Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Ecient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and ecient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

Thesis (M. Tech.(Environmental Health)) -- Central University of Technology Free State, 2005 / In developing countries such as South Africa commercial chicken farmers produce the majority of eggs, approximately 5.8kg of eggs per capita per annum. Despite this, many people, especially in rural and marginal-urban areas, still consume eggs produced by backyard systems. Backyard systems are characterised by fragmented and small-scale production units that require minimal management and chickens are often unhoused or poorly housed. In most cases, eggs from backyard systems are laid in nests in poor hygienic condition. Eggs are a cheap, readily available and a good source of animal protein and are consumed by the majority of the people in the community, including the young, the old and people with HIV/AIDS. With little information available regarding the microbiological quality of eggs produced by backyard chickens in Southern Africa, the risks posed by these eggs to consumers are unknown. In this study the microbiological quality of eggs from randomly selected household near Hennenman keeping backyard chickens was determined. The study was done over three seasons which included the cold-dry (May-July), mild-dry (October- February) and the warm-wet (August-September) seasons. The following organisms were isolated: Salmonella spp., Pseudomonas spp., Staphylococcus spp., Escherichia coli and Total Coliforms. Staphylococcus spp. was further characterised to species level. Most of the species were of human origin, with the exception of only two species, S. hyicus and S. lentus, which have previously been associated with chickens. Furthermore, questionnaires were administered to the backyard chicken keepers to assess their knowledge regarding chicken keeping and nest hygiene, the proper method of egg collection and storage, and the preparation of eggs. The decrease of vitamins and Staphylococcus spp. occurring during different preparation methods (scrambling, frying and boiling) was also determined. The results obtained showed that the eggshells were more contaminated than the egg contents. This had been expected as the eggshell is more in contact with the external environment than the egg contents are. Faecal contaminants (Salmonella spp., Escherichia coli and Total Coliforms) were present in both the eggshell and the egg contents during all seasons and this could be attributed to the infrequent cleaning of chicken nests as ascertained from the questionnaires. From the vitamin analysis it was observed that backyard-produced eggs had lower concentrations of vitamins A and E compared to commercially-produced eggs. When determining the best preparation method, causing the most degradation of Staphylococcus spp., while on the other hand preserving vitamins, it was found that scrambling was the best method, followed by the frying and boiling methods respectively.

Thesis (M.Tech.: Biotechnology)- Dept. of Biotechnology & Food Technology, Durban University of Technology, 2007 xv, 102 leaves / For decades the genus Aspergillus (of fungi) has been classified based on morphological and growth criteria. Members of the Aspergillus section Flavi are economically valuable and methods of differentiating them are thus very important. Several molecular methods have been developed to distinguish these strains. Also, a number of biochemical and genetic studies have been used in order to provide a better means of classification (Lee et al., 2004). Aflatoxins, the most frequently studied mycotoxins, are produced by certain Aspergillus species/strains/isolates of fungi. The aflatoxin biosynthetic pathway studies have led to a number of discoveries. Several structural and regulatory genes (and their enzymes) involved in the biosynthesis of aflatoxins have been discovered and purified (Trail et al., 1995). Aflatoxin production and contamination of agricultural crops are major causes of economic losses in agriculture. Thus, better methods of characterization/differentiation are required for both aflatoxigenic and non-aflatoxigenic isolates. Molecular biology is one of the current tools used to differentiate between these isolates. Polymerase Chain Reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) analysis has been used successfully in the analysis of DNA relatedness of species of fungi, bacteria, plants and animals. Dendograms which evaluate/assess the likeness between different isolates has also been used (Martinez et al., 2001). Restriction fragment length polymorphism (RFLP) analysis has been applied to a number of studies to detect differences between fungi and to establish relationships between them. Therefore, the scope of this study was to investigate RAPD analysis (with dendograms) and detection of RFLPs by hybridization as molecular methods that can distinctly differentiate or characterize the aflatoxigenic and non-aflatoxigenic Aspergillus isolates.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Mastitis is considered to be the most costly disease affecting the dairy industry. Management strategies involve the extensive use of antibiotics to treat and prevent this disease. Prophylactic dosages of antibiotics used in mastitis control programmes could select for strains with resistance to antibiotics. In addition, a strong drive towards reducing antibiotic residues in animal food products has lead to research in finding alternative antimicrobial agents. Streptococcus macedonicus ST91KM, isolated from bulgarian goat yoghurt, produces the bacteriocin macedocin ST91KM with a narrow spectrum of activity against Grampositive bacteria. These include mastitis pathogens Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus epidermidis as well as Lactobacillus sakei and Micrococcus varians. Macedocin ST91KM is, according to tricine-SDS PAGE, between 2.0 and 2.5 kDa in size. The activity of macedocin ST91KM remained unchanged after 2 h of incubation at pH 2.0 to 10.0 and 100 min at 100 °C. The peptide was inactivated after 20 min at 121 °C and when treated with pronase, pepsin and trypsin. Treatment with α-amylase had no effect on activity, suggesting that the mode of action does not depend on glycosylation. Precipitation with 60 % saturated ammonium sulphate, followed by Sep-Pak C18 separation recovered 43 % of macedocin ST91KM. Amplification of the genome of strain ST91KM with primers designed from the sequence of the macedocin prescursor gene (mcdA) produced two fragments (approximately 375 and 220 bp) instead of one fragment of 150 bp recorded for macedocin produced by S. macedonicus ACA-DC 198. Strain ACA-DC 198 was not available. However, the DNA fragment amplified from strain LMG 18488 (ACA-DC 206), genetically closely related to strain ACADC 198, revealed 99 % homology to the mcdA of S. macedonicus ACA-DC 198 (accession number DQ835394). Macedocin ST91KM may thus be a related bacteriocin described for S. macedonicus. The peptide adsorbed equally well (66 %) to L. sakei LMG13558 and insensitive cells, e.g. Enterococcus faecalis BFE 1071 and FAIR E92, and Streptococcus caprinus ATCC 700066. Optimal adsorption of macedocin ST91KM was recorded at 37 °C and 45 °C and at pH of 8 - 10. Addition of solvents decreased adsorption by 50%, suggesting that the receptors to which the bacteriocin binds have lipid moieties. The addition of MgCl2, KI and Na2CO3 completely prevented adsorption of macedocin ST91KM to the target cells, possibly due to competitive ion adsorption on the bacterial cell surface. The peptide has a bacteriocidal mode of action, resulting in lysis and the release of DNA and β-galactosidase. Atomic force microscopy of sensitive cells treated with macedocin ST91KM have shown deformation of the cell structure and developing of irregular surface areas. Antimicrobial susceptibility patterns were evaluated against eighteen mastitis pathogens. All isolates tested were resistant to methicillin and oxacillin, but had minimum inhibitory concentrations (MICs) falling in the intermediate and susceptible range against erythromycin. S. agalactiae and S. epidermidis had the highest sensitivity to macedocin ST91KM. A teat seal preparation containing macedocin ST91KM effectively released bacteriocin inhibiting the growth of the bacterial pathogen. Macedocin ST91KM could form the basis for an alternative dry cow therapy to prevent mastitis infections in dairy cows, as it is effective against pathogens that display resistance to conventional antibiotic therapy.

Thesis (PhD(Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Pathogenic Flavobacterium spp. cause serious disease outbreaks in a variety of farmed fish, which lead to large economic losses in the aquaculture industry on an annual basis. The ability of Flavobacterium johnsoniae-like isolates to grow as surface-associated communities (biofilms) in aquaculture systems poses a threat to fish health over extended periods of time. The biofilmforming ability of 28 F. johnsoniae-like isolates obtained from diseased fish were correlated with their chitin-degrading abilities and extracellular carbohydrate complexes (ECC) and their pulsed-field gel electrophoresis (PFGE) genotypes. Physiological changes in the proteome of 5 day planktonic, biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were analyzed by two-dimensional (2-D) gel electrophoresis and 17 differentially expressed and 14 uniquely expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Thirty-two differentially expressed genes in 5 day biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were identified using suppression subtractive hybridization (SSH). Significant negative correlations were observed between the chitin-degrading abilities and ECC and the biofilmforming capacity of 24 h biofilm cultures of F. johnsoniae-like isolates. Genetic heterogeneity was displayed by the F. johnsoniae-like isolates following PFGE. A significant positive correlation was observed between PFGE types and fish host species. Differentially and uniquely expressed proteins identified in planktonic, biofilm and agar surface-associated phases by 2-D/MS as well as differentially expressed genes identified in the biofilm and agar surface-associated phases by SSH were categorized as being involved in adaptation/protection, metabolic processes, membrane/transport/ motility and transcription/ translation. As far as we know, this is the first report on the characterization of differentially expressed genes and gene products of F. johnsoniae-like isolates obtained from diseased fish in South Africa. / AFRIKAANSE OPSOMMING: Patogene Flavobacterium spp. veroorsaak ernstige infeksie uitbrake in ’n verskeidenheid gekweekte vissoorte, wat jaarliks tot groot ekonomiese verliese in die akwakultuur bedryf lei. Die vermoë van Flavobacterium johnsoniae-tipe isolate om as oppervlak-gehegde gemeenskappe (biofilms) in akwakultuur sisteme te groei bedreig visgesondheid oor verlengde periodes. Die vermoë van 28 F. johnsoniae-tipe isolate om biofilms te vorm is vergelyk met hul vermoë om chitien te degradeer, die profiel van hul ekstrasellulêre koolhidraat komplekse (EKK) en bandpatrone verkry met puls-veld jel elektroforese (PVJE). Fisiologiese veranderinge in die proteoom van 5-dagoue planktoniese-, biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 is met twee-dimensionele (2-D) jel elektroforese geanaliseer. Sewentien differensieël uitgedrukte en 14 uniek uitgedrukte proteïene is deur middel van matriks-geassisteerde laser desorpsie ioniserings-tyd van vlug-massa spektrometrie (MGLDI-TVV MS) geïdentifiseer. Twee-en-dertig differensieël uitgedrukte gene in 5-dag-oue biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 was deur middel van suppressie afgetrokke hibridisasie (SAH) geïdentifiseer. Beduidende negatiewe korrelasies is tussen die chitin-degraderings vermoë en EKK en die biofilm-vormings kapasiteit van 24-uur-oue biofilm kulture van F. johnsoniae-tipe isolate waargeneem. Resultate verkry met PVJE het die heterogene samestelling van F. johnsoniae-tipe isolate uitgewys. ‘n Beduidende positiewe korrelasie is tussen PVJE groeperings en vis gasheer spesie waargeneem. Differensieël en uniek uitgedrukte gene geidentifiseer in die planktoniese-, biofilm- en agar oppervlak-geassosieerde fases is deur middel van 2-D/MS asook differensieël uitgedrukte gene geïdentifiseer in die biofilm en agar oppervlakgeassosieerde fases deur middel van SAH was as betrokke by aanpassing/beskerming, metaboliese prosesse, membraan/vervoer/ beweeglikheid en transkripsie/translasie gekategoriseer. Sover bekend is hierdie die eerste beskrywing van differensieël uitgedrukte gene en geenprodukte van F. johnsoniae-tipe isolate afkomstig van geinfekteerde vis in Suid Afrika.

Η πιο απλή μορφή γενετικής διαφοροποίησης στον άνθρωπο είναι οι μονονουκλεοτιδικοί πολυμορφισμοί (Single Nucleotide Polymorphisms - SNPs). Ο αριθμός αυτού του είδους πολυμορφισμών που έχουν βρεθεί στο ανθρώπινο γονιδίωμα και επηρεάζουν την παραγόμενη πρωτεΐνη αυξάνεται συνεχώς, αλλά η αντιστοίχηση τους σε πιθανές ασθένειες με πειραματικές μεθόδους είναι ασύμφορη από θέμα χρόνου και κόστους. Για αυτό τον λόγο έχουν αναπτυχθεί διάφορες υπολογιστικές μέθοδοι με σκοπό να ταξινομήσουν τους μονονουκλεοτιδικούς πολυμορφισμούς σε παθογόνους και μη. Οι περισσότερες από αυτές τις μεθόδους χρησιμοποιούν ταξινομητές, οι οποίοι παίρνοντας σαν είσοδο ένα σύνολο δομικών, λειτουργικών, ακολουθιακών και εξελικτικών χαρακτηριστικών, επιχειρούν να προβλέψουν αν ένας μονονουκλεοτιδικός πολυμορφισμός είναι παθογόνος ή μη. Για την εκπαίδευση αυτών των ταξινομητών, χρησιμοποιούνται δύο σύνολα μονονουκλεοτιδικών πολυμορφισμών. Το πρώτο αποτελείται από μονονουκλεοτιδικούς πολυμορφισμούς που έχει βρεθεί πειραματικά ότι οδηγούν σε παθογένεια και το δεύτερο από μονονουκλεοτιδικούς πολυμορφισμούς που έχει αποδειχθεί πειραματικά ότι είναι αδρανείς. Οι μέθοδοι αυτές διαφέρουν στα χαρακτηριστικά των μεταλλάξεων που λαμβάνουν υπόψη στην πρόβλεψη τους, καθώς επίσης και στην εκπαίδευση και τη φύση των τεχνικών ταξινόμησης, που χρησιμοποιούν για τη λήψη των αποφάσεων. Το βασικότερο προβλήματα τους ωστόσο έγκειται στο γεγονός ότι καθορίζουν τα χαρακτηριστικά, που θα χρησιμοποιήσουν σαν είσοδο στους ταξινομητές τους με τρόπο εμπειρικό και μάλιστα διαφορετικές μέθοδοι προτείνουν και χρησιμοποιούν διαφορετικά χαρακτηριστικά, χωρίς να τεκμηριώνουν επαρκώς τις αιτίες αυτής της διαφοροποίησης. Δύο ακόμα προβλήματα που δεν έχουν καταφέρει να αντιμετωπίσουν οι υπάρχουσες μεθοδολογίες είναι το πρόβλημα της ανισορροπίας των δύο κλάσεων ταξινόμησης και των ελλιπών τιμών σε πολλά από τα χαρακτηριστικά εισόδου των ταξινομητών, ώστε να επιτυγχάνουν πιο ακριβή και αξιόπιστα αποτελέσματα. Από τα παραπάνω είναι ξεκάθαρο πως υπάρχει μεγάλο περιθώριο βελτίωσης των υπάρχουσων μεθοδολογιών για το συγκεκριμένο πρόβλημα ταξινόμησης. Στην παρούσα διπλωματική εργασία προτείνουμε μια νέα υβριδική μεθοδολογία υπολογιστικής νοημοσύνης, που ξεπερνά πολλά από τα προβλήματα των υπάρχοντων μεθοδολογιών και βελτιώνει με τον τρόπο αυτό την απόδοσή τους. Δύο είναι τα βασικά βήματα που ακολουθήσαμε για την επίτευξη του στόχου αυτού. Πρώτον, συγκεντρώσαμε από τις διαθέσιμες δημόσιες βάσεις δεδομένων, τους μονονουκλεοτιδικούς πολυμορφισμούς που χρησιμοποιήθηκαν για την εκπαίδευση και τον έλεγχο των μοντέλων μηχανικής μάθησης. Συγκεκριμένα, συλλέχθησαν και φιλτραρίστηκαν τα θετικά και αρνητικά σύνολα εκπαίδευσης και ελέγχου, που αποτελούνται από μονονουκλεοτιδικούς πολυμορφισμούς που είτε οδηγούν σε παθογένεια, είτε είναι ουδέτεροι. Για κάθε πολυμορφισμό των δύο συνόλων υπολογίσαμε χρησιμοποιώντας υπάρχοντα διαθέσιμα εργαλεία όσο το δυνατό περισσότερα δομικά, λειτουργικά, ακολουθιακά και εξελικτικά χαρακτηριστικά. Για εκείνα τα χαρακτηριστικά, για τα οποία δεν υπήρχε κάποιο διαθέσιμο εργαλείο υπολογισμού τους, υλοποιήσαμε τον κατάλληλο κώδικα για τον υπολογισμό τους. Το δεύτερο βήμα της διπλωματικής αφορούσε το σχεδιασμό και την υλοποίηση της κατάλληλης υβριδικής μεθόδου για την επίλυση του προβλήματος που μελετάμε. Χρησιμοποιήσαμε μια νέα μέθοδο ταξινόμησης την EnsembleGASVR. Πρόκειται για μια ensemble μεθοδολογία, που συνδυάζει σε ένα ενιαίο πλαίσιο ταξινόμησης οκτώ διαφορετικούς ταξινομητές. Κάθε ένας από αυτούς τους ταξινομητές βασίζεται στον υβριδικό συνδυασμό των Γενετικών Αλγορίθμων και των μοντέλων Παλινδρόμησης Διανυσμάτων Υποστήριξης (nu-Support Vector Regression). Συγκεκριμένα ένας Προσαρμοζόμενος Γενετικός Αλγόριθμος χρησιμοποιείται για να καθοριστεί το βέλτιστο υποσύνολο χαρακτηριστικών, καθώς και οι βέλτιστες τιμές των παραμέτρων των ταξινομητών. Σαν μέθοδο ταξινόμησης των μεταλλάξεων σε ουδέτερες και παθογενείς, προτείνουμε τον nu-SVR ταξινομητή, καθώς παρουσιάζει υψηλή απόδοση, καλή γενίκευση, δεν παγιδεύεται σε τοπικά βέλτιστα, ενώ ταυτόχρονα επιτυγχάνει την ισορροπία μεταξύ της ακρίβειας και της πολυπλοκότητας του μοντέλου. Μάλιστα για να ξεπεράσουμε τα πρόβληματα των ελλιπών τιμών και της ανισορροπίας των δύο κλάσεων ταξινόμησης, αλλά και για να βελτιώσουμε τη συνολική απόδοση της μεθοδολογίας μας, επεκτείναμε τον υβριδικό αλγόριθμο, ώστε να λειτουργεί σαν μία ensemble-συλλογική τεχνική, συνδυάζοντας οκτώ επί μέρους μοντέλα ταξινόμησης. Τα πειραματικά αποτελέσματα της προτεινόμενης μεθοδολογίας ήταν εξαιρετικά ελπιδοφόρα, καθώς η EnsembleGASVR μεθοδολογία υπερτερεί σημαντικά έναντι άλλων ευρέως γνωστών μεθόδων ταξινόμησης παθογενών μεταλλάξεων. / Single Nucleotide Polymorphisms (SNPs) are the most common form of genetic variations in humans. The number of SNPs that have been found in human genome and affect protein functionality is constantly increasing. Finding matches between SNPs and diseases using experimental techniques, is excessive disadvantageous in terms of time and cost. For this reason, several computational methods have been developed. These methods classify polymorphisms as pathogenic and non-pathogenic. Most of them use classifiers, which take as input a set of structural, functional, sequential and evolutionary features and predict whether a single nucleotide polymorphism is pathogenic or neutral. For training these classifiers use two sets of SNPs. The first one consists of SNPs that have been experimentally proven as pathogenic, whereas the second set consists of SNPs that have been experimentally characterized as benign. These methods differ in the classification methods they deploy and in the features they use as inputs. However, the main problem is the determination of an empirically verified set of features for training. Specifically, different methods suggest different feature sets, without adequately documenting the causes of this differentiation. In addition, the existing methodologies do not tackle efficiently the class imbalance problem between positive and negative training sets and the problem of missing values in the datasets. In this thesis a new hybrid computational intelligence methodology is proposed, that overcomes many of the problems of existing methodologies. The proposed method achieves high classification performance and systematizes the selection of relevant features. In the first phase of this study the polymorphisms were gathered from the available public databases and they were used for training and testing of the machine learning models. Specifically, the positive and negative training and test sets were collected and filtered. They consist of single nucleotide polymorphisms that lead to either pathogenesis or are neutral. For each polymorphism of the two sets, using existing available tools, a wide range of structural, functional, sequential and evolutionary features were calculated. For those features for which there was no available tool, the suitable program (code) was developed in order to compute them. In the second step a new embedded hybrid classification method called EnsembleGASVR is designed and implemented. The method uses an ensemble methodology, based on hybrid combination of Genetic Algorithms and nu-Support Vector Regression (nu-SVR) models. An Adaptive Genetic Algorithm is used to determine the optimal subset of features and the optimal values of the parameters of classifiers. We propose the nu-SVR classifier, since it exhibits high performance, good generalization ability, it is not trapped in local optima and achieves a balance between accuracy and complexity of the model. In order to overcome the problem of missing values and class imbalance, we extended the above algorithm to function as a collective ensemble-technique, combining eight individual classification models. In overall, the method achieves 87.45% accuracy, 71.78% sensitivity and 93.16% specificity. These priliminary results are very promising and shows that EnsembleGASVR methodology significantly outperforms other well-known classification methods for pathogenic mutations.

Hälsa är ett begrepp som har avsaknad av en bestämd definition. Det finns flertalet erkända definitioner såsom WHO:s men det finns även de definitioner som skapats av den enskilda individen. De flesta definitioner som finns kring och om hälsa har sitt ursprung i den fysiska aktivitetens värld, än dock menar flera personer att om hälsa enbart skall ses som god utifrån den fysiska aktiviteten så blir begreppet hälsa inte särskilt innehållsrikt. Syftet med studien var att undersöka hur lärarna ser på begreppet hälsa. Enligt bakgrund och tidigare forskning så är hälsa ett personligt och svårdefinierat begrepp, vilket de sex lärare som intervjuades i denna kvalitativa studie bekräftade. Lärarnas kunskap om hälsa har inhämtats från tidigare idrottsliga erfarenheter och utbildning. Resultatet i denna studie visar att hälsoundervisningen stundtals blir personlig då lärarna utgår från sina tidigare idrottsliga erfarenheter när de talar och undervisar om hälsa. Resultatet har analyserats utifrån två hälsomodeller, kontinuummodellen samt den kliniska modellen. Resultatet som framkommer är att hälsa många gånger ses ur ett kontinuumperspektiv och sällan ur ett dikotomt perspektiv. Sammanfattat så handlar denna studie om hur lärarna ser på begreppet hälsa och hur de önskade att hälsoundervisningen skulle kunna se ut. / Health defines a person’s general condition, both mind and body. Being healthy or have “good health” is something that people, on a daily basis, can read about in the newspaper or on the internet, but what is health or being healthy? Health is often taken for granted and is also often associated with training, but many claim that health is not only about being fit or train hard. There are different definitions of health; world health organization (WHO) has one that has been acknowledged for many years: “Health is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity”. This definition has been controversy because it says that a person only can have good health if the person is free from illness, injury or pain which can seems quite possible to achieve. WHO:s definition about health, is not the only one, there are  many more definitions that has been created by dissimilar people. The aim with this study was to see how teachers look at health (and health studies) and where they got their knowledge about health. If you look at previous studies it says that health is a personal thing, each individual has their own opinion what health is, something that the teacher in this study agreed about. This is a qualitative study and sex teachers were interviewed. The result has been analyzed by two health models, the continuum model and the clinic model. The result showed that the teachers look at health from a continuum perspective and the teachers also said that their athletic background were the main reason for there value of health and how they look at health overall.

<p>Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets. </p><p>In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention. </p><p>I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release. </p><p>Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways. </p><p>Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.</p> / Dissertation

<p>Млеко магарице је тек последњих година доспело у фокус научних истраживања, иако су његова благотоворна терапеутска и козметичка својства позната још од античких времена. Упркос томе што знање о функционалности млека магарице константно расте, његов антимикробни потенцијал остаје најмање истражен. Литературни подаци о микробиологији и евентуалној антимикробној активности млека магарице балканске расе која егзистира на подручју Србије и околних балканских земаља су јако лимитирани. Ово млеко се традиционално конзумира без претходне топлотне обраде, што покреће низ питања која се односе на здравствену безбедност конзумирања млека у сировом стању. Предмети истраживања ове докторске дисертације били су утврђивање микробиолошког квалитета сировог млека магарице балканске расе, као и испитивање његове антибактеријске активности према одабраним сојевима Escherichia coli, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes и Klebsiella pneumoniae. У циљу утврђивања главних носилаца антибактеријског дејства, као и самог модела антибактеријског деловања, у одабраним узорцима овог млека намењеним извођењу антибактеријских испитивања, извршено је одређивање садржаја антимикробних протеина - лизозима и лактоферина, као и садржаја калцијума. У узорцима млека магарице предвиђеним за испитивање антибактеријске активности према S. aureus и L. monocytogenes, одређен је садржај масних киселина са документованом антибактеријском активношћу према Грам<br />позитивним бактеријама. Присуство L. monocytogenes и Salmonella spp. није утврђено ни у једном од 137 испитаних узорака сировог млека магарице, док је број E. coli, колиформних бактерија, Enterobacteriaceae, квасаца и плесни и суспектног B. cereus био испод границе детекције методе. Према резултатима добијеним у овој докторској дисертацији сви испитани узорци сировог млека магарице балканске расе су задовољавали захтеве европских регулатива 92/46/ЕЕC и EC 853/2004 и са микробиолошког аспекта су у потпуности били безбедни за директно конзумирање без претходне топлотне обраде. Сирово млеко магарице балканске расе је показало различиту антибактеријску активност према Грам позитивним и Грам негативним бактеријским сојевима тестираним у оквиру ове докторске дисертације, чији интензитет је зависио од нивоа контаминације узорака млека тестираним патогеном и температуре инкубирања. Температура инкубирања од +15 &deg;C је у односу на +9 &deg;C била генерално повољнија за активност антибактеријских супстанци у млеку магарице према свим тестираним патогенима, али је антибактеријски потенцијал узорака млека магарице на +15 &deg;C био лимитиран бржим умножавањем испитаних бактерија на овој температури. То је посебно важило за узорке са већим нивоима контаминације (103 и 104 cfu/ml). Антибактеријска активност сировог млека магарице према L. monocytogenes и S. aureus се нa свим радним температурама састојала од продужeња лаг фазе и инхибирања бактеријског раста, док се у случају S. Enteritidis, S. Typhimurium, E. coli АTCC 8739, E. coli АTCC 10536 и K. pneumoniae она огледала у редукцији њиховог почетног броја и/или продужењу лаг фазе. Резултати ове докторске дисертације су указали на калцијум зависну природу антибактеријске активности сировог млека магарице према S. Enteritidis, S. Typhimurium и оба тестирана соја E. coli. Најснажнију антибактеријску активност према K. pneumoniae показали су узорци млека магарице, који су поред великог садржаја лизозима имали и велики садржај лактоферина, па би у овом случају основни механизам антибактеријског деловања млека магарице могао бити синергизам лизозима и лактоферина. Лизозим је највероватније главни носилац антибактеријске активности млека магарице балканске расе, обзиром да је у односу на други антимикробни агенс &ndash; лактоферин у тестираним узорцима млека магарице детектован у вишеструко већој концентрацији. Известан допринос антибактеријској активности млека<br />магарице балканске расе према L. monocytogenes и S. aureus могле би да дају и масне киселине са документованим антибактеријским дејством према Грам позитивним бактеријама (линолна, лауринска и олеинска киселина), које су у анализираним узорцима млека магарице у збиру чиниле од 40,3 до 54,7% од укупних масних киселина.</p> / <p>Mleko magarice je tek poslednjih godina dospelo u fokus naučnih istraživanja, iako su njegova blagotovorna terapeutska i kozmetička svojstva poznata još od antičkih vremena. Uprkos tome što znanje o funkcionalnosti mleka magarice konstantno raste, njegov antimikrobni potencijal ostaje najmanje istražen. Literaturni podaci o mikrobiologiji i eventualnoj antimikrobnoj aktivnosti mleka magarice balkanske rase koja egzistira na području Srbije i okolnih balkanskih zemalja su jako limitirani. Ovo mleko se tradicionalno konzumira bez prethodne toplotne obrade, što pokreće niz pitanja koja se odnose na zdravstvenu bezbednost konzumiranja mleka u sirovom stanju. Predmeti istraživanja ove doktorske disertacije bili su utvrđivanje mikrobiološkog kvaliteta sirovog mleka magarice balkanske rase, kao i ispitivanje njegove antibakterijske aktivnosti prema odabranim sojevima Escherichia coli, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes i Klebsiella pneumoniae. U cilju utvrđivanja glavnih nosilaca antibakterijskog dejstva, kao i samog modela antibakterijskog delovanja, u odabranim uzorcima ovog mleka namenjenim izvođenju antibakterijskih ispitivanja, izvršeno je određivanje sadržaja antimikrobnih proteina - lizozima i laktoferina, kao i sadržaja kalcijuma. U uzorcima mleka magarice predviđenim za ispitivanje antibakterijske aktivnosti prema S. aureus i L. monocytogenes, određen je sadržaj masnih kiselina sa dokumentovanom antibakterijskom aktivnošću prema Gram<br />pozitivnim bakterijama. Prisustvo L. monocytogenes i Salmonella spp. nije utvrđeno ni u jednom od 137 ispitanih uzoraka sirovog mleka magarice, dok je broj E. coli, koliformnih bakterija, Enterobacteriaceae, kvasaca i plesni i suspektnog B. cereus bio ispod granice detekcije metode. Prema rezultatima dobijenim u ovoj doktorskoj disertaciji svi ispitani uzorci sirovog mleka magarice balkanske rase su zadovoljavali zahteve evropskih regulativa 92/46/EEC i EC 853/2004 i sa mikrobiološkog aspekta su u potpunosti bili bezbedni za direktno konzumiranje bez prethodne toplotne obrade. Sirovo mleko magarice balkanske rase je pokazalo različitu antibakterijsku aktivnost prema Gram pozitivnim i Gram negativnim bakterijskim sojevima testiranim u okviru ove doktorske disertacije, čiji intenzitet je zavisio od nivoa kontaminacije uzoraka mleka testiranim patogenom i temperature inkubiranja. Temperatura inkubiranja od +15 &deg;C je u odnosu na +9 &deg;C bila generalno povoljnija za aktivnost antibakterijskih supstanci u mleku magarice prema svim testiranim patogenima, ali je antibakterijski potencijal uzoraka mleka magarice na +15 &deg;C bio limitiran bržim umnožavanjem ispitanih bakterija na ovoj temperaturi. To je posebno važilo za uzorke sa većim nivoima kontaminacije (103 i 104 cfu/ml). Antibakterijska aktivnost sirovog mleka magarice prema L. monocytogenes i S. aureus se na svim radnim temperaturama sastojala od produženja lag faze i inhibiranja bakterijskog rasta, dok se u slučaju S. Enteritidis, S. Typhimurium, E. coli ATCC 8739, E. coli ATCC 10536 i K. pneumoniae ona ogledala u redukciji njihovog početnog broja i/ili produženju lag faze. Rezultati ove doktorske disertacije su ukazali na kalcijum zavisnu prirodu antibakterijske aktivnosti sirovog mleka magarice prema S. Enteritidis, S. Typhimurium i oba testirana soja E. coli. Najsnažniju antibakterijsku aktivnost prema K. pneumoniae pokazali su uzorci mleka magarice, koji su pored velikog sadržaja lizozima imali i veliki sadržaj laktoferina, pa bi u ovom slučaju osnovni mehanizam antibakterijskog delovanja mleka magarice mogao biti sinergizam lizozima i laktoferina. Lizozim je najverovatnije glavni nosilac antibakterijske aktivnosti mleka magarice balkanske rase, obzirom da je u odnosu na drugi antimikrobni agens &ndash; laktoferin u testiranim uzorcima mleka magarice detektovan u višestruko većoj koncentraciji. Izvestan doprinos antibakterijskoj aktivnosti mleka<br />magarice balkanske rase prema L. monocytogenes i S. aureus mogle bi da daju i masne kiseline sa dokumentovanim antibakterijskim dejstvom prema Gram pozitivnim bakterijama (linolna, laurinska i oleinska kiselina), koje su u analiziranim uzorcima mleka magarice u zbiru činile od 40,3 do 54,7% od ukupnih masnih kiselina.</p> / <p>Donkey&#39;s milk has recently become a very popular topic among scientific community although it has been used for therapeutic and cosmetic purposes since ancient times. Even though the number of investigations concerning donkey&#39;s milk functionality has increased, the studies on its antimicrobial potential are limited. Especially, there is a lack in literature data on microbiological quality and antibacterial activity of donkey&#39;s milk obtained from Domestic Balkan breed originated in Serbia and other countries in Balkan region. The fact that this milk is traditionally consumed without heat treatment launches a series of issues concerning its safety.<br />The aim of this thesis was to determine microbiological quality of Domestic Balkan donkey&#39;s milk, as well as to investigate its antibacterial activity against the selected strains of Escherichia coli, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes and Klebsiella pneumonia. In order to determine the major antibacterial substances, as well as the model of antibacterial activity, the content of antimicrobial proteins (lysozyme and lactoferrin) and calcium was determined. The amount of fatty acids which are known to be effective against Gram-positive bacteria was determined in milk samples tested for antibacterial activity against S. aureus и L. monocytogenes. The presence of L. monocytogenes and Salmonella spp. was not detected in any of the 137 raw donkey&#39;s milk samples, while the levels of E. coli, coliform bacteria, Enterobacteriaceae, yeast and molds and suspect Bacillus cereus were below the limit of detection of the assays. According to obtained results, all examined Domestic Balkan donkeys&#39; raw milk samples were<br />in accordance with the EU regulations 92/46/ЕЕC and EC 853/2004 in terms of their<br />microbiological quality, thus being completely safe for direct consumptions, without thermal treatment. Raw Domestic Balkan donkey&#39;s milk has expressed different antibacterial activities against Gram-positive and Gram-negative bacteria strains tested in this thesis, with the intensities which depended on contamination level and incubation temperature. In comparison to temperature of +9 &deg;C, the incubation temperature of +15 &deg;C was more favourable for activity of antibacterial substances in donkey&#39;s milk against all tested pathogens. However, the antibacterial potential of donkey&#39;s milk samples at +15 &deg;C was limited with rapid multiplication of bacteria at this temperature. This was especially pronounced in samples characterized with higher contamination levels (103 and 104 cfu/ml). Antibacterial activity of raw donkey&#39;s milk against L. monocytogenes and S. aureus at all tested temperatures reflected in prolonged lag phase and inhibition of bacterial growth, while the activity against S. Enteritidis, S. Typhimurium, E. coli АTCC 8739, E. coli АTCC 10536 and K. pneumonia comprised reduction of initial bacterial numbers and/or prolongation of lag phase.<br />Results obtained in this thesis have indicated on calcium dependent antibacterial activity of raw donkey&#39;s milk against S. Enteritidis, S. Typhimurium and both E. coli strains. The most pronounced antibacterial activity against K. pneumonia was found in donkey&#39;s milk samples which besides high content of lysozyme had high content of lactoferrin, thus indicating that the basic antibacterial mechanism of donkey&#39;s milk might be the synergism between lysozyme and lactoferrin. Lysozyme was probably the substance with the highest antimicrobial activity in Domestic Balkan donkey&#39;s milk, since it was detected in significantly higher concentrations than lactoferrin. The fatty acids which are known to be effective against Gram-positive bacteria (linoleic, lauric and oleic) and which comprised from 40.3 to 54.7% of total fatty acids in tested samples, may also contribute to antimicrobial activity of Domestic Balkan donkey&#39;s milk against L. monocytogenes and S. aureus.</p>