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During embryonic vasculogenesis, endothelial cells form in the mesoderm , assemble into cord-like structures and then undergo tube formation. Previous studies have shown that signaling by members of the hedgehog family of secreted growth factors is essential for normal development of embryonic blood vessels. Embryos lacking hedgehog function show the presence of abundant endothelial cells but the cells fail to assemble into vascular cords and lumenized endothelial tubes do not form. At present it is not known whether active hedgehog signaling is required for both cord and tube formation or only for the initial step. To address this question, we have used small molecule inhibitors and agonists to the alter activity of the hedgehog signaling pathway in the chick embryo. If development is allowed to proceed until endothelial cells of the future dorsal aortae have assembled into cords, subsequent inhibition of hedgehog signaling, using cyclopamine, does not prevent aortal cells from forming endothelial tubes, however, it does lead to a reduction in cross sectional area of the aorta and to a loss of density of the adjacent vascular plexus. In contrast, activation of the hedgehog pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. Very little, if any, of the observed effects are due to differences in number of endothelial cells in the treated embryos. Examination of endothelial cells during vascular plexus formation shows that inhibition of hedgehog signaling with cyclopamine inhibits formation of filopodia while treatment with SAG increases the number of filopodial extensions. These studies show that hedgehog signaling levels must be tightly regulated for normal vascular patterning to be achieved.

Mucins, synthesised and secreted by goblet cells, possess potential binding sites for both commensal and pathogenic organisms, and may perform a defensive role during establishment of the intestinal barrier in newly hatched chickens. Increasing interest has been directed toward bacterial interactions within the mucus layer, and the mechanisms by which bacterial colonisation can influence mucus composition during early development. This is important, firstly, as a means to understand initiation of infection and secondly, to optimise the gut microflora for enhanced animal production. Currently, information on mucosal-bacterial interactions in poultry is limited. In order to observe the effects of bacterial exposure on intestinal goblet cell mucin production during early development, differences in the small intestine of conventionally-raised (CV) and low bacterial load (LBL) broiler chicks were examined during the first 7 days post-hatch. The initial aim of the study was to construct a small-scale, economical isolator system to hatch and raise chicks in a bacterial-free environment as a means to observe bacterial interactions with the intestinal mucosa in chickens exposed to normal environmental conditions. The design and construction of flexible plastic isolators for incubation and brooding are described, along with methodologies for preparation of eggs for entry into the isolators, incubation and hatching. Two trials were conducted, the first in August 2005 and the second in March 2006. It was found that the isolator system was successful in producing low bacterial load chicks for comparative studies with conventionally raised chicks, without compromising body weight. A histological study was then conducted whereby ileal and jejunal goblet cells were stained with either periodic acid-Schiff or high iron diamine/alcian blue pH 2.5 to discriminate between neutral, sulphated and sialyated acidic mucins. Total goblet cell numbers and goblet cell and villous/crypt morphology were also examined. Bacterial colonisation of CV animals induced an increase in sialic acid moieties in both ileal and jejunal goblet cell such that initiation of these changes occurred at day 3-4 post-hatch. Differences in intestinal morphology were also consistent with other germ-free animal studies. In order to further understand the extent to which bacteria affected mucin composition, purified, isolated oligosaccharide fractions from ileal mucin at d 4 and 7 post-hatch were collected and analysed using mass spectrometry techniques to determine any structural differences in chain length or chain number between LBL and CV animals. No differences in chain length or number were observed between CV and LBL animals at either d 4 or 7 post-hatch with both groups equally displaying chain lengths of both low and high molecular weights. Although structural differences in mucin oligosaccharides were not observed between LBL and CV animals, bacterial binding assays utilising whole ileal sections were employed to determine whether or not the differences in mucin composition between LBL and CV animals during early development may have deterred or enhanced binding of certain bacterial species. Escherichia coli and Lactobacillus salivarius were selected for the experiment. Binding of L. salivarius to ileal sections was very low whereas E. coli binding was greater, and more pronounced in LBL animals, especially at d 7 post-hatch. No statistically significant differences were observed in binding of E. coli to purified ileal mucin from LBL and CV animals at either d 4 or d 7 post-hatch. Correlations between E. coli and L. salivarius adherence to ileal tissue and mucin samples, and goblet cell parameters, were not statistically significant when fitted as co-variates. It was concluded that the changes in mucin composition played a minor role in bacterial adhesion of L. salivarius and this E. coli serotype. In summary, this thesis explores the physiological changes in goblet cell mucin production in response to bacterial exposure post-hatch. The thesis outlines the complexity of mucosal-bacterial interactions which would benefit from the employment of specialised techniques such as nuclear magnetic resonance spectroscopy and microarray technologies to examine a greater range of mucin structures and gene expression. This thesis provides support for future investigations into the influence of intestinal microflora on mucosal and mucin dynamics of poultry and the potential development of prebiotics for use in animal production. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297640 / Thesis (Ph.D.) -- School of Agriculture, Food and Wine, 2007

abstract: This dissertation features a compilation of studies concerning the biophysics of multicellular systems. I explore eukaryotic systems across length scales of the cell cytoskeleton to macroscopic scales of tissues. I begin with a general overview of the natural phenomena of life and a philosophy of investigating developmental systems in biology. The topics covered throughout this dissertation require a background in eukaryotic cell physiology, viscoelasticity, and processes of embryonic tissue morphogenesis. Following a brief background on these topics, I present an overview of the Subcellular Element Model (ScEM). This is a modeling framework which allows one to compute the dynamics of large numbers of three-dimensional deformable cells in multi-cellular systems. A primary focus of the work presented here is implementing cellular function within the framework of this model to produce biologically meaningful phenotypes. In this way, it is hoped that this modeling may inform biological understanding of the underlying mechanisms which manifest into a given cell or tissue scale phenomenon. Thus, all theoretical investigations presented here are motivated by and compared to experimental observations. With the ScEM modeling framework I first explore the passive properties of viscoelastic networks. Then as a direct extension of this work, I consider the active properties of cells, which result in biological behavior and the emergence of non-trivial biological phenotypes in cells and tissues. I then explore the possible role of chemotaxis as a mechanism of orchestrating large scale tissue morphogenesis in the early embryonic stages of amniotes. Finally I discuss the cross-sectional topology of proliferating epithelial tissues. I show how the Subcellular Element Model (ScEM) is a phenomenological model of finite elements whose interactions can be calibrated to describe the viscoelastic properties of biological materials. I further show that implementing mechanisms of cytoskeletal remodeling yields cellular and tissue phenotypes that are more and more biologically realistic. Particularly I show that structural remodeling of the cell cytoskeleton is crucial for large scale cell deformations. I provide supporting evidence that a chemotactic dipole mechanism is able to orchestrate the type of large scale collective cell movement observed in the chick epiblast during gastrulation and primitive streak formation. Finally, I show that cell neighbor histograms provide a potentially unique signature measurement of tissue topology; such measurements may find use in identifying cellular level phenotypes from a single snapshot micrograph. / Dissertation/Thesis / Ph.D. Physics 2011

Οι πρωτεογλυκάνες αλληλεπιδρούν μεταξύ τους και με άλλα μορφορυθμιστικά μόρια όπως γλυκοπρωτεΐνες και ιντεγκρίνες, καθώς επίσης και με αυξητικούς παράγοντες. Μεγάλο μέρος των ποικίλων λειτουργιών των πρωτεογλυκανών συνδέεται με τις γλυκοζαμινογλυκανικές αλυσίδες (GAGs) τους. Για να μελετήσουμε τη λειτουργία των πρωτεογλυκανών κατά την ανάπτυξη του πρώιμου εμβρύου όρνιθας χρησιμοποιήσαμε το β-D-xyloside, έναν εξειδικευμένο αναστολέα της βιοσύνθεσης των πρωτεογλυκανών και ειδικότερα της πρόσδεσης των αλυσίδων γλυκοζαμινογλυκανών στον πρωτεϊνικό κορμό των πρωτεογλυκανών. Χαμηλές συγκεντρώσεις β-xyloside που είναι γνωστό ότι αναστέλλουν την προσθήκη θειϊκής χονδροϊτίνης αλλά όχι της θειϊκής ηπαράνης στον πρωτεϊνικό κορμό, αποτελούν ένα χρήσιμο εργαλείο για τη μελέτη του ρόλου των πρωτεογλυκανών. Τα πρότυπα των πρωτεϊνών στα έμβρυα που μεταχειρίστηκαν με β-xyloside έδειξαν μετατόπιση των ραδιενεργών κορυφών σε μικρότερη μοριακή μάζα που φαίνεται να οφείλεται στη μείωση του μεγέθους πρωτεογλυκανών. Ήταν αξιοσημείωτο στα αποτελέσματά μας ότι το β-xyloside μετέβαλλε τη μορφή της πρωτεογλυκάνης θειϊκής χονδροϊτίνης decorin προς χαμηλότερα μοριακά βάρη ενώ δε φάνηκε να επηρεάζει το μέγεθος της πρωτεογλυκάνης θειϊκής ηπαράνης perlecan. Στα έμβρυα που μεταχειρίστηκαν με β-xyloside περισσότερη πρωτεϊνη συντέθηκε στα έμβρυα του σταδίου ΧΙΙ (μορίδιο), σε σχέση με αυτά του σταδίου ΗΗ2 (αρχή πρωτογενούς αύλακας/πρώιμο γαστρίδιο), συγκρινόμενα με τα αντίστοιχα έμβρυα μάρτυρες. Αυτό θα μπορούσε να αντανακλά μια επιταχυνόμενη κινητοποίηση και/ή μια μετάφραση των ωογενετικών μεταγράφων στα έμβρυα του σταδίου ΧΙΙ, όταν διαταράχτηκε ο μεταβολισμός των πρωτεογλυκανών. Η απορρύθμιση πρωτεογλυκανών, τροποποιώντας τη λειτουργικότητα και επηρεάζοντας το επίπεδο έκφρασής τους, είχε ως αποτέλεσμα την αδυναμία του πρώιμου εμβρύου να δομήσει την εξωκυττάρια ύλη φυσιολογικά. Το αποτέλεσμα ήταν η κατάρρευση της τυπικής αρχιτεκτονικής του πρώιμου εμβρύου στα πειράματά μας. Πρωτεογλυκάνες θειϊκής χονδροϊτίνης φαίνεται να χρειάζονται για την οργάνωση του σταδίου του βλαστιδίου της όρνιθας. Για την επαγωγή του νευρικού συστήματος φάνηκε να χρειάζονται πρωτεογλυκάνες θειϊκής χονδροϊτίνης/δερματάνης που έχουν συγκεντρωθεί από το στάδιο πριν την έναρξη των μορφογενετικών κινήσεων της γαστριδίωσης και η συνεχής βιοσύνθεση των πρωτεογλυκανών είναι απαραίτητη για τη μορφογένεση του νευρικού σωλήνα. Μελετήσαμε επίσης το πρότυπο κατανομής της συνδετικής πρωτεϊνης πρωτεογλυκανών με ανοσοφθορισμό και ανοσοκατακρήμνιση και το ρόλο αυτής της γλυκοπρωτεϊνης με τη χρήση αντισωμάτων έναντι αυτής. Η συνδετική πρωτεΐνη μεσολαβεί στην πρόσδεση στο υαλουρονικό οξύ πρωτεογλυκανών όπως οι aggrecan, neurocan, versican και brevican δημιουργώντας σταθερά σύμπλοκα στην εξωκυττάρια ύλη. Η αναγνώριση των μορφών της συνδετικής πρωτεΐνης 1 (LP1, 48kDa) και συνδετικής πρωτεΐνης 2 (LP2, 44kDa) στο πρώιμο έμβρυο ήταν επίσης ένα ενδιαφέρον εύρημα των πειραμάτων μας. Είναι γνωστό ότι συνδυασμοί των LP1 και LP2 δημιουργούν πιο σταθερά σύμπλοκα απ’ ότι μόνο του το καθένα από τα δύο μόρια αυτά. Αυτό φαίνεται και από τα πειράματά μας που δείχνουν ότι η aggrecan (180kDa) φαίνεται να συν-κατακρημνίζεται με τις συνδετικές πρωτεΐνες LP1 και LP2. Τα πειράματα ανοσοφθορισμού έδειξαν ότι η συνδετική πρωτεΐνη αρχίζει να εκφράζεται στο στάδιο του βλαστιδίου και επιδεικνύει μια διαφορική έκφραση χωρο-χρονικά κατά την ανάπτυξη του πρώιμου εμβρύου υποδεικνύοντας ότι είναι σημαντική στην κυτταρική μετανάστευση και διαφοροποίηση κυττάρων και στη μορφογένεση ιστών και οργάνων. Αυτό επιβεβαιώθηκε και από τη μελέτη του ρόλου της, στην επόμενη σειρά πειραμάτων μας με τη χρήση μονοκλωνικού αντισώματος έναντι αυτής. Η αναστολή της λειτουργίας της συνδετικής πρωτεϊνης με τη χρήση αντισωμάτων έναντι αυτής έδειξε ότι αυτή η πρωτεϊνη φαίνεται να είναι σημαντική στην οργάνωση της νευρικής πλάκας και τη μορφογένεση του νευρικού σωλήνα, στη μορφογένεση του καρδιακού σωλήνα, του εντέρου, στο σχηματισμό της ραχιαίας αορτής και στην επιθηλιοποίηση των σωμιτών. / Proteoglycans participate in cellular interactions via modulating the effects of growth factors or with other mechanisms in early embryo. The majority of the functions of proteoglycans are associated with the glycosaminoglycan (GAG) chains. We used β-D-xyloside, an inhibitor of proteoglycan synthesis and specifically of GAG attachment to proteoglycan core proteins, to study proteoglycan functions in early chick embryo development. Low concentrations of β-xyloside which are known to affect differentially chondroitin but not heparan sulfate proteoglycan biosynthesis have provided a convenient tool for altering proteoglycan production. The protein patterns of xyloside-treated embryos showed a shift of radioactive peaks to lower molecular mass which could be attributed to the reduction of proteoglycan size as was demonstrated by chondroitinase ABC/AC II treatments. It was notable in our data that β-xyloside altered the chondroitin sulfate proteoglycan decorin to lower molecular mass while it did not seem to affect the size of the heparin sulfate proteoglycan perlecan. More protein was synthesized from xyloside-treated embryos at stage XII (morula) than from embryos at stage HH2 (initial primitive streak/early gastrula) when compared to the controls. This could have reflected an accelerated translation and/or mobilization of oogenetic transcripts in embryos at stage XII when proteoglycan metabolism was disrupted. Misregulation of proteoglycans by modulating the functionality of the protein and by influencing their expression level resulted in an inability of the early embryo to assemble a stable extracellular matrix that would have been normally produced. These changes were associated with the collapse of the typical blastula architecture and inhibition of the induction of mesoderm in the chick embryo. Induction of neuroectoderm required proteoglycans assembled before the initiation of gastrulation movements. However, sustained proteoglycan biosynthesis was required for the morphogenetic movements to form the neural tube and the rest of the embryonic axis. We also studied the spatiotemporal distribution pattern of link protein by immunofluorescence and immunoprecipitation and the role of this glycoprotein by blocking antibodies in the early chick embryo. The recognition of the link protein 1 (LP1, 48 kDa) and link protein 2 (LP2, 44 kDa) types was an important finding of our study. Link protein links several proteoglycans, such as aggrecan to hyaluronan, creating stable aggregates in the extracellular matrix and has a general function in the organization of the extracellular matrix. It is known that combinations of LP1 and LP2 create more stable complexes than the individual link protein molecule. This was also shown in our experiments, in that aggrecan (180kDa) co-precipitated with LP1 and LP2. Our immunofluorescent experiments showed that link protein expression was first detectable at the blastula stage (st. XIII) and its presence may be fundamental as the first extracellular matrix starts to assemble before the initiation of the first major cellular migrations during the gastrula stage. Link protein influorescence was strong in the cells ingressing through the primitive streak and in the migrating cells in embryos at stage HH3 (intermediate streak/mid-gastrula). At stage HH4 (definitive streak/late gastrula), link protein fluorescence was strong at the apical surface of the neural plate. At stage HH4-5 (head process), link protein fluorescence was strong at the apical surface of the neural folds, notochord and endoderm. At stage HH13 (19 somites), link protein fluorescence was intense in the encephalic vesicles, in the extracellular matrix, in the lumen of encephalic vesicles, intense in migrating neural crest cells, neural tube and in notochord, strong in gut lower wall, hard tube and dorsal aorta wall, intense in dermomyotome and strong in sclerotome in somites. By stage HH17 (29 somites), link protein fluorescence was strong in neuroepithelium and extracellular matrix in the lumen of the diencephalon, strong in neural crest cells, in the intraretinal space in the eye, in myocardium and endocardium, in dorsal aorta, in dermomyotome, the outer surface of pharyngeal arches wall of aortic arches and intense in thyroid rudiment. Inhibition of function of link protein by blocking antibodies showed that link protein was important in neuroepithelial tissue organization and neural tube closure, in normal differentiation of the neural tube to form the brain, in the morphogenesis of the heart tube, the dorsal aorta and gut and in somite epithelialization.

Made available in DSpace on 2014-12-17T15:37:19Z (GMT). No. of bitstreams: 1 MarceloCR.pdf: 216453 bytes, checksum: 003d93ffae0499641acc9c223d20b61c (MD5) Previous issue date: 2006-10-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Parental care s costs increase with the time spent due to incapacity of parents give assistance to increasing offspring food s requirement. This parental care deficits is crucial to offspring s emancipation that involve abilities development to they survive independently. In this work we observe 16 Red-footed boobies chick (Sula sula) on different stages of development, at area of Parque Nacional Marinho of Fernando de Noronha s archipelago, from August to October of 2005. Our data show the parents presence decrease during chick development and an activity frequency modification, in move and fly, which suggest the emancipation s chick development. We observe also the action of Kleptoparasits and the influence with offspring parental care. Our data show consistency with the literature. However, we consider that studies carried through during the peak of the reproductive station can support the hypothesis of that the conflicts, together with the initial period of development of the flight, can be representative in terms of mortality of chicks for the studied species / Os custos do cuidado parental tendem a aumentar com o decorrer do tempo, principalmente devido ? incapacidade dos pais atenderem ?s crescentes necessidades alimentares da prole. Esse d?ficit no cuidado parental ? crucial para a emancipa??o da prole no que se refere ao desenvolvimento de suas habilidades para sobreviver independentemente. Neste trabalho observamos 16 filhotes de Atobas-de-p?s-vermelhos (Sula sula) em diferentes est?gios de desenvolvimento durante o per?odo de agosto at? outubro de 2005, na ?rea do Parque Nacional Marinho do Arquip?lago de Fernando de Noronha / PE. Os dados apontaram a diminui??o da presen?a dos pais ao longo dos est?gios de desenvolvimento do filhote, juntamente com altera??es nas freq??ncias de atividades como deslocamento e v?o, que sugerem o desenvolvimento da emancipa??o do filhote. Ainda observamos a a??o de cleptoparasitas e sua influ?ncia no cuidado com a prole. Os dados mostraram-se consistentes com a literatura. Contudo, propomos que estudos realizados durante o pico da esta??o reprodutiva podem sustentar a hip?tese de que os conflitos, juntamente com o per?odo inicial de desenvolvimento do v?o, podem ser representantes em termos de mortalidade de filhotes para a esp?cie estudada

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