The natural ability of filamentous fungi to synthesize, glycosylate, and secrete high levels of protein products has made them potentially attractive hosts for heterologous protein production. Advances in fungal genetics enabled the expression of several high value proteins in filamentous fungi. Particularly the genus, Aspergillus has proven to be potentially useful for the expression of eukaryotic gene products. This thesis pertains to the optimization of recombinant protein production by the fungal host, Aspergillus niger. The target recombinant protein of interest is hen egg white lysozyme (HEWL). This protein encoded in the genome resulting in relatively stable gene construct; however, it is subject to extracellular protease attack. The objective of the proposed research is the development and application of engineering methodology for the analysis and optimization of a fungal bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. To accomplish this, during the first phase of this study a statistically based experimental method was used to systematically elucidate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl₂.2H₂O) on hen egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2⁵⁻¹ fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other medium components were not important within the levels tested. Then, the method of steepest ascent was employed to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g/L, peptone 34 g/L, ammonium sulfate 11.9 g/L, yeast extract 0.5 g/L, and CaCl₂.2H₂O 0.5 g/L. This medium was projected to produce theoretically 212 mg/L lysozyme. Using this optimized medium, an experimentally observed maximum lysozyme concentration of 209±18 mg/L verified the applied methodology. A second optimization approach was based on metabolic flux analysis (MFA). A comprehensive metabolic network comprising three intracellular compartments (cytoplasm, mitochondrion and peroxisome) was developed for Aspergillus niger. The metabolic flux network included carbohydrate and amino acid metabolism in both anabolic and catabolic reactions. According to experimental observations, the time course of fermentation was divided into five phases, each with unique physiological properties. The network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consists of 137 metabolites and 287 processes, of which 181 represent biochemical conversions and 106 represent transport processes between the different compartments and the extracellular environment. In addition, due to the physiological evidence some biochemical reactions considered to be active only in one direction. Linear programming was used for optimizing of the specific growth rate as the objective function in combination with 37 measured input and output fluxes of the key metabolites to evaluate corresponding intracellular flux distributions throughout the batch fermentations. The general applicability of the methodology was evaluated by establishing commonality to optimize recombinant HEWL production. The proposed model was able to predict correctly the specific growth rate, oxygen uptake rate, and carbon dioxide evolution rate with good precision. The results of the metabolic flux and sensitivity analysis were employed for medium design. Growth was biphasic; glucose was utilized initially as the carbon source and was followed by its oxidation product, gluconate, later. Logarithmic sensitivity analysis revealed that the addition of proline, alanine and glutamate benefited growth in defined media. The experimental observations and flux analysis showed that tyrosine was a potential candidate for biomass production improvement. The two amino acids, namely proline and tyrosine benefited biomass production during the initial growth phases. Glutamate and alanine were particularly important during the latter stages of the batch process. A series of growth studies were conducted with the identified amino acids added in the medium. In these preliminary nutritional experiments the contribution to growth enhancement was 46% for proline, 23% for glutamate, and 22% for tyrosine. Model predictions were further verified by conducting batch and fed-batch fermentations in a 7- liter bioreactor. The programmed addition of four amino acids (proline, glutamate, alanine, and tyrosine) according to a predetermined schedule resulted in a 44% improvement in biomass and 41% improvement in recombinant protein production. The experiments also confirmed the model prediction that extra amount of amino acids besides the identified ones would not significantly enhance biomass and the recombinant protein production. A computer-based control system was developed for the on-line monitoring and control of the major state variables (e.g., temperature, pH, and DO) during the time course of fermentation. The graphical programming environment, LabVIEW was used to acquire and integrate these variables in a supervisor computer. The temperature of the bioreactor during sterilization and fermentation was controlled using a cascade methodology. The controller parameters of the master and slave loops were determined experimentally to yield a smooth response with minimum overshoot of both the bioreactor and jacket temperatures. The program scheduled various required steps in an established order during the fermentation. This feature of the software guarantees that every necessary operation will be met. The graphical representation of the process is displayed on the screen and helps the user to follow the process and perform the required adjustments. Furthermore, different variables can be observed simultaneously and saved in text or spreadsheet files for further analysis.

This study investigated factors associated with two mortality events: avian botulism in waterfowl and mortality associated with hatching asynchrony in prefledgling Franklins gulls (Larus pipixcan). The initial focus of my research was on the spatiotemporal relationship between mortality of Franklins gulls and the onset of botulism outbreaks in waterfowl, and the suitability of gull carcasses for proliferation and toxigenesis of Clostridium botulinum. From 1999 to 2001, dead hatch-year Franklins gulls were by far the most abundant carcasses, and the only source of toxin-laden maggots found on transects prior to the occurrence of avian botulism in waterfowl. Nest density was a significant predictor of hatch-year gull carcass density. High density of toxic material from gull carcasses prior to the onset of botulism in waterfowl coincided with high densities of susceptible birds; hence, mortality of Franklins gulls has the potential to be a major initiating factor for botulism outbreaks at Eyebrow Lake, Saskatchewan. The causes of gull mortality were conditions or diseases associated with starvation, stress, or immunosuppression, and most mortality occurred in third-hatched chicks. To separate effects of laying order from effects of hatching asynchrony on prefledgling survival, a cross-fostering experiment was conducted to create clutches containing asynchronously hatching eggs of the same laying order, and of similar egg mass, egg volume, and female quality. Hatching order, independent of laying order, significantly affected survival to fledging, whereas laying order had no observable effect, indicating that intraclutch variation in egg quality does not predetermine the fate of prefledglings, and may be less important than hatching asynchrony for survival of prefledgling Franklins gulls. Relationships among hatching asynchrony, laying order, mass, corticosterone, immune function, growth, and survival at two stages of development were complex. Hatching asynchrony significantly affected early and late prefledgling survival, and was directly or indirectly associated with mass, corticosterone level, and cell-mediated immune responses at early and later stages of development. Both hatching asynchrony and mass appeared to play key roles in mediating life history trade-offs among cell-mediated immune function, growth, and survival. In contrast to cell-mediated immune responses, primary humoral immune response was not directly affected by hatching order or mass, nor was it associated with survival to fledging. Rather, it was associated with laying order, neonatal testosterone, corticosterone at 2 weeks, growth of leg length, and clutch initiation date, illustrating the importance of examining more than one branch of the immune system in studies of life history trade-offs. This study is a step toward using a multipronged and multidisciplinary approach to demonstrate interactions and trade-offs among life history traits, the physiological mechanisms that produce these relationships, and how these relationships may change depending on stage of development.

This thesis explores the complex relationship between food protein structure and digestibility. Food proteins are important nutrients that play a central role in controlling the textural properties of many foods. Processing of food proteins may alter the protein aggregate structure and digestibility. The degree of protein aggregation during food processing depends on the denaturing conditions and the presence of other food components. Sugars and lipids may contribute to protein glycation and protein cross-linking via the Maillard reaction. Furthermore, amino acid residues of food proteins may be chemically modified during processing, thereby influencing both the structure and the nutritional value of proteins. An in vitro digestibility assay was used to investigate the relationship between protein aggregate structure and protein digestibility. Raw and boiled egg whites were exposed to a wide range of conditions: pH 2 - 12, in the presence and absence of 200 mM NaCl. It was found that pH and NaCl treatment prior to in vitro digestion resulted in significantly different protein ultrastructures, but did not markedly influence protein digestibility under the tested conditions. Raw egg white was less digestible than boiled egg white under all test conditions. The inclusion of Maillard reaction partners caused protein cross-linking concurrent with a decrease in digestibility. The digestibility decreased with the reactivity of the Maillard reaction partner and with increasing heating time. Proteomic analysis, using tandem mass spectrometry, of raw and heated egg white showed an increase in hydrothermally induced amino acid modifications. In the presence of glucose and methylglyoxal, a Maillard reaction specific increase in arginine modification to hydroimidazolone was observed with increasing heating times. The observed modifications are likely to contribute to a change in the nutritional quality of egg white. Aggregation kinetics of the major egg white protein, ovalbumin, were studied by dynamic light scattering, small angle X-ray scattering, and transmission electron microscopy. Shape determination was only possible for ordered aggregates, but not for disordered aggregates. Prior to heating, ovalbumin molecules in the presence of water and glucose repelled each other in concentrated solution. The presence of NaCl shielded electrostatic repulsion, leading to early onset dimerisation and disordered aggregation upon heating. Methylglyoxal treated ovalbumin formed more ordered aggregates. The scattering of these structures was able to be fitted to cylindrical shape models showing an increase of cylinder length with time while the cylinder diameter remained near constant over 24 hours of heating. In addition, food protein derived amyloid fibril aggregates were characterised. Amyloid fibrils are a common ordered protein fold that has been linked to neurodegenerative diseases. In the recent literature, amyloid fibrils have been proposed as new functional macromolecules in proteinaceous foods because of their desirable textural properties. Food fibrils formed from whey, egg white, soy bean and kidney bean protein were tested to establish whether they are protease resistant or display toxicity to human Caco-2 cells (a model intestinal cell line). The food fibrils were compared to insulin amyloid fibrils, a well characterised amyloid system. It was shown that the food fibrils displayed some resistance towards in vitro hydrolysis and were not found to be toxic. This work contributes to the understanding of food protein aggregation and digestibility under relevant conditions. It highlights the relationship of aggregate structure and digestibility and the particular role of the Maillard reaction. Moreover, evidence is provided that food protein derived amyloid fibrils may be safe ingredients in consumables. These findings may contribute to optimising industrial food processes and creating safe new food products.

Στην παρούσα εργασία πραγματοποιήθηκε εκτίμηση της αναπαραγόμενης βιομάζας του ευρωπαϊκού γαύρου, Engraulis encrasicolus, στην περιοχή του Βορειοανατολικού Αιγαίου (Θρακικό Πέλαγος, Κόλπος Καβάλας, Στρυμωνικός Κόλπος, Λήμνος) τα έτη 2003 και 2004 με τη Μέθοδο Ημερήσιας Παραγωγής Αβγών (DEPM). Για την εφαρμογή της μεθόδου πραγματοποιήθηκαν δύο ωκεανογραφικά ταξίδια με το Ε/Σ «ΦΙΛΙΑ» κατά το μέγιστο της ωοτοκίας του γαύρου τον Ιούνιο του 2003 και του 2004. Στη διάρκεια τους συλλέχθηκαν δείγματα ιχθυοπλαγκτού για την εκτίμηση της ημερήσιας παραγωγής αβγών, ενώ πραγματοποιήθηκαν και λήψεις κατακόρυφων διατομών θερμοκρασίας και αλατότητας σε εκτεταμένο δίκτυο σταθμών. Παράλληλα, πραγματοποιήθηκαν δειγματοληψίες ενήλικων ατόμων γαύρου είτε επί του επαγγελματικού στόλου των γρι-γρι της περιοχής, είτε με την πελαγική τράτα του «ΦΙΛΙΑ», τα οποία χρησιμοποιήθηκαν για την εκτίμηση της αναλογίας φύλου, της γονιμότητας ομάδας, της συχνότητας ωοτοκίας και του μέσου βάρους των θηλυκών. Όσον αφορά στις περιβαλλοντικές συνθήκες, το 2003 παρατηρήθηκε αυξημένη στρωματοποίηση των υδάτων, χαμηλότερη επιφανειακή αλατότητα και υψηλότερες τιμές χλωροφύλλης-α, διαφορές που πιθανώς οφείλονται σε αυξημένη εκροή νερού της Μαύρης Θάλασσας. Και τα δύο έτη η βιομάζα του ζωοπλαγκτού ήταν περίπου ίδια. Μέσω απλής ανάλυσης πηλίκου για το χαρακτηρισμό του αναπαραγωγικού ενδιαιτήματος του γαύρου, βρέθηκε ότι και τις δύο χρονιές η ωοτοκία πραγματοποιήθηκε σε νερά με χαμηλή αλατότητα (<34.5), πλούσια σε χλωροφύλλη-α και ζωοπλαγκτό. Αντίθετα, τα θερμοκρασιακά εύρη κατά τις δύο χρονιές διέφεραν, γεγονός που φαίνεται να αντανακλά περισσότερο τις διαφορές θερμοκρασίας ανάμεσα στα δύο έτη παρά διαφορετική προτίμηση για ωοτοκία. Επιπλέον, το 2004 η παραγωγή αβγών ήταν μειωμένη, το πεδίο αναπαραγωγής είχε συρρικνωθεί και η ωοτοκία ήταν επικεντρωμένη στην περιοχή του Θρακικού. Για την εκτίμηση της συχνότητας ωοτοκίας πραγματοποιήθηκε ιστολογική ανάλυση των θηλυκών γονάδων του γαύρου, από την οποία προέκυψε ότι ενώ τα στάδια ανάπτυξης των υγιών ωοκυττάρων ήταν παρόμοια με περιγραφές για το είδος Engraulis mordax, τα στάδια της ατρησίας παρουσίασαν ορισμένες διαφορές. Αυτές αφορούσαν στην εμφάνιση καφε-κίτρινων χρωστικών (χαρακτηριστικό γνώρισμα της δ-ατρησίας) στο τέλος της β-ατρησίας. Επιπλέον, η απορρόφηση των κενών ωοθυλακίων διαρκούσε δύο ημέρες σε αντίθεση με παρατηρήσεις για το Engraulis mordax, όπου παρατηρούνταν και κενά ωοθυλάκια τριών ημερών, διαφορά που οφείλεται στις υψηλότερες θερμοκρασίες του ΒΑ. Αιγαίου σε σχέση με περιοχές αναβλύσεων. Οι παράμετροι των ενηλίκων που προέρχονταν από δείγματα της επαγγελματικής και πειραματικής αλιείας δεν εμφάνισαν σημαντικές διαφορές μεταξύ τους. Αντίθετα, διαφορές παρατηρήθηκαν ανάμεσα στα δύο έτη όσον αφορά στις παραμέτρους του μέσου βάρους, της συχνότητας ωοτοκίας και της γονιμότητας. Συγκεκριμένα, το 2004 τα ψάρια ήταν βαρύτερα, πιο εύρωστα και απελευθέρωναν μεγαλύτερο αριθμό αβγών ανά μικρότερα χρονικά διαστήματα. Αν λάβει κανείς υπ’όψιν ότι το 2004 η αναπαραγόμενη βιομάζα ήταν σημαντικά μικρότερη ενώ η βιομάζα του ζωοπλαγκτού παρέμεινε η ίδια, οι παραπάνω διαφορές μπορούν να εξηγηθούν από φαινόμενα εξάρτησης των παραμέτρων αυτών από την πυκνότητα του πληθυσμού (density dependence). Η αναπαραγόμενη βιομάζα το 2004 (6251t) ήταν σημαντικά μειωμένη και αντιστοιχούσε σχεδόν στο 1/3 της βιομάζας του 2003 (17600t). Η μείωση αυτή πιθανώς να οφείλεται σε συνδυασμό έντονης αλιευτικής πίεσης και χαμηλών επιπέδων στρατολόγησης της ηλικιακής κλάσης του 2003. / The spawning biomass of the European anchovy, Engraulis encrasicolus, stock in the N.E. Aegean Sea was estimated by means of the Daily Egg Production Method (DEPM) for the years 2003 and 2004. Two oceanographic surveys were conducted with the R/V “PHILIA” during the maximum reproductive activity of the anchovy population in June 2003 and 2004. Ichthyoplankton sampling and vertical profiles of temperature and salinity were performed over an extensive grid of stations. At the same time adult anchovy samples were collected either on board the commercial purse-seine fleet or by means of an experimental pelagic trawl operated by “PHILIA”. The adult samples were used to estimate parameters of the DEPM: sex ratio, mean female weight, batch fecundity and spawning frequency. Significant interannual differences were found in the environmental conditions. In June 2003 the water column was more stratified, less saline (5m) and richer in chlorophyll-α, which probably were due to larger outflow of Black Sea Water (BSW). The zooplankton biomass remained the same during 2003 and 2004. A simple quotient rule analysis was applied to characterize the spawning habitat of anchovy. In both years anchovy spawning appeared to take place in less saline waters (34.5), rich in chlorophyll-α and zooplankton. On the contrary, anchovy spawning appeared to take place over different temperature range in the two years. This rather reflects different temperature values in 2003 and 2004 than different selection for spawning. In 2004 the daily egg production was reduced, the spawning area was limited and the spawning activity took place mainly in the Thracian Sea. Histological analysis of the female anchovy gonads was carried out in order to estimate the spawning frequency. The developmental stages of healthy oocytes were similar to those of the species Engraulis mordax. However, the atresia stages were different with regard to the appearance of brown-yellow pigments at the end of beta stage atresia instead of the end of delta stage atresia. Moreover, the absorption of the postovulatory follicle lasted two days instead of three days. The higher temperatures in the N.E. Aegean Sea were responsible for the shorter duration of the postovulatory follicle absorption. There were no statistically significant differences between DEPM adult parameters calculated from purse-seine samples compared to pelagic trawl samples. On the contrary, mean female weight, fecundity and spawning frequency showed statistically significant differences between the two years. In 2004 the anchovies were in better condition and produced numerous eggs in short interspawning intervals. Since the estimated biomass was lower in 2004 while the zooplankton biomass remained stable, it seems that density-dependence phenomena could justify the interannual differences. The estimated spawning biomass in 2004 (6251t) was significantly lower compared to that of 2003 (17600t). Intense fishing effort and low levels of recruitment of the 2003 cohort are probably responsible for this decrease.

Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.

Three dimensional printing (3DP) is one of the most important solid freeform fabrication (SFF) methods that can produce any material with desired 3D shape by using suitable powder-binder formulations. It differs from the standard molding operations in that it can produce a complicated shapes by a software driven instrument in a laminated fashion and the cost is lower. This method can be applied in a very wide area including drug release operations, biomaterial production especially for bone fixation, prototype production for all purposes, wound dressing etc. It can also be used in obtaining edible objects by using natural polysaccharides with water based binders. In this study, it is aimed to understand the gelling behaviour of some of the gelling materials, which are alginates, pectins and carageenans, and effect of various factors on the production of confectionary objects by means of 3DP process. Effect of multivalent cations, especially Ca2+ ion, on the gelling behaviour of these materials are investigated. The egg-box structure obtained between the polymer segments increases the water holding capacity of the materials and much more chewy structures can be obtained. The molecular changes are followed by Fourier Transform infrared spectroscopy (FTIR). In 3DP applications, the composition of powder and binder, pH, temperature, relative humidity (RH) and machine parameters are important factors affecting the texture of the final object. The texture of the produced specimens is examined by using a texture analyzer and maximum force values are given as g/cm at failure. Alginate and carrageenans are found to be more effective in obtaining chewy textures with Ca2+ ion content in sugar based matrices and optimization of machine parameters are performed to obtain a higher resolution on the specimens.

Aurora A (AurA) is an important mitotic kinase mainly studied for its involvement in cell cycle progression, centrosome maturation, mitotic spindle pole organization and bipolar spindle formation. It localizes to duplicated centrosomes and spindle microtubules (MTs) during mitosis where it regulates various factors participating in metaphase spindle formation. AurA is degraded late in mitosis suggesting that it might also have a function in anaphase. In this study we focused in understanding AurA function during anaphase in two different experimental systems. First, we kept AurA active in cycled Xenopus egg extracts and found that MTs maintained their mitotic organization longer throughout mitotic exit. We also observed chromosome segregation defects and problematic nuclear envelope formation. These observations indicate that AurA activity needs to be down-regulated for the transition from metaphase back to interphase. To get insights into the role of AurA during metaphase-anaphase transition we initially asked whether its kinase activity is still necessary for the maintenance of the metaphase spindle. We saw that the inhibition of AurA kinase activity in metaphase resulted to a collapse of the established metaphase spindle in HeLa cells. Indicating that AurA activity is necessary for the metaphase spindle maintenance. Then, we looked whether AurA kinase activity is still necessary during anaphase. We inhibited AurA at the onset of anaphase in Hela cells and found that anaphase spindles were smaller. We also observed that the MT structure responsible for anaphase spindle elongation, the central spindle, was defectively assembled and organized. Moreover, in cells where AurA was inhibited segregation of chromosomes was defective. These results indicate that AurA kinase activity is necessary for anaphase spindle elongation, central spindle assembly and organization and chromosome segregation. To understand further how AurA regulates anaphase spindle formation we looked known AurA substrates. We depleted TACC3, a known AurA substrate involved in MT formation earlier in mitosis and observed that TACC3 depletion phenocopied AurA inhibition. This indicates that TACC3 has a function in MT organization and chromosome segregation during anaphase and this function could possibly be regulated by AurA. In this study we have demonstrated that AurA activity is essential for metaphase spindle maintenance. We also found that during anaphase when AurA is either maintained active or inhibited MT organization is greatly affected and chromosome segregation is defective. Suggesting that AurA activity needs to be tightly controlled during anaphase for a correct completion of mitosis. / Aurora A (AurA) es una quinasa mitótica importante que se ha estudiado principalmente en su papel durante la progresión del ciclo celular, la maduración del centrosoma, la organización y la formación del polo y del huso mitótico. Durante la mitosis, AurA se localiza en los centrosomas duplicados y en los microtúbulos (MTs) del huso y se ha observado que regula varios factores que participan en la formación del huso mitótico. AurA se degrada al final de la mitosis indicando que pueda tener una función durante la anafase. En este estudio nos hemos centrado en la comprensión de la función de AurA durante la anafase en dos sistemas experimentales diferentes. En primer lugar, utilizando extractos de huevos de Xenopus hemos mantenido AurA activa durante la transición de metafase a anafase y hemos visto que los MTs del huso mitótico mantienen su organización durante más tiempo. También hemos observado que cuando AurA se mantiene activa existen defectos en la segregación cromosómica y la formación de la membrana nuclear. Esto indica que la actividad de AurA tiene un papel regulador sobre los MTs y la chromatina durante la transición de la metafase a la interfase. Para entender cual es la función de AurA durante la transición de metafase a anafase primero hemos estudiado si la actividad de la quinasa es necesaria para el mantenimiento del huso mitótico. Hemos visto que la inhibición de la actividad quinasa AurA resultó en el colapso del huso durante la metafase en células HeLa. Esto indica que la actividad de AurA es necesaria para el mantenimiento del huso mitótico de metafase. A continuación hemos analizamos si la actividad quinasa de AurA sigue siendo necesaria para la anafase. Para ello hemos inhibido AurA en células Hela al inicio de la anafase. En estas condiciones los husos de la anafase son más pequeños y la estructura de los MTs responsable del alargamiento del huso mitótico durante la anafase, el huso central, se organiza defectuosamente. Además, se encontraron errores durante la segregación de los cromosomas. Estos resultados indican que la actividad quinasa de AurA es necesaria para el alargamiento del huso durante la anafase y la organización y segregación cromosómica. Para entender el mecanismo de la función de AurA durante la anafase hemos estudiado a sustratos de AurA. Al estudiar TACC3 , un sustrato conocido de AurA que participa en la formación de MTs en las fase iniciales de la mitosis hemos encontrado que su eliminación de células HeLa produce el mismo fenotipo que la inhibición de AurA. Esto indica que TACC3 tiene una función en la organización de MT y la segregación de cromosomas durante la anafase y que esta función podría estar regulada por la quinasa AurA. En este estudio hemos demostrado que la actividad quinasa de AurA es esencial para el mantenimiento del huso mitótico. También hemos encontrado que durante la anafase cuando la quinasa AurA se mantiene activa o se inhibe la organización de los MTs del huso mitótico se ve muy afectada y los cromosomas se segregan defectuosamente. Por tanto los resultados de este estudio indican que la actividad quinasa de AurA está estrechamente controlada durante la anafase para el correcto cumplimiento de la mitosis.

Common measures of egg quality have been survival to specific developmental stages, higher hatching rate of fertilized eggs and final production of fry. Determinants of egg quality are variable among and between teleost species and no common unified criteria have been established. Maternally inherited genes influence egg quality and early embryo development is partially programmed by the messenger ribonucleic acid (mRNA). Among the genes, the insulin family is important for growth functions and the presence of their transcripts in the ovary, oocytes and embryos implies their involvement during the reproductive process and their relevance to egg quality. The insulin-like growth factor (IGF) system has three components, the ligands IGF-I and II, the IGFBPs (insulin-like growth factor binding proteins) and the IGF receptors that mediate biological activity of the ligands. Vitellogenin (Vtg) is the major source of nutrients for the developing embryo and elevated levels in female fish plasma signals gonadal development preceding spawning. In oviparous fish where the developing embryo is dependent on the stored food in the yolk, vitellogenin levels in the egg could indicate its capability to support embryonic growth. This study aimed to develop molecular tools, specifically probes for IGF-I, IGF-II and IGF-IR, for the evaluation of fish egg quality. These probes would be used to determine expression levels of IGF-I, IGF-II and IGF-IR during egg development to assess their potential as molecular indicators for egg quality. In addition, this study also aimed to establish an enzyme-linked immunoassay (ELISA) for quantifying Vtg in fish eggs and determine if differences in Vtg levels could be linked to fertilization and hatching success. Through reverse-transcription polymerase chain reaction (RT-PCR) putative complementary deoxyribonucleic acid (cDNA) fragments of IGF-I, IGF-II and IGF-IR were cloned and sequenced from mullet (Mugil cephalus) and grouper (Epinephelus coioides). The relative expression ratio of the three genes in the eggs of mullet and grouper were assayed by quantitative PCR (QPCR) and calculated using the Pfaffl method (Pfaffl, 2001). Levels of vitellogenin in different batches of mullet eggs were quantified by ELISA. Spawned eggs of grouper were grouped into low (<60%) or high (>60%) fertilization rate (FR) and the fertilized eggs that were incubated until hatching were grouped into medium (>90%) or high (>90%) hatching rate (HR). Samples were categorized into sinking eggs, late embryo and hatched larvae. Relative expression ratio of IGF-II was significantly high (P<0.01) compared to IGF-I and IGF-IR in all samples examined. All three genes were strongly expressed in sinking eggs compared to either late embryo or hatched larvae. However, there was no significant interaction effect between the genes and the samples analyzed. Mullet samples all came from a high FR and high HR group and were categorized into sinking, multicell stage, blastula, gastrula, late embryo and hatched larvae. There was a significant interaction effect (P<0.01) between gene and stage, showing that genes are differentially expressed during embryonic development. IGF-II was strongly expressed relative to the other genes in all stages examined and was highest during the gastrula stage. Vtg levels were examined in mullet oocytes and egg samples that were grouped into 4; oocytes from females that subsequently spawned, had fertilized eggs which hatched (Group A); oocytes from females that did not spawn, therefore no fertilization and no hatching (Group B); eggs that were stripped, artificially fertilized but no hatching (Group C); and eggs that were spawned, assumed to be fertilized but did not hatch (Group D). Group A showed a trend of higher Vtg levels than the other three but this result was not statistically significant.

Vitamin B₁₂ deficiency has been difficult to diagnose, mainly due to the vitamin's lack of biological significance in serum in which it is usually assayed. This research has investigated the marker of vitamin B₁₂/cobalt (Co) deficiency in sheep, methylmalonic acid (MMA), in comparison with serum and liver vitamin B₁₂ concentrations in farm situations where vitamin B₁₂ deficiency is expected in order to establish more accurate reference ranges for serum and liver vitamin B₁₂, and MMA. In addition, an attempt was made to ascertain the vitamin B₁₂ requirements of preruminant (PR) lambs, and to determine whether metabolic demand for vitamin B₁₂ influences tissue concentrations. Furthermore, since the vitamin is active in biological tissues in form of its coenzymes, 5’ -deoxyadenosylcobalamin and methylcobalamin, a preliminary assessment of variation in the distribution of these coenzymes in liver in different situations has been sought. The first trial was set up to find out if the addition of propionate to the PR lamb's diet stimulated the uptake and/or storage of vitamin B₁₂ in the liver as a reflection of the need to deal with the incoming propionate. Sixteen ten day old lambs (Dorset Down/Coopworth cross-bred) were housed indoors soon after birth and fed on milk replacer. For half of the lambs 7.5 % (w:w) of the milk powder was replaced by propionate. Within each group, four lambs were treated with 250 µg vitamin B₁₂ twice weekly. Supplementation with vitamin B₁₂ increased liver concentrations from ~250 to ~900 nmol/kg fresh tissue, but there was no effect of propionate. Propionate addition did, however, result in increased plasma vitamin B₁₂ concentrations in vitamin B₁₂ supplemented groups, values being 3323 and 2355 pmol/l in propionate supplemented and control groups, respectively. This suggested that diet could influence plasma vitamin B₁₂ concentrations. An attempt was made to quantify the PR lamb's ability to absorb vitamin B₁₂ from the alimentary tract by comparing the ability of intra-muscular (IM) and oral vitamin B₁₂ to raise plasma and liver vitamin B₁₂ concentrations. Twenty-seven three to four day old lambs from a farm with marginal Co status were housed indoors and fed on milk replacer. They were divided into three groups: control (n=3), IM treatment (n=12) and oral treatment (n=12). The two treatment groups were further subdivided into five sub-groups. These received, respectively, 0.2 (n=3), 0.4 (n=2), 0.8 (n=2), 1.6 (n=2) and 3.2 µg OH-cbl/d (n=3). The oral groups received tenfold the amount of the comparable IM groups, on the assumption that if oral absorption of the vitamin is about 10 % both groups would show similar increases in plasma and liver vitamin B₁₂ concentration. None of the IM groups showed any significant change in plasma or liver vitamin B₁₂. In the oral groups only the group on the highest dose of vitamin B₁₂, viz 32 µg/d, showed increases in plasma and liver concentrations. It was concluded that either absorption of vitamin B₁₂ was greater than 10 % or that the vitamin was retained better when administered orally. The amount retained in the livers of the lambs in the highest oral group was calculated to represent ~ 7.5 % of the dose. In a follow-up 24 h trial, 14 of the above lambs were divided into three groups: Control (n=3), oral (n=6) and IM (n=5) treatment. The IM group received 3.2 µg OH-cbl and the oral group tenfold the amount as single doses at 0800 h. Blood samples were taken at regular intervals throughout the 24 h period and assayed for vitamin B₁₂, Vitamin B₁₂ concentrations in the IM group rose steeply within the first hour after injection to a concentration that was calculated to reflect 100 % uptake of the vitamin. It rose more slowly over about 8 h in the oral group. From the area under the curve absorption of the oral dose was estimated to be ~ 7 %. The next experiment involved a farm where Co deficiency had been reported previously. In the first year, 50 pregnant two-tooth Half-bred ewes were divided randomly into two groups of 25. One group received a Co bullet plus 1000 µg OH-cb1 IM, the other group remained unsupplemented. In the following year the trial was repeated. Ewes from the previous year's trial (by then four-tooths) were augmented by a new cohort of pregnant two-tooths to make up numbers to 75. After lambing the lambs were divided into four groups: first by their dams' vitamin B₁₂ treatment, then half of each group received injections of vitamin B₁₂ at approximately three weekly intervals while the other half remained untreated. The trials lasted about five months, from mid-pregnancy until weaning. Pasture Co was at its lowest at lambing in both years, 0.09 and 0.10µg/g DM, respectively. In the first year, vitamin B₁₂ concentrations in the untreated ewes rose from 340 to 950 pmol/l in plasma and decreased in liver from 330 to 170 nmol/kg fresh tissue. In the Co treated group, vitamin B₁₂ concentrations in plasma rose from 500 to 1550 pmol/l and in liver from 310 to 560 nmol/kg fresh tissue. In the second year, vitamin B₁₂ concentrations in serum in the unsupplemented groups fell from 500 to 260 pmol/l around lambing before rising again to starting values at weaning, and liver vitamin B₁₂ concentrations fell from 450 at the start to 230 nmol/kg fresh tissue at the end of the trial. Serum vitamin B₁₂ concentrations in the two-tooth supplemented group rose from < 500 to > 3000 pmol/l whereas in the four-tooth supplemented group serum vitamin B₁₂ levels started at ~2800 and rose to nearly 5000 pmol/l. The supplemented four-tooths maintained higher liver vitamin B₁₂ concentrations throughout compared to the supplemented two-tooths, viz 680 compared to below 400 at the start and 900 versus 650 nmol/kg fresh tissue at weaning, respectively. MMA in the untreated groups rose to 15 and to 8 µmol/l during early lactation in the first and second years, respectively, whereas MMA in the treated groups stayed below 3 µmol/l in the first season and below 1.5 µmol/l in the second season. There was a live weight response to treatment in the ewes as the unsupplemented groups showed a significantly lower weight gain during the trials than the supplemented groups, viz 10.0 versus 13.6 kg in the first year, and 10.6 versus 13.3 kg in the four-tooths and 9.9 versus 12.1 kg in the two-tooths in the second year. There was also a significant difference in faecal egg count (FEC) in the first year. FEC in the untreated group was higher during lactation than in the treated group, viz 590 versus 170 eggs per gram wet faeces (epg), respectively. In the second year, the two-tooths had a higher FEC than the four-tooths, viz 120 versus 40 epg during the same time span, respectively. While there was a trend for treatment having an effect on FEC similar to that in the first year it was not significant. Supplementation of ewes in the first year increased mean milk vitamin B₁₂ concentrations at lambing from 800 to 1400 pmol/l and at weaning from 1750 to 4000 pmol/l. In the second year, Co bullet treatment increased milk vitamin B₁₂ concentrations in the four-tooths and two-tooths from 1500 and 2300 to 4000 and 2900 pmol/l at lambing, and from 1800 and 1400 to 6200 and 4500 pmol/l at weaning, respectively. Treatment of ewes increased vitamin B₁₂ concentrations in the lambs which were not themselves supplemented. Plasma values in the first year increased from 160 to 325 pmol/l soon after birth and from 650 to 900 pmol/l at weaning, and liver values from 75 to 140 nmol/kg fresh tissue soon after birth and from 150 to 240 nmol/kg fresh tissue at weaning. In the second year, plasma vitamin B₁₂ concentrations increased from 160 to 380 pmol/l soon after birth and from 500 to 700 pmol/l at weaning, and in liver from 130 to 260 nmol/kg fresh tissue soon after birth and from 220 to 340 nmol/kg fresh tissue at weaning. There was also a significant effect of ewe supplementation on lamb MMA in 1997/1998 when values decreased from 19 to 8 µmol/l around the time of rumen development. MMA in the second year stayed below 3 µmol/l throughout in all groups of lambs. There was no difference in LWG between any groups of lambs. FEC was lowest in the group where both ewes and lambs were supplemented and highest in the group where neither ewes nor lambs were treated. Further investigations were conducted on farms in Southland with lambs post-weaning in order to compare changes in serum and liver vitamin B₁₂ with serum MMA and LWG to determine the critical time and level of deficiency. In the first year, three farms with 50 lambs each participated. Lambs from each farm were allocated to five groups of 10 animals each. The first group received a Co bullet at weaning, and each month another group was treated with a Co bullet. The lambs were weighed monthly, and blood and liver samples were taken prior to treatment and each subsequent month from five lambs of the first supplemented group. The trial lasted about four months. Serum vitamin B₁₂ concentrations in lambs at weaning were between 500 and 1000 pmol/l. Although supplementation increased serum levels for the first month this was followed by a drop to near or below starting concentrations. An exception was Farm 3 where serum vitamin B₁₂ concentrations rose again at the end of the trial. Liver vitamin B₁₂ concentrations also showed an overall decline from starting levels (200 to 300 nmol/kg fresh tissue) to the end of the trial (100 to 200 nmol/kg fresh tissue). MMA started around 2 µmol/l and reached between 6 and 7 µmol/l in the untreated lambs on Farms 1 and 3 two months after weaning before decreasing to around 3 µmol/l at the end of the trial, whereas the treated lambs maintained MMA concentrations around 2 µmol/l. On Farm 2 MMA started just below 5 µmol/l, decreased to around 1 µmol/l for treated and untreated lambs one month later and rose again to between 2.5 and 4 µmol/l, respectively, at the end of the trial. LWG was below average for all lambs (between 0.20 and 0.04 kg/d except for Farm I in the first month after weaning) but no significant differences were noted between treated and untreated lambs on any of the farms. Another trial was conducted on one of these farms in the following year. One hundred lambs were divided into two groups of 50 each at weaning and sampled monthly for about six months. One group was treated with two Co bullets, the other group remained untreated. Pasture Co was between 0.04 and 0.07 µg/g DM, yet serum levels for the untreated group stayed ~500 pmol/l throughout the trial. Serum vitamin B₁₂ concentrations for the treated group started at ~500 pmol/l, rose to ~2500 pmol/l before falling back to ~2000 pmol/l. Liver vitamin B₁₂ concentrations for the untreated and treated groups were 529 and 427 nmol/kg fresh tissue at weaning, respectively. This decreased for both groups to ~350 nmol/kg fresh tissue one month after weaning. In the untreated lambs liver values decreased further to ~290 nmol/kg fresh tissue whereas they increased to ~450 nmol/kg fresh tissue for the treated group at the end of the trial. MMA concentrations started between 2 and 3 µmol/l for both groups and increased to 4.5 µmol/l for the untreated group one month later before falling back to 3.2 µmol/l. In the treated group MMA decreased to ~1µmol/l and stayed at that level throughout the trial. There was no difference in weight gain. In order to obtain an understanding of the distribution of corrinoids in biological tissues a High Performance Liquid Chromatography method was developed. The sensitivity of the analytical method meant that it was only practical to assay mainly liver samples because of the higher vitamin B₁₂ concentrations than in other tissues. The general finding was that the coenzyme 5’ –deoxyadenosylcobalamin (ado-cbl) constituted the highest proportion of corrinoids in liver (45 %), followed by analogues (28 %), OH-cbl (24 %) and lastly methy1cobalamin (3 %). Ado-cbl did tend to be proportionately higher in supplemented than in unsupplemented animals (56 and 42 %, respectively), whereas biologically non-active analogues tended to be higher in untreated than in treated sheep (29 and 21 %, respectively). It was concluded that in the farm trials Co deficiency was only mild or not present although deficiency would have been predicted from the low vitamin B₁₂ concentrations in serum and liver and from raised MMA values. Therefore, currently used thresholds in New Zealand appear to be too high for vitamin B₁₂, and overseas values for MMA do not seem to be appropriate. Revised marginal ranges of 100 to 200 pmol/l for serum, 100 to 200 nmol/kg fresh tissue for liver and 10 to 20 µmol/l for MMA are suggested. Further, this work shows that Co bullets were effective in elevating blood and liver vitamin B₁₂concentrations for longer than one year. In the trials with preruminant lambs it was found that maintenance requirements were met by the vitamin B₁₂ content of milk replacer. There is evidence from indoor and farm trials that vitamin B₁₂ from milk was much more readily absorbed than vitamin B₁₂ from supplements. It was estimated that suckling lambs probably require between 1200 and 4000 pmol vitamin B₁₂/d, depending on age.

A study was carried out at Poultry Science Laboratory LAVIC at the Federal University of Santa Maria UFSM. The aim was avaluated the effect of different feeding schedules on the performance and eggs quality of broilers breeders Cobb 500. The experimental period was separated into two phases, the first from 28th to 40th week (Phase I) and the second from 40th to 60th week (Phase II) of hen s age. The feeding schedules evaluated were: a single feeding at 08:00 am; twice daily feeding (50% at 08:00 am and 50% at 3:00 pm) and a single feeding at 3:00 pm. At the Phase I, was used 546 females and 63 males allocated in a completely randomized design (CRD) with 7 pens with 26 females and 3 males per repetition and, to the Phase II was used 330 females and 45 males assigned in a CRD with 5 pens with 22 females and 3 males by repetition. The diets were based on corn and soybean meal. At the Phase I, the egg production of hens fed at 3 pm was reduced (P=0.0002). These hens had higher egg and yolk weight than hens fed at 8 am. Hens fed at 3 pm showed better shell quality (egg specific gravity, weight and thickness). The bacterial contamination of the shell was not affected by the diffirent feeding schedules, as well as hatchability, fertility, contaminated and pipped eggs. Embryonic mortality was the lowest in eggs came from hens fed at 8 am. The hatchability of fertile shown to be higher in hens fed once daily in the morning than broiler breeders fed twice daily. At the Phase II, the egg production was not affected by feeding schedules. The hens fed in the afternoon had higher egg and shell weight tham others. The hens fed at 3 pm had higher egg specific gravity and eggshell thickness than eggs come from hens fed at 8 am. Calcium (Ca) and phosphorus (P) plasma levels (21 hours after laying) were higher in hens fed at 3 pm than hens fed at 8 am. The tibia weight was higher in hens fed 8 am than hens fed twice daily feeding. A single feeding at 3:00 pm showed lowest egg production of the 28th to 40th weeks of age. Broiler breeders fed in the afternoon had better egg quality and bacteriological differences were not found in the eggs of hens fed at different schedules. / Um estudo foi conduzido no Laboratório de Avicultura LAVIC da Universidade Federal de Santa Maria (UFSM) com o objetivo de avaliar o efeito de diferentes horários de arraçoamento sobre o desempenho e qualidade de ovos de matrizes de frangos de corte da linhagem Cobb 500. O período experimental foi dividido em duas fases, a primeira da 28ª a 40ª semanas (Fase I) e a segunda da 40ª a 60ª semanas (Fase II) de idade das aves. Avaliaram-se três horários de arraçoamento: único - 8hs; duas vezes ao dia (50% às 8hs e 50% às 15hs) e único - 15hs. Na Fase I foram utilizadas 546 fêmeas e 63 machos distribuídos em um deliamento inteiramente casualizado (DIC) composto por 7 repetições de 26 fêmeas e 3 machos. Na Fase II utilizou-se 330 fêmeas e 45 machos distribuídos em um DIC composto por 5 repetições de 22 fêmeas e 3 machos. As dietas foram à base de milho e farelo de soja. Na Fase I, a taxa de postura de aves alimentadas às 15hs foi reduzida (P=0.0002). Estas aves apresentaram maior peso de ovo e gema em comparação às aves alimentadas às 8hs e, aves alimentadas às 15hs apresentaram melhor qualidade de casca (gravidade específica, peso e espessura) em relação às demais. A contaminação bacteriológica da casca não foi afetada pelos diferentes horários de arraçoamento, bem como eclodibilidade, fertilidade, ovos contaminados e bicados. A mortalidade embrionária foi menor em ovos resultantes de matrizes arraçoadas às 8hs. Estas aves produziram ovos que apresentaram maior taxa de eclodibilidade de ovos férteis em relação às aves alimentadas duas vezes ao dia. Na Fase II, a taxa de postura não foi afetada pelos horários de arraçoamento. Matrizes alimentadas à tarde apresentaram maior peso de ovo e casca em relação às demais. Estas aves apresentaram maior gravidade específica e espessura de casca em relação às aves alimentadas às 8hs. Cálcio (Ca) e fósforo (P) plasmáticos (21 horas após a postura) foram maiores em matrizes arraçoadas às 15hs em relação às aves alimentadas apenas às 8hs. Matrizes alimentadas às 8hs apresentaram maior peso de tíbia do que aquelas alimentadas duas vezes ao dia. O arraçoamento único às 15hs demonstrou menor produção de ovos da 28ª a 40ª semanas de idade. Matrizes alimentadas a tarde apresentam melhor qualidade de ovos, sendo que não foram encontradas diferenças bacteriológicas nos ovos das aves alimentadas em diferentes horários.