*Edited by Mustafa Demetgul and Muhammet Ünal*

Mass production companies have become obliged to reduce their production costs and sell more products with lower profit margins in order to survive in competitive market conditions. The complexity and automation level of machinery are continuously growing. This development calls for some of the most critical issues that are reliability and dependability of automatic systems. In the future, machines will be monitored remotely, and computer-aided techniques will be employed to detect faults in the future, and also there will be unmanned factories where machines and systems communicate to each other, detect their own faults, and can remotely intercept their faults. The pioneer studies of such systems are fault diagnosis studies. Thus, we hope that this book will contribute to the literature in this regard.

Photo by v\_alex / iStock

Fault Diagnosis and Detection

Fault Diagnosis and Detection

*Edited by Mustafa Demetgul*

*and Muhammet Ünal*


*2.4. LGF Modulates Microglia Activation and Reduces Inflammation in APPswe Mice*

Recent reports suggest that microglia play an important role in the evolution of AD. The immunohistochemical analysis of the hippocampus and cerebral cortex of APPswe mice showed that the number of microglial cells that expressed the Ionized Calcium Binding Protein-1 (Iba1) was significantly higher than in WT control mice (Figure 4A). Most of the Iba1-positive cells associated with the plaques were located around them (Figure 4B), and a few showed Iba1-positive processes

penetrating into the plaques (Figure 4C). Iba1 protein levels were also up-regulated in both structures (Figure 4D,H), suggesting the presence of activated microglia in these mice. Increased proliferation has also been associated with microglia activation. As shown in Figure 4E, the expression of PCNA was significantly increased in the hippocampus and cerebral cortex of APPswe mice. Similar to this finding, BrdU incorporation was higher in these mice than in the WT group in both structures (Figure 4F), and more than 61 ± 4% (*n* = 8) of the proliferating cells were Iba1-positive microglia (Figure 4G).

**Figure 3.** Effects of LGF in tau pathology, ubiquitinated protein levels and HSP70 heat shock protein expression. Panel (**A**), shows the relative optical density of phospho-tau/tau ratio analyzed by Western blot and expressed as % vs. WT. Note how LGF treatment reduces the expression of phosphorylated tau in the hippocampus and cerebral cortex in APP mice. Panels (**B**,**C**) show the quantification of accumulated ubiquitinated proteins (**B**) and HSP-70 (**C**) in the hippocampus and cerebral cortex of APPswe mice. Note how LGF reduces the accumulation of ubiquitinated proteins (**B**) and increases HSP70 expression (**C**). Panel (**D**) shows representative Western blot images of phospho-tau, total tau, Hsp-70 and β-actin as charge control. Results represent the mean ± SEM of 4 to 8 independent mice. The statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. \* *p* ≤ 0.05, \*\* *p* ≤ 0.01, \*\*\* *p* ≤ 0.001 vs. WT. + *p* ≤ 0.05, ++ *p* ≤ 0.01, +++ *p* ≤ 0.001 vs. APPswe mice.

The APP-LGF group of animals showed a significant reduction in Iba1 (Figure 4D,H) and PCNA protein expression (Figure 4E,H), and a slight but reduced number of BrdU-positive cells in comparison with APPswe mice (Figure 4F). Besides, LGF treatment increased from 6 ± 2.9 (*n* = 3) to 19 ± 11 (*n* = 4) the percentage of Iba1-positive cells, which prolongations penetrated profoundly in the plaques in the hippocampus.

**Figure 4.** Liver growth factor modulates microglia cell activity in the hippocampus and cerebral cortex of APPswe mice. Panel (**A**) shows the quantification of Iba1-positive microglia cells in APPswe mice. Panels (**B**,**C**) show different responses of Iba1-positive cells: in (**B**) microglial prolongations do not penetrate inside the deposit, while in (**C**) they actively phagocytize the plaque (scale bar: 50 μm). Panels (**D**,**E**) show quantitative Western blot analysis of Iba1 (**D**) and PCNA (**E**) protein expression. Note how LGF administration significantly reduces the over-expression of Iba1 (**D**) and PCNA (**E**) in the hippocampus and cerebral cortex. LGF also reduces BrdU cell incorporation (**F**) as an index of cellular proliferation. Note how BrdU (**G**, red) is associated with Iba1-positive cells (**G**, green, white arrows). Panel (**H**) show representative Western blots of Iba1 and PCNA and their respective β-actin as charge control. The results represent the mean ± SEM of 4 (**A**,**F**) and 8 to 10 (**D**,**E**). independent mice. The statistical analysis was performed by one-way ANOVA followed by Newman–Keuls test. \* *p* ≤ 0.05, \*\* *p* ≤ 0.01, \*\*\* *p* ≤ 0.001 vs. WT. + *p* ≤ 0.05, ++ *p* ≤ 0.01, +++ *p* ≤ 0.001 vs. APPswe mice.

GFAP is a protein expressed by astrocytes for which up-regulation has been associated with inflammatory states in chronic processes as AD and aging. As shown in Figure 5A, GFAP was over-expressed in the hippocampus and cerebral cortex of APPswe mice, and LGF treatment significantly reduced its levels in both structures. On the other hand, LGF decreased ASC protein levels that were up-regulated in the hippocampus and the cerebral cortex of APPswe aged mice (Figure 5B,D). ASC is involved in the production of Interleukin-1beta (Il-1beta) but, neither APP nor APP-LGF treated mice showed any significant change in the levels of this pro-inflammatory cytokine in the hippocampus (113 ± 6 (*n* = 6) and 101 ± 7 (*n* = 6) % of IL-1beta expression vs. WT in APP and APP-LGF treated mice, respectively) and the cerebral cortex (102 ± 11 (*n* = 5) and 92 ± 12 (*n* = 6) % of IL-1beta expression vs. WT in APP and APP-LGF treated mice, respectively). Similar results were observed when we analyzed the potential effects of LGF in Tumor Necrosis Factor-alpha (TNF-alpha) protein expression in the hippocampus (92 ± 6 (*n* = 6) and 88 ± 10 (*n* = 8) % of TNF-alpha expression vs. WT in APP and APP-LGF treated mice, respectively) and cerebral cortex (95 ± 6 (*n* = 5) and 107 ± 9 (*n* = 5) % of TNF-alpha expression vs. WT in APP and APP-LGF treated mice, respectively). This latter cytokine mediates LGF-induced neuroregeneration/neuroprotection [6,20].

LGF also modulated the expression of the transcription factor Nrf2, and the class B scavenger receptor CD36, two proteins that regulate oxidative stress and inflammatory responses in AD. As shown in Figure 5D, Nrf2 protein levels were reduced in the hippocampus and cerebral cortex of APPswe mice, and LGF treatment up-regulated Nrf2 protein expression to reach control values. LGF treatment reduced the levels of CD36 that is over-expressed in the hippocampus of APPswe mice (Figure 5C,E). Besides, LGF up-regulated CD36 protein expression in the hippocampus of WT mice (Figure 5C,E), but it lacked activity in the cerebral cortex, a brain structure where neither APP nor APP-LGF mice showed any alteration in CD36 protein levels (Figure 5C).


**Fermented Alfalfa Brown Juice Significantly Stimulates the Growth and Development of Sweet Basil (***Ocimum basilicum* **L.) Plants**

**Szilvia Kisvarga 1, Döme Barna 2,\*, Szilvia Kovács 2, Gábor Csatári 2, Ibolya O. Tóth 2, Miklós Gábor Fári 2, Péter Makleit 2, Szilvia Veres 2, Tarek Alshaal 2,3 and Nóra Bákonyi 2**

Received: 17 April 2020; Accepted: 2 May 2020; Published: 7 May 2020

**Abstract:** Fertilization management is a key issue in plant nutrition to produce plants with good quality and quantity. Deproteinized leaf juice or brown juice (BJ) is a by-product during the isolation of leaf protein from biomass crops such as alfalfa. The idea of using BJ as a biostimulant fits well in the aspect of circular economy since BJ is currently a problematic issue of the leaf protein production approach. Fractionation of one-kilogram fresh biomass results in approximately 500 cm3 BJ. Due to fast spoil of fresh BJ, if left on room temperature, it is found that fermentation of fresh BJ using lactic acid bacteria and reducing its pH increases its stability and storage on room temperature. In the present study, we examined the effect of fermented alfalfa BJ on vegetative, physiological, and anatomical properties of the versatile sweet basil (*Ocimum basilicum* L. 'Bíborfelh˝o') plants. Sweet basil seedlings were sprayed at different doses of fermented alfalfa BJ (i.e., 0.5%, 1.0%, and 2.5%) and tap water served as a control (0.0% BJ). The results revealed that foliar application of fermented alfalfa BJ significantly improved the biometrical features of sweet basil plants. Plants treated with fermented BJ showed significantly higher values of all the measured parameters compared to the control (0.0%), except for the number of leaves per plants where control plants (0.0%) had more leaves. However, the leaves of control plants (0.0%) were smaller than treated plants as data of leaf area showed. Fermented alfalfa BJ significantly increased the content of photosynthetic pigments (chl a and chl b), relative chlorophyll (SPAD value), lengths of stem and root, fresh masses of stem, root, and leaves, volumes of stem and root, and leaf area. Despite all rates of fermented BJ displayed higher values over control plants (0.0%), the rate of 0.5% was the best one supported by results. Application of fermented alfalfa BJ influenced the anatomical parameters as well. These findings demonstrate the possible use of fermented alfalfa BJ as a promising novel plant biostimulant.

**Keywords:** sweet basil; alfalfa brown juice; fermentation; biostimulation; chlorophyll pigments; histological changes

#### **1. Introduction**

The increase in the world's population leads to several issues that we already have to face, and we must find sustainable solutions for them to save our life on the planet. One of these concerns

is the sustainable supply of high-quality protein. To solve the protein issue, several approaches were proposed to identify novel protein sources or alternatives [1–3]. Alfalfa (*Medicago sativa* L.) leaf protein concentrate (LPC) is a promising element either in human or animal diet as the human population of the Earth (7.2 billion) is growing rapidly causing a high demand for animal protein [4,5]. The isolation of leaf protein resulted in four products, i.e., green juice, fiber, leaf protein concentrate, and deproteinized juice or referred to as brown juice (BJ). The amount of produced BJ during the isolation of leaf protein and the utilization of this product is a huge obstacle for the wider recognition of LPC production. Fractionation of one-kilogram fresh biomass results in 450–550 mL BJ [6–8]. Therefore, disposal of BJ is a big challenge that the leaf protein isolate approach faces. Another reason making disposal of BJ risky is the high content of several bioactive components such as sugars, free amino acids, minerals and vitamins that BJ contains [7,9,10]. Therefore, finding an alternative use of BJ is urgent due to environmental concerns, besides maximizing the benefit from this waste that is very rich in several valuable compounds and nutrients. Instead of dumping it we could make a valuable product, thus making a step towards the circular economy concept [11,12]. The BJ contains about 40% carbohydrates (mainly monosaccharides, like glucose and fructose) and 3% nitrogen-based on dry mass [12]; additionally, numerous biologically active components like phenols, amino acids, macro- and microelements and biostimulators, etc. [7,11,13]. However, fresh BJ cannot be stored at room temperature, after a few days it gets spoiled at pH 5–6 due to its high sugar content. In our previous study, inoculation of BJ with lactic acid bacteria was not only found to increase BJ stability at room temperature but also substantially improved the nutritional characteristics of BJ [7] through converting sugars into organic acids decreasing the pH to almost 4.5. Therefore, BJ seems to be an ideal component in animal feeding programs as well as plant nutrition and soil stabilization. Several authors have previously suggested BJ as a plant fertilizer, fodder, and growth medium for microbes [10,14–16]. Fermented BJ can be applied as a very effective foliar biostimulant. We have observed remarkable effects of fermented alfalfa BJ on the growth dynamic of plumed cockscomb [7]. Additionally, lactic acid bacteria as a plant growth-promoting bacteria represents an additional benefit of fermentation of BJ as it promotes plant growth [12,17].

Sweet basil (*Ocimum basilicum* L.) is a well-known annual herb, member of the Lamiaceae family. It is one of the 150 species of the Ocimum genus, a very important medicinal, spice and fresh vegetable, culinary herb and industrial plant, cultivated for aromatic and medicinal use on large areas in many countries [18–23]. It is native to India, Asia and Africa, but grows in many regions of the world, including Italy, Thailand, Vietnam and Laos [24]. Sweet basil is popularly used in traditional Chinese medicine, because of its selected purified components and antiviral activity [25]. The latest scientific developments revealed the strong pharmacological action and nutritional aspects because of antioxidant content [25], additionally, it is a rich source of acylated and glycosylated anthocyanins being a valuable source for the food industry [26].

The aim of this study was to examine the impact of the fermented BJ on the biometrical, physiological and anatomical features of sweet basil plants.

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

#### *2.1. Sources of Brown Juice and Plant Materials*

The fresh alfalfa BJ was obtained from the Proteomill Green Protein Biorefinery Factory (Tedej Ltd., Hajdúnánás, Hungary). The fresh BJ was fermented using lactic acid bacteria to avoid its fast spoiling. The physicochemical properties of fresh and fermented BJ as well as the fermentation process were described by Bákonyi et al. [7]. The seeds of sweet basil (*Ocimum basilicum* 'Bíborfelh˝o') were obtained from the National Agricultural Research and Innovation Center (NARIC, Budapest, Hungary).

#### *2.2. Experimental Study*

A pot experiment under greenhouse conditions was carried out at the NARIC to assess the possible growth stimulation effect of fermented BJ using the multipurpose sweet basil as a plant model. The experimental design was the Randomized Complete Block design (RCB) with 15 replicates. A polyethylene pot (7 × 7 × 8 cm) was filled with white peat for young plants (Klassman-Deilmann TS 3 FINE type, Geeste, Germany). The characteristics of growth medium are as follows: fine structure, pH (H2O) 6, N 140 mg L−1, P (P2O5) 100 mg L−1, K (K2O) 180 mg L−1, Mg 100 mg L−1, S 150 mg L−1. The sweet basil seeds were sown in the nursery substrate on 16 July 2018. The germinated seedlings were treated once a week with fermented BJ at different rates (i.e., 0.5, 1, and 2.5%) in the early stage of development (stage 1 BBCH) [27]. On 1 August 2018, the seedlings turned to stage 2 BBCH, all identical and healthy, were transferred to the pots. One pot contained one seedling and each treatment contained 15 pots. Fermented BJ was sprayed on the plants twice a week (on Tuesdays and Fridays) from 15 August till the end of the experiment (11 September) at rates of 0.5, 1.0, and 2.5%. Final application volume of 250 mL BJ was equally shared among all replicates of the same treatment (15 plants). The control plants (0.0%) were sprayed with the equivalent amounts of tap water. The experiment was terminated on 11 September (stage 5 BBCH) and plants were harvested, and samples were collected for the further biometric, physiological and anatomical analyses.

#### 2.2.1. Analysis of Biometric Features of Basil Plants

At the end of the experiment, before flowering (BBCH stage 5), all plants in each treatment were harvested and the following vegetative parameters were measured: root and stem length (cm), root and stem volume (mL), root and stem fresh mass (g plant−1), the number of leaves (pcs plant−1) and leaf area. Roots were carefully removed from the pots and washed by tap water on a sieve to remove the adhered growth medium particles. Length was measured with a measuring ruler, while volume was determined by a graduated cylinder, fresh mass by OHAUS Pioneer PA214 analytical balance and the number of leaves by counting. The leaf area (cm2) of sweet basil plants were measured by AreaMeter 350 (Opti-Sciences, Hudson, NY, USA). Six plants were analyzed and represented as a mean ± SD (*n* = 6). In the case of leaf area, we used the data of nine plants. All leaves of the examined plants were measured.

#### 2.2.2. Determination of Chlorophyll Contents

At the end of the experimental period, the relative chlorophyll values were measured by SPAD 502 chlorophyll meter (Minolta, Japan) using the last fully developed leaves. The chlorophyll-a and -b contents were extracted based on the method of Moran and Porath [28] and determined by the method of Wellburn [29], Vidician and Cachita-Cosma [30]. We used the following formulas: "Chlorophyll a (mg·g−1) = (11.65 a664–2.69 a647)" and "Chlorophyll b (mg·g−1) = (20.81 a647–4.53 a664)". The samples were taken from the last fully expanded leaves and chlorophyll pigments were extracted by 5 mL *N*,*N*-dimethylformamide (DMF) added to 0.05 g leaf disc. The samples were soaked in this solvent for 48 h at room temperature in the dark. After 48 h, the discs were removed and the contents of chlorophyll-a and -b were measured by METEREKSP-830 spectrophotometer. All the measurements were repeated three times for each plant making up to nine measurements for each of the treatments.

#### 2.2.3. Anatomical Analysis

We used three individual plants per treatment for the stem's histological examination. 15 different cross-sections per plant internodes (*n* = 45) were prepared as described in the following: each plant was cut into smaller pieces and the third internodes (from beneath) fixed separately in Strasburger-Flemming's solution [31], which is a mixture of glycerin:alcohol:water (1:1:1) for a week. Then, several cross-sections were prepared using blades, after clarification, they were stained with Toluidin-blue. Each analysis was performed under a light microscope (Zeiss Axioscope

2+; Zeiss International, Oberkochen, Ostalbkreis, Germany) with a compatible camera, and Scope Photo software (Scopetek, München, Germany) was used for processing the images. The measured parameters were: thickness of the epidermis, primary cortex, pith including primary and secondary vascular tissues.

#### *2.3. Statistical Analysis*

Results of the experiments were subjected to one-way ANOVA by SigmaPlot 12.0 and IBM SPSS Statistics 24 software and the means were compared by Tukey Test [32] at *p* ≤ 0.05. Before the ANOVA test, in SPSS the Levene's Test for Equality of Variances was performed in the case of anatomical data. SigmaPlot 12.0 automatically ran a check test for Equality of Variance. The Equality of Variance test for different variables at the four treatments of BJ (i.e., 0.0, 0.5, 1.0, and 2.5%) were negative, *p* ≤ 0.05, and the variances showed homogeneity.

#### **3. Results**

#### *3.1. Plant Biometric Features*

Results of root and stem length of sweet basil are presented in Figure 1. The treated plants with fermented BJ had taller stems and roots compared to the control plants (0.0%). Most importantly, fermented BJ enhanced plant development resulting in higher values of both root and stem. Despite all treated plants showed a taller stem than control plants (0.0%), increasing the rate of applied fermented BJ slightly reduced the stem length. However, the differences in stem length between treatments of fermented BJ were not significant. Treated plants with 0.5% fermented BJ showed an increase of 50.4% in stem length compared to control (0.0%) as the highest measured increase. Similarly, root length showed a relationship as root length gradually increased with increasing the rate of the applied fermented BJ. The application of fermented BJ significantly increased root length compared to the control (0.0%); however, no significant differences in root length were reported between different rates of fermented BJ (Figure 1).

Stem volume of sweet basil plants follows a dose-relationship response to fermented BJ; increasing rates of fermented BJ significantly and gradually caused an increase in stem volume. The highest increase in stem volume (38.6%) compared to untreated control plants (0.0%) was recorded by 2.5% BJ (Figure 2). Similar effect was noticed for root volume, as it increased upon spraying plants with increased rates of fermented BJ. Nevertheless, the highest rate of fermented BJ (2.5%) showed the same value as for treatment of 0.5% fermented BJ, which was two-fold higher than the control plants (0.0%). The highest root volume was 1.16 mL and was found when plants received 1.0% fermented BJ.

Results of stem and root fresh mass are presented in Figure 3. The fresh mass of stem gradually increased as a result of increased rates of fermented BJ. Significantly, all fermented BJ increased the stem fresh mass. While control plants (0.0%) possessed a stem fresh mass of 0.73 g plant−1, treated plants with 2.5% fermented BJ showed a stem fresh mass of 1.52 g plant−1 as the highest recorded value. The differences between fermented BJ treatments were not significant. In contrast to stem fresh mass, root fresh mass increased as rate of fermented BJ increased up to 1.0%, then decreased at 2.5% fermented BJ. However, all treatments showed significant increases in the fresh mass of root system in comparison to control plants (0.0%) (Figure 3). Plants received 1.0% of fermented BJ, which was almost three-fold higher in their root fresh mass than control plants (0.0%).

**Figure 1.** Root and stem length of sweet basil plants treated with different rates of fermented BJ. Sample size *n* = 6 (mean ± SD). Different letters above the same columns show significant differences at the level of *p* ≤ 0.05.

**Figure 2.** Stem and root volume of sweet basil plants treated with different rates of fermented BJ. Sample size *n* = 6 (mean ± SD). Different letters above the same columns show significant differences at the level of *p* ≤ 0.05.

**Figure 3.** Fresh mass of stem (**A**) and root (**B**) of sweet basil plants treated with different rates of fermented BJ. Sample size *n* = 6 (mean ± SD). Different letters above the same columns show significant differences at the level of *p* ≤ 0.05.

Figure 4 shows the results of the leaves fresh mass, number of leaves and leaf area of sweet basil plants after treating them with different rates of fermented BJ extracted from alfalfa biomass. Results showed that fermented BJ significantly improved plant growth as the leaves fresh mass of treated plants was higher than those untreated (control, 0.0%). All fermented BJ rates resulted in significantly higher values of leaves fresh mass than the control (0.0%). However, the highest applied rate of fermented BJ (2.5%) showed a lower fresh mass value than that measured at 0.5 and 1.0% of fermented BJ. The highest leaves fresh mass was recorded at treatment with 0.5 and 1.0% fermented BJ (Figure 4A). Contrarily, the number of leaves per plant gradually reduced with increasing rate of applied fermented BJ (Figure 4B). Control plants (0.0%) possessed the highest number of leaves (22 leaves per plant), while the plants sprayed with 2.5% fermented BJ had 11.7 leaves per plant. The differences among all treatments including the control (0.0%) were statistically significant. Despite control plants (0.0%) possessing a higher number of leaves than the treated plants with fermented BJ, the leaf area of plants sprayed with different rates of fermented BJ was larger than the control plants (0.0%). While the control plants (0.0%) had a leaf area of 71.4 cm2, the plants of the treatment of 0.5% fermented BJ displayed a leaf area of 131.5 cm2 (Figure 4C). Moreover, other treatments exhibited significantly higher leaf areas than control plants (0.0%) as well.

**Figure 4.** (**A**) Leaves fresh mass; (**B**) number of leaves; (**C**) leaf area of sweet basil plants treated with different rates of fermented BJ. Sample size *n* = 6 (mean ± SD). Different letters above the same columns show significant differences at the level of *p* ≤ 0.05.

#### *3.2. Contents of Photosynthetic Pigment*

The results showed that the content of chlorophylls increased upon spraying sweet basil plants with fermented BJ (Table 1). The contents of chlorophyll-a (chl a) and chlorophyll-b (chl b) in the control (0.0%) leaves were 6.90 and 1.95 mg g−1, respectively, and significantly increased to 8.04 (chl a) and 2.66 (chl b) mg g−1 after treating plants with 0.5% (chl a) and 2.5% (chl b) fermented BJ, respectively. The total content of chlorophylls ranged from 8.86 to 10.60 mg g−1, 2.5% BJ application increased the chlorophyll content by 19% compared to the control (0.0%). Chlorophyll a/b ratio in treated plants with fermented BJ ranged from 2.97 to 3.50; while the control plants (0.0%) possessed a chlorophyll a/b

ratio of 3.52 (Table 1). The SPAD value showed a significant, 28% increase when leaves were treated with 0.5% concentration of fermented alfalfa BJ in comparison to the control (0.0%).

**Table 1.** Content of chlorophyll in sweet basil leaves (mg g−1) and their relative changes (%) compared to the control plants (0.0%) (*n* = 9).

Different letters (a, b, ab) in the same columns show significant differences at the level of *p* ≤ 0.05.

#### *3.3. Anatomical Traits of Sweet Basil*

The cortex is made up of angular collenchyma (two to four cells thick) and typical parenchymatous cells. The pith consists of mainly parenchymatous cells, but the ratio of primary and secondary vascular tissues is dependent on the treatments. Treatments 0.5% and 1.0% resulted in well-developed secondary vascular tissues (Figure 5).

**Figure 5.** Anatomical sections of sweet basil stem. Subfigures show samples treated with different rates of fermented BJ such as (**A**) 0.0%, (**B**) 0.5%, (**C**) 1.0%, (**D**) 2.5%. *ep* epidermis, *co* cortex, *ang* angular collenchyma, *pi* pith, *pph* primary phloem *ca* cambium *px* primary xylem *sx* secondary xylem, after treating with different rates of fermented BJ (i.e., control (0.0%), 0.5%, 1.0%, 2.5%). Scale bar is 200 μm.

Differences which are visible in the tissues caused by the treatments were proven true by our measurements. All levels of concentration increased the thickness of the epidermis, however only two tests were significant. The impact of all treatments was a decrease in the thickness of the primary cortex. Treatments 0.5% and 1.0% increased both the extension of the pith and the proportion of vascular tissue in it, which were proven statistically significant. Growth of the secondary vascular tissue was the highest at the 0.5% treatment. It can be concluded that a greater brown juice concentration, which was 2.5% in this analysis, may block the development of secondary vascular tissues (Table 2).

**Table 2.** Anatomical traits of basil stem tissue (epidermis, cortex, pith involved primary and secondary vascular tissues) *(*μm) after treatment with fermented BJ. Sample size *n* = 45 (Mean ± SD).

Different letters (a, b, ab) in the same columns show significant differences at the level of *p* ≤ 0.05.

Consequently, the use of an incremental concentration of brown juice can increase the thickness of the stem and the vascular tissue in it but only to a certain extent.

#### **4. Discussion**

The BJ is largely produced during the isolation of leaf protein from several green leafy crops. The storage of fresh BJ in room temperature is the main concern due to the high content of carbohydrates particularly in the form of monosaccharaides. Lactic acid bacteria showed a considerable effect on reducing BJ pH as a result of organic acids production under an anaerobic condition and consequently increased the stability and handling of BJ at a pH of 4.5–4.8 [7].

Fermented BJ can be exploited as an organic fertilizer or growth stimulator due to its richness in free amino acids, soluble sugars, vitamins, organic acids and other nutrients. In addition, lactic acid bacteria—as plant growth promoting bacteria—represent an additional benefit for using fermented BJ as a fertilizer [7,33]. In the present study, fermented BJ proved its efficiency as a promising plant growth stimulator as it caused a significant increase in photosynthetic pigments including chl a, chl b, total chlorophyll and relative chlorophyll (SPAD value). Similar findings have been previously reported by Bergstrand et al. [34] who proved in their study on nitrogen speciation in pot experiment of sweet basil fertilized by different organic manures, i.e., blood meal + Baralith®Enslow and poultry manure, that the plant-based organic fertilizer treatment induced the chlorophyll content. Moreover, applying biofertilizers, i.e., Nitrajin (including Azotobacter, Azospirillum and Pseudomonase), increased the amount of photosynthetic pigments and the leaf area of sweet basil [35]. Additionally, Ertani et al. [36] mentioned that the extract prepared from alfalfa biomass using enzymes contained growth stimulant compounds like triacontanol and indole-3-acetic acid, which significantly improved the relative chlorophyll and growth of maize plants under salt-stress conditions.

Noticeably, all rates of BJ resulted in higher values of stem and root length (Figure 1), stem and root volume, stem and root fresh mass, number of leaves and leaf area. This was in agreement with those documented earlier by El-Ziat et al. [37]. They cited that organic fertilization and humic acid application improved growth parameters of 'Red Rubin' basil plant, i.e., fresh weight, plant height and leaf area, in a greenhouse experiment. Organic fertilization of basil using organic NPK fertilizer (4–3–4) (Organic Fertilizer, Mighty Grow®Fruitdale, AL) resulted in changes in both fresh and dry weight, and in nutrient uptake as well [24]. Onofrei et al. [18] stated that different organic foliar applications, i.e., Fylo®, Geolino Plants&Flowers®, Cropmax®, Fitokondi®, stimulated the content of total phenolic compounds contributing to healthier vegetable production of *Ocimum basilicum* L.

Unique positive changes in secondary vascular tissues observed as a result of 0.5% and 1.0% BJ treatments (Figure 5 and Table 2), which according to our best knowledge have not been published before. However, similar effects were reported on plumed cockscomb (*Celosia argantea* var. plumosa 'Arrabona') plants by Bákonyi et al. [7] who cited significant changes in histological parameters after treating plants with fermented BJ. The tissue structure of the stem of sweet basil analyzed is a typical structure of a plant at the age of 10–12 weeks. Stems were covered by the epidermis (single row) with a thin layer of cuticle. Contrary to Venkateshappa and Sreenath [38], more types of the trichomes are identified on the surface of the stem, e.g., non-glandular uniseriate hair (composed of three cells) and glandular capitate hairs, which supports the findings of Werker et al. [39] and Nasssar et al. [40].

In a few cases, the application of fermented BJ led to a slight reduction in some measured parameters. However, all rates of fermented BJ were better than the control plants (0.0%, untreated). This beneficial role of fermented BJ is owed to its high content of phytoavailable nutrients such as N, P, K, Ca, Mg, S, Mn, Fe, Cu, Zn, and Mo [7]. Similar findings were reported earlier by Ream et al. [41]. They revealed that BJ at the rate of 1.25 cm enhanced the yield and growth of corn, alfalfa and bromegrass. However, they also stated that higher rates of BJ (2.5 cm) caused a reduction in development and yields of all crops. Another study cited similar results where the application of BJ obtained from alfalfa biomass at low rates positively improved the germination of many crops, i.e., cowpea, mung bean and groundnut. Negative impacts were reported when BJ was used at higher rates, above 10% [33]. The detrimental effect of high rates of BJ may be attributed to the existence of some phytotoxic organic compounds in BJ [6]. Moreover, the reduction in plant growth at a high applied rate of fermented BJ could be due to the high electrical conductivity of BJ [7]. Additionally, spraying plants with acidic solutions (low pH as at the high rate of fermented BJ) is known to lessen the stomatal conductance [42]; therefore, a reduction in plant growth is expected. This hypothesis was supported by the earlier findings of Long et al. [43].

The results obtained from the present study alongside with our previous published work [7] strongly support the hypothesis about prospective studies on BJ either fermented or fresh to improve soil properties through the soil application technique. In alkaline, salt-affected soils, and sandy soils the expected benefits of using fermented BJ would be more due to the high content of macro and microelements, free amino acids [7,42,44]. Additionally, the soluble sugars will facilitate and improve the growth of rhizosphere bacteria supporting them against the unflavored growth conditions [45]. Another substantial result for using fermented BJ via soil application is increasing the soil aggregates due to a high sugar content in BJ. Moreover, the low pH of fermented BJ is an advantage of using fermented BJ as a soil conditioner since it will partially modify the local soil pH around the root system facilitating the uptake of especially microelements by plant roots [43]. However, in our next studies, we are going to work on these hypotheses.

#### **5. Conclusions**

This study demonstrates the potentiality of fermented alfalfa BJ to enhance both quantity and quality of one of the most important multipurpose crops, i.e., sweet basil, which is cultivated on large areas in many countries. Our results showed a significant increase in photosynthetic pigments leading to better vegetative growth; and consequently, enhancement in important medical phytochemicals in sweet basil could occur. For a plant such as sweet basil (used as spice and fresh vegetable, and culinary herb), larger leaf area, number of leaves, and thickness of stem (including vascular tissues) as well as fresh weight are very important decisive traits. The results also indicated that concentrations of 0.5 and 1.0% fermented BJ were the most valuable and have a considerable biostimulator effect on leaves as these treatments were more effective improving leaf parameters in comparison to control plants (0.0%).

Additionally, we came to the conclusion based on the results that fermented alfalfa BJ has great potential as a biofertilizer and plant growth promoter. Future experiments are needed to justify more of these findings with other horticultural and agricultural crops.

**Author Contributions:** Conceptualization, N.B., S.K. (Szilvia Kisvarga) and M.G.F.; methodology, S.K. (Szilvia Kisvarga), D.B. and I.O.T.; software, D.B.; validation, D.B.; formal analysis, G.C. and S.K. (Szilvia Kovács); investigation, S.V. and P.M.; resources, N.B. data curation, T.A.; writing—original draft preparation, N.B., T.A. and D.B.; writing—review and editing, N.B., T.A., S.V. and D.B.; visualization, G.C. and S.K. (Szilvia Kovács); supervision, M.G.F.; project administration, N.B.; funding acquisition, N.B. and M.G.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was financed by the Higher Education Institutional Excellence Programme (NKFIH-1150-6/2019) of the Ministry of Innovation and Technology in Hungary, within the framework of the Biotechnology thematic programme of the University of Debrecen.

**Acknowledgments:** We acknowledge the financial support of the followings: "Complex Rural Economic and Sustainable Development, Elaboration of its Service Networks in the Carpathian Basin (Project ID: EFOP-3.6.2-16-2017-00001, Hungary)" research project and Széchenyi 2020 under the GINOP-2.2.1-15-2017-00051.

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

#### **References**

© 2020 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/).

MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com

*Agronomy* Editorial Office E-mail<EMAIL_ADDRESS>www.mdpi.com/journal/agronomy

MDPI St. Alban-Anlage 66 4052 Basel Switzerland

Tel: +41 61 683 77 34 Fax: +41 61 302 89 18


4. Role of platelets in APS

Thrombocytopenia is the most relevant non-criteria manifestation of APS.

≥1 clinical episode of arterial, venous or small vessel thrombosis.

≥1 unexplained death of a morphologically normal foetus ≥10 weeks of gestation

• severe preeclampsia or eclampsia defined according to standard definition;

For histopathological confirmation, thrombosis must be present without inflammation of the vessel

≥3 unexplained consecutive miscarriages <10 weeks of gestation, with maternal and paternal factors

Presence of antiphospholipid antibodies (aPLs), on two or more occasions at least 12 weeks apart and no more than 5 years prior to clinical manifestations, as demonstrated by ≥1 of the following:

• Medium to high-titre (>40 GPL or MPL, or >99th percentile) anticardiolipin IgG or IgM;

≥1 premature delivery of a morphologically normal foetus <34 weeks of gestation because of:

Thrombosis must be objectively confirmed.

• recognised features of placental insufficiency

• Lupus anticoagulant;

Table 1. Revised classification criteria for antiphospholipid syndrome [19].

(anatomic, hormonal or chromosomal abnormalities) excluded

• Anti-β2 glycoprotein-I (anti-β2GPI) IgG or IgM >99th percentile

activation, angiogenic imbalance and activation of immune cells [20].

prevalence is around 40–50 cases per 100,000 persons [24].

morbidity [16]. These data can underline the significance of APS.

diagnostic clinical criterion of APS.

wall

3. Epidemiology of APS

APS.

Vascular thrombosis

56 Thrombocytopenia

Pregnancy morbidity

Laboratory criteria

However, despite the pro-thrombotic nature of APS, thrombocytopenia is one of the most common non-criteria findings of the disease. Recently, thrombocytopenia is proposed to be a

In case of OAPS several disease processes may occur in the placentae of women with antiphospholipid syndrome due to the antiphospholipid autoantibodies, not only thrombosis and infarction, but also inflammatory events, mediated by cytokine release, complement

Presence of aPL Abs per se does not guarantee a patient will develop APS as only 8.1% of patients with aPL antibodies without a history of clinical thrombosis developed thrombosis during a 5-year follow-up period, suggesting that a patient needs an additional insult to develop the clinical disease [21, 22]. The prevalence of the antibodies increases with age [23]. Forty percent of patients with APS have SLE. The prevalence of ACA in SLE is from 12 to 30%, and LA is found in 5–34%. From the patients with SLE and aPL antibodies 50–70% progress to

The incidence of the APS is around five new cases per 100,000 persons per year, and the

The aPLs are positive in approximately 13% of patients with stroke, 11% with myocardial infarction, 9.5% of patients with deep vein thrombosis and 6% of patients with pregnancy Platelets play a key role in APS-related thrombosis due to the presence of multiple receptors that can interact with anti-β2-GPI antibodies (especially apolipoprotein E receptor 20 (apoER20 ) and glycoprotein Ibα (GPIbα)) with consequent release of different pro-coagulant mediators such as thromboxane B2, platelet factor 4 (PF4) and platelet factor 4 variant (CXCL4L1) [28].

In case of APS, thrombosis results from a hypercoagulable state caused by activation of endothelial cells, monocytes and platelets. It has been demonstrated that platelets are required for enhanced activation of the endothelium and fibrin generation by the anti-β2GP1 autoantibody/β2GP1 complex. Thus, the first event is the activation of thrombocytes, endothelial cells are activated indirectly [29].

Platelet activation, a major contributing factor of arterial thrombosis in APS, might play a role in APS-related thrombosis in at least two ways:

First, due to the presence of multiple receptors that can interact with antibodies, platelets can facilitate the dimerisation of β2-GPI enhancing the coagulation response.

Second, platelets provide a surface for coagulation reactions [30].

The role of thrombocytes in the pathogenesis of APS is supported by the fact that circulating platelet- and endothelial-derived microparticle level are elevated in patients with primary APS [31].

Mouse models of APS have shown that platelets are the first target for circulating anti-β2-GPIβ2-GPI complexes, and the enhancement of endothelium activation is also platelet thrombusdependent [29].


Light-Dependent Regulation of Circadian Clocks in Vertebrates

*Izawa Junko, Yoshimi Okamoto-Uchida, Akari Nishimura and Jun Hirayama*

#### **Abstract**

Circadian clocks are intrinsic time-tracking systems that endow organisms with a survival advantage. The core of the circadian clock mechanism is a cellautonomous and self-sustained oscillator called a cellular clock, which operates via a transcription-/translation-based negative feedback loop. Under natural conditions, circadian clocks are entrained to a 24-hour day by environmental time cues, most commonly light. In mammals, circadian clocks are regulated by cellular clocks located in the central nervous system, such as the suprachiasmatic nucleus (SCN), and in other peripheral tissues. Importantly, mammals have no photoreceptors in the peripheral tissues; therefore the effect of light on peripheral clocks is indirect. By striking contrast, zebrafish peripheral cellular clocks are directly light responsive. This characteristic of the zebrafish cellular clock has contributed to the identification of molecules and signaling pathways that are involved in the lightdependent regulation of the cellular clock. Here, selected light-dependent regulatory mechanisms of circadian clocks in mammals and zebrafish are described.

**Keywords:** circadian clock, cellular clock, zebrafish, light, photolyase

#### **1. Introduction**

Circadian clocks constitute ubiquitous processes that regulate various biochemical and physiological events that occur with 24-hour periodicity, even in the absence of external cues [1]. The exact timing of this rhythm is established by cell-autonomous mechanisms, called cellular clocks, which are controlled by a transcription-/translation-based negative feedback loop [2, 3]. In both vertebrates and invertebrates, cellular clocks are scattered throughout their bodies; thus, the circadian system comprises both central and peripheral oscillators [4].

To guarantee that an organism's behavior remains tied to the rhythms of its environment, the circadian clock must respond to environmental stimuli to be reset [5]. The main cue for animals is light, which is provided by the day-night cycle. It has been proposed that in mammals the light-induced resetting of the circadian clock is dependent on transcription activation in the suprachiasmatic nucleus (SCN), where the central clock is located [6]. The mammalian route for the regulation of the circadian clock by light uses the retinohypothalamic tract (RHT), which connects directly to the central clock located in the SCN [7]. This makes it difficult to understand the mechanisms underlying light regulation of the circadian clock at a cellular level. Thus, although changes in gene expression have been implicated

**20**

978-0-471-56802-5

2008]

*Chronobiology - The Science of Biological Time Structure*

Philadelphia, US: Saunders; 2005. ISBN

[10] Dzogang F, Lightman S, Cristianini N. Diurnal variation of psychometric indicators in twitter content. PLoS One. 2018;**13**(6). DOI: 10.1371/journal.

Massachusetts: Sinauer Associates, Inc.;

978-1-4160-2769-0

2005. p. 587

pone.0197002. eCollection

[11] Nelson RJ. An Introduction to Behavioral Endocrinology.

[12] Frank DW, Evans JA, Gorman MR. Time-dependent effects of dim light at night on re-entrainment and masking of hamster activity rhythms. Journal of Biological Rhythms. 2010;**25**(2):103-112. DOI:

10.1177/0748730409360890

ISBN 0-8493-2233-2

edu/~halberg/

[13] Refinetti R. Circadian Physiology. 2nd ed. Boca Raton, FL: CRC Press, Taylor & Francis Group; 2006. p. 700.

[14] Refinetti R, Cornélissen G, Halberg F. Procedures for numerical analysis of circadian rhythms. Biological Rhythm Research. 2007;**38**(4):275-325. DOI: 10.1080/09291010 600903692

[15] Cornelissen G, Halberg F. Introduction to chronobiology. In: Medtronic Chronobiology Seminar #7; April 1994. Library of Congress Catalog Card #94-060580; 1994. p. 52. Available from: http://www.msi.umn.

[1] Kreitzman L, Foster RG. Rhythms of Life: The Biological Clocks that Control the Daily Lives of every Living Thing. New Haven, Connecticut: Yale University Press; 2004. ISBN

[2] Chandrashekaran MK. Biological rhythms research: A personal account. Journal of Biosciences. 1998;**23**:545

Methodological problems. Acta Medica

[3] Halberg F. Chronobiology:

[4] Halberg F. Quo vadis basic and clinical chronobiology: Promise for health maintenance. The American Journal of Anatomy. 1983;**168**:543-594

[5] DeCoursey PJ, Dunlap JC, Loros JJ. Chronobiology. Sunderland,

[6] Fuller PM, Lu J, Saper CB. Differential rescue of light- and food-entrainable circadian rhythms. Science. 2008;**320**(5879):1074-1077. DOI: 10.1126/science.1153277. PMC 3489954. PMID 18497298. Bibcode: 2008Sci.320.1074F [Accessed: 30 May

[7] Rossi EL, Lloyd D. Ultradian Rhythms in Life Processes: An Inquiry into Fundamental Principles of Chronobiology and Psychobiology. Springer, Berlin, Heidelberg: Springer-Verlag Berlin and Heidelberg GmbH & Co. K; 1992. ISBN 978-3-540-19746-1

[8] Hayes DK. Chronobiology: Its Role in Clinical Medicine, General Biology, and Agriculture. Hoboken, New Jersey, US: John Wiley & Sons; 1990. ISBN

[9] Postolache TT. Sports Chronobiology, an Issue of Clinics in Sports Medicine.

Massachusetts, USA: Sinauer Associates Inc.; 2003. ISBN 978-0-87893-149-1

Romana. 1980;**18**:399-440

0-300-10969-5

**References**

in the light-induced phase shift of the circadian clock [6, 8], the induction of the expression of clock genes by light and the exact mechanism by which these gene products work remain to be elucidated at the cellular level.

Zebrafish peripheral clocks display a striking characteristic in that they are directly light responsive [9, 10]. Light induces the expression of clock genes and the circadian expression of several clock-related genes in zebrafish peripheral cells [11]. In addition, zebrafish embryonic cell lines can recapitulate the light-response characteristics of a vertebrate clock. In these cell lines, the oscillations of clock gene expression can be entrained to a new light-dark cycle, showing that cultured zebrafish cells have the clock components required for light-induced circadian clock resetting, and the cultured cell system thus provides a valuable tool for studying the light-dependent regulation of the circadian clock at a cellular level [12, 13]. Zebrafish cellular clocks can be studied in cultured cells, which facilitate the study of the photic responses of clock genes encoding cellular-clock regulators, and have revealed cellular signaling pathways that are involved in the light-dependent regulation of the cellular clock [14–18]. Additionally, an increased understanding of light-dependent cellular-clock regulation in zebrafish has suggested intriguing associations among the circadian clock, DNA repair, and cell cycle control [19–23].

Here we describe selected light-dependent regulatory aspects of vertebrate circadian machinery.

### **2. Cellular-clock regulation in mammals**

In mammals, the cellular clock comprises the CLOCK, NPAS2, BMAL1, BMAL2, PER1, PER2, CRY1, and CRY2 proteins [1, 24]. These cellular clock components are called clock proteins. CLOCK or NPAS2 proteins heterodimerize with BMALs to form an active transcription complex that transactivates clock-controlled genes, including *Crys* and *Pers*. Once the CRY and PER proteins have been translated, they are translocated to the nucleus, where they inhibit CLOCK (NPAS2):BMAL-mediated transcription through a direct protein-protein interaction, setting up the rhythmic gene expression that drives the circadian clock. The CLOCK(NPAS2):BMAL complex also stimulates expression of the clock-controlled genes (Ccgs) to regulate various elements of physiology. This accounts in part for the presence of circadian rhythms in a variety of physiological processes [25]. Although the relatively straightforward mechanism of positive and negative feedback loops is necessary to establish and maintain circadian clocks, cellular clocks have further levels of complexity, including posttranscriptional regulation, posttranslational modification, chromatin remodeling, availability and stability of clock proteins, and regulation of intracellular localization. These regulatory mechanisms provide an interface that can be used as an entry point for stimuli that can reset or control the clock. In addition, genetic studies of genes encoding cellular-clock regulators have revealed distinct roles for clock proteins in regulating circadian clocks, as well as direct links between the circadian clock and various pathologies [26–28].

#### **3. Photoreceptors for circadian-clock regulation in mammals**

Circadian clocks regulate various biochemical, physiological, and behavioral processes with a periodicity of approximately 24 hours. Under natural conditions, circadian rhythms are entrained to this 24-hour day by environmental time cues,

**23**

**Figure 1.**

*Light-Dependent Regulation of Circadian Clocks in Vertebrates*

hypothalamic tract to the SCN central clock in mammals [33, 34].

**ipRGC**

**Light**

Strong

Gap junction

G ju

with light level being the most important [5]. The eye is the principal mediator of light input to the central clock in mammals. Rods and cones receive visual information within the retina [29, 30] (**Figure 1**). These cells, however, are dispensable for photoreception of circadian clocks. Indeed, rodents that lack classical visual responses are still capable of circadian photoentrainment [31]. Retrograde tracing experiments have identified retinal cells projecting to the SCN through the RHT, but not to the visual centers of the brain [32]. These cells constitute a small subset of retinal ganglion cells (RGCs) localized in the ganglion cell layer (GCL), and they have been shown to display intrinsic phototransduction abilities, with photic properties matching those of clock entrainment [33]. The main candidate for the circadian photoreceptor is melanopsin, which is an opsin found in the eye and other photoreceptive structures in amphibians and exclusively in retinal RGCs in primates and rodents. Photic information received by RGCs is conveyed through the retino-

**SCN**

**RHT**

**Cone cell**

*DOI: http://dx.doi.org/10.5772/intechopen.86524*

Biopolar cells

**Rod cell** Weak

*Retinal cells responsible for vision and photoreception for circadian clock regulation.*

RGCs

#### *Light-Dependent Regulation of Circadian Clocks in Vertebrates DOI: http://dx.doi.org/10.5772/intechopen.86524*

*Chronobiology - The Science of Biological Time Structure*

**2. Cellular-clock regulation in mammals**

circadian machinery.

various pathologies [26–28].

products work remain to be elucidated at the cellular level.

in the light-induced phase shift of the circadian clock [6, 8], the induction of the expression of clock genes by light and the exact mechanism by which these gene

Zebrafish peripheral clocks display a striking characteristic in that they are directly light responsive [9, 10]. Light induces the expression of clock genes and the circadian expression of several clock-related genes in zebrafish peripheral cells [11]. In addition, zebrafish embryonic cell lines can recapitulate the light-response characteristics of a vertebrate clock. In these cell lines, the oscillations of clock gene expression can be entrained to a new light-dark cycle, showing that cultured zebrafish cells have the clock components required for light-induced circadian clock resetting, and the cultured cell system thus provides a valuable tool for studying the light-dependent regulation of the circadian clock at a cellular level [12, 13]. Zebrafish cellular clocks can be studied in cultured cells, which facilitate the study of the photic responses of clock genes encoding cellular-clock regulators, and have revealed cellular signaling pathways that are involved in the light-dependent regulation of the cellular clock [14–18]. Additionally, an increased understanding of light-dependent cellular-clock regulation in zebrafish has suggested intriguing associations among the circadian clock, DNA repair, and cell cycle control [19–23]. Here we describe selected light-dependent regulatory aspects of vertebrate

In mammals, the cellular clock comprises the CLOCK, NPAS2, BMAL1, BMAL2, PER1, PER2, CRY1, and CRY2 proteins [1, 24]. These cellular clock components are called clock proteins. CLOCK or NPAS2 proteins heterodimerize with BMALs to form an active transcription complex that transactivates clock-controlled genes, including *Crys* and *Pers*. Once the CRY and PER proteins have been translated, they are translocated to the nucleus, where they inhibit CLOCK (NPAS2):BMAL-mediated transcription through a direct protein-protein interaction, setting up the rhythmic gene expression that drives the circadian clock. The CLOCK(NPAS2):BMAL complex also stimulates expression of the clock-controlled genes (Ccgs) to regulate various elements of physiology. This accounts in part for the presence of circadian rhythms in a variety of physiological processes [25]. Although the relatively straightforward mechanism of positive and negative feedback loops is necessary to establish and maintain circadian clocks, cellular clocks have further levels of complexity, including posttranscriptional regulation, posttranslational modification, chromatin remodeling, availability and stability of clock proteins, and regulation of intracellular localization. These regulatory mechanisms provide an interface that can be used as an entry point for stimuli that can reset or control the clock. In addition, genetic studies of genes encoding cellular-clock regulators have revealed distinct roles for clock proteins in regulating circadian clocks, as well as direct links between the circadian clock and

**3. Photoreceptors for circadian-clock regulation in mammals**

Circadian clocks regulate various biochemical, physiological, and behavioral processes with a periodicity of approximately 24 hours. Under natural conditions, circadian rhythms are entrained to this 24-hour day by environmental time cues,

**22**

with light level being the most important [5]. The eye is the principal mediator of light input to the central clock in mammals. Rods and cones receive visual information within the retina [29, 30] (**Figure 1**). These cells, however, are dispensable for photoreception of circadian clocks. Indeed, rodents that lack classical visual responses are still capable of circadian photoentrainment [31]. Retrograde tracing experiments have identified retinal cells projecting to the SCN through the RHT, but not to the visual centers of the brain [32]. These cells constitute a small subset of retinal ganglion cells (RGCs) localized in the ganglion cell layer (GCL), and they have been shown to display intrinsic phototransduction abilities, with photic properties matching those of clock entrainment [33]. The main candidate for the circadian photoreceptor is melanopsin, which is an opsin found in the eye and other photoreceptive structures in amphibians and exclusively in retinal RGCs in primates and rodents. Photic information received by RGCs is conveyed through the retinohypothalamic tract to the SCN central clock in mammals [33, 34].


**5. Working principle of EDFA**

The invention of the Erbium Doped Fiber Amplifier (EDFA) in the late eighties was one of the major events in the history of optical communication systems. It provided new life to the research of technologies that allow high bit rate transmission over long distances. EDFA has a narrow high gain peak at 1532nm and a broad peak with a lower centered at 1550nm. The use of an increasing number of channels in the present day DWDM optical networks requires a flat gain spectrum across the whole usable bandwidth. Owing to their versatility, useful gain bandwidth, high pumping efficiency and low intrinsic noise, EDFAs are the amplifier of choice for most of the network applications. They are based on single mode optical fibers with cores that have been doped, typically to a few hundred part per million, with the trivalent erbium ion, Er3+. The gain is provided through stimulated emission, as in laser. The Er3+ ion acts mostly as a three level system, in which the main participants are the 4I15/2 ground state, the 4I13/2 first excited level and the 4I11/2second excited level. The energy level diagram of Er3+ is shown in figure 4 ) [Keiser 2009;Mynbaev 2003].

Fig. 4. Energy Level Diagram of Er3+

Hybrid Fiber Amplifier 111

The optical fiber can be doped with any of the rare earth element, such as Erbium (Er), Ytterbium (Yb), Neodymium (Nd) or Praseodymium (Pr), Thulium (Tm). The host fiber material can be either standard silica, a fluoride based glass or a multicomponent glass. The operating regions of these devices depend on the host material and the doping elements. Fluorozirconate glasses doped with Pr or Nd are used for operation in the 1300nm window, since neither of the ions can amplify 1300nm signals when embedded in silica glass. The next popular material for long haul telecommunication applications is a silica fiber doped with Thulium, which is known as Thulium Doped Fiber Amplifier (TDFA). In some cases as Yb is added to increase the pumping efficiency and the amplifier gain. The TDFA are used in S-band (1460-1530nm). The energy state diagram of Tm3+ is shown in figure 7 [Aozasa et.al.2008].

Fig. 6. Gain Spectrum of EDFA

Fig. 7. Energy Level Diagram of Tm3+

**7. Role of TDFA in communication systems**

Fig. 5. Absorption and Emission Spectra of EDFA

EDFAs are of particular interest in telecommunications, because their emission spectrum shows a gain of more than 20dB over the range of 1530-1560nm. This is also the third window used in optical communication. The absorption spectrum reveals that good absorption takes place around 380nm, 520nm, 800nm, 980nm, and 1480nm. The absorption bands at shorter wavelengths are not of interest owing to the non- availability of semiconductor laser diodes at these wavelengths. At 980nm and 1480nm, efficient laser diodes are available and therefore used as pump sources.


**Application of Finite Symmetry Groups to Reactor Calculations**

Yuri Orechwa1,\* and Mihály Makai2 *1NRC, Washington DC 2BME Institute of Nuclear Techniques, Budapest 1USA 2Hungary*

#### **1. Introduction**

284 Nuclear Reactors

Zinkle, S. J., N. Hashimoto, et al. (2003). "Microstructures of irradiated and mechanically

Zinkle, S. J. and Y. Matsukawa (2004). "Observation and analysis of defect cluster

p.3-12 792: 3-12.

333(1-3 PART A): 88-96.

deformed metals and alloys: Fundamental aspects." Materials Research Society Symposium -Radiation Effects and Ion-Beam Processing of Materials; 2003; v.792;

production and interactions with dislocations." Journal of Nuclear Materials 329-

Group theory is a vast mathematical discipline that has found applications in most of physical science and particularly in physics and chemistry. We introduce a few of the basic concepts and tools that have been found to be useful in some nuclear engineering problems. In particular those problems that exhibit some symmetry in the form of material distribution and boundaries. We present the material on a very elementary level; an undergraduate student well versed in harmonic analysis of boundary value problems should be able to easily grasp and appreciate the central concepts.

The application of group theory to the solution of physical problems has had a curious history. In the first half of the 20th century it has been called by some the "Gruppen Pest" , while others embraced it and went on to win Noble prizes. This dichotomy in attitudes to a formal method for the solution of physical problems is possible in light of the fact that the results obtained with the application of group theory can also be obtained by standard methods. In the second half of the 20th century, however, it has been shown that the formal application of symmetry and invariance through group theory leads in complicated problems not only to deeper physical insight but also is a powerful tool in simplifying some solution methods.

In this chapter we present the essential group theoretic elements in the context of crystallographic point groups. Furthermore we present only a very small subset of group theory that generally forms the first third of the texts on group theory and its physical applications. In this way we hope, in short order, to answer some of the basic questions the reader might have with regard to the mechanical aspects of the application of group theory, in particular to the solution of boundary value problems in nuclear engineering, and the benefits that can accrue through its formal application. This we hope will stimulate the reader to look more deeply into the subject is some of the myriad of available texts.

The main illustration of the application of group theory to Nuclear Engineering is presented in Section 4 of this chapter through the development of an algorithm for the solution of the neutron diffusion equation. This problem has been central to Nuclear Engineering from the very beginning, and is thereby a useful platform for demonstrating the mechanics of bringing group theoretic information to bear. The benefits of group theory in Nuclear Engineering are

\*The views expressed are those of the authors and do not reflect those of any government agency or any part thereof.

An extensive discussion of the mathematics and application of group theory to engineering problems in general and nuclear engineering in particular is presented in (Makai, 2011).

Application of Finite Symmetry Groups to Reactor Calculations 287

Although the basic mathematical definition of a group and much of the abstract algebraic machinery applies to finite, infinite, and continuous groups, our interest for applications in nuclear engineering is limited to finite point groups. Furthermore, it should be kept in mind that most of the necessary properties of the crystallographic point groups for applications, such as the group multiplication tables, the class structures, irreducible representations, and characters are tabulated in reference books or can be obtained with modern software such as

An abstract group *G* is a set of elements for which a law of composition or "product" is defined. For illustrative purposes let us consider a simple set of three elements {*E*, *A*, *B*}. A law of composition for these three elements can be expressed in the form of a multiplication table, see Table 1. In position *i*, *j* of Table 1. we find the product of element *i* and element *j* with the numbering 1 → *E*, 2 → *A*, 3 → *B*. From the table we can read out that *B* = *AA* because element 2, 2 is *B* and the third line contains the products *AE*, *AA*, *AB*. The table is symmetric therefore *AB* = *BA*. Such a group is formed for example by the even permutations of three objects: *E* = (*a*, *b*, *c*), *A* = (*c*, *a*, *b*), *B* = (*b*, *c*, *a*). The multiplication table reflects four necessary **E A B E** E A B **A** A B E **B** B E A

conditions that a set of elements must satisfy to form a group *G*. These four conditions are: 1. The product of any two elements of *G* is also an element of *G*. Such as for example *AB* = *E*.

3. *G* contains a unique element *E* called the identity element, such that for example *AE* =

4. For every element in *G* there exists another element in *G*, such that their product is the identity element. In our example *AB* = *E* therefore *B* is called the inverse of *A* and is

The application of group theory to physical problems arises from the fact that many characteristics of physical problems, in particular symmetries and invariance, conform to the definition of groups, and thereby allows us to bring to bear on the solution of physical

For example, if we consider a characteristic of an equilateral triangle we observe the following with regard to the counter clockwise rotations by 120 degrees. Let us give the operations

The group operation is the sequential application of these operations, the leftmost operator should be applied first. The reader can easily check the multiplication table 2. applies to the

3-rotation by 240*o*.

the following symbols: *E*-no rotations, *C*3-rotation by 120*o*, *C*3*C*3 = *C*2

**2. Basic group theoretic tools**

**2.1 Group definition**

denoted *B* = *A*−1.

MAPLE or MATHEMATICA for example.

Table 1. Multiplication table for elements {*E*, *A*, *B*}

problems the machinery of abstract group theory.

2. Multiplication is associative. For example (*AB*)*E* = *A*(*BE*).

*EA* = *A* and the same holds for every element of *G*.

not restricted to solving the diffusion equation. We wish to also point the interested reader to other areas of Nuclear Engineering were group theory has proven useful.

An early application of group theory to Nuclear Engineering has been in the design of control systems for nuclear reactors (Nieva, 1997). Symmetry considerations allow the decoupling of the linear reactor model into decoupled models of lower order. Thereby, control systems can be developed for each submodel independently.

Similarly, group theoretic principles have been shown to allow the decomposition of solution algorithms of boundary value problems in Nuclear Engineering to be specified over decoupled symmetric domain. This decomposition makes the the problem amenable to implementation for parallel computation (Orechwa & Makai, 1997).

Group theory is applicable in the investigation of the homogenization problem. D. S. Selengut addressed the following problem (Selengut, 1960) in 1960. He formulated the following principle: If the response matrix of a homogeneous material distribution in a volume *V* can be substituted by the response matrix of a homogeneous material distribution in *V*, then there exists a homogeneous material with which one may replace *V* in the core. The validity of this principle is widely used in reactor physics, was investigated applying group theoretic principles (Makai, 1992),(Makai, 2010). It was shown that Selengut's principle is not exact; it is only a good approximation under specific circumstances. These are that the homogenization recipes preserve only specific reaction rates, but do not provide general equivalence.

Group theory has also been fruitfully applied to in-core signal processing (Makai & Orechwa, 2000). Core surveillance and monitoring are implemented in power reactors to detect any deviation from the nominal design state of the core. This state is defined by a field that is the solution of an equation that describes the physical system. Based on measurements of the field at limited positions the following issues can be addressed:

The solution to these problems requires a complex approach that incorporates numerical calculations incorporating group theoretic considerations and statistical analysis.

The benefits of group theory are not restricted to numerical problems. In 1985 Toshikazu Sunada (Sunada, 1985) made the following observation: If the operator of the equation over a volume *V* commutes with a symmetry group *G*, and the Green's function for the volume *V* is known and volume *V* can be tiled with copies tile *t* (subvolumes of *V*), then the Green's function of *t* can be obtained by a summation over the elements of the symmetry group *G*. Thus by means of group theory, one can separate the solution of a boundary value problem into a geometry dependent part, and a problem dependent part. The former one carries information on the structure of the volume in which the boundary value problem is studied, the latter on the physical processes taking place in the volume. That separation allows for extending the usage of the Green's function technique, as it is possible to derive Green's functions for a number of finite geometrical objects (square, rectangle, and regular triangle) as well as to relate Green's functions of finite objects, such as a disk, or disk sector, a regular hexagon and a trapezoid, etc. Such relations are needed in problems in heat conduction, diffusion, etc. as well.

An extensive discussion of the mathematics and application of group theory to engineering problems in general and nuclear engineering in particular is presented in (Makai, 2011).