Promoting Central Nervous System Regeneration by Targeting Intracellular Pathways

Adult central nervous system neurons regenerate poorly after injury. One reason for this regenerative failure is that a developmental change occurs where essential growth-molecules become excluded from axons with maturation. In this thesis, two strategies were employed in order to improve axon regeneration: 1) restoring the axonal transport of growth-molecules in mature axons via manipulation of transport machinery; 2) reverting mature neurons to an earlier developmental state where growth-molecules are abundant in the axon. In order to restore axon transport of growth-molecules, protrudin - a membrane-associated protein involved in directional membrane trafficking, was studied. Phosphorylated protrudin preferentially binds Rab11, a small GTPase involved in axon transport of growth receptors. This association is required for neurite outgrowth and for anterograde movement of this complex. We found that endogenous protrudin is excluded from mature axons similar to other growth-related machinery and cargo. It was hypothesised that introducing a phosphomimetic form of protrudin to mature cortical neurons, could increase the phospho-protrudin/Rab11 interaction resulting in improved anterograde transport of growth-molecules and enhanced axon regeneration. We found that overexpression of two constitutively phosphorylated forms and also wild-type protrudin increased the proportion of regenerating axons after $\textit{in vitro}$ laser axotomy. Furthermore, overexpression of wild-type and phospho-protrudin in the retina resulted in enhanced axon regeneration in a mouse model of optic nerve crush 2 weeks after injury. Live-cell imaging experiments revealed that this increased regenerative ability is accompanied with increased transport of Rab11 endosomes as well as growth receptors into the axon. Importantly, by further studying protrudin’s mechanisms of action, we identified novel players in the process of axon regeneration, including an exciting new role for the endoplasmic reticulum. In summary, protrudin is a promising intervention which improves axon regeneration $\textit{in vitro}$ and $\textit{in vivo}$ and due to its participation in multiple molecular pathways, new targets for aiding and understanding axon regeneration could be uncovered. Another approach to aid axon regeneration is to rejuvenate mature neurons. We hypothesised that overexpressing different combinations of transcription factors that are developmentally down-regulated could bring neurons to an earlier developmental stage. Four maturity markers were identified – doublecortin as an early maturity marker and 68-kDa neurofilament, calcitonin, tubulin-4a as late maturity markers. Some transcription factors showed promising results in rejuvenating primary cortical neurons. Further studies are needed to identify correct combinations of transcription factors to achieve maximum effect and to examine the effects of this treatment on axon regeneration.Gates Cambridge - PhD funding award Medical Research Council and Christopher Reeve Foundation - funded researc

Modelling Study With MIKE 21 And Analysis Of Data On Non-Fish Marine Resources

This ecological modelling study is focused on the development of scenarios for the management of the industrially exploited shellfish species and the recovery of the black mussel population.A two-dimensional hydrodynamic model has been developed for a selected region of the Bulgarian Black Sea coast - between Cape Kaliakra and Cape Emine, that allows simulation of sea currents based on several input parameters - wind, water temperature and salinity, etc. This hydrodynamic model forms the basis for subsequent ecological modelling and future projections of the development and mortality of the black mussel (Mytilus galloprovincialis) and Rapa whelk (Rapana venosa) in the selected sector. The ecological model is based on the EcoLab software product and includes three different scenarios of shellfish resource development. The ecological model and generated scenarios could be helpful for the elaboration of management measures and reduction of the pressure on the black mussel population

Selective rab11 transport and the intrinsic regenerative ability of CNS axons.

Neurons lose intrinsic axon regenerative ability with maturation, but the mechanism remains unclear. Using an in-vitro laser axotomy model, we show a progressive decline in the ability of cut CNS axons to form a new growth cone and then elongate. Failure of regeneration was associated with increased retraction after axotomy. Transportation into axons becomes selective with maturation; we hypothesized that selective exclusion of molecules needed for growth may contribute to regeneration decline. With neuronal maturity rab11 vesicles (which carry many molecules involved in axon growth) became selectively targeted to the somatodendritic compartment and excluded from axons by predominant retrograde transport However, on overexpression rab11 was mistrafficked into proximal axons, and these axons showed less retraction and enhanced regeneration after axotomy. These results suggest that the decline of intrinsic axon regenerative ability is associated with selective exclusion of key molecules, and that manipulation of transport can enhance regeneration

Protrudin functions from the endoplasmic reticulum to support axon regeneration in the adult CNS

Funder: Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation); doi: https://doi.org/10.13039/100000865Funder: Christopher and Dana Reeve Foundation (Christopher & Dana Reeve Foundation); doi: https://doi.org/10.13039/100001305Funder: International Foundation for Research in Paraplegia (Internationale Stiftung für Forschung in Paraplegie); doi: https://doi.org/10.13039/501100001708Funder: Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.); doi: https://doi.org/10.13039/100000009Funder: Christopher and Dana Reeve Foundation (Christopher & Dana Reeve Foundation)Abstract: Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury has permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules and organelles. We achieved this by expressing the adaptor molecule Protrudin which is normally found at low levels in non-regenerative neurons. Elevated Protrudin expression enabled robust central nervous system regeneration both in vitro in primary cortical neurons and in vivo in the injured adult optic nerve. Protrudin overexpression facilitated the accumulation of endoplasmic reticulum, integrins and Rab11 endosomes in the distal axon, whilst removing Protrudin’s endoplasmic reticulum localization, kinesin-binding or phosphoinositide-binding properties abrogated the regenerative effects. These results demonstrate that Protrudin promotes regeneration by functioning as a scaffold to link axonal organelles, motors and membranes, establishing important roles for these cellular components in mediating regeneration in the adult central nervous system

OpenBIM-Tango integrated virtual showroom for offsite manufactured production of self-build housing

As a result of progressive use of BIM in the AEC sector, the amount of diverse project information is increasing rapidly, thus necessitating interoperability of tools, compatibility of data, effective collaboration and sophisticated data management. Media-rich VR and AR environments have been proven to help users better understand design solutions, however, they have not been quite advanced in supporting interoperability and collaboration. Relying on capabilities of openBIM and IFC schema, this study posits that this shortcoming of VR and AR environment could be addressed by use of BIM server concept allowing for concurrent multiuser and low-latency communication between applications. Successful implementation of this concept can ultimately mitigate the need for advanced technical skills for participation in design processes and facilitate the generation of more useful design solutions by early involvement of stakeholders and end-users in decision making. This paper exemplifies a method for integration of BIM data into immersive VR and AR environments, in order to streamline the design process and provide a pared-down agnostic openBIM system with low latency and synchronised concurrent user accessibility that gives the “right information to the right people at the right time”. These concepts have been further demonstrated through development of a prototype for openBIM-Tango integrated virtual showroom for offsite manufactured production of self-build housing. The prototype directly includes BIM models and data from IFC format and interactively presents them to users on both VR immersive and AR environments, including Google Tango enabled devices. This paper contributes by offering innovative and practical solutions for integration of openBIM and VR/AR interfaces, which can address interoperability issues of the AEC industry

Estabilización proteica de vinos blancos mediante adsorción en columnas de relleno

La estabilización proteica del vino blanco es una etapa importante en su elaboración, y tiene una relación muy estrecha con la comercialización del producto final. Los métodos tradicionales utilizados en la mayoría de las bodegas consisten en operaciones discontinuas con adición de material adsorbente, que requieren mucha mano de obra, hay pérdida de producto y se generan residuos que tienen un impacto ambiental elevado.En este trabajo se ha estudiado la alternativa de utilizar materiales adsorbentes que permitan disminuir o eliminar las desventajas existentes. Después de una revisión bibliográfica detallada se eligieron el óxido de zirconio y el óxido de aluminio, en diferentes granulometrías y grados de acidez, para estudiar la posibilidad de transformar el proceso de estabilización proteica tradicional discontinuo, en un proceso continuo mediante adsorción con óxidos metálicos empacados en una columna. Los resultados obtenidos muestran que dicha transformación es posible, y que el óxido de zirconio es un adsorbente que muestra una selectividad con las fracciones proteicas del vino, se puede regenerar, se puede empacar en una columna sin causar problemas técnicos y no modifica las características fisicoquímicas del vino tratado, cumpliéndose así todos los requisitos buscados, obteniendo finalmente un vino blanco estabilizado.Wine is one of the most popular low-alcohol drinks, thus many research projects have focused on making wine easier to produce or on improving the final product. The macromolecules in wine can form precipitates or cause turbidity, which affects its stability. The white wine protein stabilization is an important stage in its elaboration, and has a very narrow relationship with the commercialization of the product. The colloidal fractions of the wine, such as proteins and polysaccharides, have a considerable influence on some of the basic operations (for example, filtration, clarification, and cold stabilization).There are a lot of nonselective models for reducing the protein content in wine. The traditional methods used in most of the cellars consist on discontinuous operations with addition of adsorbent materials that require a lot of manpower, there is loss of product and residuals are generated that have a high environmental impact. In this work has been studied to use alternative adsorbent materials, like metal oxides, that allow to diminish or to eliminate the existent disadvantages. Two types of adsorbent material were used: zirconium and alumina. The used materials were in different particle size and acidity degrees.The obtained results show that these materials can be used in packed bed. The protein fractions were removed from a white wine in a packed bed of ZrO2 and showed an improvement in the protein stability. The affinity of the ZrO2 depended on the molecular weight of the wine protein fraction. The physicochemical properties of the wine were not modified after the treatment. Finally, the zirconium oxide in the both form can be regenerated

In Vitro Comparison of Several Methods for Initial Proximal Caries Detection

Introduction: Initial proximal caries is both diagnostic and therapeutic challenge. The disadvantages of the conventional methods for caries detection and the development of technologies led to the creation of contemporary optical devices for early caries detection.Aim: In vitro comparison of the diagnostic accuracy of several methods for early proximal caries detection – visual-tactile, bitewing radiography and laser fluorescence device (DIAGNOdent pen).Materials and methods: Fifty-eight proximal surfaces of extracted human permanent premolars and molars were examined by two examiners using visual inspection, bitewing radiography, DIAGNOdent with proximal contact, and DIAGNOdent directly in the lesion. Results were compared with the histological gold standard. Statistical analysis with ROC curve, sensitivity, specificity and diagnostic accuracy of each detection method was performed. Analysis was conducted in 3 diagnostic thresholds – initial, developed and advanced demineralization.Results: Sensitivity of visual inspection was 16%–33%, specificity 93.3%–100%, sensitivity of bitewing radiography 54%–67%, speci­ficity 93%–94%, sensitivity of DIAGNOdent with proximal surfaces in contact 88%–91%, specificity 79%–89%, sensitivity of DIAG­NOdent directly 89%–92.5%, specificity 81.29%–93%. The highest diagnostic accuracy, increasing with the rise of the level of demin­eralization, was shown by DIAGNOdent directly, followed by DIAGNOdent with proximal contact, bitewing radiography, and visual inspection with the lowest accuracy.Conclusion: The use of contemporary diagnostic devices significantly increases the possibility for early detection of proximal lesions. DIAGNOdent can be used as an adjunct to and increasing the diagnostic accuracy of the conventional caries detection methods

Axonal Organelles as Molecular Platforms for Axon Growth and Regeneration after Injury.

Investigating the molecular mechanisms governing developmental axon growth has been a useful approach for identifying new strategies for boosting axon regeneration after injury, with the goal of treating debilitating conditions such as spinal cord injury and vision loss. The picture emerging is that various axonal organelles are important centers for organizing the molecular mechanisms and machinery required for growth cone development and axon extension, and these have recently been targeted to stimulate robust regeneration in the injured adult central nervous system (CNS). This review summarizes recent literature highlighting a central role for organelles such as recycling endosomes, the endoplasmic reticulum, mitochondria, lysosomes, autophagosomes and the proteasome in developmental axon growth, and describes how these organelles can be targeted to promote axon regeneration after injury to the adult CNS. This review also examines the connections between these organelles in developing and regenerating axons, and finally discusses the molecular mechanisms within the axon that are required for successful axon growth