The purpose of this investigation is to study the effects of methylxanthines, catecholamines, and cyclic nucleotides on melanin synthesisand conidiation using the technique of phase contrast microscopy. Verticillium wild type (T9) strain was grown in both sucrose nitrate liquid medium and polygalacturonic acid media (SNLM and PGAM). These media allowed rapid production of conidia and synchronous development of microsclerotia respectively. Even though caffeine has a greater effect on conidiation, both caffeine and theophylline promoted melanization. Dibutyl cyclic AMP to lesser extent inhibited conidiation, while cyclic AMP had little to no effect on conidiation. Dibutyl cyclic AMP inhibited melanin production. Cyclic AMP had a stimulatory effect on melanin production. Isoproterenol a stimulant of adenylate cyclase activity, inhibited conidiation the first two days, but had no apparent effect after day 3. Isoproterenol also induced melanin production in the flask. Propranolol, an antagonist of hormonally induced adenylate cyclase activity, did not repress conidiation when compared to the drug-free SNLM. Melanin was not formed in either SNLM and PGAM, suggesting that the effect of propranolol was opposite that of isoproterenol. Propranolol, a beta blocking agent, reversed the isoproterenol induced inhibition of conidiation. This reversal indicates that there is a receptor which isoproterenol attached itself to - the binding beta receptor site. The isoproterenol effect on conidiation and melanin synthesis indicates that, there might be cyclic AMP involvement in development, since this agent affects cyclic AMP level. Since melanin is known to be associated with microsclerotia, it is possible that cyclic AMP might be involved in this development. In this study, the effects of methylxanthines, catecholamines and cyclic nucleotides on melanin synthesis and conidiation was determined. There is every possibility that cyclic AMP might be involved in the regulation of conidiation process and melanin synthesis.

Student Number : 9501020M - M Sc dissertation - School of Clinical Medicine - Faculty of Health Sciences / An exciting and novel concept of tissue engineering and morphogenesis is the generation of bone by the implantation of smart biomaterials that in their own right can induce a desired and specific morphogenetic response from the host tissues without the addition of exogenously applied bone morphogenetic and osteogenic proteins.

The topic of this thesis has been built on previous work of our group, especially Rieger et al. 2008 and Čepl et al. 2010. We examined and described the regularity of morphogenesis of S. marcescens, morphotype F colonies (from "the fountain", because of a shape it resembles). Typical colony consists of elevated red navel, low non-pigmented ring and again elevated red rim. Structured profile of the colony together with changes in pigmentation of structures during development without need of artificial dying, provides considerable advantage in observation of the morphogenesis. Aims of this thesis were (i) to find other factors that affect the morphogenesis, (ii) to characterize interactions of S. marcescens colonies with other bacterial strains (S. rubidaea and E .coli) and finally (iii) to study the phenomenon of induced resistance to the antibiotics described in (Heal and Parsons, 2002; Lu 2004; Bernier et al. 2011) in our model organisms. (i) Mutual interactions of colonies on the plate indicates that morphogenesis is affected by autocrine signals, which diffuse into the medium and the atmosphere and affect the development of surrounding colonies. We have detected changes of pH of the medium during the development of colonies in their vicinity. At ...

As glândulas salivares são estruturas essenciais para a manutenção da homeostase da cavidade oral pela síntese e secreção do fluido salivar. A disfunção ou perda permanente das glândulas salivares causadas por radioterapia, doenças inflamatórias ou desordens congênitas elevam principalmente o risco de infecções da mucosa oral e de estruturas dentárias, além de potencialmente prejudicar funções fisiológicas como fala, mastigação e paladar, diretamente interferindo na qualidade de vida dos indivíduos afetados. Os tratamentos atualmente disponíveis são apenas paliativos, ressaltando a necessidade de se compreender melhor os processos embriogênicos a fim de desenvolver novas estratégias terapêuticas capazes de regenerar as glândulas salivares. O princípio da formação das glândulas salivares baseia-se na coordenação de diversos processos morfogenéticos, e este trabalho foca particularmente em investigar a formação do espaço luminal do sistema de ductos, uma vez que a adequada abertura dos lumens é um processo essencial para a secreção salivar. Relata-se que a remoção das células centrais dos cordões sólidos epiteliais por morte celular apoptótica é o principal mecanismo de abertura do espaço luminal dos futuros ductos glandulares em camundongos. Porém, pouco se sabe sobre o controle temporal da apoptose durante o desenvolvimento glandular e sobre seu comportamento em glândulas salivares humanas. Neste trabalho, o perfil de expressão de diversas proteínas envolvidas na cascata apoptótica em glândulas salivares fetais humanas foi analisado de acordo com cada estágio morfogenético por imunoistoquímica (Bax, Bak, Bad, Bid, Bcl-2, Bcl-x, Bcl-xL, caspase-3 clivada, caspases-6, -7 e -9, apaf-1, survivina e citocromo c). As análises semi-qualitativas resultaram em negatividade apenas para as proteínas Bcl-2, Bad, Bid e caspase-3 clivada em todas as fases de desenvolvimento. A expressão nuclear de Bax e Bak foi identificada em presumidos espaços luminais em estágios precoces, enquanto Bcl-xL foi o fator antiapoptótico da família Bcl-2 que exibiu expressão nuclear mais importante. Caspases-6, -7 e -9 foram positivas em todas as fases, e a ausência de caspase-3 clivada sugere caspase-7 como principal caspase efetora da apoptose em desenvolvimento de glândulas salivares humanas. Ambos os componentes do complexo apoptossomo foram positivos durante o desenvolvimento glandular, e o inibidor survivina demonstrou mais positividade nuclear em estágios mais avançados. Ao observar a expressão de reguladores apoptóticos durante o desenvolvimento glandular humano, foram realizados experimentos funcionais com culturas de tecido glandular de camundongos para avaliar o papel das caspases durante a formação desta estrutura. Inicialmente detectou-se a atividade apoptótica em glândulas salivares de camundongos albinos no centro dos cordões epiteliais primários a partir de estágios precoces de desenvolvimento através de TUNEL e caspase-3 clivada. A partir disso, foi realizada a inibição apoptótica funcional in vitro durante o mesmo período, que resultou em ductos significativamente mais amplos e em defeitos morfológicos importantes nas estruturas luminal e acinar. Este trabalho evidenciou portanto atividade apoptótica durante a formação de glândulas salivares humanas e de camundongo, expressando-se em fases mais precoces do que reportadas anteriormente. Além disso, a ausência de Bad e Bid indica que a via intrínseca está mais ativa que a extrínseca, e distintos perfis de expressão da maioria das moléculas sugere adicionais funções não-apoptóticas durante a morfogênese glandular. / Salivary glands are essential structures for the maintenance of homeostasis of the oral cavity by synthesizing and secreting saliva. Permanent dysfunction or loss of salivary glands caused by radiotherapies, inflammatory diseases or congenital disorders increase mainly the risk of infections of the oral mucosa and tooth surface, also impairing physiological functions as speech, mastication and taste, directly interfering in quality of life. Current treatments are only palliative-based, which highlights the need of having a better understanding of embryonic processes to develop therefore new therapeutic strategies able to regenerate salivary glands. The development of glandular secretory units and ductal system involves the coordination of several morphogenetic processes, and this study particularly focuses in investigating the formation of the lumenal space of the ductal system, as the proper lumen opening is an essential step for the salivary secretion. The clearance of the central cells of developing solid epithelial stalks by apoptotic cell death is the main mechanism of lumen space opening within presumptive ducts in mouse salivary glands. However little is known about its temporal regulation and its function in human salivary glands. Here we analysed the profile expression of several apoptosis-related proteins during human salivary gland development in correlation to each morphogenetic stage by immunohistochemistry (Bax, Bak, Bad, Bid, Bcl-2, Bclx, Bcl-xL, cleaved caspase-3, caspases-6, -7 e -9, apaf-1, survivin e citocromo c). Immunohistochemical results were analysed semi-qualitatively, and proteins Bcl-2, Bad, Bid and cleaved caspase-3 were considered completely negative at all stages of development. The nuclear expression of Bax and Bak were observed within the presumptive luminal spaces at early stages, while Bcl-xL was the antiapoptotic factor of Bcl-2 family that showed more prominent nuclear expression. Caspases-6, -7 and -9 were positive at all stages, and the absence of cleaved caspase-3 suggests caspase-7 as the main effector caspase during human salivary gland development. Both components of the apoptosome complex were also positive through all development, and the inhibitor of apoptosis survivin has shown more nuclear positivity at later stages. As the expression of apoptotic regulators was observed during human salivary gland development, functional experiments were then performed in mouse salivary gland cultures to determine the apoptotic activity of during the glandular formation. Initially, the apoptotic activity was detected in mouse salivary glands within the centre of primary epithelial stalks from early stages of development by TUNEL and cleaved caspase-3. Thus the in vitro apoptotic inhibition was performed at the same stages, which resulted in significant wider ducts and important morphological defects within luminal and acinar structures. This work has therefore evidenced the existence of apoptotic role in salivary gland lumen formation of both human and mouse models, having an earlier start point as reported before. Moreover, the absence of Bad and Bid indicates that the intrinsic pathway is more active than the extrinsic during human development, and the distinct subcellular expression of most molecules suggests additional non-apoptotic functions.

While the genetic control of ureteric bud (UB) morphogenesis has been extensively studied, the cellular basis of this process remains unclear. The renal organoid system is a novel technique in which embryonic kidneys are dissociated into single cells and then reaggregated, where they reassociate to form organotypic structures. This system may be very beneficial for investigating the cellular basis of ureteric bud development. Here, we first used a fluorescent UB marker, Hoxb7:myrVenus, and time-lapse microscopy to characterize the cellular and tissue-level events during self-organization and UB morphogenesis of E12.5 or E14.5 renal organoids. Briefly, we found that UB structures self-assembled by aggregation of individual cells that sent out long cell processes. The cellular aggregates grew and elongated into epithelial tubes that displayed characteristic ampullae, bifurcated, and appropriately expressed UB tip markers analogous to their in vivo counterparts. We also found that cap mesenchymal cells are attracted to newly formed epithelial structures early in renal organoid development, and were later found in cell clusters surrounding new branches. RET is a trans-membrane tyrosine kinase receptor (RTK), expressed in ureteric bud cells, whose expression is gradually restricted to the tips of the growing ureteric tree. We demonstrate that the renal organoid system can be used, as an alternative to the generation of in vivo chimeric embryos, to study Ret-dependent cell rearrangements previously shown to establish and maintain the UB tip progenitor domain. Chimeric renal organoids that juxtaposed wild-type cells with Sprouty1–/– mutant cells (higher Ret-signaling) or with Ret51/cre (lower Ret-signaling) mutant cells recapitulated the cell sorting pattern observed in similar in vivo chimeras. The cells with higher Ret-signaling preferentially sorted to, and were maintained in, the forming and growing tips of these mosaic ureteric bud structures, out-competing cells with lower Ret-signaling. We then used the mosaic organoid system to ask if fibroblast growth factor receptor 2 (Fgfr2), another RTK expressed in the ureteric bud and important for its development, also mediates individual cell rearrangements that generate and maintain the UB tips. UB cells null for Fgfr2 were largely unable to compete with wild-type cells for occupancy of the UB tips in chimeric renal organoids. Using the innovative MASTR (Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination) technique in vivo, mosaic homozygous deletion of Fgfr2 in newly formed ureteric buds also revealed that mutant cells were slightly deficient in their ability to contribute to Fgfr2 heterozygous UB tips. This demonstrates a novel, cell-autonomous role of Fgfr2 in ureteric bud development. Matrix metalloproteinase 14 (MMP14) is a membrane-bound protein known to participate in a wide variety of cell functions including degradation of the extracellular matrix (ECM), cell signaling, and cell-autonomous cell migration. It is expressed in the UB and was discovered to act downstream of Ret-signaling. Although needed in the ureteric epithelium for ECM degradation and proper UB morphogenesis, its specific function in the UB has not been thoroughly investigated. In generating in vivo chimeras, we discovered that Mmp14 null cells could contribute to wild-type ureteric bud tips at E12.5 and E14.5, demonstrating that, despite its documented role in UB branching, Mmp14 does not have a cell-autonomous role in the cell rearrangements observed during UB morphogenesis.

Chloride intracellular channels (CLICs), 1 and 4 are expressed in endothelial cells where they promote cell proliferation, migration and vessel morphogenesis in vitro. Clic4-/- mice exhibit defects in retinal angiogenesis suggesting CLIC4 functions as an angiogenic regulator. S1P signaling, through S1P receptors S1P1 and S1P2, is essential for endothelial cell functions during vascular development. S1P treatment promotes CLIC4 localization to cell surface suggesting a link between CLICs and S1P pathways. Here we demonstrate that CLICs function in embryonic development, retinal angiogenesis and vascular permeability regulation. Clic1-/-;Clic4-/- embryos die in utero and exhibit severe growth restriction with vascular defects prior to death. Loss of Clic4 in murine endothelium (Clic4ECKO) caused aberrant retinal angiogenesis characterized by reduced vascular outgrowth and increased vessel sprouting. Clic4ECKO mice exhibited increased vessel leakiness as assessed by a lung permeability assay. We establish that CLIC1 and CLIC4 function in distinct branches of the S1P pathway to promote angiogenesis. Knockdown of CLIC1 or CLIC4 in endothelial cells impeded S1P1-mediated induction of AKT and Rac1 and reduced endothelial cell migration and adherence junctions formation. CLIC1 knockdown alone inhibited RhoA activation and actin stress fibers downstream of S1P2. Using pharmacological perturbation of S1P signaling in Clic knockout mice we established that Clic4 is essential for S1P1-mediated regulation of retinal angiogenesis and vascular permeability. We conclude that CLIC1 and CLIC4 function as effectors in the S1P pathway, where they have overlapping functions in S1P1-PI3K signaling and CLIC1 uniquely acts as an effector in S1P2-RhoA signaling cascade. Through these findings, our work defines a molecular mechanism through which CLICs function in endothelium.

Systems biology takes a holistic approach to biological questions as it applies mathematical modeling to link and understand the interaction of components in complex biological systems. Multiscale modeling is the only method that can fully accomplish this aim. Mutliscale models consider processes at different levels that are coupled within the modeling framework. A first requirement in creating such models is a clear understanding of processes that operate at each level. This research focuses on modeling aspects of biological development as a complex process that occurs at many scales. Two of these scales were considered in this work: cellular differentiation, the process of in which less specialized cells acquired specialized properties of mature cell types, and morphogenesis, the process in which an organism develops its shape and tissue architecture. In development, cellular differentiation typically is required for morphogenesis. Therefore, cellular differentiation is at a lower scale than morphogenesis in the overall process of development. In this work, cellular differentiation and morphogenesis were modeled in a variety of biological contexts, with the ultimate goal of linking these different scales of developmental events into a unified model of development. Three aspects of cellular differentiation were investigated, all united by the theme of how the dynamics of gene regulatory networks (GRNs) control differentiation. Two of the projects of this dissertation studied the effect of noise and robustness in switching between cell types during differentiation, and a third deals with the evaluation of hypothetical GRNs that allow the differentiation of specific cell types. All these projects view cell types as highdimensional attractors in the GRNs and use random Boolean networks as the modeling framework for studying network dynamics. Morphogenesis was studied using the emergence of three-dimensional structures in biofilms as a relatively simple model. Many strains of bacteria form complex structures during growth as colonies on a solid medium. The morphogenesis of these structures was modeled using an agent-based framework and the outcomes were validated using structures of biofilm colonies reported in the literature.

No description available.

Temnospondyls are large extinct fossil tetrapods; superficially resembling crocodiles in their general size, appearance and lifestyle. Temnospondyls are a group of early tetrapods, the oldest fossils are more than 340 million years old, and they existed for more than 200 million years. This doctoral thesis examined the postcranial skeleton of temnospondyls and its evolutionary history and diversification. Standard taxonomic techniques were used to distinguish between the types of variation observed in the postcranial skeleton and for phylogenetic analysis. The thesis consists of a series of published articles, three describing the postcranial skeletons of various temnospondyls, and three summary articles, all with extensive illustrations. To provide data, the postcranial skeletons of three temnospondyl taxa were described. The articulated postcranial skeleton of a basal stereospondyl (rhinesuchid) is immature, and paedomorphism of the postcranial skeleton in stereospondyls is discussed. The robust appendicular skeleton of Eryops megacephalus is plesiomorphic, well-ossified, and terrestrially adapted. The paedomorphic postcranial skeleton of Trimerorhachis insignis is plesiomorphic, and secondarily aquatic, the description includes growth stages. This study found that extensive morphogenetic variation is present in the postcranial skeleton of temnospondyls. Many phylogenetically significant characteristics develop with morphogenesis, they may be absent in early growth stages, and may never develop even in the largest growth stages of taxa with paedomorphic postcranial skeletons. Consequently, assessment of the presence or absence of a phylogenetically significant characteristic in any taxon may be dependant on the morphogenetic stage of the specimen examined. This finding has major implications for the phylogenetic analysis of temnospondyls and other early tetrapods. An overview of phylogenetic variation in the postcranial skeleton is presented, including a large phylogenetic analysis of the Temnospondyli. The most primitive temnospondyls possess fully ossified postcranial skeletons, well adapted for terrestrial locomotion, but some of the derived clades of temnospondyls have paedomorphic postcranial skeletons and are exclusively aquatic. For the first time, the postcranial skeleton of temnospondyls is comprehensively compared with that of other early tetrapods in the largest phylogenetic analysis to date, resulting in the unexpected discovery that temnospondyls are most closely related to the ancestors of amniotes. The Temnospondyli plus Neospondyli (Seymouriamorpha plus Cotylosauria plus Lepospondyli) forms a large new clade, the Terrapoda, defined by the presence of many derived synapomorphies. Some of the cranial synapomorphies of the Terrapoda are most likely related to improvements in hearing. The postcranial synapomorphies indicate that the Terrapoda are the first vertebrates to have evolved limbs that are well adapted for terrestrial locomotion. The Terrapoda are the first truly terrestrial vertebrates, their postcranial adaptations facilitated their colonisation of the land and consequent phylogenetic radiation during the early Carboniferous. Both analyses incorporate characters from previous analyses and many new postcranial characters. The results of the phylogenetic analyses are statistically more parsimonious than previous analyses and have much lower levels of homoplasy. Comparative analyses indicate that the distinctive results are most likely due to the increased use of characters pertaining to temnospondyls, increased use of postcranial characters, and differentiation between sources of morphological variation to minimise morphogenetic and phenotypic variation and elucidate true phylogenetic signal.

The Turing mechanism for the production of a broken spatial symmetry in an initially homogeneous system of reacting and diffusing substances has attracted much interest as a potential model for certain aspects of morphogenesis such as pre-patterning in the embryo, and has also served as a model for self-organization in more generic systems. The two features necessary for the formation of Turing patterns are short-range autocatalysis and long-range inhibition which usually only occur when the diffusion rate of the inhibitor is significantly greater than that of the activator. This observation has sometimes been used to cast doubt on applicability of the Turing mechanism to cellular patterning since many messenger molecules that diffuse between cells do so at more-or-less similar rates. Here we show that stationary, symmetry-breaking Turing patterns can form in physiologically realistic systems even when the extracellular diffusion coefficients are equal; the kinetic properties of the 'receiver' and 'transmitter' proteins responsible for signal transduction will be primary factors governing this process.