Pseudomonas aeruginosa ᴅ-arginine dehydrogenase (PaDADH) is a flavin-dependent enzyme. The enzyme catalyzes the oxidative deamination of a broad range of ᴅ-amino acids to their corresponding imino-acids, which are non-enzymatically hydrolyzed to α-keto-acids and ammonia. A46, S45 and I335 residues are located in flexible loops, which form a flask-like substrate-binding pocket. In this study, I335, A46, and S45 were mutated to histidine, glycine, and alanine, respectively and individually, through site-directed mutagenesis, to investigate their role in binding and catalysis in PaDADH. The results showed that A46 and S45 residues participate in the optimal orientation of the substrate α-amino group and I335 modulate the active site flexibility.

Στην παρούσα εργασία μελετήθηκε η επίδραση των διατροφικών επιπέδων βιταμίνης Α στην ανάπτυξη των σκελετικών δυσπλασιών και των μεριστικών χαρακτήρων του λαβρακιού. Εφαρμόσθηκαν 7 διαφορετικά διατροφικά επίπεδα ρετινόλης (0, 5, 10, 15, 25, 35 και 70 mg ρετινόλης ανά Kg ξηράς τροφής) σε 21 νυμφικούς πληθυσμούς λαβρακιού (3 επαναλήψεις ανά διατροφική ομάδα). Η εφαρμογή των διαφορετικών συνθηκών έλαβε χώρα στο διάστημα των πρώτων 45 ημερών ανάπτυξης. Συνολικά μελετήθηκαν 1098 νύμφες και 1053 ιχθύδια. Τα αποτελέσματα έδειξαν πως οι διαφορετικές συγκεντρώσεις της βιταμίνης Α επιδρούν σημαντικά στην ανάπτυξη σκελετικών παραμορφώσεων στις νύμφες και τα ιχθύδια του λαβρακιού, καθώς επίσης και στους μεριστικούς χαρακτήρες. Οι διάφορες σκελετικές δομές δεν παρουσίασαν την ίδια ευαισθησία στη βιταμίνη Α. Σημαντικά επηρεάστηκαν δομές του κρανίου όπως οι βραγχιοστεγείς ακτίνες, το υοειδές τόξο και οι γνάθοι, καθώς και τα σκελετικά στοιχεία των πτερυγίων. Τα βέλτιστα επίπεδα των εξεταζόμενων συγκεντρώσεων βιταμίνης Α στα οποία παρατηρούνται οι μικρότερες συχνότητες εμφάνισης δυσπλασιών διαφοροποιήθηκαν σημαντικά, ανάλογα με την εξεταζόμενη ανατομική περιοχή. Εντούτοις, η συγκέντρωση 5-10 mg Kg-1 παρατηρήθηκε σε πολλές περιπτώσεις να αντιστοιχεί στο βέλτιστο. Στο εύρος αυτό της συγκέντρωσης ρετινόλης παρατηρήθηκε και η μεγιστοποίηση του ρυθμού σωματικής αύξησης των ατόμων, όπως αυτή εκτιμήθηκε από το ολικός μήκος σώματος στην ηλικία των 45 ημερών μετά την εκκόλαψη. Όσον αφορά στους μεριστικούς χαρακτήρες, αυξανομένης της συγκέντρωσης της ρετινόλης οι προ-αιματικοί σπόνδυλοι τείνουν να αυξηθούν ενώ ο αριθμός των ραχιαίων στοιχείων όπως και των δερματοτρίχιων φαίνεται να μειώνεται. Τα αποτελέσματα συζητούνται ως προς τα αποτελέσματα παρόμοιων εργασιών για άλλα είδη ψαριών, ως προς τους πιθανούς μηχανισμούς δράσης της ρετινόλης, καθώς και ως προς την υπόθεση της αλλαγής των διατροφικών προτιμήσεων του λαβρακιού κατά τη διάρκεια της οντογένεσης. / The goal of the current MSc Thesis was to examine the effect of Vitamin A both on the development of skeletal deformities and of the meristic characters in European sea bass. The experimental design involved 7 different dietary levels of retinol (0, 5, 10, 15, 25, 35, 70 mg retinol kg-1 of dry food) and included 3 repetitions. Due to the fact that both the meristic characters and the skeletal deformities develop during early developmental stages, the application of the experimental dietary levels took place during the first 45 days post hatching. In total 1098 larvae and 1053 juveniles were examined. Results demonstrated that the different dietary levels of Vitamin A significantly affected the development of skeletal deformities in both the larval and juvenile stage, as well the meristic characters. The different anatomical areas exhibited different sensitivity in Vitamin A. There was a significant effect on cephalic structures such as the branchiostegal rays, hyoid arch and the jaws and on the internal skeletal elements of the fins. The optimum levels of dietary Vitamin A where the lowest frequencies of skeletal deformities were observed differed between the different anatomical areas. However, the dietary level of 5-10 mg Kg-1 was identified as the common optimum. In this range of dietary retinol the growth rate was maximized, as it was estimated by the body fork length at the age of 45 days post hatching. As far as the meristic characters are concerned, the number of pre-haemal vertebra tended to increase whereas the number of dorsal elements and caudal dermatotrichia to decrease at elevated retinol levels. The results are discussed in respect to similar studies for other fish species, the presumable molecular mechanisms of retinol action, as well as the hypothesis of switching dietary preferences of sea bass during ontogeny.

No description available.

Calcific aortic valve sclerosis is characterized by focal lesions in the valve leaflet. These lesions are rich in myofibroblasts that express α-SMA and cause fibrosis. Lesions tend to occur in regions of the leaflet that are subjected to large bending loads, suggesting a mechanobiological basis for myofibrogenic differentiation and valve pathogenesis. In this thesis, a bioreactor was developed to study the effect of physiological loading on myofibrogenic differentiation of valve interstitial cells. Cyclic loading of native porcine aortic valve leaflets ex vivo resulted in increased α-SMA expression, predominantly in the fibrosa and spongiosa (similar to sclerotic leaflets). Cofilin, an actin-binding protein, was also upregulated by loading, suggesting it plays a role in mechanically-induced myofibrogenesis. Similarly, loading of a tissue engineered aortic valve leaflet model resulted in increased α-SMA transcript and protein expression. These data support an integral role for mechanical stimuli in myofibrogenic differentiation and sclerosis in the aortic valve.

Calcific aortic valve sclerosis is characterized by focal lesions in the valve leaflet. These lesions are rich in myofibroblasts that express α-SMA and cause fibrosis. Lesions tend to occur in regions of the leaflet that are subjected to large bending loads, suggesting a mechanobiological basis for myofibrogenic differentiation and valve pathogenesis. In this thesis, a bioreactor was developed to study the effect of physiological loading on myofibrogenic differentiation of valve interstitial cells. Cyclic loading of native porcine aortic valve leaflets ex vivo resulted in increased α-SMA expression, predominantly in the fibrosa and spongiosa (similar to sclerotic leaflets). Cofilin, an actin-binding protein, was also upregulated by loading, suggesting it plays a role in mechanically-induced myofibrogenesis. Similarly, loading of a tissue engineered aortic valve leaflet model resulted in increased α-SMA transcript and protein expression. These data support an integral role for mechanical stimuli in myofibrogenic differentiation and sclerosis in the aortic valve.

Statement of problem: We have shown that the C677T 5,10 methylenetetrahydrofolate reductase (MTHFR) variant is associated with elevated maternal serum α-fetoprotein (MSAFP), the most common screening test for neural tube defects (NTD). Therefore, past contradictory studies of NTDs and C677T MTHFR may have been biased because of changes in case populations after prenatal diagnosis and termination of pregnancy. Further, an unexplained elevation of MSAFP is known to increase the risk for later pregnancy complications. Is the C677T MTHFR variant a predisposing genetic variant for both NTDs and later complications of pregnancy? Methods: A retrospective study of women with pregnancies resulting in NTD outcome and women with unexplained elevations of MSAFP was undertaken. Women and their partners were genotyped for the C677T MTHFR allele. Couples with a pregnancy resulting in a NTD outcome were compared to couples whose pregnancy outcome did not involve. Couples with unexplained elevations of MSAFP who did and did not have later complications of pregnancy were also compared. Allele frequencies for all groups were then compared against the previously established Manitoba population allele frequency (based on 977 consecutive newborn metabolic screening bloodspots). A review of all studies of NTDs and association with the C677T MTHFR variant was undertaken to determine if the association between the variant and MSAFP is a source of bias. NTD incidence was examined before and after folic acid food fortification introduced in Canada in 1999. Results: There is an increase in the allele frequency of the C677T MTHFR variant in parents with an unexplained elevated MSAFP followed by later complications of pregnancy. The C677T MTHFR variant is also a contributing genetic factor to NTDs worldwide. The incidence of NTDs in Manitoba has decreased by 37% since food fortification with folic acid was introduced. Conclusions: The C677T MTHFR variant is a contributing genetic factor to both later complications of pregnancy after an unexplained elevation of MSAFP and to NTDs. This variant is folate sensitive and folic acid fortification has reduced the incidence of NTDs.

Statement of problem: We have shown that the C677T 5,10 methylenetetrahydrofolate reductase (MTHFR) variant is associated with elevated maternal serum α-fetoprotein (MSAFP), the most common screening test for neural tube defects (NTD). Therefore, past contradictory studies of NTDs and C677T MTHFR may have been biased because of changes in case populations after prenatal diagnosis and termination of pregnancy. Further, an unexplained elevation of MSAFP is known to increase the risk for later pregnancy complications. Is the C677T MTHFR variant a predisposing genetic variant for both NTDs and later complications of pregnancy? Methods: A retrospective study of women with pregnancies resulting in NTD outcome and women with unexplained elevations of MSAFP was undertaken. Women and their partners were genotyped for the C677T MTHFR allele. Couples with a pregnancy resulting in a NTD outcome were compared to couples whose pregnancy outcome did not involve. Couples with unexplained elevations of MSAFP who did and did not have later complications of pregnancy were also compared. Allele frequencies for all groups were then compared against the previously established Manitoba population allele frequency (based on 977 consecutive newborn metabolic screening bloodspots). A review of all studies of NTDs and association with the C677T MTHFR variant was undertaken to determine if the association between the variant and MSAFP is a source of bias. NTD incidence was examined before and after folic acid food fortification introduced in Canada in 1999. Results: There is an increase in the allele frequency of the C677T MTHFR variant in parents with an unexplained elevated MSAFP followed by later complications of pregnancy. The C677T MTHFR variant is also a contributing genetic factor to NTDs worldwide. The incidence of NTDs in Manitoba has decreased by 37% since food fortification with folic acid was introduced. Conclusions: The C677T MTHFR variant is a contributing genetic factor to both later complications of pregnancy after an unexplained elevation of MSAFP and to NTDs. This variant is folate sensitive and folic acid fortification has reduced the incidence of NTDs.

Parkinson’s disease is a neurodegenerative disease resulting primarily from loss of dopaminergic innervation in the striatum subsequent to cell loss in the substantia nigra pars compacta. The abnormal accumulation of the normal pre-synaptic protein α-synuclein (αsyn) forms intraneuronal inclusions known as Lewy neurites and Lewy bodies. The origins of central Lewy pathology have been suggested to lie in the enteric nervous system, ascending through the vagus nerve to the dorsal motor nucleus of the vagus. To ascertain gastrointestinal regions most likely to be the source of central Lewy pathology, αsyn expression was evaluated in the neural elements of gastrointestinal regions receiving the densest vagal innervation. The vermiform appendix was found to have the densest αsyn-immunoreactive innervation in all layers of the gut wall. In addition, macrophages in the appendiceal mucosa were laden with αsyn within lysosomes, consistent with attempts to prevent the spread of disease or to correct synaptic dysfunction.

Glucose metabolism in pancreatic α- and β-cells is believed to regulate secretion of glucagon and insulin, respectively. In β-cells, ATP links glucose metabolism to electrical activity and insulin secretion. In α-cells, ATP has been attributed various roles in glucose-regulated glucagon release, but the underlying mechanisms are poorly understood. Despite its importance in insulin and glucagon secretion little is known about ATP kinetics in α- and β-cells. In this thesis, the novel fluorescent ATP biosensor Perceval was used to monitor physiologically relevant ATP concentrations with little influence of ADP. Glucose stimulation of β-cells within mouse and human pancreatic islets induced pronounced rise of ATP with superimposed oscillations. Simultaneous measurements of the sub-plasma membrane ATP and Ca2+ concentrations revealed glucose-induced oscillations in opposite phase. ATP increased further and the oscillations ceased when voltage-dependent Ca2+ influx was prevented. In contrast, ATP promptly decreased in response to K+-depolarization-induced elevation of Ca2+. Also mobilization of Ca2+ from intracellular stores lowered ATP, but the negative effect was not due to increased ATP consumption by the sarco/endoplasmic reticulum Ca2+-ATPase. Store-operated Ca2+ entry alone had little effect but markedly elevated ATP when combined with muscarinic receptor activation. When comparing ATP and Ca2+ responses in α- and β-cells within the same islet, glucose-induced ATP generation was much less pronounced and the dose-response relationship left-shifted in the α-cells. At basal glucose, individual α-cells showed Ca2+ and concomitant ATP oscillations in opposite-phase with variable frequency. These oscillations largely cancelled out when averaging data from several α-cells. At high glucose, the Ca2+ and ATP oscillations in α-cells tended to synchronize with the corresponding β-cell oscillations. Since β-cell Ca2+ oscillations drive pulsatile insulin secretion, which is antiparallel to pulsatile glucagon secretion, there seems to be an inverse relationship between changes in α-cell Ca2+ and glucagon release. This paradox is attributed to paracrine inhibition overriding Ca2+ stimulation, since somatostatin receptor blockade potently stimulated glucagon release with little effect on α-cell Ca2+ signalling. The data indicate that complex ATP-Ca2+ interactions in α- and β-cells underlie cell-intrinsic regulation of glucagon and insulin secretion and that paracrine inhibition of glucagon release becomes important in hyperglycaemia.

A new analytical technique to measure the chemical composition distribution (CCD) of polyolefins, crystallization elution fractionation (CEF), was introduced in 2006 during the First International Conference on Polyolefin Characterization. CEF is a faster and higher resolution alternative to the previous polyolefin CCD analytical techniques such as temperature rising elution fractionation (TREF) and crystallization elution fractionation (CRYSTAF) (Monrabal et al., 2007). Crystallization elution fractionation is a liquid chromatography technique used to determine the CCD of polyolefins by combining a new separation procedure, dynamic crystallization, and TREF. In a typical CEF experiment, a polymer solution is loaded in the CEF column at high temperature, the polymer is allowed to crystallize by lowering the solution temperature, and then the precipitated polymer is eluted by a solvent flowing through the column as the temperature is raised. CEF needs to be calibrated to provide quantitative CCD results. A CEF calibration curve consists of a mathematical relationship between elution temperature determined by CEF and comonomer fraction in the copolymer that could be estimated by Fourier transform infrared spectroscopy (FTIR) and carbon-13 nuclear magnetic resonance (13C NMR). Different comonomer types in ethylene/α-olefin copolymers will have distinct calibration curves. The main objective of this thesis is to obtain CEF calibration curves for several different ethylene/α-olefin copolymers and to investigate which factors influence these calibration curves. A series of homogeneous ethylene/α-olefin copolymers (1-hexene, 1-octene and 1-dodecene) with different comonomer fractions were synthesized under controlled conditions to create CEF calibration standards. Their average chemical compositions were determined by 13C NMR and FTIR and then used to establish CEF calibration curves relating elution temperature and comonomer molar fraction in the copolymer.