**6. Conclusion**

cosides which were secondary metabolites showed various biological activities and act in plant defense mechanisms. Flavonoids usually occur as glycosides and aglycones in plant tissue which have significant antioxidant properties and antimicrobial and insect‐repel‐ lent properties as well [15]. Flavonoids and their antimicrobial effect are useful as a food preservative to extend the shelf life and safety of foods. Flavonoids play important roles in biological activities, including antiallergenic, antiviral and antifungal effects [23]. It is also present in various common fruits and vegetables (apples, grapes, lemons, tomatoes, onions, lettuce and broccoli). The following flavonoids are antifungal agents in plants: iso‐ flavonoids, flavans flavanones. However, the antifungal activity of flavonoid compounds plays an important role between plant‐microorganism and host plant's defensive systems [8]. Saponin and flavonoids are found in fruits and vegetables and in general they form a soapy lather after extracted from parts of plants [5]. Mandalari et al. [24] reported that Bergamot fruit extract which is rich in flavonoid has an effective on the yeast *Saccharomyces*

180 Superfood and Functional Food - An Overview of Their Processing and Utilization

Okwu et al. [25] also showed that the antifungal activity of both citrus species and grape has an important effect against *Fusarium oxysporum* (**Table 1**). Thiosulfinates come from hydrolysis products of garlic and onion. They have a strong potential of producing antimi‐ crobial effects against pathogenic microorganisms [5]. Broccoli, Brussels' sprouts, cabbage mustard and horseradish have glucosinolates that also have a wide range of antibacterial effects. Moreover, olive leaves (*Olea europaea*) are rich in phenolic compounds, with dem‐ onstrated strong antimicrobial effects and can be potentially used in food processing [5]. Al‐Zoreky [29] reported that phenolics and flavonoids present in pomegranate fruit peels demonstrated strong antimicrobial activity against some food pathogen microorganisms. In addition, *Psidium guajava* has phenolic, flavonoid, carotenoid, terpenoid and triterpenes that demonstrated strong antimicrobial activity [30]. Salas et al. [31] reported that flavonoids extracted from citrus species, not only naringin, hesperidin and neohesperidin but also enzy‐ matically modified derivatives of these compounds, have strong antifungal activity [31]. The limonoid compounds have important antibacterial and antiviral activity as shown in **Table 1** [1]. Vikram et al. [32] reported that seeds of grapefruits have significant inhibitory effect on pathogenic *Escherichia coli* O157:H7. Black raspberry and Chardonnay seed extracts have also antibacterial activity to inhibiting growth of some food pathogen microorganisms [33]. Tehranifar et al. [26] reported that high percentage of phenolic content in the peel and seed of pomegranate fruit has high antifungal activity especially on postharvest fungi (*Penicillium italicum*, *Rhizopus stolonifer* and *Botrytis cinerea*). Another study showed that berry extracts exhibit selective inhibitory properties against intestinal bacteria [1]. Recently, antifungal activity has been found in all tissue types of strawberry fruit due to the phenolic compounds

*cerevisiae* (**Table 1**).

that inhibit the growth of fungi [23].

**5. Other beneficial properties of plant phytochemicals**

In the last decade, the results of many research have shown the positive effects of phytochemicals in human health. There is a strong correlation of antioxidant consumption In a conclusion, potentially a great number of phytochemicals including some of the vitamins, flavonoids, terpenoids, carotenoids, phenolics, phytoestrogens, minerals and antioxidants in plant materials are used as alternative preservative agents for controlling postharvest physi‐ ological disorders or microbial pathogen injuries of both fresh fruit and vegetables in the food industry. Many publications have focused on the potential protective nature of these natural phytochemical compounds against fungal and bacterial attacks. Moreover, these natural com‐ pounds have become interesting candidates not only for plant protection but also human and animal health protection from fungal and bacterial diseases because of their lower toxicity or absence of toxicity.


**5. DCM Caused by Disrupted NaV1.5 Interaction with Partner Proteins**

On the other hand, it is possible that the interaction of the defective NaV1.5 with its partner proteins of the cytoskeleton and intercalated disc, is responsible for the structural phenotype [28]. Interestingly, pathogenic *SCN5A* variants have been described in rare forms of left-ventricular non-compaction with a high arrhythmic burden [29]. Studies on induced pluripotent stem cell-derived cardiomyocytes have shown that the ARVC-associated missense variant *SCN5A-*R1898H leads to a significant reduction in peak *I*Na current, and of the abundance of NaV1.5 and N-cadherin clusters at the intercalated disc [13]. Other studies have shown that *SCN5A*-positive Brugada syndrome patients have significant cardiomyopathic changes, primarily in the right ventricular outflow tract, such as fatty wall replacement, fibrosis, and reduced expression of connexin 43 [30–32]. Moreover, missense *PKP2* variants identified in *SCN5A-*negative Brugada syndrome patients were shown to cause a loss of expression of desmosomal protein plakophilin-2, which was associated with decreased *I*Na, reduced number of NaV1.5 channels at the intercalated disc, and increased separation of microtubules from the cell end [33]. These interactions between the cardiac sodium channel complex and the intercalated disc likely underline mechanisms relevant to *SCN5A*-medicated ARVC and *PKP2*-mediated Brugada syndrome. Could an abnormal NaV1.5 channel result in reduced ventricular contractility through disrupting the function of its interacting proteins and/or those already implicated in DCM, such as proteins of the sarcomere, cytoskeleton, or the dystrophin-associated glycoprotein complex?

To date, several proteins interacting with the NaV1.5 channel have been shown to contribute to the *SCN5A*-mediated phenotypes through their alteration of the sodium channel availability or biophysical properties. NaV1.5 regulatory proteins caveolin 3 (*CAV3*), alpha 1 syntrophin (*SNTA1*), and cardiac sodium channel beta subunit 4 (*SCN4B*) have been reported in association with rare subtypes of congenital long QT syndrome. Pathogenic variants in cardiac sodium channel beta subunit 3 (*SCN3B*) and glycerol 3 phosphate dehydrogenase 1-like protein have been identified in patients with Brugada syndrome. Interestingly, cardiac sodium channel beta subunit 1 (*SCN1B*) has been linked to Brugada syndrome, atrial fibrillation (also *SCN2B*), and cardiac conduction disease. A number of other proteins have been shown to interact with and regulate the NaV1.5 channel. These include anchoring adaptor proteins ankyrin-G, syntrophins, MOG1, plakophilin-2, enzymes, such as nedd4-like enzymes, calmodulin kinase II δc, and protein tyrosine phosphatase H1, and other proteins that modulate the channel biophysical properties, such as 14-3-3η, calmodulin, telethonin, GPD1L, and FHF1B [34]. However, strong associations between pathogenic variants in these protein genes and development of DCM have not been reported.

The sarcolemmal membrane-associated protein (SLMAP) is localized at T-tubules and sarcoplasmic reticulum. Pathogenic variants in *SLMAP* have been shown to cause Brugada syndrome via modulating the intracellular trafficking of the NaV1.5 channel [35]. A recent report showed that transgenic mice with cardiac-specific expression of the SLMAP isoform 3 (SLMAP3) develop a significant decrease in fractional shortening and in cardiac output without notable hypertrophy, fibrosis, or fetal gene activation [36]. Electrocardiography identified increased PR interval and a decreased R amplitude. Western blot analysis revealed a decreased protein levels of NaV1.5 and calcium transport system of the sarcoplasmic reticulum (SERCA2a/PLN), suggesting a selective regulatory role of SLMPA3 in ion transport proteins at the level of gene expression. It is, however, unclear whether SLMAP3 is a contributor of DCM phenotype in humans or whether any of the reported DCM-associated *SCN5A* variants disrupts an interacting domain of its partner proteins or other DCM-associated genes/proteins. The list of proteins interacting with NaV1.5 is also not conclusive, and many aspects require further research. It is expected that these patterns will become clearer with further experimental evidence and with more genotype-phenotype analyses on DCM and related disorders. As a first step, *SCN5A* disruption has been demonstrated to result in TGF-β1-mediated fibrosis in a murine model of sinus node dysfunction. It is therefore possible that *SCN5A* variants can influence the pro-fibrotic milieu associated with other protein variants, and thereby contribute to the development of DCM [37].

#### **6. DCM Resulting from Long-Standing and Frequent Arrhythmias**

Alternatively, high arrhythmic burden may lead to ventricular dysfunction over time. This theory was primarily based on the finding that in several cases of MEPPC and DCM due to gain-of-function *SCN5A-*R222Q variant, therapy with hydroquinidine, flecainide, or amiodarone (in addition to standard treatment of heart failure) rapidly and effectively reduced the number of multifocal premature ventricular contractions and reversed the LV remodeling [10,16,38]. Following the initial report of R222Q, similar phenotypes have been reported for R222P, I141V, and G213D [39–41]. Nevertheless, whether the recovery of ventricular function relates to the premature ventricular contraction burden reduction or to intracellular mechanisms secondary to pharmacological blockade of the defective sodium channel, remain to be elucidated. Furthermore, some *SCN5A*–DCM patients, such as those carrying the D1275N variant, lack a history of long-lasting ventricular arrhythmias [14], and thus the LV dysfunction in these patients is unlikely to be a consequence of ventricular arrhythmias. It is therefore more likely that this is a contributing mechanism rather than a primary cause.


**3. Results**

#### *3.1. The Trophoblast of Chorionic Villi in Human Placentae Expressed E-cadherin and a Wide Array of TJ Proteins*

In this study, we analyzed the expression of TJ proteins in the placentae from control and ZIKV-infected women. The clinical data and perinatal outcomes of control and ZIKV-infected women during pregnancy and their newborns are summarized in Tables 1 and 2. With regards to the newborn of ZIKV-infected donor D8, that carries a 22q11 deletion, all the reported phenotypic characteristic of the child conform with 22q11 deletion syndrome.

**Table 1.** Clinical data of women with ZIKV during pregnancy and physical findings of their newborns.

> GW, Gestational week; HC, Head circumference; F, Female.

**Table 2.** Clinical data of control women during pregnancy and physical findings of their newborns.

GW, Gestational week; F, Female; M, Male; HC, Head circumference.

In the trophoblast of chorionic villi in placentae derived from control and ZIKV-infected women during pregnancy, we explored the expression of the AJ protein E-cadherin and of the following TJ proteins: claudins -1, -2, -3, -4, -5, -7, and -10; JAMs -A, -B, and -C; occludin, ZO-1, and ZO-2 (Table 3). All these proteins, with the exception of claudins -2, -10, and JAM-A (data not shown), were expressed in the STB of chorionic villi.

**Table 3.** Expression of E-cadherin and TJ proteins in the trophoblast of placental chorionic villi derived from control and ZIKV-infected women.

BD, barely detected; ND, not detected. −, absent; ±, barely detected; +++ highly abundant; =, no change; ↓, decrease.

#### *3.2. E-cadherin Stained the Basal Membrane of the STB and Its Expression was not A*ff*ected in ZIKV-Infected Placentae*

In chorionic villi of human placentae, E-cadherin was observed not at the apical surface of the STB layer in contact with the intervillous space, which in situ is occupied by maternal blood, but in the basal membrane of the STB in contact with remnants of the CTB layer and the chorionic mesenchyma (Figure S1a). In this and most of the subsequent images, the trophoblast layer was identified with cytokeratin 18, a canonical marker of epithelial cells [28]. The intensity of the fluorescent signal of E-cadherin in the STB layer was not affected in placentae derived from ZIKV-infected women (Figure S1b). Likewise, by Western blot, no difference in the amount of E-cadherin was observed in placental lysates from control and ZIKV-infected women (Figure S1c,d).

#### *3.3. The Expression of Claudin-1 Slightly Increased in Placentae Derived from ZIKV-Infected Women*

By immunofluorescence, we observed that claudin-1 stained fetal endothelia within the chorionic villi parenchyma as well as the STB layer. The relative mean fluorescence intensity of the signal in the STB layer slightly increased in placentae from ZIKV-infected women in comparison to control placentae, albeit not at a significant level (Figure S2a,b). By Western blot, we observed that the amount of claudin-1 was not significantly higher in placentae derived from ZIKV-infected women in comparison to control placentae (Figure S2c,d).

#### *3.4. The Expression of Claudin-3 was Strong in Placental Vessels and Faint at the STB Layer of Both Control and ZIKV-Infected Placentae*

*Claudin-3* was strongly expressed in the vessels of the parenchyma in chorionic villi, while staining at the STB cell layer was weak in both control and ZIKV-infected placentae (Figure S3a,b). By Western blot, we detected no difference in the amount of claudin-3 between control and ZIKV-infected placentae (Figure S3c,d).

#### *3.5. The Expression of Claudin-4 at the Basolateral Membrane of STB Diminished in ZIKV-Infected Placentae*

In the chorionic villi of control human placentae, claudin-4 was observed at the basolateral membrane of the STB in contact with the chorionic parenchyma. The same pattern was observed in the placentae from ZIKV-infected women; however, in this case, the fluorescent signal at the STB layer was less intense (Figure 1a,b). Western blot analysis confirmed the decreased expression of claudin-4 in ZIKV-infected placentae in comparison to those of the control (Figure 1c,d).

**Figure 1.** Claudin-4 present at the basal surface of the STB layer diminished in ZIKV-infected placentae. (**a**) Frozen sections of human placentae derived from women infected or not (control) with ZIKV were processed for immunofluorescence with a rabbit antibody against claudin-4 (Cl-4) and a mouse antibody anti cytokeratin 18 (K18). DNA of nuclei was stained with DAPI. Apical surface of STB cell layer (arrow); basolateral surface of STB cell layer (arrowhead); intervillous space (asterisk). Bar, 50 μm; magnification bar, 25 μm. (**b**) Measurements of mean fluorescence intensity of the trophoblast layer were done on three independent images from each condition. Since the variances were equal among the two groups, we applied a Student's *t*-test. To include effect size estimation, since both groups have similar standard deviations, we used Cohen's *d*. The three values obtained per donor are represented by dots with the same color. \*\* *p* = 0.0054, Cohen's *d* = 3 indicating that 99.9% of the values from the ZIKV group are below the mean value of the control group. (**c**) Representative Western blot of three independent experiments. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as loading control. (**d**) Densitometric analysis of Western blots. The three values obtained per donor are represented by dots with the same color. \*\* *p* = 0.00092 Cohen's *d* = 2.67 indicating that 99.9% of the values from the ZIKV group are below the mean value of the control group.

#### *3.6. Claudin-5 Strong Expression in the Vessels of Chorionic Villi and Faint Stain in STB Cells Did not Change with ZIKV Infection*

Claudin-5 was previously identified as an endothelial claudin [29], and accordingly, strongly stained the vessels within the chorionic parenchyma. In addition, claudin-5 faintly stained the STB cell layer in both ZIKV-infected and in control placentae (Figure S4a,b). Western blot analysis also revealed that ZIKV infection induced no change in the amount of claudin-5 expressed in the placentae (Figure S4c,d). The Western blot was done with a placental lysate, and hence, contains claudin-5 from both the vessels and the STB. In the case of claudin-5, this situation is particularly relevant, as this claudin is more abundant in vessels than in the STB cell layer.

#### *3.7. Claudin-7 Expression at the Basal Surface of STB Cells was not Altered by ZIKV Infection*

In the chorionic villi of control and ZIKV-infected placentae, claudin-7 was preferentially expressed in the basal membrane of STB, although a faint staining was also detected at the apical surface (Figure S5a,b). Immunofluorescence quantitation of claudin-7 at the STB cell layer and Western blot analysis showed no change in claudin-7 abundance in ZIKV-infected placentae (Figure S5c,d).

#### *3.8. Occludin Expression in the STB was Low and not A*ff*ected by ZIKV Infection*

In chorionic villi, occludin was strongly expressed in parenchymal vessels and faintly stained the STB cell layer in both control and ZIKV-infected placentae (Figure 2a,b). Western blot analysis showed a decrease in occludin expression in response to ZIKV infection (Figure 2c,d). These Western blots, however, most likely reflect the amount of occludin present in endothelial TJs rather than in the STB cell layer.

#### *3.9. The Expression of ZO-1 was Higher in Chorionic Vessels than in the STB and was not A*ff*ected by ZIKV Infection*

ZO-1 strongly stained the vessels in the chorionic villi and delineated the STB layer in a moderate manner, although in some placentae from both control (D19) and ZIKV-infected women (D10), labeling at the STB was more intense (Figure S6a,b). Western blot analysis revealed a similar amount of ZO-1 in both group of placentae, and most likely reflects the content of ZO-1 at endothelia, due to the higher expression of this protein in vessels in comparison to the STB (Figure S6c,d).

#### *3.10. In Chorionic Villi, the Expression of ZO-2 in the STB was Much Higher than in the Mesenchymal Vessels*

*ZO-2* in the chorionic villi of human placentae was clearly expressed in the STB, while it was barely present in the vessels of the mesenchyme. By immunofluorescence, only in one ZIKV-infected placentae (D8), the amount of ZO-2 increased in comparison to the rest of the placentae (Figure S7a,b) and the Western blot analysis showed no change in ZO-2 expression induced by ZIKV infection (Figure S7c,d).

#### *3.11. ZIKV Infection Had no Impact on the Expression of JAMs -B and -C in the STB of Chorionic Villi*

JAM-B stained with a similar intensity the STB cell layer of both control and ZIKV-infected placentae. Dots of JAM-B were also present in the chorionic mesenchyme, but not along the TJs of chorionic vessels, which were conspicuously stained with ZO-1 (Figure S8a,b). By Western blot, similar amounts of JAM-B were found between control and ZIKV-infected placentae (Figure S8c,d). In contrast, JAM-C was not observed at the STB cell layer and profusely stained the endothelial vessels in the chorionic mesenchyme. In the control and ZIKV-infected placentae, the same JAM-C staining pattern was observed (Figure S9a,b), and no change in the amount of JAM-C was detected by Western blot after ZIKV infection (Figure S9c,d).

In summary, the exploration of E-cadherin and a wide variety of TJ proteins revealed that E-cadherin and claudins -4 and -7 were present in the basal membrane of the STB, while all the other TJ

proteins studied localized in both the apical and basal membranes of the STB. The pattern of expression of these proteins was preserved in ZIKV-infected placentae, although in this pathological condition, the amount of claudin-4 diminished in the STB.

**Figure 2.** Occludin expression at chorionic vessels is stronger than in the cell layer. (**a**) Frozen sections of human placentae derived from women infected or not (control) with ZIKV were processed for immunofluorescence with a mouse antibody against occludin (Occ) and a rabbit antibody anti E-cadherin (E-cadh). DNA of nuclei were stained with DAPI. Chorionic vessels (arrows); STB (arrowhead); intervillous space (asterisks). Bar, 50 μm. (**b**)Measurements of mean fluorescence intensity of trophoblast layer were done on three independent images from each condition. (**c**) Representative Western blot of three independent experiments. GAPDH was employed as loading control. (**d**) Densitometric analysis of Western blots. \* *p* = 0.0286. The data were further analyzed to include effect size estimation with Glass'delta since each group had a different standard deviation. The value of Glass'delta = 1.955 indicates that 99.9% of the values from the ZIKV group are below the mean value of the control group.

#### *3.12. The STB Layer of Placentae of ZIKV-Infected Women is Permeable to Ruthenium Red*

Since claudin-4 functions as a cationic barrier [30] or an anion pore [31] that increases TER in cationic and anion selective cell lines and decreases paracellular permeability in the cationic selective cell line MDCK [32], we next determined whether the TJs of ZIKV-infected placentae were leaky. For this purpose, placental tissue was fixed and processed for TEM in the presence of ruthenium red, an electron-dense paracellular marker. In all the ZIKV-infected placentae, we observed ruthenium red staining in the paracellular pathway bellow the TJ region in the STB cell layer, in contrast to control placentae, where, as we had previously shown [9], staining was restricted to the apical membrane of the STB (Figure 3).

**Figure 3.** The STB in the placentae of ZIKV-infected women was permeable to ruthenium red. Placental tissue was fixed and processed for TEM in the presence of ruthenium red. Ruthenium red staining in the paracellular pathway (arrows). Bar, 1 μm.

#### *3.13. Hofbauer Cell Hyperplasia, an Increased Diameter of Microvilli and Intravillous Calcifications were Observed in ZIKV-Infected Placentae*

The chorionic villi derived from women infected with ZIKV during pregnancy displayed several histological alterations, including: (1) Hofbauer cell hyperplasia, evaluated by counting the number of CD68+ cells in the parenchyma of floating chorionic villi stained with hematoxylin (Figure 4a,b); (2) intravillous calcifications observed in hematoxylin and eosin-stained samples, which show a tendency to increase in ZIKV placentae in comparison to control, but whose difference is not statistically significant (Figure 4c,d); and (3) a higher diameter of chorionic microvilli in hematoxylin and eosin-stained samples (Figure 4e,f).

**Figure 4.** The chorionic villi of ZIKV-infected placentae display Hofbauer cell hyperplasia and a higher mean diameter. (**a**) Hofbauer cells (arrows) in chorionic villi were detected with an antibody against CD68 in slides stained with haematoxylin. Bar, 100 μm. (**b**) The number of Hofbauer cells was evaluated counting CD68+ positive cells in five optical fields per placenta. Each dot corresponds to the mean value of Hofbauer cells/field present in each placenta. \* *p* = 0.0294. (**c**) Calcification (arrow) present in a chorionic villus detected in a slide stained with haematoxylin and eosin. Bar, 50 μm. (**d**) Calcifications were counted in five optical fields per placenta. Each dot corresponds to the mean value of calcifications/field present in each placenta. (**e**) The diameter of chorionic microvilli is higher in ZIKV-infected placentae than in the control condition. Bar, 200 μm. (**f**) The diameter of chorionic villi was measured using the image analysis software Zen (version ZEN 2.3 lite, Carl Zeiss Microscopy, Jena, Germany) in five optical fields of samples from placentae stained with haematoxylin and eosin. Each dot corresponds to the mean diameter of chorionic villi per placenta. \* *p* = 0.0286.

Some placentae infected with ZIKV also displayed chorionic villi edema, heterogeneous maturation of chorionic villi, characterized by the co-existence of villi with different diameters; increased syncytial knots due to premature aging; and karyorrhexis, the irregular distribution of chromatin in the cytoplasm due to the destructive fragmentation of the nucleus of dying cells (Figures S10–S13). In contrast, tissue sections of chorionic villi from the placentae of control women displayed a homogeneous maturation of chorionic villi, an absence of inflammatory cells, and a parenchyma without abundant Hofbauer cells (Figures S14–S17).

We also performed a Masson's trichrome stain in the chorionic villi of D1 placenta from a ZIKV positive woman in order to detect pathological changes involving the connective tissue. Supplemental

Figure S18 reveals perivascular fibrosis and abundant intravilli collagen, as well as mesenchymal edema and karyorrhexis, which were previously observed with the hematoxylin and eosin stain.

#### *3.14. ZIKV Added to the Basolateral Surface of the Trophoblast-Derived Cell Line BeWo Reduces the Transepithelial Electrical Resistance and Claudin-4 Expression*

To further explore the e ffect of ZIKV on TJs of the trophoblasts, we incubated ZIKV at a MOI of 1 with the trophoblast-derived choriocarcinoma cell line BeWo cultured on Transwell filters. We worked with BeWo cells because they constitute a human cell culture model of placental villous trophoblast, and because the STB layer of human placenta is poorly susceptible to infection by ZIKV [33]. Figure 5a shows that the apical administration of ZIKV immediately increases TER by 34%, reaches 40% above control after 6 h, and diminishes to 27% above control after 24 h. Instead, the administration of ZIKV to the basolateral surface of BeWo cells induced a fast drop of TER of 20% in comparison to control monolayers at 1.5 h. However, after 10 h, the values of TER had recovered and were undistinguishable from those of control monolayers. Hence, these results reveal that ZIKV in contact with the basolateral surface of trophoblast cells is able to reduce the degree of sealing of TJs.

**Figure 5.** ZIKV added to the basolateral surface of BeWo cells transiently diminishes the transepithelial electrical resistance and claudin-4 expression. Confluent monolayers of BeWo cells that had achieved a stable value of TER were incubated with ZIKV (MOI = 1) added to the apical or basolateral surface. (**a**) TER was continuously measured in the cellZscope system in three inserts per condition. Results are shown with the corresponding standard deviation. (**b**) Immunofluorescence for claudin-4, occludin and ZO-1 in BeWo monolayers done after 1.5 h of incubation with ZIKV. Bar, 50 μm. X-Y, en face view; X-Z, lateral view.

By immunofluorescence, we observed no change in the expression of occludin or ZO-1 in BeWo cells, 1.5 h after the addition of ZIKV to the apical or basolateral surfaces (Figure 5b). Instead, the expression of claudin-4 diminishes 1.5 h after ZIKV is added to the basolateral surface. In addition, while occludin and ZO-1 concentrate as spots at the TJ region in immunofluorescence z-sections, claudin-4 distributes along the basolateral membrane, as had been previously observed in intestinal Caco-2 cells [34] These results thus confirm the capacity of ZIKV to diminish the expression of claudin-4 in trophoblasts.


**1. Introduction**

The amount of personal data being collected is rapidly proliferating. Enterprises and governments use this data to profile individuals and to predict and control their attitudes and behavior. This can result in customized experiences, personalized services, and more efficient use of resources. It can also result in misinformation and exploitation by the entity that collected the data or by others that purchase or steal it. In response to increases in cybercrime and growing consumer concern, legislation to protect personal data is being proposed and implemented. Organizations trading in personal data face increasing costs associated with managing and securing data. They also face increasing risks that data will be misused or stolen, and that they will face legal or financial consequences, as well as damage to both their reputation and to relationships with customers and other stakeholders.

In this chapter, we explore how blockchain and artificial intelligence can offer solutions for protecting and securing personal data. Decentralized and federated identify systems provide users control over what, when and how much of their personal information can be shared and with whom. These systems can also reduce cybersecurity threats. Artificial intelligence complements blockchain-based privacy solutions by enabling users to better manage their data and by ensuring that data and models derived from the data are more accurate, fair, and reliable.


4. MEMS SINS-enhanced PIG navigation system by PJ detection in smalldiameter pipeline

In the above sections, the constant azimuth and pitch angles could be used to estimate the SINS errors in SPS. Therefore, this section would introduce an MEMS SINS-enhanced pipeline navigation system by PJ detection.


**1. Introduction**

Discussions on climate change have become increasingly more relevant in the general scientific community, particularly since the creation of the Intergovernmental Panel on Climate Change (IPCC), which is composed of a diverse group of worldwide researchers, focusing on climate change studies and its impacts on society. Since the industrial revolution, the concentration of carbon dioxide (CO2) in the atmosphere has increased with the use of fossil fuels, deforestation, the use of nitrogen in agriculture and livestock farming, which are reported to be the main uses responsible for the anthropic greenhouse effect. Studies have already shown the relevance of biosphere–atmosphere interactions in Brazilian biomes regarding planetary climate regulation due, for example, to water, energy, and carbon exchanges with the atmosphere [1–6]. However, there are still uncertainties regarding these processes due to the remarkable diversity of physiognomies, landscapes and other biophysical aspects that might play a role in differentiating atmospheric patterns from one place to another within each biome.

To reduce these uncertainties, in situ measurements are needed to better understand the particularities of each environment. Furthermore, these observed data can also be used to assess soil–vegetation–atmosphere models [7–10] and to analyze satellite-derived estimates of water and/or CO2 balance components [11–16]. Both these models and remote sensing data are extremely important in providing reliable information on CO2 exchanges over tropical forests where flux tower coverage is scarce or non-existent, such as in many parts of Brazil.

Additionally, certain forest physiognomies are not endemic to Brazil, but occur in several other regions of the globe, and therefore, their particularities regarding biophysical patterns need to be understood in detail. Wetlands in tropical rainforests such as the Amazon are environments where organic production rates are high and anoxic conditions are frequent, and therefore, they represent crucial zones for the global balance of greenhouse gases in the atmosphere. Cerrado ecosystems (savannas), however, are located at the tropics and subtropics and are characterized by marked wet and dry seasons. They cover approximately 60%, 50% and 45% of the total area of Africa, Australia and South America, respectively [17]. These ecosystems play an important role in the cycle of several greenhouse gasses, as reported by the studies [18–20] among others, and on energy fluxes (latent and sensible heat) in the context of climate change, since they cover approximately 20% (2.7 billion ha) of the global surface [17].

Carbon dynamics and biophysical evapotranspiration controls over tropical Brazilian ecosystems and in other regions of the world have also been studied because of the need to better understand the effects of land use change on regional and global biogeochemical cycles [13,15,21]. Evidently, it also allows us to estimate the contribution of tropical biomes to the regional and global climate controls, also comprising the relationship between precipitation, evapotranspiration, plant productivity and greenhouse gas exchanges within the atmosphere [22]. These changes motivated the development of various research fields within climate sciences focusing on the debate over greenhouse gases cycles and anthropic influence over these cycles. CO2 concentration in the atmosphere surpassed 405 ppm [23] and continues increasing each decade, as well as that of other greenhouse gases. Therefore, it is necessary to develop methods and techniques to quantify these emissions and the fluxes of these gases, improving the understanding on how different environments function and respond to land-use changes.

The eddy covariance method (EC) directly and non-intrusively estimates the vertical transport of CO2 and other greenhouse gases. Information measured through EC allows for the better comprehension of biogeochemical cycles, energy fluxes and other atmospheric controls. It also provides clues to other pivotal questions regarding ecosystem (in this study, tropical ecosystems) controls over climate. However, due to the lack of an extensive EC measurement network throughout Brazil, other data sources are required, such as satellite-derived data. Our hypothesis is that both gross primary production (GPP) and water-use efficiency (WUE) data respond differently to varied water availability conditions on the main tropical ecosystems in Brazil. Thus, our objective is to analyze the dynamics of GPP and WUE in the Amazon, Cerrado (savanna), Pantanal and Caatinga biomes through data collected through EC systems and to assess the performance of data derived from the Moderate Resolution Imaging Spectroradiometer (MODIS) sensor for the quantification of CO2 balance components over these environments.

#### **2. Materials and Methods**

#### *2.1. Data Policy and Use License*

The Ameriflux platform compiles data monitored in three Brazilian biomes: BR-Sa1 (Amazon), BR-CST (Caatinga) and BR-Npw (Pantanal). Data from the Cerrado site (BR-BI, Bananal Island—Javaés) are available at https://daac.ornl.gov (accessed on 25 April 2022). Data provided by the ORNL DAAC can be accessed for free, without restrictions and in accordance with NASA's Earth Science Program. Ameriflux data are shared under CC-BY-4.0 data usage license (Creative Commons by Attribution 4.0 International). This license states that the use of data is free to share (copy and redistribute the material in any medium or format) and/or adapt (remix, transform, and build upon the material). The scientific literature references describing each of the data sites are: BR-Sa1 [24], BR-CST [25] and BR-Npw [26].

#### *2.2. Description of Study Area*


**4. Relationship between rural tourism and natural-cultural environment**

Tourism and environment are representing common relationship concepts. Successful tourism activity needs clean and orderly environment (Kiper 2006). Rural tourism is both entagled with rural settlements and is based on natural resources (Soykan 2003).

Rural tourism, natural resources, cultural heritage, rural lifestyle and an integrated tourism is a type of local economic activities. Therefore, rural tourism in rural areas was carried out with a number of elements in their natural landscape and cultural landscape (water, vista, topography, vegetation, clean air), as well as in the variety of recreational activities suitable for all kinds of environments. Therefore, rural tourism and its natural assets and raw materials to create, as well as directing people to travel is an attractive force (Kiper, Özyavuz & Korkut 2011).

Natural and cultural landscape values form a basis for rural tourism. These values are geographical position, micro-climatic conditions, existence of water, natural beauties, existence of natural vegetation, existence of wildlife, surface features, geomorphologic structure, local food, festivals and pageants, traditional agricultural structure, local handicrafts, regional dress culture, historical events and people, heritage appeals, architectural variety, traditional music and folk dance, artistic activities and so on (Gerry, 2001; Lane, 1993; Lanquar, 1995; Soykan, 1999; Brassoulis, 2002; Catibog-Sinha & Wen, 2008; Mlynarczyk, 2002; Drzewiecki, 2001, Kiper, T. 2006; Kiper, Korkut & Ylmaz, 2011).

Relationship between rural tourism and natural - cultural environment in Figure 3.

Adventure- emphasis on activity

Nature based- primarily viewing of natural landscape

Wildlife- primarily viewing of wildlife

Ecotourism- includes educative and conservation supprting elements Rural tourism- primarily viewing of natural and cultural landscape

Fig. 3. An overview of tourism (Dowling, Moore, Newsome 2002)

Tourism Planning in Rural Areas and Organization Possibilities 133

Fig. 5. Natural vegetation cover

Fig. 6. Natural vegetation structure

According to Weaver and Opperman (2000) tourism attractiveness is form of two: natural and cultural.

Natural attractiveness: the topography (mountains, canyons, beaches, caves, volcanoes, fossil sites, etc..), (Fig.4), climate (temperature, rainfall, humidity and so on. ), hydrology (lakes, rivers, waterfalls, hot springs, etc..), wildlife (mammals, birds, insects, etc..), the natural vegetation structure (Fig.5, Fig.6)

Cultural attractiveness: Archeological heritage (castle, cave, bridge etc.), religious structures (mosque, monastery etc.), conventional architecture, folkloric values, gastronomy, etc.

Rural tourism resources vary with the geographical, it mostly with natural style, toiling morphology, farm life and tradition primarily. It is affected by the climate and the seasons, so can satisfy tourist various needs. Rural tourism cultural resources include folk festivals, crafts, folk architecture, folk art, marriage customs, fun legends, and so on (Fan &Yang, 2011).

According to Tane and Therhemer tourism in natural environments is mostly practiced by nature lovers that prefer isolation instead of urban crowds, and silence instead of the noise of tourist resorts. Being directly connected to rural tourism, it takes place in partially or completely isolated areas (mountain, sea, delta), being mostly destined to young or adult tourists.

Fig. 4. Topographic structure

According to Weaver and Opperman (2000) tourism attractiveness is form of two: natural

Natural attractiveness: the topography (mountains, canyons, beaches, caves, volcanoes, fossil sites, etc..), (Fig.4), climate (temperature, rainfall, humidity and so on. ), hydrology (lakes, rivers, waterfalls, hot springs, etc..), wildlife (mammals, birds, insects, etc..), the

Cultural attractiveness: Archeological heritage (castle, cave, bridge etc.), religious structures (mosque, monastery etc.), conventional architecture, folkloric values, gastronomy, etc.

Rural tourism resources vary with the geographical, it mostly with natural style, toiling morphology, farm life and tradition primarily. It is affected by the climate and the seasons, so can satisfy tourist various needs. Rural tourism cultural resources include folk festivals, crafts, folk architecture, folk art, marriage customs, fun legends, and so on (Fan &Yang, 2011). According to Tane and Therhemer tourism in natural environments is mostly practiced by nature lovers that prefer isolation instead of urban crowds, and silence instead of the noise of tourist resorts. Being directly connected to rural tourism, it takes place in partially or completely

isolated areas (mountain, sea, delta), being mostly destined to young or adult tourists.

and cultural.

natural vegetation structure (Fig.5, Fig.6)

Fig. 4. Topographic structure

Fig. 5. Natural vegetation cover

Fig. 6. Natural vegetation structure

Tourism Planning in Rural Areas and Organization Possibilities 135

access to resources of welfare and livelihood services. Optimum usages of environmental, economical and production potentials of rural areas for improving income and welfare can be efficient in reaching the goal of reducing the rural immigration. Rural tourism is a part of tourism market and is a source of employment and income. Also it can be presented as a

Declining economic activity, restructuring of the agricultural sector, dwindling rural industrialisation and out-migration of higher educated youth, has led to the adoption, in many western nations, of tourism as an alternative development strategy for the economic and social regeneration of rural areas (Pompl & Lavery, 1993; Williams & Shaw, 1991;

Rural tourism in one of the forms of sustainable development that through promoting productivity in rural zones, brings about employment, income distribution, preservation of village environment and local culture, raising host community's participation and presenting appreciate methods to conform beliefs and traditional values with new

Sustainable development is a process having economic, social, cultural and environmental–ecological dimensions. This process is perceived as a development in all respects for both urban and rural societies. Yet, in most of the developing countries rural population is gradually diminishing, notwithstanding the agricultural lands that are losing productivity are increasing. While this situation primarily results in increasing impoverishment of rural society, it also causes problems such as deforestation, erosion and productivity loss with the misuse of resources. On the other hand, damaging the natural resources emerge problems such as migration, poverty and hunger. These problems primarily affect rural people. Most affected ones by these problems are women and children. Overcoming these problems would be possible by sustainable planning and management of rural areas in accordance with their resource potential (Golley and Bellot,

Rural tourism has a positive impact on agricultural development, farmers' incomes and the standard of living in rural areas. However, one important issue is how to reach a consensus among local government, farmers' associations, and individual farmers on what kind of

Agro-tourism represents a real opportunity for the local economy, the main motivation in training and development initiatives, the traditional activities that have long been neglected, of crafts, the strengthening and development of local artistic creations, linking to friends, material needs and spiritual needs of tourists, local economic activities stimulated life.

Rural household translates offer accommodation and services and creates motivation agro household to prepare and arrange inside and outside the household to obtain revenue, stimulate peasant to invest in their own household, to develop complementary activities of its concerns. The villager will compete, will carry from now on business in a competitive environment and will be forced to become competitive, increase the quality of their services

Today, Villages are one of the resources, attracting the attention of tourism planners more than ever. Those with some specific cultural, natural or social appeal have a very strong

projects to promote, and how they should be funded and operated (Hong, 1998).

significant tool for the socio-economical development of rural areas.

Hannigan, 1994; Dernoi, 1991; Wickens, 1999)

and products to be applied (Munteanu, 2007).

circumstances (Kanaani, 2005).

1999).

Tourism in natural environments creates the possibility for tourists to practice climbing, fishing, hunting or other sports like dirt track, canoe racing, sky diving etc.

The relationship between rural tourism and other tourism forms practiced in the rural environment is extremely important, because it establishes the connections between natural, human, economical and social parameters, in order to insure all factors that lead to tourist growth and durable development (Tane Thierheimer, 2009).

According to Butler and Hall, to speak about rural tourism in a place, an economically viable rural population sustaining rural culture and identity through being engaged in rural activities is a prerequisite.

For, the target group will be the local community who is both the most influenced by tourism in the region and is the most influential on it. Certain states such as participation and embracing are prerequisites for sustainability (Kiper, 2011).


*4.5. Reputation Management System*

A reputation management system was added to the system to ensure the quality of service. After receiving the charging service, the EV user can leave feedback about the received service. A rating from one to five can be provided, where five (5) indicates the best service and one (1) means the service is the worst possible. The server will calculate the mean of overall ratings for each HCS, and when suggesting nearby HCSs to the EVs, HCSs with relatively higher ratings will get priority on the list. Additionally, the EV users will be able to provide a comment describing the service received. After receiving the feedback from the EV, all the fields of the transaction are completed, and the server will generate the block from the transaction.

This will help HCSs to decide on their pricing, as they have the authority to fix their service rates; an HCS with a higher rating may ask for a higher price, and a newcomer may ask for a lower price to get good review scores. In this way, the reputation system will also be useful to create competition between the service providers, i.e., HCSs.