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Electric vehicles are a mobility innovation that can help significantly reduce greenhouse gas emissions and mitigate climate change. However, increasing numbers of electric vehicles require the construction of a dense charging infrastructure with a sufficient number of chargers. Based on the identified requirements for existing electric vehicle users and potential new customers, the paper proposes a charging point model for an urban area equipped with a local transformer station and a sufficient number of low-power chargers. In particular, the model focuses on efficient use of chargers throughout the day, considering private rental of chargers paid by residents in the evening. The model uses an optimization method that compares the non-covered fixed costs due to unsold electricity to nonresidents and the annualized costs of building an additional transformer. The proposed optimal charging point solution was tested in a case study using real data capturing users’ habits and their arrivals in and departures from the car park. As our model results show, the great benefit of a park-and-ride car park equipped with chargers consists of a simple increase in car park efficiency, ensuring sufficient numbers of private charging lots, optimizing operating costs, and supporting the development of electromobility.

Electric vehicles represent an innovation in mobility that can help significantly reduce greenhouse emissions and mitigate climate change. However, replacing internal combustion with electric vehicles is not enough. This replacement needs to be complemented with a change in the energy mix of individual countries towards renewable energy sources and efficient use of electricity generated as a secondary product. Recuperative braking energy from trains can serve as one source of such secondary energy. Following an analysis of recuperative energy generated and analysis of charging requirements of individual electric vehicles, the paper proposes a model of a charging site near train stations. Using this energy to charge electric vehicles helps to reduce energy consumption from the electricity grid and thus reduce carbon emissions. Compared to other articles, the proposed model ensures the efficient use of recuperative braking energy from trains by using the variable charging power function; thereby, the installation of additional battery storage is eliminated. Our model results show that the benefits of a car park with a reservation system near train stations increase the car park efficiency, provide a sufficient number of private charging points, contribute to efficient use of recovered energy, and reduce carbon emissions.

This dissertation investigates and provides comprehensive insights into three key areas of electric vehicle charging infrastructure optimization, the integration of recuperated braking energy from trains into electric vehicle charging stations, and the influence of transaction costs on the implementation and operation of electric vehicle charging infrastructure. The study aims to address the following research questions: (1) How can electric vehicle charging points be optimized for efficient use while maintaining cost-effectiveness? (2) How can the incorporation of recuperated braking energy from trains into electric vehicle charging stations impact their energy consumption and operating costs? (3) How do transaction costs affect the implementation and operation of electric vehicle charging stations, and how can they be reduced?To optimize electric vehicle charging points, the research reveals several effective strategies. Firstly, leveraging existing parking lots equipped with low-power chargers during off-peak hours, when they are underutilized, proves to be a viable approach. By reserving parking spaces in these lots, electric vehicle owners without private garages or chargers can access charging facilities, maximizing the potential of existing substations. Power dividing between individual charging stations and deferred charging functions are proposed to balance the load on local substations and mitigate peak demand. Integration of battery storage is explored as a solution to enhance stability and support both the local substation and the broader power grid. Additionally, the research highlights the importance of installing a sufficient number of AC (Alternating Current) chargers in urban and suburban areas, rather than focusing solely on the higher charging power of DC (Direct Current) chargers, based on real-world data analysis. Quantity over higher power output is emphasized when expanding charging facilities in public parking lots.The incorporation of recuperated braking energy from trains into electric vehicle charging stations is investigated as a means to improve energy efficiency and reduce the strain on the distribution grid. By capturing and utilising reclaimed brake energy and using it to charge electric vehicles, it helps to minimise energy waste and reduce the load on the electricity grid. Storage of excess recuperated braking energy in battery or capacitor energy storage systems and implementing charging power management functions at individual stations are identified as effective strategies. The study also reveals that companies responsible for powering train lines have an economic opportunity to sell recuperated braking energy for electric vehicle charging at a significantly higher value compared to selling it back to the grid. This presents a potential revenue boost for the company while potentially offering electric vehicle owners lower per kWh prices.Transaction costs are recognized as crucial factors influencing the successful implementation of electric vehicle charging stations, and more generally sustainable mobility. Contrary to common assumptions, the research finds that transaction costs tend to increase over time due to changes in programme administration, internal programme conditions, and information loss. To mitigate the impact of transaction costs, the dissertation proposes various strategies. Streamlining programme administration processes, improving information management systems, fostering collaboration and knowledge sharing among stakeholders, and implementing standardized procedures for infrastructure development are identified as effective approaches.The findings of this dissertation provide valuable insights and practical solutions for optimizing electric vehicle charging infrastructure, incorporating recuperated braking energy, and reducing transaction costs. The research contributes to the development of efficient and sustainable electric vehicle charging systems, facilitating the transition to cleaner transportation and supporting global efforts in mitigating climate change.

Somatic JAK2 mutations are the main molecular cause of the vast majority of polycythemia vera (PV) cases. According to a recent structural model, the prevalent acquired V617F mutation improves the stability of the JAK2 dimer, thereby enhancing the constitutive JAK2 kinase activity. Germline JAK2 mutations usually do not largely alter JAK2 signaling, although they may modulate the impact of V617F. We found an unusual germline JAK2 mutation L604F in homozygous form in a young PV patient, along with a low allele burden JAK2 V617F mutation, and in her apparently healthy sister. Their father with a PV-like disease had L604F in a heterozygous state, without V617F. The functional consequences of JAK2 L604Fmutation were compared with those induced by V617F in two different in vitro model systems: (i) HEK293T cells were transfected with plasmids for exogenous JAK2-GFP expression, and (ii) endogenous JAK2 modifications were introduced into HeLa cells using CRISPR/Cas9. Both mutations significantly increased JAK2 constitutive activity in transfected HEK293T cells. In the second model, JAK2 modification resulted in reduced total JAK2 protein levels. An important difference was also detected: as described previously, the effect of V617F on JAK2 kinase activity was abrogated in the absence of the aromatic residue F595. In contrast, JAK2 hyperactivation by L604F was only partially inhibited by the F595 change to alanine. We propose that the L604F mutation increases the probability of spontaneous JAK2 dimer formation, which is physiologically mediated by F595. In addition, L604F may contribute to dimer stabilization similarly to V617F.

P21-activated kinases (PAKs) regulate processes associated with cytoskeletal rearrangements, such as cell division, adhesion, and migration. The possible regulatory role of PAKs in cell metabolism has not been well explored, but increasing evidence suggests that a cell metabolic phenotype is related to cell interactions with the microenvironment. We analyzed the impact of PAK inhibition by small molecule inhibitors, small interfering RNA, or gene knockout on the rates of mitochondrial respiration and aerobic glycolysis. Pharmacological inhibition of PAK group I by IPA-3 induced a strong decrease in metabolic rates in human adherent cancer cell lines, leukemia/lymphoma cell lines, and primary leukemia cells. The immediate effect of FRAX597, which inhibits PAK kinase activity, was moderate, indicating that PAK nonkinase functions are essential for cell metabolism. Selective downregulation or deletion of PAK2 was associated with a shift toward oxidative phosphorylation. Alternatively, PAK1 knockout resulted in increased glycolysis. However, the overall metabolic capacity was not substantially reduced by PAK1 or PAK2 deletion, possibly due to partial redundancy in PAK1/PAK2 regulatory roles or to activation of other compensatory mechanisms. Competing Interest Statement The authors have declared no competing interest. Footnotes * The revised version contains additional experiments (Fig.6 and Supplementary Figs S2 and S6). Data presentation was slightly modified (Figs 2 and 4). The discussion was completed and the manuscript was checked for grammatical errors.

One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase), which was recently assigned in vitro as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon. We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress. We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.

The ability to form persisters has been observed in many microorganisms, including Staphylococcus aureus, mainly in the context of chronic infections and the pathogenicity of these microbes. In our research, we have demonstrated that salt or oxidative stress could play a role in the formation of S. aureus persisters outside the host's intracellular interface. We pre-exposed planktonic growing bacterial culture to an oxidative or salt stress and monitored the dynamics of persister formation after ciprofloxacin and gentamicin treatment. In parallel, using the quantitative PCR (qPCR) approach, we determined the expression level of the stress sigma factor SigB. The pre-exposure of bacteria to salt stress caused a 1–2.5 order of magnitude increase in persister formation in the bacterial population after antibiotic exposure, depending on the type and concentration of the antibiotic used. In contrast, oxidative stress only minimally influenced the formation of persisters, without correlation to the antibiotic type and concentration. We have demonstrated that both stress and antibiotic exposure increase the expression of sigB in bacterial populations from very early on. And that the expression level of sigB differs with the type of antibiotic and stress, but no correlation was observed between persister formation and sigB expression. The method used could be helpful in testing the ability that strains can have, to form persisters.

Background One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase), which was recently assigned in vitro as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon. Methods and Results We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress. Conclusion We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.