has been achieved, including stationary ELMy H-mode operation at 4.5 MA. The high confinement hybrid scenario has been extended to high triangularity, lower ρ*and to pulse lengths comparable to the resistive time. The steady-state scenario has also been extended to lower ρ*and ν*and optimized to simultaneously achieve, under stationary conditions, ITER-like values of all other relevant normalized parameters. A dedicated helium campaign has allowed key aspects of plasma control and H-mode operation for the ITER non-activated phase to be evaluated. Effective sawtooth control by fast ions has been demonstrated with3He minority ICRH, a scenario with negligible minority current drive. Edge localized mode (ELM) control studies using external n = 1 and n = 2 perturbation fields have found a resonance effect in ELM frequency for specific q95values. Complete ELM suppression has, however, not been observed, even with an edge Chirikov parameter larger than 1. Pellet ELM pacing has been demonstrated and the minimum pellet size needed to trigger an ELM has been estimated. For both natural and mitigated ELMs a broadening of the divertor ELM-wetted area with increasing ELM size has been found. In disruption studies with massive gas injection up to 50% of the thermal energy could be radiated before, and 20% during, the thermal quench. Halo currents could be reduced by 60% and, using argon/deuterium and neon/deuterium gas mixtures, runaway electron generation could be avoided. Most objectives of the ITER-like ICRH antenna have been demonstrated; matching with closely packed straps, ELM resilience, scattering matrix arc detection and operation at high power density (6.2 MW m-2) and antenna strap voltages (42 kV). Coupling measurements are in very good agreement with TOPICA modelling. \ua9 2011 IAEA, Vienna