*2.9. Measurement of Photochemical Quenching, Electron Transfer Rate, and Absorbed Energy of the PSII Reaction Center*

After dark adaptation of the leaves for 30 min, chlorophyll fluorescence quenching analysis was performed using a pulse modulation fluorimeter (FMS-2, Hansatech, King's Lynn, UK). In this procedure, the leaf adapts to the light for the first 30 s, and the light intensity is the same as the ambient light intensity before the measurement of the leaf (1000 μmol·m−2·s−1). The steady-state fluorescence parameter (*F*s) and electron transfer rate (ETR) under the light-adapted condition was measured, and the saturated pulsed light (8000 μmol·m−2·s−1) was used to determine the photochemical quenching (qP), the maximum fluorescence value (*F*m ). Then, the method of Hendrickson et al. [37] was followed to determine the light energy absorbed by the PSII reaction center into each of four parts [38]. That is, the following parameters were inferred: quantum yield (*Φ*PSII) for the photochemical reaction, quantum yield (*Φ*NPQ) dependent on the proton gradient lutein cycle on both sides of the thylakoid membrane, quantum yield of fluorescence and heat energy dissipation (*Φ*f,D), and the thermal dissipation of the quantum yield (*Φ*NF) of the deactivated PSII reaction center. These parameters were inferred using the following formulae:

> *Φ*PSII = [1 − (*F*s/*F*m )] [(*F*v/*F*m)/(*F*v/*F*mM)] *Φ*NPQ = [(*F*s/*F*m ) − (*F*s/*F*m)] [(*F*v/*F*m)/(*F*v/*F*mM)] *Φ*f,D = (*F*s/*F*m) [(*F*v/*F*m)/(*F*v/*F*mM)] *Φ*NF = 1 − [(*F*v/*F*m)/(*F*v/*F*mM).

The sum of each of these parameters is 1, namely *Φ*PSII + *Φ*NPQ + *Φ*f,D + *Φ*NF = 1 (Figure 1).

**Figure 1.** Energy allocation pathways of photosystem II (PSII): proportion of light energy used in the quantum yield of PSII photochemistry (*Φ*PSII), xanthophyll-mediated thermal dissipation (*Φ*NPQ), quantum yield used in thermal dissipation in non-functional PSII (*Φ*NF), and quantum yield of fluorescent and heat energy dissipation (*Φ*f,D).

#### *2.10. Statistical Analysis*

Excel and SPSS software were used to conduct statistical analyses on the measured data. The data in the figure shows means of four plants ± standard deviation. Tukey Multiple Comparisons test was adopted to compare the differences between treatments. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### **3. Results**

#### *3.1. Effects of N Metabolism Indicators*

NO2 fumigation affected nitrogen metabolism in mulberry. The nitrate nitrogen content of mulberry leaves fumigated with NO2 increased by 56.10% (*p* < 0.05) at 4 h after the start of the treatment, and when the fumigation time lasted for 8 h, it was nearly doubled (*p* < 0.05) compared to the control. The amino acid content in mulberry was not significantly increased (*p* > 0.05) when NO2 was fumigated for 4 hours; however, it increased by 35.17% (*p* < 0.05) after 8 hours of fumigation.

Nitrate reductase and nitrite reductase activities increased significantly (*p* < 0.05) by 4 h after the start of the treatment, and when the fumigation time lasted for 8 h, the activities enhanced more (Figure 2a–d).

**Figure 2.** Effects of N Metabolism Indicators. (**a**) NO3 −-N content, (**b**) amino acid content, (**c**) nitrate reductase activity, and (**d**) nitrite reductase activity in leaves of mulberry seedlings exposure to 4 μL·L−1 NO2 for 0 h, 4 h (4 h·d−1, for one day), and 8 h (4 h·d−1, for 2 days). Date represent means of four plants ± standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### *3.2. Effects of Gas Exchange Parameters*

NO2 fumigation changed the *Pn*-*C*i curve of mulberry leaves, and *Pn* showed an upward trend with the increase of CO2 concentration. When the CO2 concentration was lower than 400 μmol·mol−1, *Pn* increases approximately linearly with the increase of CO2 concentration. Subsequently, as the CO2 concentration continued to increase, the rate of *Pn* increase slowed. When the CO2 concentration reached 1200 μmol·mol−1, the *Pn*-*C*i gradually flattened (Figure 3a). Under the same CO2 concentration, the *Pn* of mulberry leaves with NO2 fumigation was significantly higher than that of the control, indicating that NO2 fumigation improved the carbon assimilation capability of mulberry leaves. Stomatal conductance (*G*s), the maximum carboxylation rate (*V*cmax), and dark respiration rate (*R*d) of the mulberry leaves increased significantly (*p* < 0.05) after 4 h and 8 h of fumigation compared with the control (Figure 3b–d).

**Figure 3.** Effects of Gas Exchange Parameters. (**a**) *Pn*-Ci curve, (**b**) conductance of H2O, (**c**) the maximum carboxylation rate, and (**d**) dark respiration rate in leaves of mulberry seedlings exposure to 4 μL·L−1 NO2 for 0 h, 4 h (4 h·d−1, for one day), and 8 h (4 h·d−1, for 2 days). Date represent means of four plants ± standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### *3.3. Effects of Distribution of Light Absorbed by PSII*

Of the light energy absorbed by PSII, the fraction allocated to photochemical conversion (*Φ*PSII), increased slightly, while the fraction dissipated nonphotochemically in a manner dependent on the trans-thylakoid proton-gradient and the xanthophyll cycle (*Φ*NPQ) increased after NO2 fumigation. By contrast, the proportion of basic fluorescence quantum yields and heat-dissipated quantum yields (*Φ*f,D) and heat-dissipation quantum yields of inactive PSII reaction centers (*Φ*NF) declined after NO2 fumigation. The proportions of *Φ*NPQ and *Φ*PSII increased by 2.49% (*p* < 0.05), 36.23% (*p* < 0.05) at 4h and increased by 5.67% (*p* < 0.05), 41.07% (*p* < 0.05) at 8h. By contrast, the proportions of *Φ*f,D and *Φ*NF decreased by 5.01% (*p* < 0.05), 60.41% (*p* < 0.05) at 4h and decreased by 10.34% (*p* < 0.05) and 75.74% (*p* < 0.05) at 8 h (Figure 4). The quantum yield for heat dissipation in mulberry leaves fumigated with 4 μL·L−1 NO2 was reduced to increase the photosynthetic energy used for photochemical reaction. As shown in Figure 7, the partial energy from total energy absorbed by mulberry leaves fumigated by NO2 was used for NO2 metabolism; whether this affects the activity of PSII and PSI is discussed in the following sections.

**Figure 4.** Light energy used for quantum yield of PSII photochemistry (*Φ*PSII), xanthophyll-mediated thermal dissipation (*Φ*NPQ), basic fluorescence quantum yield and heat dissipation quantum yield (*Φ*f,D), and quantum yield used in thermal dissipation in non-functional PSII (*Φ*NF) in leaves of mulberry seedlings exposure to 4 μL·L−1 NO2 for 0 h, 4 h (4 h·d−1, for one day), and 8 h (4 h·d−1, for 2 days). Date represent means of four plants ± standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### *3.4. Effects of Chlorophyll A Fluorescence Transient*

NO2 fumigation significantly changed the shape of OJIP curves. The fluorescence intensity at points O and J (*F*o and *F*J, respectively) decreased, and the fluorescence intensity at points I and P (*F*I and *F*p, respectively) increased significantly. The OJIP curves of the leaves were normalized to the span O-P. The variable fluorescence at point J on the OJIP curve of leaves was significantly increased by NO2. The difference between the normalized O-P curve and the control was assessed, and the difference between ΔVt and the control at point J was most significant at 2 ms (Figure 5a–d).

**Figure 5.** Effects of Chlorophyll A Fluorescence Transient. (**a**) OJIP transmission, (**b**) fluorescence intensity at points O, J, I, and P, (**c**) standardized OJIP transmission, (**d**) standardized OJIP transmission and CK in leaves of mulberry seedlings exposure to 4 μL·L−1 NO2 for 0 h, 4 h (4 h·d−1, for one day), and 8 h (4 h·d−1, for 2 days). Date represent means of four plants ± standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### *3.5. Effects of PSII activity*

NO2 fumigation had a significant effect on PSII potential photochemical activity (*F*v/*F*o), PSII maximum photochemical efficiency (*F*v/*F*m), photochemical quenching (qP), electron transport rate (ETR), and number of active reaction centers per unit area (*RC*/*CS*m), which is shown in Table 1.

**Table 1.** PSII potential photochemical activities (*F*v/*F*o), PSII photochemical efficiency (*F*v/*F*m), photochemical quenching (qP), electron transport rate (ETR), and the number of reactive centers per unit area (*RC*/*CS*m) in leaves of mulberry seedlings exposure to 4 μL·L−1 NO2 for 0 h, 4 h (4 h·d−1, for one day), and 8 h (4 h·d−1, for 2 days). Date represent means of four plants ± standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### *3.6. Effects of PSI Activity*

The relative drop in 820 nm light signals during far-red light illumination reflects the activity of PSI Δ*I*/*I*o. NO2 fumigation increased the drop in the 820 nm optical signal, and the difference increased significantly at 8 h, indicating that NO2 fumigation increased the PSI activity of mulberry leaves (Figure 6a). PSI activity was not significantly raised (*p* > 0.05) for 4 h fumigation, but when fumigated for 8 h, the PSI activity was improved by 20.22% (*p* < 0.05) compared with the control (Figure 6b).

**Figure 6.** Effects of PSI activity. (**a**) Changes in light transmission at 820 nm, (**b**) Relative values of Δ*I/I*o in leaves of mulberry seedlings exposure to 4 μL·L−1 NO2 for 0 h, 4 h (4 h·d−1, for one day), and 8 h (4 h·d−1, for 2 days), where the Δ*I*/*I*o values at 8 h were taken as 100%. Date represent means of four plants ± standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### **4. Discussion**

The present study revealed that the absorption of NO2 by mulberry leaves is not only involved in nitrogen metabolism in vivo, but also increases the leaf content of nitrate nitrogen and amino acids, enhances the activity of nitrate reductase (NR) and nitrite reductase (NiR), and improves the nitrogen metabolism capacity (Figure 2). Zeevaart et al. [39] fumigated pea seedlings with ammonium nitrogen as the only nitrogen source condition and found that NO2 induces NR activity, in perhaps the earliest study on NO2 and nitrogen metabolism. Subsequent studies have since found that atmospheric NO2 has a significant effect on NR. Lower concentrations of NO2 increase NR activity in barley (*Hordeum vulgre*) [40], Scots pine (*Pinus sylvestris*) [41], and red spruce (Picea rubens) [42]. Similarly, when Hisamatsu et al. [43] fumigated squash (*Cucurbita maxima*) seedlings with NO2, NR activity in cotyledon was significantly reduced. In the present study, NR activity had exhibited a small increase at 4 h but increased sharply at 8 h. Enhanced NR activity promotes the improvement of nitrogen metabolism. It not only increases the consumption of excess light energy, but also provides necessary enzymes for carbon metabolism and accelerates carbon metabolism.

The 4 μL·L−1 NO2 fumigation treatment significantly increased the net photosynthetic rate of mulberry leaves (Figure 3a), accelerating photosynthetic electron transport and enhancing phosphorylation activity in leaves. Additionally, the increase of stomatal conductance promoted the degree of CO2 acquisition and transportation (Figure 3b), thereby accelerating the production and accumulation of organic matter in plants [44]. At the same time, the maximum carboxylation rate, which is an important parameter for characterizing photosynthetic capacity of plants, also increased (Figure 3b), indicating that NO2 fumigation increased Rubisco activity of mulberry leaves, thereby enhancing the fixation ability of CO2 [45]. Moreover, the dark respiration rate was significantly increased (Figure 3d), indicating that its fumigated leaves consumed excess light energy through respiration to protect the photosynthetic system and increase the photosynthetic rate.

Accordingly, the quantitative study of the final destination of light energy absorbed by plant leaves is an important part of research on photosynthesis. Earlier studies on light energy are more directly expressed by actual photochemical efficiency (*Φ*PSII) and non-photochemical quenching (NPQ) [46,47]. However, in higher plants, NPQ is only determined by the establishment of proton gradients on both sides of the thylakoid membrane and the xanthophyll cycle [48]. Thus, NPQ does not represent all non-photochemical quenching processes. In addition, plant leaves absorb light energy via mechanisms other than photochemical reactions, including physiological processes such as photorespiration, the water–water cycle, and xanthophyll cycle, which can be areas in which light energy is distributed as the final destination [49–51]. The theory developed by Hendrickson et al. [50] clarifies these processes. In this framework, excitation energy can be divided into the light energy absorbed by the PSII reaction centers and used for the quantum yield of photochemical reaction (*Φ*PSII), the quantum production dependent on the trans-thylakoid proton-gradient for xanthophyll cycle quantum yield (*Φ*NPQ), fluorescence quantum yield and heat dissipation (*Φ*f,D), and heat dissipation quantum yield in inactive PSII reaction centers (*Φ*NF) [51]. In our experiment, mulberry leaves fumigated with NO2 showed proportional increases in *Φ*PSII and *Φ*NPQ, indicating that NO2 fumigation promoted the establishment of light-induced proton absorption in the chloroplast H+ concentration gradients on both sides of the thylakoid. In other words, the presence of ΔpH on both sides of the thylakoid membrane increased the power of ATP synthesis in chloroplasts and promoted the photoprotective mechanism based on the xanthophyll cycle. Additionally, *Φ*f,D and *Φ*NF proportionally decreased, indicating that the proportion of heat dissipation and inactivation reaction centers decreased and that the absorbed light energy was mostly used for photosynthetic carbon assimilation, thereby improving the photochemical efficiency of the mulberry leaf (Figure 4).

In this experiment, the changes in PSII of mulberry leaves were analyzed using the JIP test. NO2 changed the structure and function of PSII and the photosynthetic primary reaction process in mulberry leaves. However, because the OJIP curve is greatly influenced by the environment, its relative fluorescence intensity is affected by various environmental factors. Therefore, the OJIP curve was often normalized to the span O-P. The relative variable fluorescence of *V*J and *V*I at points J (2 ms) and I point (30 ms) for NO2-fumigated leaves was significantly decreased (Figure 5), indicating that the PSII reaction center receptor-side electronic primary quinone receptors (*Q*A) to the secondary quinone receptor (*Q*B) transmission capacity and plastoquinone (PQ) accept electronic ability were enhanced (Figure 7). The potential photochemical activity of PSII (*F*v/*F*o), PSII maximum photochemical efficiency (*F*v/*F*m), photochemical quenching (qP), electron transfer rate (ETR), and the number of active reaction centers per unit area (*RC*/*CS*m) were significantly higher under fumigation than in the control treatment (Table 1), indicating that NO2 fumigation enhances the activity of PSII reaction centers and the degree of openness of reaction centers, which promotes the electron transport of leaves, photosynthetic primary reaction process, and the rate of light photons received by PSII reaction centers. The proportion of light energy used in photochemical reactions increased, thus accelerating the synthesis of NADPH and ATP, as well as the conversion efficiency of light energy and carbon assimilation processes, which increased the photochemical efficiency. However, Hu et al. [31] quantified the photosynthetic responses of hybrid poplar cuttings to 4 μL·L−1 NO2. It was found that significant declines in *F*v/*F*m, indicating inhibition of and even damage to photosynthetic apparatus. The study of the maximum redox capacity (Δ*I*/*I*o) of PSI

was also increased (Figure 6) as well as the ability to receive electrons; the ability of PSII to supply electrons in the photosynthetic apparatus was matched by the ability of PSI to receive electrons. Thus, not only can electron transfer be promoted efficiently, but electrons can also be transferred to ferredoxin, which distributes electrons to the nitrogen metabolism, photorespiration, and NO2 metabolic pathways (Figure 7).

**Figure 7.** Photosynthetic electron flow distribution. The electrons produced by water splitting in photosynthesis pass through photosystem II (PSII), primary quinone receptor (*Q*A), secondary quinone receptor (*Q*B), plastoquinone (PQ), cytochrome *b6f* (Cyt *b6f*), plastid blue pigment (PC), and photosystem I (PSI) to ferredoxin (Fd) were assigned to four pathways: CO2 assimilation, nitrogen metabolism, photorespiration, and NO2 metabolic pathways (new pathway).

#### **5. Conclusions**

The metabolism of atmospheric NO2 utilized and consumed light energy and photosynthetic electrons absorbed by leaves of mulberry fumigated with NO2. However, this part of the photosynthetic energy consumption did not reduce the photosynthetic capacity of the mulberry leaves, but instead increased the photosynthetic efficiency of the plant leaves. This is because the mulberry leaves absorbed NO2 and conducted nitrogen metabolism and respiration, which consumed excess light energy, and thus protected the photosynthetic apparatus. However, the light energy absorbed by the PSII reaction center in the form of heat dissipation in mulberry plants fumigated with 4 μL·L−1 NO2 was also reduced, such that the absorbed light energy was more effectively used in photosynthetic carbon assimilation. Therefore, in the case where the concentration of the atmospheric pollutant NO2 is lower than the concentration that can damage the mulberry, NO2 can be absorbed by the mulberry to reduce the haze in the air.

**Author Contributions:** Y.W. and G.S. conceived and designed the study. Y.W., W.J., Y.C., and D.H. performed the experiments. Y.W., J.W., and M.Z. contributed to the sample measurement and data analysis. Y.W. and G.S. wrote the paper.

**Funding:** This research was funded by the National Natural Science Foundation of China, grant number 31870373, and the Natural Science Foundation of Heilongjiang Province, grant number ZD201105 and the Applicant and Developmental Project for Agriculture of Heilongjiang Province, grant number GZ13B004.

**Acknowledgments:** The authors want to appreciate Wah Soon Chow from the Australian National University for revising the manuscript and our colleague Yanbo Hu for his advice and great comments to improve the paper.

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

#### **References**

*Forests* **2019**, *10*, 312

51. Hendrickson, L.; Förster, B.; Pogson, B.J.; Chow, W.S. A simple chlorophyll fluorescence parameter that correlates with the rate coefficient of photoinactivation of photosystem II. *Photosynth. Res.* **2005**, *84*, 43–49. [CrossRef]

© 2019 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 (http://creativecommons.org/licenses/by/4.0/).


*Edited by Jonathan Glazzard*

Dyslexia is biological in origin, but environmental factors also play a role. It affects people differently, but typical difficulties include reading, writing, and spelling, limited working memory capacity, and difficulties with organization, sequencing, and concentration. Early identification is crucial so that learners can receive the support that they need. This book explores contemporary perspectives on dyslexia. It starts by outlining approaches to support dyslexic students drawn from the application of cognitive science. It moves on to consider treatments, the role of technology, and approaches for working with teaching assistants.

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Oral Surgical Procedures

*Oral Diseases*

2004;**30**:289-301

2010;**36**:609-617

endodontically treated teeth: A

[48] Tang W, Wu Y, Smales RJ. Identifying and reducing risks for potential fractures in endodontically treated teeth. Journal of Endodontia.

[49] Onay EO, Ungor M, Ari H, et al. Push-out bond strength and SEM evaluation of new polymeric root canal fillings. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. 2009;**107**:879-885

literature review. Journal of Endodontia.

**192**

**Chapter 14**

**Abstract**

**1. Introduction**

**195**

Surgery

*Randa Abdulmoein AlFotawi*

rich vascularity and good final aesthetic outcome.

their fundamental principles are highlighted.

**Keywords:** flap principle, applications, classification, flap technique

Oral surgical flap by definition is the operation in which a portion of the mucoperiosteal tissue is surgically detached from the underlying bone for better access and visibility. Common principles have been applied for all flap designs. First, the base of the flap should be broader than the free end to ensure adequate blood supply. Second, the incision should be performed at a right angle to the underlying bone, avoiding any anatomical structures, and it should provide adequate visualization. Third, the flap should be wider than the anticipated underlying bone defect and delicately handled without tension. Fourth, the vertical releasing incision should start from the buccal vestibule and end up mesial or distal to the interdental papilla. Different flaps have been proposed for various intraoral surgeries, that is, third molar surgery, canine exposure, various periodontal surgery, dental implant preparation, endodontic surgeries, and repair of oroantrual communications. The review will focus on oral anatomy, classification, indications, complications of common oral flap techniques; common flap designs are illustrated, and

Flap Techniques in Dentoalveolar

Most dentoalveolar procedures involve the reflection of mucosal flaps. This step

is crucial for exposure or removal of impacted teeth, implant bed preparation, exposure of the alveolar bone for augmentation, periodontal surgeries, and repair of mucosal soft tissue defects, such as oroantral fistula. Because of the rich vascularity of the oral mucosa, great freedom is allowed for flap design, but it tends to result in carelessness and lack of thoughtful planning, which may lead to uneventful outcomes or/and complications. In this chapter, we review oral anatomy, classification, indications, and complications of common oral flap techniques; common flap designs are illustrated, and their fundamental principles are highlighted. The review has covered various flap designs based on their indications. Yet the common flap's principles are fundamental for all types of flaps regardless of their application, namely, it should provide wide exposure, clear vision, good access, and assure


Author details

Anna Antonenko1 \*, Olena Vavrinevych1 , Maria Korshun2 and Sergiy Omelchuk3

1 Hygiene and Ecology Department № 1 of O.O. Bogomolets National Medical University, Kyiv, Ukraine

2 Hygiene and Ecology Department № 3 of O.O. Bogomolets National Medical University, Kyiv, Ukraine

3 Hygiene and Ecology Institute of O.O. Bogomolets National Medical University, Kyiv, Ukraine

\*Address all correspondence to<EMAIL_ADDRESS>
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**4.2 CY regulates cytokine network released by Mf**

One of the most strongly expressed of CY and their derivatives treatment of macrophages is the activation of IL-6 production (Bryniarski 1996, 2009) . Our results do not address directly the questions by which mechanisms NM modifies the macrophages to produce more IL-6. IL-6 gene expression can be induced by a variety of physiological (cytokines, growth factor, bacterial products) and non-physiological stimuli (certain toxins, medicaments, prostaglandin E1), by at least three different signals pathways (diacyloglycerol, cAMP- and Ca2+- activated pathways). At the DNA level three functional promoter domains were described in conserved region of IL-6 promoter (MRE, NF-IL6 and NFB). Exactly how transduction pathways are assigned seems unknown with the possible exception of protein kinase C signal which seems to focus on MRE region (Bryniarski et al. 1996). Since NM

The Influence of Cyclophosphamide on Immune Function of Murine Macrophages 155

receives a proper signal (e.g. phagocytosis), translocate to form an active enzyme. We propose a possible explanation that under our experimental conditions, ACR, in contrast to NM, does not bind efficiently to important docking proteins to trigger the increased production of ROI. Conflicting results regarding ROI production were also published by other groups. Some reports describe the inhibitory activity of ACR, and others indicate an increased production of radicals. In effect, one can conclude that experimental conditions

Our experimental data showed that untreated and in vivo CY treated populations of peritoneal macrophages produce the similar level of nitrogen oxide (Marcinkiewicz et al. 1994) which does not allow to speculate on its function in the immune regulatory system

Our data show that chemotherapy by CY or its products may activate the immune system by modulating cytokine networks and activation of Mf. This may lead to an enhancement of antigen-specific cell mediated immunity but also to activation of mechanisms of innate immunity mediated by Mf, like the production of ROI. Additionally, in animal models derivatives of different mustards led to decreased secretion of IL-10 and TGF- by tumor cells and to their elimination. These and other similar experiments in humans support the notion that, at a correct dosage, CY and its metabolites can be a promising accessory tool in

The mechanism of CY influences macrophage immune function in as was shown previously in case of peritoneal Mf and TMf and seems to be the effect of network of different related factors. The analysis shows as the most important the influence of CY on the secretory activity of Mf which is the inhibition of IL-10 (in case of peritoneal Mf) and TGF- (mainly in case of TMf) with parallel activation of proinflammatory cytokines secretion mainly IL-6, and to a lesser degree IL-12. Both cytokine signals lead to activation of antigen presentation in Mf. The other important factor mediated by CY treatment is an influence on the activation of a cell surface markers expression responsible for uptaking antigen into APC (FcR I, FcRII, CD23 – FcRII/III) and their following presentation to T lymphocytes

Although our experiments indicate that in the testis – immune privileged organ – some subpopulations of Mf are potentially able to present antigen if they would sneak through the blood-testis barrier, they also suggest how this potential activity is under control of other Mf and Sertoli cells. Our previous observations have shown that TMf are poor producers of oxygen radicals and nitric oxide both involved in the mechanisms of natural immunity which may be an evolutionary adaptation to diminish the risk of DNA mutations during spermatogenesis. Additionally we showed that specific immune responses controlled by the male gonads minimize the risk of development of autoimmune reactions and are potentially deleterious to testicular functions (Bryniarski 2004). Testicular Mf are good producers of TGF-, which allows them to play an important functional population of testicular interstitial tissue cells that preserves state of tolerance in testes an immune privilege organs.

were the key (Bryniarski at al. 2009).

anti-tumor therapy.

mediated by macrophage stimulated with CY.

**4.4 Immunomodulation in chemotherapy with low doses of CY**

subpopulations (CD80/CD86, MHC class II, CD14-LPS receptor).

**4.5 Influence of CY on testicular macrophages**

binds to both DNA and proteins, it could stimulate IL-6 production directly by alkylating any domain of promoter region, or indirectly, by the alkylation of cell surfaces or by both mechanisms simultaneously.

IL-6 is one of the major mediators of the immune response, with pleotropic and sometimes opposed effects on many different targets. It has been shown for instance that IL-6 enhances the cytotoxic activity of NK cells, thus may potentially augment the host defenses and contribute to anti-tumor effects of alkylating agents. Nonetheless, in case of the IL-6 dependent tumours like myelomas or plasma cell leukemias increased IL-6 level could be deleterious. The increased production of IL-6 may be responsible for observed paradoxical effects of CY which under certain conditions enhances, rather than suppresses both the humoral and cell mediated immune responses (Bryniarski et al.1996).

Apart from the influence of CY and its metabolites on the IL-6 production the inhibition of IL-10 and TGF- production by macrophages was also observed. The results presented above in Table 4 and Fig.3 clearly suggest that the state of tolerance or unresponsiveness observed after TNP-Mf i.v. injection seems to be mediated by the network of pro- and antiinflammatory cytokines secreted from macrophages and also tentatively delivered by natural regulatory cells. It is highly unlikely that low concentration of cytokine metabolites (10-7 and 10-6M) have a direct cytotoxic effect on the Treg-inducing Mf since the cell viability remains unchanged during 24 h culture. Our interpretation is also supported by finding that shifting the balance between pro- and anti-inflammatory cytokines allows for deliberate manipulation of the outgoing response. IL-10 and TGF-, which are anti-inflammatory cytokines, inhibit the activity of Th1 cells and Mf and down-regulate their function. As shown in Figure 3, administration of anti-IL-10 and/or anti-TGF- mAbs into animals which received non-immunogenic TNP-Mf restores their immune potential although to different degrees. It indicates that the key suppressive cytokine is IL-10, a finding that is supported by other groups (Bryniarski et al. 2009). In a symmetrical situation, as we have shown previously, administration of anti-IL-12 antibodies inhibits the function of immunogenic TNP-Mf *in vivo* (Bryniarski et al. 2009). The increased production of IL-12 and IL-6 by macrophages indicates that the cell surface signal delivered by ACR or NM activates the transcription factor NF-B required for the release of inflammatory cytokines (Bryniarski et al. 2009). As reported by other groups, ACR, when allowed free access to the interior of the cell, can either block or enhance the activity of NF-B in alveolar macrophages depending on the design of cell treatment (Bryniarski et al. 2009).

#### **4.3 CY modulates oxygen radicals formation by macrophages**

Our results indicate that CY upregulates not only the specific immune response, by converting non-immunogenic (tolerogenic) Mf into antigen-presenting cells but also positively influences a typical parameter of innate immunity – production of oxygen radicals. In up-regulating the immune function of Mf, ACR and NM had much the same effect. This was however not the case with regard to the production of ROI's by these cells. Using the low concentrations of metabolites, NM was highly stimulatory while ACR did not influence the formation of oxygen radicals above the level observed in control Mf (high concentration of both metabolites were inhibitory). One possibility is that the ACR and NM bind to different targets on the cell surface. NADPH oxidase catalyzing the generation of ROI is composed of several cytosolic and membrane-bound proteins which, after the cell

binds to both DNA and proteins, it could stimulate IL-6 production directly by alkylating any domain of promoter region, or indirectly, by the alkylation of cell surfaces or by both

IL-6 is one of the major mediators of the immune response, with pleotropic and sometimes opposed effects on many different targets. It has been shown for instance that IL-6 enhances the cytotoxic activity of NK cells, thus may potentially augment the host defenses and contribute to anti-tumor effects of alkylating agents. Nonetheless, in case of the IL-6 dependent tumours like myelomas or plasma cell leukemias increased IL-6 level could be deleterious. The increased production of IL-6 may be responsible for observed paradoxical effects of CY which under certain conditions enhances, rather than suppresses both the

Apart from the influence of CY and its metabolites on the IL-6 production the inhibition of IL-10 and TGF- production by macrophages was also observed. The results presented above in Table 4 and Fig.3 clearly suggest that the state of tolerance or unresponsiveness observed after TNP-Mf i.v. injection seems to be mediated by the network of pro- and antiinflammatory cytokines secreted from macrophages and also tentatively delivered by natural regulatory cells. It is highly unlikely that low concentration of cytokine metabolites (10-7 and 10-6M) have a direct cytotoxic effect on the Treg-inducing Mf since the cell viability remains unchanged during 24 h culture. Our interpretation is also supported by finding that shifting the balance between pro- and anti-inflammatory cytokines allows for deliberate manipulation of the outgoing response. IL-10 and TGF-, which are anti-inflammatory cytokines, inhibit the activity of Th1 cells and Mf and down-regulate their function. As shown in Figure 3, administration of anti-IL-10 and/or anti-TGF- mAbs into animals which received non-immunogenic TNP-Mf restores their immune potential although to different degrees. It indicates that the key suppressive cytokine is IL-10, a finding that is supported by other groups (Bryniarski et al. 2009). In a symmetrical situation, as we have shown previously, administration of anti-IL-12 antibodies inhibits the function of immunogenic TNP-Mf *in vivo* (Bryniarski et al. 2009). The increased production of IL-12 and IL-6 by macrophages indicates that the cell surface signal delivered by ACR or NM activates the transcription factor NF-B required for the release of inflammatory cytokines (Bryniarski et al. 2009). As reported by other groups, ACR, when allowed free access to the interior of the cell, can either block or enhance the activity of NF-B in alveolar macrophages depending

humoral and cell mediated immune responses (Bryniarski et al.1996).

on the design of cell treatment (Bryniarski et al. 2009).

**4.3 CY modulates oxygen radicals formation by macrophages**

Our results indicate that CY upregulates not only the specific immune response, by converting non-immunogenic (tolerogenic) Mf into antigen-presenting cells but also positively influences a typical parameter of innate immunity – production of oxygen radicals. In up-regulating the immune function of Mf, ACR and NM had much the same effect. This was however not the case with regard to the production of ROI's by these cells. Using the low concentrations of metabolites, NM was highly stimulatory while ACR did not influence the formation of oxygen radicals above the level observed in control Mf (high concentration of both metabolites were inhibitory). One possibility is that the ACR and NM bind to different targets on the cell surface. NADPH oxidase catalyzing the generation of ROI is composed of several cytosolic and membrane-bound proteins which, after the cell

mechanisms simultaneously.

receives a proper signal (e.g. phagocytosis), translocate to form an active enzyme. We propose a possible explanation that under our experimental conditions, ACR, in contrast to NM, does not bind efficiently to important docking proteins to trigger the increased production of ROI. Conflicting results regarding ROI production were also published by other groups. Some reports describe the inhibitory activity of ACR, and others indicate an increased production of radicals. In effect, one can conclude that experimental conditions were the key (Bryniarski at al. 2009).

Our experimental data showed that untreated and in vivo CY treated populations of peritoneal macrophages produce the similar level of nitrogen oxide (Marcinkiewicz et al. 1994) which does not allow to speculate on its function in the immune regulatory system mediated by macrophage stimulated with CY.

#### **4.4 Immunomodulation in chemotherapy with low doses of CY**

Our data show that chemotherapy by CY or its products may activate the immune system by modulating cytokine networks and activation of Mf. This may lead to an enhancement of antigen-specific cell mediated immunity but also to activation of mechanisms of innate immunity mediated by Mf, like the production of ROI. Additionally, in animal models derivatives of different mustards led to decreased secretion of IL-10 and TGF- by tumor cells and to their elimination. These and other similar experiments in humans support the notion that, at a correct dosage, CY and its metabolites can be a promising accessory tool in anti-tumor therapy.

The mechanism of CY influences macrophage immune function in as was shown previously in case of peritoneal Mf and TMf and seems to be the effect of network of different related factors. The analysis shows as the most important the influence of CY on the secretory activity of Mf which is the inhibition of IL-10 (in case of peritoneal Mf) and TGF- (mainly in case of TMf) with parallel activation of proinflammatory cytokines secretion mainly IL-6, and to a lesser degree IL-12. Both cytokine signals lead to activation of antigen presentation in Mf. The other important factor mediated by CY treatment is an influence on the activation of a cell surface markers expression responsible for uptaking antigen into APC (FcR I, FcRII, CD23 – FcRII/III) and their following presentation to T lymphocytes subpopulations (CD80/CD86, MHC class II, CD14-LPS receptor).

#### **4.5 Influence of CY on testicular macrophages**

Although our experiments indicate that in the testis – immune privileged organ – some subpopulations of Mf are potentially able to present antigen if they would sneak through the blood-testis barrier, they also suggest how this potential activity is under control of other Mf and Sertoli cells. Our previous observations have shown that TMf are poor producers of oxygen radicals and nitric oxide both involved in the mechanisms of natural immunity which may be an evolutionary adaptation to diminish the risk of DNA mutations during spermatogenesis. Additionally we showed that specific immune responses controlled by the male gonads minimize the risk of development of autoimmune reactions and are potentially deleterious to testicular functions (Bryniarski 2004). Testicular Mf are good producers of TGF-, which allows them to play an important functional population of testicular interstitial tissue cells that preserves state of tolerance in testes an immune privilege organs.

The Influence of Cyclophosphamide on Immune Function of Murine Macrophages 157

A single administration of low dose of CY (50 mg/kg) into either donors or recipients restores the ability of Mf to induce significant CS reaction as a result of: i.) elimination of suppressive properties of Mf; ii.) and/or depletion of population of regulatory T cells in recipients or iii.) elimination of their suppressive activities. In vitro studies with metabolites of CY in contrast to studies in vivo allow identifying the factors which express direct action on selected populations of cells in contrary to experimental research in vivo which is able to identify parallel with direct also indirect effects of cyclophosphamide action on other than macrophages cell populations ( T reg cells) that may change and modulate the activity of macrophages and their influence on the immune response. We propose the schemes which summarize the influence of low doses of CY on the immune response in mice

Figure 6 a-c. The network of the CY influence on the macrophage and regulatory T cells in

\* Induction of T suppressor factor (see the reference by Bryniarski et al. 2-nd European Congress of

Fig. 6.a. Activation of unresponsiveness in recipients after i.v. injection of TNP substituted

(**Figures 6a-6c**).

Immunology Berlin 2009)

Mf – lack of CHS reaction.

mice.

That state of tolerance eliminates the cellular immune response from the testis and in consequence makes an extremely dangerous the viral infection as well as in malignances taking place in testis. CY treatment often change TMf activity from unresponsiveness into actively antigen presenting cells which in consequence help to undertake anticancer response but also often can leads to activation an autoimmune response and immunological infertility as a consequences of chemotherapy.

#### **5. Conclusions**

The influence of CY on Mf can be summarized as a sum of several different mechanisms mediated by macrophages such as secretion of a specific pattern of cytokines and enhances expression of cell surface markers that can stimulate antigen presenting function by macrophages and last not least production of ROI's. On the other side there are several observations that low dose CY treatment has a direct influence of the different regulatory cells in immune system. One of them is a negative activation of CD8+ T lymphocytes leading to elimination of their effector mediators - suppressor cytokines secretion mainly TGF- and IL-10, which negatively regulate the cellular immune response, but do not express any negative effect of humoral response. That state of abrogation of unresponsiveness is also observed experimentally when the TNP-substituted Mf obtained from oil-induced donors are injected into CY treated recipients of cells (see **Figure 5**) group C. In that case instead of unresponsiveness expressed by control group (group A) strong CHS response appears 24 h after challenge. This phenomenon clearly shows the influence of CY on the suppressor network of T reg cells. This also clearly shows that CY-manipulation leads to manifold effects in which manifold can be described as wiped out or misdirected. In the literature there are two papers suggesting depletion activity of CY on Treg CD4 CD25 T lymphocytes and Treg CD4 CD25 FoxP3+ lymphocytes (Ghiringhelli et al. 2004 & Zhao et al. 2010)

1x106 TNP substituted Mf (groups A and C) or TNP-Mf CY (groups B and D) were injected i.v. into naive (groups A and B) or treated with low dose of CY CBA/J mice. Seven days later the CHS response was measured (see legend to Table 2). Statistical significance (a posteriori Bonferroni test) Group A ve groups B, C and D p<0.001.

Fig. 5. Alleviation of suppression of contact sensitivity response induced by low dose treatment with cyclophosphamide applied either to macrophage donors or recipients.

That state of tolerance eliminates the cellular immune response from the testis and in consequence makes an extremely dangerous the viral infection as well as in malignances taking place in testis. CY treatment often change TMf activity from unresponsiveness into actively antigen presenting cells which in consequence help to undertake anticancer response but also often can leads to activation an autoimmune response and immunological

The influence of CY on Mf can be summarized as a sum of several different mechanisms mediated by macrophages such as secretion of a specific pattern of cytokines and enhances expression of cell surface markers that can stimulate antigen presenting function by macrophages and last not least production of ROI's. On the other side there are several observations that low dose CY treatment has a direct influence of the different regulatory cells in immune system. One of them is a negative activation of CD8+ T lymphocytes leading to elimination of their effector mediators - suppressor cytokines secretion mainly TGF- and IL-10, which negatively regulate the cellular immune response, but do not express any negative effect of humoral response. That state of abrogation of unresponsiveness is also observed experimentally when the TNP-substituted Mf obtained from oil-induced donors are injected into CY treated recipients of cells (see **Figure 5**) group C. In that case instead of unresponsiveness expressed by control group (group A) strong CHS response appears 24 h after challenge. This phenomenon clearly shows the influence of CY on the suppressor network of T reg cells. This also clearly shows that CY-manipulation leads to manifold effects in which manifold can be described as wiped out or misdirected. In the literature there are two papers suggesting depletion activity of CY on Treg CD4 CD25 T lymphocytes

and Treg CD4 CD25 FoxP3+ lymphocytes (Ghiringhelli et al. 2004 & Zhao et al. 2010)

1x106 TNP substituted Mf (groups A and C) or TNP-Mf CY (groups B and D) were injected i.v. into naive (groups A and B) or treated with low dose of CY CBA/J mice. Seven days later the CHS response was measured (see legend to Table 2). Statistical significance (a posteriori Bonferroni test)

Fig. 5. Alleviation of suppression of contact sensitivity response induced by low dose treatment with cyclophosphamide applied either to macrophage donors or recipients.

infertility as a consequences of chemotherapy.

Group A ve groups B, C and D p<0.001.

**5. Conclusions**

A single administration of low dose of CY (50 mg/kg) into either donors or recipients restores the ability of Mf to induce significant CS reaction as a result of: i.) elimination of suppressive properties of Mf; ii.) and/or depletion of population of regulatory T cells in recipients or iii.) elimination of their suppressive activities. In vitro studies with metabolites of CY in contrast to studies in vivo allow identifying the factors which express direct action on selected populations of cells in contrary to experimental research in vivo which is able to identify parallel with direct also indirect effects of cyclophosphamide action on other than macrophages cell populations ( T reg cells) that may change and modulate the activity of macrophages and their influence on the immune response. We propose the schemes which summarize the influence of low doses of CY on the immune response in mice (**Figures 6a-6c**).

Figure 6 a-c. The network of the CY influence on the macrophage and regulatory T cells in mice.

\* Induction of T suppressor factor (see the reference by Bryniarski et al. 2-nd European Congress of Immunology Berlin 2009)

Fig. 6.a. Activation of unresponsiveness in recipients after i.v. injection of TNP substituted Mf – lack of CHS reaction.

The Influence of Cyclophosphamide on Immune Function of Murine Macrophages 159

The work is supported by grant No K/ZDS/001429 to KB. The author expresses gratitude to Ms. Katarzyna Nazimek M.Sc. for precious help in preparing the Figures and Tables.

Adjalle, R. Plouin, P. F. Pacak, K. & Lehnert, H. (2009). Treatment of malignant

Audia, S. Nicolas, A. Cathelin, D Larmonier N, Ferrand C, Foucher P, Fanton A, Bergoin E,

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P.W. Ptak, W. (2009) Role of low molecular weight RNA in contact sensitivity

trophoblastic tumors: case of resistant pulmonary metastasis. *Nature, Clinical*

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115.

**6. Acknowledgments**

**7. References**

Fig. 6.b. The network of the CY influence on the macrophage and regulatory cells in mice. The i.v. injection into naïve recipient of TNP substituted Mf harvested from CY treated donors results in a state of high CHS reaction 24 h after challenge.

Fig. 6.c. The network of the CY influence on the macrophage and regulatory cells in mice. The i.v. injection of TNP substituted Mf harvested from naïve donors into recipients previously treated with low dose of CY results in expression of high CHS reaction 24 h after challenge with PCL hapten. The activation of CHS is the effect of blocking of natural reg T cells or antigen specific Ts cells.

## **6. Acknowledgments**

The work is supported by grant No K/ZDS/001429 to KB. The author expresses gratitude to Ms. Katarzyna Nazimek M.Sc. for precious help in preparing the Figures and Tables.

#### **7. References**

158 Pharmacology

Fig. 6.b. The network of the CY influence on the macrophage and regulatory cells in mice. The i.v. injection into naïve recipient of TNP substituted Mf harvested from CY treated

Fig. 6.c. The network of the CY influence on the macrophage and regulatory cells in mice. The i.v. injection of TNP substituted Mf harvested from naïve donors into recipients

cells or antigen specific Ts cells.

previously treated with low dose of CY results in expression of high CHS reaction 24 h after challenge with PCL hapten. The activation of CHS is the effect of blocking of natural reg T

donors results in a state of high CHS reaction 24 h after challenge.

**8**

*1Indonesia 2The Netherlands*

**Modification of Interleukin-10 with Mannose-**

**6-Phosphate Groups Yields a Liver-Specific**

*2University of Groningen, Dept. of Pharmacokinetics, Toxicology and Targeting,*

Cytokines and other biological compounds are considered as future drugs and they are of particular interest for the treatment of chronic diseases. These endogenous compounds, that normally mediate local cellular communications, are very promising candidates to generate new drugs because of their high potency (pM-nM concentrations) and their fundamental roles in pathological processes. However, the therapeutic application of cytokines is limited, because several problems are encountered with their application *in* vivo (Schooltink and Rose-John 2002; Standiford 2000; Vilcek and Feldmann 2004). For instance, some cytokines are efficiently degraded in plasma by various enzymes and cytokines are rapidly excreted by the kidneys. Consequently their residence time in the body and thus the exposure to the diseased cells is short (plasma half life is often minutes), which does not favour an optimal biological efficacy. Another major problem is the occurrence of side effects. Because cytokine receptors are ubiquitously expressed in all organs, unusual high plasma concentrations of

To overcome these problems, we use drug targeting techniques to selectively deliver the cytokine to a specific (diseased) cell (Allen and Cullis 2004; Beljaars, Meijer, Poelstra 2002). The challenge is to improve its distribution within the body and direct the cytokine to a cell of interest, while maintaining the biological activity of that particular cytokine after chemical modification. A conventional way to modify proteins is conjugation with polyethylene glycol (PEG) (Jevsevar, Kunstelj, Porekar 2010). The attachment of PEG moieties improves the pharmacokinetics. That is, PEG substitution prevents rapid renal elimination which results in compounds with prolonged plasma concentrations, thereby making a reduced number of doses possible. For instance, PEGasys (PEGylated interferon 2a), an example of a PEGylated cytokine that is now commonly used to treat patients infected with viral hepatitis, is dosed once a week while the unmodified interferon is dosed daily. This leads to an improvement in the compliance and quality of life in patients with chronic

the cytokine can lead to (unwanted) effects in various organs.

diseases. The side effects, however, are not diminished after PEGylation.

**1. Introduction**

**Cytokine with Antifibrotic Activity in Rats**

Catharina Reker-Smit2, Klaas Poelstra2 and Leonie Beljaars2

*1Pharmaceutics, Bandung Institute of Technology, Bandung*

Heni Rachmawati1,2, Adriana Mattos2,

immunotherapy of established tumors to be curative. *European Journal of Immunology*. Vol. 34 pp.336-344.


**Conflict of interest**

*Quality of Life - Biopsychosocial Perspectives*

and loss of energy or increased fatigue [57].

respectively [58].

It can be classified on (i) major depression, when the presence of depressive mood and anhedonia (reduced positive affect) for 2 weeks is accompanied by at last five symptoms such as depressed mood, uncontrolled weight for no apparent reason, lack of motivation, psychomotor disorder, loss of energy or increased fatigue, alteration in sleep, difficulty thinking and thoughts of dead or suicide; (ii) minor depression, which is similar to major depression, but the patient has few than five symptoms; and (iii) dysthymia, when the patient experiences an acute depressive mood for most of the day which persists for at least 2 years. However, at least two of the following symptoms should be present: changes in appetite, low self-esteem, sleep alteration, difficulty thinking, discouragement,

The nine-item patient health questionnaire is a valid and reliable screaming tool frequently used for the diagnosis of depression. It is an easy and brief self-report questionnaire and can be used in patient with T2D. It consists of nine questions based in the depressive symptoms with score of "0" (not at all) to "3" (nearly every day). It calculated a total score, and it ranges from 0 to 27. A cutoff score of 10–14, 15–19, and 20–27 indicates moderate, moderately severe, and severe depression,

Referring to anxiety, it is defined as a subjective feeling of fear, worry, and discomfort, for no reason at all or derived from anticipation of something [59]. Results from a recent meta-analysis support scientifically evidence that people with T2DM exhibit an increased likelihood of having anxiety disorder and anxiety symptoms than people without diabetes, and on the other side, it is associated with poor glycemic control and increased diabetes complications [53]. Those patients can experience physiologic sensation such as tachycardia, dizziness, sweating, headaches, and gastrointestinal disorder and also avoid places, people, and events. The diagnosis of the disease may induce anxiety because the patient has to adopt a severe lifestyle change in function of the disease treatment with a daily management of diabetes and worry of the long-term micro- and macrovascular complication [53]. It is common to diagnose anxiety using self-report symptom scales, mainly the Spielberger Trait Anxiety Questionnaire, the general health questionnaire for anxiety subscale, Hospital Anxiety and Depression

It is important to note that the idea that physical exercise has positive effects on depression and anxiety of patients with T2D in some cases is not unanimous. For instance, results from a systematic review of intervention studies [60] concluded that the evidences of physical exercise on psychological outcomes are conflicting. In this review, aerobic exercise shows to improve symptoms of anxiety. Significant difference in depression was found only in resistance training. For quality of life, among 6 studies (478 participants examined), only 2 reported significant effect of aerobic training compared to control group. Previous studies examined (361 participants), a mixed effect of resistance training on the mental domain of the SF-36 and SF-12 questionnaire was found. The authors stand out that heterogeneity of the studies was reflected on the mixed results found [60]. On the other side, a randomized controlled trial of 218 inactive patients with T2D found that no exercise was superior to resistance or combined exercise of 22 weeks, three times per week for improving mental health status [61]. Against the mixed evidence of physical exercise to improve mental health in T2D, it is necessary for future studies to confirm some findings. The literature showed that physical activity can mitigate mental disorder in active patient with T2D [15], and in this sense, an important benefit of physical activity or physical

For these patients, it is expressly recommended to perform at least 150 min of aerobic exercise of moderate-to-vigorous intensity and at least 2 sessions of resistance exercise. The compliance with these recommendations may have a positive

Scale for anxiety (HADS), and Hopkins symptom checklist [59].

exercise for mental health on those patients could be speculated.

**74**

Authors have no conflict of interest to disclose.


**6. Arrhythmogenic right ventricle cardiomyopathy**

Arrhythmogenic right ventricle cardiomyopathy (ARVC) is characterised by structural abnormalities and cardiac dysfunction of mainly the right ventricle, but it can also involve the left ventricle. It is a rare type of cardiomyopathy. It occurs if the muscle tissue in the right ventricle dies and is replaced by a scar tissue. This disrupts the pathway of the heart's electrical signals leading to arrhythmias.

ARVC usually affects teenagers or young adults. ARVC has a prevalence of 1 in 5000 among healthy young people. ARVC is seen in up to 20% of all causes of sudden death in young people. It is a complex genetic condition due to its genetic variation.


In addition to orthogonality, one desirable property for wavelets is symmetry, which requires all filters in the filter bank to possess exactly linear phase, because the symmetric extension method is generally used to treat the boundaries of images [5, 6]. It is known in [1–4] that finite impulse response (FIR) filters (corresponding to the compactly supported wavelets) can easily realize exactly linear phase. However, it is widely appreciated that the only FIR solution that produces a real orthogonal symmetric wavelet basis is the Haar wavelet, which is not continuous and the corresponding filter is of order 1 only that is not enough for many practical applications. To obtain wavelet filter banks with higher degrees of freedom, infinite impulse response (IIR) filters have been used to construct wavelet filter banks with some of the desired properties [7–12].  Allpass filter possesses unit

#### **Chapter 13**


**5.1.1 Chlorinated insecticides**

The first synthetic insecticide was DDT with a wide spectrum of insecticidal action that was used in agriculture and against insect vectors of deadly diseases. DDT solubility in water is very low, about 0.006 mg/l, which makes it one of the most hydrophobic insecticides. DDT residues either as parent compound or its metabolites DDD and DDE are stable and have high persistence in the environment. It has a great tendency to be stored in fatty tissue of different organisms. After the introduction of DDT, HCH was marketed. HCH has eight isomeric forms of which γ-isomer is called Lindane. Lindane is a volatile insecticide and was used against agricultural and households pests. Lindane is less persistent than the other organochlorine insecticides especially under moist conditions. The cyclodiens are stable organochlorine soil applied insecticides. These included aldrin, dieldrin, endrin, chlordane, heptachlor and endosulfan. Cyclodiens are environmentally persistent compounds that have raised concern about adverse effects on human health and wildlife. Residues of DDT and its metabolites DDD and DDE, dieldrin and heptachlor epoxide were detected in high percentage of soil and water samples from agricultural areas decades after their use were banned. Extensive studies on organochlorine pesticides has shown the environmental persistence of these Compounds (LeaMond et al. 1992; Reiser & O'Brien, 1999).

#### **5.1.2 Organophosphorus Insecticides**

Organophosphorus insecticides replaced persistent organochlorine compounds. Utilization of these insecticides increased rapidly and for several decades comprised high proportion of total insecticide use. Organophosphates are unstable compounds, however some of these insecticides are more acutely toxic to invertebrate than chlorinated insecticides. Parathion was the first marketed product that was effective against a wide variety of pests. Some organophosphates caused severe toxicity associated with many deaths especially in developing country, whereas a few compounds such as malathion are relatively safe to mammals and degrade fairly rapidly in the environment. Most organophosphates are harmful to beneficial arthropods, though few compounds such as phosalone and dimethoate are considered as harmless compounds.

The occurrence and movement of some organophosphate pesticides are reported in rivers and streams. Several studies conducted to find out the presence of organophosphate residues in California rivers during 1993-1994. Diazinon, methidathion, dimethoate and chlorpyrifos residues were detected in water samples. The detection occurred mostly during rainy season, showing how run off influences the presence of pesticide residues in rivers and streams (Ganapathy et al. 1997).

#### **5.1.3 Carbamate Insecticides**

156 Pesticides in the Modern World - Risks and Benefits

The environmental persistence is different from pesticide to pesticide. Some are persistent and remain in the environment either as a parent compound or transferred products. The fate of pesticides in soil depends on the value of Koc, carbon sorption coefficient. High values of Koc indicate a pesticide that strongly adsorbs to the soil particles and less likely to move with water. Moreover, soil composition, pH, moisture content and microbial activity

The most toxic and environmentally persistent compounds are found among insecticides;

The first synthetic insecticide was DDT with a wide spectrum of insecticidal action that was used in agriculture and against insect vectors of deadly diseases. DDT solubility in water is very low, about 0.006 mg/l, which makes it one of the most hydrophobic insecticides. DDT residues either as parent compound or its metabolites DDD and DDE are stable and have high persistence in the environment. It has a great tendency to be stored in fatty tissue of different organisms. After the introduction of DDT, HCH was marketed. HCH has eight isomeric forms of which γ-isomer is called Lindane. Lindane is a volatile insecticide and was used against agricultural and households pests. Lindane is less persistent than the other organochlorine insecticides especially under moist conditions. The cyclodiens are stable organochlorine soil applied insecticides. These included aldrin, dieldrin, endrin, chlordane, heptachlor and endosulfan. Cyclodiens are environmentally persistent compounds that have raised concern about adverse effects on human health and wildlife. Residues of DDT and its metabolites DDD and DDE, dieldrin and heptachlor epoxide were detected in high percentage of soil and water samples from agricultural areas decades after their use were banned. Extensive studies on organochlorine pesticides has shown the environmental

therefore, the emphasis is on groups of insecticides that have been studied in detail.

persistence of these Compounds (LeaMond et al. 1992; Reiser & O'Brien, 1999).

Organophosphorus insecticides replaced persistent organochlorine compounds. Utilization of these insecticides increased rapidly and for several decades comprised high proportion of total insecticide use. Organophosphates are unstable compounds, however some of these insecticides are more acutely toxic to invertebrate than chlorinated insecticides. Parathion was the first marketed product that was effective against a wide variety of pests. Some organophosphates caused severe toxicity associated with many deaths especially in developing country, whereas a few compounds such as malathion are relatively safe to mammals and degrade fairly rapidly in the environment. Most organophosphates are harmful to beneficial arthropods, though few compounds such as phosalone and

The occurrence and movement of some organophosphate pesticides are reported in rivers and streams. Several studies conducted to find out the presence of organophosphate residues in California rivers during 1993-1994. Diazinon, methidathion, dimethoate and chlorpyrifos residues were detected in water samples. The detection occurred mostly during rainy season, showing how run off influences the presence of pesticide residues in rivers

affect pesticide persistence.

**5.1.1 Chlorinated insecticides**

**5.1.2 Organophosphorus Insecticides**

dimethoate are considered as harmless compounds.

and streams (Ganapathy et al. 1997).

**5.1 Insecticides**

Carbamate insecticides are a group of synthetic compounds derived from carbamic acid. The first carbamate carbaryl was an N-methyl carbamate with high insecticidal activity against many insect pests and ectoparasites of animals. Carbamates especially N-mathyl carbamates are extremely toxic to hymenoptera and are lethal to exposed foraging bees. Carbamates biodegradation in environment is relatively rapid.

Oxime carbamates are a group of carbamate with systemic action. Aldicarb, an oxime carbamate is the most potent toxic substance (LD50=0.9 mg/kg) ever used in crop protection. Because of high toxicity it is used as granular formulation. Aldicarb sulfoxide is its oxidative metabolite that may undergo further oxidation to the sulfone. Oxidative residues and its parent compound (Tatal aldicarb) are toxic and highly mobile in the environment. Total aldicarb is detected especially in shallow ground water since 1979. Ground water quality monitoring has shown that many samples contain aldicarb residues and some of them exceeded maximum acceptable concentration (Priddle et al. 1989; Marade & Weaver 1994).


**1. Introduction**

### *1.1. Literature Review*

The crucial and most obvious features of economic systems are cooperation and interaction, e.g., supply chains, ownership dependencies, cooperation networks, and financial networks. The companies and their activities form complex networks of relationships and dependencies [1–3]. The idea that the economy can be considered as a complex system can be found at the end of the XX century in economic literature, e.g., [4]. The features of the network depend on various parameters, such as technology, law, regulations, culture, climate, weather, resources, and many other parameters. Among various aspects of economic activity, the financial market plays a special role. Besides fundraising for the business, they allow monitoring the condition of enterprises and even whole economic sectors. The stock market indices are regularly published and are considered to be not only a measure of the change of companies' group values, but also a test of the economy's status [5]. Therefore, within this study, the cross-correlations among stock time series of S&P 500 index components are analysed. This index was chosen because it is based on quotations of 500 shares of the largest stocks that trade on the New York Stock Exchange and NASDAQ. Considering the importance of those companies, as well as their variety and number, this set can be considered to be representative of the USA's economy and, therefore, can be the basis of the analysis of cross-correlations among them. Particularly, these stocks can be analysed from the point of view of cross-correlation network structures formed by those companies. The cross-correlation analysis consists of two steps: the distance (usually based on linear correlation [6]) matrix calculation and its analysis by constructing a network of random matrix approaches [7–9]. The alternative cross-correlation analysis should be here mentioned. Recently, detrended cross-correlation analysis based on detrended fluctuation analysis (DFA) [10] and their modifications have been very popular; see the recent review [11], or the power law classification scheme [12]. However, the most popular strategy is based

on the network construction and its structure analysis [13,14]. The most popular choice is the minimum spanning tree (MST) analysis [3,9,15–21]. MST application results primarily from the portfolio optimisation problem [22], but is also due to the proper recovering of the industrial sectors [3,6]. Besides MST analysis, the second most commonly used group of methods are those which construct networks based on assumed threshold [23]. This approach is used also in this paper—the distance matrix is filtered assuming that nodes are connected when the distance fulfills a given condition. Within this analysis, the properties of the most typical, weak, strong or significant correlations are investigated. The network generation procedures are described in detail in Section 3. The main aim of the paper is the description of the structural changes observed during the COVID-19 pandemic and their comparison to the changes in cross-correlation network properties during other, recently observed crises.

Another important aspect is the influence of the external parameters on the network structure. It is a truism to say that the state of the market depends on the macroeconomic situation. Particularly, the reaction of the market to crashes is also widely discussed from the point of view of network structure, e.g., [9,24,25], or in the analysis of globalization processes [26–28]. However, the present pandemic situation should not be considered as the typical shock but rather a change of the "economic environment". The most important fact is that the pandemic was expected and induced serious changes in the economy. The government, due to the pandemic situation, introduced special rules lasting a relatively long time. The restrictions form special conditions which are expected to change the structure of cross-correlations among companies. The network structure of shares' cross-correlations is the subject of interest of various studies [9,21,29]. A natural extension of time series analysis through the network methods is evolving network analysis, since the economy time series are non-stationary, and thus, should be described by an evolving network rather than a static network. Evolving network theory was initially applied to systems naturally described by network structures, such as social networks [30], scientific collaboration networks [31] and economy time series [8,25,32,33], to mention a few examples.

#### *1.2. COVID-19 History*

Although the COVID-19 epidemic is a contemporary event, for the convenience of future readers, a short description of the epidemic key points in the USA is presented below.

**December 2019** The first known cases have been identified in Wuhan, China.

**January 2020** The epidemic spreads to other provinces of China.

**February 2020** Italy is affected with a rapidly growing number of infected and fatal cases. **March 2020** The USA overtakes China and Italy with the highest number of confirmed cases in the world.

The present situation is the subject of various studies. More detailed history and discussion on the influence of pandemic on stock markets from the standard time series analysis point of view can be found at [34–38].

#### *1.3. Paper Structure*

The present paper is organised as follows. Section 2 describes the data analysed. The Section 3 defines the methods used: the statistical distance, the network construction algorithms and the network parameters (node entropy, cycle entropy, averaged clustering coefficient and transitivity) calculated and analysed in the study. Section 4 presents the obtained results, including the evolution of the node and cycle entropy and the averaged clustering coefficient and transitivity. It is worth stressing that the parameters introduced here (the node and the cycle entropy) are sensitive to economic crises. Moreover, the performed analysis shows that the structure of the defined networks changes significantly in the crisis. The main outcome of the paper is the analysis of a representative group of USA companies and the observation of their reaction to changing economic situations. A very promising outcome of the study is that four periods during the COVID-19 pandemic are distinguished, which shows that the reactions to various factors are different and that this analysis is capable of observing this.


In fact, such plague-ridden soldiers and infected military units were soon to prove an increasingly pressing problem. For independently of each other, the French and imperial units deployed to the theatre of war, imported plague into Northern Italy.¹⁹⁷ From 1630 onward, the region thus experienced epidemic outbreaks of unprecedented intensity. Not only was the population of Northern Italy plague stricken, however, but also the French army itself suffered massively from it.¹⁹⁸ A good illustration of this quandary would be how the capture of the town of Pinerolo, taken by French units in March 1630, initially represented a strategic coup for the French forces.¹⁹⁹ And yet, some three months later, the plague outbreak had transformed Pinerolo into a deserted site, so much so that Richelieu mulled over whether and how such a locality could still be held.²⁰⁰ Against such an ominous backdrop, the cardinal minister also considered deploying troops close to the town. He entertained doubts, however, as to whether a contagion-free space could be found anywhere in the region.²⁰¹ The northern Italian theatre of war, which Louis XIII and Richelieu had hitherto regarded as the arena for the rise of a glorious and powerful France they had so longed for, suddenly turned into a theatre of action which could scarcely provide quarters for several hundred soldiers.

195 See Avenel, Lettres, vol. 3, p. 787. See also, on this point, Lacour-Gayet/Lavollée, Mémoires du cardinal de Richelieu, vol. 10, p. 383.

196 See Avenel, Lettres, vol. 3, p. 290.

197 See Cipolla, Cristofano and the Plague, pp. 15–17.

198 See BnF, Dupuy 27, f° 18r –22r ; Pagès, Autour du "grand orage", p. 80.

199 See, on this point, ibid. pp. 82–84. Here, Pagès does not limit himself to a strategic appraisal, he also attributes a quasi-outstanding political significance to the capture of Pinerolo.

200 See Avenel, Lettres, vol. 3, p. 813.

201 See ibid., p. 790, 830.

# V Conseils du roi: Governing between Crisis and Catastrophe

The impending reality that scope for manoeuvre in Northern Italy's theatre of war was diminishing, both figuratively and geographically, was not least the upshot of a policy that consistently placed the aspiration to bolster the king's glory above the needs of his subjects. An examination of the Royal Councils' files makes it clear how excessive demands were often foisted upon the populations of the Ancien Régime amid the ongoing crisis. In concrete terms, this sense of over-taxing expressed itself across the innumerable petitions bearing witness to the wish to obtain relief from other sources in view of the burdens triggered by famine, war and disease. Whether by way of arrêts simples or arrêts en commandement, the Royal Councils' rulings thus reached were already the result of an administrative coping mechanism. In all likelihood, it was largely on account of this process' inherent logic that the Conseils du roi rulings exposed, as can be deduced from the relevant local and regional studies, a crisis that was often truly catastrophic in scale.

In scrutinising the many thousands of resolutions that have survived, primarily those pronounced by the Conseil d'État et des finances or by the Conseil de direction des finances, but also those by the other royal councils, this article will not only enable us to draw conclusions about how this royal institution was transformed. It can also divulge reliable answers concerning the critical question of how the French Crown responded to outbreaks of plague that had kept the Ancien Régime society at large on tenterhooks at the end of the 1620s and the turn of the 1630s. Andrea Iseli's statement made some years ago that the Conseils du roi only started assuming an active or proactive role during the so-called plague of Marseilles of 1720–1722 is, to some extent, implicitly true. Because nearly all the Conseils du roi's rulings falling within the scope of this study and concerning the socalled plague of La Rochelle were triggered by corresponding external petitions by the king's subjects for financial compensation for damages incurred due to war, famine and contagion. Scarcely a century before the devastating epidemic that wrought havoc on Marseilles, as well as further afield in Provence and Languedoc, and in view of the even more devastating nation-wide epidemic, one must therefore refer to the Conseils du roi's passive role.

This was already evident in the case of the municipalities which routinely had to bear the brunt of coping with epidemic outbreaks. Many municipal authorities were also forced to heavily invest in the epidemic police during those years between 1628 and 1631. The royal councils did compensate them, at least in part, for this investment by way of channelling financial support to the city in question or by allowing them to levy an equivalent tax. Such a tax empowered the municipalities to rustle up the money they had previously spent on ensuring healthcare for the plague-stricken.

The fermiers, whose core function was to collect the indirect taxes levied by the French Crown, could also look forward to some financial compensation. As matters transpired, however, some of these officials did not get their money's worth on account of the dire socio-economic conditions prevailing throughout the late 1620s and early 1630s. In this instance, too, the Conseils du roi proved themselves amicable and often accommodated the fermiers, at least partially, by reducing, for example, the amount that these tax collectors had to pay to the Crown.

Matters were somewhat more complex, however, for the numerous officiers in the service of the Crown who had purchased their office from the king. The droit annuel could have been particularly significant notably in times of crisis such as the late 1620s and early 1630s. For, during periods of increased mortality, the droits annuel opened a pathway for the potential inter-generational transfer of the office within the family concerned. There was no guarantee, however, either for the family of the deceased that the office would remain their prerogative, or for the Crown that the office would not revert to them. Indeed, in the latter scenario, the office could have been re-sold and thus monetised by the Crown. The fact, however, that the king's representatives would die in large numbers during such periods as the mortality crisis of 1629–1631 could scarcely have been in the interest of either Louis XIII or Cardinal Richelieu. While such offices were at times primarily prestigious in character, those holding them also occupied prominent positions in the functioning of the monarchy, as in the judicial sphere, for example. Perhaps, this consideration also came into play during those challenging years as the royal councils endeavoured to ensure that offices did not simply lapse, but rather continued to be manned.

To conclude, it is remarkable how, on one hand, the Conseils du roi reacted quite flexibly to the requêtes submitted to them across the years 1629–1631. On the other hand, the often uniform content characterising the Conseils' rulings following such questions and petitions is equally striking. Based on the evidence of the large number of copies of resolutions by the councils, we can draw the conclusion that the already severely strained royal finances were further overburdened at that juncture. To some extent, this was the result of the sometimes significant shortfalls in revenue, which occurred repeatedly in the field of taxation, for example, on account of the overall exceptional plight during those years characterized by epidemics, famine and wars. It could equally be explained, however, by the compensation the Conseils du roi granted to those individuals and institutions incurring financial damages as a result of the prevailing crisis or to those using their own financial resources in a bid to manage that situation. The councils' resolutions per se, however, could indicate the purpose of the altogether quite extensive compensatory measures, for they repeatedly report on uprisings erupting over and again during those years of the Ancien Régime. Notwithstanding the prevailing circumstances, the French Crown did, to a certain extent, demonstrate responsibility in policing epidemics by contributing, albeit indirectly in most cases, to the financial costs incurred by its subjects in grappling with epidemics. The Conseils du roi neither ignored nor relativised the highly infectious diseases which were to claim the lives of hundreds of thousands in France during the late 1620s and early 1630s. Rather, they had reckoned with such an outcome.

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