The contribution of many neuronal kinases to the adaptation of nerve cells to ischemic damage and their effect on functional neural network activity has not yet been studied. The aim of this work is to study the role of the four kinases belonging to different metabolic cascades  in the adaptive potential of the neuron-glial network for modeling the key factors of ischemic damage. We carried out a comprehensive study on the effects of kinases blockade on the viability and network functional calcium activity of nerve cells under ischemic factor modeling in vitro. Ischemic factor modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). The most significant neuroprotective effect was shown in the blockade of FLT4 kinase in the simulation of hypoxia. The studies performed revealed the role of FLT4 in the development of functional dysfunction in cerebrovascular accidents and created new opportunities for the study of this enzyme and its blockers in the formation of new therapeutic strategies. Ischemic stroke is one of the most common diseases in the world, characterized by high disability and death rates of patients and is especially significant in terms of life expectancy. In the coming decades, an increase in the average age of the population and additional risk factors such as hypertension, being overweight, and a sedentary lifestyle will contribute to the increase of the prevalence of pathologies related to the acute impairment of blood supply to the brain.Ischemic damage triggers signaling pathways that lead to nerve cell death and destruction of the brain tissue. Oxygen and energy starvation activate pathogenetic reactions in various types of cells of the central nervous system (CNS), including neurons, astrocytes, microglia, and vascular endothelial cells. The most important of these reactions include excitotoxicity, mediated by the excessive release of excitatory neurotransmitters , as wellVarious kinases are components of almost all these molecular cascades; they can be considered potential molecular targets for the development of neuroprotection methods in ischemic stroke. For example, the neuroprotective role of phosphatidylinositol-3-kinase (PI3K)/Akt cascade has been widely studied ,11,12. PIn our recent study , we perfUnlike other tissues and organs, brain functionality depends not on individual cells, but rather on their functional ensembles, which make up neuron-glial networks. Neural networks are considered as the minimum functional unit that ensures the implementation of higher cognitive functions, including memory, consciousness, emotional reactions, and the ability to respond to environmental changes. To understand the pathogenesis and to develop methods of therapy for diseases of the central nervous system, it is essential to study the functional network activity of nerve cells.The functional network activity is spontaneous spatiotemporal patterns of bioelectric signaling between nerve cells in neuronal assemblies. These rhythmic modulations in neuronal activity have been linked to various important brain functions, including attention, sensory or computational processing, decision-making and consciousness . CalciumThe functions of the studied kinases in the nervous system are so far almost undescribed. However, in our recent work, it was shown that their blockade has a positive effect on the viability of nerve cells in a deficiency of energy substrates, which indicates the important role of the selected kinases in the regulation of adaptation of nerve cells to stress-factors . We carrWe have shown that the effect of ischemic factors on cells of the cerebral cortex does not significantly affect the level of mRNA expression of the studied kinases. When modeling hypoxia, the expression of the considered kinases did not change; when modeling glucose deprivation (GD), an increase in the expression of SRC kinase mRNA by 1.44 ± 0.36 times was noted.It is interesting to note that the level of expression of all studied kinases in hippocampal cell cultures significantly changed when both ischemic factors were simulated . It was Thus, for further research, we chose hippocampal cell cultures as an object since they demonstrated a strong response to ischemic exposure by the kinome.We evaluated the neuroprotective effect of the blockade of the studied kinases in the modeling of glucose deprivation and acute normobaric hypoxia in vitro in primary hippocampal cultures. It is important to note that, under normal conditions, the blockade of the selected kinases did not affect the viability of nerve cells, except for the blockade of the FLT4 kinase. The use of the inhibitor SAR-131675 under normal conditions led to a decrease in the viability of nerve cells .Modeling of both damaging factors led to a strong decrease in viability on the 7th day after exposure compared to the “Intact” group . To veriInhibitors of SRC, Ikkb, and eEF2K kinases significantly increased the resistance of cells to the action of both key ischemic factors . ACHP, aWe assessed the effect of blockade of the studied kinases on the calcium activity of primary hippocampal cultures under physiological conditions. It is important to note that, although the blockade of Ikkb and eEF2K kinases did not affect the viability of nerve cells, it modulated their calcium activity. The eEF2K blockade led to significant inhibition of calcium activity and a decrease in the number of cells in which calcium events were recorded from by 3.8 times .The inhibition of Ikkb and FLT4 kinases led to a decrease in the duration of calcium oscillations. The SRC kinase was of particular interest because its blockade did not significantly affect the main characteristics of calcium activity under normal conditions. Representative examples of recordings of calcium activity together with kinase blockade are shown in Next, we examined the calcium activity of primary hippocampal cultures when simulating ischemic factors together with the inhibition of kinases. It has been shown that both glucose deprivation and hypoxia led to inhibition of calcium activity. Hypoxia has a stronger negative impact on nerve cells. The number of cells with the recorded calcium events decreased by 23% when modeling GD, and by 42% when modeling hypoxia . FurtherThe blockade of eEF2K kinase aggravated the negative effect of damaging factors on calcium dynamics in cells of primary hippocampal cultures. The number of active cells in the “GD + eEF2K” and “Hypoxia + eEF2K groups was significantly lower than in the “GD” and “Hypoxia”. The blockade of Ikkb kinase led to the aggravation of the disorders caused by glucose deprivation and had no effect on hypoxic damage.The effects of SRC and FLT4 kinase blockers are of the greatest interest. With the blockade of SRC kinase, the number of cells exhibiting calcium activity in the presence of energy substrates deficiency did not differ from the intact group, however, under hypoxia, no protective effect was revealed for this parameter. The use of the SAR-131675 blocker, which inhibits the FLT4 kinase, maintained the number of cells in which calcium events were recorded under the influence of both hypoxia and glucose deprivation. However, in the “GD + FLT4” group, a decrease in the frequency and duration of calcium oscillations was noted. The number of active elements is fundamental for maintaining the functional activity of the neuron-glial network. Therefore, the effect of the FLT blocker can be considered protective.We have studied the effect of FLT4 kinase blockade in modeling stress factors on primary hippocampal cultures’ cellular composition. The results are presented in The key aspect in the characterization of the dynamic interactions of neuron-glial networks in our study is the neural network parameters of calcium activity, which characterize the degree of connectivity of network elements after exposure to ischemic factors . ApplicaThe impact of ischemic factors leads to a significant decrease in the connectivity of neural-glial networks and loss of functional connections B,C,E,F. When modeling glucose deprivation of the studied kinases, only the blockade of FLT4 makes it possible to partially maintain the degree of correlation of the calcium activity of cells. The level of correlation of all cells does not differ from the indices of the intact group and is 0.56  . The lev.Under the influence of hypoxia, the destruction of network interactions is expressed more significantly in terms of the correlation of the activity of functional network elements. The most significant neuroprotective effect was shown in the blockade of FLT4 kinase in the simulation of hypoxia. The degree of correlation between adjacent pairs of cells and all cells in the culture, the rate of calcium signal propagation, and the proportion of functionally significant connections from their maximum possible number in the “Hypoxia + FLT4” group did not differ from the parameters of intact cultures .Under hypoxic exposure, partial preservation of the network characteristics of calcium activity was observed in the “Hypoxia + Ikkb” group. The level of correlation of calcium signals was 0.33 , which is significantly higher than in the “Hypoxia” group . However, it should be noted that the correlation of activity between adjacent cells was significantly lower than in the intact group and did not differ from the “Hypoxia” group . Moreover, the percentage of correlated connections from the total number of possible connections did not differ from the indices of the intact group D.2+] are directly related to synaptic activity [Our work was focused on identifying new targets for correcting the consequences of the impairment of cerebral circulation. To this end, we focused on studying the molecular and cellular mechanisms of action of kinome representatives with previously undescribed neuroprotective functions. When modeling individual ischemic factors, the blockade of all the kinases considered in the work preserves the viability of nerve cells effectively. However, in some cases, the functional activity of neuron-glial networks is not maintained, for example, in the blockade of eEF2K kinase. It is the preservation of a functionally complete interaction between the components of neuron-glial networks that is the basis for effective neuroprotection and is necessary to maintain the cognitive and amnestic functions of the brain. To assess the neuroprotective effect of blockade of various molecular targets, it is not enough to assess the survival of nerve cells. A key aspect for understanding the state of neuron-glial networks is the assessment of functional network activity. Calcium imaging is one of the relevant methods for recording the spatiotemporal patterns of neural network activity. Traditional electrophysiological methods make it possible to record either the activity of single neurons (patch-clamp) or small neuronal ensembles (multielectrode arrays), the individual cellular elements of which can be included in various functional networks. Their total activity does not allow making unambiguous conclusions about the functional network architecture. Visualization of calcium dynamics in the cytoplasm of nerve cells is an extremely informative approach for assessing neural network metabolic activity since it allows visualizing the architecture and mapping the activity of neural networks with cellular resolution since changes in the content of cytoplasmic transient-free calcium [Caactivity .Among the kinase blockers studied, the most promising molecular targets for the development of methods for correcting and maintaining functional neural network activity are ACHP, which inhibits Ikkb kinases, and SAR-131675, which inhibits FLT4 kinase. On the other hand, the very fact of maintaining viability, without maintaining activity, could in the short term be considered as a favorable factor that allows cells to be preserved for possible stimulation of functional regeneration.The choice of kinases as potential targets for the activation of nerve cells’ adaptive resources was determined by the results of screening of the effect of selective and nonselective blockers of more than 50 kinases, the effect of which on the viability of nerve cells in the modeling of ischemic factors in vitro has not been previously studied . The fouThe studied kinases belong to various metabolic pathways. The IκB kinase enzyme complex is part of the upstreaming NF-κB signal transduction. The effects of the activation of this nuclear transcription factor are multidirectional, which may be associated with the neuroprotective effect of Ikkb kinase blockade . Our datThe SRC kinase was of great interest for us. We have shown that its inhibition makes it possible to maintain the viability of cells in primary cultures of the hippocampus when modeling the key factors of ischemia, significantly higher than it was in the control group. Kinases of the SRK family of nonreceptor kinases are involved in the regulation of many normal and pathological processes in the nervous system, including cell survival and proliferation, and angiogenesis. Although studies of the effect of SRC kinase on the resistance of the brain to ischemia have not been carried out, some works have shown the neuroprotective effect of inhibition of c-SRC kinase under the neurotoxic effects of kainic acid and oxidative stress . The neuThe data on the role of FLT4 kinase in ischemic injury are of the greatest interest. There is little information available about its functions in the nervous system. FLT4 kinase  acts as a cell surface receptor for VEGFC and VEGFD  and promotes proliferation, survival, and migration of endothelial cells and regulates angiogenesis. Signaling with activated FLT4 results in enhanced production of VEGFC and to a lesser extent VEGFA, thereby creating a positive feedback loop that amplifies FLT4 signaling. It mediates the activation of the MAPK1/ERK2, MAPK3/ERK1 signaling pathway, the MAPK8 and JUN signaling pathway, and the AKT1 signaling pathway, which are involved in maintaining cell viability. It is known that VEGF has neurotrophic and neuroprotective activity in both the peripheral and central nervous systems, exerting a direct effect on neurons, Schwann cells, astrocytes, neural stem cells, and microglia. The involvement of FLT4 in the cascades initiating the maintenance of cell survival, neurogenesis, and angiogenesis, suggests that modulating the activity of VEGF receptors can be effective in the therapy of several neurodegenerative diseases and ischemic stroke. However, there are, so far, few data on this in the scientific literature. When simulating focal ischemia in rats, it was shown that blockade of FLT4 (VEGFR3) reduces activation of the lymphatic endothelium, reduces the level of activation of proinflammatory macrophages, and reduces the volume of cerebral infarction . It has FLT4 blockade can likely reduce the level of inflammation and reactivity of glial cells, since VEGFR-3 could be involved in the development of the inflammatory response of astrocytes in ischemic strokes ,29. At pOur model made it possible to assess the neuroprotective effect of FLT4 blockade using the SAR-131675 inhibitor when modeling isolated ischemic factors. A significant result is the preservation of correlation characteristics and functional network architecture when using this blocker together with modeling ischemic injury. We were the first to describe the effect of FLT4 blockade on the functional calcium activity of nerve cells. Preservation of a functionally complete dynamic interaction between the components of neuron-glial networks allows us to consider FLT4 as a promising target for the development of methods of protection against cerebral ischemia and is of interest for future research.The studies performed revealed the role of FLT4 in the development of functional dysfunction in cerebrovascular accidents and exposed new opportunities for the study of this enzyme and its blockers in the formation of new therapeutic strategies.All experimental procedures were approved by the Bioethics Committee of Lobachevsky University and carried out in accordance with Act 708n (23 082010) of the Russian Federation National Ministry of Public Health, which states the rules of laboratory practice for the care and use of laboratory animals, and Council Directive 2010/63 EU of the European Parliament (22 September 2010) on the protection of animals used for scientific purposes. Pregnant C57BL/6 mice (day of gestation 18) were killed by cervical vertebra dislocation.2+- and Mg2+-free phosphate-buffered saline (PBS) with subsequent enzymatic digestion with 0.25% trypsin-ethylenediaminetetraacetic acid  for 20 min. After centrifugation (800 rpm for 3 min), the pellet of dissociated cells was seeded on coverslips at an approximate initial density of 7000–9000 cells/mm2. The primary neuronal cultures were grown in neurobasal medium , supplemented with 2% B27 , 0.5 mM L-glutamine , and 0.4% fetal bovine serum  under constant conditions of 35.5 °C, 5% CO2, and a humidified atmosphere in a Binder C150 incubator . A half-replenishment of the medium was performed once every three days [Neuronal cells were obtained from mice embryos and cultured on coverslips (18 × 18 mm) pretreated with polyethyleneimine solution (1 mg/mL)  according to the previously developed protocol described in . Isolatiree days .To verify the cellular content, primary hippocampal cultures were subjected to immunocytochemical staining on day 21 of culture development in vitro (DIV). The cultures were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by incubation with a solution of 0.2% Triton X-100/PBS for effective cell permeabilization. For immunofluorescence reactions, the cultures were then incubated for 2 h in the presence of a polyclonal mouse anti-GFAP  primary antibody  and polyclonal rabbit anti MAP2 (a marker of differentiated neurons) primary antibody . Next, the cultures were subjected to a 45-min incubation in the following secondary antibody mixture: goat anti-mouse Alexa 647  and chicken anti-Rabbit Alexa Fluor 488 . The stained material was observed using a Zeiss LSM 800 fluorescence confocal microscope .Modeling of glucose deprivation in vitro was performed on the 14th day of cultivation (DIV) of primary cultures by replacing the normal culture medium with a medium that did not contain glucose, lactate, and pyruvate  for one hour, followed by a reverse replacement of the medium with a standard one.Acute normobaric hypoxia was modeled on day 14 of culture development in vitro (DIV) by replacing the normoxic culture medium with a medium containing low oxygen for 10 min. The oxygen was displaced from the medium in a sealed chamber with an inert gas (argon). The oxygen concentration in the culture medium was decreased from 3.26 mL/L to 0.37 mL/L . After 1Total RNA was extracted from primary dissociated neuronal cultures of the mouse cerebral cortex and hippocampus using the ExtractRNA reagent ; reverse transcription was performed using the MMLV RT kit and the random primer mixture . RT-qPCR reactions were performed using the qPCRmix-HS SYBG Green kit  using an Applied Biosystems 7500 RT-PCR amplifier. The reaction was set up in four repetitions using the following cycle protocol: 50 °C for two minutes, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 62 °C for one minute, then 95 °C for 15 s, 60 °C for one minute, and 95 °C for 30 s. The primers used are shown in Oaz1 gene was used as a housekeeping gene to normalize the findings.Data processing was carried out using the ΔΔC method and a control sample, in which the level of the target gene was taken as a unit. The Application of kinase inhibitors was carried out on the 14th day of cultivation, 20 min before the modeling of the ischemia factor, and immediately during the modeling at a concentration of 1 μM. The chemical structure of the inhibitors is shown in The viability of primary hippocampal cultures was expressed as a ratio of the number of nuclei of dead cells stained with propidium iodide  to the total number of cells stained with bis-benzimide . Propidium iodide and bis-benzimide at concentrations of 5 μg/mL and 1 μg/mL, respectively, were added to the culture 30 min before registration. The stained cultures were observed using a ZEISS Observer A1 inverted fluorescence microscope  with a 20×/0.2 objective.2-incubator. The OGB1 fluorescence excitation wavelength was set at 488 nm with an argon laser, and the emission was recorded using a 500–530 nm filter. To evaluate the dynamics of changes in the intracellular calcium concentration, a time-series of confocal images was recorded. The registration rate used was two frames per second. The spontaneous calcium activity of primary hippocampal cultures was recorded on day 7 post-ischemic damage. The following main parameters were analyzed: duration of calcium events , frequency of calcium oscillations (average number of oscillations per min), and percentage of active cells  [To characterize the dynamics of calcium homeostasis in the hippocampal cells, functional calcium imaging was performed with a fluorescent calcium-sensitive dye Oregon Green 488 BAPTA-1 AM (OGB-1)  on an LSM 510 confocal laser scanning microscope  with a W Plan-Apochromat 20 ×/1.0 objective. The calcium sensor, 0.4 μM OGB-1 , was dissolved in DMSO  with 4% pluronic F-127 , added to the examined cultures and incubated for 30 min in a COmber, %) ,21.Calcium network analysis was performed using a previously developed algorithm . The appIn the paper by , severalThe propagation speed of calcium waves in the cell culture is associated with spatiotemporal delays. To estimate propagation speed, the average propagation speed of delays was introduced based on two parameters: the distance between corresponding somas and cross-correlation time delay between fluctuations in the calcium level of cell pairs. The average ratio of these quantities shows how far the wave peaks travel per unit of time.Quantitative results are presented as a mean ± standard mean error (SEM) for normal distributions, or as a median value and second and third interquartile range. To compare the two unrelated groups, the Mann-Whitney U test and ANOVA was used. The Tukey post hoc test was used as a post hoc test following ANOVA. At least three independent biological replicates were used for all experiments. The difference between the groups was considered significant if the p-value was less than 0.05.The studies performed revealed the role of FLT4 in the development of functional dysfunction in cerebrovascular accidents and exposed new opportunities for the study of this enzyme and its blockers in the formation of new therapeutic strategies.


In Caucasians, regular consumption of cocoa induces a drop in arterial blood pressure via an increase in nitric oxide (NO) production. However, black individuals have a different NO biodisponibility compared to Caucasians. The aim of this study was to determine, in black Africans, the physiological variations in arterial blood pressure among cocoa consumers.In total, 49 male black African volunteers, aged between 18 and 30 years old, were randomized into two groups; those consuming 10 g of cocoa powder per day  for 3 weeks (consumer group), and those not consuming cocoa (control group). Systolic (SBP) and diastolic blood pressures (DBP), and heart rate (HR) were measured in the morning on an empty stomach (fasting), on day (D) 1 (without cocoa), D8, D15, and D22. Data were collected by groups and by subgroups established according to the level of SBP, DBP, or HR on D1. The means and variations of the means (between D1 and the subsequent days) of the recorded parameters were calculated and compared between groups and between subgroups.p = 0.02). Between the control and consumer subgroups according to SBP, no statistical difference in the means or variations in SBP was noted. On D8 and D22, the variations in the SBP of consumers with SBP ≥ 110 mmHg on D1 were significantly different from those of other consumers . In the subgroups with a DBP < 75 mmHg on D1, the mean DBP of the consumers was significantly lower than that of the controls on D8 .On D8, the variations in SBP in the consumer group were significantly different from the control group (−3.72 ± 6.01 versus 0.57 ± 6.66 mmHg; In young black African men living in Côte d’Ivoire, regular consumption of cocoa resulted in a decrease in SBP and DBP. The decrease in SBP appeared to be greater the higher the baseline SBP was. High blood pressure is a public health problem around the world . AccordiFor many years, to prevent an abnormal rise in blood pressure, the learned cardiology societies have recommended some hygieno-dietetic measures such as the regular consumption of fruits and vegetables as a first line of treatment . SeveralRegarding the study population, these previous studies were mainly carried out in mixed gender populations , 2017, eThis study was approved by the Ethics Committee of the University Teaching Hospital of Yopougon  and followed the guidelines of the Declaration of Helsinki. All patients were informed on the purpose of the study protocol and gave their written consent.The population consisted of volunteer students from both the Félix Houphouët-Boigny and Nangui Abrogoua universities .2. Non-inclusion criteria were smoking, regular alcohol consumption, diabetes, dyslipidemia, a cardiovascular , respiratory, hematological or infectious symptomatology or disease, unexplained fatigue, a physical activity score greater than 35 points (corresponding to very active subjects) according to Ricci and Gagnon scoring system, recent or current intake of a drugs that can modify the blood pressure or heart rate (HR), and regular consumption of all cocoa-based products or products rich in flavonoids .Those included in the study were of black African origin, male, aged between 18 and 30 years, and had a body mass index (BMI) between 18.5 and 29.9 kg/m‘These inclusion and non-inclusion criteria were assessed using a survey sheet organized into two parts: a questionnaire and a record of anthropometric measurements. The questionnaire portion was performed by the participant in the presence of an investigator. The height of participants was measured, without shoes, using a vertical measuring rod graduated in centimeters . Participants stood, knees straight and looking ahead, with their arms alongside their body, with their feet together and their heels against the measuring rod. The weight of participants was measured with an electronic scale . For measuring, participants wore light clothes, without a belt, and with empty pockets. The weight was displayed in kg with a margin of error of 0.1 kg.p = 0.47), and a mean BMI of 21.5 ± 2 kg/m2 and 21.6 ± 2.1 kg/m2, respectively (p = 0.94).Among 411 volunteer students, 65 were selected according to our selection criteria  and wereAn electronic scale  was used for the weight measurement in kilograms. Electronic sphygmomanometers  were used to measure SBP, DBP, and HR.Three brands of 100% cocoa powder available in supermarkets in Abidjan (Côte d’Ivoire) were selected: TAFISSA, NESTLE, and RYAN’S. The analysis of polyphenols and flavonoids in these powders were carried out by the Laboratory of Industrial Processes, Synthesis, Environment and New Energies (LAPISEN) of the National Polytechnic Institute Houphouët-Boigny of Yamoussoukro (Côte d’Ivoire). The method of A follow-up form filled out at each meeting made it possible for participants to notify us about the presence of acute stress, symptoms, or use of medications during the 7 days preceding the current visit. A food sheet given to each subject at the beginning of each week was used for the daily recording of the types of food consumed and for recording any medication taken. On this sheet, the instructions to be followed were provided, including foods to avoid and directing participants to consume cocoa in the morning on an empty stomach thirty minutes before breakfast.This was an experimental, prospective, randomized, single-blind study (the investigators at the measuring station were blinded to the grouping of the participant). The protocol was performed in two steps.The subjects included in the study were informed of the foods to avoid for the 7 days before the date set for their entry into the 2nd stage. This included food products containing a significant amount of flavanols .The 2nd stage lasted 3 weeks, during which participants were assessed four times: On day (D)1 , on D8 [8th day corresponding with the end of the 1st week (W1)], on D15 [15th day corresponding with the end of the 2nd week (W2)], and D22 [22nd day corresponding with the end of the 3rd week (W3)].The follow-up protocol at this second stage was the same on each of the four visits. Participants were directed to fast for ten hours before arriving for assessment between 7 am and 8 am. An individual follow-up sheet looked for specific dates of major stress, symptoms, or drug intake. Body weight was measured as described previously and participants were then placed in groups of 3, in a seated position (back and arms supported with the feet on the ground). Each participant faced toward one of the corners of the quiet, semi-lit and air-conditioned room at 22°C. Blood pressure cuffs were placed on the non-dominant arm of each subject. After 5 min of rest, the arterial blood pressure was measured according to the recommendations of the ESH and ESC . SBP andAfter leaving the blood pressure measurement room, the participants were received, in turn, in another room by a single investigator appointed for the duration of the study. During the first visit (D1), this investigator communicated the study group (consumer or control) to each participant while specifying that this information should not be communicated to a third person (neither participant nor investigator). The investigator then diluted two sticks of 5 g of cocoa powder in water that the participant consumed in his presence. The investigator then gave the participant the 12 other sticks of cocoa powder for the remaining 6 days of the week with a food sheet to be completed by the participant. The control participants were also received individually like the consumer participants. They received neither cocoa nor a placebo and were given the same confidentiality instructions. Throughout the study, no participant had to change his usual level of physical activity or diet.For each participant at each of the four visits, the individual averages of SBP, DBP, and HR were obtained from the three measurements taken at the 5th, 7th, and 10th minute at rest . In eachThe data obtained were imported, cleaned, categorized, and analyzed with R4.0.2 software. Since the distribution of groups and subgroups does not always follow a normal distribution, comparative analyzes of the means of groups and subgroups were made using the Wilcoxon–Mann–Whitney test for comparisons of means between the two groups or two subgroups, and Friedman’s test was applied to compare the weekly evolution of parameters over the 3 weeks in groups and subgroups. The tests evaluating the changes in parametric values over time were one-sided. Multiple linear regression was used to estimate the effect of cocoa consumption on changes in SBP, DBP, and HR. The analyses were carried out with a type I error of 0.05.t-test formula for paired populations, to detect a change of at least 1 mmHg in arterial pressure in cocoa consumers with a standard deviation of 1.5 mmHg, taking into account a previous study in a similar population who consumed 5 g of TAFISSA cocoa powder for 3 weeks . At the end of the 2nd (W2) and 3rd (W3) weeks, this decrease was 2.63 and 2.2 mmHg, respectively, without any statistical significance . When comparing the subgroups, no statistical difference in the means or the mean variations was observed between the control and consumer subgroups with an SBP at D1 < 110 mmHg  and 3rd (W3) weeks of follow-up, the negative variations in SBP (reflecting a decrease in SBP) in the consumer subgroup with an SBP at D1 ≥ 110 mmHg were significantly greater than the variations of the consumer subgroup with an SBP at D1 ≤ 110 mmHg . HoweverDuring the follow-up period, there were no significant changes in the mean or the mean variations of DBP in the control and consumer groups . The meap = 0.21, 0.24, and 0.74).With the multiple linear regression analysis, the increase in DBP of 1 mmHg at D1 resulted in a decrease in the DBP of 1.68, 1.74, and 0.42 mmHg, respectively at the end of the 1st week (W1), the 2nd week (W2), and the 3rd week (W3) of follow-up in the consumers in comparison with control subjects, with no significant difference  compared to those of control subjects not receiving any form of cocoa. This work demonstrated significantly greater weekly reductions in SBP and DBP in consumers than in controls. In the consumer group, the reductions in SBP were significantly greater among those with the highest SBP at entry into the study. This observation was not found in the control group.The comparison of the variations in SBP between the cocoa consumers and the controls found a greater decrease in SBP in consumers during the 3 weeks, with a significant difference only at the end of the 1st week. Likewise, multiple linear regression analysis showed that the increase in SBP of 1 mmHg at D1 was accompanied by a greater decrease in SBP in the consumer group compared to the control group at each time point over the 3 weeks, with statistical significance only at the end of the 1st week. In an earlier study  conducteIn our population of normotensive black African subjects, a weekly reduction in the consumer SBP (between −4.16 and −3 mmHg) was included within the range of SBP variations in the studies conducted in black normotensive consumers  and CaucTaking into account the level of SBP at D1 (<110 mmHg or ≥110 mmHg), there was no statistical difference between the weekly variations in SBP of the consumers  and those of the controls, with both groups having the same SBP at D1. These results were identical to those of In our group of cocoa consumers, the decreases in the SBP of those with an SBP ≥ 110 mmHg at D1 were statistically greater than those of consumers at D1 with a lower SBP (<110 mmHg) at W1 and W3 of follow-up. These statistical differences were not noted in the control group. These results, depending on the level of SBP of the consumers, were found in other studies carried out in black  or CaucaThe comparison of the DBP between the consumers and controls revealed a statistical difference in the means of the DBP from D1  but without statistical difference in the mean variations over the 3 weeks of follow-up. The consumers with a DBP < 75 mmHg at D1 exhibited a significantly lower DBP than the controls with the same baseline level of DBP at D8.p = 0.98). On the other hand, a study by Some non-significant variations in the DBP between consumers and controls were also found by In the consumer group, the weekly decreases in DBP obtained during the 3 weeks of follow-up (between −2.74 and −1.49 mmHg) were comparable to those found in the Caucasian populations . In factp = 0.007). The differences between these studies could be explained by the quantity of cocoa received. As cocoa is rich in minerals, including potassium, a study focusing on the relationship between the amount of potassium in cocoa and HR variations would help form a better understanding of the variations in HR when consuming cocoa.The present study did not find a statistical difference between the reductions in HR of the control subjects and those of cocoa consumers. The same was observed amongst the subgroups of the controls and the cocoa consumers according to the HR at baseline. These results are in agreement with those of Among consumers of the present study, no statistical difference was noted according to the HR at D1. These results were not in agreement with those of Taking into account the genetic and environmental aspects of the populations, our results cannot be extrapolated to black Africans in general. Studies comparing black African populations will be needed in the future.The selection of males only for this study was due to the fact that hormonal fluctuations in women during the menstrual cycle might influence the arterial blood pressure . TherefoChoosing participants aged 18–30 was motivated by the data in the literature, according to which the drop in the systolic and the diastolic arterial blood pressures induced by cocoa consumption would be greater in subjects under 50 years of age .The single-blind study design could constitute a bias because it tends to induce a greater reduction in arterial blood pressures compared to double-blind studies, although not statistically significant according to the data in the literature .We did not perform blood tests to measure the metabolites of NO nor the phenols of the cocoa or their metabolites responsible for stimulating the NO production and lowering the blood pressure during consumption of cocoa powder. The results of this work will allow us to program the dosages of NO in another study.In our population of young male, normotensive, black African subjects living in Côte d’Ivoire (West Africa), the daily consumption of 10 g of 100% cocoa powder  demonstrated some significantly greater weekly reductions in SBP and DBP in the consumer group compared to the control group. Additionally, the drop in SBP in the consumer group increased with the baseline SBP unlike in the control group. In view of these results, a study focusing on the black male and female population, investigating regular consumption of cocoa and performing blood analysis to assess NO metabolites, converting enzyme, endothelin-1, and cocoa flavanols would make it possible to establish the mechanisms behind the lowering of arterial blood pressure in this black African population.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.This study was approved by the Ethics Committee of the University Teaching Hospital of Yopougon  and followed the guidelines of the Declaration of Helsinki. All patients were informed on the purpose of the study protocol and gave their written consent.ES-B and SO contributed to the conception of the study, data analysis and interpretation, drafting of the manuscript, and approval of the final version of the manuscript, and agreed to be accountable for all aspects of the work. HA involved in approval of the final version of the manuscript and agreed to be accountable for all aspects of the work. TY, FG, and KY involved in data acquisition and agreed to be accountable for all aspects of the work. ME involved in drafting of the manuscript and agreed to be accountable for all aspects of the work. CD involved in revising the manuscript critically and approval of the final version of the manuscript, and agreed to be accountable for all aspects of the work. PB involved in revising the manuscript critically, drafting of the manuscript, and approval of the final version of the manuscript, and agreed to be accountable for all aspects of the work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study evaluates changes in power and strength after implementing two different models of 9-week strength training in elite women’s football players. A group of 13 players  underwent either a complex (the intermittent load type) or combined  model of training. The training load was tailored to each athlete. Results showed that the complex model of training improved power  and height of vertical jump , weight of 1 Repeat Maximum (1RM) which was , power and speed in the acceleration phase of barbell half squats (BHS) at weights from 20 to 60 kg, and the number of repetitions in BHS . The combined model of training improved the time of shuttle run , weight of 1RM in BHS  and BP , power in the acceleration phase of BHS at weights from 50 to 60 kg, the number of repetitions in BP , BHS , barbell bench pulls  and sit-ups . These findings indicate that the complex model of training improves explosive abilities, whereas the combined model is effective for developing strength at weights close to players’ 1RM and for repeatedly overcoming resistance. Therefore, coaches should choose the training model based on the needs of individual players. Developing and loading strength abilities in football come from knowledge of game performance and its particular components and limitation factors . Some ofPeriodization of strength training mostly depends on the period of the season. Football players should achieve their peak as part of preparatory training and then maintain it for rest periods of up to 35 weeks . Many auBuzek  states tThis study evaluates the effect of complex and combined models of traditional strength training on different strength abilities: maximal, endurance and explosive strength. The first model has been set up based on football actions in the game, basic principles of strength training and energy coverage of these actions. Players in the match need to use different kinds of strength abilities and muscle contractions and our aim was to train them based on the requirements of the game. Football actions during the game have a certain duration and intensity, and players overcome certain resistances. We wanted to incorporate these factors and imitate similar conditions in the strength training and determine if this kind of training with light weights and high volume would be effective. The second model was selected based on its effectiveness for developing several strength abilities. Comparison of the complex and combined models of the training could add valuable information about building and periodization of strength training in collective games. We hypothesized that the complex model of training improves explosiveness and endurance strength significantly more than the combined model, whereas maximal strength increases more after applying the combined model of training.Thirteen professional soccer players  volunteered to participate in the study. None of the participants had any musculoskeletal disorders, and all of them have played in the first national football league of Slovakia. The length of playing activity was 6.3 ± 1.9 years. Four players were in the junior national U19 team and six of them were in the senior national team. During the research period, it was recorded that all 13 players fully completed the study. At the beginning of the study 21 players were involved, but because of injury, sickness, and transfers to other clubs some of them did not finish both models of strength training. All 13 participants first completed the complex model and after one year in the same period completed the combined model of training. The procedures followed were in accordance with the ethical standards on human experimentation stated in compliance with the 1964 Helsinki Declaration and its later amendments. The project was approved by the ethics committee of the Faculty of Physical Education and Sports, Comenius University in Bratislava (1/0824/17 and 1/0089/20).Application of the complex and the combined model for the development of strength abilities took place during the transition period I (five weeks) and in the beginning of preparatory period II (four weeks). Both models were implemented for nine weeks, with 27 training units (TU) strength training, 13 TU football training and 2 TU aerobics. Duration of each TU was from 60 to 90 minutes . FootbalBoth strength models have the same principles: circuit organization form; three sets; 3 min rests between sets; and eight exercises which were focused on main muscle groups. Four exercises were always the same: sit-ups, barbell half squat (BHS), bench press (BP) and barbell bench pull (BBP). The next four exercises were performed with own weight and we used many of their variations based on the strength level of the players . These four exercises were performed on time (30s) or number of repetition (20×). All players had set up their individual weights on these exercises BHS, BP, BBP according to the input measurements of maximal strength 1RM at the beginning of the training program, and they increased their individual weight of resistance about 4%–6% every two weeks. The frequency of TU in both models was three times per week .The first experimental factor was the complex model of strength training, which is based on changing the contractions and varying the intensity during workout. Every football player started working out with a weight of 30% of their one repeat maximum (1RM). The weight was raised every two weeks by 4%–6% from 1RM until the end of the monitored period, so the players worked out the last week with 42%–45% of their 1RM. The speed of contractions was implemented in this order: the first 6 s were on velocity strength at maximum intensity; followed by isometric contractions in 4-second intervals in the middle position, endurance strength was applied for 40 s (without stopping movement and without emphasis on intensity of movements). The 4-second isometric contraction was then repeated and finally there were 16 s focusing on explosive strength (in their subjective maximum intensity during the concentric phase) in any number of repetitions (minimum one). Duration of one exercise was then 1 min 10 s. After the players finished the first exercise, they had 20 s to go to another station. In this circuit form they changed eight exercises in three sets with a rest interval of 3 min between the sets. This model simulated intermittent football actions in the football game (sprint–tackle–jogging–tackle–jump–rest). The second experimental factor was the combined model of strength training. This model was applied in the following season in the same period. In this stimulus was included two methods for developing strength abilities in one training unit (TU). The circuit form was used as in the previous year with the same eight exercises. In the first set, the maximal strength method was used with 3–6 repetitions, 80% from 1RM (concentric), with an interval of rest period between exercises which was under 20 s. In the second and third sets the dynamic method was used with 15–30 repetitions, with 30% of 1RM (concentric). The number of repetitions depended on the players’ subjective feelings. If they felt that the speed of the concentric phase was decreasing because of tiredness of muscles, then they were instructed to stop and move to the next exercise. In both these methods, players tried to hold maximal intensity in the concentric phase of movements. The load was raised every two weeks in the same way as in the complex model about 4%–6% from 1RM (concentric) from the input maximal strength test. Players worked out three times per week with a progression up to 92%–95% from 1RM in the first set and up to 42%–45% in the second and third set. This model was chosen based on its high effectiveness in a previous study .Standing long jumpShuttle run (10 × 5 m)Vertical jumps (Jumper)Selected motor tests: Players went through general warm up (10’) and then performed one standard standing long jump (SLJ) to determine the explosive power of their lower limbs. After 5 mins of break, this was followed by one set of 10 s jumping, continued on the FiTRO Jumper device . The procedure was jumping as high as possible (without using arms and bending knees) and staying on the mat as short a time as possible. The three highest jumps were selected and their mean results calculated ,29. The Barbell bench pulls (2 × 20 s) 16 kgSit-ups (2 × 20 s)Barbell half squats (2 × 20 s) 20 kgBench presses (2 × 20 s) 16 kgInterval control exercise:After the shuttle-run test, players executed interval control exercises where an effort was made to achieve as many repetitions (n) as possible during 2 × 20 s periods with a passive rest of 20 s. The players always started from the low position, except for in the barbell half squat, and also rested in the same position between two intervals of loads. It was necessary to execute these exercises with a full range of motion. The weight of the barbell on the pretest and post-test was the same. These interval control exercises measure the strength and endurance capacity of muscles.Tests for determining maximal performance and maximal strength:The players went through a general warm up (5’), after which followed a stepwise diagnostic series for detecting their one repeat maximum (1RM) with the following exercises: barbell half squats (BHS), bench press (BP) and barbell bench pull (BBP). The players did this series with an effort to achieve a maximal speed in the concentric phase with different weights from 20 kg up. After every successful attempt an increase in weight of about 5 kg followed. All strength ability parameters were registered until the 1RM was found .For gaining the strength ability factors a monitoring machine, FITROdyne Premium  was used. This device can record the vertical velocity and length of motion. This pc-based system, FiTROdyne, can capture biomechanical parameters during workout . The device contains a sensor unit with a precise encoder and a reel. While dragging the tether (which is attached by means of a hook to the bar axis) the reel rotates and computes speed. Information from a sensor is transferred to the PC by a USB cable. The process works on Newton’s second law of motion and Newton’s law of gravitation constant. The force is calculated for moving a bar in a vertical direction as the sum of gravitational force and acceleration force. Acceleration of vertical movements is gained by derivation of vertical velocity. Power is measured as a result of force and velocity and the actual position by integration of speed. Extensive computer software permitted the collection, calculation, and online display of the elementary biomechanical parameters involved in the workout. Previous studies have acknowledged that measurements of peak and mean power during resistance exercises using the FiTRO Dyne Premium system provide reliable data ,34.≤ 0.05 and statistically significant differences were marked with a symbol (*). In order to interpret the practical significance of the research results, the effect size (ES) was reported. Cohen [Data is presented as means and standard deviations (SD). The Shapiro–Wilk Test for normality was performed on all variables. Data showed a normal distribution. A paired Student’s T Test was employed to find the significant differences from pre to post- tests in complex model 1 and combined model 2. For determination of changes between the two models of training, two-way repeated measures ANOVA and a Bonferroni post-hoc test were used. The level of significance was set at p d. Cohen  defined The combined model players significantly decreased the time of the shuttle run  but did not significantly change the length of the standing long jump. The complex model of training failed to improve the standing long jump length and the time in the shuttle run test .After using the complex model of training, the power (P) and height of jump (h) increased significantly. On the other hand, their values significantly decreased after the combined model of training.The best values of power in the concentric phase of take-off and height of the jump were achieved during the pretest measurement of the second model, while the lowest values were observed during the pretest measurement of the first model. Power increased by 10 W/kg  and jump height about 5.3 cm  at the post-test of model 1. However, the power decreased about 7.8 W/kg  and jump height about 2.9 cm  in the post-test model 2 .The weight during the half squat with a barbell on the top of the back increased after both the combined models of training by 9.6 kg , and the complex model of training by 5.8 kg . The lifting weight also increased during the bench press after the second model of training by 4 kg , whereas no significant changes were found in the weight of 1RM BBP .The mean speed and power in the concentric phase of BHS increased  after the complex model of training . Their vPeak values of power were achieved at 45 kg prior to the training and increased to 55 kg after the combined model of training. The mean power and speed in the acceleration phase of BHS significantly increased at weights of 50 kg , 55 kg  and 60 kg .Maximal values of power in a diagnostic series of BHS improved from 313W to 345W  after the complex model of training. Furthermore, the mean work increased from 229 J to 283J . Additionally, there was an increase in the weights, from 41 to 49 kg , and mean force, from 400 N to 483 N . Players reached their maximum values of power at 41 kg, which corresponded to 68.33% of their 1RM concentric contraction, prior to the training and at 49 kg, which corresponded to 74.24% their 1RM concentric contraction, after the complex model of training. The maximum power improved about 5.91% to higher values of 1RM. After the combined model of training, the maximum values of power increased from 322 to 348 W . This change, as well as the range and acceleration of movement, was not significant. However, force improved from 411 to 475N , weight from 42 to 48 kg  and work from 234 to 269 J . In contrast, the speed of movement decreased from 79 to 74 cm/s . The maximum power was achieved at a weight of 42kg which corresponded to 71.2% 1RM concentric contraction, prior to the training, and increased to 48kg, which corresponded to 69.6% of 1RM, after the second model of training. The maximum power decreased about 1.63% to lower values from 1RM .After applying the combined model, the players improved in barbell bench pulls about five repetitions , sit-ups about three repetitions , barbell half squats about three repetitions  and bench press about five repetitions . After using the complex model of training, players improved just in barbell half squats about three repetitions ; no significant changes were observed in the amount of repetitions in bench press, barbell bench pull or sit-ups .The results of this study supported the hypothesis that both models would significantly improve performance in selected motor tests. While the complex model of training increased vertical jump height, the combined model of training decreased the time in the shuttle run. In the maximum concentric contraction, significant improvements were noticed after both models of training in the BHS, and combined model also improved the BP exercise. Both models were effective in improving different factors in the maximal power in BHS. Players improved significantly in power, weight, force and work after the first model as well as in weight, force and work after the second model of training. Players also achieved significant changes in power and speed during diagnostic series (20–60kg) on lower weights after the complex model of training in contrast to the combined model when they achieved significant results on heavy weights. The results from the post-tests of interval control exercises do not fully support the hypothesis that the complex model of training significantly improves more strength endurance abilities. In the combined model training, the players significantly improved in all exercises. The complex model positively influenced only exercise BHS.It should be noted that, in football, the gains in muscular strength should not compromise velocity of movement. The aim of strength training is to enhance muscle strength so that acceleration and speed in football—specific movements, such as sprinting, turning and changing direction—may be improved ,37.≤ 0.05). This could have happened because of a different methodology of evaluation during the vertical jump test or inefficient activation of the stretch shortening cycle during this period [The results from 1RM of exercise BHS do not fully correspond with the results of other authors ,32,38,39s period . ResultsHarris  applied Better results in maximal strength are commonly linked with improvements in relative strength and in power-related abilities ,43,44. TThe main limiting factors of this study are that both interventions were done on one small group of players (without selections and randomizations and a control group). The effectiveness of the second model could also have been influenced by experiences from the previous year, and because three players have not had any experience with working out in the gym before the first model was applied. For more precise diagnostic results, it is recommended doing the control exercise (2 × 20s) with weights derived from a certain percentage of the maximum possible overcoming resistance 1RM. Our results confirm positive effects of both models of training. However, further research may be focused on adjusting entry training loads about 10%–15% from 1RM in both models or change time intervals in the complex model of training.These findings demonstrate the positive effect of both complex and combined models of training on the strength abilities of football players. However, each model of training has a different impact on the development of muscle power and strength. The new approach in the complex model of training was effective in improving explosiveness and producing power during exercises and could be used for improvement of specific strength abilities in football. The combined model was mostly effective for developing strength in exercises at resistance close to 1RM, which could be important for players as a precondition for future achievement of a high level of explosiveness. Each model of training should be chosen accordingly, based on the needs of a group or of individual athletes. For players who started in the gym or in the first phase of preparation, it would be better starting with the complex method of training to maintain good habits during their workouts and also because this type of training, based on our results, was more specific to football players. Furthermore, it can prepare individuals for the second stage of booster blocks, and, subsequently, use (either in one preparation period or in the following year) of the combined model of training, which will ensure the necessary development of maximum power parameters and thus create the necessary basis to possibly transfer to specific activities.For players who have a well-mastered technique of exercises but without the necessary strength base, it is recommended to start with the combination model of training. In this example, it would be used to develop a sufficiently fundamental strength level and then finish with the complex model of training. Based on the order of the preparatory period, it would move from general to specific exercises.For the third group of players, who have a sufficiently developed strength base and correct technique for executing individual exercises, it is recommend to work mainly on developing limiting factors during a strength preparation period and, thus, use the complex model of training.


The size (diameter) of the nanoparticles , because it can be effective in completely destroying tumors while maintaining low toxicity or even without toxicity on the healthy tissues. Superparamagnetic hyperthermia uses the natural thermal effect in the destruction of cancer cells, obtained as a result of the phenomenon of superparamagnetic relaxation of the magnetic nanoparticles (SPMNPs) introduced into the tumor; SPMNPs can heat the cancer cells to 42–43 °C under the action of an external alternating magnetic field with frequency in the range of hundreds of kHz. However, the effectiveness of this alternative method depends very much on finding the optimal conditions in which this method must be applied during the treatment of cancer. In addition to the type of magnetic nanoparticles and the biocompatibility with the biological tissue or nanoparticles biofunctionalization that must be appropriate for the intended purpose a key parameter is the size of the nanoparticles. Also, establishing the appropriate parameters for the external alternating magnetic field (AMF), respectively the amplitude and frequency of the magnetic field are very important in the efficiency and effectiveness of the magnetic hyperthermia method. This paper presents a 3D computational study on specific loss power ( At present, the most frequently used techniques in cancer therapy are the well-known chemo- and radiotherapy. However, these techniques have a high level of toxicity on the body and in the case of more advanced tumors may even prove ineffective. Therefore, given the high toxicity of current techniques used for treating cancer and the increasing incidence of cancer cases affecting the population , it is aMagnetic hyperthermia (MHT) using magnetic nanoparticles for cancer therapy is one of the alternative methods to classical chemo- and radiotherapy techniques, which looks to be the most promising in the field due to its non-invasive and low-toxicity characteristics when treating malignant tumors ,27,28,29+2Y2+3O4−2 where X is generally a bivalent metal ion  and Y is trivalent Fe O4−2, where the iron ions in the right parenthesis are found in the octahedral lattice and the Fe+3 ions outside the parentheses are found in the tetrahedral lattice, is the most suitable for supraparamagnetic hyperthermia [The most suitable nanoparticles for this type of therapy are the ferrimagnetic ones, spinel type, Xalent Fe ,35. Howealent Fe ,43,44,45rthermia ,53,54,55rthermia . Also, mrthermia , the nanrthermia ,58,59,60rthermia  make it rthermia ,62, or b3O4 nanoparticles, which is a critical parameter. In addition to this parameter, the shape and distribution of nanoparticles are important, as well as the dipole magnetic interactions when they exist, as well as the intrinsic magnetic observables characteristic of the magnetic material. Also, the parameters of the external magnetic field (AMF) are very important, respectively the amplitude , which led to poor results in the application of magnetic hyperthermia. In addition, the lack of a systematic study in the field for optimal conditions in which to obtain the maximum specific loss power and the efficient heating of the nanoparticles, this paper comes to complete that lack and to focus on a systematic study in the filed using a powerful 3D tool. Establishing optimal conditions in SPMHT for nanoparticle size and AMF parameters are key observables in making SPMHT efficient and effective in destroying tumor cells (SPMHT optimization) without affecting healthy tissues. Thus, given that SPMHT is more suitable for obtaining the hyperthermic effect in magnetic nanoparticles, and that there are many experimental studies in the field of magnetic hyperthermia with FeDMop), the optimal amplitude of the AMF (Hop) applied from the outside and the optimal limit frequency (flop) that can be used in magnetic hyperthermia to obtain maximum efficiency in SPMHT, by reaching the maximum specific loss power (PMop) and, at the same time, the maximum efficiency in destroying tumor cells by increasing the temperature (ΔT) of magnetic nanoparticles to the one required in hyperthermia of 42–43 °C (ΔTop) in a short period of time. Having this in view, a 3D computational study was done taking into account the allowable biological limit, in order to efficiently apply SPMHT in vitro, in vivo experiments and later in clinical trials in optimal conditions, for maximum efficacy.The aim of this study is precisely to find the optimal parameters in SPMHT, regarding the optimal diameter of magnetic nanoparticles (M) of superparamagnetic nanoparticles in an external magnetic field (H) in the absence of interactions between them (dispersed nanoparticle system) is done according to the Langevin function [ζ is the argument of the Langevin function, given by the following relationThe magnetization mNP=VIn this relation D asIn the approximation of spherical nanoparticles the magnetic moment can be expressed as a function of the diameter of the nanoparticle For low amplitudes of the magnetic field H, when the condition ζ≪1  is fulfiReplacing the Langevin function (Equation (6)) in Equation (1), taking into account the Equation (3), the magnetization of the nanoparticle system in low fields is obtained,Considering the magnetic volume fraction in the case of dispersed magnetic nanoparticles =Msat/Ms , the magH is the amplitude and f is the frequency of AMF, and In an alternating magnetic field (AMF) with a frequency of the order of hundreds of kHz the magnetic nanoparticles heat up, and the volume loss power (power density) is given by the relation ,32(12)p formula (14)χ″=χK is the magnetic anisotropy constant, and D) is:According to Neel’s theory the magnetic relaxation time (Néel) has the expression ,70(15)τe 10−9 s . For sphm) of the heating magnetic nanoparticles system. In low magnetic fields the static magnetic susceptibility D (size) of the nanoparticles and the magnetic anisotropy constant (Equation (16)) characteristic of the magnetic nanoparticles.In magnetic hyperthermia the specific loss power The measure of the heating of magnetic nanoparticles  as a function of the variables: the diameter of the magnetic nanoparticles D, the amplitude of the magnetic field H, and the frequency f of AMF.In conclusion, Equation (17), together with Equations (11) and (16), and Equation (18) are used in our 3D study to calculate the specific loss power (3O4 (magnetite) nanoparticles which are the most commonly used in magnetic and superparamagnetic hyperthermia, having the characteristic observables given in H) and the frequency (f) of the field considered for this study. All these values were established taking into account the real conditions in which the magnetic hyperthermia experiments are performed, based on the previous results.Our study on the specific loss power in SPMHT, which leads to the heating of magnetic nanoparticles, was done for approximately spherical FeA professional software was used for the 3D study and calculation of the observables of interest in SPMHT, and a 3D graphical representation of the results was generated. The 3D representation allows observation at the same time of the dependence of the observables of interest as a function of two variables, thus providing more information than in a simple 2D representation for a function of a single variable. Also, the possibility of studying for a wide range of values allows an overview of the variation of the respective observables, so that the optimal values can be extracted. Thus, the 3D representation is a powerful tool for a much more complete study.3O4 nanoparticles (D), which is a critical parameter in SPMHT, as well as the amplitude of magnetic field (H) and the frequency of AMF (f). For the magnetic volume fraction (ε), the usual values in the case of magnetic nanoparticle dispersions were used. The study was done at room temperature, considering the temperature value of 298 K.The observables pursued in our study, which are of particular interest in the efficient practical application of SMHT, are the specific loss power (sP) given by Equation (17) (or specific absorption rate (SAR) for an adiabatic system), and then the heating rate  can be fulfilled. Calculating the power sP under these conditions as a function of the nanoparticle diameter D and the AMF frequency f, s = PsP, considering a field of 2 kA/m, we obtain the result shown in In agreement with the theory presented in (i)sP critically depends on the diameter D of Fe3O4 nanoparticles, having a high maximum at the values of diameters (MD) ~16–17 nm, these depending slightly on the frequency (MD diameter decreases slightly with increasing frequency). For diameters D less than or greater than MD, respectively D less than ~15 nm at 100 kHz and ~13 nm at 500 kHz, and for D greater than ~20 nm at 100 kHz and ~18 nm at 500 kHz, the specific loss power becomes almost zero. This variation of specific loss power shows that it strongly depends on the diameter of the nanoparticles, and that the diameter of the nanoparticle is a critical parameter, sP having a maximum value (sMP) at the diameter of MD. Also, the maximum specific loss power sMP increases approximately in proportion to the increase in AMF frequency. This result clearly shows that in SPMHT the nanoparticles cannot be of any size, because at larger or smaller nanoparticle sizes than MD, the specific loss power decreases rapidly to zero, and the hyperthermic effect can no longer be obtained. In other words, before starting any experiment with magnetic hyperthermia, the first thing to do is to determine the value of the critical diameter MD  of the nanoparticles, which will lead to a maximum dissipated power, and as an effect, to an efficient heating of magnetic nanoparticles. Otherwise, for other values of the diameter (sizes) of the nanoparticles the hyperthermic effect will be weak or will even be lacking. In the scientific literature are reported many experiments of magnetic hyperthermia where inappropriate diameters (sizes) of magnetic nanoparticles have been used, which have led to poor results [The specific loss power  results ,22,55,60(ii)sMP power will be quite low, below the value of 0.7 W/g, a value that is unsuitable for magnetic hyperthermia.In low magnetic fields, although Equation (6) is well satisfied, the maximum specific loss power at the critical diameter is very small, so that under these conditions, the heating of the nanoparticles will also be reduced and inefficient in magnetic hyperthermia of tumors. For the case presented even at the highest frequency of 500 kHz the The 3D representation in the figure shows two very important aspects:sMP is needed, that will lead to an efficient heating of magnetic nanoparticles with MD diameter, so that the temperature of 42–43 °C required in hyperthermia can be reached, and also, in a short time, so as not to affect healthy cells. For the fixed parameters of the magnetic nanoparticles the increase of the maximum power sMP for a MD diameter can be obtained efficiently by increasing the H amplitude of magnetic field and/or the frequency f of the AMF. Also, another parameter is ε, but this is limited to maximum values of up to 0.15 in the case of magnetic nanoparticles. Thus, in order to increase the efficiency in magnetic hyperthermia a high incease of the maximum specific loss power H increases from 15 kA/m (a) to 30 kA/m (b), considering for ε the value of 0.15. According to the obtained results, and using Equation (17), results are more efficient to increase the maximum specific loss power by increasing the amplitude of magnetic field (sMP) obtained was represented for MD of 16.5 nm as a function of the amplitude of magnetic field in the range 1–70 kA/m. Furthermore, from a practical and technical point of view it is easier to obtain larger magnetic fields at lower frequencies than large fields at high frequencies.ic field b and notlf) AMF depending on the values established for the magnetic field in the range 5–50 kA/m, and the pairs of values for , respectively that can be used in the range 100–1000 kHz for the limit value lH × f given by Equation (19).However, for in vitro, in vivo, and in clinical trials magnetic hyperthermia experiments, the biological limitation for the magnetic field and the frequency AMF imposed by the following formula (19)H · However, the use of a large magnetic field, although advantageous in terms of dissipated power, means that Formula (17) for calculating the specific loss power under linear conditions of magnetization of nanoparticles in low amplitude of AMF can no longer be used. This formula must be modified taking into account the magnetization of nanoparticles in the large field. But in these conditions the questions naturally arise: (a) how will the large magnetic fields affect the maximum dissipated power? (b) can high values for the magnetic field be used in practice? The answers to these questions will be given in the next section.sP) (Equation (17)) will no longer be a constant, and equal to the initial magnetic susceptibility, but will be given (by definition When applying a high amplitude AMF (greater than a few kA/m) the magnetization of the nanoparticles will no longer be linear , but wilExpressing the static magnetic susceptibility , (14), (16) and (21), will have the formula:H (Equation (2)). Thus, the heating temperature in this case will be given by the same Formula (18), but with Ps given by the new Equation (22).In this case the calculations for the specific loss power will be made using Equation (22), with sMP can be observed, so at the frequency (maximum) of 1000 kHz and MD ~15 nm the maximum for sP decreases from ~500 W/g to ~300 W/g for the field of 15 kA/m, and from ~2125 W/g to ~700 W/g for the field of 30 kA/m. At the same time, there is also a greater decrease of the maximum specific loss power (sMP) when the magnetic field has a higher value of 30 kA/m, respectively.Doing the calculations in the same conditions as in the previous case shown in sP were calculated in both cases for the values of the magnetic field of 0.5; 1; 2.5; 5; 10; and 15 kA/m (as in MD ~16 nm that gives the maximum specific loss power (sMP). From the curves thus obtained in both cases, for each value of the magnetic field the maximum values of the specific loss power sMP, were extracted, obtaining the curves from H > 5 kA/m). At low values of the magnetic field, up to 2.5 kA/m, the powers in both cases are the same , and then Equation (22) the specific loss powers /m as in . The calthe same a, and thMD diameter in the range 15–17 nm . Thus, as seen in sMP with increasing magnetic field over 5 kA/m is due to decreasing magnetic susceptibility with increasing AMF amplitude.In large magnetic fields, static magnetic susceptibility To conclude, the realistic formula for calculating the specific loss power at large magnetic fields is that given by Equation (22). This is reduced to the formula given by Equation (17) only for low magnetic fields.However, in a high magnetic field another important aspect from the point of view of SPMHT is the following: what is the maximum value for the magnetic field that can be used? At first appearance, from the point of view of magnetic hyperthermia (maximum specific loss power) according to the data in H and frequency f in Calculating the maximum specific loss power using Equation (22), from its 3D representation as in sMP and the diameters MD that the nanoparticles must have to obtain the maximum power, the following question arises: what are the optimal values for the magnetic field, frequency and diameter of magnetic nanoparticles, within the biological permissible limit, in order to obtain the optimal maximum for specific loss power (sMopP) for the efficient heating of the magnetic nanoparticles, and finally for obtaining the optimal temperature required in SPMHT, of 42–43 °C, in the shortest time, so as not to affect the healing tissues. For a practical implementation of the SPMHT method it is essential to know all these aspects so that the method can be used effectively and with maximum efficiency in the destruction of tumor cells. Outside the optimal conditions poor results will be obtained in the superparamagnetic hyperthermia of the tumors, depending on the values that the parameters D, H and f will have compared to the optimal ones.Having in view the results obtained in MopD) and the optimal limit frequency (flop) for different values of the magnetic field in the range considered for analysis, 5–50 kA/m (sMopP) for superparamagnetic hyperthermia, and finally to determine the optimal heating of the nanoparticles and obtaining the optimal temperature necessary for the destruction of the tumor cells, without affecting the healthy cells around them, the 3D representations of the specific loss power Ps were used, as in MopD and sMopP were determined.In order to determine the optimal values of the nanoparticle diameter . With the obtained data, the curve representing sMP as a function of the magnetic field H from From all 3D representations as in (1)Not all values for the magnetic field and the frequency limit can be used to obtain an optimal specific loss power: for example, a power high enough to efficiently heat magnetic nanoparticles, but not to use unnecessary resources in the production of the magnetic field. Thus, from the variation of the maximum power specifically it results at magnetic fields higher than 25–30 kA/m, for the limit values of the AMF frequency specified in (2)For magnetic fields lower than 10 kA/m, although the specific loss power increases rapidly with increasing magnetic field, it will still have lower values than in the range 10–25/30 kA/m. In addition to this, it is also very important that for low fields below 10 kA/m the frequency limit AMF must increase from 500 kHz to over 1000 kHz, which raises possible problems of affecting healthy tissues at high frequencies, especially at over 1000 kHz. It is also technically more difficult to obtain the required magnetic fields at frequencies higher than 500 kHz. In addition, the power being significantly reduced in this interval will not obtain an efficient (fast) heating of the nanoparticles.From the obtained results  some versMopP), and for the efficient practical application of SPMHT with Fe3O4 nanoparticles, is the one included in the magnetic field range of 10–25 kA/m, delimited in opH and the optimal limit frequency (lopf) 200–500 kHz. The optimal MopD diameter of the nanoparticles, corresponding to each pair (Hf)op are shown in green color in the table. Example, for the magnetic field of 15 kA/m (opH) the applicable optimal limit frequency (lopf) will be 334 kHz, and the Fe3O4 nanoparticles must have the optimal diameter (MopD) of 16.3 nm in which case an optimal specific loss power (sMopP) is obtained of 108.66 W/g, which will lead to the optimal, efficient/fast heating of the nanoparticles, these reaching in a very short time the value of the temperature used in magnetic hyperthermia (42–43 °C). In the next section this issue will be presented and discussed.In conclusion, considering these results, the recommended interval for the optimal maximum specific loss power (sMopP is significantly lower (being of ~108 W/g) compared to the case shown in sMopP power shown in At the end of this section, we must also mention the fact that at the field value of 15 kA/m, within the admissible biological limit, the power 3O4 nanoparticles in the optimal conditions previously determined, for the magnetic field in the optimal region of 15 kA/m, the optimal lopf frequency limit of 334 kHz and the optimal diameter MD of ~16 nm of the nanoparticles, which gives the optimal maximum of the specific loss power (sMopP). In the calculations the unfavorable case used in practice, when ε is 0.017, was considered for the magnetic volume fraction in order to see if an efficient heating can be obtained even in these conditions. Also, the calculations took into account the correction brought to the temperature in the Equations (2), (11) and (16), when it increases above the room temperature of 298 K. The obtained result is shown in Using the specific loss power given by Equation (22) for large magnetic field and then Equation (18) for the heating rate, we calculated the heating temperature of FeThe obtained result confirms the correct approach of our optimization in SPMHT by establishing the optimal parameters, so that SPMHT can be applied efficiently in hyperthermia experiments to obtain the maximum effect: efficient heating at 42–43 °C in a short time.MopD diameter of the nanoparticles, the opH magnetic field amplitude, the optimal lopf limit frequency of the AMF, and the ε volume fraction of the nanoparticles can be correctly established for the efficient application of SPMHT in vitro, in vivo, and in clinical trials, with maximum efficacy on tumors.Following our optimization study the optimal parameters required in suparparamagnetic hypertermia, the MopD ~16 nm), the values of the optimal amplitude and frequency of AMF (opH and lopf) to obtain the highest maximum of the specific loss power (sMopP) within the admissible biological limit. In Refs. [t/Δt) depending on different parameters: radius particles [sP in our case, respectively) for a model with cubic AC susceptibility in the nonlinear regime was recently presented in Ref. [Other relevant previous computational studies ,74,75,76In Refs. ,72,75 prarticles ,72, viscarticles  or tempearticles  indicatearticles ,35. An iiT) in time, where the aim is to reach the temperature of 43 °C required in tumor hyperthermia, is much more suggestive than the heating rate for the practical application of SPMHT. Also, by using the 3D power tool, compared to other previous 2D studies, it is possible to know the specific loss power dependence simultaneously by two variables of high interest in SPMHT such as nanoparticle size and frequency of AMF, and this for a very wide range of values which allows the capture of other variations of finesse that in 2D can go unnoticed. Thus, our study will obtain more complete information on the observables and processes that contribute to the increase of specific loss power, so that the optimization is done accordingly in order to obtain the maximum effect in SPMHT when implementing this technique.In addition, in our case the presentation of the direct dependence of heating temperature (Δ3O4 nanoparticles (MD ~16 nm) that leads to the maximum effect in magnetic hyperthermia, it is in good agreement with the one already used in magnetic hyperthermia experiments in clinical trials where used nanoparticles with a diameter of 15 nm [® clinical therapy approved in Germany (Berlin) for treating brain tumors using magnetic hyperthermia [Compared to some relevant experimental data that have given good results in vivo cancer therapy and in clinical trials, our theoretical results are in good agreement. Thus, regarding the size of Feof 15 nm ,11,12,14rthermia . Also, trthermia ,46,78,79In addition, by considering the optimal conditions and parameters determined by us, it will certainly increase the effectiveness in treating cancer by magnetic hyperthermia.3O4, with maximum efficacy in tumor therapy, the following optimization conditions in SPMHT must be met simultaneously:(1)sMP);Ferrimagnetic nanoparticles must have the optimal diameter  in the range 16.0–16.8 nm, depending on the AMF frequency used in magnetic hyperthermia: 16.0 nm for the frequency of 500 kHz and 16.8 for the frequency of 200 kHz, which leads to a maximum of specific loss power ((2)The optimal magnetic fields used must be in the range of 10–25 kA/m, depending on the power that is required in the superparamagnetic hyperthermia experiment, in which case the specific loss power given by Equation (22) will be used;(3)The optimal frequency of the AMF must be located in the range of 200–500 kHz, depending on the amplitude of the magnetic field, with the fulfillment of the condition for the admissible biological limit (Equation (19)): 200 kHz for the field of 25 kA/m and 500 kHz for the 10 kA/m field;(4)sMopP MopD of 16.3 nm, Hop of 15 kA/m, optimal limit frequency of 334 kHz, at a volume fraction of 0.017, for Fe3O4 nanoparticles the heating temperature of 42–43 °C required in hyperthermia for the destruction of tumor cells is obtained in a very short time of only 4.1–4.3 s, with major benefits in the practical implementation of the SPMHT method, in order not to affect the healthy tissues;Under optimal conditions, For the efficient practical implementation of SPMHT with ferrimagnetic nanoparticles of FMopD, magnetic field amplitude opH and AMF frequency lopf in different cases are found in The exact values for the optimal parameters, nanoparticle diameter Our study will allow the use of SPMHT in in vitro, in vivo and then in clinical trials experiments of SPMHT in conditions of maximum efficiency in order to obtain the maximum specific loss power, and with maximum efficacy on malignant tumor cells in alternative cancer therapy, by obtaining the necessary temperature in hyperthermia in a very short time.


The paper presents an elaboration the stress concentration mechanism of pores affecting the tensile properties. According to a mathematical analysis of a sample under uniaxial stress, the greater the radius of the pore, the higher the stress value is at the pore perimeter. When the absolute pressure drops from 1013 mbar to 100 mbar, the porosity decreases from 6.8% to 2.8%, and the pore number and mean size decreases. In tensile tests, the pore sizes of the fracture surface decrease with decreasing absolute pressure, and the pore distribution becomes uniform. The tensile properties and extensibility of the sample are improved, and the microscopic fracture surface of the sample changes from cleavage fracture to quasi-cleavage fracture. The number, size and distribution of pores in die casting collectively affect the properties of the sample. Large-size or complex pores or pores with concentrated distributions produce large stress concentrations, decreasing the strength of the metal.The absolute pressure strongly affects the porosity and mechanical properties of castings produced by vacuum high-pressure die casting (V-HPDC) technology. The pore size, quantity and distribution of AlSi The technology of high-pressure die casting (HPDC) is a special kind of casting method in modern metal processing technology involving little or no cutting. Due to its high productivity and its high shape and dimensional accuracy, HPDC has been widely used in the automobile, naval and aerospace fields, among other fields. The aluminum-silicon alloy, due to its light weight, good comprehensive mechanical properties, high wear resistance and low thermal expansion coefficient, is considered the best material for engine cylinder blocks ,2,3. HPD9Cu3 die castings produced under different degrees of vacuum [9Cu3 alloy castings under three different absolute pressures of 500 mbar, 200 mbar and 100 mbar. The studies showed that as the absolute pressure decreased from 500 to 100 mbar, the average porosity decreased, and the elongation and tensile strength increased substantially. M. Koru et al. studied the effects of heat, dynamic parameters and vacuum application on porosity in HPDC of the A383 aluminum alloy [9Cu3 alloy castings [Cao et al. studied the porosity and mechanical properties of AlSif vacuum . In thatum alloy . Flow-3Dcastings . Three kcastings ,24,25,26The above research indicates that the application of a vacuum system in the production process of HPDC has a great impact on the quality of die castings, and substantial research on the pores and mechanical properties of aluminum (magnesium) alloy die castings in vacuum applications has been conducted by scholars worldwide. Specifically, vacuum application can reduce the porosity of die castings and improve their mechanical properties. However, the specific relationship between the absolute pressure, porosity and mechanical properties in the die casting process and the underlying mechanism of material fracture are not clear and need further study. 9Cu3 die castings are evaluated in a comprehensive way under three different absolute pressures to reveal potential relationships between the absolute pressure, porosity and mechanical properties. In addition, the mechanism of influence of die casting pores on the mechanical properties of die casting samples is analyzed from the perspective of the material fracture mechanism. Through this study, we can better understand the effect of absolute pressure on the porosity and mechanical properties of die castings.In this paper, using experimental methods, the porosity and mechanical properties of AlSi9Cu3 (9Cu3) in Chongqing University. The parts were produced by a horizontal cold chamber die casting machine  equipped with a vacuum-assistance system. The vacuum pump and the vacuum valve in this system work in conjunction to discharge the air contained in the injection cylinder, as well as the air remaining in the mold cavity of the castings. To ensure the feasibility of the experiment, the following process parameters were controlled: The die temperature and the casting temperature were set to 250 °C and 650 °C, respectively. The maximum wall thickness and the minimum wall thickness were 30 mm and 3.5 mm, respectively. The velocities of slow injection and fast injection were set to 0.2 m/s and 5 m/s, respectively. The absolute pressure was set to one of three values: 1013 mbar, 200 mbar or 100 mbar.The die casting parts studied in this experiment were engine blocks, and the material was AlSi9Cu3 . X-ray fSamples  were all taken from the produced engine cylinder blocks by wire-electrode cutting. The three-dimensional diagram of engine cylinder blocks is shown in The dimensions of samples for the density measurements are 10 mm × 7.5 mm × 5 mm. The porosity rate of the casting was evaluated according to the standard BN-75/4051-10 . The denNext, the porosity of the specimen is calculated by Equation (2):A tensile testing machine  was used to measure the tensile strength of the die casting samples at room temperature. The tensile rate is 1 mm/min. The dimensions of the tensile sample with rectangular cross-section are shown in The dimensions of samples for OM were 10 mm × 7.5 mm × 5 mm. The samples were used for OM after coarse grinding, accurate grinding and buffing . The microstructure and pore defects were visualized by assembling a composite image of 16 OM images captured at a magnification of 50×, as shown in The number and size of the pores of each sample were counted with a NanoMeasurer 1.2 software quantitative analysis system for metallographic characterization . The porThe porosity and density of the castings under the three different vacuum levels of 1013, 200 and 100 mbar are shown in Cao et al. discussed the effect of the absolute pressure in the cavity on the pore defects of die castings . In the The pore defects were observed by OM and X-ray imaging . The porThe measurement data for the tensile tests are shown in The fracture morphology and microstructure of tensile samples after fracture are shown in It can be observed from The mechanical effects related to the fracture in the tensile test also merit study. The presence of pores inside the metal microstructure cause stress concentrations in the areas surrounding the pores. Idealizing the pore as a circle, G. N. Savin  discusseThe stress function is introduced, and the former equation can be represented in the form of a two-component function, as shown in Equation (5).Re represents the real part of the expression in square brackets; According to the Kolosoff–Muskhelishvili equation, The area S is mapped within the unit circle (or outside the circle) by the mapping function The stress function is shown in Equation (8):Here, z axis, the stress function is as shown in Equation (9).If a plate with a circular pore with a radius of R is stretched by an external force P along the OHere, the larger the radius R of the circular pore, the greater is the corresponding stress value. The initial stress cracks occur in the region where the stress is concentrated during the stretching process , which leads to a fracture.Substituting the values of related functions in Equation (9) into Equation (7), the stress functions When The principal stress trajectory is plotted with Equation (10).According to Equations (10)–(12), the isostress parameters As can be seen from It has been noted in the literature ,34,35,36To study the influence law of die casting pores on the mechanical properties of die castings, OM and X-ray were used to capture a metallographic map and analyze the number, size and distribution of die casting pores at different absolute pressures. SEM was used to obtain micrographs of tensile fracture surfaces. Tensile experiments were conducted on samples. Then, the stress concentration mechanism of the tensile fracture was evaluated.(1) From the test results, the density of the die castings increased with decreasing absolute pressure, and the porosity of the die castings and the average pore size decreased.(2) From the tensile test results, the tensile strength and elongation of the die casting samples increased with decreasing absolute pressure. With decreasing absolute pressure, the surface appearance of the sample fracture surface became flatter, the sizes of the surface pores were reduced, the pore distribution became more uniform and the fracture type changed from a cleavage fracture to quasi-cleavage fracture.(3) From the results of mathematical model analysis, pore defects had an important effect on the tensile performance of die castings. The larger the pore size, the greater the corresponding stress value in the horizontal direction of the pore boundary. The stress concentration mechanism near pores caused initial cracks and crack propagations around the pores during the tensile process, which led to the decrease of tensile strength.