*Article* **Albumin Might Attenuate Bacteria-Induced Damage on Kupffer Cells for Patients with Chronic Liver Disease**

**Hao Lin 1, Yuhui Fan 1, Andreas Wieser 2,3,4, Jiang Zhang 5, Ivonne Regel 1, Hanno Nieß 6, Julia Mayerle 1, Alexander L. Gerbes 1 and Christian J. Steib 1,\***

**Abstract:** Chronic liver diseases (CLDs) are complex diseases that cause long-term inflammation and infection, which in turn accelerate their development. The usage of albumin in patients with CLDs has been debated for years. Human serum albumin (HSA) plays a key role in immunomodulation during the process of CLDs. The correlation between albumin and C-reactive protein (CRP) in CLD patients was analyzed by linear regression with the Pearson statistic. The damage of THP-1 and primary cells was evaluated by measuring the lactate dehydrogenase (LDH) in the supernatant. Immunofluorescence staining was performed to determine underlying pathways in Kupffer cells (KCs). Albumin negatively correlated with infection in patients with CLDs. In vitro experiments with THP-1 cells and KCs showed that albumin reduced LDH release after stimulation with bacterial products, while no differences in hepatic stellate cells (HSCs) and sinusoidal endothelial cells (SECs) were detected. Moreover, immunofluorescence staining revealed an increase of p-ERK and p-NFkB p65 density after albumin treatment of KCs stimulated by bacterial products. In conclusion, albumin could assist CLD patients in alleviating inflammation caused by bacterial products and might be beneficial to patients with CLDs by securing KCs from bacteria-induced damage, providing a compelling rationale for albumin therapy in patients with CLDs.

**Keywords:** hepatic non-parenchymal cells; albumin; chronic liver diseases; bacteria

#### **1. Introduction**

Chronic liver diseases (CLDs) are a long-term pathological process involving continuous destruction and regeneration of liver parenchyma that leads to cirrhosis at its most advanced stage. The mechanism of the progression of CLDs is complicated and remains unsettled, but unceasing inflammation and bacterial infections may play an important role in this process [1,2]. Spontaneous bacterial peritonitis (SBP) is one of the most common infections during CLDs [3]. Gram-negative aerobic or facultative aerobic organisms such as *Klebsiella pneumoniae* (*K. pneumoniae*), *Escherichia coli* (*E. coli*), *Pseudomonas aeruginosa* (*P. aeruginosa*) and *Enterobacter cloacae* (*E. cloacae*) are the most common cause of SBP patients; other common organisms include the Gram-positive species *Enterococcus faecium* (*E. faecium*), *Streptococcus pneumoniae* (*S. peneumoniae*) and *Staphylococcus aureus* (*S. aureus*) [4].

**Citation:** Lin, H.; Fan, Y.; Wieser, A.; Zhang, J.; Regel, I.; Nieß, H.; Mayerle, J.; Gerbes, A.L.; Steib, C.J. Albumin Might Attenuate Bacteria-Induced Damage on Kupffer Cells for Patients with Chronic Liver Disease. *Cells* **2021**, *10*, 2298. https://doi.org/ 10.3390/cells10092298

Academic Editors: Silvia Fischer and Alexander E. Kalyuzhny

Received: 2 August 2021 Accepted: 1 September 2021 Published: 3 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Kupffer cells (KCs) are resident liver macrophages as well as the largest population of innate immune cells in the liver [5]. The major function of KCs is to scavenge and phagocyte cell debris, small particles, protein complexes and senescent red blood cells through an interplay with pattern recognition receptors. In addition, gut-derived toxic materials including pathogens from the intestinal flora and endotoxic lipopolysaccharide (LPS) are removed by KCs [5–7]. Thus, KCs play an essential role in maintaining the homeostasis to protect the host and in prompting immunogenic and tolerogenic immune responses.

Human serum albumin (HSA) is a crucial plasma protein often administered in the therapies of patients with CLDs. However, there still are debates on the benefit of albumin in patients with cirrhosis over the long term. Notably, a recent clinical trial presented that the long-term albumin administration to patients with cirrhosis reduced systemic inflammation [8] and the cumulative incidence of complications of cirrhosis, including SBP and non-SBP bacterial infections [9,10]. Among the reasons of benefits of albumin in patients with cirrhosis, expander function depending on its known oncotic properties is well known, although it is now irrefutably clear that the immunomodulatory function of albumin turns to be more important during the therapies for liver cirrhosis [11–13]. HSA is capable of binding inflammatory factors and mediators including LPS, lipoteichoic acid and peptidoglycan, which are the surface components of Gram-negative and Gram-positive bacteria capable of activating the innate immune system through Toll-like receptor 2/4 (TLR 2/4) and initiating inflammation [12–15]. Albumin preconditioning abrogated the LPS-mediated inflammation through the Nuclear Factor-κB (NF-κB) activation [16] and enhanced monocyte interleukin-6 (IL-6) gene expression via the extracellular signal-regulated kinase (ERK) and NF-κB pathways [17]. Albumin can modulate innate immune responses to sepsis and cirrhosis-associated prostaglandin E2-mediated immune dysfunction following albumin infusion [18]. Nevertheless, the role of albumin on KCs, the first barrier against bacteria and a vital role in bacteria-induced immune responses, has not been investigated.

Hence, our study underscores a protective role of albumin in patients with CLDs and in KCs stimulated by the bacterial products, suggesting that albumin therapies aim to not only improve plasma osmolality for patients with CLDs but benefit KCs in reducing the damage from microbial products, providing a convincing rationale for albumin application in patients with CLDs.

#### **2. Materials and Method**

#### *2.1. Study Cohort*

From July 2016 to March 2019, 138 outpatients or consecutive hospitalized patients with CLDs were obtained to be included in this study. The types of CLDs included in this study are: chronic viral hepatitis B (*n* = 26), autoimmune hepatitis (*n* = 21), chronic viral hepatitis C (*n* = 18), liver cirrhosis (*n* = 33, including 29 hepatitis-induced, 3 alcohol-induced and 1 Alagille syndrome-induced), primary sclerosing cholangitis (*n* = 8), primary biliary cholangitis (*n* = 6), steatosis hepatitis (*n* = 5), nonalcoholic steatohepatitis (NASH, *n* = 4), alcoholic liver disease (*n* = 3), cryptogenic cirrhosis (*n* = 3), cystic liver disease (*n* = 2), Budd–Chiari Syndrome (*n* = 2), hemochromatosis (*n* = 2), liver adenoma (*n* = 2), M. Wilson disease (*n* = 1), sarcoidosis (*n* = 1) and toxic liver disease (*n* = 1). The study protocol was approved by the ethics committee of the Faculty of Medicine at the Ludwig-Maximilians University (LMU) (approval number 17-756), and patients provided written informed consent. All indicators were measured by the clinical chemistry laboratory of the university hospital of LMU using an automatic analyzer (cobas®8000 modular analyzer series, Roche, Switzerland) and standardized operating procedures according to the manual. A score was calculated using the following formula: MELD Score = 10 × (0.957 × ln (Creatinine) + 0.378 × ln (Bilirubin) + 1.12 × ln (INR) + 0.643).

#### *2.2. Human Tissue Specimens*

Double-coded liver tissue samples and corresponding data used in this study were provided by the Biobank of the Department of General, Visceral and Transplant Surgery of LMU. This Biobank operates under the administration of the Human Tissue and Cell Research (HTCR) Foundation. The framework of HTCR Foundation [19], which includes obtaining written informed consent from all donors, has been approved by the ethics committee of the Faculty of Medicine at the LMU (approval number 025-12) as well as the Bavarian State Medical Association (approval number 11142) in Germany. A total of 10 human liver tissues were included in our experiments and were from patients with liver metastasis.

#### *2.3. Primary Hepatic Non-Parenchymal Cells Isolation and Culture*

Liver tissues were preserved in RPMI 1640 medium (Gibco, Karlsruhe, Germany) on ice immediately after surgical removal, and cells were isolated within 6 h. Nycodenz (Axis Shield, Rodelokka, Norway) gradients density centrifugation was performed to isolate primary liver KCs and hepatic stellate cells (HSCs). In brief, liver samples were cut into 1–5 mm thick slices and rinsed by phosphate-buffered saline (PBS). The tissue slices were digested with 15 mg/mL pronase (Sigma, St. Louis, USA) at 37 ◦C for 20 min. After two times of 80 μm and 60 μm nylon mesh filtration, the tissue solution was mixed with Nycodenz solution (16.7% for KCs and 28.7% for HSCs) and centrifuged (1400× *g*, 20 min) without the brake, dividing into three layers. We collected the middle layer and interfaces. To isolate sinusoidal endothelial cells (SECs), CD146 MicroBeads (Miltenyi Biotec, Teterow, Germany) and a magnetically activated cell sorting system were employed to filtrate the tissue digestive solution instead of Nycodenz. Cells were seeded in multiple well plates and cultured at 37 ◦C in 5% CO2 in RPMI 1640 with 10% fetal calf serum (FCS, PAN, Aidenbach, Germany) and 1% Penicillin-Streptomycin (Sigma, St. Louis, MO, USA). Detailed procedures were described previously [20–22].

#### *2.4. Cell Culture and Treatment*

The THP-1 cell line (American Type Culture Collection, reference number TIB-202™) was a gift from Prof. Peter Nelson. Cells were cultured at 37 ◦C in 5% CO2 in RPMI 1640 with 10% FCS and 1% Penicillin–Streptomycin. To differentiate THP-1 into adherent macrophages, 20 ng/mL phorbol myristate acetate (PMA) was used to treat cells in a complete medium for 48 h. The medium was changed into a serum-free medium before the stimulation of bacterial products. Cells were treated with albumin at the same time with (peri-treatment) or 24 h before (pre-treatment) the stimulation of bacterial products. The supernatant was harvested after 24 h of the microbial isolate stimulation.

#### *2.5. Bacterial Products Isolation and Stimulation*

Bacterial strains including *K. pneumoniae*, *E. coli* and *E. cloacae*, *P. aeruginosa*, *E. faecium*, *S. peneumoniae* and *S. aureus* were isolated from patients with SBP (different groups of patients from the study cohort). The isolates were cultured on Columbia 5% sheep blood media (Becton Dickinson, Heidelberg, Germany) at 37 ◦C under aeration. Bacterial colonies were taken off the solid media with caution not to include any parts of the media. The bacterial pellet was resuspended in phosphate-buffered saline (PBS pH 7.4) by pipetting up and down and vortex mixing. Cells were washed in PBS three times to remove any residual media or debris. After the last washing steps, the bacterial cell mass was resuspended in PBS buffer and sterilized using heat inactivation [23]. Bacterial inactivation was confirmed twice by plating the heat-inactivated extracts on media and cultivating them for 48 h at 37 ◦C. Bacterial product solutions were measured for protein content in serial dilutions (Bradford) and were subsequently divided into aliquots and stored frozen until use. The bacterial products solutions were diluted to various concentrations (1, 8, 16 μg/mL) with serum-free medium and then were used to stimulate cells at different concentrations with or without HSA.

#### *2.6. Evaluation of Cell Damage*

The lactate dehydrogenase (LDH) was used to assess the cell damage of THP-1 and primary cells. LDH catalyzes the reversible conversion of lactate to pyruvate with the reduction of NAD+ to NADH. Thus, the production of NADH was employed to determine the LDH activity indirectly, which was measured by the absorbance value at 365 nm [20,24,25]. 5% Triton-X-100, which is inducing cell damage, was applied as a positive control.

#### *2.7. Immunofluorescence Staining*

Cells were fixed with 4% paraformaldehyde for 10 min (Roth, Karlsruhe, Germany), then they were blocked in 10% donkey serum for 30 min, and were permeabilized with 0.1−0.5% Triton X-100 in PBS for 10 min [26]. Subsequently, cells were incubated with the primary antibodies (1:250 p-ERK and 1:1000 p-NF-kB p65, Cell Signaling Technology) overnight at 4 ◦C and fluorescent-labeled secondary antibodies at room temperature for 1 h the next day. The images were captured by immunofluorescence microscopy (Leica, Germany).

#### *2.8. Statistical Analyses*

Results are presented as mean ± standard deviation (SD) or median and interquartile range (IQR). Normality of data distribution was tested by Kolmogorov–Smirnov Z test (Table 1). Statistical comparisons by using the unpaired two-tailed Student's *t*-test and Mann–Whitney U test were performed. Correlations between variables were calculated using linear regression with the Pearson statistic. A *p*-value less than 0.05 was considered significant. SPSS was used for data analysis.

**Table 1.** Normality test of laboratory parameters.

K-S test: Kolmogorov–Smirnov Z test. Data are expressed as mean ± SD.

#### **3. Results**

#### *3.1. Albumin Negatively Correlated with Infection in Patients with CLDs*

All 138 patients with CLDs were divided into two groups according to their HSA levels: the normal albumin group (≥3.5 g/dL) and the low albumin group (<3.5 g/dL). Table 2 displayed that CLD patients with lower albumin had higher bilirubin, aspartate aminotransferase (GOT), Gamma-GT, LDH, international normalized ratio (INR), and creatinine, C-reactive protein (CRP), MELD Score, while NaCl, KCl and Zinc serum levels were remarkably lower. Pearson correlation analysis and linear regression were performed to investigate the association between these indicators and albumin levels. As exhibited in Table 3 and Figure 1, high serum albumin levels correlated with a low concentration of CRP in patients with CLDs showing an r value of 0.565 (*p* < 0.001). Which indicated that patients with CLDs with high serum albumin had less infection. In addition, the levels of NaCl, KCl and Zinc positively correlated with albumin levels in patients with CLDs

while bilirubin, GOT, Gamma-GT, LDH, INR, and creatinine had a negative correlation with albumin levels (Table 3 and Figure S1).

**Table 2.** Characteristic analysis based on different albumin concentrations.

Data are expressed as mean ± SD or median and interquartile range (IQR); a Student's *t*-test.; b Mann–Whitney U test.

R and *p* values were obtained using the Pearson correlation test.

The CRP levels were negatively correlated with albumin levels in patients with CLDs (r = −0.565, *p* < 0.001).

#### *3.2. Albumin Reduces the Cell Damage Caused by a Bacterial Infection in THP-1 Cells*

It is well-known that a CRP test could be used to monitor conditions that cause inflammation, including bacterial infections, fungal infections and inflammatory bowel diseases. CLD patients would most likely have SBP during the development of CLDs, and the most common reasons for SBP are bacterial invasions [3,4]. Besides this, the sensitivity and specificity of CRP are high enough to evaluate the condition of bacterial infection in patients with CLDs [27]. Together, the increase of CRP levels in CLD patients are mainly attributed to the bacterial infection in our study; therefore, we used bacterial products to stimulate THP-1 and primary cells in the subsequent experiments. Our previous publication indicated that Zinc protects KCs from microbial infection [28]. As shown in

Table 3 and Figure S1, the concentration of albumin had a strong positive correlation with Zinc levels in patients with CLDs (r = 0.752, *p* < 0.001). In this study, we postulated that albumin protects macrophages from the damage of bacterial infection. Instead of KCs, we selected the macrophage cell line THP-1 to perform in vitro validation experiments, because the amounts of KCs from human liver specimens were limited. First, we optimized the concentration of bacterial products that causes LDH release, as a marker for cell damage. We treated THP-1 cells with different concentrations of bacterial products and demonstrated increased LDH levels when using 8 and 16 μg/mL of bacterial isolates (Figure 2A) Next, we tested the cell damage caused by 8 bacterial strains associated with CLDs including regular Gram-negative and Gram-positive bacteria, such as *K. pneumoniae*, *E. coli* and *E. cloacae*, *P. aeruginosa*, *E. faecium*, *S. peneumoniae* and *S. aureus*. Figure 2B showed that all of the eight most ubiquitous bacteria in CLDs can cause considerable increases in LDH levels when THP-1 cells were treated with 8 μg/mL of the bacterial products.

**Figure 1.** The correlation between albumin and CRP.

Moreover, we analyzed whether albumin might show toxic effects on THP-1 macrophages when treated with increasing concentrations. Notably, in a range from 0.01−0.4 mg/mL, albumin treatment of THP-1 cells did not result in a meaningful increase of LDH levels. To investigate if albumin protects macrophages from bacterial damage, we treated THP-1 cells with 8 μg/mL bacterial products of four bacteria strains including *E. coli TOP10* and *E. cloacae* (Gram-negative bacteria strains) and *S. peneumoniae* and *S. aureus* (Gram-positive bacteria strains) in combination with 0.4 mg/mL albumin. Therefore, THP-1 cells either were treated with albumin 24h before (pre-treatment) or at the same time (peri-treatment) with microbial stimulation. Our data demonstrated, consistent with our previous results, that bacterial products increased LDH release in THP-1 cells compared to the control group ( # *p* < 0.05, Figure 2D). Furthermore, both the pre-treatment and peri-treatment of albumin significantly decreased LDH release in four bacterial product groups compared to noalbumin (blank) treatment (\* *p* < 0.05, Figure 2D). These results demonstrated that albumin protected THP-1 cells from bacteria-associated damage, irrespective of the pre-treatment or peri-treatment conditions with albumin.

**Figure 2.** Effects of albumin on the injury induced by bacteria in THP-1. (**A**) The cell damage caused by bacteria products at various concentrations. (**B**) Measurement of the cell damage caused by bacterial components (8 μg/mL) isolated from indicated bacteria strain from patients with CLDs. (**C**) Toxic measurement of different doses of albumin in THP-1. (**D**) Effects of albumin treatment (0.4 mg/mL) on THP-1 stimulated with bacterial isolates (8 μg/mL). All LDH data are normalized to a positive control (5% Triton-X-100). The 5% Triton-X-100 was set as a positive control in all LDH measurement assays. Data expressed as means ± SD, \* *p* < 0.05 vs. blank treatment in their own group, # *p* < 0.05 vs. blank treatment in the control group, *n* = 6 in each group from three independent experiments.

#### *3.3. Albumin Safeguards KCs, Rather Than HSCs and SECs from Bacteria-Induced Cell Damage*

To investigate whether albumin has identical effects on primary hepatic non-parenchymal cells as it was shown above for the THP-1 cell line, KCs, HSCs and SECs isolated from human liver specimens were examined. Like the THP-1 cells, primary KCs, HSCs and SECs were treated with albumin and stimulated by bacterial products. LDH was measured to assess cell damage. Albumin treatment of KCs remarkably reduced LDH release caused by bacterial isolates in both pre-and peri-treatment conditions. Interestingly, for HSCs, the bacterial products notably increased LDH levels (# *p* < 0.05); however, no significant changes in LDH levels were found after albumin treatment (Figure 3B). In addition, bacterial product stimulation and albumin treatment did not induce any notable changes in LDH levels in SECs (Figure 3C). From these findings, we conclude that albumin has a protective role on KCs preventing KC damage during a bacterial invasion in CLD patients.

**Figure 3.** Different effects of albumin on different hepatic non-parenchymal cells. (**A**) Effects of albumin (0.4 mg/mL) on the cell damage induced by bacterial products (8 μg/mL) for KCs. No significant effects of albumin on HSCs (**B**) and SECs (**C**) were found in both pre-treatment and peri-treatment. Data expressed as means ± SD, \* *p* < 0.05 vs. blank treatment in their own group, # *p* < 0.05 vs. blank treatment in the control group, *n* = 6 in each group from three independent experiments with a total of three different human livers.

#### *3.4. ERK and NF-kB Pathways Were Involved in the Protective Effects of Albumin*

ERK and NF-kB pathways are the most important pathways participating in the TLR/MyD88/IRAK4 axis that is activated by Gram-positive and Gram-negative bacteria in livers [29–31]. To determine the activation of the ERK and NF-kB pathway in KCs, we performed immunofluorescence staining of phosphor-ERK (p-ERK) and phosphor-NF-kB (p-NF-kB) under control, *E. coli* and *E. coli* plus albumin treatment. The results showed that albumin intensifies the staining density of p-ERK and p-NF-kB compared to the control group and *E. coli* group in KCs (Figure 4). These findings provided preliminary evidence of the involvement of ERK and the NF-kB pathway in the process of albumin protection on liver macrophages.

**Figure 4.** The potential mechanism of the protective effects of albumin in KCs. Immunofluorescence labeling of p-ERK and p-NF-kB protein (green) of KCs treated with albumin and *E. coli*. The nuclei were stained with 4- -6-diamidino-2 phenylindole (blue). Scale bars, 100μm. The relative fluorescence intensity compared with DAPI intensity in the staining experiments was measured. Data expressed as means ± SD, \* *p* < 0.05 vs. blank group, # *p* < 0.05 vs. *E. coli* group, *n* = 6 in each group from three independent experiments with a total of three different human livers.

#### **4. Discussion**

In the present study, we described for the first time the beneficial effects of albumin reducing the damage of KCs caused by bacteria infections. Albumin is applied regularly to improve osmotic pressure for patients with cirrhosis, but there is only little information on further functions of albumin. This study highlighted the relationship of albumin and inflammation after bacteria invasion in patients with CLDs, the immune-related role of albumin in patients with CLDs, and its underlying mechanism. Novel findings in this study were: (1) In patients with CLDs, albumin levels were found to be inversely correlated to CRP levels, a marker for infection. (2) Albumin protects KCs against bacterial-induced cell injury, but no effects were observed in HSCs. (3) The ERK and NF-kB pathway may be involved in the protective effects of albumin treatment in KCs.

In patients with CLDs, the clinical benefits of albumin therapy have been debated for years. Several reports have failed to show any favorable effect on the administration of albumin in CLDs except for delaying the onset of renal failure [32,33]. Nevertheless, the wide range of potential benefits of albumin administration, such as anti-inflammatory activity, antioxidant function, immunomodulation and the transportation of many endogenous and exogenous substances, still need to be further investigated [34]. Recently, there were some important clinical trials conducted to supply more convincing evidence of the benefits of albumin administration in patients with CLDs. The data indicated that longterm albumin supplementation to patients with cirrhosis and ascites improved survival, lowered hospitalizations and prevented complications [8,9,35]. In our present study, we found that patients with lower albumin levels had more severe inflammation (Figure 1), suggesting that albumin eased the inflammation in patients with CLDs and participated in decreasing the immune responses due to the bacterial invasion. Albumin has the ability to bind and inactivate many inflammatory mediators, such as pathogen-associated molecular patterns, reactive oxygen species, bioactive lipid metabolites and nitric oxide, which could be the underlying reason for improving the inflammation for patients with CLDs with normal albumin.

The immunomodulatory function of albumin has been increasingly studied recently. Decreased serum albumin in patients with cirrhosis binds fewer prostaglandin E2 (PGE2) resulting in increased bioavailability of PGE2, which dampens the macrophage response to LPS [18], indicating that albumin may promote immune functions in some ways. KCs are one of the most important immune cells participating in the immune reaction. To explore the relative mechanism of albumin on reducing the inflammation presented in Figure 1, we treated KCs with albumin in our study. Another study showed no positive changes under the administration of albumin on patients with cirrhosis [36], even though there were three clinical trials to prove the benefit of albumin on patients with CLDs referred above. Interestingly, a loading dose of albumin administration in the other three studies has been considered to be the reason behind this difference [12], which means that the concentration of albumin administration is critical to show its advantages. In addition, it has previously been observed that 0.4 mg/mL albumin could improve PGE2 mediated immunosuppression [18]. Taken together, 0.4 mg/mL albumin was used in our cell experiments. KC isolation from the human liver is a tricky technique, and the number of cells was limited. THP-1 is a human monocytic cell line obtained from a patient with acute monocytic leukemia, and the cells differentiated with PMA are commonly utilized as a model for human macrophage function and biology [36]. Therefore, we used them rather than KCs to measure the damage from different strains and various concentrations of bacterial products. Our results presented that 8 μg/mL and 16 μg/mL of bacterial product stimulation had a significant injury on THP-1 cells (Figure 2A). Bacterial products of all tested strains damaged THP-1 cells regardless of Gram-negative or Gram-positive bacteria (Figure 2B), whereas albumin with increasing concentrations had no toxic effects on cells (Figure 2C).

Next, we treated KCs and THP-1 with albumin to examine its protective effects on primary macrophages. Our data showed that albumin treatment before and during the stimulation with bacterial products protected KCs and THP-1 from injury by microbial isolates (Figures 2D and 3A), which might be caused by the capability of albumin to bind inflammatory factors as mentioned above. Another possible explanation for this is that the albumin might strengthen the phagocytosis of KCs, and increase the albumin levels

inside cells, and this might have a synergistic effect on the protection of KCs. Besides, we confirmed our previous conclusion that microbial isolates did damage to KCs and HSCs rather than SECs (Figure 3B,C) [28]. However, one unanticipated finding was that albumin treatment had no notable protective effects on HSCs (Figure 3B). The receptor of polymerized albumin was found on macrophages [37] and its role for endocytosis of albumin has been described [38]. Similarly, stellate cells can actively take up albumin from extracellular sources [39]. Nonetheless, the phagocytosis of macrophages is stronger than stellate cells, which might be a possible mechanism behind this unexpected result.

Taken together, we initially had speculated that patients with CLDs with normal albumin could inactive inflammatory factors and bacterial products. Furthermore, it allows KCs to function normally and facilitate an immune response to foreign bacteria. We hypothesized that at the beginning of immune responses, CLD patients with normal albumin might have more intense inflammation and higher CRP for a short time (acute inflammation responses) due to the response of a healthy immune system, then finally return to normal with the development of the effective immune responses maintained by the protective effect of albumin on KCs, which can explain the relationship between our CRP data in patients with CLDs in Figure 1 and the cell damage assay in Figures 2D and 3A. Thus, we conjectured that the protective effect of albumin on KCs might confer some benefits of diminishing the inflammation in patients with CLDs.

Our previous publication revealed that Zinc protects KCs from this damage caused by bacterial isolates [28]. Considering similar protective effects of Zinc and albumin, we hypothesized that a potential interacting mechanism of these effects might exist. Bacteria or bacterial products stimulate TLRs which are the essential molecules involved in bacteria-induced inflammation in macrophages. Activation of TLRs triggers ERK and NF-κB signaling pathways, which are critical for a normal immune response [29,30]. A previous study demonstrated that albumin treatment produces a dose-dependent increase in pro-inflammatory gene expression (acute inflammation responses) in vitro through activation of ERK and NF-κB pathways [40–43]. Thus, we speculated that the protective effects of albumin on liver macrophages are dependent on the ERK and NF-kB pathways. An amplifying effect of albumin on the activation of the ERK and NF-kB pathways caused by bacteria products was observed compared to the *E. coli* stimulation (Figure 4). Together, our findings provided possible mechanisms for the effect of albumin and aided in understanding the therapeutic benefits of albumin on patients with CLDs.

In conclusion, our results revealed that albumin might help patients with CLDs to better overcome the effects caused by bacterial infection and defend KCs from the injury caused by bacteria in patients with CLDs (Figure 5), implying immunomodulatory-related benefits of albumin administration on patients with CLDs. Thus, we conjectured that albumin supplementation might mitigate bacterial infection in patients with CLDs.

Bacteria raised higher CRP levels in patients with CLDs with low albumin than CLD patients with high albumin. HSCs and SECs instead of SECs could be demolished by bacteria. However, albumin could protect KCs rather than HSCs from the cell damage caused by bacteria.

**Figure 5.** A proposed role of albumin in patients with CLDs and different hepatic non-parenchymal cells.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/cells10092298/s1, Figure S1: Correlation between albumin and various clinical characteristics.

**Author Contributions:** H.L., A.W., J.Z. and C.J.S. conceived and designed the experiments; H.L., Y.F. and J.Z. performed the experiments; H.L., Y.F. and C.J.S. analyzed the data; A.W., I.R., H.N., J.M., A.L.G. and C.J.S. contributed reagents/materials/analysis tools; J.Z., A.W. and C.J.S. wrote the manuscript; I.R., A.L.G. and C.J.S. provided guidance for the research. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Deutsche Forschungsgemeinschaft under Grant Numbers DFG STE 1022/2-3 and DFG STE 1022/4-1, and the Human Tissue and Cell Research Foundation, a non-profit foundation regulated by German civil law, which facilitates research with human tissue through the provision of an ethical and legal framework for prospective sample collection.

**Institutional Review Board Statement:** The framework of HTCR Foundation has been approved by the ethics committee of the Faculty of Medicine at the LMU (approval number 025-12) as well as the Bavarian State Medical Association (approval number 11142) in Germany.

**Informed Consent Statement:** Written informed consent has been obtained from the patient(s) to publish this paper.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors thank Elisabeth-Ingrid Liß for her excellent technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

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**5. Conclusions and outlook**

For WACC 0%, the system profits of 14.1 k USD are distributed to the domestic

Altogether, there is a strong impact of the costs of capital on the financial and technical parameters of the system. The maximum field capacity is utilized up to the WACC 20% scenario, and even for WACC 30%, the food production of a leastcost system would be sufficient to meet the domestic demand. An increase in the cost of capital by 1% leads to a decrease in system profits by 880 USD. The required funds to finance long-term assets amount to 139.0 k USD for WACC 0% and decrease from there on as CapEx-intensive technologies such as PV are increasingly

The costs, revenues, and profit for scenario S3 are depicted in **Figure 12**. For WACC 0%, the cost-minimizing system is designed in a dimension that the maximum farmland capacity is utilized, regardless of the available power generation technologies. Since revenues are directly proportional to the maize grain production, annual revenues are constant at 52.6 k USD. It can be clearly seen that system costs rise with the constraints on combination of power generation technologies. While the total annual costs for the fully fledged system amount to 38.5 k USD, the omission of biogas leads to a cost increase by 29%, while the omission of biogas and photovoltaics leads to an increase by 74% to 66.8 k USD. Hence, a system in which electricity is exclusively generated from diesel is not even net-profitable in a fully socially financed scenario and thus cannot sustainably provide the domestic community with energy and water free of charge. In order to cover the net losses, 46.4 USD per capita and year or 0.34 USD/kWh are charged for electricity. As the WACC increases to 15%, only the fully fledged EWF system operates at full food production, while the omission of biogas reduces the agricultural productivity by 16% and the absence of both renewable energy sources reduces the productivity by 68% to 70 tons per year, which is just sufficient to feed the domestic community. While the fully fledged EWF system breaks even, the unavailability of biogas prevents the systems from being profitable. Net losses for the DG + PV EWF system of 15.4 k USD and 17.3 k USD for the pure DG EWF system imply annual electricity and water expenses of 50 USD and 56.3 USD per capita, respectively; allocated to power consumption, this equals 0.36 USD/kWh and 0.41 USD/kWh, respectively. In the WACC 30% scenario, none of the EWF systems utilizes the maximum field capacity. While the fully fledged system still produces enough maize grain to provide for the domestic community (104 tons), the DG + PV EWF system and the pure DG EWF system produce just 13 tons and 8 tons, respectively. As the trend of declining profitability with an increase in WACC continues to proceed, even the

*Costs and revenues variation in power generation technology choice for WACC = 0, 15, and 30%.*

substituted with investment light technologies such as diesel.

**4.5 Results of technology variation scenario S3**

community, corresponding to 45.61 USD per capita—0.10 more than the total expenses required for food. The market-based financing scenario (WACC 15%) breaks even (net loss of 1.8 USD). A finance system with WACC 30% generates a loss of 10.4 k USD, which implies an electricity price of 0.25 USD/kWh or annual costs of 33.84 USD per capita for electricity and total costs of 79.45 USD per capita

for energy, water, and food.

*Regional Development in Africa*

**Figure 12.**

**112**

This contribution presents an economic analysis of decentralized energy-waterfood systems and their capability to provide economic-feasible solutions for rural electrification and thus the potential to enable economic development of the rural population in sub-Saharan Africa. Their decentralized design avoids the financial and governmental obstacles coming with electrification through grid extension. Biogas motors as controllable power generators substitute the costly and environmental unfriendly use of diesel generators. Although the deployment of water pumps increases the system investment costs, they lead to two major advantages compared to micro-grids without their utilization. Firstly, they are flexible loads opposite to most private power consumers (e.g., light bulbs). The water pumps are powered by cheap solar power during daytime with little or even without use of costly battery storage. Secondly, water pumps are productive power consumers opposite to private consumption, because their utilization enables year-round agriculture, which increases local productivity. Hence the local population is enabled to pay back the investment costs despite their formerly low purchase power. The leastcost modeling on the case study of the rural community Kpori, a 300-inhabitant farming village in northern Ghana, confirmed this hypothesis. The system integration of biogas generators and water pumps to closed-loop energy-water-food systems reduces the costs significantly compared to current electrification approaches with diesel generators only or diesel generators combined with solar photovoltaics and batteries. The decreased demand of costly batteries and diesel and increased profits from year-round agriculture lead to annual costs of 2 USD for the for electricity and water supply of the community compared to 17,326 USD for power supply just with diesel, assuming WACC of 15% and that the profits from agricultural sales subsidize the power supply. The cost analysis of these modeling results shows that 37% of the costs are spent for farming salaries and just 9% on CAPEX but 21% on capital costs due to the WACC of 15%. The remaining costs result from costs for fuel and other operation costs such as maintenance. The conducted variation of WACC showed on the one hand that this has a strong impact on the LCOE, which are 0.08 USD/kWh for WACC of 0%, 0.22 USD/kWh for WACC of 15%, and 0.29 USD/kWh for WACC of 30%. On the other hand, increasing WACC leads to significant reduction of installed PV capacities and increased share of power from diesel generators. The utilization of biogas is almost independent of the WACC

because of its low CAPEX and constrained maximum capacity due to shortage of livestock manure as input. Based on this model results, decentralized energy-waterfood systems have shown their potential to enable LCOE below state-of-the-art offgrid systems and local job creation through improved agricultural productivity.

In order to prove the potential of decentralized energy-water-food systems, they must be implemented on-ground including research on the optimal management and ownership structures; professional requirements for its managers, technicians, and farmers; as well as possible investment strategies. Also, the least-cost model shall be improved regarding more detailed modeling of groundwater availability, nutrients in the soil, water consumption of different crops, and biogas digestion of various inputs. After adding these improvements of the model, it shall be disseminated to and used by interested NGOs and social enterprises. Thereby, decentralized energy-water-food systems could prove their potential to improve access to reliable energy, water, and food supply, to create local jobs, and thus to fight extreme poverty of the population in rural sub-Saharan Africa.

**Unit Value**

**Unit Value**

**Unit Value**

**Unit Value**

**Unit Value**

Technology — Lead-acid Depth of discharge — 60 Energy investment cost capacity USD/kWh 350 Power investment costs USD/kW 300 Energy fixed costs USD/kWh/year 10 Power fixed costs USD/kW/year 30 Variable costs USD/kWh 0 Round-trip efficiency % 85 Lifetime Year 10

Load efficiency % 29 Minimum load % 40 Investment costs USD/kW 675 Fixed costs USD/kW/year 10 Variable costs USD/kWh 0.01 Lifetime Year 15

Maximum installed capacity ton/h 0.0153 Investment costs USD/ton/h 788.4 k

Variable costs USD/ton 2.1 Lifetime Year 20

Fixed costs % 3.5% of investment costs

Material — Plastic (PVC) Investment costs USD/m3 60

Fixed costs USD/kW/year 20 Variable costs USD/kWh 0 Lifetime Year 25

*Economic Development of Rural Communities in Sub-Saharan Africa through Decentralized…*

**Table A3.**

**Table A4.**

**Table A5.**

**115**

**Table A2.**

*Techno-economic parameters for battery.*

*Techno-economic parameters for biogas generator.*

*Techno-economic parameters for biogas digester.*

*Techno-economic parameters for solar photovoltaics.*

*DOI: http://dx.doi.org/10.5772/intechopen.90424*


**6.1.3 Effects of the shear rate on the liposome properties**

*Mean diameter -* Additional data allowed the construction of the curve presented in Figure 8A. The curve shows a clear relationship between liposome comminution and shear rate, with the mean diameter exponentially decaying with applied shear rate. The error bars are higher at lower shear rates due to the poor homogenization of liposomes provided by the lower pumping capabilities of the mechanical systems.

The microchannel microfluidizer, working in the pressure range of 200 to 1500 bars, provided shear rates in the range of 2×105 to 6×105 s-1 .The data from the microchannel microfluidizer were obtained for pre-treated liposomes using Ultra-Turrax at shear and feed flow rates of 5600 to 24000 s-1 and 0.09 to 0.96 mL.s-1, respectively. The results show that the pre-formed liposomes reached the nanometric range (100 nm) in only one passage using Ultra-Turrax and the high shear rate range of the microchannel microfluidizer.

This comminution behavior agrees with the results reported by Diat et al. (1993a). Through a balance between elastic and viscous forces in the liposomes, the mean diameter is reduced according to the square root of the applied shear rate (Equation 21).

Technological Aspects of Scalable Processes for the

0.0 0.2 0.4 0.6

q [Å-1]

**Sample Period[Å] Bilayer**

η

various shear treatments compared to control (Bangham's method).

A B C

Fig. 10. Transmission electronic microscopy of liposomes obtained using (A) Bangham's method followed by extrusion in membranes, (B) ultra-turrax under a shear rate of 21430 s-1, (C) microchannel microfluidizer under a shear rate 6 961 000 s-1. Bars :200nm

liposomes.

I(q) [arb. u.]

*N is the average number of bilayers and*

**<IP_ADDRESS> Morphology**

Production of Functional Liposomes for Gene Therapy 285

lipids, and stiffness is an extensive property dependent on the structure and packing of the aggregate. Therefore, we associated the changes in the slope of the straight lines in Figure 8B with changes in the elastic constants as a consequence of the changes in packing in the

> -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

Δρ [arb. u.]

 no shear extrusion microfluid turrax

**thickness[Å] N**η

r[Å]

turrax microfluid

extrusion

no shear

Fig. 9. Left: Small angle X-ray scattering (SAXS) profiles determined for the liposomes treated under moderate shear (extruded through polycarbonate membranes) and high shear produced by Ultra-Turrax*®* (21430 s-1) and microchannel microfluidizer devices (6 961 000 s-1).

Right: Electron density profile across the bilayer obtained from the fitting (see Table 3).

*no shear* 76.7±0.2 59,9±0.1 3.2±0.1 0.18±0.02 *Extrusion* 73.4±0.1 59,3±0.1 3.4±0.1 0.26±0.01 *Turrax* 72.5±0.2 59,1±0.1 4.1±0.1 0.26±0.01 *Microfluid* 72.3±0.1 59,2±0.2 2.3±0.1 0.09±0.01

 *is the Caillé parameter.*

Figure 10 presents transmission electronic microscope images of the liposomes under

Table 3. Structural parameters obtained from full curve modeling of SAXS data.

Fig. 8. Cumulative hydrodynamic mean diameter as a function of the shear rate provided by Caules stirrer, Ultra-Turrax®, or microchannel microfluidizer. (A) Z-average values obtained from light scattering measurements. (B) Linear relationship from Equation 21 proposed by Diat et al. (1993a).

$$R = \sqrt{\frac{4.\pi.\left(2.k + \overline{k}\right)}{\mu.d.\dot{\gamma}}}\tag{21}$$

Where R is the liposome radius at equilibrium, k and k are the average and Gaussian elastic constant of the membrane, respectively, µ is the viscosity of the liposome dispersion, ߛሶ is the shear rate, and d is the interlamellar distance. The different slopes for the straight lines in Figure 9B were obtained for the shear rate ranges of the devices used, agreeing with the mass balance between elastic and viscous forces in the liposomes described by Diat et al. (1993b). Higher shear rates produce higher slopes, indicating the presence of liposomes with higher elastic constant, lower viscosity, and shorter interlamellar distance.

#### **<IP_ADDRESS> Viscosity, surface tension, and zeta potential**

The shear rate also reduced the viscosity of the liposome dispersion from 5 to 2 mPa, but no significant changes were observed in the surface tension as a consequence of the reduction in size. However, the reduction in size also resulted in rearrangement of the lipids in the external layer, changing the zeta potential from -50 to -40 mV.

#### **<IP_ADDRESS> Lamellar packing**

Small angle X-ray scattering (SAXS) characterization showed changes in the interlamellar distances for the applied shear rate ranges (Figure 9). The distance decreased with higher shear rates. The decreasing interlamellar distance was a consequence of the loss of interlamellar water, calculated to be up to 12% for the highest level of shear rate, as well as of lipid packing in the liposome, found to be less than 2%. The water layer is calculated as the difference between the full period and the bilayer thickness (see Table 4). These factors may explain the observed changes in zeta potential and the different slopes of the straight lines obtained for the studied shear rate ranges. In addition to the interlamellar distance and packing, the elasticity of the bilayer may also be influenced by the shear rate and changes in the elastic constants determined. In general, elastic modulus is not the same as stiffness. Elastic modulus is a property of the constituent material, whereas stiffness is a property of the structure. In the case of liposomes, the elastic modulus is an intensive property of the

A B

Cumulative mean hydrodynamic diameter (nm)

Fig. 8. Cumulative hydrodynamic mean diameter as a function of the shear rate provided by Caules stirrer, Ultra-Turrax®, or microchannel microfluidizer. (A) Z-average values obtained from light scattering measurements. (B) Linear relationship from Equation 21

π

*R*

higher elastic constant, lower viscosity, and shorter interlamellar distance.

**<IP_ADDRESS> Viscosity, surface tension, and zeta potential**

0 5000<PHONE_NUMBER>2000025000 2500000 5000000 7500000

 Ultra-Turrax Cowles Microfluidizer

Shear rate (s-1 )

external layer, changing the zeta potential from -50 to -40 mV.

4. . 2. ( ) . . *k k*

*d*

+ = (21)

0,000 0,005 0,010 0,015 0,020 0,025 0,030

(Shear rate)-1/2 (s1/2)

 Ultra-Turrax Cowles Microfluidizer

μ γ

Where R is the liposome radius at equilibrium, k and k are the average and Gaussian elastic constant of the membrane, respectively, µ is the viscosity of the liposome dispersion, ߛሶ is the shear rate, and d is the interlamellar distance. The different slopes for the straight lines in Figure 9B were obtained for the shear rate ranges of the devices used, agreeing with the mass balance between elastic and viscous forces in the liposomes described by Diat et al. (1993b). Higher shear rates produce higher slopes, indicating the presence of liposomes with

The shear rate also reduced the viscosity of the liposome dispersion from 5 to 2 mPa, but no significant changes were observed in the surface tension as a consequence of the reduction in size. However, the reduction in size also resulted in rearrangement of the lipids in the

Small angle X-ray scattering (SAXS) characterization showed changes in the interlamellar distances for the applied shear rate ranges (Figure 9). The distance decreased with higher shear rates. The decreasing interlamellar distance was a consequence of the loss of interlamellar water, calculated to be up to 12% for the highest level of shear rate, as well as of lipid packing in the liposome, found to be less than 2%. The water layer is calculated as the difference between the full period and the bilayer thickness (see Table 4). These factors may explain the observed changes in zeta potential and the different slopes of the straight lines obtained for the studied shear rate ranges. In addition to the interlamellar distance and packing, the elasticity of the bilayer may also be influenced by the shear rate and changes in the elastic constants determined. In general, elastic modulus is not the same as stiffness. Elastic modulus is a property of the constituent material, whereas stiffness is a property of the structure. In the case of liposomes, the elastic modulus is an intensive property of the

proposed by Diat et al. (1993a).

Cumulative mean hydrodynamic diameter (nm)

**<IP_ADDRESS> Lamellar packing**

lipids, and stiffness is an extensive property dependent on the structure and packing of the aggregate. Therefore, we associated the changes in the slope of the straight lines in Figure 8B with changes in the elastic constants as a consequence of the changes in packing in the liposomes.

Fig. 9. Left: Small angle X-ray scattering (SAXS) profiles determined for the liposomes treated under moderate shear (extruded through polycarbonate membranes) and high shear produced by Ultra-Turrax*®* (21430 s-1) and microchannel microfluidizer devices (6 961 000 s-1). Right: Electron density profile across the bilayer obtained from the fitting (see Table 3).

*N is the average number of bilayers and* η *is the Caillé parameter.*

Table 3. Structural parameters obtained from full curve modeling of SAXS data.


**5. Conclusions**

The research deepened the debate on sustainable development by relating the Quintuple Helix Innovation Model (QHIM) to the Sustainable Development Goals (SDGs). The literature review identified knowledge gaps in the implementation of the SDGs. For this reason, the study proposed 20 indicators related to the QHIM through a case analysis in Latin American countries to respond to the identified gaps. The study also applied a Fuzzy Logic algorithm to identify forgotten effects that may affect the SDGs' achievement, confirming the case study's findings. The results revealed the importance of correctly identifying cause-effects by seeking harmony between systems.

As theoretical contributions, the research advanced the frontier of knowledge by reducing the identified gaps, and at the same time, contributes to sustainable development with the proposed models. As practical contributions, the applied study offers governments, society, academia, and companies solutions adjusted to the problems identified, such as the lack of integration and systemic vision to achieve SDGs. Also, the research involved the participation of stakeholders in decision making.

Therefore, the present study is novel and useful for various stakeholders. A limitation of the research may be the number of variables used. Finally, the study results indicate promising research lines on sustainable development and decision making in uncertain environments applied to other countries. The study also opens up new research opportunities in prioritization models that facilitate decision-making.

**Author Contributions:** Conceptualization, L.B.-P. and I.D.l.V.; methodology, L.B.-P. and A.M.G.-L.; software, A.M.G.-L.; validation, L.B.-P., I.D.l.V., and A.M.G.-L.; formal analysis, I.D.l.V.; investigation, L.B.-P., and I.D.l.V.; resources, L.B.-P. and A.M.G.-L.; writing—original draft preparation, L.B.-P.; writing—review and editing, L.B.-P. and I.D.l.V.; supervision, A.M.G.-L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors wish to thank The Royal Academy of Economic and Financial Sciences and the University of Barcelona from Spain, and CENTRUM Católica Graduate Business School, Peru.

**Conflicts of Interest:** The authors declare no conflict of interest.


**2.2 Cloud system and big data analysis technology**

This research is based on the fact that FTIR operates continuously for 24 hr and captures signals every second to obtain data throughout the year as a basis for big data. The main research significance is not to grasp the huge data information, but to professionally process these meaningful data, so that FAB factory managers can know whether the exhaust emissions meet the alert or warning potential under the sensing trend. Most of the research uses the big data platform for deployment, debugging and maintenance. This study is connected to the Chief Cloud eXchange (CCX) cloud database platform and the Spark big data platform. At the same time, these two platforms are also more suitable for primary systems to implement systems. **Figure 6** is the research cloud computing system and database architecture.

For the big data of the chip process exhaust obtained by RFID, full consideration should be given to (1) big data life cycle, (2) big data technology ecology, (3) big data acquisition and preprocessing, (4) big data storage and management, (5) Big data computing model and system.

The big data analysis method used in this study is described as follows. U is defined as the non-empty initial universe of the object. Then define E as a set of parameters related to the object in U. Let P (U) be the power set of U, and A ⊂ E.  (U). In other words, the soft set on U is a parameterized family of Universe U subsets.

In addition, if the universe set U is a non-empty finite set, and σ is the equivalent relationship on U. Then (U, σ) is called approximate space. If X is a subset of U, then X can be written as a union of equivalent classes of U or not. If X can be written as a union of equivalent classes of U, then X is definable, otherwise it is undefinable. If X is undefinable, it can be approximated as two definable subsets, called the upper and lower approximation of X, as shown below Eq. (6) [22, 23].

$$\underline{\operatorname{app}}(X) = \cup \{ [\chi]\_{\sigma} : [\chi]\_{\sigma} \subseteq X \},$$

$$\overline{\operatorname{app}}(X) = \cup \{ [\chi]\_{\sigma} : [\chi]\_{\sigma} \cap X \neq \mathcal{O} \}. \tag{6}$$

**Figure 5.** *The absorption spectrum of several gas molecules in the infrared range.*

intensity of the light beam in this wavelength band is weakened, and the ratio of light intensity before and after absorption is The concentration of the gas is directly related. The absorption band and intensity of the gas sample can be measured to know the composition and concentration contained in the gas. For a maximum path difference d adjacent wavelengths λ1 and λ2 will have n and (n + 1) cycles respectively in the interferogram. The corresponding frequencies are ν1 and ν2, and the

FTIR mainly emits a beam of light to the measurement area and measures the intensity change of the beam after passing the gas to be measured. Since each gas molecule has its specific infrared light absorption coefficient, when the light beam passes through the measurement area, the specific gas molecule will absorb light of a specific wavelength, so that the intensity of the light beam in this band is reduced, and the ratio of the light intensity before and after absorption The concentration of the gas is directly related, and the absorption band and intensity of the gas sample

d ¼ nλ1 and d ¼ ð Þ n þ 1 λ2 (1) λ1 ¼ d*=*n and λ2 ¼ d*=*ð Þ n þ 1 (2) ν1 ¼ 1*=*λ1 and ν2 ¼ 1*=*λ2 (3) ν1 ¼ n*=*d and ν2 ¼ ð Þ n þ 1 *=*d (4)

ν2 � ν1 ¼ 1*=*d (5)

membership function in the following Eqs. (1)�(5) [20, 21]:

*Linked Open Data - Applications,Trends and Future Developments*

*The instrument configuration of the pumped FTIR.*

**Figure 4.**

**84**

**Figure 6.** *The research cloud computing system and database architecture.*

The process of data decomposition is defined as follows: Let X is defined as the number of groups and Y as several data, as shown below Eqs. (7)�(8).

$$\mathbf{X} = (\mathbf{Y}/\mathbf{10}, \mathbf{000})\tag{7}$$

Find k, for which ck = max ci.

*DOI: http://dx.doi.org/10.5772/intechopen.92849*

hk is the optimal choice of value for the selected object. If k has more than

*Study on IoT and Big Data Analysis of 12" 7 nm Advanced Furnace Process Exhaust Gas Leakage*

Forming up the n � n discernibility matrix. The elements of S table is defined as d(x, y) = a ∈ A | f (x, a) ̸= f (y, a), d(x, y) is an attribute. set distinguishing x and y. For each attribute a ∈ A, if d(x, y) = a1, a2, ...,

Formulate the Boolean function a1 ∨ a2... ∨ ak or discernibility function which represented by Σ d(x, y) as indicated: F (A) = Π (x,y)∈U � U) Σ d

If d(x, y) = ∅, constant 1 will be assigned to the Boolean function. Execute the attribute reduction process based on the simplified Boolean

In the reactor, chemical reaction is used to form the reactant (usually a gas) into a solid product, and a thin film is deposited on the surface of the wafer. This process is called CVD (Chemical Vapor Deposition). This process has (1) good step coverage, (2) energy with high aspect ratio gap filling, (3) good thickness uniformity, (4) high pure and dense, (5) when ratio can be controlled, (6) low stress for high film quality, (7) good electrical properties, (8) base plate and excellent film adhesion characteristics. **Figure 7** shows the system architecture of CVD

The precipitation of products during the CVD process can be divided into the following steps: (1) the source gas diffuses to the substrate surface, (2) the substrate adsorbs the source gas, (3) the substances adsorbed on the substrate react chemically on its surface, (4) The precipitated material diffuses on the surface of the substrate, (5) The reaction product is separated from the gas-phase reactant, (6) The precipitated non-volatile material is deposited on the substrate surface by diffusion and the like. The composition, structure and performance of the products obtained in this chemical reaction can be controlled by changing the parameters of the reaction. The reaction parameters mainly include the type of gas, the gas reaction concentration, the delivery method of the reactant, the gas flow rate, the total gas pressure and the Area pressure, heating method, substrate material, substrate surface state, substrate reaction temperature, temperature distribution and

When entering the 7-nanometer process, the channel material of the semiconductor PN junction must also be changed. Since the electron mobility of silicon is 1500 cm2 / Vs, and germanium can reach 3900 cm2 / Vs, and the implementation

*j*

*hi*,*j* and hi,j is the

one value, any one of the benefits could be chosen. ci is the choice of value of an object hi where *ci* ¼ P

entries in the table of the reduct soft set. Algorithm (3): Rough set parameter reduction algorithm.

f is an information function that maps an. object in U to exactly one value in V. Output: Simplified reduct sets. Input the information Table S.

New optimized reduct sets are generated.

Input: An information system S = (U, A, V, f). U is a finite nonempty set object. A is a finite nonempty set of attributes.

V is a nonempty set of values.

Discretization of data.

ak ̸= ∅.

(x, y).

function.

in FAB.

gradient, etc.

**87**

**2.3 Process design and discussion**

If X contains remainder, then

$$\mathbf{X} = \mathbf{X} + \mathbf{1} \tag{8}$$

Where the number of groups will be added to 1.

Algorithm (1): The most optimized attribute set searching algorithm.

Select the highest number of attribute values, HR if HR does not have the same number with attribute value AND HR has more than one value then.

Select the first reduction set, FR of attribute values.

else

Proceed to the next process

else

Proceed to the next process

Algorithm (2): Soft set parameter reduction algorithm.

In tabular representation, let (F, P) represent the soft set. If Q is the reduction of P, the soft set reduction set is defined as (F, Q) of the soft set (F, P) where P ⊂ E. Input: A soft set (F, E), set P.

Output: Optimal decision.

Input the set P of choice parameters.

Find all reducts of (F, P).

Select one reduct set (F, Q) of (F, P).

Find weighted table of soft set (F, Q) according to the decided weights.

Find k, for which ck = max ci. hk is the optimal choice of value for the selected object. If k has more than one value, any one of the benefits could be chosen. ci is the choice of value of an object hi where *ci* ¼ P *j hi*,*j* and hi,j is the entries in the table of the reduct soft set. Algorithm (3): Rough set parameter reduction algorithm. Input: An information system S = (U, A, V, f). U is a finite nonempty set object. A is a finite nonempty set of attributes. V is a nonempty set of values. f is an information function that maps an. object in U to exactly one value in V. Output: Simplified reduct sets. Input the information Table S. Discretization of data. Forming up the n � n discernibility matrix. The elements of S table is defined as d(x, y) = a ∈ A | f (x, a) ̸= f (y, a), d(x, y) is an attribute. set distinguishing x and y. For each attribute a ∈ A, if d(x, y) = a1, a2, ..., ak ̸= ∅. Formulate the Boolean function a1 ∨ a2... ∨ ak or discernibility function which represented by Σ d(x, y) as indicated: F (A) = Π (x,y)∈U � U) Σ d (x, y). If d(x, y) = ∅, constant 1 will be assigned to the Boolean function. Execute the attribute reduction process based on the simplified Boolean function.

New optimized reduct sets are generated.


**8. Conclusion**

Citrus fruit is an evergreen tree that needs all the essential nutrients for proper metabolic functioning. A balance between macronutrients and micronutrients is needed to optimize the yield of high-quality fruit and maintain healthy trees that are tolerant to pests, diseases, and other unfavorable conditions. Citrus fruits are valuable sources of potassium, which is needed to ensure water and electrolyte balance in the body. More so, increase consumption of citrus fruits and products provide virtually all the mineral elements needed by the body to maintain good health and prevention of degenerated diseases.


**8. Remediation of Hg-contaminated soils and water**

Increasing Hg emissions due to anthropogenic activities caused severe soil pollution issues [44–46]. As a crucial link between the atmosphere and water, soil plays

#### *Perspective Chapter: The Toxic Silver (Hg) DOI: http://dx.doi.org/10.5772/intechopen.111464*

a central role in the global Hg cycle [47]. Soil is not only an Hg sink, receiving Hg input from the environment but also reemitting it to the atmosphere [48, 49], water [42, 50], or the plants grown thereon [39].

Mercury, like other potentially toxic elements, is not biodegradable, and therefore, its remediation should encompass either removal from soil or immobilization [32]. The main Hg removal technologies are physical and chemical remediation methods, as well as bioremediation technology. Adsorption of Hg2+ and Hg(0) from water on surfaces of high surface area and high porosity such as chitosan derivatives, synthesized thioether-functionalized covalent triazine nanospheres, pentasil zeolite (type ZSM-5), and utilized silica-coated magnetic nanoparticles are the most common physical approaches for remediating Hg-contaminated soil [51]. Other techniques could help such as soil replacement, physical separation, soil vapor extraction, fixed/stabilized soil, vitrification, thermal desorption, and electrokinetic remediation technology [6]. The latter technique (electrokinetic remediation) depends on passing a direct current between electrodes through the soil to make the Hg ions move through an ion exchange membrane from the soil to the electrodes. The addition of chelating agents to soil could effectively increase the solubility and removal efficiency of Hg [6]. Recently, He and his research group introduced a novel *in situ* immobilization technology by injecting stabilized iron sulfide nanoparticles into soil to immobilize Hg [32].

The *in situ* thermal desorption is a promising technique of Hg remediation that does not need to dig up the contaminated soil; instead, thermal conductive heating (TCH) elements are inserted into the soil in order to directly transfer heat to above 600°C to volatilize various species of mercury, such as HgO, HgS, HgCl2, and mercury associated with organic matter and thus achieve an acceptable decontamination level [51].

Biological remediation/bioremediation depends on plant and microbial in remediation soils [6]. In particular, genetically engineered plants can change methylmercury complexes, and mercury ions into metallic forms of lower toxicity, and then extract, detoxify, and/or sequester this contaminant from soil and water [10]. Phytoremediation is an umbrella term, which refers to the different low cost and ecofriendly technologies that utilize plants in decontaminating areas [52]. This includes: phytostabilization, phytoextraction, and phytovolatilization. This *in situ* application of phytoremediation lessens the disturbance of the surrounding environment and also declines the spread of contamination *via* air and water [32]. There are many other technologies such as the use of nanoparticles to remove/absorb Hg from soil, water, and flue gas, owing to their high adsorption capacity, small dimension, and other unique electrical, mechanical, and chemical properties [53].

Continuous monitoring of Hg levels in air, soils, water, and foods is necessity to ensure their sustainable safe use in order to protect human health and the surrounding ecosystem. In addition, increasing the awareness of humans about the danger of Hg is a proactive step to prevent and reduce the danger of Hg pollution. Furthermore, remediation protocols should be followed in Hg-contaminated areas to lessen its toxicity.


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## Meet the editor

Mário Franco is an Associate Professor of Entrepreneurship and SME Administration at the Department of Management and Economics, Beira Interior University, Portugal. He received his Ph.D. in Management from Beira Interior University in 2002. In 1997, he was a doctoral candidate and participated in the European Doctoral Programme in Entrepreneurship and Small Business Management in Spain and Sweden. He is the director

of the 2nd Cycle (Master) of Management. His research focuses on strategic alliances, business networks, innovation, and business creation. He is a member of a research unit at the Center for Advanced Studies in Management and Economics of the University of Beira Interior (CEFAGE-UBI) and is currently involved in several research projects on SMEs. He has authored several articles published in journals such as Long Range Planning, R&D Management, International Entrepreneurship and Management Journal, and Management Decision, and some books and book chapters published by international publishers.

Contents

**Section 1**

*by Elif Bilginoğlu*

the Theory X Style

*and Maureen Metcalf*

Enterprises (SMEs)

**Section 2**

**Preface XI**

Leadership Guided by Theoretical Perspectives **1**

**Chapter 1 3**

**Chapter 2 27**

**Chapter 3 39**

Leadership and Innovation and SMEs **55**

**Chapter 4 57**

**Chapter 5 85**

**Chapter 6 101**

A Contemporary Approach to Learning Transfer from the Perspective of Constructivist Theory: Marathon Running as a Leadership Development Strategy for Millennial Leaders

Turning Bad Leaders into Good Leaders: Diminishing

Humane Leadership in Islamic Approaches

*by Iffah Budiningsih and Tjiptogoro Dinarjo Soehari*

Optimizing Innovative Leadership and Followership *by Neil E. Grunberg, Erin S. Barry, Michael Morrow-Fox*

*by Ahmad Rafiki, Miftahuddin and Atika Rizki*

Spirituality Leadership Applications in Small and Medium

*by Mohd Faridh Hafez Mhd Omar*

*by Lindall Elaine Adams, Marion Williams and Ibrahim Al Zaabi*

Strengthening Innovative Leadership in the Turbulent Environment

### Contents

Preface

Organizations are part of increasingly volatile societies marked by rapid digital and technological transformations. The technological life cycle tends to be shorter now than it has been in the past and this is impacting organizational performance. In the face of this rapid evolution, the obsolescence of business technology is a short-term

In this context, leadership is viewed as an important issue because the continuity of organizations and their success depends on their leaders and the behaviours they demonstrate. How a leader behaves or how they access and "use" the various leadership styles can impact all stakeholders. Leadership responsibility can enable organizations to achieve a successful future. Thus, leadership plays an important role in how workers interpret and carry out their activities. Leadership and its influence on organizations are key concepts in how organizations/companies work and how they are insighted. Leadership is a process that influences the behaviour of

In this sense, understanding leadership is extremely important for an organization as well as the environment around it, since organizations are inserted in a social, economic, and environmental context where actions have effects and impacts. In addition, leadership should align with the goals and objectives of organizations to facilitate the achievement of those same goals and objectives. The leadership style that a leader presents can have a considerable impact on organizations because the market in which they are located is volatile and there are too many factors that condition their survival. Leadership, when linked with the organization's strategy, can influence the organization's entire process, as it establishes a path towards better

However, leadership has become a complex, difficult, and multi-faceted issue for organizations because it entails challenges and issues, for example, determining what is the best type of leader. Therefore, research in the area of leadership has been obscured rather than discussed. Despite the significant body of literature on the subject, leadership remains quite misunderstood in the organizational world.

Leadership has given rise to several studies and involves a wide spectrum of relevant frameworks to explain the components of leaders' behaviour; however, there is still a vast field for research in search of more conceptual transparency, intrinsic characteristics, and the impact it has on everything that surrounds it. Therefore, the importance of leadership has been widely recognized in the literature, evidenced by the notable increase in relevant publications in the past few years. As such, this book is devoted to

the study of different perspectives, contexts, and new insights into leadership.

for Millennial Leaders" by Elif Bilginoğlu, presents a literature review of the

Chapter 1, "A Contemporary Approach to Learning Transfer from the Perspective of Constructivist Theory: Marathon Running as a Leadership Development Strategy

leaders to perform proposed tasks and achieve defined objectives.

involvement and performance of the parties involved.

The book includes four sections and eleven chapters.

reality if organizations are not in tune with reality.


*3.3. Effect of System Temperature on Power Conversion*

To determine how the system temperature affects the power-generation efficiency of this BIPV system, monthly power conversion and system temperature were compared and analyzed, and tabulated in Table 4. Figure 8 shows that the trends of system temperature and power conversion are opposite to each other. The system temperature trends followed the seasonal changes in South Korea, i.e., the system temperature increased in spring and summer and decreased in autumn and winter. Conversely, power conversion decreased in spring and summer and increased in autumn and winter.

**Table 4.** Power conversion from January 2020 to June 2022.

**Figure 8.** Power conversion and system temperature in (**a**) 2020, (**b**) 2021, and (**c**) 2022.

As mentioned previously, the system temperature is one of the factors affecting the power generation performance of a BIPV system [21,26–33,35–37]. In this BIPV roofing system, it was apparent that the system temperature affected the performance of the PV modules. The highest efficiency of 11.42% was achieved in December 2020, when the system temperature was the lowest. In contrast, the lowest efficiency of 5.24% occurred in July 2021, when the system temperature was 10 ◦C above the ambient temperature. This correlation shows how a system temperature increase negatively impacts the PV module efficiency, dropping to half its maximum value at the peak system temperature. A PV temperature increase leads to an increase in carrier concentration, thereby enhancing the rate of carrier recombination and leading to decreased open-current voltage (VOC), fill factor (FF), and thus performance [38].

Figure 9 shows that during the 2.5-year observation period, there was no significant decline in system performance regarding power conversion. The largest drop in power conversion was in July 2021, which decreased by ~1% compared to the previous year. Such a large decline in efficiency was not evident in the rest of the months when efficiency decreased on average only by 0.25% compared to the values in the previous years. These findings confirm that this particular BIPV system can maintain its performance with a negligible effect on efficiency over 2.5 years.

**Figure 9.** Comparison of monthly power conversion from January 2020 to June 2022.

### **4. Conclusions**

The long-term performance of a BIPV system of 160 Wp was evaluated using outdoor monitoring in South Korea for 2.5 years. This study revealed the intercorrelations between power generation, solar irradiance, and system temperature. While solar irradiance mainly affected power generation, system temperatures contributed to power generation fluctuations, following the seasonal changes from cold to warm and again to cold temperatures throughout the year. Results showed that half of the BIPV system's performance was lost due to temperature fluctuations. Therefore, addressing this issue is crucial, particularly in hot regions. Factors such as wind direction, ventilation, and the type of PV technology could play important roles in achieving better economic value for the installed PV system. The temperature behind the PV modules was lower than that behind the roof tiles, implying that installing such a BIPV system will not increase the temperature of the roofing system. This finding suggests that the comfort of the room underneath the PV modules may be uncompromised by using this configuration in the climate of South Korea. Therefore, expanding the use of PV modules as an entire roofing system by using this configuration has great potential. This study proves that one of the generally associated temperature concerns—the roofing temperature—is not necessarily concerned with this BIPV system configuration.

**Author Contributions:** Conceptualization, W.K.K., M.H.A.A., S.A. and S.J.; Methodology, W.K.K., M.H.A.A., S.A. and S.J.; Data curation, M.H.A.A., S.A. and S.J.; Formal analysis, M.H.A.A., S.A. and S.J.; Resources, W.K.K., J.K. (Jitaek Kim) and J.K. (Jungtaek Kim); Funding acquisition, W.K.K.; Supervision, W.K.K.; Writing—original draft, M.H.A.A., S.A. and S.J.; Writing—review and editing, W.K.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the 2021 Yeungnam University Research Grant.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.


**Author details**

*Human Blood Group Systems and Haemoglobinopathies*

As part of the quality assurance tools, the processes of prospective sample collection, stabilization and processing, conservation, and distribution in multicenter research studies are managed under a global approach based on risk analysis, aimed at preventing undesired results. Since incidents are treated in the format of corrective and preventative action (CAPA) and root cause analysis (RCA) documentation,

So, the implementation by the biobanks of a comprehensive quality control for sample collection kits before shipment to recruitment sites is a result from this risk analysis. The potential consequences (inadequate preservation of samples due to inappropriate devices, containers, or additives, lower amount of derivatives for analysis, or loss of traceability) and costs of this process (new shipment of replacement kits and time of technician dedicated to kit checking), in addition to the number of nonconforming kits detected, have been evaluated. Quality control of kits is performed with a checklist, and follow-up of kit preparation and shipments is made with a specific record. Similarly, a checklist for the recruitment sites is included to

In addition, a training activity for the staff from the recruitment sites is organized before initializing the donors' recruitment to present the procedures for sample handling and the kit composition and usage. On the other hand, a pilot in a recruitment site is necessary to validate the model designed and to propose

The biobank-supported workflow, specifically designed and implemented for each research study, allows obtaining prospective, harmonized, and quality samples avoiding pre-analytical bias and contributing to the validity of the analytical results. Through biobanking processes and continuous and overlapped checkpoints, quality derivatives are obtained based on common and evidence-based standard operating procedures and supervised materials, with associated traceability information in relation with their collection, processing, conservation, and distribution. The effective workflow established is valid for large and complex multicenter

This work was supported by Consejería de Salud y Familias-Junta de Andalucía, Ministerio de Economía y Competitividad-Gobierno de España, and Instituto de Salud Carlos III (grant number PT17/0019/0004 co-financed by FEDER). Thanks go to the staff from the Andalusian Public Health System Biobank for their continuous effort and improvements that make these supporting activities possible. Several projects have been satisfactorily attended following the

The authors have declared no conflict of interest.

**4.2 Risk management tool**

these records are used as reference.

track the reception of kits.

improvement measures.

**5. Conclusions**

projects.

**Acknowledgements**

previous premises.

**Conflict of interest**

**174**

Verónica Valdivieso-Gómez, Javier Garrancho-Pérez, Inés Aroca-Siendones and Rocío Aguilar-Quesada\* Andalusian Public Health System Biobank, Coordinating Node, Granada, Spain

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**1. Introduction**

Bridges have always fascinated humanity. They symbolize links between people, communities, and nations [1]. Many bridges were built during the large infrastructural boom between the sixties and the eighties of the last century. The automotive industry has rapidly grown during this period, driving developed countries to construct many new bridges. An overview over the period from 1925 to 2004, which shows when bridges were built in the United States (US), Japan, Germany, and the Netherlands, is shown in Figure 1 [2–5].

A recent analysis [6] of data from the US Federal Highway Administration (FHWA) and the US National Bridge Inventory (NBI) indicates a strong shift in nationwide spending from building new bridges towards rehabilitation and replacement (Figure 2). Out of the 611,845 public road bridges in the US, 58,791 (9.6%) were classified as structurally deficient in 2015, and another 84,124 (13.7%) as functionally obsolete. These figures have led to claims that the US is about to experience a crisis concerning infrastructure [7]. To some extent, a similar situation is expected in many European countries. Many of the existing bridges have now reached the age of 50 years, the originally designed

service life. Although this certainly does not mean that these bridges are unsafe, an increasing number will need to be replaced in the future. The replacement can be either due to deterioration or increased traffic loading, but also because of functional requirements. It is clear that, apart from rehabilitation, society will face a large infrastructural replacement task in the future.

**Figure 1.** Historical overview of built bridges in Japan [2], the US [3], Germany [4], and the Netherlands [5].

**Figure 2.** US bridges capital outlay and age (routine maintenance costs not included) [6].

A very common bridge type is the overpass (Figure 3). Traditionally, overpasses in the Netherlands were mainly built as cast in-situ reinforced concrete plate bridges. An advantage of a plate bridge with multiple intermediate supports compared to a single span bridge is the possibility to achieve higher slenderness due to a more favorable moment distribution. On the other hand, its main disadvantage is the increased on-site construction time needed for mounting the formwork, placing the reinforcement, and pouring and hardening of the concrete. In the past, traffic hindrance due to construction was not an issue, because these bridges were built in the open field, in the new highways. Nowadays, the Dutch highway network is one of the busiest in the world. With 3,075 km of highway in total and 73 km of highway per 1,000 km2 [8], the Netherlands has the highest highway density in the European Union. The busiest highways accommodate over 200,000 vehicles daily. As a result, the availability of adequate infrastructure is of the utmost importance for society.

**Figure 3.** Example of a common four-span plate bridge type in the Netherlands: Bridge Zijlweg of the highway A59 in the province Brabant.

Traffic jams cause major problems in highly populated areas. Besides increased stress and time delays, an increase in traffic congestions may cause stalling of the economy. The cumulative combined costs of traffic congestion for the national economies of the US, United Kingdom (UK), France, and Germany are estimated to reach a staggering 4.4 trillion dollars by the year 2030 [9]. A report by the TNO [10] shows that Dutch companies lose around 1.1 billion euros annually due to traffic jams. These losses are equal to 18% of the Dutch annual infrastructure budget of around 5.9 billion euros. Therefore, one of the main demands of owners, contractors, and society in the modern construction industry is to minimize traffic hindrance. Therefore, for the next generation of road infrastructure, rapid and low hindrance bridge replacement will be a governing task.


**5. Role of flavonoids in plants**

Plants are the key source of natural products and plants had already yielded a vast number of phytochemicals and still continue to a major source of biologically active molecules. Numerous plants have already established their potentiality as a source of naturally occurring insecticides, pesticides, fungicides and agro-chemicals as an alternative to toxic and hazardous synthetic chemicals. Owing to ever-increasing awareness to the hazardous side effects of synthetic chemicals, more and more emphasis is being given on the use of products obtained from natural sources so that ecological balance is well-maintained. The WHO has already called for an immediate ban on the use of many synthetic chemicals viz. endosulfan is a dangerous synthetic pesticide that causes severe damage to the eyes, kidneys, and liver. The Government of India had already banned the use of 12 highly toxic and hazardous pesticides and imposed restriction on the use of many others to prevent environmental pollution. To minimize the hazardous effects and to control environmental pollution, attempts are now being made to develop naturally occurring plant-based pesticides. Many phytochemicals such as phytoecdysones, and azadirachtin (from Indian neem) have been reported to possess pesticidal and insecticidal properties and are being widely used in protecting the loss of crop from the attack of insects and parasites in place of their synthetic analogues because of their non-toxic, non-pollutant, readily bio-degradable character and harmless nature of their residues.

As a result of changes in plant growth, conditions, and maturity, there are more than 10,000 types of flavonoid compounds found in vascular plants, which vary in type and quantity according to a variety of factors. There is still a lack of systematic analysis of the flavonoid content of many plant species, which makes it difficult to identify and quantify all the flavonoids humans consume [57]. In order to defend themselves against herbivores, pathogens, oxidative cell damage, and fungal parasites, plants have evolved to synthesize flavonoids [58]. On the other hand, flavonoids act as a stimulant that aids in pollination and guides insects on their way to food sources. For example, flavonoid compounds anthocyanins are responsible for the pink, blue red, light purple and violet colors in flowers, fruits, and vegetables [56, 59].

#### **5.1 Combating oxidative stress of flavonoids**

It has long been reported that flavonoids have a variety of functions in plants [60]. Both abiotic and biotic factors contribute to oxidative stress in plants as a result of ROS being generated in plants. A high level of oxidative stress commonly enhances the synthesis of flavonoids in plants. The pigments are capable of absorbing the most energetic rays of the sun (i.e., UV-B and UV-A), inhibiting ROS production, and quenching ROS once they have been generated [61]. When plants moved from water to soil, flavonoids were primarily responsible for screening UV-B. Different flavonoids have different antioxidant capacities and UV-wavelength-absorbing capabilities based on their substitutions. There is an increase in antioxidant capacity in flavonoids with dihydroxy B rings substituted for these rings, whereas flavonoids with monohydroxy B rings have a greater ability to absorb UV wave lengths. Glycosylation is generally the hallmark of the most reactive hydroxyl groups of flavonoids (7-OH in flavones or 3-OH in flavanols). Flavonoids can be transported from the endoplasmic reticulum to various cellular compartments and secreted from their plasma membrane and cell wall through glycosylation, thereby increasing their solubility in the aqueous cellular environment, protecting the reactive hydroxyl groups from auto-oxidation [62]. Studies have shown that antioxidant flavonoids are found in cells of the mesophyll and in chloroplasts, which generate ROS. Using this method, they are able to easily quench H2O2, hydroxyl radicals, and singlet oxygen [61, 63]. Conditions that restrict CO2 diffusion to carboxylation sites and carboxylation efficiency may exacerbate oxidative stress caused by an excessive amount of excitation energy in chloroplasts [61, 64]. A number of environmental factors can restrict CO2 assimilation, including drought/ salinity, temperature fluctuations, and nutrient scarcity. This can decrease the activity of ROS detoxifying enzymes in the chloroplast [65], which increases the production of antioxidant flavonoids. Flavonoids are highly important for plants under severe stress conditions because of their reducing properties. In addition to their functional roles, dihydroxy B ring substitutes are also highly concentrated [66]. It has been suggested that flavonoids represent a secondary antioxidant defense system in plants under stress [61]. In response to oxidative stress, lipid peroxidation occurs, causing cell membrane degradation. It has been suggested that quercetin-3-*O*-rutinoside (Rutin) interacts with phospholipids polar head at the water lipid interface, increasing membrane inflexibility and thereby protecting membranes from oxidative damage [67].

#### **5.2 Role of flavonoids as growth regulator**

Role of Flavonoids as Growth Regulator: in plant-environment interactions, flavonoids play an essential role. There is evidence that flavonoids control auxin catabolism


**2. Materials and methods**

#### **2.1 Artificial Intelligence and integrated management of the water cycle**

Water, as a primary source of life and as a natural, cultural, economic and political resource, requires *intelligent management* and the same artificial intelligence can assist in the involvement of the collective intelligence dispersed in citizenship, now evolved into a true *Smart Communities*, to ensure the protection, conservation and rational and optimal use in an *Adaptive Water Management* regime.

The management of water resources requires the formulation of new paradigms capable of combining, on the one hand, the protection of water resources, through new systems and intelligent technologies, capable of increasing the efficiency in the use of resources and the performances of networks and treatment plants present in the territory and on the other hand the development of new monitoring systems distributed and easy to access for widespread control of the quality status. In both cases, the AI plays an extraordinarily important role, especially in the presence of massive amounts of data: an increasingly recurring situation due to the strengthening of water and environmental monitoring systems. The development of interoperable technologies capable of promoting the dissemination and exchange of large volumes of information between decision-makers, managers and citizens, can lead to the creation of *widespread knowledge* capable of feeding artificial intelligence systems and aimed at supporting better environmental protection, ending with a direct impact on the educational and behavioral side. In this direction, the *ubiquity of water*, in every declination of social and productive life, constitutes a natural element to channel information and to consolidate a new culture that can combine the expressions of AI favoring growth, the sharing of structured expert knowledge and not and increasing the sense of belonging to one's territory and to the natural resources it expresses. The recognition of water, as a human right, passes through the acceptance of the sense of widespread (public) ownership and responsibility that must guide both the small daily choices and the big planning, management, political and administrative decisions.

In line with the definitions of the Water Framework Directive 2000/60/EC and the updates in progress, and in general with the articulated Community, national and regional regulatory framework, it is necessary to pursue the objectives of safeguarding, protecting and improving the environmental quality of water bodies, as well as the prudent and rational use of natural resources based on management that is not only sustainable but adaptable to the circumstances that arise also as a result of global changes: all elements of high complexity that find in AI an indispensable ally. In this vision, participatory processes that can also be activated through AI are crucial for triggering paths that lead to the construction of the economic and social vocation of smart cities.

#### *IoT Applications Computing*

The keyword underlying the concept of AI is "*integration*" to be achieved at various levels of knowledge: both in the management of the entire " *water supply chain* " but also with the active involvement of citizens, management bodies, research bodies and universities, companies, supervisory authorities, to achieve management of water resources capable of facing complexities, in line with the needs of environmental sustainability and reduction of impacts.

A correct understanding of the management of water resources can certainly not be limited to the simple government of only one of the components such as, procurement, distribution networks, purification, etc., but it requires a broader perspective that allows the analysis and definition of coordinated and integrated strategies that affect the entire water cycle (**Figure 1**).

Furthermore, even the potentially most efficient strategies have no chance of success if they are not supported by an "awareness" of citizens who must be directly involved as actors within a system that cannot ignore virtuous behavior at the macro level and micro-communities.

It must be emphasized that the constantly growing demographic evolutions, the consequent increased use of the intensification of crops, the effects of climate change with the increase in the frequency of extreme events, determine an extreme urgency in implementing every possible solution (including technological and "intelligent") that can make the resource management system as a whole more efficient, both in quantitative and qualitative terms.

In this context, the research activities on AI that envisage water management in the implementation of environmental policies, in close connection with the Europe 2020 strategy which has identified smart growth, sustainable growth and inclusive growth, as engines of the relaunch of the economy.

#### **Figure 1.**

*Water cycle and pressure factors and areas of application of AI [7]. Legend: 1) climatic change, 2) pollution, 3) physical alterations, 4) over exploitation.*

*Artificial Intelligence and Water Cycle Management DOI: http://dx.doi.org/10.5772/intechopen.97385*

The applications of AI in the water management sector, operating on huge amounts of data, concern the monitoring and management of extreme events also interface with the "*Territory security*" area, while others concern the collection and storage of data, their dissemination and their interoperable using interfaces with the "*Home automation and Smart Grids*" area, in particular concerning aspects relating to the improvement of the quality of life in domestic environments, the reduction of management costs and the transmission of information through *Power Line Communication* (PLC) and their storage using Cloud technology [8].

By way of example, a monitoring system based on AI technologies makes it possible to more effectively target control actions on diffused loads generated as a result of an overflow of the network and on production ones, in order to reduce the presence of metal contaminants and organic and maximize nutrient recovery. The AI itself, combined with innovative devices for controlling the efficiency of urban sewers, allows immediate intervention, reducing the risk of contamination of the unsaturated and groundwater [9]. Precisely for these reasons, these technologies are particularly functional for achieving *good ecological and chemical status* in water bodies, envisaged by the European Directive 2000/60/EC. In response to what is strongly desired by administrators, managers and citizens, the AI itself uses *early warning indicators* that make it possible to identify and suggest mitigation strategies on a local scale of extreme events attributable to natural factors (e.g. climate change and consequent changes in the regime rainfall) or anthropogenic (eg illicit disposal or accidental spills).

AI can be decisive in identifying and managing adaptation guidelines in relation to the climate changes underway. In particular, it can be useful for:

	- **control of leaks,** orienting measurement strategies and priorities and the most effective types of intervention to reduce water dispersion;
	- the definition of **investments in water networks and infrastructures**, supporting a holistic water policy that takes into account an extremely large number of technical, managerial, social and economic variables;
	- the promotion of the **natural conservation of water** by orienting the areas in which to favor it both for employment opportunities and for the reduction of hydraulic risk;
	- **the aggregation** of fragmented **surveillance activities** between the different management and control bodies, also in order to improve the quality and use of information;
	- support **capacities in adapting to extreme climatic events**, in particular as regards the control of floods and drought;
	- **the efficiency of water use in all sectors** and guaranteeing sustainable withdrawal and supply of freshwater also to reduce conflicts of use and to address water scarcity in the short, medium and long term;

As better specified below, in urban and semi-urban areas, AI can also intervene effectively in the urban wastewater purification sector, orienting technological applications to improve the efficiency and versatility of plants and favoring low environmental impact technologies, in terms of occupied surfaces, production of sludge and odor emissions, aimed at maximizing energy recovery and the recovery of raw materials and in particular nutrients and biofuels [10].