*<IP_ADDRESS>.1. Abietic acid*

*3.1.2. Labdanes*

116 Terpenes and Terpenoids

described in this chapter as well (**Figure 4**).

**3.2. Tricyclic diterpenoids**

etane as standard (**Figure 6**).

*<IP_ADDRESS>. Tricyclic abietatrienes*

**Figure 4.** Skeleton of labdanes.

See **Figure 5**

*3.2.1. Abietanes*

The labdane-related diterpenoids are a special group, consisting of over 7000 members, which are distinguished by their unique biosynthesis. Gibberellin phytohormones as well as antibiotics such as some of phytoalexins and phytoanticipins fall into this family [34]. Labdanolic acids have been identified as biomarkers for the botanical origin of French ambers [35], while copalic acid and its relatives have been associated with the biological activity of the resins from *Copaifera* species. The lanceolatins are a group of **labdanes** and **abietanes** which were obtained [40] from *Cephalotaxus lanceolata* (Cephalotaxaceae). Some of the abietanes were

Abietane is a class of diterpenoids with excellent metabolic profile. Compounds of this class showed broad spectrum antiviral, antibacterial, and antifungal activity [36, 37]. Abietane diterpenoids are extracted from few fungal species [38]. The intervention of quinone methides in the antioxidant activity of the phenolic diterpenoids ferruginol and carnosic acid has been discussed. The antifungal activity of some abietic acid esters in the context of their use as wood preservatives and the antiviral activity of podocarpic acid derivatives have been examined. In case of human cells, antiproliferative effect on tumor cells has been reported [39].

About 200 compounds of this family have been identified commonly known as dehydroabietic derivatives (dehydroabietanes) [40] assuming 20-carbon saturated aromatic ring I, abi-

This group of abietane terpenes includes a tricyclic ring, three double bonds on B or C rings. Carboxylic acids are representatives of this group, of which the earliest example is the biologi-

cally active dehydroabietic acid (**Figure 6**), which possess an acid group at C-18 [42].

Abietic acid was extensively studied in various organisms for its biological activities. Current data also suggest that abietic acid is an important compound for synthesis of novel metabolites. They contribute to the body of knowledge related to compound 1 and deepen the understanding of the potential and properties of 1 and its derivatives (**Figure 7**).


**Author details**

Roberto Gambari1,2, Giulia Breveglieri1,2, Francesca Salvatori1,2, Alessia Finotti1 and Monica Borgatti1\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Life Sciences and Biotechnology, Biochemistry and Molecular Biology Sec‐ tion, University of Ferrara, Ferrara, Italy

2 Biotechnology Center, University of Ferrara, Ferrara, Italy


*2.4. Statistical Analysis*

Data were analyzed by using the following linear mixed model, following the rules suggested by Onofri et al. [27]. In particular, the field (two levels), the year (four levels) and the system (three levels) were added as fixed e ffects with all their two- and three-way interactions. It should be noted that the 'year × field' interaction corresponds to the crop e ffect, as the crops are univocally identified by one specific field in one specific year. The blocks within fields and the plots within blocks within fields were added as random e ffects to account for blocking units and repeated measures. The 'field × year × system' interaction was always significant and the corresponding means were compared by using a generalized multiple comparison procedure with multiplicity adjustment [28]. All analyses were conducted by using the R statistical environment [29].


*3.1. Sc Induces miRNA Expression in HepaRG Cells*

First, the miRNA expression profile in HepaRG cells was analyzed with a customized liver-specific miCURY LNA Array covering 84 miRNAs. Compared to the control group, the number of deregulated miRNAs above 1 log2 Fold Change (FC) or below 1 log2 FC tended to be highest at 250 μM after 8 h and at 35 μM after 24 h, respectively (see Supplementary Figure S1). Among the top deregulated miRNAs, a selection of five downregulated and four upregulated miRNAs were chosen to be investigated within senecionine (Sc) treated HepaRG cells. Sc was used for qPCR in order to have greater certainty for general PA effects and to exclude very specific effects of one single PA. To this end, HepaRG cells were treated with 35 μM of Sc and miRNA expression was analyzed after five different time points. Seven out of nine miRNAs proved to be differentially expressed after Sc treatment. The results are summarized in a heat map in Figure 1. Interestingly, all but one miRNA showed an increased expression compared to the solvent control in a time-dependent manner and for three miRNAs, the upregulation was highest after 8 h. *miRNA-122-3p* (alias miRNA-122\*) was the only one which showed significant downregulation after 24 h.

**Figure 1.** miRNA expression in differentiated HepaRG cells after exposure to 35 μM Sc at 5 different time-points. The results were evaluated using the 2−ΔΔCt method [40]. Results are shown as log2 fold changes (log2 FC) and as mean of three independent biological replicates. Gene expression values were referred to the solvent control (ctrl) of the respective time point but, for clarity, only the ctrl values of the 2 h time point are depicted here. Up- or downregulation of gene expression is indicated in green or red, respectively. The higher the values, the stronger the coloration. Mean values and standard deviations (SD) are summarized in the Supplementary Materials Table S2. Statistical analysis was performed using Student's *t*-test as this is a case of single comparison analysis. That is, each miRNA expression value of one time point was compared to the ctrl of this respective time point. Subsequently, *p*-values were subjected to FDR correction. Significant differences are depicted as follows: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p*< 0.001.

#### *3.2. Prediction of Biological Consequences of PA-Mediated miRNA Level Alterations*

Subsequently, the target genes of seven miRNAs that were identified to have significantly altered expression log2 FC values in HepaRG cells (see Figure 1) were predicted with IPA software (see Figure 2 for workflow).

The predicted target genes (773 in total) were compared to the gene expression dataset from a whole genome microarray conducted with primary human hepatocytes incubated with 100 μM Sc for 24 h [44]. Matched and prioritized targets (143 target genes) were submitted to a so-called IPA expression analysis to predict affected diseases and functions. In Figure 3 the top "diseases and bio functions" of the deregulated target genes of seven selected miRNAs predicted in Sc-treated primary human hepatocytes are shown. "Cancer" and "organismal injury and abnormalities" are the two main diseases and disorders that are regulated by the highest number of predicted targets. Among molecular and cellular functions, "cell death and survival" has the highest number of target genes with the most pronounced statistical significance. "Liver hyperproliferation" is the top hepatotoxicity function with 57 target genes involved, followed by "hepatocellular carcinoma" with 16 target genes.

Based on the expression analysis, 21 target genes involved in cellular growth and development, apoptosis and inflammation were chosen to be further investigated by qPCR in HepaRG cells incubated with 35 μM Sc for five different time points. The results are depicted in Figure 4 as a heat map. A total of 11 out of 21 genes showed an opposing expression pairing (indicated by superscript number 1); that is, significant downregulation, while their attributed miRNAs were upregulated. For the miRNA-4434 in particular, interaction with 5 out of 11 targets was predicted. These five targets comprised Growth Arrest-Specific protein 2 (*GAS2*), P21-Activated Kinase-1 (*PAK1*), LEPtin Receptor Overlapping Transcript (*LEPROT*), POZ/BTB and AT hook containing Zinc finger 1 (*PATZ1*) and

**Figure 2.** Workflow of miRNA target gene prediction with IPA. The target genes of 7 differentially and significantly expressed miRNAs in HepaRG cells were predicted with IPA (773 predicted targets) and compared to the dataset of a whole genome microarray conducted with primary human hepatocytes [44]. Among the 8623 Differentially Expressed Genes (DEGs) of the primary human hepatocytes after Sc-treatment (100 μM, 24 h), 143 targets were found to overlap with the predicted miRNA target genes. Out of these, 21 targets that simultaneously showed both a significant deregulation in the microarray as well as a high (predicted) confidence of miRNA-target interaction, were selected to be further investigated in HepaRG cells exposed to 35 μM of Sc for 5 different time points. A total of 11 out of 21 target genes showed an opposing expression pairing to their annotated miRNAs (indicated by superscript number 1), as verified by qPCR in HepaRG cells. IPA analysis revealed that 5 out of 11 targets were regulated by one miRNA (miRNA-4434).

**Figure 3.** Top 3 pathways in 3 "diseases and bio functions" of deregulated target genes in primary human hepatocytes listed in IPA after expression analysis with 7 miRNAs. IPA target gene prediction of 7 significantly deregulated miRNAs in HepaRG cells treated with 35 μM of Sc for 24 h was compared to the gene expression data from Sc-treated primary human hepatocytes (100 μM, 24 h) [44]. The numbers of deregulated target genes (number of molecules) predicted to be involved in diseases and bio functions are depicted as bar charts and their lower range of the *p*-value is shown in −log2 FC (orange graph).

ST6 beta-GALactoside alpha-2,6-sialyltransferase 1 (*ST6GAL1*). Therefore, this miRNA was chosen for further experiments.

**Figure 4.** Target gene expression in differentiated HepaRG cells after exposure to 35 μM Sc at 5 different time points. The results were evaluated using the 2−ΔΔCt method [40]. Results are shown as log2 FC and as mean of three biological replicates. Gene expression values were referred to the solvent control (ctrl) of the respective time point but, for clarity, only the ctrl values of the 2 h time point are depicted here. Up- or downregulation of gene expression is indicated in green or red, respectively. The higher the values, the stronger the coloration. Mean values and standard deviations are summarized in the Supplementary Materials Table S3. Superscript number 1 indicates the target genes that show an opposite regulation to their annotated miRNAs and the black arrows indicate the predicted regulatory miRNAs, respectively. Statistical analysis was performed using Student's *t*-test as this is a case of single-comparison analysis. That is, each target gene expression value of one time point was compared to the solvent control of this respective time point. Subsequently, *p*-values were subjected to FDR correction. Significant differences are depicted as follows: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p*< 0.001.

#### *3.3. MiR-4434 Negatively Regulates PAK1 Gene Expression in HepaRG Cells*

In order to investigate whether it is indeed the PA-mediated induction of miR-4434 levels that leads to the downregulation of the pre-selected target genes, differentiated HepaRG cells were treated with 35 μM of the PA Sc and, subsequently, transiently transfected either with the synthetic miRNA antagonist antagomiR-4434 or with an antagomiR-NC (both at 50 and 75 nM concentrations) or with transfection reagent and water only, as a negative treatment control. The miRNA inhibitor does not degrade its target, but forms a stable complex with it, resulting in an inhibition of miRNA-4434 function. The antagomiR-NC is non-homologous to any mammalian gene and was used to examine if the results of the antagomiR-4434-mediated inhibition were specific. That is, results achieved with the antagomiR-NC should be similar to results from negative treatment control (transfection reagent and water only). It was ensured that the combined incubation with antagomiR and Sc was non-cytotoxic (see Supplementary Materials Figure S2). The gene expression of all 21 target genes was assessed after 48 h and the values of the Sc-treated cells were always referred to their respective solvent control (antagomiR-4434, antagomiR-NC, or negative treatment control) (see Supplementary Materials Figure S3). When comparing antagomiR-4434-transfected cells with the negative control (antagomiR-NC) within the Sc-treatment group, the antisense effect of the antagomiR and its subsequent effect on miR-4434 target gene expression becomes evident. Here, the inhibition of miR-4434 led to significantly attenuated downregulation of *PAK*1 gene expression, with increased significance at the higher antagomiR concentration (Figure 5).

**Figure 5.** *PAK1* gene expression in differentiated HepaRG cells after exposure to 35 μM Sc and antagomiR-mediated inhibition of miR-4434 after 48 h. The results were evaluated according to the 2−ΔΔCt method [40]. (**a**) After housekeeper normalization, the values of the Sc-treated cells were referred to the respective solvent control (0.35% ACN). Noteworthy, the different treatments (antagomiR-4434, antagomiR-NC and negative treatment control) were referred to their respective solvent controls. AntagomiR/antagomiR-NC treatment was applied in the two concentrations 50 and 75 nM. Results are shown as log2 FC and as mean of three replicates. Mean values and standard deviations are summarized in the Supplementary Materials Table S4. (**b**) For normalized *PAK1* expression analysis within the Sc-treated cells, antagomiR-4434 treatment was referred to antagomiR-NC treatment. Again, antagomiR/antagomiR-NC treatment was applied in the two concentrations 50 and 75 nM. Additionally, the negative treatment control was referred to antagomiR-NC to ensure specific antagomiR-mediated inhibition. Statistical analysis was performed using Student's *t*-test as this is a case of single-comparison analysis. That is, the value of *PAK1* gene expression with antagomiR-4434 treatment (50 and 75 nM, respectively) was compared to the value of *PAK*1 gene expression with antagomiR-NC treatment (50 and 75 nM, respectively). Significant differences are depicted as follows: \* *p* < 0.05, \*\*\* *p* < 0.001.

#### **4. Discussion**

This study aimed to investigate the PA-induced effects on miRNA expression. Furthermore, miRNA-mediated effects on potential target genes were considered as an additional, however indirect, molecular regulator in PA-mediated hepatotoxicity. In the last years, miRNAs have emerged as very important players in the regulation of a variety of biological processes in many cell types, including those in the liver [32]. miRNA function ensures fine tuning of target gene expression, and subsequently protein abundance and protein distribution under constantly changing cellular conditions [47]. For example, in the liver intracellular miRNA levels regulate lipid and glucose metabolism [48], inflammation [49–51], apoptosis and necrosis [52–54], cell cycle and proliferation [55], as well as epithelial–mesenchymal transition during liver regeneration [56] and liver fibrosis [57,58]. Alterations in physiological miRNA levels correlate with various liver diseases such as viral hepatitis, alcoholic and nonalcoholic steatohepatitis, drug-induced liver injury, and autoimmune liver disease [32]. In metazoan cells, it is generally assumed that translational repression, presumably occurring during translation initiation, is the predominant mechanism by which miRNAs negatively regulate their target genes [29]. Therefore, it could be concluded that protein analysis is the investigative method of choice. However, transcript degradation of the target mRNA is an unfailing secondary effect triggered by the initial event of translational repression [59–61]. Therefore, qPCR analysis of target mRNA expression is a reliable and widely used method to assess the effects of miRNA-mediated gene silencing and was also applied here.

To elucidate the regulatory effects of miRNAs on their target genes is rather challenging because of its sheer complexity. For example, one miRNA can regulate many targets, and one target can be regulated by many different miRNAs in turn. Furthermore, miRNAs can directly bind their target gene, or they can indirectly regulate a target gene via binding to regulatory molecules like transcription factors, which have their own mode of action [62]. Thus, miRNAs can actually decrease, increase or not change the expression levels of their target genes [63]. This study aimed to achieve the first insights into the mechanistic of miRNA expression in HepaRG cells to understand its implication in PA-mediated hepatotoxicity. Therefore, for simplicity we only considered target genes that showed reverse correlation in gene expression in relation to their assigned miRNAs. Thus, target genes potentially showing a positive correlation or no correlation at all could have been overlooked. In general, miRNA upregulation upon PA-treatment has been described before. For example, *miRNA-34a*, which is considered to be a biomarker for exposure to genotoxic compounds, showed significant upregulation after chronic PA ingestion in a feeding study with rats [35]. Furthermore, blood samples from patients with HSOS showed elevated levels of miRNA-148a-3p, miRNA-362-5p, and miRNA-194-5p, which could be correlated to the severity of the PA-induced liver injury [37].

Upon Sc-incubation, seven liver-specific miRNAs could be verified to be significantly deregulated in HepaRG cells. All but one showed increased gene expression in a timedependent manner. For *miRNA-4301, miRNA-5100* and *miRNA-4454*, upregulation was highest after 8 h, showing the early responsiveness of miRNA levels. miRNA-4301 and miRNA-5100 have been observed to regulate proliferation and apoptosis in lung and breast cancer cells [64–67], but according to our assessment there are no reports on their implication in hepatic diseases. For *miRNA-4454*, it was reported that upregulation positively enhances hepatic carcinoma progression [68], miR-223 is a common regulator in various liver diseases [69], and *miRNA-3663-3p* was shown to be downregulated in hepatocellular carcinoma cells, thus positively regulating cell proliferation of cancer cells [70]. The involvement of these three miRNAs in liver diseases might pose an interface between their PA-induced upregulation and possible carcinogenic properties that have been described for PAs [71–73]. *miRNA-4434* had the highest upregulation of all miRNAs investigated. This miRNA plays a role in different tumors, either as promoter or inhibitor of proliferation [74–76]. However, reports on its relevance in liver function and disease are rare. One study observed miRNA-4434 to be inhibited by a long non-coding RNA

(lncRNA) called Long Stress Induced Non-Coding Transcripts 5 (LSINCT5) in hepatocellular carcinoma progression, potentially resulting in inhibited miRNA-4434 induced apoptosis [77]. *miRNA-122-*3p (alias miRNA-122\*, derived from the antisense strand of the precursor (pre)-miRNA; [78]) was the only miRNA that showed a significant downregulation only after 24 h. Noteworthy, *miRNA-122-*3p downregulation has been observed during early and advanced liver fibrosis [79]. As fibrosis is a disease which is also observed after chronic PA-intoxication, this could point towards a connection between downregulated *miRNA-122-3p* levels and PA-mediated toxicity resulting in fibrosis. The most abundant miRNA in the liver, *miRNA-122-5p* (alias miRNA-122a, derived from the sense strand of the pre-miRNA), did not show a statistically relevant deregulation in gene expression after Sc-treatment at any time-point and was therefore excluded from further analysis.

The comparison analysis between IPA-predicted mRNA targets and differentially expressed genes obtained from a whole genome microarray [44] revealed a target match of 143 genes that are involved in many diseases and disorders such as cancer in general and hepatocellular carcinoma in particular, with cellular growth and proliferation as underlying molecular and cellular functions. Of course, this analysis is a rather generalized evaluation. Therefore, out of these targets, a set of 21 genes regulating processes in cellular growth, development, apoptosis and inflammation was chosen to be verified in Sc-treated HepaRG cells and subjected to further IPA analysis. For cAMP-dependent PRotein Kinase type I-Alpha Regulatory subunit (*PRKAR1A*), a miRNA-dependent regulation was also observed in Mc-treated mice for type II-alpha subunit (*Prkar2a*). Moreover, in the same study, target gene V-type proton ATPase subunit e 2 (*Atp6v0e2*) also showed a significant upregulation in a miRNA-dependent way [36]. Here, another subunit type (*ATP6V1H*) was selected. Both *PRKAR1A* and *ATP6V1H* showed upregulation of gene expression, as was observed in the study mentioned above. A small subgroup of 11 genes showed reverse correlation in gene expression in comparison to their assigned miRNAs. These five miRNAs out of the initial set of seven were miRNA-223, miRNA-3663-3p, miRNA-4301, miRNA-4434, and miRNA-5100. According to IPA analysis, the 11 dysregulated genes generally promote apoptosis and necrosis and decrease cellular survival. Interestingly, miRNA-4434 was predicted to be an upstream regulator of 5 out of the 11 genes, which was the highest number of annotated targets for one miRNA. Surprisingly, it was not a well-established hepatic miRNA such as miRNA-223 or miRNA-122-5p, but the rather less-known miRNA-4434. The five target genes are involved in processes such as cell cycle progression (*GAS2* and *PAK1*), growth hormone signaling (*LEPROT*), apoptosis (*PATZ1*) and immunity (*ST6GAL1*), suggesting a correlation between PA-induced upregulation of miRNAs and subsequent downregulation of these five target genes, presumably resulting in disturbed cellular function. Ultimately, antagomiR-mediated inhibition of miRNA-4434 resulted in significantly altered gene expression pattern of the target gene *PAK*1, strongly indicating a biological connection. The *PAK1* gene encodes one family member of the serine/threonine-specific intracellular protein kinases that are involved in a number of cellular functions including cell cycle regulation, apoptosis, and cytoskeletal motility, usually through substrate phosphorylation [80]. PAK1 is a regulator in key signaling pathways which are relevant for cell cycle progression and proliferation. On the one hand, *PAK1* is overexpressed in many cancers and positively correlates with promotion of cell survival, invasion and metastasis, and drug resistance [81]. In the context of tumor therapy, for example, PAK1 triggers DNA repair caused by genotoxic therapeutic agents [82]. On the other hand, *PAK1* hinders cell cycle progression upon inhibition [83]. Additionally, miRNA interaction with *PAK1* expression has been described previously: in hepatocellular carcinoma development, miRNA-485-5p was observed to suppress *PAK1* levels, and lncRNA-mediated binding of miRNA-485-5p resulted in the upregulation of *PAK1* during hepatocellular carcinoma progression [84]. Here, a PA-induced upregulation of miRNA-4434 can be assumed to negatively regulate *PAK1* expression, presumably resulting in cell-cycle arrest. Notably, an implication of PAs in cell cycle regulation, which is closely linked to DNA damage, has been observed before [85]. This effect was observed to be higher for the more toxic PAs in

comparison to the less toxic PAs. In conclusion, we suggest that the identification of the regulatory mechanism of miRNA-4434-initiated and *PAK1*-induced dysregulation of cell cycle signaling may help to understand the molecular mode of action of some hepatotoxic and carcinogenic effects of Sc.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods11040532/s1: Table S1: miRNAs investigated with miR-CURY LNA miRNA custom PCR array, including 84 liver-specific miRNAs, 7 housekeeping genes, 1 interplate calibrator, 3 RNA isolation efficiency spike-in controls and 1 reverse transcription efficiency spike-in control; Figure S1: miRNA expression profile in differentiated HepaRG cells after exposure to Lc and determined with miRCURY LNA miRNA Array; Table S2: miRNA expression in differentiated HepaRG cells after exposure to 35 μM Sc at 5 different time-points; Table S3: Target gene expression in differentiated HepaRG cells after exposure to 35 μM of Sc for 5 time points; Figure S2: Cytotoxicity of differentiated HepaRG cells after antagomiR/antagomiR-NC- and Sc-incubation; Figure S3: Target gene expression in differentiated HepaRG cells after exposure to 35 μM Sc and antagomiR-mediated inhibition of miR-4434 after 48 h; Table S4: Target gene expression in differentiated HepaRG cells after exposure to 35 μM Sc and antagomiR-mediated inhibition of miR-4434 after 48 h.

**Author Contributions:** A.-M.E. conceptualization, investigation, visualization, writing. H.S. conceptualization, evaluation, writing—review and editing. A.B. conceptualization, supervision, writing review and editing. S.H.-P. conceptualization, supervision, funding acquisition, project administration, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the GERMAN FEDERAL INSTITUTE FOR RISK ASSESS-MENT (grant number 1322-624).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article (or Supplementary Material).

**Acknowledgments:** We thank Claudia Luckert for graciously providing additional documents and Beatrice Rosskopp for her excellent technical work.

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

#### **Abbreviations**


**Part 3**

**Optimization**

308 Serial and Parallel Robot Manipulators – Kinematics, Dynamics, Control and Optimization

Yager, R. & Filev, D.P. (1994). *Essentials of Fuzzy Modelling and Control*. John Wiley and Sons,

Zadeh, L.A. (1968). Probability Measures of Fuzzy Events. *Journal of Mathematical Analysis*

Zadeh, L.A. (1975). The Concept of a Linguistic Variable and its Applications to

Approximate Reasoning –I. *Information Sciences*, Vol.8, pp. 199-249

*and Applications*, Vol.23, No.2, (August 1968), pp. 421-427

New York, USA

**16**

*Turkey*

Pakize Erdogmus1 and Metin Toz2 *1Duzce University, Engineering Faculty, Computer Engineering Department*

*Computer Education Department, Duzce*

*2Duzce University, Technical Education Faculty,*

**Heuristic Optimization Algorithms in Robotics**

Today, Robotic is an essential technology from the entertainment to the industry. Thousands of articles have been published on Robotic. There are various types of robots such as parallel robots, industrial robots, mobile robots, autonomous mobile robots, health-care robots, military robots, entertainment robots, nano robots and swarm robots. So, this variety brings a lot of problems in Robotic. Inverse kinematic for serial robots, forward kinematic for parallel robots, path planning for mobile robots and trajectory planning for industrial robots are some of the problems in Robotic studied a lot. Some of the problems are solved easily with some mathematical equations such as forward kinematic problem for serial robots and inverse kinematic problem for parallel robots. But the problems consisting of nonlinear equations and higher order terms can't be solved exactly with the classical methods. The forward kinematics problem of the 6 degrees of freedom (DOF) 6x6 type of Stewart Platform can be given as an example to such problems. It has been shown that there are 40 distinct solutions for this problem (Raghavan, 1993). Some of the unsolved problems with classical methods are optimization problems and heuristic optimization techniques are an alternative way for the solution of such problems. The heuristic optimization techniques produce good solutions for the higher order nonlinear problems in an acceptable solution time, when the

problems aren't solved with the classical methods (Lee & El-Sharkawi, 2008).

problems in Robotic are solved with GA and PSO.

In this chapter, first in Section 2, optimization is shortly described. The introduction and some well-known heuristic algorithms including, Genetic Algorithms(GA), Simulated Annealing(SA), Particle Swarm Optimization(PSO) and Gravitational Search Algorithm(GSA) are reviewed in Section 3. In Section 4, two well-known optimization

Optimization is of great importance for the engineers, scientists and managers and it is an important part of the design process for all disciplines. The optimal design of a machine, the minimum path for a mobile robot and the optimal placement of a foundation are all

A constrained optimization problem has three main elements; design variables, constraints and objective function/functions. Design variables are independent variables of the

**1. Introduction**

**2. Optimization**

optimization problems.


*3.8.2 The transportation of these fragmented or detached crystal may occur by*

To sum up the formation of the equiaxed zone consisting of equiaxed grains, is promoted by the following:

Obviously, the above form certain factors which influence the crystallographic morphology of the casting.


**3. Gamma irradiation on textile fabrics**

Gamma rays are high-energy electromagnetic radiations having energies above 100 keV and wavelengths less than 10 picometers. Surface modification of textiles using gamma ray is one of the promising methods.

The excellent mechanical properties, heat and oxidation resistance, and environmental stabilities make carbon fiber fabrics ideal reinforcing materials in the advanced composite fields, such as solar panel of space station and electric vehicle body. All these excellent properties depend largely on the interfacial adhesion which finally affects the overall property of the resulting composites. Recently, radiation-induced grafting has been extensively applied as a competitive methodology to develop new functional materials. An easy method to evenly functionalize the fabric surfaces by *γ*-ray irradiation grafting was reported. This novel technique is simple, green, and versatile. The functionalization was much more uniform compared with the traditional electrochemical method. The interfacial strength of the compo‐ sites had a dramatic increase [8].

Presently, polymeric membranes are extensively employed in the field of biomedical materials contacting with blood. Sulfonated polypropylene nonwoven fabric (PPNWF) has been successfully prepared via gamma-ray preirradiation-induced graft polymerization of sodium styrenesulfonate (SSS) and acrylamide (AAm) by li et al (**Figure 2**).

**Figure 2.** Schematic diagram of preparing sulfonated PP NWF via gamma-ray preirradiation method [9].

Gamma ray preirradiation-induced cograft polymerization has been extensively applied to modified polymer materials because of its simple procedure and even grafting. Acrylonitrile (AN) has been widely used in the modification of materials and an amidoxime group can be obtained by further chemical treatments from the nitrile group to increase adsorption effi‐ ciency for heavy ions. In a research that has been done in 2013 by Liu and his coworkers, a preirradiation-induced emulsion cograft polymerization method was used to introduce AN and AA onto a PE nonwoven fabric. The use of AA is meant to improve the hydrophilicity of the modified fabric. The modified nonwoven fabric is ready for further amidoximation to the application of heavy metal ion extraction [10].

In the other research, SSS was grafted onto PPNWF via *γ*-ray coirradiation method with the existence of *N*-vinyl-2-pyrrolidone. The modified PPNWFs presented good blood compatibil‐ ity, such as lower hemolysis rate and lower platelet adhesion. Besides, the modified PPNWFs prolonged the clotting time and presented excellent anticoagulant effect [11].

The textile industries are one of the major sources of water pollution in terms of releasing highly colored waste stream in surface water bodies. The wastewater generated in textile processing plants is contaminated with toxic synthetic colorants and various perilous chemicals. The main objectives of the study by Bhuiyan et al. were to degrade the dye molecules and organic pollutants of textile wastewater by using gamma irradiation followed by the investigation of physicochemical parameters of the irradiated water as well as looking into the scope for using treated wastewater for irrigation and dyeing purposes. The wastewater samples were submitted to Cobalt-60 gamma radiation source.

The excellent mechanical properties, heat and oxidation resistance, and environmental stabilities make carbon fiber fabrics ideal reinforcing materials in the advanced composite fields, such as solar panel of space station and electric vehicle body. All these excellent properties depend largely on the interfacial adhesion which finally affects the overall property of the resulting composites. Recently, radiation-induced grafting has been extensively applied as a competitive methodology to develop new functional materials. An easy method to evenly functionalize the fabric surfaces by *γ*-ray irradiation grafting was reported. This novel technique is simple, green, and versatile. The functionalization was much more uniform compared with the traditional electrochemical method. The interfacial strength of the compo‐

Presently, polymeric membranes are extensively employed in the field of biomedical materials contacting with blood. Sulfonated polypropylene nonwoven fabric (PPNWF) has been successfully prepared via gamma-ray preirradiation-induced graft polymerization of sodium

styrenesulfonate (SSS) and acrylamide (AAm) by li et al (**Figure 2**).

**Figure 2.** Schematic diagram of preparing sulfonated PP NWF via gamma-ray preirradiation method [9].

application of heavy metal ion extraction [10].

Gamma ray preirradiation-induced cograft polymerization has been extensively applied to modified polymer materials because of its simple procedure and even grafting. Acrylonitrile (AN) has been widely used in the modification of materials and an amidoxime group can be obtained by further chemical treatments from the nitrile group to increase adsorption effi‐ ciency for heavy ions. In a research that has been done in 2013 by Liu and his coworkers, a preirradiation-induced emulsion cograft polymerization method was used to introduce AN and AA onto a PE nonwoven fabric. The use of AA is meant to improve the hydrophilicity of the modified fabric. The modified nonwoven fabric is ready for further amidoximation to the

In the other research, SSS was grafted onto PPNWF via *γ*-ray coirradiation method with the existence of *N*-vinyl-2-pyrrolidone. The modified PPNWFs presented good blood compatibil‐ ity, such as lower hemolysis rate and lower platelet adhesion. Besides, the modified PPNWFs

The textile industries are one of the major sources of water pollution in terms of releasing highly colored waste stream in surface water bodies. The wastewater generated in textile

prolonged the clotting time and presented excellent anticoagulant effect [11].

sites had a dramatic increase [8].

312 Radiation Effects in Materials

The irradiated wastewater was found to be recyclable in textile wet processing and reusable for irrigation purposes [12, 13].

The presence of toxic metals and pathogenetic microbes in drinking water is a potential health risk. Consequently, numerous investigations have been carried out on the functionalized polymer membrane with AgNPs as effective antimicrobial agents for water treatment. Unfortunately, the AgNPs were commonly inert with polymer surfaces so that silver releases into water filtrate in an overdose compared to the permitted limit of standard at maximum of 0.1 mg/L, according to the US Environmental Protection Agency (EPA) and World Health Organization (WHO).

A new method to immobilize AgNPs onto the acrylic grafted polyethylene nonwoven (PE) fabric by gamma Co-60 irradiation for drinking water treatment has been described in 2013.

The PE fabric pieces were treated by a mixture of acetone/H2O solution and dried before irradiation. The PE fabric samples were irradiated at the required doses up to 50 kGy by γ ray from Co-60 source. Thereafter, the graft reactions were carried out at ∼90°C in a flask con‐ taining 1 g preirradiated PE, 0.05 g Mohr's salt and AA with concentrations of 10–50% (v/v) in 100 ml aqueous solution. The PE-g-PAAc samples were soaked overnight at room temperature in Ag NPs colloidal solutions and then squeezed to remove the excess Ag NPs, rinsed with pure water and dried in an oven at 70 C for 2 h. The dried fabrics were then annealed at 120 C for 1 h for esterification of –COOH group of PAAc with –OH group of Polyvinyl alcohol (PVA). The prepared fabrics contained about 10,000 ppm Ag NPs showing strong bactericidal efficiency against *Escherichia coli*. Based on the strong bactericidal efficiency and under permitted limit of silver release into water filtrate, PE-g PAAc/Ag NPs fabrics can be used for the treatment of drinking water. Also, this kind of filters can be used in air cleaners and have other applications [14].

In the other research, the silver ions have been reduced effectively by gamma irradiation and immobilized on the cotton fabrics by in situ synthesis. The Ag NPs content deposited on the fabrics was of 1696 mg/kg, when the fabric sample was irradiated in 1.5 mM Ag NO and 1.0% chitosan solution at the dose of 13.8 kGy at 30°C.

Antibacterial efficacy of the Ag NPs fabrics after washing 40 cycles of washing was about 99.99% for *Staphylococcus aureus* and *E. coli*. The AgNPs/cotton fabrics washing from 1 to 40 cycles were innoxious to skin (k = 0). These results confirm that gamma irradiation of cotton fabrics in the presence of AgNO and chitosan solution is a promising approach for preparation of stable, safe, and efficacious antibacterial fabrics [15].

In the past decades, the materials with high water and oil repellency have attracted much attention from researchers and industries. The perfluoroalkyl phosphate acrylates have been grafted onto a cotton fabric via gamma-ray irradiation to improve the hydrophobic and oleophobic properties.

**Figure 3.** Photographs of water and sunflower oil drops on the grafted sample [16].

The results show that the fabric became highly hydrophobic and oleophobic with the contact angles of above 150o and 140o for water and sunflower oil, respectively (**Figure 3**) [16].

In the other research, a novel coating formulation for improving the UV protection property on cotton, PET, and cotton/PET fabrics was prepared, and gamma rays were applied for surface curing. Aluminum potassium sulfate (Alum) was used individually and in binary coat with Zinc Oxide (ZnO), to induce the UV-blocking properties [17].


**1. Introduction**

The immunocompetence handicap hypothesis [1] suggests that the expression of secondary sexual traits honestly signals male quality because testosterone—needed to develop these traits—has immunosuppressive e ffects. However, many studies in vertebrates have failed to reveal a clear relationship between the expression of secondary sexual ornamentation and immune defense. This might be because the physiological relationship between these traits is not as simple as originally thought and because of the alterations of stress hormones, which may involve trade-o ffs between sexual ornamentation and immunity [2]. However, many studies in insects and spiders have found that the expression of males' secondary sexual characteristics correlates positively with their immune defense [3–7]. Since insects lack male-specific hormones such as testosterone, it has been suggested that in these animals, the immunocompetence handicap mechanism would be mediated by juvenile hormone (JH) [8].

Juvenile hormone is synthesized in the corpora allata, and it is known to play a crucial role in many aspects of development, reproduction, aging, and behavior in insects. For example, it seems that juvenile hormone type III is associated with sex pheromone production in cockroaches [9]. Similarly, it was found that the administration of juvenile hormone increased the attractiveness of male pheromones in the mealworm beetle, *Tenebrio molitor*. Still, it reduced the strength of the encapsulation response and phenoloxidase activity of hemolymph [8]. Interestingly, it has been found that while JH increases male attractiveness, it reduces the size of the testis and sperm viability, suggesting another cost of high juvenile hormone levels [10]. In the territorial damselfly, *Calopteryx virgo*, it has been found that the administration of methoprene acid (an analog of JH) increased aggression and occupation time in territories but decreased phenoloxidase activity of hemolymph [11]. Juvenile hormone has also been shown to affect genes related to antibacterial peptide expression [12,13]. In the diamondback moth, *Plutella xylostella*, it was shown that juvenile hormone inhibited hemocyte-spreading behavior [13], suggesting that JH induces immune suppression because effective hemocyte-spreading is important for phagocytosis, nodulation, and encapsulation [14]. Furthermore, they found that pyriproxyfen (a JH analog) enhanced the pathogenicity of *Bacillus thuringiensis* subsp. *kurstaki* [13].

Juvenile hormone may also be associated with a trade-off between reproduction and immunity in insects. For example, mating reduced the activity of phenoloxidase enzyme in the hemolymph of both sexes of *T. molitor* [15], but there was no effect on the hemocyte load. It has been found that the observed decrease in phenoloxidase levels was caused by an increase in juvenile hormone levels due to mating, which indicates that juvenile hormone might indeed mediate the trade-off between mating and immunity [15]. Previously, it was shown that mating enhances resistance against entomopathogenic fungi, *Beauveria bassiana,* infection and that the effect was stronger in males than among females of *T. molitor* [16]. This shows that the effect of juvenile hormone on the immune system may be more complicated than previously thought. Overall, these results suggest that studies testing the effect of juvenile hormone on the resistance against highly virulent parasites and pathogens are needed.

Entomopathogenic fungi (EPF), including entomophthoralean fungi, offer environmentally friendly alternatives to conventional synthetic chemicals for arthropod pest control. There are over 750 different species of EPF identified so far [17]. Although entomophthoralean fungi are highly efficacious, much attention has focused on researching species belonging to the order Hypocreales because they are more amenable for mass production and have a relatively wide host range. Approximately 80% of the commercially available EPF products are based on the *Metarhizium robertsii, M. anisopliae, M. brunneum,* and *B. bassiana*.

The study aimed to test whether juvenile hormone affects the resistance of *T. molitor* against a real pathogen and whether there is a sex difference in the pathogen resistance. We tested juvenile hormone-related effects on the immunity of *T. molitor* against *M. robertsii*.

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