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Inom laboratoriemedicin finns utrustning som spektralfotometern som kan mäta absorption i en lösning för att bestämma koncentrationen av proteiner, aminosyror och liknande. Detta är en ofta en dyr och svårtillgänglig utrustning. Avdelningen Medicinsk Teknik - Forskning och Utveckling (MT-FoU) vid Norrlands Universitetssjukhus har fått förfrågan om att undersöka möjligheterna till att konstruera en enkel och billig UV-absorptionsfotometer. Målet var att ta fram en prototyp av en UV-absorptionsfotmoter för att kunna mäta koncentrationen av SAICA-r och S-Ado vilket förekommer i urinet vid sjukdomen Adenolosuccinat lyase deficiency. För detta examensarbete valdes Adenosine triphosphate (ATP) som mätsubstans, eftersom ATP är ett stabilt protein som är lösligt i vatten och som tål att frysa och tinas. ATP har dessutom ett absorbansspektra som ligger väldigt nära SAICA-r och S-Ado. En fungeradeprototyp av UV-absorptionsfotometern har tagits fram. Prototypen påvisar en god linjäritet (R2 = 0.9956) för koncentrationer mellan 0-50 mg/L samt en hög noggrannhet för absorbans σ=0.0006. Med dessa resultat bedöms prototyp vara kapabel till att mäta halten SAICAr och S-Ado. / In laboratory medicine there is equipment such as the spectralphotometer that can measure absorption in a solution to determine the concentration of proteins, amino acids and the like. This is an often expensive equipment and often not readily available. The Department of Biomedical Engineering - Research and Development (BE-RD) at University hospital of Umeå has been asked to investigate the possibilities of constructing a simple and cheap UV-absorption photometer. The aim of this study was to produce a functioning prototype of a UV-absorption photometer that would be able to measure the concentration of SAICA-r and S-Ado which occurs in the urine of the rare disease Adenolosuccinate lyase deficiency (ASL). For this bachelor thesis, adenosine triphosphate (ATP) was chosen as measuring substance, since ATP is a stable protein which is soluble in water and which can be frozen and defrosted. It also has an absorbance spectra which is very close to SAICA-r and S-Ado. A functioning prototype of the UV-absorption photometer has been developed. The prototype demonstrates a good linearity(R2 = 0.9956) for concentrations between 0-50 mg/L and a high accuracy for absorbance σ=0.0006. With these results, the prototype is judged to be capable of measuring the content of SAICAr and S-Ado in a urine sample.

This dissertation focuses on the development of hand-portable capillary liquid chromatography (LC) instrumentation. In this work, battery-operable nano-flow pumping systems (isocratic and gradient) were developed and integrated with portable UV-absorption detectors for capillary LC. The systems were reduced in size to acceptable weights and power usage for field operation. A major advantage of the pumps is that they do not employ a splitter, since they were specifically designed for capillary column use, thereby greatly reducing solvent consumption and waste generation. UV-absorption detectors were specifically designed and optimized for on-column detection to minimize extra-column band broadening. Initially, an isocratic nano-flow pumping system with a stop-flow injector was integrated with an on-column UV-absorption detector (254 nm). The pumping system gave excellent flow rate accuracy (<99.94%) and low percent injection carry-over (RSD 0.31%) suitable for quantitative analysis. Using sodium anthraquinone-2-sulfonate, the detector gave an LOD (S/N = 3) of 0.13 µM, which was 12 times lower than a commercial UV-absorption detector. Reversed-phase separations of a homologous series of alkyl benzenes was demonstrated. Further miniaturization of UV-absorption detection was accomplished using a 260 nm deep UV LED. The detector was small in size and weighed only 85 g (without electronics). No optical reference was included due to the low drift in the signal. Two ball lenses, one of which was integrated with the LED, were used to increase light throughput through the capillary column. Stray light was minimized by the use of a band-pass filter and an adjustable slit. Signals down to the ppb level (nM) were easily detected with a short-term noise level of 4.4 µAU, confirming a low limit of detection and low noise. The detection limit for adenosine-5'-monophosphate was 230 times lower than any previously reported values. Isocratic separations of phenolic compounds were performed using a poly(ethylene glycol) diacrylate monolithic capillary column. Finally, a novel nano-flow gradient generator integrated with a stop-flow injector was developed. Gradient performance was found to be excellent for gradient step accuracy (RSD < 1.2%, n = 4) and linear gradient reproducibility (RSD < 1.42%, n = 4). Separations of five phenols were demonstrated using the nano-flow gradient system. Efforts to develop a 405 nm laser diode-based UV-absorption detector for hemoglobin analysis were described.

Voltage-dependent anion-selective channels (VDAC), or mitochondrial porins,regulate the flow of metabolites across the mitochondrial outer membrane. They presumably span the membrane as β-barrels, but the residues forming the individual β-strands are unknown. This information is essential for understanding the structure and function of the protein. Using Neurospora VDAC as a template, published data were reassessed to delineate a unified model for porin structure Bay and Court 2002, which was subsequently refined in collaboration with Greg Runke Runke et al. 2006. The focus of this work was the development and analysis of systems for maintaining high levels of folded porin for the acquisition of high resolution data needed for model testing. The conformation of hexahistidinyl-tagged Neurospora porin in detergent was probed by fluorescence, near-UV circular dichroism and ultraviolet absorption spectroscopy. Derivatives of tryptophan and tyrosine were also examined by fluorescence spectroscopy and UV absorbance spectroscopy to model the interactions between the detergents and the amino acid side chains in the protein. Detergent-specific levels of β-strand and tyrosine exposure were observed. In all cases, the two tryptophan residues reside in weakly asymmetric, hydrophobic environments, suggesting transient tertiary interactions. Porin solubilized in these detergents forms functional channels in liposomes and membrane insertion is accompanied by increased levels of β-strand and loss of protease sensitivity. These data were used to develop mixed detergent folding systems. A mixture of SDS and dodecyl-β-D-maltopyranoside (DDM)supports a β-strand rich conformation at high protein concentrations. The tertiary contacts and protease resistance of the SDS/DDM solubilized porin are very similar to those of the protein following reconstitution into liposomes. Finally, the role of sterols in porin folding was examined, as the addition of sterols to detergent-solubilized VDAC is required for channel formation in artificial membranes. Sterols do not alter the secondary structure of VDAC, and subtle alterations to tertiary interactions were detected, suggesting that sterols do not promote an insertion-competent structure, but rather facilitate insertion into artificial bilayers. In summary, this analysis of the folded states of detergent-solubilized porin has revealed a system that maintains high concentrations of mitochondrial porin in a state that is very promising for structural studies. / February 2007

Voltage-dependent anion-selective channels (VDAC), or mitochondrial porins,regulate the flow of metabolites across the mitochondrial outer membrane. They presumably span the membrane as β-barrels, but the residues forming the individual β-strands are unknown. This information is essential for understanding the structure and function of the protein. Using Neurospora VDAC as a template, published data were reassessed to delineate a unified model for porin structure Bay and Court 2002, which was subsequently refined in collaboration with Greg Runke Runke et al. 2006. The focus of this work was the development and analysis of systems for maintaining high levels of folded porin for the acquisition of high resolution data needed for model testing. The conformation of hexahistidinyl-tagged Neurospora porin in detergent was probed by fluorescence, near-UV circular dichroism and ultraviolet absorption spectroscopy. Derivatives of tryptophan and tyrosine were also examined by fluorescence spectroscopy and UV absorbance spectroscopy to model the interactions between the detergents and the amino acid side chains in the protein. Detergent-specific levels of β-strand and tyrosine exposure were observed. In all cases, the two tryptophan residues reside in weakly asymmetric, hydrophobic environments, suggesting transient tertiary interactions. Porin solubilized in these detergents forms functional channels in liposomes and membrane insertion is accompanied by increased levels of β-strand and loss of protease sensitivity. These data were used to develop mixed detergent folding systems. A mixture of SDS and dodecyl-β-D-maltopyranoside (DDM)supports a β-strand rich conformation at high protein concentrations. The tertiary contacts and protease resistance of the SDS/DDM solubilized porin are very similar to those of the protein following reconstitution into liposomes. Finally, the role of sterols in porin folding was examined, as the addition of sterols to detergent-solubilized VDAC is required for channel formation in artificial membranes. Sterols do not alter the secondary structure of VDAC, and subtle alterations to tertiary interactions were detected, suggesting that sterols do not promote an insertion-competent structure, but rather facilitate insertion into artificial bilayers. In summary, this analysis of the folded states of detergent-solubilized porin has revealed a system that maintains high concentrations of mitochondrial porin in a state that is very promising for structural studies.

Voltage-dependent anion-selective channels (VDAC), or mitochondrial porins,regulate the flow of metabolites across the mitochondrial outer membrane. They presumably span the membrane as β-barrels, but the residues forming the individual β-strands are unknown. This information is essential for understanding the structure and function of the protein. Using Neurospora VDAC as a template, published data were reassessed to delineate a unified model for porin structure Bay and Court 2002, which was subsequently refined in collaboration with Greg Runke Runke et al. 2006. The focus of this work was the development and analysis of systems for maintaining high levels of folded porin for the acquisition of high resolution data needed for model testing. The conformation of hexahistidinyl-tagged Neurospora porin in detergent was probed by fluorescence, near-UV circular dichroism and ultraviolet absorption spectroscopy. Derivatives of tryptophan and tyrosine were also examined by fluorescence spectroscopy and UV absorbance spectroscopy to model the interactions between the detergents and the amino acid side chains in the protein. Detergent-specific levels of β-strand and tyrosine exposure were observed. In all cases, the two tryptophan residues reside in weakly asymmetric, hydrophobic environments, suggesting transient tertiary interactions. Porin solubilized in these detergents forms functional channels in liposomes and membrane insertion is accompanied by increased levels of β-strand and loss of protease sensitivity. These data were used to develop mixed detergent folding systems. A mixture of SDS and dodecyl-β-D-maltopyranoside (DDM)supports a β-strand rich conformation at high protein concentrations. The tertiary contacts and protease resistance of the SDS/DDM solubilized porin are very similar to those of the protein following reconstitution into liposomes. Finally, the role of sterols in porin folding was examined, as the addition of sterols to detergent-solubilized VDAC is required for channel formation in artificial membranes. Sterols do not alter the secondary structure of VDAC, and subtle alterations to tertiary interactions were detected, suggesting that sterols do not promote an insertion-competent structure, but rather facilitate insertion into artificial bilayers. In summary, this analysis of the folded states of detergent-solubilized porin has revealed a system that maintains high concentrations of mitochondrial porin in a state that is very promising for structural studies.

miRNAs are short non-coding RNA molecules which play role in post-transcriptional regulation of gene expression. Thus, miRNAs are related to a variety of physiological processes and diseases, e.g. some tumours or viral infections. In this diploma thesis we studied temperature-induced dissociation of complexes that simulated pairing of miRNA with target mRNA. For these purposes we used UV absorption spectroscopy and Raman spectroscopy. Measured spectra were then treated by means of a factor analysis. The aim of the thesis was to determine thermodynamic parameters of the complexes dissociation and to clarify how inner loops and bulges affect the stability of oligonucleotide duplexes. Also, we have tested the suitability of the used approach for study of oligonucleotide complexes. We have shown that factor analysis of temperature dependent UV absorption spectra is a proper base to determine thermodynamic parameters of the duplex dissociation. Raman spectra were affected by large errors, nevertheless they enabled to observe some structural changes related to the transition. It has been shown that the bulge loop present in the studied complex reduces both its flexibility and the energetic preference of its formation.

Syftet med examensarbetet har varit att hitta lämpliga textila material för en klimatsmart rullgardin med flera olika skikt skapad av företaget Climate Curtains, samt att ta fram en väl genomtänkt design som är anpassad för miljön den är tänkt att hänga i, nämligen skolor och kontor. Den klimatsmarta rullgardinen ska ha ett absorberande mattsvart material närmast glasfönstret för att fånga upp UV-strålning och därmed bli varmt. Värmen ska sedan föras in i rummet med hjälp av fläktar som sitter monterade inuti gardinstången. En solcellsfilm ska även fästas på skiktet närmast fönstret och har därför tagits med i beräkningarna, dock är solcellsfilmen fortfarande under utveckling. Tanken med solcellsfilmen är först och främst att den ska förse fläktarna med energi men även webbelysning eller laddning av mobil och dator vid överflöd av energi ska vara möjligt. Rullgardinen ska även ha ett vitt reflekterande material in mot rummet för att möjligheten att vända gardinen ska finnas. Under sommartid kan den vända gardinen istället reflektera bort UV-ljus. Mellan dessa två skikt ska ett antal mellanskikt hänga för att erhålla bättre isoleringsegenskaper samtidigt som buller och oljud minskas. Det har även varit relevant att undersöka om flamskyddsmedel bör användas eftersom att gardinen ska hänga i offentliga miljöer. Examensarbetet har bestått av litteraturstudier och praktiska experiment. Experimenten som utfördes var nöthärdighets-, flexibilitets-, reflektionstest samt säkerställning av färg. Materialen som har undersökts har alla varit gjorda av 100% polyester och två alternativa material valdes för vardera skikt. Polyester valdes till alla skikt dels för att det är billigt och dels för att polyester visade sig ha mycket goda egenskaper för de krav som ställdes. Ytterskiktet som är närmast glasfönstret samt innerskiktet in mot rummet valdes till vävar med olika bindningar. Till ytterskiktet jämfördes en tvåskaftsväv med en kypertväv där tvåskaftsväven uppvisade bäst resultat i alla testerna. Kypertväven färgade av sig och nöttes ner i nöthärdighetstestet vilket inte tvåskaftsväven gjorde. Flexibilitets- och reflektionstestet visade goda resultat för båda vävarna. I reflektionstestet pikade tvåskaftsvävens reflektionskurva en aning i det röda spektrat, vilket innebar att den hade en viss röd nyans i sig. Säkerställning av färg gick inte att utföra för de svarta vävarna då svart färg finns i oändligt många olika nyanser. Till det vita innerskiktet jämfördes en tvåskaftsväv med en satinväv. Båda vävarna hade relativt lika resultat i nöthärdighets- samt flexibilitetstestet men satinväven fick bättre resultat i reflektionstestet vilket var avgörande. Vid säkerställning av färg testades den vita färgen i ett ljusskåp i olika ljus för att kontrollera dess vithet. Det visade sig att båda vävarna innehöll optiskt vitmedel vilket även framkom i reflektionstestet då de reflekterade över 100 %. Till mellanskikten jämfördes två olika nonwovenmaterial som består av ihåliga polyesterfibrer. Ihåliga fibrer ha bra ljudabsorption vilket kan vara bra i miljöer såsom skolor och kontor där det kan vara mycket buller och höga ljudnivåer. Det ena materialet vägde 100g/m2 och det andra 250g/m2. Ett nöthärdighets- och flexibilitetstest utfördes på dessa nonwovenmaterial. De båda fick godkänt resultat i flexibilitetstestet men nonwoven 100 höll inte måttet i nöthärdighetstestet varav nonwoven 250 är mest lämpat för rullgardinen. Litteraturstudiens resultat tyder på att det inte finns några krav på flamskyddsmedel i gardiner i offentlig miljö utan endast rekommendationer. Det är alltså upp till företag att bestämma om de vill flamskydda gardinerna. De finns en mängd olika flamskyddsmedelsvarianter men de har alla en sak gemensamt och det är att de är fettlösliga och långlivade, vilket gör att de är svåra att bryta ned. Gardinen designades i en stilren och klassisk design. En kornisch utformades dels för att dölja gardinstången och dels för att erhålla ett snyggare helhetsintryck. Gardinen kommer att ha en fåll längst ned på det yttre och det inre skiktet. Inuti fållen kommer en plastskena läggas för att få tyngd i materialet och därmed ett bättre fall. Fållen ger även gardinen en lyxigare känsla. Resterande kanter på alla materialen för alla skikt valdes att lasras. Med laserskärning erhålls fina kanter som inte fransar sig alls och laserskärning garanterar även att det inte blir någon nötning på produkten under skärningen vilket är fördelaktigt på en rullgardin då den rullas upp och ner mycket. Skisser ritades upp i Kaledo Style för att få en tydligare bild av hur rullgardinen kan komma att se ut. Eventuella tillval, mönster på det vita skiktet samt solceller i olika storlekar, kan även göras på gardinen beroende på kundens önskemål. För att kunna applicera mönstret på det textila materialet undersöktes tre alternativa tryckmönstermetoder; rollerprinting, roterande screentryck samt digital inktryck. De två sistnämnda lämpar sig bäst för ändamålet. / The aim of the thesis was to find the optimal textile materials for a climate-smart blind with multiple layers created by the company Climate Curtains. The aim was also to develop a well thought out design that is adapted to the environments it is supposed to hang in, which are schools and offices. The climate-smart blind should have an absorbing black matte material closest to the glass window to capture UV radiation and thereby become warm. The heat will then be brought into the room with the help of fans that are mounted inside the curtain rod. A solar cell film should also be attached to the layer closest to the window and have therefore been included in the calculations, however, the solar film is still under development. The idea of a solar cell film is first and foremost that it will provide fans with energy but also charging of mobile and computer at the abundance of energy. The blind must also have a white reflective material into the room for the possibility of turning the curtain in the summer. This leads to a reflection of the UV light instead of absorption. Between these two layers, a number of intermediate layers will hang to provide isolation of air and noise reduction. The amount of flame retardants have also been relevant to examine because the curtain will hang in public places. There are no requirements in public places when it comes to flame retardants, but there are recommendations published. A solar cell film should also be attached to the layer closest to the window and have therefore been included in the calculations. However, the solar film is still under development. The work has consisted of literature studies, laboratory work and compilation of discussion and results. The laboratory work included abrasion resistance-, flexibility-, reflection test and a test to secure the colors. The materials studied have all been made of 100% polyester and two alternative materials were chosen for each layer. Polyester was chosen to all layers partly because it is cheap and secondly because polyester was found to have very good qualities for the demands made. The outer layer closest to the window and the inner layer into the room consists of fabrics made of weaves with different constructions. For the outer layer a plain weave and a twill weave were chosen to be compared against each other. The plain weave got the best results in all of the tests. For the inner layer a satin weave and a plain weave were compared, and the satin weave got the best results in all the tests. Furthermore, the layers in between the weave textiles are made of hollow polyester fibers as a nonwoven material. Hollow fibers have good sound absorption, which can be useful in environments such as schools and offices where there may be much noise and high sound levels. One material weighing 100 g/m2 and the other 250 g/m2. An abrasion resistance- and flexibility test was conducted on these nonwoven materials. They both got approved results in the flexibility test. However, nonwoven 100 was not good enough in the abrasion resistance test and therefor nonwoven 250 is the most suitable middle layer for the blind. The curtain was designed in a stylish and classic design. A cornice designed partly to hide the curtain rod and partly to obtain a more attractive overall impression. The blind will have a hem at the bottom of the outer and the inner layer. Inside the hem a plastic splint is added to give better stability. The hem also gives the curtain a luxurious feel. The remaining edges on all materials for all layers were chosen to be lasered. With laser cutting fine edges are obtained.4Sketches were drawn up in Kaledo Style to get a clearer picture of how the blind could look like. Any optional patterns on the white layer and the solar cells of different sizes, can also be made on the curtain, depending on customer requirements. In order to apply the pattern to the textile material three alternative methods was studied; roller printing, rotary screen printing and digital ink print. Screenprinting and digital printing was best suited for the purpose.

L'anomalie isotopique de l'ozone observée au début des années 1980 a été la première manifestation de fractionnement isotopique indépendant de la masse d'origine chimique. Attribuée au départ, essentiellement au processus de formation de l'ozone, les travaux récents mettent en évidence d'autres contributions telles que la photodissociation. Cette thèse utilise une approche théorique basée sur des calculs ab initio de chimie et dynamique quantique pour déterminer les sections efficaces d'absorption et leurs variations isotopiques. Ces sections efficaces permettent d'étudier la photodissociation de l'ozone par irradiation du soleil (flux actinique) ; ce qui permet une évaluation précise du processus de photodissociation à l'enrichissement isotopique de l'ozone. Ces résultats pourront être intégrés dans un modèle global prenant en compte à la fois les processus de formation et de destruction de l'ozone. / The ozone isotopic anomaly observed in early 1980s has been the first occurance of mass independent fractionation of chemical origin. Assigned first, essentially to the ozone formation process, recent works show additional contributions such as the photolysis process. This thesis uses a theoretical approach based on ab initio quantum chemistry and dynamics computations to obtain the absorption cross sections and their isotopic variations. The absorption cross sections allow to study ozone's photodissociation by solar irradiation (actinic flux) ; which enables an accurate evaluation of the photodissociation process in ozone's isotopic enrichment. Obtained results could be integrated in a global model that takes into account both ozone's formation and destruction processes.

Highly profitable black caviar market and the depletion of wild sturgeon stocks warrant improvements in sturgeon aquaculture. Therefore, chromosomal manipulations, particularly gynogenesis, are focused on for increasing the ratio of females over males in progeny. The present study focused on optimizing chromosomal manipulations in sturgeons, particularly gynogenesis. The reasons of low survival rates were analyzed and the critical steps of gynogenesis induction processes were optimized. In addition, alternative ways of DNA inactivation in sperms were investigated, as well as the influence of native light-dependent DNA repair mechanisms on gynogenesis induction. Methods of interspecific gynogenesis usage for simplifying gynogenetic progeny separation were also proposed. Spectrophotometry analysis was used to investigate the ability of UV light, as the most common DNA inactivating agent, to penetrate into sperm. In addition, investigation of UV-irradiated sperm motility and results of partial gynogenesis induction showed that low transparency of sperms for UV-light can cause significant heterogeneity of UV-irradiation. As a result, a proper dilution of sperm was suggested as a critical step for homogeneous UV-irradiation of samples. Gynogenesis in sterlet was induced with chemical agents that damage sperm DNA, as an alternative to UV irradiation for applied in large-scale production of gynogenotes. All tested substances showed ability to inactivate DNA in spermatozoa, and thus producing gynogenotes. Negative impact of treatments with chemical agents on the sperm motility was observed. Subsequently, these treatments had a low efficiency of gynogenesis induction. The highest percentage of produced gynogenetic larvae 19.8 ? 8.9% was obtained by treatment with aminomethyl-4,5?,8-trimethylpsoralen (AMT) at 50 ?M followed by UV-A (360 nm) irradiation at dose of 900 J/m2. Therefore, this treatment could be used as a substitute for commonly used UV-C irradiation, e.g., in the case of large volumes of sperm. Detailed investigation of photoreactivation in sturgeon sperm revealed a significant level of light-dependent DNA restoration in sperms irradiated with high doses of UV-C light. Induction of gynogenesis with UV-C irradiation followed by exposure to visible light resulted in significant deviations from the typical Hertwig effect. In contrast, the red light with a wavelength of more than 600 nm did not result in decreased DNA damage, instead a moderate increase in damage was observed, i.e., it did not induce photoreactivation. Therefore, the use of infrared light to illuminate work stations during the induction of gynogenesis is suggested. The use of interspecific gynogenesis, particularly gametes of sturgeon species with different ploidy levels, was suggested as a way to simplify the separation of gynogenotes. In addition, application of this method allowed studying the effectiveness of DNA-inactivation and ploidy restoration treatments separately, as well as evaluation of fitness parameters and survival rates in each group of progeny without the physical separation of fish. Finally, the protocol for tetraploidization in sterlet was optimized for the prospective using tetraploid individuals for the induction of gynogenesis and androgenesis with diploid eggs and sperm. In conclusion, the described methods and protocols allowed gynogenesis induction in sturgeons with a survival rate sufficient for aquaculture, taking into consideration their high fertility, although further studies of the consequences of this treatment on fish is required.