Immune thrombocytopenia (ITP) is an autoimmune hematologic disorder characterized by a low platelet count due to increased platelet destruction or decreased production. In primary ITP, the patient can have a low platelet count (<100 billion cells/L) for clinically unknown reasons. ITP is a rare disease that affects approximately 3/100 000 adults each year and some patients may experience bleeding symptoms. Autoantibody-mediated autoimmunity plays a role in the destruction of platelets by targeting platelet glycoproteins (GPs). Autoantibodies against platelet membrane GPIIbIIIa and GPIbIX are observed in about 50% of patients through direct antigen-capture assays, and 18% in patients through indirect antigen-capture assays. It is possible that some antibodies may not be detectable due to affinity or titre, or there may be other factors involved in platelet destruction. Currently, there is no definitive diagnostic test available for ITP, as a result of low assay sensitivity and different mechanisms involved in disease pathogenesis. The objective of this study was to use a novel approach to increase autoantibody detection unique to ITP patients. Total IgG was purified from patient and control plasma samples. A streptavidin-based antigen-capture assay was optimized to test the effect of biotinylation on the detection of anti-GPIIbIIIa and anti-GPIbIX autoantibodies in primary ITP patients (n=14), secondary ITP patients (n=3), non-immune thrombocytopenic controls (n=2) and healthy controls (n=16). Streptavidin-coated biosensors were used in an optimized biolayer interferometry (BLI) assay to study autoantibodies binding to biotinylated GPIIbIIIa and GPIbIX. Detection of anti-GPIIbIIIa autoantibodies in the streptavidin antigen-capture assay had a sensitivity of 24% and anti-GPIbIX autoantibodies had a sensitivity of 25%. BLI showed binding of autoantibodies in approximately 5% of ITP samples for both GPIIbIIIa and GPIbIX. The samples that had detectable autoantibodies in the antigen-capture assay did not have detectable antibodies in the BLI assay. BLI was not able to confirm antibody detection found in enzyme immunoassays. / Thesis / Master of Science (MSc) / Platelets are blood cells involved in clotting at sites of injury. Immune thrombocytopenia (ITP) is a disease defined by a low platelet count that can lead to bleeding. ITP is a rare disease that affects 3 in 100 000 adults every year. ITP is thought to be caused by proteins known as antibodies that bind self-platelets and lead to their destruction. These antibodies are directly found on approximately 50% of patients’ platelets, and only 18% of patients have antibodies in circulation. It is possible in many patients, antibodies are present at a low concentration, or are too weak to be detected in antibody tests. In this study, a new technology known as biolayer interferometry was employed to find antibodies in a higher percentage of patients. Results showed only 6% of ITP patients had detectable antibodies in their circulation. This research will improve our understanding of antibodies in ITP.

In 2015, the United nations presented the 17 Global Goals that would put an end to extreme poverty, inequality and climate change by 2030. One of these goals was clean water and sanitation. In 2015 1.8 billion people did not have access to clean water. Because of the contaminated water, one million people die every year worldwide. Africa, and especially Ghana, has had a high development in the recent years. The population has grown and more resources are needed. Clean water in Ghana is not a given matter, three million people live without access to clean water. To work towards the Global Goal water can be clean locally. A simple and cheap way is to build slow sand filters, which also are the purpose of this project. These filters purify the water mechanically, chemically and biologically. The biologically purification takes place in the biolayer that grows on the sand inside the filter and it consumes contaminants in the water. It takes about a month for the biolayer to be fully developed and clean the water to its full potential. The positive aspects with sand filters are that people get healthier and can save money that can be invested in education or business. It can also reduce the need for water in plastic bags or bottles and would reduce littering. The companies that produce this water could end their business and air pollutions would be reduced as well.   During this project, slow sand filters have been tested and evaluated in Sweden and Ghana with the purpose to develop a theoretical filter that benefits the biolayer under local conditions in Ghana, this was of the one aims. Experiments in Sweden showed that the flow decreased with increased sand height and decreased hydraulic head. In Ghana three filters were built with the sand heights 30, 50 and 80 cm to clean 7 litres of drinking water for a family of four. None of these produced drinkable water by WHO’s and EU’s standards.   The next aim was to understand which chemical and physical factors that effected the development of the biolayer. The detected relations were absolute conductivity, total alkalinity, coliform bacteria and oxidantial reduction potential which were between the biolayer in the 30 and 50 filters.   The flow rate in Ghana was too high and to lower it, a new diffuser with smaller holes would be built to get the recommended flow of 0,4 m3/m2/h. A too high flow broke the bound between the biolayer and made an uncomfortable environment. A sedimentation should be installed before the sand filter to reduce the variations of the incoming water such as turbidity, suspended solids etc., so the biolayer would flourish. It was not enough dissolved oxygen in the water so the pause period would be decreased to 12 hours to get more oxygen in the filter each day. For a sand filter to work as planned a lot of attention should be given to the filter. It is a system that should be used all the time for the best purification. To build a filter takes a lot of time and it also takes time for the biolayer to develop. If it is not going to be used much, another treatment method should be used.   The last aim was to evaluate the cost of the materials that could be bought locally to the filter. One filter cost about 130 GHS. / 2015 tog Förenta nationerna fram de 17 globala målen för att få ett slut på extrem fattigdom, ojämlikhet och klimatförändringen till år 2030. Ett av dessa mål handlar om rent vatten och sanitet. 2015 var det 1,8 miljarder människor som inte hade tillgång till rent vatten. På grund av det förorenade vattnet dör en miljon människor i hela världen varje år. Afrika, och speciellt Ghana, har haft en snabb utveckling de senaste åren. Folkmängden har ökat och mer naturresurser behövs. Rent vatten i Ghana är inte en självklarhet, tre miljoner människor lever idag utan tillgång till rent vatten i Ghana. Ett sätt för att jobba mot det globala målet är rening av vatten lokalt. Ett enkelt och billigt sätt är att bygga långsamsandfilter, vilket även var syftet med denna studien. Dessa filter renar vattnet mekaniskt, kemiskt och biologiskt. Den biologiska reningen sker av en biofilm som växer på sanden inuti filtret som konsumerar föroreningar i vattnet. Det tar ungefär en månad för biofilmen att bli färdigutvecklad och rena vattnet till sin fulla potential. Det positiva med sandfilter är att människorna skulle bli friskare och spara pengar som kan investeras på utbildning eller företag. Ur miljöpunkt skulle reduktionen av köpt vatten i plastpåsar och flaskor minska nedskräpningen och företagen som producerar dessa kan avsluta produktionen och därmed minska luftföroreningar.    Under detta projekt har långsamsandfilter utvärderats både i Sverige och Ghana för att utveckla ett nytt teoretiskt filter som gynnar tillväxten av biofilm under lokala förhållanden i Ghana, vilket var ett mål. Experimenten i Sverige visade att flödet sjönk med ökad sandhöjd, men även med minskat hydrauliskt tryck. I Ghana byggdes tre filter med sand höjderna 30, 50 och 80 cm för att rena 7 liter dricksvatten till en familj på fyra. Ingen av dessa lyckades producera drickbart vatten enligt WHO:s och EU:s standarder.   Nästa mål var att förstå vilka av de kemiska och fysiska faktorer som påverkade biofilmstillväxten. Det förhållanden som upptäcktes var absolut konduktivitet, total alkalinitet, coliform bacteria och oxidential reduction potential vilket fanns i 30 och 50 filtret.   Flödet i Ghana var för högt, så för att minska det skulle en diffusör med mindre hål byggas för att få det rekommenderade flödet 0,4 m3/m2/h. Ett för högt flöde gjorde sönder bindingen mellan biofilmen och skapade en otrivsam miljö. En sedimentation skulle installeras innan sandfiltret för att minska variationer på ingående vatten i filtret för att få biofilmen att trivas bättre. Det fanns för lite löst syre i vattnet och om pausperioden minskas till 12 timmar skulle mer syre i filtret varje dag. För att ett sandfilter ska fungera som planerat måste mycket tid läggas på filtret. Sandfilter är ett system som bör används ofta för bästa rening. Att bygga ett filter kräver mycket tid, samt att det tar tid innan biofilmen har utvecklats. Om sandfiltret inte kommer används mycket föreslås att en annan metod används istället.   Det sista målet var att utvärdera kostnaden av materialen som kunde köpas lokalt till filtret. Ett filter kostade runt 130 GHS.

En dépit d’importants progrès dans le domaine de la thérapie anti-cancéreuse, les traitements actuels restent controversés, notamment en raison de la quantité importante d'effets secondaires induits. L'immunothérapie ciblée a récemment émergée en tant qu'alternative, afin d'améliorer les modalités de traitement des patients atteints du cancer. Malgré tout, seul un nombre limité d’approches sont aujourd’hui disponibles, et une grande partie des problèmes demeurent actuellement sans solution. C'est dans ce contexte que nous nous sommes intéressés à la conception de structures biomoléculaires innovantes et bifonctionnelles, capables de rediriger des anticorps endogènes, présents naturellement dans la circulation sanguine de l'homme, contre les tumeurs et, ce, sans immunisation préalable. Les anticorps naturels circulant étant polyspécifiques et ayant la capacité d’interagir avec des antigènes glycosylés, nous nous sommes plus particulièrement concentrés sur la conception de glycoconjugués multivalents, ligands d’anticorps endogènes. Une première partie de notre étude a consisté à synthétiser différents glycodendrimères multivalents, reposant sur des châssis peptidiques et obtenus par ligations chimiosélectives, tout en variant la nature du motif glycosylé et des plateformes, ainsi que la valence du conjugué. Puis, dans un second temps, des tests d’interaction par biopuce ont été mis en place avec une lectine modèle, la lectine Helix Pomatia Agglutinin (HPA). Des protocoles expérimentaux visant à calculer des constantes de dissociation de surface, ainsi que des IC50 ont été mis en place, permettant d’identifier de bons ligands de HPA avec des affinités de l’ordre du nanomolaire. Les tests par biopuce ont ensuite été confirmés avec d’autres méthodes d’analyses (BLI, ELLA). Finalement, afin d'identifier des architectures tri-dimensionnelles permettant une affinité optimale avec des anticorps, les tests d’interaction ont été adaptés au criblage de séra humains. Un large panel de glycoconjugués a alors été criblé par biopuce avec une vingtaine de séra, permettant la détermination de structures glycosylés prometteuses, qui pourront par la suite être utilisées dans le cadre de notre approche anti-cancéreuse. / Despite significant progress in anti-cancer therapy, current treatments are still controversial due to numerous side effects. Targeted immunotherapy recently emerged as an ideal alternative to improve treatment modalities for cancer patients. However, very limited approaches are available today and major issues remain to be addressed. In this context, we are interested in the design of biomolecular structures, innovative and bifunctional, able to hijack endogenous antibodies - which are naturally present in the human blood stream - toward cancer cells without pre-immunisation. Since natural circulating antibodies are polyspecific and have the ability to interact with multiple carbohydrate antigens, we focused on the design of multivalent glycodendrimers, as ligands for endogenous antibodies. The first part of our study consisted in synthesizing several multivalent glycoconjugates, based on peptide scaffolds and obtained by chemoselective ligations. To evaluate their influence on antibodies, the nature of both the carbohydrate and the scaffold, and the valency were varied. Then, in a second part of the study, microarray assays were developed with a model lectin, the Helix Pomatia Agglutinin (HPA). Experimental procedures were designed to determine surface dissociation constant and IC50 values, leading to the identification of high affinity ligands for HPA in the nanomolar range. Microarray assays were confirmed by other analytical methods (BLI, ELLA). Finally, the assays on slides were adapted to human sera screening, in order to identify tridimensional architectures highly affine to sera antibodies. A large panel of glycoconjugates were screened by microarray with around twenty sera, leading to the determination of promising glycosylated structures, as antibody ligands. The latter could be subsequently used for our anti-cancer approach.