Erythropoietin (EPO) is a glycoprotein hormone, the molecule comprises a single polypeptide chain of 165 aminoacids with two disulfide bonds, 1 O-linked (Ser-126), and 3 N-linked (Asn-24, 38, 83) glycans representing about 40 % of the total mass (30 kDa). It is secreted primarily by adult kidneys in response to tissue hypoxia and it is involved in the maturation and ultimately regulation of the level of red blood cells. The recombinant analogue (rhEPO), available since 1989 has found widespread use in the treatment of different diseases. Besides, rhEPO is illicitly used by athletes to boost the delivery of oxygen to the tissue and enhance performance in endurance sports. Current tests to differentiate between endogenous EPO and its recombinant analogues are based on differences in their isoelectric focussing (IEF) profiles and on differences in their molecular weight (SDS-PAGE). In this study, different methods to facilitate the detection of recombinant EPOs and analogues in antidoping control have been developed: A plasmatic EPO immunopurification method; a new screening method based on immunoaffinity techniques to detect the abuse of recombinant erythropietins in urine; and a liquid chromatography-mass spectrometry method that allows to detect the unambiguous differing structure between exogenous EPOs and endogenous, the N-glycolyl-neuraminic acid. / La eritropoetina (EPO) és una hormona glicoproteica formada per una cadena peptídica de 165 aminoàcids que conté dos ponts disulfur, un O-glicà (Ser-126) i tres N-glicans (Asn-24, 38, 83) que representen al voltant d’un 40% de la seva massa molar (~ 30kDa). Es produeix principalment en el ronyó, en resposta a la reducció d’oxigen en el teixits, i estimula l’eritropoesi a la medul·la òssia. La EPO recombinant (rhEPO) s’administra com a fàrmac pel tractament de diferents malalties. També s’ha observat la seva utilització en esportistes amb l’objectiu d’augmentar el nivells d’oxigen als teixits i així incrementar el seu rendiment. Els mètodes que s’utilitzen per diferenciar la EPO orinaria endògena de l’exògena estan basats en diferencies dels seus perfils isoelectroforètics (IEF) o en els seus pesos moleculars (SDS-PAGE). El problema d’aquests mètodes és que són llargs, costosos i només poden utilitzar la orina com a matriu biològica. En aquest estudi, s’ha dut a terme el desenvolupament dels següents mètodes que faciliten la detecció d’EPOs recombinants i anàlegs en el control antidopatge: Un mètode d’immunopurificació d’EPO en plasma; un mètode d’screening ràpid basat en tècniques d’immunoafinitat per detectar l’abús d’ eritropoietines recombinants en orina; i un mètode de cromatografia liquida acoblada a espectrometria de massa que permet detectar una clara diferencia estructural entre la majoria de les EPOs exògenes i la endògena, el N-glicolil-neuraminic àcid.

Wild birds are the natural reservoirs of Influenza A viruses (IAVs) which cause occasional pandemics and seasonal epidemics. Avian IAVs can be transmitted from wild birds to domestic poultry, low mammals, and humans. It is well accepted that avian IAVs prefer to sialic acids (Sia) α2,3-linked galactose (SAα2,3-Gal), whereas human IAVs to α2,6-linked galactose (SAα2,6-Gal). However, SAα2,3-Gal in wild bird tissues is widely distributed with little variation while some subtypes of avian IAVs have species preference. The different isolation rates among wild bird species cannot be explained by avian IAVs binding to SAα2,3-Gal alone. Specifically, this dissertation had the following aims: Firstly, to determine distribution of glycan receptors across respiratory and gastrointestinal tissues of wild birds and domestic poultry; Secondly, to determine the viral-receptor binding specificity of avian IAVs; Thirdly, to understand the role of glycan motifs in shaping virus evolution during the natural history of IAVs, especially from wild bird to poultry transmission. We found that avian H7 and H10 viruses acquired the binding ability to SAα2,6-Gal without adaptation, furthermore, we evaluated one of these H10 virus that possess the ability of binding to SAα2,6-Gal in ferret model and found it could cause aerosol and contact transmissions. On the other hand, H7 viruses have strong binding avidity to SLex which are present widely in epithelial cells of chicken trachea, which could facilitate the transmission of avian H7 viruses from waterfowl to poultry. Lastly, we found that H7 viruses from waterfowl bound both Neu5Ac and Neu5Gc while chicken isolates from China only prefer to Neu5Ac. Of interest, we found Neu5Gc was found in mallards but not in chickens, which indicated that viruses reduce or lost binding ability to Neu5Gc by adapting in chicken. In summary, this dissertation focused on certain subtypes of avian IAVs, which have caused threats to domestic poultry and public health, and primary avian species for influenza risk. The glycan substructures may play an important role in avian IAVs transmission and adaptation. The knowledge derived from this dissertation will help identify species for influenza surveillance in wild birds and facilitate risk assessment of avian IAVs.