From molecules to organs : Microscopy and multi-scale nature of development

Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development.

Epidermis differentiation in Salmo irideus Gilb. development [Translation from: Archo ital.Anat.Embriol. 76, 19-30, 1971]

Recent histochemical and histophysiological research on the skin of fish has posed interesting problems both with regard to the presence of specialized types of cell and with regard to the function of this organ. The present paper intends to study the development and the functional differentiation of the trout's skin, both from the histomorphological and the histochemical point of view. The skin of Salmonids is devoid of specialized cells; the granular cells of Petromyzonti, the serous cells of Selacii and the clavate cells described in many other teleosts and it lacks keratin. As such it can be considered a good working model which can be used to show the eventual histomorphological and histochemical changes occurring both in the transformation from the endovular to the aqueous environment. Histomorphological observations were carried out demonstrate the great structural simplicity of the trout's skin at all stages. The article concludes that glycogen increases greatly when the epidermis thickens and therefore when it becomes necessary to guarantee resistance, amongst which is a support mechanism.

Structural capital, innovation capability, and company performance in technology-based colombian firms

[EN] In today s economy, innovation is considered to be one of the main driving forces behind business competitiveness, if not the most relevant one. Traditionally, the study of innovation has been addressed from different perspectives. Recently, literature on knowledge management and intellectual capital has provided new insights. Considering this, the aim of this paper is to analyze the impact of different organizational conditions i.e. structural capital on innovation capability and innovation performance, from an intellectual capital (IC) perspective. As regards innovation capability, two dimensions are considered: new idea generation and innovation project management. The population subject to study is made up of technology-based Colombian firms. In order to gather information about the relevant variables involved in the research, a questionnaire was designed and addressed to the CEOs of the companies making up the target population. The sample analyzed is made up of 69 companies and is large enough to carry out a statistical study based on structural equation modelling (partial least squares approach) using PLS-Graph software (Chin and Frye, 2003). The results obtained show that structural capital explains to a great extent both the effectiveness of the new idea generation process and of innovation project management. However, the influence of each specific organizational component making up structural capital (organizational design, organizational culture, hiring and professional development policies, innovation strategy, technological capital, and external structure) varies. Moreover, successful innovation project management is the only innovation capability dimension that exerts a significant impact on company performance.

Development of nucleic acid vaccines: use of self-amplifying RNA in lipid nanoparticles

Self-amplifying RNA or RNA replicon is a form of nucleic acid-based vaccine derived from either positive-strand or negative-strand RNA viruses. The gene sequences encoding structural proteins in these RNA viruses are replaced by mRNA encoding antigens of interest as well as by RNA polymerase for replication and transcription. This kind of vaccine has been successfully assayed with many different antigens as vaccines candidates, and has been shown to be potent in several animal species, including mice, nonhuman primates, and humans. A key challenge to realizing the broad potential of self-amplifying vaccines is the need for safe and effective delivery methods. Ideally, an RNA nanocarrier should provide protection from blood nucleases and extended blood circulation, which ultimately would increase the possibility of reaching the target tissue. The delivery system must then be internalized by the target cell and, upon receptor-mediated endocytosis, must be able to escape from the endosomal compartment into the cell cytoplasm, where the RNA machinery is located, while avoiding degradation by lysosomal enzymes. Further, delivery systems for systemic administration ought to be well tolerated upon administration. They should be safe, enabling the multiadministration treatment modalities required for improved clinical outcomes and, from a developmental point of view, production of large batches with reproducible specifications is also desirable. In this review, the concept of self-amplifying RNA vaccines and the most promising lipid-based delivery systems are discussed.

Energetics and Structural Characterization of the large-scale Functional Motion of Adenylate Kinase

Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins.

Development of a universal stress sensor for graphene and carbon fibres

Carbon fibres are a significant volume fraction of modern structural airframes. Embedded into polymer matrices, they provide significant strength and stiffness gains by unit weight compared with competing structural materials. Here we use the Raman G peak to assess the response of carbon fibres to the application of strain, with reference to the response of graphene itself. Our data highlight the predominance of the in-plane graphene properties in all graphitic structures examined. A universal master plot relating the G peak strain sensitivity to tensile modulus of all types of carbon fibres, as well as graphene, is presented. We derive a universal value of - average - phonon shift rate with axial stress of around -5ω0 -1 (cm -1 Mpa-1), where ω0 is the G peak position at zero stress for both graphene and carbon fibre with annular morphology. The use of this for stress measurements in a variety of applications is discussed. © 2011 Macmillan Publishers Limited. All rights reserved.

Preliminary histological study of the ovarian development of the giant tiger prawn, Penaeus monodon

This study was undertaken to determine structural characteristics within the gonads which might serve as an index to age and longevity and furnish information on the frequency of spawning of an individual.

Ultra-structural defects cause low bone matrix stiffness despite high mineralization in osteogenesis imperfecta mice.

Bone is a complex material with a hierarchical multi-scale organization from the molecule to the organ scale. The genetic bone disease, osteogenesis imperfecta, is primarily caused by mutations in the collagen type I genes, resulting in bone fragility. Because the basis of the disease is molecular with ramifications at the whole bone level, it provides a platform for investigating the relationship between structure, composition, and mechanics throughout the hierarchy. Prior studies have individually shown that OI leads to: 1. increased bone mineralization, 2. decreased elastic modulus, and 3. smaller apatite crystal size. However, these have not been studied together and the mechanism for how mineral structure influences tissue mechanics has not been identified. This lack of understanding inhibits the development of more accurate models and therapies. To address this research gap, we used a mouse model of the disease (oim) to measure these outcomes together in order to propose an underlying mechanism for the changes in properties. Our main finding was that despite increased mineralization, oim bones have lower stiffness that may result from the poorly organized mineral matrix with significantly smaller, highly packed and disoriented apatite crystals. Using a composite framework, we interpret the lower oim bone matrix elasticity observed as the result of a change in the aspect ratio of apatite crystals and a disruption of the crystal connectivity.