*3.3. Loading Scheme*

Assuming that the roof pressure acting on the crushed rocks in the gob area remains unchanged during the two caving activities of roof strata, it was considered necessary that the axial pressure acting on the crushed rocks in the gob area to be increased in a stepped manner [22]. Thus, the tests were conducted under incremental loading as shown in Figure 6. In GERRF method, the support structures of gangue rib deformed in response to lateral stress generated by crushed rocks in gob side. In engineering practice, the support structures of gangue rib are not allowed to produce large deformations. So, in the tests, the maximum axial pressure was set at a value corresponding to when the support structure will begin to deform, which was set as 1.5 MPa according to preliminary experiments results. In order to observe the periodic deformation behavior of crushed rocks in greater detail, the loading increment Δ*P* was designed as 0.15 MPa.

**Figure 6.** Loading path in the tests.


**Abstract**

The pandemic year 2020 has led to significant changes in all areas of life. Education was no exception. Due to the closure of schools and universities during the lockdown, educational systems worldwide had to be switched overnight, from face-to-face to a completely virtual education model without prior preparation. The study conducted in Montenegro, based on interviews and a survey, collected data on the basis of which it provided an insight into how the teaching staff of Montenegrin universities coped with the newly created circumstances. The aim of this primary research is to determine the challenges faced by teachers and students in the Montenegrin higher education system regarding their implementation of online teaching during the COVID-19 pandemic in 2020. It focuses on the digital competence of the teaching staff, challenges in applying digital technology, opinions regarding online assessments and students' experiences with online learning. Despite the presented challenges all teachers experienced, the research confirmed that this was a unique experience that brought new opportunities and contributed to improving the teachers' ability to use new technologies. It is to be expected that this unfortunate situation will trigger the development of various mechanisms for modernizing the way of providing knowledge in the future.

**Keywords:** higher education, online teaching, digital literacy, technological challenges, online assessments, student engagement, student experience, academic integrity, quality assurance


**Analog‐to‐Digital Conversion for Cognitive Radio: Subsampling, Interleaving, and Compressive Sensing**

José Ramón García Oya, Fernando Muñoz Chavero and Rubén Martín Clemente

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69241

#### Abstract

This chapter explores different analog-to-digital conversion techniques that are suitable to be implemented in cognitive radio receivers. This chapter details the fundamentals, advantages, and drawbacks of three promising techniques: subsampling, interleaving, and compressive sensing. Due to their major maturity, subsampling- and interleavingbased systems are described in further detail, whereas compressive sensing-based systems are described as a complement of the previous techniques for underutilized spectrum applications. The feasibility of these techniques as part of software-defined radio, multistandard, and spectrum sensing receivers is demonstrated by proposing different architectures with reduced complexity at circuit level, depending on the application requirements. Additionally, the chapter proposes different solutions to integrate the advantages of these techniques in a unique analog-to-digital conversion process.

Keywords: analog-to-digital conversion, cognitive radio, compressive sensing, interleaving, multistandard receivers, software-defined radio, spectrum sensing, subsampling

#### 1. Introduction

Analog-to-digital conversion (ADC) stage is one of the main bottlenecks of the high-speed telecommunications systems. This chapter presents a survey of different feasible analog-todigital conversion techniques that are suitable to overcome these difficulties and to get the software-defined radio (SDR) paradigm [1], where most functionalities, instead of being performed in the analog domain (i.e., filters and mixers), are performed in the digital domain. In SDR, the analog-to-digital conversion is implemented immediately after the antenna, and the radio frequency (RF) signal is directly converted to digital without any previous mixing

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

stage. Since it is not possible to approach this idea with traditional analog-to-digital conversion from current commercial devices, this chapter describes some techniques that may be employed instead. Although the proposed systems have more restrictive specifications, these solutions reduce the final complexity, as will be detailed in this chapter. This work explores three different promising techniques: subsampling, interleaving, and compressive sensing (CS).

The proposed techniques are an appealing solution to approach the cognitive radio (CR) objectives [2, 3], which are conditioned by the physical implementation of the SDR receiver. Due to the necessity of several wireless standards coexisting in the same device, a high flexibility and programmability will be an important requirement for the proposed architectures, with the objective of being employed in multistandard receivers.

With these objectives in mind, this chapter describes in detail three different approaches for implementing the analog-to-digital conversion stage. The choice of one or other of these approaches will depend on the environment, the properties of the received signals, and the parameters that have to be optimized. For receivers where the main requirements are a highresolution and a high-analog bandwidth that covers a maximum number of communication standards, we propose a system based on subsampling techniques. For applications where the main requirement is to maximize the data acquisition rate, the proposed system is based on interleaving techniques, that is, the interconnection of several analog-to-digital converters in parallel. Finally, compressive sensing techniques will be preferred for scenarios where the spectrum can be considered sparse, that is, for a wideband spectrum with a low spectral occupancy, where it will be possible to recover the received signal and implement an estimation of the radio channel by using a reduced number of samples from the ADC. This emerging technology will be presented at architectural level, so that it will be studied from the point of view of its integration with the two main techniques detailed in this chapter, that is, subsampling and interleaving techniques, with the objective of exploiting their advantages for sparse spectrum sensing applications.


**4.1 Chrysin**

188 Basic and Clinical Endocrinology Up-to-Date

To understand how the receptor antagonists enhanced StAR protein expression and steroidogenesis, their effects on StAR gene transcription were investigated. Luciferase assays of StAR promoter activity indicated that blocking the TBX A2 receptors with the antagonists enhanced StAR promoter activity in a concentration-dependent manner in the MA-10 cells incubated with 0.1 mM cAMP. The increase in the promoter activity was associated with a similar increase in StAR mRNA level (Pandey et al., 2009). The observations indicated that blocking the TBX A2 receptor enhanced StAR gene transcription, similar to the observations on the cells treated with TBXAS inhibitor (Wang et al., 2008). Further study showed that blocking the TBX A2 receptors significantly reduced DAX-1 protein and increased StAR protein in MA-10 mouse Leydig cells. The results were confirmed with the Leydig cells isolated from aged rats. The aged Leydig cells expressed high levels of DAX-1 protein, but blocking the TBX A2 receptors dramatically reduced the DAX-1 protein, which was associated with the increases in StAR protein and testosterone production (Pandey et al., 2009). These observations indicated that the signaling through COX2-TBXAS-TBX A2-receptor inhibits StAR gene expression and testosterone biosynthesis

In addition, while sub-threshold levels of cAMP were unable to stimulate significant increases in StAR gene expression, interrupting the signaling through this pathway at any step, by inhibiting COX2 activity (Wang et al., 2005; Wang et al., 2003b) or TBXAS activity (Wang et al., 2008) or blocking the TBX A2 receptors (Pandey et al., 2009), reduced the threshold, with sub-threshold levels of cAMP being able to induce maximal levels of StAR protein and steroidogenesis. These studies indicated that the signaling through COX2- TBXAS-TBX A2-receptor pathway plays an important role in regulating sensitivity of Leydig cells to LH or cAMP stimulation. Therefore, when COX2 increases in Leydig cell aging, the increase in COX2 enhances the negative signaling through this pathway, which in turn reduces the sensitivity of Leydig cells and inhibits the LH-induced StAR gene expression

**4. Flavonoid intervention in the COX2-dependent inhibition of StAR gene**

To study the possibility of delaying the decline in blood testosterone by intervention in the mechanism, aged rats were fed with increasing concentrations of a selective COX2 inhibitor mixed in their diet. After 30 days, StAR protein in their Leydig cells increased in a concentration-dependent manner. The blood testosterone concentrations increased up to 120% over control (Wang et al., 2005). The studies suggest a possibility of delaying the agerelated declines in StAR protein and testosterone using COX2 inhibitors. However, longterm application of the COX2 inhibitors currently used in the clinical practice is limited by their side effects. Therefore, alternative approaches are needed for the health of aging males. In the recent years, steroidogenic effects of natural flavonoids have been studied with Leydig cells. Flavonoids are a group of the polyphenolic compounds that are widely distributed in various food and food supplements, especially in fruits and vegetables. Previous studies have reported the activities of flavonoids in anti-inflammation, anti-cancer, and anti-oxidation (Cardenas et al., 2006; Chen et al., 1990; Ferrandiz & Alcaraz, 1991). One of the important mechanisms for these activities is the inhibition of COX2 expression and blocking the COX2-dependent signaling by flavonoids, which enables flavonoids to enhance StAR gene expression and testosterone biosynthesis in Leydig cells. A group of flavonoids

by regulation of DAX-1 expression.

and testosterone biosynthesis.

**expression and testosterone biosynthesis**

Chrysin is present in plants (Williams et al., 1997), honey and propolis (Gambelunghe et al., 2003; Jiang et al., 2008; Kassim et al., 2010). It blocks the COX2-dependent signaling principally by inhibition of COX2 expression. The reduction of COX2 by chrysin is due to its inhibitory effects on the activities of several transcription factors that regulate COX2 gene transcription in different tissues. Chrysin is able to inhibit the activity of NF-kB and reduce COX2 promoter activity (Ha et al., 2010; Li et al., 2010). In macrophages, chrysin suppressed lipopolysaccharide (LPS)-induced COX2 expression by inhibiting activity of nuclear factor for IL-6 (NF-IL6) (Woo et al., 2005). NF-IL6 is a member of the C/EBP family that binds to human COX2 promoter region at -124/-132 and induces COX-2 expression. Another member of C/EBP family is C/EBPβ that is activated by c-Jun N terminal kinase (JNK) (Cho et al., 2003). Chrysin inhibited JNK activation and reduced LPS-induced COX2 expression (Ha et al., 2010). In addition, the cytokines, such as IL-1β and TNF-α, are able to induce COX2 expression (Chen et al., 2001a; Ishikawa et al., 2005). It was observed that chrysin significantly reduced the LPS-released IL-1β and TNF-α (Ha et al., 2010; Romier et al., 2008). In addition to the inhibition of COX2 gene expression, chrysin and its derivatives are able to inhibit COX2 activity. A group of chrysin derivatives was synthesized by modification of its molecular structure. The modification significantly improves the activities of the derivatives in inhibiting COX2. Some of the chrysin derivatives are able to bind to COX2 molecule and selectively inhibit COX2 activity (Cho et al., 2004; Dao et al., 2004).

Chrysin has been described as a testosterone-increasing agent that blocks further testosterone metabolism by inhibiting aromatase activity (Dhawan et al., 2002; Kellis & Vickery, 1984). This testosterone-increasing activity of chrysin was confirmed in the recent studies with Leydig cells isolated from mouse and MA-10 mouse Leydig cell line, with the steroid hormone productions of the Leydig cells being significantly enhanced by chrysin

Natural Flavonoids in StAR Gene Expression

Leydig cells (Li et al., 2011).

**4.3 Luteolin**

and Testosterone Biosynthesis in Leydig Cell Aging 191

from mouse. The binding of apigenin to the TBX A2 receptors was examined. The binding competition between apigenin and the selective receptor antagonist 3H-SQ29548 indicated that this flavonoid specifically blocked the TBX A2 receptors on MA-10 mouse Leydig cells. Blocking the receptors with apigenin significantly increased StAR protein and testosterone production in mouse Leydig cells (Li et al., 2011). In MA-10 Leydig cells, apigenin induced concentration-dependent increases in StAR promoter activity, StAR mRNA, StAR protein and steroid hormone production. Further study indicated that this flavonoid enhanced StAR gene transcription by reduction of the transcriptional repressor, DAX-1 protein. While the mechanism for the reduction of DAX-1 protein in apigenin-treated cells is unknown, the results from the study suggest that minimal levels of PKA and protein kinase C (PKC) activities are essential for the effects of apigenin on DAX-1, StAR and steroidogenesis in

Luteolin is found in fruits and vegetables (Harnly et al., 2006). It was detected in bird chili (1035.0 mg/kg, in dry weight), belimbi leaves (464.5 mg/kg), onion leaves (391.0 mg/kg), belimbi fruit (202.0 mg/kg), dried asam gelugur (107.5 mg/kg), local celery (80.5 mg/kg), broccoli (74.5 mg/kg), carrot (37.5 mg/kg), limau purut leaves (30.5 mg/kg), French bean (11.0 mg/kg), and white radish (9.0 mg/kg) (Miean & Mohamed, 2001). Similar to the effects of apigenin, luteolin is able to inhibit COX2 expression and block the TBX A2 receptors. It was reported that luteolin reduced COX2 expression by inhibiting NF-kB activity (Chen et al., 2007a; Gutierrez-Venegas et al., 2006; Kim & Jobin, 2005), through the mechanism involving the luteolin-reduced IkB kinase (IKK) activity, IkB degradation, nuclear translocation of NF-kB p65 subunit, and NF-kB-binding to the promoter DNA. Luteolin may reduce COX2 expression by inhibiting activity of MAPK and subsequently reducing the activities of the transcription factors that are essential for COX2 transcription (Choi & Lee, 2010; Gutierrez-Venegas et al., 2006). It is known that COX2 expression is up-regulated by inflammatory cytokines, such as IL-1β and TNF-α. Luteolin was reported being able to reduce these cytokines, which may contribute to its inhibitory effect on COX2 expression (Wu et al., 2009). In addition, luteolin is also a natural antagonist of the TBX A2 receptors. It blocked the COX2-dependent signaling through COX2-TBXAS-TBX A2-receptor pathway

The inhibitory effect of luteolin on COX2-dependent signaling suggests a possibility of using this flavonoid to enhance StAR gene expression and steroidogenesis in Leydig cells. This possibility was examined by the experiments with MA-10 mouse Leydig cells cultured for 6 hours in the medium containing increasing concentrations of luteolin and a cAMP analog (dbcAMP). StAR and DAX-1 proteins, steroid production, StAR mRNA and StAR promoter activity were analyzed as described in the previous study (Jana et al., 2008). As shown in Fig. 2A, the treatments with luteolin induced a concentration-dependent increase in steroid hormone production. Progesterone concentrations in culture medium were increased from 4.9 to 124.7ng/ml, as the levels of luteolin in the culture were increased from 0 to 12 µM. In the presence of 22(R)hydroxycholesterol(22R), there was no significant difference in steroid production among the treatments, suggesting that luteolin increase steroidogenesis by improving mitochondrial cholesterol transfer in Leydig cells. The results were enhanced by the luteolin-increased StAR protein expression. Similar increases were observed in the analyses of StAR promoter activity and StAR mRNA levels (Fig. 2B), indicating a regulatory effect of luteolin on StAR gene transcription. In addition, a synergistic interaction between

by binding to the receptors (Guerrero et al., 2005; Guerrero et al., 2007).

(Jana et al., 2008). However, it failed to induce the significant increase in steroid hormone when MA-10 Leydig cells were co-incubated with 22(R)hydroxycholesterol, suggesting that this flavonoid increase steroidogenesis mainly by improving the cholesterol transfer to the mitochondrial inner membrane. This was proved by the results from Western blot analyses that showed a significant increase in StAR protein in the Leydig cells treated with chrysin. The observations were corroborated by the significant increases in StAR promoter activity and StAR mRNA levels in the cells, indicating that chrysin acted on StAR promoter and enhanced StAR gene transcription. The transcription factors that act on StAR promoter were examined in the Leydig cells incubated with chrysin. It was found that this flavonoid significantly reduced the transcriptional repressor, DAX-1 protein (Jana et al., 2008). As DAX-1 protein and DAX-1 mRNA were reduced, StAR protein and steroid hormone production dramatically increased. Similar to the effect of COX2 inhibitors, chrysin did not affect PKA activity. In the absence of cAMP, this flavonoid alone was not able to increase StAR protein and steroidogenesis. However, in the presence of 10 µm chrysin, sensitivity of Leydig cells dramatically increased, with sub-threshold levels of cAMP being able to induce maximal levels of StAR protein and steroidogenesis. The studies indicated that chrysin enhanced steroidogenesis in Leydig cells mainly by enhancing cAMP-induced StAR gene expression.

#### **4.2 Apigenin**

Apigenin is present in various plants at different concentrations (Miean & Mohamed, 2001). It was found in parsley at high level (Nielsen et al., 1999). This flavonoid blocks COX2 dependent signaling at two separated steps, by inhibiting COX2 expression and blocking the TBX A2 receptors. It inhibits COX2 expression at the levels of transcription and translation. At the transcriptional level, apigenin inhibits activities of the transcription factors that are important for COX2 gene expression. Previous studies reported that incubation of mouse macrophages with apigenin reduced COX2 expression by inhibition of NF-kB activation, through a mechanism involving the apigenin-reduced degradation of the inhibitor kB (IkB) (Liang et al., 1999). This observation was enhanced by the study on the apigenin-reduced COX2 expression, in which apigenin-inhibited IkBα degradation resulted in reductions in NF-kB-binding to the promoter DNA and the TNF-α-induced COX2 expression (Shukla & Gupta, 2004). In addition, apigenin is able to inhibit the activation of MAPK. It is known that MAPK activation is essential for the activities of several transcription factors, such as CREB, NF-kB, and C/EBP, that induce COX2 expression in various tissues (Chun & Surh, 2004; Tsatsanis et al., 2006). The inhibitory effects of apigenin on MAPK activation significantly reduced COX2 gene transcription (Ha et al., 2008; Yi Lau & Leung, 2010). At the translational level, apigenin increased the localization to cytoplasm of two proteins, HuR and T-cell-restricted intracellular antigen 1-related protein (TIAR). HuR and TIAR then bound to the AU-rich elements in the 3'-untranslated region of COX2 mRNA and inhibited COX2 translation (Tong et al., 2007). In addition to its inhibitory effects on COX2 expression, apigenin acts as a natural antagonist of the TBX A2 receptors. Binding of apigenin to the receptors blocked the signaling through COX2-TBXAS-TBX A2-receptors pathway (Guerrero et al., 2007; Navarro-Nunez et al., 2008).

It was reported that blocking the signaling at any step of the COX2-TBXAS-TBX A2 receptors pathway significantly increased StAR gene expression and testosterone biosynthesis in Leydig cells (Pandey et al., 2009). Therefore, the effects of apigenin on steroidogenesis were studied using MA-10 mouse Leydig cell line and Leydig cells isolated

from mouse. The binding of apigenin to the TBX A2 receptors was examined. The binding competition between apigenin and the selective receptor antagonist 3H-SQ29548 indicated that this flavonoid specifically blocked the TBX A2 receptors on MA-10 mouse Leydig cells. Blocking the receptors with apigenin significantly increased StAR protein and testosterone production in mouse Leydig cells (Li et al., 2011). In MA-10 Leydig cells, apigenin induced concentration-dependent increases in StAR promoter activity, StAR mRNA, StAR protein and steroid hormone production. Further study indicated that this flavonoid enhanced StAR gene transcription by reduction of the transcriptional repressor, DAX-1 protein. While the mechanism for the reduction of DAX-1 protein in apigenin-treated cells is unknown, the results from the study suggest that minimal levels of PKA and protein kinase C (PKC) activities are essential for the effects of apigenin on DAX-1, StAR and steroidogenesis in Leydig cells (Li et al., 2011).

#### **4.3 Luteolin**

190 Basic and Clinical Endocrinology Up-to-Date

(Jana et al., 2008). However, it failed to induce the significant increase in steroid hormone when MA-10 Leydig cells were co-incubated with 22(R)hydroxycholesterol, suggesting that this flavonoid increase steroidogenesis mainly by improving the cholesterol transfer to the mitochondrial inner membrane. This was proved by the results from Western blot analyses that showed a significant increase in StAR protein in the Leydig cells treated with chrysin. The observations were corroborated by the significant increases in StAR promoter activity and StAR mRNA levels in the cells, indicating that chrysin acted on StAR promoter and enhanced StAR gene transcription. The transcription factors that act on StAR promoter were examined in the Leydig cells incubated with chrysin. It was found that this flavonoid significantly reduced the transcriptional repressor, DAX-1 protein (Jana et al., 2008). As DAX-1 protein and DAX-1 mRNA were reduced, StAR protein and steroid hormone production dramatically increased. Similar to the effect of COX2 inhibitors, chrysin did not affect PKA activity. In the absence of cAMP, this flavonoid alone was not able to increase StAR protein and steroidogenesis. However, in the presence of 10 µm chrysin, sensitivity of Leydig cells dramatically increased, with sub-threshold levels of cAMP being able to induce maximal levels of StAR protein and steroidogenesis. The studies indicated that chrysin enhanced steroidogenesis in Leydig cells mainly by enhancing cAMP-induced StAR gene

Apigenin is present in various plants at different concentrations (Miean & Mohamed, 2001). It was found in parsley at high level (Nielsen et al., 1999). This flavonoid blocks COX2 dependent signaling at two separated steps, by inhibiting COX2 expression and blocking the TBX A2 receptors. It inhibits COX2 expression at the levels of transcription and translation. At the transcriptional level, apigenin inhibits activities of the transcription factors that are important for COX2 gene expression. Previous studies reported that incubation of mouse macrophages with apigenin reduced COX2 expression by inhibition of NF-kB activation, through a mechanism involving the apigenin-reduced degradation of the inhibitor kB (IkB) (Liang et al., 1999). This observation was enhanced by the study on the apigenin-reduced COX2 expression, in which apigenin-inhibited IkBα degradation resulted in reductions in NF-kB-binding to the promoter DNA and the TNF-α-induced COX2 expression (Shukla & Gupta, 2004). In addition, apigenin is able to inhibit the activation of MAPK. It is known that MAPK activation is essential for the activities of several transcription factors, such as CREB, NF-kB, and C/EBP, that induce COX2 expression in various tissues (Chun & Surh, 2004; Tsatsanis et al., 2006). The inhibitory effects of apigenin on MAPK activation significantly reduced COX2 gene transcription (Ha et al., 2008; Yi Lau & Leung, 2010). At the translational level, apigenin increased the localization to cytoplasm of two proteins, HuR and T-cell-restricted intracellular antigen 1-related protein (TIAR). HuR and TIAR then bound to the AU-rich elements in the 3'-untranslated region of COX2 mRNA and inhibited COX2 translation (Tong et al., 2007). In addition to its inhibitory effects on COX2 expression, apigenin acts as a natural antagonist of the TBX A2 receptors. Binding of apigenin to the receptors blocked the signaling through COX2-TBXAS-TBX A2-receptors pathway

It was reported that blocking the signaling at any step of the COX2-TBXAS-TBX A2 receptors pathway significantly increased StAR gene expression and testosterone biosynthesis in Leydig cells (Pandey et al., 2009). Therefore, the effects of apigenin on steroidogenesis were studied using MA-10 mouse Leydig cell line and Leydig cells isolated

expression.

**4.2 Apigenin**

(Guerrero et al., 2007; Navarro-Nunez et al., 2008).

Luteolin is found in fruits and vegetables (Harnly et al., 2006). It was detected in bird chili (1035.0 mg/kg, in dry weight), belimbi leaves (464.5 mg/kg), onion leaves (391.0 mg/kg), belimbi fruit (202.0 mg/kg), dried asam gelugur (107.5 mg/kg), local celery (80.5 mg/kg), broccoli (74.5 mg/kg), carrot (37.5 mg/kg), limau purut leaves (30.5 mg/kg), French bean (11.0 mg/kg), and white radish (9.0 mg/kg) (Miean & Mohamed, 2001). Similar to the effects of apigenin, luteolin is able to inhibit COX2 expression and block the TBX A2 receptors. It was reported that luteolin reduced COX2 expression by inhibiting NF-kB activity (Chen et al., 2007a; Gutierrez-Venegas et al., 2006; Kim & Jobin, 2005), through the mechanism involving the luteolin-reduced IkB kinase (IKK) activity, IkB degradation, nuclear translocation of NF-kB p65 subunit, and NF-kB-binding to the promoter DNA. Luteolin may reduce COX2 expression by inhibiting activity of MAPK and subsequently reducing the activities of the transcription factors that are essential for COX2 transcription (Choi & Lee, 2010; Gutierrez-Venegas et al., 2006). It is known that COX2 expression is up-regulated by inflammatory cytokines, such as IL-1β and TNF-α. Luteolin was reported being able to reduce these cytokines, which may contribute to its inhibitory effect on COX2 expression (Wu et al., 2009). In addition, luteolin is also a natural antagonist of the TBX A2 receptors. It blocked the COX2-dependent signaling through COX2-TBXAS-TBX A2-receptor pathway by binding to the receptors (Guerrero et al., 2005; Guerrero et al., 2007).

The inhibitory effect of luteolin on COX2-dependent signaling suggests a possibility of using this flavonoid to enhance StAR gene expression and steroidogenesis in Leydig cells. This possibility was examined by the experiments with MA-10 mouse Leydig cells cultured for 6 hours in the medium containing increasing concentrations of luteolin and a cAMP analog (dbcAMP). StAR and DAX-1 proteins, steroid production, StAR mRNA and StAR promoter activity were analyzed as described in the previous study (Jana et al., 2008). As shown in Fig. 2A, the treatments with luteolin induced a concentration-dependent increase in steroid hormone production. Progesterone concentrations in culture medium were increased from 4.9 to 124.7ng/ml, as the levels of luteolin in the culture were increased from 0 to 12 µM. In the presence of 22(R)hydroxycholesterol(22R), there was no significant difference in steroid production among the treatments, suggesting that luteolin increase steroidogenesis by improving mitochondrial cholesterol transfer in Leydig cells. The results were enhanced by the luteolin-increased StAR protein expression. Similar increases were observed in the analyses of StAR promoter activity and StAR mRNA levels (Fig. 2B), indicating a regulatory effect of luteolin on StAR gene transcription. In addition, a synergistic interaction between

Natural Flavonoids in StAR Gene Expression

by reduction of DAX-1 expression.

DAX-1 protein

formation (Garcia-Saura et al., 2005; Sheu et al., 2004).

and Testosterone Biosynthesis in Leydig Cell Aging 193

signaling. While LPS activates the MAPKs, quercetin inhibited the activation of each of them, and subsequently reduced IκB phosphorylation and COX2 expression (Gutierrez-Venegas et al., 2007). The observations were enhanced by the study on the inhibitory effects of quercetin on ROS-induced MAPK activation and COX2 expression (Huang et al., 2006). The inhibition of MAPKs by quercetin was associated with the reductions in inflammatory cytokines, such as IL-1β and TNF-α (Overman et al., 2011). In addition, quercetin is able to block the COX2-TBXAS-TBX A2-receptor signaling pathway by inhibition of TBX A2

The steroidogenic effect of quercetin was studied with MA-10 mouse Leydig cells. It was reported that quercetin increased StAR mRNA levels, StAR promoter activity and steroid hormone production (Chen et al., 2007c). The observations were corroborated by the results shown in Fig. 3., which indicated the quercetin-enhanced StAR gene transcription and translation in MA-10 cells cultured for 6 hours (Fig. 3 A and B). Following these studies, the transcription factors in StAR gene transcription were examined. It was found that the incubation of MA-10 Leydig cells with quercetin reduced DAX-1 protein, similar to the effects of other flavonoids. The reduction in DAX-1 protein was associated with a dramatic increase in StAR protein (Fig. 3C), suggesting that quercetin enhance StAR gene expression

Fig. 3. Effect of quercetin on StAR gene expression and steroidogenesis in MA-10 mouse Leydig cells. MA-10 cells were incubated with quercetin for 6 hours, and then collected for analyses: A, StAR protein and steroidogenesis; B, StAR gene transcription; C, Reduction in

luteolin and cAMP was observed (Fig. 2C), in which luteolin reduced the threshold of cAMP-induced StAR gene expression and increased the sensitivity of Leydig cells to cAMP stimulation. Further study indicated that luteolin enhanced StAR gene transcription by inhibiting DAX-1 expression (Fig. 2D), similar to the observation with apigenin.

Fig. 2. Effect of luteolin on StAR gene expression and steroidogenesis in MA-10 mouse Leydig cells. MA-10 cells were incubated with luteolin for 6 hours, and then collected for analyses: A, StAR protein and steroidogenesis; B, StAR gene transcription; C, Interaction between luteolin and cAMP; D, Reduction in DAX-1 protein.

#### **4.4 Quercetin**

Quercetin was reported as one of the major flavonoids in the plants (Miean & Mohamed, 2001), especially in onions (Slimestad et al., 2007). It blocks the COX2-dependent signaling by inhibiting COX2 expression and TBX A2 production. Similar to other flavonoids, quercetin depresses COX2 expression by inhibiting activities of NF-kB. It was observed that this flavonoid reduced the activity of NF-kB by inhibiting IKK/IκB signaling cascade (Garcia-Mediavilla et al., 2007), which in turn reduced NF-kB nuclear translocation, its binding to promoter, and COX2 expression (Crespo et al., 2008; Puangpraphant & de Mejia, 2009). Another signaling pathway regulating COX2 expression is PI3K-Akt pathway. This signaling pathway may activate CREB (Alique et al., 2011) or NF-kB (Yang et al., 2009), and induce COX2 expression. A recent study found that quercetin directly bound with PI3K to inhibit PI3K activity and Akt phosphorylation, resulting in a reduction in COX2 expression (Lee et al., 2010). In the LPS-induced COX2 expression, the members of MAPK, including extracellular signal related kinase (ERK), p38 and JNK, are involved in the LPS-induced

luteolin and cAMP was observed (Fig. 2C), in which luteolin reduced the threshold of cAMP-induced StAR gene expression and increased the sensitivity of Leydig cells to cAMP stimulation. Further study indicated that luteolin enhanced StAR gene transcription by

inhibiting DAX-1 expression (Fig. 2D), similar to the observation with apigenin.

Fig. 2. Effect of luteolin on StAR gene expression and steroidogenesis in MA-10 mouse Leydig cells. MA-10 cells were incubated with luteolin for 6 hours, and then collected for analyses: A, StAR protein and steroidogenesis; B, StAR gene transcription; C, Interaction

Quercetin was reported as one of the major flavonoids in the plants (Miean & Mohamed, 2001), especially in onions (Slimestad et al., 2007). It blocks the COX2-dependent signaling by inhibiting COX2 expression and TBX A2 production. Similar to other flavonoids, quercetin depresses COX2 expression by inhibiting activities of NF-kB. It was observed that this flavonoid reduced the activity of NF-kB by inhibiting IKK/IκB signaling cascade (Garcia-Mediavilla et al., 2007), which in turn reduced NF-kB nuclear translocation, its binding to promoter, and COX2 expression (Crespo et al., 2008; Puangpraphant & de Mejia, 2009). Another signaling pathway regulating COX2 expression is PI3K-Akt pathway. This signaling pathway may activate CREB (Alique et al., 2011) or NF-kB (Yang et al., 2009), and induce COX2 expression. A recent study found that quercetin directly bound with PI3K to inhibit PI3K activity and Akt phosphorylation, resulting in a reduction in COX2 expression (Lee et al., 2010). In the LPS-induced COX2 expression, the members of MAPK, including extracellular signal related kinase (ERK), p38 and JNK, are involved in the LPS-induced

between luteolin and cAMP; D, Reduction in DAX-1 protein.

**4.4 Quercetin**

signaling. While LPS activates the MAPKs, quercetin inhibited the activation of each of them, and subsequently reduced IκB phosphorylation and COX2 expression (Gutierrez-Venegas et al., 2007). The observations were enhanced by the study on the inhibitory effects of quercetin on ROS-induced MAPK activation and COX2 expression (Huang et al., 2006). The inhibition of MAPKs by quercetin was associated with the reductions in inflammatory cytokines, such as IL-1β and TNF-α (Overman et al., 2011). In addition, quercetin is able to block the COX2-TBXAS-TBX A2-receptor signaling pathway by inhibition of TBX A2 formation (Garcia-Saura et al., 2005; Sheu et al., 2004).

The steroidogenic effect of quercetin was studied with MA-10 mouse Leydig cells. It was reported that quercetin increased StAR mRNA levels, StAR promoter activity and steroid hormone production (Chen et al., 2007c). The observations were corroborated by the results shown in Fig. 3., which indicated the quercetin-enhanced StAR gene transcription and translation in MA-10 cells cultured for 6 hours (Fig. 3 A and B). Following these studies, the transcription factors in StAR gene transcription were examined. It was found that the incubation of MA-10 Leydig cells with quercetin reduced DAX-1 protein, similar to the effects of other flavonoids. The reduction in DAX-1 protein was associated with a dramatic increase in StAR protein (Fig. 3C), suggesting that quercetin enhance StAR gene expression by reduction of DAX-1 expression.

Fig. 3. Effect of quercetin on StAR gene expression and steroidogenesis in MA-10 mouse Leydig cells. MA-10 cells were incubated with quercetin for 6 hours, and then collected for analyses: A, StAR protein and steroidogenesis; B, StAR gene transcription; C, Reduction in DAX-1 protein

Natural Flavonoids in StAR Gene Expression

testosterone biosynthesis.

**6. Acknowledgment**

**7. References**

expression and testosterone biosynthesis.

No.2, pp. 147-153.

mesangial cells. *Am J Physiol Cell Physiol*.

**5. Summary**

and Testosterone Biosynthesis in Leydig Cell Aging 195

(Setchell et al., 2001; Setchell et al., 2002). Specifically in the flavonoid-enhanced StAR gene expression and testosterone production, the following factors have been discussed recently: 1) Biphasic effects of flavonoids on StAR gene expression. As mentioned above, some of the flavonoids are able to block L-type calcium channels. It was reported that blocking L-type calcium channel produced biphasic effects on StAR gene expression in Leydig cells (Pandey et al., 2010). While these flavonoids might enhance StAR gene expression by reducing the transcriptional repressor DAX-1 protein, they might inhibit the positive effect of calciumdependent signaling on StAR gene expression; 2) Inhibitory effects of flavonoids on steroidogenic enzymes. It was reported that some of the flavonoids are able to inhibit activities of the enzymes involved in testosterone biosynthesis (Figueiroa et al., 2009; Hu et al., 2010). These inhibitory effects may reduce the efficacy of flavonoid-enhanced testosterone production; 3) Levels of cAMP in Leydig cells. The levels of cAMP-PKAphosphorylation are critical for the flavonoid-enhanced StAR gene expression in Leydig cells. As shown in Fig. 2C, in the absence of cAMP, flavonoid alone is not able to induce significant increases in StAR protein expression and steroid hormone production (Jana et al., 2008; Li et al., 2011). In the studies without exogenous cAMP, the endogenous cAMP becomes important for the steroidogenic effect of flavonoids. When the levels of endogenous cAMP or PKA activity in Leydig cells are not sufficient, flavonoid might not be able to induce significant increase in StAR gene expression. Therefore, minimal level of cAMP or PKA activity should be considered for the flavonoid-enhanced StAR gene expression and

In summary, LH-stimulation of Leydig cells induces both positive and negative signals in the regulation of StAR gene expression and testosterone biosynthesis. In the aging process, the increase in COX2 enhances the negative signaling, resulting in the declines in StAR gene expression and testosterone biosynthesis in Leydig cells. It was found that some of the flavonoids are able to block the COX2-dependent signaling and enhance StAR gene expression. While further investigations are needed, the studies suggest a potential for application of the natural flavonoids in delaying the age-related declines in StAR gene

Abayasekara, D.R., Band, A.M., & Cooke, B.A. (1990). Evidence for the involvement of

Alique, M., Calleros, L., Luengo, A., Griera, M., Iniguez, M.A., Punzon, C., Fresno, M.,

phospholipase A2 in the regulation of luteinizing hormone-stimulated steroidogenesis in rat testis Leydig cells. *Molecular Cellular Endocrinology,* Vol.70,

Rodriguez-Puyol, M., & Rodriguez-Puyol, D. (2011). Changes in extracellular matrix composition regulate cyclooxygenase- 2 (COX-2) expression in human

The author would like to acknowledge the support of NIH Grant AG025349.

#### **4.5 Potential application of natural flavonoids in delaying the declines in StAR gene expression and testosterone biosynthesis**

The studies reviewed above suggest that flavonoids are potential resources for alternative medicine to delay the decline in StAR gene expression. Among the various compounds of natural flavonoids, many of them are able to reduce COX2 expression, such as Kaempferol (Garcia-Mediavilla et al., 2007), tectorigenin, tectoridin (Kim et al., 1999), wogonin (Chen et al., 2008) and Silibinin (Kim et al., 2009). Some of the flavonoids reduce the inflammatory factors that induce COX2 expression (Garcia-Lafuente et al., 2009; Khanna et al., 2007; Kim et al., 2004). Another group of flavonoids was reported as receptor antagonists that inhibit the COX2-dependent signaling by blocking the TBX A2-binding to the receptors (Navarro-Nunez et al., 2009). In addition to the COX2-dependent signaling, StAR gene expression is affected by the signaling through different pathways. For example, calcium influx through L-type calcium channels regulates the threshold of cAMP-induced StAR gene expression, so that blocking the L-type calcium channels reduced the threshold and enhanced StAR gene expression in Leydig cells (Pandey et al., 2010).  The steroidogenic effects of the flavonoids in this group were described (Yu et al., 2010). It is possible to identify different flavonoids that might enhance StAR gene expression and testosterone biosynthesis through different mechanisms.

The potential for application of natural flavonoids in delaying the decline in testosterone is supported by the animal studies on the flavonoid-enhanced testosterone production and reproductive function. A study with 2-year-old male rats reported that supplementation with chrysin improved their reproductive functions, with their sperm count, fertilization potential and litter size being increased when they were allowed to interact with female rats (Dhawan et al., 2002). It was also observed that lifetime exposure to the flavonoids, including daidzein, glycitein and genistein, increased serum and testicular testosterone concentrations of rats (McVey et al., 2004). The observation was enhanced by the study on the effects of catechins on testosterone biosynthesis in rats, in which injection with catechins or its derivatives increased testosterone in blood (Yu et al., 2010). A recent study reported the beneficial effects of quercetin in diabetic rats, with their sperm viability and motility being improved by the flavonoid. These effects of quercetin are associated with an increase in serum total testosterone (Khaki et al., 2010). In addition, some flavonoids in herbal medicine are able to enhance testosterone production in rats. For example, icariin, a flavonoid in the plants in *Epimedium* family, has been extracted from traditional Chinese medicine (Du et al., 2002). It was observed that supplementation of icariin to 15-month-old male rats significantly increased their blood testosterone levels (Zhang & Yang, 2006).

For application of natural flavonoids to improve the health of aging males, further studies, especially long-term animal studies and clinical studies, are needed. Two of the important considerations in the studies are mechanism and efficacy of the flavonoid-enhanced testosterone biosynthesis. The mechanisms reported for the inhibitory effects of flavonoids on COX2-dependent signaling are mostly based on the studies with the cells from various tissues. These mechanisms need to be verified with Leydig cells. In addition to the COX2 dependent signaling, other mechanisms might be involved in the flavonoid-enhanced StAR gene expression and testosterone production. Further studies on the mechanisms may improve the understanding of the steroidogenic effects of flavonoids. Regarding the efficacy, bioavailability is an important factor being discussed in the studies on flavonoids (Setchell et al., 2001; Setchell et al., 2002). Specifically in the flavonoid-enhanced StAR gene expression and testosterone production, the following factors have been discussed recently: 1) Biphasic effects of flavonoids on StAR gene expression. As mentioned above, some of the flavonoids are able to block L-type calcium channels. It was reported that blocking L-type calcium channel produced biphasic effects on StAR gene expression in Leydig cells (Pandey et al., 2010). While these flavonoids might enhance StAR gene expression by reducing the transcriptional repressor DAX-1 protein, they might inhibit the positive effect of calciumdependent signaling on StAR gene expression; 2) Inhibitory effects of flavonoids on steroidogenic enzymes. It was reported that some of the flavonoids are able to inhibit activities of the enzymes involved in testosterone biosynthesis (Figueiroa et al., 2009; Hu et al., 2010). These inhibitory effects may reduce the efficacy of flavonoid-enhanced testosterone production; 3) Levels of cAMP in Leydig cells. The levels of cAMP-PKAphosphorylation are critical for the flavonoid-enhanced StAR gene expression in Leydig cells. As shown in Fig. 2C, in the absence of cAMP, flavonoid alone is not able to induce significant increases in StAR protein expression and steroid hormone production (Jana et al., 2008; Li et al., 2011). In the studies without exogenous cAMP, the endogenous cAMP becomes important for the steroidogenic effect of flavonoids. When the levels of endogenous cAMP or PKA activity in Leydig cells are not sufficient, flavonoid might not be able to induce significant increase in StAR gene expression. Therefore, minimal level of cAMP or PKA activity should be considered for the flavonoid-enhanced StAR gene expression and testosterone biosynthesis.


Eventually, naturalized resettlement beneficiaries may want to return to their home country. In this light, the question arises whether and how returning to the initial home country impacts US citizenship. In *Afroyim v Rusk*, the US Supreme Court interpreted Section 1 of the Fourteenth Amendment to the US Constitution as giving every citizen "*a constitutional right to remain a citizen* [...] *unless he voluntarily relinquishes that citizenship*".1265 Still, Section 349 lit a INA lists specific reasons for expatriation. These reasons were subject of the 1990 Announcement of the US Department of State. The State Department declared in its Announcement that the expatriation grounds did not entail a loss of citizenship *ex lege*.

1261 Stephen H Legomsky and David B Thronson, *Immigration Law and Policy*, 1547.

1262 See ibid 1547.

1263 See International Rescue Committee, 'How immigrants and refugees become US citizens' (3 July 2018).

1264 See Stephen H Legomsky and David B Thronson, *Immigration Law and Policy*, 1622.

1265 Ibid 1605.

Rather, all individuals would be presumed to "*intend to retain United States citizenship when they obtain naturalization in a foreign state, subscribe to routine declarations of allegiance to a foreign state, or accept non-policy level employment with a foreign government*". With that Announcement, the State Department clarified that in order to surrender US citizenship, individuals either had "*to affirm that intention in writing to a US consular officer or to formally renounce* [...]".1266 This reflects a general discomfort of the US government towards expatriation, not only in cases where it would result in statelessness.1267

### Analysis 5.4.3

Laws and practice in Europe and the US indicate that resettlement beneficiaries face various challenges to access and maintain long-term residence and citizenship status. On that basis, there are three key challenges to stimulate long-term integration *de lege ferenda*: First, the reduction of differential treatment where there is no objective and reasonable justification; second, the elimination of excessive examination requirements and/or fees; third, the reconsideration of rules on the loss of legal status.

### Temporary approach versus long-term integration <IP_ADDRESS>

It is evident from the outlined European and US resettlement policies that long-term integration approaches significantly differ among EUMS and even amongst American states. The major issue comprises the conceptualization of resettlement either as a temporary protection tool versus a long-term integration measure, or durable solution.

The issue is of political nature and can be explained as follows: Governments are seemingly more prone to justify temporary admissions towards their electorate, i.e. the receiving community, than long-term integration and naturalization of third-country nationals. This traces back to the fact that the electorate feels less threatened by foreigners when they are only temporarily admitted.1268 Furthermore, governments appear to disregard

1266 Ibid 1614.

1267 See ibid 1614f.

1268 See Jürgen Fijalkowski, 'Optionen und Spielraum europäischer Zuwanderungspolitik — Ein Essay' in Alfredo Märker and Stephan Schlothfeldt

the negative impacts of temporary protection on the lives of protection seekers who might suffer from a so-called warehouse effect. This was critically addressed by *Bruce-Jones*: 1269

*To keep newly arrived people separate from the labour economy and other facets of social citizenship and participation, is ultimately a form of warehousing. Temporary protection, whilst it arguably coaxes states to provide certain forms of relief up-front, would trap refugees into lives 'on hold'. The eventual forced return of migrants to countries of origin would threaten to break apart supportive networks and family bonds accrued in host countries, which will have, in the meantime, become home for these people*.

As some governments became aware of these negative impacts, High Commissioner *Grandi* expressed hope for a decisive shift towards a sustainable long-term approach in the course of the Global Refugee Forum in Geneva in December 2019.1270

### Economic benefits <IP_ADDRESS>

Beneficial aspects of long-term integration in the receiving country are predominantly linked to an economic rationale. In other words, from the receiving country's perspective, economic arguments speak in favor of long-term integration. The US practice relies on self-sufficiency and labor market entry, which has proven to be economically beneficial. For example, in 2016, labor force participation and employment rates of resettle-

(eds), *Was schulden wir Flüchtlingen und Migranten?: Grundlagen einer gerechten Zuwanderungspolitik* (Springer 2002) 19-44; see also e.g. call by Chancellor Angela Merkel for asylum seekers and refugees to return to their home country, Marcel Leubecher, 'Warum die meisten Flüchtlinge Merkels Appell nicht folgen werden' (*Die Welt*, 1 February 2016) <https://www.welt.de/politik/deuts chland/article151744427/Warum-die-meisten-Fluechtlinge-Merkels-Appell-nic ht-folgen-werden.html> accessed 27 March 2021.

1269 Eddie Bruce-Jones in Satvinder Singh Juss (ed), *Research Handbook on International Refugee Law*, 77.

1270 See UNHCR, 'Global Refugee Forum pledges collective action for better refugee inclusion, education, jobs' (*Press release*, 18 December 2019) <https:/ /www.unhcr.org/news/press/2019/12/5dfa56b54/global-refugee-forum-pledges -collective-action-better-refugee-inclusion.html> accessed 27 March 2021.

ment refugees in the US exceeded those of the overall US population.1271 Furthermore, a 2015 study conducted in the area of Ohio revealed that added economic value emerged from high self-employment rates among refugees.1272 Nonetheless, the short period of assistance and the related time pressure to enter the labor market deprived refugees of the opportunity to search for jobs matching their qualifications. Even though past employment rates showed that refugees were more likely to be employed than US-born people, refugees were also more likely to accept low-skilled jobs despite holding a bachelor's degree.1273

From the US experience, the following conclusion can be drawn for future EU resettlement legislation: Granting resettlement beneficiaries a more relaxed transition period would allow them to prepare for and take job opportunities according to their profile, which would in turn result in an overall more beneficial outcome – not only for the resettlement beneficiaries themselves but also for the economy of the receiving country and EU's internal market as a whole – thus creating a win-win situation. This corresponds to the previous statement of the Commission that the aim should be to enable economic productivity of migrants.1274 The Com-

1273 See Michael Fix, Kate Hooper and Jie Zong, 'How Are Refugees Faring: Integration at US and State Levels' (June 2017) 18.

1271 See Donald Kerwin, 'The US Refugee Resettlement Program – A Return to First Principles: How Refugees Help to Define, Strengthen, and Revitalize the United States' in (2018) 6 Journal on Migration and Human Security 3, 213.

1272 See US Together, Community Refugee & Immigration Services, World Relief Columbus and City of Columbus, 'Impact of Refugees in Central Ohio' (2015) <https://www.crisohio.org/wp-content/uploads/2016/04/IMPACT-OF-REF UGEES-ON-CENTRAL-OHIO\_2015-SP\_I.pdf> accessed 27 March 2021; see also Initiative of the Harvard Immigration and Refugee Clinical Program, 'Fulfilling US Commitment to Refugee Resettlement: Protecting Refugees, Preserving National Security, & Building the US Economy through Refugee Admissions' in (2017) 5 Texas A&M Law Review, 183.

1274 See Yuval Dvir, Paul Morris and Miri Yemini, 'What kind of citizenship for whom? The 'refugee crisis' and the European Union's conceptions of citizenship' in (2019) 17 Globalization, Societies and Education 2, 214. Specifically, in its 2016 Action Plan on the Integration of Third Country Nationals, the Commission made it clear that social integration had to be realized by offering migrants meaningful opportunities to participate in the economy and society of the receiving EUMS. See Commission, Communication 'Action Plan on the Integration of Third Country Nationals', COM(2016) 377 final, 5.

mission re-emphasized this goal in the 2020 Recommendation in a New Pact on Migration and Asylum.1275

#### Harmonization of permanent residence status <IP_ADDRESS>

*De lege lata*, Art 4 Long-term Residents Directive states that EUMS shall grant long-term residence status to third-country nationals, including refugees and persons eligible for subsidiary protection,1276 after five years of legal and uninterrupted stay in an EUMS. Five years are a comparatively long period given that in the US, refugees have to (or may) apply for an adjustment to LPR status already after one year. As regards consistency within EU law, the five-year requirement under the Long-term Residents Directive aligns with the requirements for EU citizens to gain permanent residency in another EUMS under Art 16 para 1 EU Citizenship Directive.

*De lege ferenda*, valid arguments speak in favor of introducing a waiting period shorter than five years for resettlement beneficiaries to become permanent residents of an EUMS. First, earlier recognition of long-term residence status corresponds to the very character of resettlement as a durable solution. Second, refugees, unlike EU citizens, do not usually have social support from their home country. Thus, they depend on the long-term residence status because this status usually implies social rights. Finally, the prospect of earlier long-term residence status can be an incentive for beneficiaries of international protection to refrain from unauthorized secondary movement. To that effect, the Commission proposed in its New Pact on Migration and Asylum to amend the Long-term Residents Directive so that beneficiaries of international protection would obtain long-term residence status after three years of legal and continuous residence instead of the usual five years.1277

Furthermore, Art 5 Long-term Residents Directive sets out additional requirements for permanent residence status, such as a stable and regular source of income, health insurance and, if so required by an EUMS, inte-

1275 See Commission, Communication on a New Pact on Migration and Asylum, 27.

1276 In 2011, the Long-term Residents Directive was amended to include persons eligible for international protection.

1277 See Commission, Communication on a New Pact on Migration and Asylum, 6.

gration measures.1278 As indicated above, the CJEU upheld the Dutch law provision requiring a civic integration examination1279 (see 5.4.1). By comparison, refugees in the US do not have to undergo examination to obtain an adjustment of their residence status to LPR. They only have to do so for naturalization. Still, there is no compelling reason for condemning the CJEU's approach to accept examination of *basic* civic and/or language knowledge as a requirement for permanent residence status, hence before naturalization – unless costs are excessive.

In terms of costs, the US fees of more than USD 1,000 for adjusting to LPR status are relatively high compared to the EU average, although the fees to apply for permanent residency significantly vary throughout the EU; by the end of 2019, costs for citizenship applications varied from less than EUR 100 (Hungary, Spain, Latvia, Estonia, Luxembourg, the Czech Republic, Portugal, and Slovenia) up to EUR 1,100 in Lithuania – the fees in the former EUMS UK even amounted to EUR 1,345.1280 Given these variations among EUMS, the question of EU harmonization arises. Such harmonization must be reflected in light of the principle of subsidiarity. Is the EU really in a better position than the EUMS to determine these costs than EUMS? Very strong arguments against EU regulation are the differences in social assistance, living costs, and the general economic situations in the EUMS. The EUMS themselves are arguably in the best position to determine the application costs according to these specifics.

To conclude, valid policy arguments speak in favor of (i) shortening the waiting period for resettlement beneficiaries to become permanent residents of an EUMS to less than five years; and (ii) harmonizing the requirements to obtain long-term residence status through future EU resettlement legislation. Concerning the latter, the CJEU has already established that there are limits for national particularities. Accordingly, examination must be kept to a basic level, must not involve excessive costs and must account for the individual situation of the applicant.

1278 Art 5 para 2 Long-term Residents Directive states: "*Member States may require third-country nationals to comply with integration conditions, in accordance with national law*", Koen Lenaerts and Piet van Nuffel, *European Union Law*, 325, para 10-014.

1279 See Case C-257/17 *C, A v Staatssecretaris van Veiligheid en Justitie*, paras 63f.

1280 See Andrew Henderson, '5 Most Expensive Residency Applications in Europe' (last updated 26 December 2019) <https://nomadcapitalist.com/2017/11/06/mo st-expensive-residency-applications/> accessed 27 July 2022.

### Naturalization <IP_ADDRESS>

In the ideal case, the long-term residence status of a resettlement beneficiary ends with his or her naturalization in the receiving country. As highlighted above, international refugee law, namely Art 34 Refugee Convention, instructs states to facilitate access to citizenship for refugees. The refugees' special need for access to citizenship is rooted in the very definition of a refugee as being unable or unwilling to avail him or herself of the protection of his or her home country.

It follows from *Owen*'s findings that in general, EUMS facilitate the access to citizenship for refugees compared to other third-country nationals. Nevertheless, they follow different, i.e. more or less restrictive policies. In this light, the CJEU clarified that national authorities must conduct individual assessments of the implications of denial of EU citizenship rights when deciding upon national citizenships.

Similar to most EUMS, the US prioritizes the naturalization of refugees in comparison to other immigrants. A 2018 study on naturalization rates assessing the full population of refugees resettled in the US between 2000 and 2010 showed that resettlement refugees in the US were significantly more likely to acquire citizenship than immigrants entering from other programs.1281 In general, statistics disclosed that resettlement refugees had a relatively high naturalization rate compared to other immigrants to the US, which reflects a less restrictive citizenship policy towards refugees. For instance, resettled refugees benefit from a shorter waiting period: Unlike for other immigrants, already their first year in the US, before adjustment to LPR status, counts.1282

### Re-resettlement <IP_ADDRESS>

Naturalization impacts the freedom of movement, i.e. the right to leave and enter a country. The freedom of movement not only becomes an issue when an individual wishes to leave the country of (first) refuge in order to be resettled to a receiving country, but also *vice versa*, i.e. in case the naturalized resettled individual wishes to return to his or her initial home

1281 See Nadwa Mossaad et al, 'Determinants of refugee naturalization in the United States' in (11 September 2018) 115 PNAS 37, 9178.

1282 See Stephen H Legomsky and David B Thronson, *Immigration Law and Policy*, 1546.

country. The relevant question in terms of the latter is: To what extent can a receiving country justifiably interfere with the right to leave of resettled and naturalized individuals?

Art 12 para 2 ICCPR determines that any country, including the "*own country*", must grant a right to leave. After and arguably before naturalization, a receiving country can be considered as one's "*own country*". This holds particularly true where the respective resettlement beneficiary has established special ties to that country.1283 If, notwithstanding such special ties to the receiving country, the (naturalized) resettlement beneficiary wants to return to his or her initial home country, he or she is likely to contravene the interests of the former. Especially, the receiving country as the new "*own country*" may have an interest in restricting a resettled naturalized individual's right to leave, because it has invested in his or her long-term integration. Since the right to leave is not absolute, the receiving country may restrict it under certain conditions and in line with the principle of proportionality (see 3.3.2). Under Art 12 para 2 ICCPR, such restrictions can only be based on the grounds of national security, public order, public health or morals, or the rights and freedoms of others. The mere reason that the receiving country "*invested*" in the integration of resettlement beneficiaries and has an interest to keep them as contributors to its economy and society would therefore not be sufficient.

Unjustified derogations from the right to leave may, amongst others, be induced through the expatriation policy of the country of new citizenship. The US constitutes a liberal example in this regard. From an EU law perspective, the above-mentioned CJEU case law makes it clear that the potential loss of national citizenship of an EUMS needs to be assessed in the light of EU citizenship, and with due consideration of risks for the safety and liberty of the individual concerned.

Finally, not all resettlement beneficiaries actually pursue the goal of naturalization. For instance, Palestinians actively denied resettlement offers,

1283 See Rutsel Martha and Stephen Bailey, 'The right to enter his or her own country' (*EJIL: Talk!*, 23 June 2020) <https://www.ejiltalk.org/the-right-to-en ter-his-or-her-own-country/> accessed 27 March 2021; "*The scope of "his own country" is broader than the concept 'country of his nationality'. It is not limited to nationality in a formal sense, that is, nationality acquired at birth or by conferral; it embraces, at the very least, an individual who, because of his or her special ties to or claims in relation to a given country, cannot be considered to be a mere alien*", OHCHR, 'General Comment No 27: Article 12 (Freedom of Movement)', UN Doc CCPR/C/21/Rev1/Add9 (2 November 1999) para 20.

invoking their collective rights as people because they feared never being able to return to their initial home country.1284

### Preliminary conclusion 5.4.4

Economic arguments speak in favor of a policy approach that preserves the originally intended long-term integration character of resettlement. A future policy that introduces clear and harmonized requirements for long-term residence status as well as naturalization would help to make resettlement beneficiaries contribute to the local communities. Moreover, harmonization likely decreases discriminatory practice, which is necessary to foster compliance with international law. Notwithstanding, from an EU law perspective, subsidiarity considerations have to be taken into account, especially when it comes to the regulation of specific requirements such as the determination of costs or the content of examinations. Eventually, even naturalized resettlement beneficiaries may want to leave their new home country to return to their prior home country. In such situations, the receiving country as the new home country must not restrict the right to leave of the resettled refugee unless on the basis of legitimate grounds and in line with the principle of proportionality.

1284 See Anne Irfan, 'Rejecting resettlement: the case of Palestinians' in (2017) 54 Forced Migration Review, 68 (70f).

### Conclusion 6

The second Chapter of this monography elaborated on the concept of resettlement and revealed that resettlement constitutes a means of responsibility sharing. Yet, a binding international resettlement mechanism has not evolved. Also, at the EU level, a binding obligation to conduct resettlement does not exist and is not provided for in the Proposal for a Union Resettlement Framework Regulation.

There is no binding definition of resettlement under international law. Despite conceptualization efforts of the UNHCR, its resettlement definition has not reached the status of costumery international law. The Commission has defined resettlement at the EU level. Only lately, in the 2021 AMIF regulation, an explicit reference to resettlement as durable solution can be found. The US understanding of resettlement reflects the idea of providing a durable solution to refugees. The firm resettlement bar takes account of the conditions in the country of (first) refuge and makes eligibility for resettlement dependent on whether firm resettlement has already been provided or is accessible in that country.

History confirms that resettlement has been a vital tool in various contexts where countries of (first) refuge were unable to offer firm resettlement to refugees (e.g. Austria in the course of the influx of Hungarian refugees and Thailand with regard to Vietnamese refugees). It also derives from history that global resettlement efforts have depended on the willingness of prospective receiving countries to resettle. Indeed, US and European policy-makers have pursued similar motives when engaging in resettlement. Besides the humanitarian purpose of resettlement, foreign policy, security and economic interests have impacted their decisions. In this light, critics pointed to the discrepancy between a humanitarian measure and a migration control mechanism. When resettlement entails controlling (the entry of) people and discriminating against particular groups, it not only departs from its humanitarian nature, but also entails potential human rights violations. It is up to the states as the main actors in the resettlement process as well as the UNHCR and other non-state actors to uphold the humanitarian nature of resettlement. This predominantly applies to states facing certain responsibilities under international law towards resettlement beneficiaries.

The first major legal question underlying the third Chapter of this monography referred to the international obligations that must be respected in the course of (EU) resettlement. When states engage in resettlement, they must consider obligations under universal and regional human rights treaties as well as the Refugee Convention and its Protocol. With regards to universal human rights treaties and the ECHR, the analysis revealed that the application of these treaties can exceptionally be triggered by the exercises of jurisdiction in the course of targeted extraterritorial actions during selection missions, namely when a receiving country acts through its state officials and implements its resettlement policy on foreign territory. The basis of the jurisdictional link thereby lies in the control over the targeted actions of policy implementation, and is established only with regards to those rights affected by the specific actions in furtherance of the respective policy and/or application of the domestic law of the receiving country.

As opposed to human rights treaties, most rights under the Refugee Convention are territorially limited and even require a certain level of attachment with the receiving country. Nonetheless, Art 3 (non-discrimination) and Art 33 (*non-refoulement*) Refugee Convention apply extraterritorially if the threshold to establish extraterritorial jurisdiction as applied in human rights treaties is met.

If jurisdiction is established, substantive rights under the respective treaties must be granted to resettlement beneficiaries. First, when receiving countries select resettlement beneficiaries on foreign territory, they may face extraterritorial *non-refoulement* obligations arising from implicit and explicit *non-refoulement* provisions under the CAT, ICCPR, CRC, ECHR and the Refugee Convention. Yet, effective control over *non-refoulement*  rights in the course of resettlement selection will only be established in exceptional cases and the *non-refoulement* obligations are primarily left to the state on whose territory resettlement beneficiaries are located. Moreover, resettlement beneficiaries must be granted the right to leave, meaning that migration control policies of receiving countries implemented by countries of (first) refuge must not unjustifiably interfere with such right. In addition, potential resettlement beneficiaries have a right to review a negative selection decision when there is an arguable claim of violation of a right under the ICCPR and/or the ECHR. Besides, the Refugee Convention guarantees any refugee access to courts in all Contracting States. However, refugees are limited to rely on judicial remedies in the respective domestic law. Furthermore, international human rights law offers refugees protection in cases where they are discriminated against other refugees, e.g. in the resettlement selection process or with regards to the legal status in the receiving country. However, when it comes to issues of discrimination between refugees and nationals of the receiving country, there is no comprehensive protection under international human rights law. Also, the Refugee Convention does not account for equal treatment between refugees and nationals in a comprehensive manner since only a few rights in this Convention postulate such treatment. Upon arrival on a receiving country's territory, reception conditions must correspond to the rights and freedoms guaranteed under general international and European human rights law as well as the Refugee Convention. Ultimately, receiving countries may not arbitrarily refuse naturalization of a resettled refugee.

When the actors involved in the resettlement process violate the outlined obligations under international law, the ARSIWA and the ARIO provide rules for the attribution of responsibility. The assessment showed that in the context of the resettlement process, certain requirements for attribution, especially the knowledge threshold for receiving countries to establish derivative responsibility due to aid or assistance, are difficult to prove.

The identified pertinent international obligations are also relevant under EU law. In this light, Chapter 4 dealt with the EU legal framework of resettlement. So far, EUMS have engaged in resettlement on the basis of discretionary choices. Still, Art 78 para 1 TFEU requires that EU resettlement legislation must be developed and interpreted in conformity with international refugee law as well as with pertinent international and European human rights. In addition, the principle of solidarity and responsibility sharing (Art 80 TFEU) as well as the principle of consistency (Art 21 para 3 TEU and Art 7 TFEU) apply when regulating and implementing EU resettlement. The relevant rules of EU competence to adopt legislation on resettlement are Art 78 para 2 lit d and lit g TFEU. Art 78 para 2 lit d TFEU allows for the establishment of procedural rules on the resettlement process, including extraterritorial processing. However, this Article does not cover procedures on centralized EU assessment. Art 78 para 2 lit g TFEU can be used in a complementary manner for third-country support to ensure the effective application of international protection obligations.

From an institutional perspective, the EU has financially supported resettlement operations of EUMS through the AMIF. Besides, the EU has provided operational support through the EUAA and its predecessor EA-SO. The expanded mandate of the EUAA includes binding decision-making power, which can also be relevant for resettlement selection decisions. Conferring such power to the EUAA is covered by the criteria set out in the *Meroni* judgement. While former EASO's advisory opinions lacked means of review, binding decisions of the EUAA can be subject to review by the CJEU under Art 263 TFEU. Moreover, the empowerment of the EUAA has introduced a shift from assisted processing to common processing.

Eventually, the Proposal for a Union Resettlement Framework Regulation reflects the strive for a permanent resettlement framework. However, similar to the EU-Turkey Statement, the Proposal shows inconsistencies regarding the principle of non-discrimination as well as potential violations of Art 31 Refugee Convention (when penalizing those who tried to enter the EU irregularly by excluding them from resettlement).

In the long term, the arguments in favor of a permanent EU resettlement framework prevail to ensure continued resettlement contributions in compliance with international law. Whether such EU framework will set out a mandatory resettlement quota remains a highly political question. The concept of flexible solidarity has been proposed as a way out of the current political deadlock on the issue of mandatory quotas. In the end, flexible solidarity does not relieve EUMS from obligations under international human rights and refugee law.

Lastly, the comparative analysis of European and US resettlement practices identified the following legal issues throughout the resettlement process that demand contemplation *de lege ferenda*. Starting with the pre-selection of potential resettlement beneficiaries, EUMS and the US rely on the UNHCR as major referral entity. In addition to ordinary UNHCR pre-selection, a future increase of cooperation with NGOs as referral entities as well as the involvement of the EUAA and civil society could open up resources for a more comprehensive and diversified case identification system. Furthermore, the harmonization of EUMS national selection practices would enable more comparable and consistent (high) procedural standards.

Concerning the scope of resettlement beneficiaries, individuals who do not qualify as Convention refugees, in particular people fleeing from war, may equally be in need for resettlement. To that effect, relying on the category of 'forced migrants' in the future scope of resettlement beneficiaries would include "*any individual who, owing to the risk of serious harm, is compelled to leave or unable to return to her or his country of origin*".1285 In addition, IDPs, who have not been able to leave their country of origin, are relevant target groups *de lege ferenda*. So far, the US, as opposed to most

1285 Kiran Banerjee, 'Rethinking the Global Governance of International Protection' in (2018) 56 Columbia Journal of Transnational Law, 319.

EUMS, has considered IDPs for resettlement, but the groups of eligible IDPs have not been determined based on vulnerability and protection needs. If future EU legislation followed the US approach granting resettlement to IDPs, the eligibility of IDPs for resettlement to the EU would have to be based on objective criteria to avoid interference with the principle of non-discrimination under international law. Precisely, the criterion of integration potential as applied by some EUMS is problematic because it lacks objectivity and fails to account for actual humanitarian needs. It constitutes an additional requirement that has no basis in the Refugee Convention and thus could be a source of discrimination between and amongst (groups of) refugees.

For selection decisions, the comparative analysis found that instruments of appeal are scarce. This contradicts international law, where the ICCPR and the ECHR require effective means to appeal decisions when there is an arguable claim of violation of rights under the respective Treaty. Moreover, the right to good administration as stipulated in Art 41 Charter entails that potential resettlement beneficiaries must be granted the right to be heard.

Regarding pre-departure orientation programs, the comparison revealed divergent practices. Future EU regulation could contribute to evening out existing shortfalls and inequalities not only by imposing comparably high standards of pre-departure orientation, but also by ensuring that orientation programs are equally accessible to all refugees. Considerations *de lege ferenda* also need to account for continuity between pre-departure and post-arrival assistance because this kind of practice has already proven successful.

Furthermore, a lesson can be learned from the US public-private partnerships with voluntary agencies, the Volags. With their experience and network, Volags are particularly well suited to match refugee profiles with conditions in the receiving communities. Besides, Volags have supported resettlement beneficiaries arriving in the US in achieving self-sufficiency. The US focus on fast self-sufficiency and labor market entry, combined with meaningful time limits for assistance deserves to be taken into consideration when drafting future EU resettlement legislation. What is more, references in the Refugee Convention and in EU law indicate that the will of resettlement beneficiaries has legal weight in the placement process.

In addition, the analysis of the US' centralized resettlement approach pointed to shortfalls in information sharing and funding. Even if a fully centralized approach is not covered by the current EU Constitutional Framework, similar shortfalls can be avoided *de lege ferenda* in EU resettlement legislation by enhancing information sharing with the local communities as well as ensuring proactive and tailor-made funding to respond to local needs. In particular, direct EU-level funding for those municipalities willing to admit individuals in need for resettlement is necessary to enable efficient use of open capacities.

Moreover, future EU resettlement should be designed in a way to foster EUMS compliance with their obligations (*de lege lata*) concerning reception conditions upon arrival. In addition, sources of unequal treatment among and between (groups of) refugees, particularly in terms of their legal status, need to be addressed *de lege ferenda* in order to avoid violations of non-discrimination obligations under international human rights law.

Facilitated access to long-term residence status corresponds to resettlement's character as a durable solution. The failure of support from their home countries makes refugees so dependent on long-term residence status. Thus, the recommendation *de lege ferenda* consists of reducing disproportionate hurdles to access long-term residence status by harmonizing the requirements for such status in the EU within the limits established by CJEU case law, i.e. keeping examination to a basic level, refraining from excessive examination costs and accounting for the individual situation of the applicant. Still, EUMS must be afforded the opportunity to have their national values reflected in the content of language and civic integration tests.

Concerning the access to citizenship, CJEU case law determines that EUMS authorities must assess the implications of EU citizenship on the individual applicant when deciding on the granting or refusing of national citizenship of resettlement beneficiaries.

Finally, successful integration and naturalization in the receiving country imply that this country becomes the "*own country*" of a resettlement beneficiary. Ideally, the interests of the resettlement beneficiary and the receiving country become blended during the integration process. On the one hand, the resettlement beneficiary should achieve a durable solution and self-sufficiency, and on the other hand, this country gains interest in keeping the resettlement beneficiary, as he or she has become a beneficial contributor to the receiving economy and society.

Ultimately, it seems too idealistic to assume that increased EU regulatory involvement in the field of resettlement will entirely eliminate human rights abuses. Likely, there will be opposing EUMS preferring to build a fortress instead of setting a sign of solidarity with third countries and protection seekers. Nevertheless, within the scope of its competences, the

### *6 Conclusion*

EU has regulatory power1286 to foster human rights compliance in EU resettlement, a previously legally grey area. Supposing the claim that the EU "*affects the lives of many people in ways they perceive as profoundly unjust*" 1287 contains a grain of truth, the EU now has the chance to prove the opposite by developing an EU resettlement policy that positively affects the lives of people within and beyond its external borders.

1286 See Anu Bredford, *The Brussels Effect: How the European Union Rules the World*  (Oxford University Press 2020) 288.

1287 Dimitry Kochenov, Gráinne de Búrca and Andrew Williams, 'How just is the EU, or: is there a 'new' European deficit?' (*Verfassungsblog*, 10 June 2015) <https://verfassungsblog.de/how-just-is-the-eu-or-is-there-a-new-european-defici t-2/> accessed 27 March 2021.

## General

### Europe

US

Danilo ZAK, 'Bill Summary: The Afghan Afjustment Act' (*National Immigration Forum*, 11 August 2022) <https://immigrationforum.org/article/bill -summary-the-afghan-adjustment-act/> accessed 3 September 2022

### Cases

## ICJ

### ECtHR and European Commission on Human Rights

*Fábián v Hungary* App No 78117/13 (ECtHR 5 September 2017)

*Petropavlovskis v Latvia* App No 44230/06 (ECtHR 13 January 2015)

*Tarakhel v Switzerland* App No 29217/12 (ECtHR 4 November 2014)

*Al-Skeini and Others v United Kingdom* App No 55721/07 (ECtHR 7 July 2011)

*Al-Jedda v the United Kingdom* App No 27021/08 (ECtHR 7 July 2011)

# CJEU

Case C‑432/20*, Landeshauptmann von Wien* [2022] EU:C:2022:39

Joined Cases C-391/16, C-77/17 and C-78/17 *M v Ministerstvo vnitra, X and X v Commissaire général aux réfugiés et aux apatrides* [2019] EU:C:2019:403

Case C-257/17 *C, A v Staatssecretaris van Veiligheid en Justitie* [2018] EU:C:2018:876

### US Supreme Court

*California et al v Texas et al* 593 US \_\_ (2021)

*Acting Commissioner, Immigration and Naturalization Service, et al v Haitian Centers Council, Inc* 509 US 155 (1993)

### Other courts

Legal texts and International Treaties

## EU secondary legislation

US legislation


**7. Conclusion**

Ever-increasing population has paved the way to agricultural land depletion which needs to be controlled by adopting sustainable crop production practices. Direct incorporation of crop residues into agricultural soil conserves soil nutrients as well as organic carbon contentbut initiates considerable crop management problems by delayingthe decomposition process. Conversion of crop residues to biochar by thermo-chemical process (slow pyrolysis) is an attractive, economical alternative approach for effective management and disposal of these excess crop residues, whichotherwise are being used inefficiently. Addition of biochar to soil is one of the best practices to overcome any biotic/abiotic stress caused, such as heavy metal toxicity, soil acidity, nutrient unavailability etc. and to increase the crop productivity. From the agriculture point of view, application of biochar as a soil conditioner generates numerous benefits, such as improvement of the physical, chemical and biological properties of soils, and this in turn contributes to an increased crop yield. Owing to their physicochemical properties, biochar can be used for soil carbon sequestration, reduction of the bioavailability of contaminants affecting living organisms as well as for water treatment. The persistence of biochar effects on soil processes and mechanisms remains to be resolved under realistic field conditions. Therefore, it is recommended to use biochar as a soil amendment for enhancing soil health and environmental condition as well as long-term carbon sequestration.

*Biochar: A Sustainable Approach for Improving Soil Health and Environment DOI: http://dx.doi.org/10.5772/intechopen.97136*


**Appendix A**

Based on the proposed iterative method, the iterative constants for the martensite phase with stress and strain conditions are displayed in Tables A1 and A2, respectively.

**Table A1.** Constants *am*, *bm*, and *R*2 *m* (m = 1, 2, 3, 4) for the martensite phase of linear equation with stress condition by the proposed iterative method.

**Table A2.** Constants *am*, *bm*, and *R*2 *m* (m = 1, 2, 3, 4) for the martensite phase of linear equation with strain condition by the proposed iterative method.

**Table A2.** *Cont.*


**2.2 Bryan Cervical Disc Prosthesis**

Bryan Cervical Disc Prosthesis (Medtronic Sofamor Danek, Memphis, Tennessee) consists of a nucleus made of polyurethane between two titanium alloy endplates in a clamshell configuration. (Figure 2) There are two bearing surfaces in the arthroplasty at the interfaces between the nucleus and the endplates. A polyurethane sheath attaches to the endplates and surrounds the nucleus. Sterile saline is injected between the outer sheath and the nucleus as lubricant. The endplates have a titanium porous coating for bone ingrowth and a small flange anteriorly to prevent posterior migration.

(Courtesy of Medtronic Sofamor Danek, Memphis, Tennessee)

Fig. 2. Bryan Cervical Disc Prosthesis


*2.7. Minimum Inhibitory Concentration (MIC) and Minimum Bactericide Concentration (MBC) Assays*

Minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) were determined according to the method described by Elshikh et al. with minor adjustment [25]. Briefly, 100 μL of MH broths were added into 96-well plate and then another 100 μL of dissolved samples, whose initial concentrations were 100 mg/mL, were added in first wells. Serial two-fold dilutions were made with final concentrations ranging from 50 mg/mL to 0.39 mg/mL. Afterwards, 100 μL of the standardized bacteria suspensions (1 <sup>×</sup> 106 CFU/mL) was added to each test well, so that the final

volume in each well was 200 μL. Plates were incubated at 37 ◦C for 24 h. After incubation, 30 μL of the freshly prepared resazurin (0.015%) was added to all test wells, and further incubated for 2 h to allow the viable microorganism to metabolize the blue resazurin dye into pink resorufin. MIC was defined as the concentration at which the corresponding well showed no color change. Afterwards, the contents of wells with concentrations equal or higher than MIC values were directly incubated onto the MH plate, and the lowest concentration at which there was no colony growth was defined as MBC.


**Section 1**

**Fuel Properties as Factors Affecting Injection Process and Systems**

4 Fuel Injection in Automotive Engineering

as IV generation injectors) provides the possibility of application of variable injection rate and fuel injection pressure around 250 MPa. The introduction of such injector is signalized

The automotive market, throughout the last few years, is subject to conspicuous changes in the scope of clients' preferences. The sales of diesel engine cars gradually grow. Compared to the engine with spark-ignition, the diesel engine has a higher efficiency, lower fuel consumption and longer operation life. A diesel engine is much more easily adaptable to supercharging, which enables obtaining significant power gain without changing the stroke, displacement volume or engine speed. In a diesel engine that works with higher air-to-fuel ratio, the combustion is more efficient with lower carbon and hydrocarbons content in the exhaust gases. From the end user's point of view, the most important effect is the operation cost economy as the diesel engines consume less fuel. To meet the present and future demands, the automotive manufacturers work over new designs and technologies in diesel engines and offer a wide range of car models fitted with such drive units. The development of high-speed diesel engines is explicitly guided by the direct injection strategy and growth of the fuel injection pressure, exceeding 200 MPa. Such tendency forced intensive research and development works over new generations of injection systems. They must provide such parameters of mixture and the rate of the combustion process that would be able to meet the future requirements related to pollutant emissions in the exhaust gases and reduction of CO2. These are the basic criteria determining the trends in the development and improvement of high-speed diesel engines and their injection systems but, in other hand, they signalize the scale of issues that the engineers and manufacturers of such devices must face. The classic injection systems cannot provide the proper rate of the injection process. Fulfilling the future requirements related to exhaust emissions and noise reduction combined with the fuel economy increase requires the absolute application of electronic control systems. This, in turn, is conditioned by the introduction of extended injection rate algorithms that may only be described by complicated 3D functional surfaces. The algorithms must also take into account the additional control parameters and functions and

by Bosch (so-called HADI system – Hydraulically Amplified Diesel Injector).

everything requires the strict application of adjusted values feedbacks.

directions of the injection systems remains still an open question.

Undoubtedly, the present state-of-the-art accumulation fuel systems provide great features and control precision of the injection process, but this advantage alone would not be able to meet further demands for green engines; it seems that development of direct injection strategy is closed to the systematic limitations. It is necessary to combine various progression trends, both in design and technology domains. As anticipated, the combustion engine shall be the basic drive source in the next 30-40 years, so the issue of development

**4. Conclusion**

**1**

M. P. Ashok

*India*

**Role of Emulsified Fuel in**

**for Fuel Injection System**

*Department of Mechanical Engineering, FEAT,*

 *Annamalai University, Annamalai Nagar, Tamil Nadu,*

**the Present IC Engines – Need of Alodine EC**

The present environment gets polluted day by day, due to the increase in industries, automobiles, etc., Eventhough, government has given strict rules and regulations against the environment pollution, practically there are lot of problems to implement the same by the users and production units, which leads to the result of global warming, acid rain, damaging of ozone layer affecting the green house effect and human health, etc. The pathetic information is that, the harmful pollutant does not allow the children also from

Due to the depletion of fossil fuel and its dangerous harmful emissions, the entire universe has given much attention for identifying an alternate fuel for the current existing engine. Also the fuel to be identified must have the property of fuel to the current existing engine and much free from the harmful pollution, emitted by IC (Internal Combustion) engine.

Present days emulsified fuel is much familiar for the IC Engines. Particularly ethanol based emulsion process fuels are much familiar, due to the depletion of fossil fuels. Entire universe has given much attention & many research works are going on based on the alternate fuel to

Based on that, emulsification technique is one of the possible approaches to identify an alternate fuel. Particularly /presently, the bio fuel from ethanol addition to the fossil fuels, play a vital role to run the current existing IC Engine, with best performance and fewer

Presently the maximum blending of 50% Ethanol to 50% Diesel (50E: 50D), research work has been carried out by M.P.Ashok, academician, researcher and scientist {(in the field of

Based on the test result of Phase-I, it is possible to run the engine at 50% blending of ethanol, in the normal fuel injection system for a single cylinder, DI (Direct Injection) oriented engine.

Water-in-Oil) W/O–Emulsion} at Annamalai University, India, during the year 2007.

Based on that, many research works have been carried out.

**1. Introduction**

their birth itself.

the IC engines.

harmful emissions.

**Ethanol Corrosion Resistant Coating**