L'alpha-1 antitrypsine (A1AT) est un inhibiteur de sérine protéase sécrété principalement par le foie et libéré dans la circulation où sa concentration physiologique est de 1,5 à 3,5 g/L. La principale fonction de l'A1AT est d'inhiber l'activité de l'élastase des neutrophiles (NE) afin de maintenir l'équilibre protéase/anti-protéase dans les poumons. Son déficit (A1ATD) touche plus de 3,4 millions d'individus dans le monde chez qui l'élastase des neutrophiles décompose l'élastine, provoquant ainsi une diminution de l'élasticité du poumon ainsi qu'une dégradation de son tissu conjonctif. En conséquence, l'A1ATD entraîne des troubles respiratoires tels que l'emphysème ou la maladie pulmonaire obstructive chronique et ceux qui en sont atteints nécessitent des injections fréquentes d'A1AT purifiée à partir du sang d'un donneur. Cependant, l'A1AT plasmatique est hétérogène dans son état de glycosylation et sa qualité varie d'un lot à l'autre. De plus, il y a un risque, même très faible, de transmission d'agents pathogènes avec l'administration d'A1AT purifiée par plasma. Par conséquent, il existe un besoin pour une version recombinante. La glycoprotéine mature possède trois sites de N-glycosylation comprenant principalement des structures de type complexe bi-antennaires afucosylées et α-2,6-di-sialylées, A2G2S2 (6,6). Bien que la glycosylation ne soit pas essentielle à l'activité inhibitrice de l'A1AT, il a été démontré qu'elle a un impact significatif sur sa demi-vie in vivo. Notamment, l'acide sialique, un monosaccharide terminal chargé négativement présent sur les N-glycanes, aide à prolonger la demi-vie de l'A1AT dans le sérum en empêchant l'interaction entre l'avant-dernier galactose (Gal) du N-glycane et les récepteurs hépatiques des asialoglycoprotéines (ASGPRs), composés de deux sous-unités appelées lectines hépatiques (HL) 1 et 2, qui se lient aux glycoprotéines asialylées contenant un Gal terminal et conduisent à leur dégradation. Par conséquent, il est important de produire A1AT dans un système d'expression qui peut effectuer les modifications post-traductionnelles (PTM) appropriées à des fins thérapeutiques. Jusqu'à présent, la production d'A1AT recombinante (rA1AT) a été tentée dans différents systèmes d'expression cellulaire avec un succès limité. Malgré la disponibilité de diverses lignées cellulaires, les cellules ovariennes de hamster chinois (CHO) ont été largement utilisées pour la production de glycoprotéines thérapeutiques car ces cellules sont compatibles avec des stratégies de glyco-ingénierie pour produire des glycoprotéines recombinantes composées de glycanes de type humain. Cependant, ces cellules synthétisent des N-glycanes de type complexe comprenant de la fucosylation centrale et de l'acide sialique lié en α-2,3. Par conséquent, dans ce projet, l'objectif était de développer une version recombinante d'A1AT avec un profil de glycosylation humaine exprimée en cellules CHO modifiées et qui se prête à des utilisations thérapeutiques. À cette fin, dans notre étude, nous avons d'abord empêché l'α-2,3 sialylation ainsi que la fucosylation centrale en éliminant les gènes responsables via la technologie CRISPR/Cas9, suivie de la surexpression de l'α-2,6‐sialyltransférase humaine à l'aide d'un système d'expression inductible au cumate. Nous avons ensuite montré la supériorité du promoteur inductible CR5 pour l’expression de A1AT par rapport à cinq promoteurs constitutifs forts couramment utilisés dans l'industrie. En utilisant le promoteur CR5, nous avons généré des populations de CHO stables modifiées par glyco-ingénierie produisant plus de 2,1 g/L pour la forme native et 2,8 g/L pour la version mutée d'A1AT avec des N-glycanes analogues au produit clinique dérivé du plasma, la Prolastin-C. L'effet bénéfique de la supplémentation en N‐acétylmannosamine du milieu de culture cellulaire sur la glycosylation de l'A1AT a également été démontré. Enfin, nous avons montré que l'activité anti‐élastase des rA1ATs est comparable à celle de la Prolastin-C, et que la substitution des résidus méthionines critiques par des valines rendait A1AT significativement plus résistante à l'oxydation. Nous avons ensuite étudié l'impact de la glycosylation d'A1AT sur son interaction avec les orthologues d'ASGPR. Pour cela, nous avons initialement utilisé un test d'internalisation cellulaire basé sur la lignée cellulaire hépatique humaine HepG2 connue pour exprimer les ASGPRs à sa surface et avons examiné leur interaction avec les rA1ATs possédant divers profils de glycosylation. Comme le test d'internalisation basé sur les cellules HepG2 a démontré un faible rapport signal sur bruit (SNR) ainsi qu'un niveau élevé de signal de fond d'internalisation, nous avons cherché à développer un nouveau test basé sur des cellules CHO surexprimant des orthologues ASGPR recombinants. Alors que la sous-unité HL-1 humaine seule était suffisante pour lier et internaliser l'A1AT asialylée, les sous-unités HL-1 et HL-2 étaient nécessaires pour former des récepteurs fonctionnels et ayant une forte affinité pour les ASGPR de rat et de souris. Afin d'améliorer le SNR de notre test cellulaire d'internalisation, le tri cellulaire activé par fluorescence (FACS) a été utilisé pour enrichir les populations de cellules CHO pour celles exprimant des niveaux élevés d'orthologues ASGPR. Enfin, en utilisant des structures de glycanes remodelés par voie enzymatique de Prolastin-C, nous n'avons observé aucune internalisation lorsque les glycanes sont terminés avec α-2,6-Neu5Ac ni α-2,8-Neu5Ac-α-2,6-Neu5Ac par l’ASGPR de l'humain, du rat et de la souris. D'autre part, l'absorption de Prolastin-C portant des glycanes bi-antennaires avec une branche terminée par de l'acide sialique α-2,3 et l'autre par du galactose terminal, par l'ASGPR de souris a été statistiquement plus élevée que celle de l'humain et du rat. En somme, l'A1AT recombinante résistante à l'oxydation décrite dans ce projet pourrait représenter un meilleur médicament biothérapeutique tout en offrant une alternative sûre et plus stable pour la thérapie d'augmentation. Nous avons également contribué à une meilleure compréhension de l'impact de la sialylation de l'A1AT sur son internalisation cellulaire par les orthologues ASGPR. / Alpha-1 antitrypsin (A1AT) is a serine protease inhibitor secreted primarily by the liver, and released in the circulation where its physiological concentration is 1.5-3.5 g/L. The main physiological function of A1AT is to inhibit the activity of neutrophil elastase (NE) to maintain the protease/anti-protease balance in the lung. The A1AT deficiency (A1ATD) is affecting more than 3.4 million individuals worldwide where neutrophil elastase breaks down elastin, thereby causing a decrease in the elasticity of the lung as well as a degradation of its connective tissue. As a result, A1ATD leads to respiratory disorders such as emphysema or chronic obstructive pulmonary disease. Treatment of this health condition requires frequent injections of A1AT purified from donor blood. However, plasma A1AT is heterogeneous in its glycosylation state and its quality varies from batch to batch. Moreover, there is a risk, however very low, of pathogen transmittance with plasma-purified A1AT administration. Therefore, there is a need for recombinant version. The mature glycoprotein has three N-glycosylation sites possessing mostly afucosylated, α-2,6-di-sialylated bi-antennary complex-type structures, A2G2S2 (6,6). Though glycosylation is not essential for A1AT's inhibitory activity, it has been shown to have a significant impact on its in vivo half-life. Notably, sialic acid, a terminal negatively charged monosaccharide present on N-glycans, helps to prolong the half-life of A1AT in serum by preventing the interaction between the penultimate galactose (Gal) of the N-glycan and the hepatic asialoglycoprotein receptors (ASGPRs), composed of two subunits termed hepatic lectin (HL) 1 and 2, which bind to asialylated glycoproteins containing terminal Gal and lead to their degradation. To this extend, it is important to produce A1AT in an expression system that can carry out the appropriate post-translational modifications (PTMs) for therapeutic purposes. Thus far, the production of recombinant A1AT (rA1AT) has been attempted in different cell expression systems with limited success. Despite the availability of various cell lines, Chinese hamster ovary (CHO) cells have been widely used to produce therapeutic glycoproteins as these cells can tolerate glycoengineering strategies to produce recombinant glycoproteins with human-like glycans. However, these cells synthesize complex-type N-glycans with core-fucosylation along with α-2,3-linked sialic acid. Therefore, in this research project, the aim was to develop a recombinant version of A1AT with human glycosylation pattern expressed in genetically engineered CHO cells that would be amenable to therapeutic uses. To this end, in our study, we first prevented α-2,3 sialylation as well as core-fucosylation by eliminating the corresponding genes via CRISPR/Cas9 technology, followed by overexpressed human α-2,6‐sialyltransferase using a cumate‐inducible CHO expression system. We then showed superiority of the CR5 inducible promoter compared to five strong constitutive promoters commonly used in the industry. Using the CR5 promoter, we generated glycoengineered stable CHO pools producing over 2.1 g/L of the wild-type and 2.8 g/L of the mutein forms of A1AT, with N‐glycans analogous to the plasma‐derived clinical product, Prolastin-C. The effect of N‐acetylmannosamine supplementation to the cell culture media on the A1AT glycosylation was also demonstrated. Finally, we showed that the anti‐elastase activity of rA1ATs is comparable to that of Prolastin-C, and that substitution of critical methionine residues with valines rendered A1AT significantly more resistant to oxidation. We then studied the impact of A1AT glycosylation on its interaction with ASGPR orthologs. For this, we initially used a cell-based internalization assay based on the human HepG2 hepatic cell line known to express ASGPRs at its surface and examined their interaction with rA1ATs possessing various glycosylation profiles. As HepG2 cell-based internalization assay demonstrated poor signal-to-noise ratio (SNR) as well as high level of background internalization signal, we then aimed at developing a new assay based on CHO cells overexpressing recombinant ASGPRs orthologs. While human HL-1 subunit alone was sufficient to bind and internalize asialylated A1AT, both HL-1 and HL-2 subunits were required to form functional and high affinity receptors for the rat and mouse ASGPRs. To enhance SNR of our cell-based uptake assay, fluorescence-activated cell sorting (FACS) was used to enrich the CHO pools for cells expressing high levels of ASGPR orthologs. Finally, using enzymatically remodelled glycan structures of Prolastin-C, we observed no uptake when glycans are terminated with α-2,6-Neu5Ac nor α-2,8-Neu5Ac-α-2,6-Neu5Ac by human, rat, and mouse ASGPR orthologs. On the other hand, the uptake of Prolastin-C bearing bi-antennary glycans with one branch terminated with α-2,3 sialic acid and the other with terminal galactose, by mouse ASGPR was observed to be statistically higher than that by human and rat ASGPR orthologs. Collectively, the oxidation-resistant recombinant A1AT described in this project could represent a viable biobetter drug while offering a safe and more stable alternative for augmentation therapy. We also contributed a better understanding of the impact of A1AT sialylation on its cellular uptake by ASGPR orthologs.

Wood is a viscoelastic composite material that has been historically prominent in the construction of buildings and continues to see widespread use. When used for exterior applications, wood is exposed to dynamic environmental conditions and can degrade if left untreated. Previous research by Lemaire-Paul et al. (2022) has proven that vacuum impregnation of the wood cell structure with a silica (SiO₂) nanoparticle colloid under a vacuum pressure of -90 kPa can enhance the viscoelastic properties, increase the density, and reduce the water uptake of white spruce wood. However, the behaviour of SiO₂-treated wood under different environmental conditions over time has yet to be fully explored. This research aims to examine the durability and performance of SiO₂-treated spruce wood samples subjected to accelerated aging conditions under high temperature and humidity as well as freeze-thaw cycling. Spruce wood samples were treated with 40% SiO₂ nanoparticle colloid under a vacuum pressure of -90 kPa. One set was placed in a hydrolytic aging chamber at 90°C and 80% RH. Another set was placed in a freeze-thaw cycling chamber that cycled from 25°C to -18°C and back at a rate of 6 cycles per day. The samples were removed at regular intervals and thermogravimetric analysis, dynamic mechanical analysis, tensiometry, X-Ray diffraction, and scanning electron microscopy were performed. When compared to the results obtained from a set of non-treated samples, it was found that the SiO₂-treated samples exhibited lower water uptake values that stabilized over time, as well as a lower rate of decrease in peak cellulose degradation temperatures under hydrolytic aging and a slight increase in peak cellulose degradation temperature over time under freeze-thaw aging. The effects of both aging conditions on the viscoelastic properties of the samples were also found to be insignificant. Both types of samples under both types of aging also exhibited an increase in crystallinity over time. These results indicate that the durability and properties of wood can be improved through nano-SiO₂ impregnation as the material remains relatively stable when subjected to high temperature and humidity conditions as well as freeze-thaw cycling over time.

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Bovine milk-derived extracellular vesicles (EVs) hold promises as oral drug delivery systems. Since EV bioavailability studies are difficult to compare, key factors regarding EV uptake and intestinal permeability remain little understood. This work aims to critically study uptake and transport properties of milk-derived EVs across the intestinal barrier in vitro by standardization approaches. Therefore, uptake properties were directly compared to liposomes in intestinal Caco-2 cells. Reliable staining results were obtained by the choice of three distinct EV labeling sites, while non-specific dye transfer and excess dye removal were carefully controlled. A novel fluorescence correction factor was implemented to account for different labelling efficiencies. Both EV and liposome uptake occurred mainly energy dependent with the neonatal Fc receptor (FcRn) providing an exclusive active pathway for EVs. Confocal microscopy revealed higher internalization of EVs whereas liposomes rather remained attached to the cell surface. Internalization could be improved when changing the liposomal formulation to resemble the EV lipid composition. In a Caco-2/HT29-MTX co-culture liposomes and EVs showed partial mucus penetration. For transport studies across Caco-2 monolayers we further established a standardized protocol considering the distinct requirements for EVs. Especially insert pore sizes were systematically compared with 3 µm inserts found obligatory. Obtained apparent permeability coefficients (Papp) reflecting the transport rate will allow for better comparison of future bioavailability testing.

Introduktion Längdskidor involverar såväl underkropps som överkroppsarbete i varierad intensitet och terräng. Tävlingarna är allt ifrån 1km upp till 30km för damer respektive 50km för herrar. Detta ställer flera olika fysiologiska krav på en längdskidåkare. Som exempelvis hög maximal aerob effekt, hög aerob kapacitet, hög anaerob kapacitet, styrka, kraftutveckling i överkropp samt hög andel fettfri massa. Tidigare studier visar att det finns en skillnad i fysiologiska egenskaper mellan sprint- och distansåkare. Det finns hittills ingen strukturerad litteraturundersökning som jämför den aktuella forskningen (från och med 2012) på skillnaderna i vilka fysiologiska egenskaper som är kopplade till sprint- respektive distansprestation. Syftet var att undersöka skillnaderna mellan vilka fysiologiska egenskaper som kan kopplas till sprintprestation och vilka fysiologiska egenskaper som kan kopplas till distansprestation inom elitlängdskidåkning. Metod En strukturerad litteraturundersökning utfördes i databaserna: Pubmed, SPORTDiscus och Sport Medicine and Education Index mellan 2012 och 2022-04-18. Även en manuell sökning i relevanta artiklars referenslistor utfördes. Resultat  Studien inkluderade 13 artiklar. Resultatet visade att sprintprestation är kopplat till högt VO2max (l/min). Distansprestation är kopplat till högt VO2max (l/min och ml/kg/min). Sprintåkare har högre VO2max uttryckt i l/min medan distansåkare har högre VO2max uttryckt i ml/kg/min vid jämförelse mellan disciplinerna. Hastighet på obla4mmol är kopplat till distansprestation men inte till sprintprestation. Hög anaerob kapacitet och större fettfri massa uttryckt i absoluta mått är starkare kopplat till sprintprestation än distansprestation.   Konklusion Distansprestation är kopplat till VO2max/VO2peak (l/min och ml/kg/min), aerob kapacitet samt hög andel fettfrimassa. Sprintprestation är kopplat till hög VO2max/VO2peak (l/min), anaerob kapacitet samt hög fettfri massa. Skillnaderna var att distansprestation var starkare kopplat till det aeroba egenskaperna i jämförelse med sprintprestation medan sprintprestation var starkare kopplat till det anaeroba egenskaper i jämförelse med distansprestation. / Introduction Cross-country skiing involves both upper- and lower body work in varied intensity and terrain. The distances are from 1km to 30km for women respectively 50km for men. There are different physiological demands on a cross-country skier. For example, high maximum aerobic power, high aerobic capacity, high anaerobic capacity, strength, upper body power and a high proportion of lean mass. Earlier studies have seen differences in physiological characteristics between sprint- and distance skiers. But there is no structured literature study that compares the current research (from 2012) on differences in physiological characteristics between sprint and distance performance. The aim was to investigate differences between physiological characteristics that can be linked to sprint and distance performance in elite cross-country skiing.  Method A structured literature review was conducted in the databases: PubMed, SPORTDiscus and Sports Medicine And Education Index between 2012 and 2022-04-18. A manual search in the reference lists of relevant articles was conducted. Results 13 articles were included in the study. The results showed that sprint performance is linked to high VO2max (l/min). Distance performance is linked to high VO2max (l/min and ml/kg/min). Sprint skiers have higher VO2max expressed in l/min while distance skiers have higher VO2max expressed in ml/kg/min in comparison with each other. Speed ​​at obla4mmol is linked to distance performance but not to sprint performance. High anaerobic capacity and greater lean mass are more strongly linked to sprint performance than distance performance. Conclusion Distance performance is linked to VO2max/VO2peak (l/min and ml/kg/min), aerobic capacity and high proportion of lean mass. Sprint performance is linked to high VO2max/VO2peak (l/min), anaerobic capacity and high lean mass. The differences were that distance performance was more strongly linked to the aerobic characteristics compared to sprint performance while sprint performance was more strongly linked to anaerobic characteristics compared to distance performance.

Soil degradation is a common occurrence in Haiti that is mainly caused by the cultivation of marginal lands and deforestation, which both contribute to the excessive erosion rate seen in the country today. The Central Plateau of Haiti is a mountainous region in which a majority of the population is rural and practices subsistence agriculture on hillsides and steeply-sloping land. Essential plant nutrients, such as nitrogen (N) and phosphorus (P), are commonly a limiting factor in crop production, yet fertilizer is unavailable or is too expensive for smallholder farmers to purchase. This study was conducted to a) evaluate organic matter and nutrient stocks of various soils in the Central Plateau region, along with other chemical and physical characteristics and b) to evaluate the phosphorus-scavenging ability of commonly-grown crops to isolate those that may benefit subsequent smallholder yields. Soils from four locations in the Central Plateau were assessed for organic matter in labile and non-labile fractions as well as for cation exchange capacity (CEC), total organic carbon (C) and N, pH, texture, and other characteristics. Results indicated that most of the soil (92%) was contained within aggregates, and organic matter was mainly present in stable, slowly-decomposing fractions. Seven species were evaluated in a controlled-environment pot experiment for bulk and rhizosphere soil P and pH, plant dry weight, and above- and below-ground P tissue content as indicators of the species' ability to solubilize P from the soil. Velvet bean (Mucuna pruriens (L.) DC) produced the most biomass and was able to take up the most P, though lablab (Lablab purpureous (L.) Sweet), took up comparable amounts of P. / Master of Science / LTRA-6 (A CAPS program for the Central Plateau of Haiti)