Extracellular Vesicles (EVs) are small membranous particles secreted by the cells. They play an important role in intercellular communication and can transport a variety of biomolecules, including proteins, lipids, and nucleic acids, to target cells. The scientific community recently considered EVs attractive candidates for developing targeted drug delivery systems (DDSs), given their biocompatibility, low immunogenicity, stability in biofluids, and capability to cross biological barriers. Most studies have shown the feasibility of incorporating desired moieties at the EV surface through the genetic modification of EV-producing cells, exploiting the fusion with proteins enriched at the EV membrane. Tetraspanins are transmembrane proteins enriched in EVs, already exploited for EV isolation or tracking upon fusion with fluorescent reporters. CD81 is a well-characterized tetraspanin with ubiquitous protein expression, overexpression tolerance and a limited number of encoded protein isoforms with respect to other EV-associated tetraspanins. Here, I have explored a CD81-based approach for EV targeting. CD81, in full-length or truncated form, was used to guide the expression into EVs of an anti-HER2 moiety, namely the light chains of trastuzumab, within three different constructs, including turbo-GFP (tGFP) as a reporter: CD81-tGFP as master control, CD81-antiHER2-tGFP and CD81delta-antiHER2-tGFP. The first part of the project was dedicated to the characterization of chimeric proteins at cellular and vesicular levels. CD81-based constructs were successfully expressed in HEK239T cells with a preferential enrichment in organelle fractions, underlying the expected involvement in the intracellular vesicular trafficking. Next, chimeric proteins were also found in the derived EVs, with a similar expression trend, corroborated by imaging flow cytometry. Nanoparticle tracking analysis and cryogenic electron microscopy acquisitions confirmed that CD81-fusion proteins boosted EV release without altering the size distribution. Subsequently, I tested the binding capacity of the chimeric proteins to HER2 receptor through orthogonal techniques, such as AlphaLISA and immunoprecipitation. Confocal imaging, also on live cells, confirmed EV internalization into breast cancer cells, depending on the recipient cell type and the presence of HER2 receptor. Moreover, chimeric EVs loaded with doxorubicin were able to mediate a concentration-dependent cytotoxic effect on recipient breast cancer cells. Of note, messenger RNA provided a valuable readout of the in vivo delivery capability of the CD81-engineered EVs, since detected by digital droplet PCR in breast cancer tumour xenografts from mice treated with chimeric EVs. The results presented in this thesis highlighted the feasibility of using CD81 fusion proteins for cell targeting and cargo delivery, ultimately opening new perspectives for the development of EV-based therapeutics.

<p dir="ltr">The rising demand for EVs, motivated by their environmental benefits, is generating increased need for EV charging infrastructure. Also, it has been recognized that the adequacy of such infrastructure helps promote EV use. Therefore, to facilitate EV adoption, governments seek guidance on continued investments in EV charging infrastructure development. The high cost of these investments motivates governments to seek optimal decisions on EV-related investments including EV charging infrastructure, and such decisions include locations, capacities, and deployment scheduling of such infrastructure. Additionally, uncertainties in travel demand prediction and EV driving range constraints need to be considered in EV infrastructure investment planning. To help address these questions, this thesis developed a framework to establish optimal schedules and locations for new charging stations and for decommissioning gasoline refueling stations for any given network over a long-term planning horizon, considering uncertainties in travel demand forecasts and EV driving-range heterogeneity. To address the uncertainties, the proposed framework is formulated as a robust mathematical model that minimizes the worst-case total system travel cost and the total penalty for unused charging station capacity. This study uses an adaption of the cutting-plane method to solve the proposed model. In the numerical analyses, the performance of the robust framework and its deterministic counterpart are compared. The results show that the optimal robust plan outperforms the deterministic plan by yielding savings in the costs of travel and electricity charging. The thesis also investigates the effects of investment budget levels of robust planning. The numerical results throw light on the relationships between higher investment levels and electric charging station deployment levels and consequently, the savings in travel costs and impacts on unused charging capacity. The outcomes of this thesis can help road agencies and related private sector entities enhance preparations towards infrastructure investments to support electric charging stations in an efficient manner.</p>

Yes / This paper presents a robust framework for active and reactive power management in distribution networks using electric vehicles (EVs). The method simultaneously minimizes the energy cost and the voltage deviation subject to network and EVs constraints. The uncertainties related to active and reactive loads, required energy to charge EV batteries, charge rate of batteries and charger capacity of EVs are modeled using deterministic uncertainty sets. Firstly, based on duality theory, the max min form of the model is converted to a max form. Secondly, Benders decomposition is employed to solve the problem. The effectiveness of the proposed method is demonstrated with a 33-bus distribution network.

Von regenerativen Zellen freigesetzte vesikuläre Strukturen mit einer Lipiddoppelmembran, sogenannte extrazelluläre Vesikel (EVs), stellen einen vielversprechenden Ansatz dar zukünftig Herz-Kreislauf-Erkrankungen zu behandeln. In diesem Zusammenhang untersuchte die vorliegende Arbeit die Eignung von EVs allogener humaner Herzzellen (CardAP-Zellen) als mögliches Therapeutikum für Erkrankungen des Herzens. Zu diesem Zwecke wurden die EVs durch differentielle Zentrifugation aus dem konditionierten Medium von CardAP-Zellen nach Kultivierung mit oder ohne pro-inflammatorische Zytokine gewonnen. Die so isolierten EV- Präparationen beider Konditionen zeigten vergleichbare Konzentrationen und verfügten sowohl über charakteristische vesikuläre Strukturen als auch transportierte Moleküle, wie die Tetraspanine. Allerdings wiesen stimulierte EVs im Gegensatz zur nichtstimulierten Vergleichsgruppe ein größeres Repertoire an miRNAs und kleinere Durchmesser auf. In verschiedenen in vitro Analysen konnte zudem nachgewiesen werden, dass EVs von CardAP-Zellen i) die Angiogenese fördern, ii) die Apoptose von Herzzellen vermindern, iii) nur schwach immunogen sind und iv) induzierte Immunreaktionen verringern können. Dabei wurden teils deutliche Unterschiede zwischen den induzierten Effekten von EVs aus stimulierten und nichtstimulierten Konditionen dokumentiert, die darauf schließen lassen, dass verschiedene Mechanismen in der Empfängerzelle angeregt werden durch die Interaktion mit den jeweiligen EVs. Insbesondere konnte in der vorliegenden Arbeit gezeigt werden, dass CD14+ Immunzellen eine essentielle Rolle bei der immunmodulierenden Wirkung der EVs in induzierten Immunreaktionen besitzen. Zusammenfassend stellen EVs von allogenen CardAP-Zellen ein aussichtsreiches therapeutisches Werkzeug für Herz-Erkrankungen dar und zukünftige Studien werden klären, ob eine Anwendung im Menschen möglich ist. / From cells released vesicular structures with a lipid bilayer, the so-called extracellular vesicles (EVs), cannot reproduce but can affect important processes in a recipient cell. EVs of regenerative cells represent a promising therapeutic approach. In this context, the present work investigated whether heart diseases could be treated in the future by using EVs from allogeneic regenerative human cardiac cells (CardAP cells). For this purpose, EVs were isolated by differential centrifugation from the conditioned medium of CardAP cells cultured with or without pro-inflammatory cytokines. These obtained EV preparations from both EV biogenesis conditions exhibited comparable concentrations, characteristic vesicular structures, and characteristic transported molecules, such as the tetraspanins. However, stimulated EVs showed a larger repertoire of miRNAs and smaller diameters in contrast to their unstimulated counterpart. Most importantly, different in vitro analysis demonstrated that the isolated EVs from CardAP cells i) promote angiogenesis, ii) decrease cardiac cell apoptosis, iii) have a low immunogenicity, and iv) can reduce induced immune responses. Interestingly, differences were documented in these beneficial features between stimulated and unstimulated EV preparations, suggesting that different mechanisms in the recipient cell are stimulated by the interaction with the respective EVs. Moreover, CD14+ cells (mainly monocytes) were shown to play an essential role in the detected immunomodulation of EVs. In summary, EVs from CardAP cells appear to be a promising therapeutic tool for cardiac diseases and further studies will clarify open questions such as the efficacy in the organism.

Les exosomes apoptotiques (ApoExo) sont des vésicules extracellulaires (EVs) dérivées de lésions vasculaires et libérées par des cellules endothéliales (ECs) apoptotiques dont la taille, les protéines, le profil en ARN et l'activité enzymatique sont différents de ceux des corps apoptotiques classiques. Notre groupe a montré que les ApoExos accéléreraient le rejet vasculaire en association avec les anti-LG3 circulants, des auto-anticorps (auto-Ac) dirigés contre le LG3, le fragment 5' du perlécan. Nous avons également démontré le rôle de biomarqueur et le rôle effecteur des anti-LG3 dans les lésions vasculaires rénales, à la fois dans les reins natifs et transplantés. La néphrite lupique (NL) est une manifestation fréquente et grave du lupus érythémateux disséminé (LED). Il n'existe pas de biomarqueurs du dysfonctionnement rénal progressif dans la NL. Nous émettons l'hypothèse que les ApoExos stimulent des cellules B spécifiques qui existent dans le répertoire immunitaire normal et que les conditions pro-inflammatoires prévalant chez les patients atteints de LED, telles que l'activation accrue des récepteurs Toll-like (TLRs), amplifient cette réponse, conduisant à la production d'anti-LG3, un auto-Ac important dans l'établissement de la NL. Des cellules B productrices d’anti-LG3 ont été trouvées dans la cavité péritonéale de souris saines et ont produit des anti-LG3 suite à une stimulation in vitro avec des agonistes des TLR1/2, TLR4, TLR7 et TLR9. Il est intéressant de noter que ces cellules sont absentes de la cavité péritonéale de souris saines ayant reçu une injection d'ApoExos. En explorant l'importance fonctionnelle des TLRs dans le déclenchement d'une réponse auto-immune dans un modèle murin lupique, nous montrons que les agonistes de TLRs connus pour contribuer à la pathogenèse du LED (TLR2, 4, 7 et 9) déclenchent une production significativement plus élevée d'IgM anti-LG3, alors que la stimulation des TLRs qui ne sont pas associés à la pathogenèse du LED (TLR3 et 5) ne le fait pas. L’injection d'ApoExo a également déclenché l'axe auto-immun IL-23/IL-17 (mesuré par ELISA et essai cytokinique), augmenté les cellules B de centres germinatifs spléniques (mesuré par cytométrie de flux), augmenté les taux circulants d’IgG totaux, d’anti-LG3 et d’auto-Ac classiques du LED (mesuré par micropuce et ELISA) par rapport à l’injection de véhicule. Des niveaux élevés d'IgG anti-LG3 circulants sont observés chez les souris prédisposées au LED par rapport aux souris saines (mesurés par ELISA), ainsi qu'une proportion accrue de cellules B1 spléniques et de cavité péritonéale (mesurés par cytométrie de flux) augmentant avec l’établissement de la maladie. Ces observations suggèrent un rôle spécifique des ApoExos dans la modulation de la production d'auto-Ac qui, à son tour, déclenche l'involution microvasculaire importante dans les maladies auto-immunes et le rejet de greffe. Ces observations suggèrent également que les cellules B spécifiques de LG3 peuvent être modulées dans des conditions pro-inflammatoires telles que celles qui prévalent chez les patients atteints de LED, conduisant à la production d'auto-Ac. Une meilleure compréhension de l'impact de ces mécanismes permettra d'améliorer l'identification, la prédiction et la prise en charge de la NL. / Apoptotic exosomes (ApoExo) are vascular injury derived extracellular vesicles (EVs) released by apoptotic endothelial cells (ECs) with distinct size, protein, RNA profile and enzymatic activity from classical apoptotic bodies. Our group showed that ApoExo accelerated vascular rejection in association with circulating anti-LG3, autoantibodies (autoAb) against LG3, the 5’ fragment of the perlecan. We have also unravelled biomarkers and effector roles of anti-LG3 in kidney vascular damage in both native and transplanted kidneys. Lupus Nephritis (LN) is a common and serious manifestation of systemic lupus erythematosus (SLE). Biomarkers of progressive renal dysfunction in LN, are lacking. We hypothesize that ApoExo stimulate specific B cells that exist in the normal immune repertoire and that the pro-inflammatory conditions prevalent in SLE patients, such as increased Toll-like Receptors (TLRs) activation, amplify this response, leading to anti-LG3 production, autoAb of importance in LN development. B cells producing anti-LG3 were found in the peritoneal cavity of healthy mice and produced anti-LG3 AutoAb when stimulated in vitro with TLR 1/2, 4, 7 and 9 agonists. Interestingly, these cells disappeared from the peritoneal cavity of healthy mice infused with ApoExo. ApoExo infusion also triggered circulating IL-23/IL-17 autoimmune axis (measured by cytokines assay), increased splenic germinal centre B cells (measured by flow cytometry), increased total circulating IgG, anti-LG3 and classical autoAb (measured by microarray and ELISA) compared to vehicle infusion. Elevated circulating anti-LG3 IgG levels are found in SLE prone mice compared to healthy ones (measured by ELISA) as well as an increased proportion of splenic and peritoneal cavity B1 cells (measured by flow cytometry). Exploring the functional importance of TLRs in triggering such a response, we show that while TLR agonists known to contribute to SLE pathogenesis (TLR2, 4, 7 and 9) triggered significantly higher IgM anti-LG3 production, stimulation of TLR that are not associated with SLE pathogenesis (TLR3 and 5) did not. These observations suggest a specific role for ApoExo in modulating the production of autoAb which, in turn, trigger microvascular involution of importance in autoimmune diseases and transplant rejection. These observations also suggest that LG3-specific B cells may be modulated under pro-inflammatory conditions such as those prevalent in lupus patients, leading to production of autoAb. A better understanding of the impact of these mechanisms will lead to improved identification, prediction, and management of LN.

The content of this work is the analysis of expert opinions on the valuation of the business or its parts in several respects. In the introduction it is mentioned adjustment of valuation by Czech methodology ZNAL, International Valuation Standards IVS and European Valuation Standards EVS. Furthermore, it is carried out the comparison of these methodologies, including the German Standard IDW S1, according to some criteria. The analytical section is dedicated to the analysis of expert opinions, to assess their level of processing, and to point out some weaknesses or strengths. The conclusion is a quantitative summary of the results of analysis of expertise, as well as pointing out the most common shortcomings and proposals for their elimination.

Li-ion batteries have gained great popularity among researchers and practitioners as an environmentally friendly energy storage solution for more environmentally friendly electric vehicles (EVs). However, because of the increased demand for Li-ion battery-powered EVs, and some issues with battery design, legislation, collection and sorting, recycling, and material recovery, achieving sustainable mobility through the circularity of Li-ion batteries is a major challenge. This study aims to identify the challenges as well as develop strategies for enhancing the circularity of Li-ion batteries in Sweden. Following a systematic literature review, two primary research questions were investigated: 1) what are the current challenges and opportunities for the circular economy in lithium-ion battery end-of-life management? 2) how the circularity of LIBs in Sweden could be enhanced? This study employed PEST and SWOT analysis, as well as 11 interviews with industry experts and researchers are performed, to determine the strengths, weaknesses, opportunities, and threats in the circularity of lithium-ion batteries in Sweden. Following that, various strategies were developed to address the identified challenges and improve the circular economy of these batteries. Finally, the developed strategies are validated through expert interviews, and various recommendations are outlined. The study's findings are significant and can assist policymakers, investors, and industry professionals concerned with the circularity of lithium-ion batteries in developing appropriate decisions and better planning for the Swedish transportation sector.

Transportation and energy play an essential role in modern society. Since the Industrial Revolution, fossil fuels have enabled great advancements in human society. Within this process, Internal Combustion Engines Vehicles (ICEVs) played a significant role in guaranteeing reliable and affordable long-distance transportation. However, the subsequent increase of the Motorized Private Transport resulted in undesired effects such as pollution. One instrument in reducing the Greenhouse Gas (GHG) emissions of the transport sector is to shift from the conventional ICEVs toward zero local emission vehicles. Electric Vehicles (EVs) are being promoted worldwide as a suitable powertrain technology that could replace the ICEVs. However, unless combined with electricity from renewable generation technologies the EVs will not effectively reduce GHG emissions. Through the simulation of future transport and energy sector scenarios in Germany, the GHG emission reductions have been analyzed. Techno-economic and environmental characteristics for several powertrain technologies under several vehicles charging strategies are evaluated. The thesis explores the impact of charging EVs on the electrical grid. The result show that EVs using smart charging strategies that support Vehicle-to-grid (V2G) are capable of fulfilling mobility needs of users while providing substantial flexibility to the electrical grid. Such flexibility can facilitate the future expansion of non-dispatchable Renewable Energy Sources (RES).

Asset managers continue to prepare physical infrastructure investments needed to accommodate the emerging technologies, namely vehicle connectivity, electrification, and automation. The provision of new infrastructure and modification of existing infrastructure is expected to incur a significant amount of capital investment. Secondly, with increasing EV and CAV operations, the revenues typically earned from vehicle registrations and fuel tax are expected to change due to changing demand for vehicle ownership and amount of travel, respectively. This research estimated (i) the changes in highway expenditures in an era of ECAV operations, (ii) the net change in highway revenues that can be expected to arise from ECAV operations, and (iii) the changes in user equity across the highway user groups (vehicle classes). In assessing the changes in highway expenditures, the research developed a model to predict the cost of highway infrastructure stewardship based on current and/ or future system usage. <div><br></div><div>The results of the research reveal that CAVs are expected to significantly change the travel patterns, leading to increased system usage which in turn results in increased wear and tear on highway infrastructure. This, with the need for new infrastructure to support and accommodate the new technologies is expected to result in increased highway expenditure. At the same time, CAVs are expected to have significantly improved fuel economy as compared to their human driven counterparts, leading to a decrease in fuel consumption per vehicle, resulting in reduced fuel revenues. Furthermore, the prominence of EVs is expected to exacerbate this problem. This thesis proposed a revision to the current user fee structure to address these impacts. This revision contains two major parts designed to address the system efficiency and equity in the near and long term. For the near term, this thesis recommended a variable tax scheme under which each vehicle class pays a different fuel tax rate. This ensures that both equity and system efficiency are improved during the transition to ECAV. In the long term, this thesis recommended supplementing the fuel tax with a distance based VMT tax, applicable to electric vehicles.<br></div>

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