Laser-TIG–hybridihitsauksen soveltuvuustutkimus

Tässä tutkimuksessa selvitetään ilman hitsauslisäainetta tapahtuvan laser–TIG–hybridihitsausprosessin soveltuvuus 6 mm ja 8 mm paksujen päittäisliitettyjen S355 K2 ja Laser 355 MC rakenneterästen hitsaukseen. Hitsien tarkastelussa huomio kiinnitetään hitsausnopeuteen, hitsien tunkeumaan, liittämistehokkuuteen, hitsien kovuuteen ja hitsausliitoksen ulkonäköön. Muita tutkittavia asioita ovat laser-TIG-hybridihitsattujen levyjen muodonmuutokset ja suuresta hitsausnopeudesta sekä pienestä t8/5 jäähtymisajasta johtuvat mahdolliset kylmähalkeamat. Laser-TIG-hybridihitsejä verrataan robotti-MAG- ja käsin MAG-hitseihin sekä kaarihitsausstandardin SFS-EN ISO 5817 hitsiluokkien mukaisiin raja-arvoihin. Laser-TIG-hybridihitsausprosessissa TIG-valokaari mahdollistaa tasaisen ja lähes roiskeettoman hitsin ja lasersäde aikaansaa syvän tunkeuman sekä tasalaatuisen juurihitsin. Laser-TIG-hybridihitsausprosessilla 6 mm paksut S355 K2 rakenneteräslevyt on mahdollista hitsata levyn yhdeltä puolelta kerralla valmiiksi. Paksummat 8 mm levyt voidaan hitsata levyn yhdeltä tai molemmilta puolilta suoritettavalla laser-TIG-hybridihitsauksella. Laser-TIG-hybridihitsausprosessilla hitsatut hitsit ovat hyvin siistejä ja lähes roiskeettomia. Verrattaessa laser-TIG-hybridihitsausprosessia muihin hitsausprosesseihin sen voidaan todeta olevan erittäin kilpailukykyinen 6 mm paksujen päittäisliitettyjen rakenneterästen hitsaamisessa, mutta se soveltuu myös 8 mm paksujen rakenneterästen hitsaamiseen. Tutkitut hitsit täyttävät kaarihitsausstandardin SFS-EN ISO 5817 B- ja D-hitsiluokkien mukaiset raja-arvot. Vertailukokeet 6 mm paksulla S355 rakenneteräksellä osoittavat, että yhdeltä puolelta suoritettavan laser-TIG-hybridihitsauksen hitsausnopeus on robotti-MAG-hitsaukseen verrattuna yli nelinkertainen ja MAG-käsinhitsaukseen verrattuna yli viisinkertainen. Laser-TIG-hybridihitsauksessa liittämistehokkuus on noin viisinkertainen robotti-MAGhitsaukseen verrattuna. Molemmilta puolilta suoritettavalla laser-TIG-hybridihitsauksella voidaan 8 mm paksulla S355 rakenneteräksellä saavuttaa noin kolminkertainen hitsausnopeus ja liittämistehokkuus robotti-MAG-hitsaukseen verrattuna. Laser-TIG-hybridihitsauksessa TIG-kaaren tuoman lisälämmön ansiosta suurillakin hitsausnopeuksilla (1 m/min) voidaan saavuttaa edulliset kovuusarvot. Kovuusmittausten tulosten perusteella 6 mm ja 8 mm paksujen S355 K2 ja Laser 355 MC rakenneterästen hitsit eivät ylittäneet kaarihitsausstandardin määrittelemää 350 HV kovuuden enimmäisrajaa. Laser-TIG-hybridihitsauksen edullisesta lämmöntuonnista johtuen levyjen pituus- ja poikittaissuuntaiset muodonmuutokset ovat noin 80 prosenttia pienemmät kuin käsin suoritettavassa MAG-hitsauksessa. Laser-TIG-hybridihitsausprosessilla käytetään I-railoa, mutta robotti-MAG- ja käsin MAG-hitsausprosesseilla joudutaan käyttämään V-railoa, jolloin lämmöntuonti ja siitä johtuvat muodonmuutokset ovat suuremmat. Korkea liittämistehokkuus ja edullinen lämmöntuonti merkitsevät vähäisempiä muodonmuutoksia ja siten merkittäviä säästöjä työ-, materiaali- ja energiakustannuksissa. 8 mm ja sitä paksummilla S355 rakenneteräksillä levyn yhdeltä puolelta suoritettava päittäisliitoksen hitsaaminen on laser-TIG hybridihitsauksella haastavaa, koska yli 200 A:n TIG-kaarivirralla suuri metallisula aiheuttaa avaimenreiän sulkeutumisen ja avaimenreiän alaosaan muodostuu kaasukuplia. Tästä voidaan tehdä sellainen johtopäätös, että päittäisliitettävien levyjen ilmarakoa pitäisi kasvattaa niin suureksi, että avaimenreiän sulavirtaus ei pääse estymään. Yli 0,25 mm:n ilmarako edellyttää lasersäteen vaaputusta tai säteen halkaisijan kasvattamista. Ilmaraon kasvattaminen edellyttää myös lisäaineen käyttöä. Tutkimustulosten perusteella laser-TIG-hybridihitsausprosessilla voidaan saavuttaa merkittäviä etuja ja kustannussäästöjä, joten sen hyödyntämistä kannattaa harkita 8 mm ja sitä ohuempien päittäisliitettävien tuotteiden konepaja- ja tehdastuotannossa. Laser-TIGhybridihitsausprosessi soveltuu esimerkiksi seuraavien tuotteiden hitsaamiseen: päittäisliitettävät levyt, palkit, koneenosat, putket, säiliöt ja erilaiset pyörähdyskappaleet.

Energy and raw material consumption analysis of powder bed fusion: case study: CNC machining and laser additive manufacturing

Laser additive manufacturing (LAM), known also as 3D printing, is a powder bed fusion (PBF) type of additive manufacturing (AM) technology used to manufacture metal parts layer by layer by assist of laser beam. The development of the technology from building just prototype parts to functional parts is due to design flexibility. And also possibility to manufacture tailored and optimised components in terms of performance and strength to weight ratio of final parts. The study of energy and raw material consumption in LAM is essential as it might facilitate the adoption and usage of the technique in manufacturing industries. The objective this thesis was find the impact of LAM on environmental and economic aspects and to conduct life cycle inventory of CNC machining and LAM in terms of energy and raw material consumption at production phases. Literature overview in this thesis include sustainability issues in manufacturing industries with focus on environmental and economic aspects. Also life cycle assessment and its applicability in manufacturing industry were studied. UPLCI-CO2PE! Initiative was identified as mostly applied exiting methodology to conduct LCI analysis in discrete manufacturing process like LAM. Many of the reviewed literature had focused to PBF of polymeric material and only few had considered metallic materials. The studies that had included metallic materials had only measured input and output energy or materials of the process and compared to different AM systems without comparing to any competitive process. Neither did any include effect of process variation when building metallic parts with LAM. Experimental testing were carried out to make dissimilar samples with CNC machining and LAM in this thesis. Test samples were designed to include part complexity and weight reductions. PUMA 2500Y lathe machine was used in the CNC machining whereas a modified research machine representing EOSINT M-series was used for the LAM. The raw material used for making the test pieces were stainless steel 316L bar (CNC machined parts) and stainless steel 316L powder (LAM built parts). An analysis of power, time, and the energy consumed in each of the manufacturing processes on production phase showed that LAM utilises more energy than CNC machining. The high energy consumption was as result of duration of production. Energy consumption profiles in CNC machining showed fluctuations with high and low power ranges. LAM energy usage within specific mode (standby, heating, process, sawing) remained relatively constant through the production. CNC machining was limited in terms of manufacturing freedom as it was not possible to manufacture all the designed sample by machining. And the one which was possible was aided with large amount of material removed as waste. Planning phase in LAM was shorter than in CNC machining as the latter required many preparation steps. Specific energy consumption (SEC) were estimated in LAM based on the practical results and assumed platform utilisation. The estimated platform utilisation showed SEC could reduce when more parts were placed in one build than it was in with the empirical results in this thesis (six parts).

Laser scribing of thin-film PV modules

This study is a literature review on laser scribing in monolithically interconnected thin-film PV modules, focusing on efficiency of modules based on absorber materials CIGS, CdTe and a-Si. In thin-film PV module manufacturing scribing is used to interconnect individual cells monolithically by P1, P2 and P3 scribes. Laser scribing has several advantages compared to mechanical scribing for this purpose. However, laser scribing of thin-films can be a challenging process and may induce efficiency reducing defects. Some of these defects can be avoided by improving optimisation or processing methods.

The effect of focal point parameters in fiber laser welding of structural steel

In this thesis the effect of focal point parameters in fiber laser welding of structural steel is studied. The goal is to establish relations between laser power, focal point diameter and focal point position with the resulting quality, weld-bead geometry and hardness of the welds. In the laboratory experiments, AB AH36 shipbuilding steel was welded in an I-butt joint configuration using IPG YLS-10000 continuous wave fiber laser. The quality of the welds produced were evaluated based on standard SFS-EN ISO 13919-1. The weld-bead geometry was defined from the weld cross-sections and Vickers hardness test was used to measure hardness's from the middle of the cross-sections. It was shown that all the studied focal point parameters have an effect on the quality, weld-bead geometry and hardness of the welds produced.

Effect of gallium-arsenide laser, gallium-aluminum-arsenide laser and healing ointment on cutaneous wound healing in Wistar rats

This study determined the effects of gallium-aluminum-arsenide laser (GaAlAs), gallium-arsenide laser (GaAs) and Dersani® healing ointment on skin wounds in Wistar rats. The parameters analyzed were: type I and III collagen fiber concentrations as well as the rate of wound closure. Five wounds, 12 mm in diameter, were made on the animals’ backs. The depth of the surgical incision was controlled by removing the epithelial tissue until the dorsal muscular fascia was exposed. The animals were anesthetized with ketamine and xylazine via intraperitoneal injection. The rats were randomly divided into five groups of 6 animals each, according to the treatment received. Group 1 (L4): GaAs laser (4 J/cm²); group 2 (L30): GaAlAs laser (30 J/cm²); group 3 (L60): GaAlAs laser (60 J/cm²); group 4 (D): Dersani® ointment; group 5 (control): 0.9% saline. The applications were made daily over a period of 20 days. Tissue fragments were stained with picrosirius to distinguish type I collagen from type III collagen. The collagen fibers were photo-documented and analyzed using the Quantum software based on the primary color spectrum (red, yellow and blue). Significant results for wound closing rate were obtained for group 1 (L4), 7.37 mm/day. The highest concentration of type III collagen fibers was observed in group 2 (L30; 37.80 ± 7.10%), which differed from control (29.86 ± 5.15%) on the 20th day of treatment. The type I collagen fibers of group 1 (L4; 2.67 ± 2.23%) and group 2 (L30; 2.87 ± 2.40%) differed significantly from control (1.77 ± 2.97%) on the 20th day of the experiment.

Low-level red laser therapy alters effects of ultraviolet C radiation on Escherichia coli cells

Low-level lasers are used at low power densities and doses according to clinical protocols supplied with laser devices or based on professional practice. Although use of these lasers is increasing in many countries, the molecular mechanisms involved in effects of low-level lasers, mainly on DNA, are controversial. In this study, we evaluated the effects of low-level red lasers on survival, filamentation, and morphology of Escherichia colicells that were exposed to ultraviolet C (UVC) radiation. Exponential and stationary wild-type and uvrA-deficientE. coli cells were exposed to a low-level red laser and in sequence to UVC radiation. Bacterial survival was evaluated to determine the laser protection factor (ratio between the number of viable cells after exposure to the red laser and UVC and the number of viable cells after exposure to UVC). Bacterial filaments were counted to obtain the percentage of filamentation. Area-perimeter ratios were calculated for evaluation of cellular morphology. Experiments were carried out in duplicate and the results are reported as the means of three independent assays. Pre-exposure to a red laser protected wild-type and uvrA-deficient E. coli cells against the lethal effect of UVC radiation, and increased the percentage of filamentation and the area-perimeter ratio, depending on UVC fluence and physiological conditions in the cells. Therapeutic, low-level red laser radiation can induce DNA lesions at a sub-lethal level. Consequences to cells and tissues should be considered when clinical protocols based on this laser are carried out.

Feasibility of industrial implementation of laser cutting into paper making machine

Laser cutting implementation possibilities into paper making machine was studied as the main objective of the work. Laser cutting technology application was considered as a replacement tool for conventional cutting methods used in paper making machines for longitudinal cutting such as edge trimming at different paper making process and tambour roll slitting. Laser cutting of paper was tested in 70’s for the first time. Since then, laser cutting and processing has been applied for paper materials with different level of success in industry. Laser cutting can be employed for longitudinal cutting of paper web in machine direction. The most common conventional cutting methods include water jet cutting and rotating slitting blades applied in paper making machines. Cutting with CO2 laser fulfils basic requirements for cutting quality, applicability to material and cutting speeds in all locations where longitudinal cutting is needed. Literature review provided description of advantages, disadvantages and challenges of laser technology when it was applied for cutting of paper material with particular attention to cutting of moving paper web. Based on studied laser cutting capabilities and problem definition of conventional cutting technologies, preliminary selection of the most promising application area was carried out. Laser cutting (trimming) of paper web edges in wet end was estimated to be the most promising area where it can be implemented. This assumption was made on the basis of rate of web breaks occurrence. It was found that up to 64 % of total number of web breaks occurred in wet end, particularly in location of so called open draws where paper web was transferred unsupported by wire or felt. Distribution of web breaks in machine cross direction revealed that defects of paper web edge was the main reason of tearing initiation and consequent web break. The assumption was made that laser cutting was capable of improvement of laser cut edge tensile strength due to high cutting quality and sealing effect of the edge after laser cutting. Studies of laser ablation of cellulose supported this claim. Linear energy needed for cutting was calculated with regard to paper web properties in intended laser cutting location. Calculated linear cutting energy was verified with series of laser cutting. Practically obtained laser energy needed for cutting deviated from calculated values. This could be explained by difference in heat transfer via radiation in laser cutting and different absorption characteristics of dry and moist paper material. Laser cut samples (both dry and moist (dry matter content about 25-40%)) were tested for strength properties. It was shown that tensile strength and strain break of laser cut samples are similar to corresponding values of non-laser cut samples. Chosen method, however, did not address tensile strength of laser cut edge in particular. Thus, the assumption of improving strength properties with laser cutting was not fully proved. Laser cutting effect on possible pollution of mill broke (recycling of trimmed edge) was carried out. Laser cut samples (both dry and moist) were tested on the content of dirt particles. The tests revealed that accumulation of dust particles on the surface of moist samples can take place. This has to be taken into account to prevent contamination of pulp suspension when trim waste is recycled. Material loss due to evaporation during laser cutting and amount of solid residues after cutting were evaluated. Edge trimming with laser would result in 0.25 kg/h of solid residues and 2.5 kg/h of lost material due to evaporation. Schemes of laser cutting implementation and needed laser equipment were discussed. Generally, laser cutting system would require two laser sources (one laser source for each cutting zone), set of beam transfer and focusing optics and cutting heads. In order to increase reliability of system, it was suggested that each laser source would have double capacity. That would allow to perform cutting employing one laser source working at full capacity for both cutting zones. Laser technology is in required level at the moment and do not require additional development. Moreover, capacity of speed increase is high due to availability high power laser sources what can support the tendency of speed increase of paper making machines. Laser cutting system would require special roll to maintain cutting. The scheme of such roll was proposed as well as roll integration into paper making machine. Laser cutting can be done in location of central roll in press section, before so-called open draw where many web breaks occur, where it has potential to improve runability of a paper making machine. Economic performance of laser cutting was done as comparison of laser cutting system and water jet cutting working in the same conditions. It was revealed that laser cutting would still be about two times more expensive compared to water jet cutting. This is mainly due to high investment cost of laser equipment and poor energy efficiency of CO2 lasers. Another factor is that laser cutting causes material loss due to evaporation whereas water jet cutting almost does not cause material loss. Despite difficulties of laser cutting implementation in paper making machine, its implementation can be beneficial. The crucial role in that is possibility to improve cut edge strength properties and consequently reduce number of web breaks. Capacity of laser cutting to maintain cutting speeds which exceed current speeds of paper making machines what is another argument to consider laser cutting technology in design of new high speed paper making machines.

Use of airborne laser scanner data in demanding forest conditions

Most of the applications of airborne laser scanner data to forestry require that the point cloud be normalized, i.e., each point represents height from the ground instead of elevation. To normalize the point cloud, a digital terrain model (DTM), which is derived from the ground returns in the point cloud, is employed. Unfortunately, extracting accurate DTMs from airborne laser scanner data is a challenging task, especially in tropical forests where the canopy is normally very thick (partially closed), leading to a situation in which only a limited number of laser pulses reach the ground. Therefore, robust algorithms for extracting accurate DTMs in low-ground-point-densitysituations are needed in order to realize the full potential of airborne laser scanner data to forestry. The objective of this thesis is to develop algorithms for processing airborne laser scanner data in order to: (1) extract DTMs in demanding forest conditions (complex terrain and low number of ground points) for applications in forestry; (2) estimate canopy base height (CBH) for forest fire behavior modeling; and (3) assess the robustness of LiDAR-based high-resolution biomass estimation models against different field plot designs. Here, the aim is to find out if field plot data gathered by professional foresters can be combined with field plot data gathered by professionally trained community foresters and used in LiDAR-based high-resolution biomass estimation modeling without affecting prediction performance. The question of interest in this case is whether or not the local forest communities can achieve the level technical proficiency required for accurate forest monitoring. The algorithms for extracting DTMs from LiDAR point clouds presented in this thesis address the challenges of extracting DTMs in low-ground-point situations and in complex terrain while the algorithm for CBH estimation addresses the challenge of variations in the distribution of points in the LiDAR point cloud caused by things like variations in tree species and season of data acquisition. These algorithms are adaptive (with respect to point cloud characteristics) and exhibit a high degree of tolerance to variations in the density and distribution of points in the LiDAR point cloud. Results of comparison with existing DTM extraction algorithms showed that DTM extraction algorithms proposed in this thesis performed better with respect to accuracy of estimating tree heights from airborne laser scanner data. On the other hand, the proposed DTM extraction algorithms, being mostly based on trend surface interpolation, can not retain small artifacts in the terrain (e.g., bumps, small hills and depressions). Therefore, the DTMs generated by these algorithms are only suitable for forestry applications where the primary objective is to estimate tree heights from normalized airborne laser scanner data. On the other hand, the algorithm for estimating CBH proposed in this thesis is based on the idea of moving voxel in which gaps (openings in the canopy) which act as fuel breaks are located and their height is estimated. Test results showed a slight improvement in CBH estimation accuracy over existing CBH estimation methods which are based on height percentiles in the airborne laser scanner data. However, being based on the idea of moving voxel, this algorithm has one main advantage over existing CBH estimation methods in the context of forest fire modeling: it has great potential in providing information about vertical fuel continuity. This information can be used to create vertical fuel continuity maps which can provide more realistic information on the risk of crown fires compared to CBH.

Real-time adaptive control of ultra-fast laser scribing process with spectrometer online monitoring

There exist several researches and applications about laser welding monitoring and parameter control but not a single one have been created for controlling of laser scribing processes. Laser scribing is considered to be very fast and accurate process and thus it would be necessary to develop accurate turning and monitoring system for such a process. This research focuses on finding out whether it would be possible to develop real-time adaptive control for ultra-fast laser scribing processes utilizing spectrometer online monitoring. The thesis accurately presents how control code for laser parameter tuning is developed using National Instrument's LabVIEW and how spectrometer is being utilized in online monitoring. Results are based on behavior of the control code and accuracy of the spectrometer monitoring when scribing different steel materials. Finally control code success is being evaluated and possible development ideas for future are presented.

Real-time monitoring of laser scribing process utilizing high-speed camera

Currently, laser scribing is growing material processing method in the industry. Benefits of laser scribing technology are studied for example for improving an efficiency of solar cells. Due high-quality requirement of the fast scribing process, it is important to monitor the process in real time for detecting possible defects during the process. However, there is a lack of studies of laser scribing real time monitoring. Commonly used monitoring methods developed for other laser processes such a laser welding, are sufficient slow and existed applications cannot be implemented in fast laser scribing monitoring. The aim of this thesis is to find a method for laser scribing monitoring with a high-speed camera and evaluate reliability and performance of the developed monitoring system with experiments. The laser used in experiments is an IPG ytterbium pulsed fiber laser with 20 W maximum average power and Scan head optics used in the laser is Scanlab’s Hurryscan 14 II with an f100 tele-centric lens. The camera was connected to laser scanner using camera adapter to follow the laser process. A powerful fully programmable industrial computer was chosen for executing image processing and analysis. Algorithms for defect analysis, which are based on particle analysis, were developed using LabVIEW system design software. The performance of the algorithms was analyzed by analyzing a non-moving image from the scribing line with resolution 960x20 pixel. As a result, the maximum analysis speed was 560 frames per second. Reliability of the algorithm was evaluated by imaging scribing path with a variable number of defects 2000 mm/s when the laser was turned off and image analysis speed was 430 frames per second. The experiment was successful and as a result, the algorithms detected all defects from the scribing path. The final monitoring experiment was performed during a laser process. However, it was challenging to get active laser illumination work with the laser scanner due physical dimensions of the laser lens and the scanner. For reliable error detection, the illumination system is needed to be replaced.

Dietary Strategies to Optimize Wound Healing after Periodontal and Dental Implant Surgery: An Evidence-Based Review

Methods to optimize healing through dietary strategies present an attractive option for patients, such that healing from delicate oral surgeries occurs as optimally as possible with minimal patient-meditated complications through improper food choices. This review discusses findings from studies that have investigated the role of diet, either whole foods or individual dietary components, on periodontal health and their potential role in wound healing after periodontal surgery. To date, research in this area has largely focused on foods or individual dietary components that may attenuate inflammation or oxidant stress, or foster de novo bone formation. These studies suggest that a wide variety of dietary components, including macronutrients and micronutrients, are integral for optimal periodontal health and have the potential to accelerate oral wound healing after periodontal procedures. Moreover, this review provides guidance regarding dietary considerations that may help a patient achieve the best possible outcome after a periodontal procedure.

Investigation of femtosecond laser technology for the fabrication of drug nanocrystals in suspension

La technique du laser femtoseconde (fs) a été précédemment utilisée pour la production de nanoparticules d'or dans un environnement aqueux biologiquement compatible. Au cours de ce travail de maîtrise, cette méthode a été investiguée en vue d'une application pour la fabrication de nanocristaux de médicament en utilisant le paclitaxel comme modèle. Deux procédés distincts de cette technologie à savoir l'ablation et la fragmentation ont été étudiés. L'influence de la puissance du laser, de point de focalisation, et de la durée du traitement sur la distribution de taille des particules obtenues ainsi que leur intégrité chimique a été évaluée. Les paramètres ont ainsi été optimisés pour la fabrication des nanoparticules. L’évaluation morphologique et chimique a été réalisée par microscopie électronique et spectroscopie infrarouge respectivement. L'état cristallin des nanoparticules de paclitaxel a été caractérisé par calorimétrie differentielle et diffraction des rayons X. L'optimisation du procédé de production de nanoparticules par laser fs a permis d'obtenir des nanocristaux de taille moyenne (400 nm, polydispersité ≤ 0,3). Cependant une dégradation non négligeable a été observée. La cristallinité du médicament a été maintenue durant la procédure de réduction de taille, mais le paclitaxel anhydre a été transformé en une forme hydratée. Les résultats de cette étude suggèrent que le laser fs peut générer des nanocristaux de principe actif. Cependant cette technique peut se révéler problématique pour des médicaments sensibles à la dégradation. Grâce à sa facilité d'utilisation et la possibilité de travailler avec des quantités restreintes de produit, le laser fs pourrait représenter une alternative valable pour la production de nanoparticules de médicaments peu solubles lors des phases initiales de développement préclinique. Mots-clés: paclitaxel, nanocristaux, laser femtoseconde, ablation, fragmentation

Pharmacologie des variations de débit sanguin oculaires chez le rat au moyen de la débitmétrie au laser par effet Doppler

La débitmétrie au laser à effet Doppler (LDF) constitue une méthode prometteuse et non-invasive pour l'étude du débit sanguin local dans l'œil. Cette technique est basée sur un changement de fréquence subi par la lumière lors du mouvement des globules rouges dans les vaisseaux. Une nouvelle sonde LDF a été testée pour sa sensibilité à évaluer la circulation rétinienne/choroïdienne sous des conditions hypercapniques et en présence de diverses substances vasoactives ou suivant la photocoagulation des artères rétiniennes chez le rat. Après dilatation pupillaire, la sonde LDF a été placée en contact avec la cornée de rats anesthésiés et parallèle à l'axe optique. L'hypercapnie a été provoquée par inhalation de CO2 (8% dans de l'air médical), alors que les agents pharmacologiques ont été injectés de façon intravitréenne. La contribution relative à la circulation choroïdienne a été évaluée à la suite de la photocoagulation des artères rétiniennes. Le débit sanguin s'est trouvé significativement augmenté à la suite de l'hypercanie (19%), de l'adénosine (14%) ou du nitroprusside de sodium (16%) comparativement au niveau de base, alors que l'endothéline-1 a provoqué une baisse du débit sanguin (11%). La photocoagulation des artères rétiniennes a significativement diminué le débit sanguin (33%). Des mesures en conditions pathologiques ont ensuite été obtenues après l'injection intravitréenne d'un agoniste sélectif du récepteur B1 (RB1). Ce récepteur des kinines est surexprimé dans la rétine des rats rendus diabétiques avec la streptozotocine (STZ) en réponse à l'hyperglycémie et au stress oxydatif. Les résultats ont montré que le RB1 est surexprimé dans la rétine chez les rats diabétiques-STZ à 4 jours et 6 semaines. À ces moments, le débit sanguin rétinien/choroïdien a été significativement augmenté (15 et 18 %) après l'injection de l'agoniste, suggérant un effet vasodilatateur des RB1 dans l'œil diabétique. Bien que la circulation choroïdienne contribue probablement au signal LDF, les résultats démontrent que le LDF représente une technique efficace et non-invasive pour l'étude de la microcirculation rétinienne in-vivo en continu. Cette méthode peut donc être utilisée pour évaluer de façon répétée les réponses du débit sanguin pendant des modifications métaboliques ou pharmacologiques dans des modèles animaux de maladies oculaires.

Development of an enzyme immobilization platform based on microencapsulation for paper-based biosensors

Un papier bioactif est obtenu par la modification d’un papier en y immobilisant une ou plusieurs biomolécules. La recherche et le développement de papiers bioactifs est en plein essor car le papier est un substrat peu dispendieux qui est déjà d’usage très répandu à travers le monde. Bien que les papiers bioactifs n’aient pas connus de succès commercial depuis la mise en marche de bandelettes mesurant le taux de glucose dans les années cinquante, de nombreux groupes de recherche travaillent à immobiliser des biomolécules sur le papier pour obtenir un papier bioactif qui est abordable et possède une bonne durée de vie. Contrairement à la glucose oxidase, l’enzyme utilisée sur ces bandelettes, la majorité des biomolécules sont très fragiles et perdent leur activité très rapidement lorsqu’immobilisées sur des papiers. Le développement de nouveaux papiers bioactifs pouvant détecter des substances d’intérêt ou même désactiver des pathogènes dépend donc de découverte de nouvelles techniques d’immobilisation des biomolécules permettant de maintenir leur activité tout en étant applicable dans la chaîne de production actuelle des papiers fins. Le but de cette thèse est de développer une technique d’immobilisation efficace et versatile, permettant de protéger l’activité de biomolécules incorporées sur des papiers. La microencapsulation a été choisie comme technique d’immobilisation car elle permet d’enfermer de grandes quantités de biomolécules à l’intérieur d’une sphère poreuse permettant leur protection. Pour cette étude, le polymère poly(éthylènediimine) a été choisi afin de générer la paroi des microcapsules. Les enzymes laccase et glucose oxidase, dont les propriétés sont bien établies, seront utilisées comme biomolécules test. Dans un premier temps, deux procédures d’encapsulation ont été développées puis étudiées. La méthode par émulsion produit des microcapsules de plus petits diamètres que la méthode par encapsulation utilisant un encapsulateur, bien que cette dernière offre une meilleure efficacité d’encapsulation. Par la suite, l’effet de la procédure d’encapsulation sur l’activité enzymatique et la stabilité thermique des enzymes a été étudié à cause de l’importance du maintien de l’activité sur le développement d’une plateforme d’immobilisation. L’effet de la nature du polymère utilisé pour la fabrication des capsules sur la conformation de l’enzyme a été étudié pour la première fois. Finalement, l’applicabilité des microcapsules de poly(éthylèneimine) dans la confection de papiers bioactifs a été démontré par le biais de trois prototypes. Un papier réagissant au glucose a été obtenu en immobilisant des microcapsules contenant l’enzyme glucose oxidase. Un papier sensible à l’enzyme neuraminidase pour la détection de la vaginose bactérienne avec une plus grande stabilité durant l’entreposage a été fait en encapsulant les réactifs colorimétriques dans des capsules de poly(éthylèneimine). L’utilisation de microcapsules pour l’immobilisation d’anticorps a également été étudiée. Les avancées au niveau de la plateforme d’immobilisation de biomolécules par microencapsulation qui ont été réalisées lors de cette thèse permettront de mieux comprendre l’effet des réactifs impliqués dans la procédure de microencapsulation sur la stabilité, l’activité et la conformation des biomolécules. Les résultats obtenus démontrent que la plateforme d’immobilisation développée peut être appliquée pour la confection de nouveaux papiers bioactifs.

Characterization of glutaraldehyde-immobilized chymotrypsin and an in-situ immobilized enzyme reactor using capillary electrophoresis-based peptide mapping

La digestion enzymatique des protéines est une méthode de base pour les études protéomiques ainsi que pour le séquençage en mode « bottom-up ». Les enzymes sont ajoutées soit en solution (phase homogène), soit directement sur le gel polyacrylamide selon la méthode déjà utilisée pour l’isolation de la protéine. Les enzymes protéolytiques immobilisées, c’est-à-dire insolubles, offrent plusieurs avantages tels que la réutilisation de l’enzyme, un rapport élevé d’enzyme-sur-substrat, et une intégration facile avec les systèmes fluidiques. Dans cette étude, la chymotrypsine (CT) a été immobilisée par réticulation avec le glutaraldehyde (GA), ce qui crée des particules insolubles. L’efficacité d’immobilisation, déterminée par spectrophotométrie d’absorbance, était de 96% de la masse totale de la CT ajouté. Plusieurs différentes conditions d’immobilisation (i.e., réticulation) tels que la composition/pH du tampon et la masse de CT durant la réticulation ainsi que les différentes conditions d’entreposage tels que la température, durée et humidité pour les particules GA-CT ont été évaluées par comparaison des cartes peptidiques en électrophorèse capillaire (CE) des protéines standards digérées par les particules. Les particules de GA-CT ont été utilisés pour digérer la BSA comme exemple d’une protéine repliée large qui requit une dénaturation préalable à la digestion, et pour digérer la caséine marquée avec de l’isothiocyanate de fluorescéine (FITC) comme exemple d’un substrat dérivé afin de vérifier l’activité enzymatique du GA-CT dans la présence des groupements fluorescents liés au substrat. La cartographie peptidique des digestions par les particules GA-CT a été réalisée par CE avec la détection par absorbance ultraviolet (UV) ou fluorescence induite par laser. La caséine-FITC a été, en effet, digérée par GA-CT au même degré que par la CT libre (i.e., soluble). Un microréacteur enzymatique (IMER) a été fabriqué par immobilisation de la CT dans un capillaire de silice fondu du diamètre interne de 250 µm prétraité avec du 3-aminopropyltriéthoxysilane afin de fonctionnaliser la paroi interne avec les groupements amines. Le GA a été réagit avec les groupements amine puis la CT a été immobilisée par réticulation avec le GA. Les IMERs à base de GA-CT étaient préparé à l’aide d’un système CE automatisé puis utilisé pour digérer la BSA, la myoglobine, un peptide ayant 9 résidus et un dipeptide comme exemples des substrats ayant taille large, moyenne et petite, respectivement. La comparaison des cartes peptidiques des digestats obtenues par CE-UV ou CE-spectrométrie de masse nous permettent d’étudier les conditions d’immobilisation en fonction de la composition et le pH du tampon et le temps de réaction de la réticulation. Une étude par microscopie de fluorescence, un outil utilisé pour examiner l’étendue et les endroits d’immobilisation GA-CT dans l’IMER, ont montré que l’immobilisation a eu lieu majoritairement sur la paroi et que la réticulation ne s’est étendue pas si loin au centre du capillaire qu’anticipée.