**2. The relevance of duplicated GGAA-motifs in the 5′-upstream regions of human genes to the regulation of biological events**

#### **2.1. The transcription factors that recognize and bind to the GGAA-containing motifs**

The most widely known transcription factors (TFs) that selectively recognize the GGAA-corecontaining sequences are the ETS (E26 transformation specific) family proteins, which consist of at least 27 members [10, 11]. Moreover, a genome-wide ChIP-seq analysis estimated that the promoter regions of human genes are very frequently occupied by ETS family or GGAA-binding proteins [12]. The duplication of the GGAA-motif could be advantageous to organisms, as it would allow the transcription of various genes to be controlled in a manner that is mainly dependent on the expression profile of the GGAA-binding proteins in the cells [13, 14]. Besides ETS family proteins, several TFs could bind to the motif. For example, some of the duplicated GGAA-motifs would be identical to IFN-stimulated response element (ISRE), the consensus sequence of which is 5′-GGAAANNGAAACT-3′ [15], if one of the Ns was G. The double-stranded sequence, 5′-AACTTT-3′, which is a core binding motif of the IRF1 [16], could be generated if CT was inserted between GGAA and TTCC. Moreover, NF-κB p65 (RELA) homodimer binds to two symmetric sequences, 5′-GGAATTTCC-3′ and 5′-GGAATTCCC-3′ [17]. IRF8 (ICSBP) either positively or negatively regulates transcription through binding to ISRE [18, 19].

Importantly, the ETS family proteins and other TFs cooperatively regulate the expression of various genes. For instance, STAT1 plays a role in the regulation of the expression of interferon (IFN) stimulated genes (ISGs) with ETS family proteins [20, 21]. Sp1 and Ets1 interact with each other to regulate mouse *Npr1* gene expression [22]. The ETS-binding consensus sequence is frequently found with a second ETS-binding sequence and with the Sp1-binding sequence, but not with a TATA element [23], implying the exclusive role of the GGAA- and TATA elements. The human *VEGFR1* promoter region, which contains overlapping GGAA-motifs, is also regulated by CREB and EGR-binding elements [24]. Furthermore, the mouse *Ppp2r1a* gene promoter, which carries duplicated TTCC motifs, is regulated by Creb, Ets1, Ap2 alpha, and Sp1proteins [25]. Thus, multiple elements adjacent to duplicated GGAA-motifs may recruit various TFs to form a pre-initiation complex on the transcription start site (TSS) of TATA-less promoters. The transcription initiation system might be advantageous for a rapid response to stresses in a TATA-independent manner.


*2.3.3 Electrolyte*

ILs have also been used as solvents in extraction processes or as electrolytes. Polymer electrolytes comprising IL [emim][Tf2N] and soluble SPI showed a high ionic conductivity and reliable mechanical strength, suitable for HPP actuators [106]. Gel polymer electrolytes (GPEs) [107–109] and solid polymer electrolytes [110–114] have been widely reported. A series of GPEs were prepared using the electrospun membranes of poly(vinylidene fluoride-cohexafluoropropylene) [P(VdF-co-HFP)] incorporating ILs, 1-alkyl-3-methylimidazolium bis(trifluoromethylsulphonyl)imide in the presence of lithium bis(trifluoromethylsulphonyl)imide (LiTf2N) [115]. An IL-GPE containing semicrystal PVDF, amorphous polyvinyl acetate (PVAc) and ionic conductive [bmim] [BF4] was prepared via the solution-casting method for solid supercapacitors [116]. A PVDF-HFP/PMMA-blended microporous gel polymer electrolyte incorporating [bmim][BF4] was fabricated for lithium-ion batteries [117]. Solid polymer electrolytes using poly(vinylidene-fluoridetrifluoroethylene) and N,N,N-trimethyl-N-(2-hydroxyethyl) ammonium bis(trifluoromethylsulphonyl)imide ([N1112(OH)] [Tf2N]) IL were fabricated for energy storage applications [118]. PI/IL composite membranes for fuel cells operating at high temperatures were prepared by impregnating a porous Matrimid® membrane with protic ILs: 1-*n*-methyl-imidazolium dibutylphosphate ([C1im][DBP]), 1-*n*-butylimidazolium dibutyl-phosphate ([C4im][DBP]) and 1-*n*-butylimidazolium bis(2-ethylhexyl)phosphate ([C4im] [BEHP]). The electrolyte membranes were used as a proton-exchange membrane fuel cell (PEMFC) [119]. An IL-polymer electrolyte film based on a low-viscosity IL (1-ethyl-3-methylimidazolium dicyanamide) incorporated into a polymer matrix

**111**

**Figure 9.**

*HFP polymer electrolyte film (maximum conductivity).*

*Progress in Ionic Liquids as Reaction Media, Monomers and Additives in High-Performance…*

**Figure 9** [120]. Quasi-solid-state electrolytes (QSEs) consisting of IL-LiTf2Nfumed silica nanoparticles were prepared for use in bulk-type all-solid-state cell

(PVDF-HFP) was prepared, exhibiting liquid-like conductivity, and the maximum

Carbon nanotubes (CNTs) consist of rolled-up graphene sheets and can be used as electronic, conductive and reinforcing fillers for polymer composites. MWCNTs possess a nanoscale diameter, a high aspect ratio, excellent mechanical properties, and good electrical conductivity. In recent years, MWCNTs have gained considerable interests of scientists and engineers, especially in polymer composites containing HPPs and ILs. A PI composite film comprising finely IL-dispersed MWCNTs in IL was obtained with a high shielding effectiveness (SE) for use in the packaging of a 2.5-Gbps plastic transceiver module with numerous applications in fibre to the home lightwave transmission systems [122]. PEI NCs consisting of bucky gels of industrial-grade MWCNTs and [bmim][PF6] were prepared; they are suitable for the aerospace and electronics industries, as shown in **Figure 10** [123]. Some PI and PEI NCs consisting of MWCNTs and polymerised ILs (PILs) were prepared, exhibiting differential function [124, 125]. The crystal structure of PVDF was modified by utilising the long alkyl chains of [C16mim][Br] and IL-modified MWCNTs, and the crystallisation kinetics of the composites was investigated [126, 127]. A series of PVDF composites with 'bucky gels' of MWNTs and ILs were obtained by simple melt compounding. According to the DSC and XRD results, the addition of ILs in the composites changed the crystallinity and crystal form of the PVDF [128]. PTFT and PVDF as the components of NCs containing HPP and ILs have received increasingly more attention. The nanomaterials with a nanoscale structure were prepared using pyridinium, imidazolium and phosphonium ILs as new synthetic building blocks in a PTFT. The cation-anion combination and functionalisation of ILs affect the ionic networks and nanostructures of materials [129, 130]. These nanomaterials show optimised thermal and mechanical properties and have numerous potential applications such as supercritical CO2 [131]. PVDF/IL/GraNCs were fabricated via the solution casting of PVDF with graphene (Gra) modified with a long alkyl chain IL [C16mim][Br], exhibiting a low loss tangent and low conductivity in the PVDF/ionic liquid-modified graphene

*Photograph of (a) assemble DSSC and (b) I-V characteristics of DSSC comprising IL-incorporated PVDF-*

S/cm, as shown in

*DOI: http://dx.doi.org/10.5772/intechopen.86472*

*2.3.4 Nanocomposite*

conductivity of PVDF-HFP + 25 wt% IL was as high as 10−3

configuration lithium-sulphur rechargeable batteries [121].

## *Progress in Ionic Liquids as Reaction Media, Monomers and Additives in High-Performance… DOI: http://dx.doi.org/10.5772/intechopen.86472*

(PVDF-HFP) was prepared, exhibiting liquid-like conductivity, and the maximum conductivity of PVDF-HFP + 25 wt% IL was as high as 10−3 S/cm, as shown in **Figure 9** [120]. Quasi-solid-state electrolytes (QSEs) consisting of IL-LiTf2Nfumed silica nanoparticles were prepared for use in bulk-type all-solid-state cell configuration lithium-sulphur rechargeable batteries [121].

## *2.3.4 Nanocomposite*

*Solvents, Ionic Liquids and Solvent Effects*

[bmim][PF6] were also obtained by spraying a suspension dispersion with an encapsulation capacity of 29% [102]. PSF microcapsules have practical use such as the removal of caprolactam from water [103]. PEEK microcapsules containing trihexyl(tetradecyl) phosphonium chloride IL was obtained in *N*,*N*-dimethylformamide (DMF) as the dispersing phase and dodecane [104]. PTFE microcapsules containing [hmim][Tf2N]

*Optical microscopic images and composition of organic phase is PSF: dichloromethane (DCM): [bmim] PF6 = 5 g: 80 ml: 3 g; continuous phase is 0.1 wt% gelatin solution; inner size of nozzle: 0.6 mm, IL microcapsules, flow rate of continuous phases (CP): 30 ml/min; and flow rate of droplet phases (DP):*

ILs have also been used as solvents in extraction processes or as electrolytes.

Polymer electrolytes comprising IL [emim][Tf2N] and soluble SPI showed a high ionic conductivity and reliable mechanical strength, suitable for HPP actuators [106]. Gel polymer electrolytes (GPEs) [107–109] and solid polymer electrolytes [110–114] have been widely reported. A series of GPEs were prepared using the electrospun membranes of poly(vinylidene fluoride-cohexafluoropropylene) [P(VdF-co-HFP)] incorporating ILs, 1-alkyl-3-methylimidazolium bis(trifluoromethylsulphonyl)imide in the presence of lithium bis(trifluoromethylsulphonyl)imide (LiTf2N) [115]. An IL-GPE containing semicrystal PVDF, amorphous polyvinyl acetate (PVAc) and ionic conductive [bmim] [BF4] was prepared via the solution-casting method for solid supercapacitors [116]. A PVDF-HFP/PMMA-blended microporous gel polymer electrolyte incorporating [bmim][BF4] was fabricated for lithium-ion batteries [117]. Solid polymer electrolytes using poly(vinylidene-fluoridetrifluoroethylene) and N,N,N-trimethyl-N-(2-hydroxyethyl) ammonium bis(trifluoromethylsulphonyl)imide ([N1112(OH)] [Tf2N]) IL were fabricated for energy storage applications [118]. PI/IL composite membranes for fuel cells operating at high temperatures were prepared by impregnating a porous Matrimid® membrane with protic ILs: 1-*n*-methyl-imidazolium dibutylphosphate ([C1im][DBP]), 1-*n*-butylimidazolium dibutyl-phosphate ([C4im][DBP]) and 1-*n*-butylimidazolium bis(2-ethylhexyl)phosphate ([C4im] [BEHP]). The electrolyte membranes were used as a proton-exchange membrane fuel cell (PEMFC) [119]. An IL-polymer electrolyte film based on a low-viscosity IL (1-ethyl-3-methylimidazolium dicyanamide) incorporated into a polymer matrix

IL lubricant with small sizes (below 10 μm) have been reported [105].

**110**

*2.3.3 Electrolyte*

**Figure 8.**

*125 μl/min.*

Carbon nanotubes (CNTs) consist of rolled-up graphene sheets and can be used as electronic, conductive and reinforcing fillers for polymer composites. MWCNTs possess a nanoscale diameter, a high aspect ratio, excellent mechanical properties, and good electrical conductivity. In recent years, MWCNTs have gained considerable interests of scientists and engineers, especially in polymer composites containing HPPs and ILs. A PI composite film comprising finely IL-dispersed MWCNTs in IL was obtained with a high shielding effectiveness (SE) for use in the packaging of a 2.5-Gbps plastic transceiver module with numerous applications in fibre to the home lightwave transmission systems [122]. PEI NCs consisting of bucky gels of industrial-grade MWCNTs and [bmim][PF6] were prepared; they are suitable for the aerospace and electronics industries, as shown in **Figure 10** [123]. Some PI and PEI NCs consisting of MWCNTs and polymerised ILs (PILs) were prepared, exhibiting differential function [124, 125].

The crystal structure of PVDF was modified by utilising the long alkyl chains of [C16mim][Br] and IL-modified MWCNTs, and the crystallisation kinetics of the composites was investigated [126, 127]. A series of PVDF composites with 'bucky gels' of MWNTs and ILs were obtained by simple melt compounding. According to the DSC and XRD results, the addition of ILs in the composites changed the crystallinity and crystal form of the PVDF [128]. PTFT and PVDF as the components of NCs containing HPP and ILs have received increasingly more attention. The nanomaterials with a nanoscale structure were prepared using pyridinium, imidazolium and phosphonium ILs as new synthetic building blocks in a PTFT. The cation-anion combination and functionalisation of ILs affect the ionic networks and nanostructures of materials [129, 130]. These nanomaterials show optimised thermal and mechanical properties and have numerous potential applications such as supercritical CO2 [131]. PVDF/IL/GraNCs were fabricated via the solution casting of PVDF with graphene (Gra) modified with a long alkyl chain IL [C16mim][Br], exhibiting a low loss tangent and low conductivity in the PVDF/ionic liquid-modified graphene

#### **Figure 9.**

*Photograph of (a) assemble DSSC and (b) I-V characteristics of DSSC comprising IL-incorporated PVDF-HFP polymer electrolyte film (maximum conductivity).*

**Figure 10.**

*Processing of PEI/bucky gel nanocomposites; the obtained composites had good toughness as the film could be curved adequately without any damage.*

(GIL) composites [132, 133]. NCs, based on a homopolymer PVDF and IL, were fabricated, and the preparation process opens up a new synthesis route for nanostructured polymer composites. Dielectric NCs based on PVDF, conductive carbon black (CB) and IL 1-vinyl-3-ethylimidazolium tetrafluoroborate [VEIM][BF4] were prepared via melt blending and electron beam irradiation (EBI) methods [134].

A nanostructured PAI was prepared in TBAB as a green medium by the step polymerisation reaction of 4,4′-methylenebis(3-chloro-2,6-diethyl trimellitimidobenzene) with 3,5-diamino-*N*-(4-hydroxy-phenyl)benzamide. Then, amino acid-functionalised multiwalled carbon nanotubes (f-MWCNTs)/PAI NCs were prepared by a solution mixing method [30].


**Author details**

Latifa Chebil1\*, Ghada Ben Rhouma1,2, Leila Chekir-Ghedira2,3 and Mohamed Ghoul1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Laboratory of Biomolecules engineering, ENSAIA, University of Lorraine, Vandoeuvre-lès-Nancy, France

2 Unity of Pharmacognosy/Molecular Biology, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia

3 Laboratory of Cellular and Molecular Biology, Faculty of Dental Medicine, University of Monastir, Monastir, Tunisia


**2.1. Drug formulation and chemicals**

**1** and **2** were synthesized by DEKK-TEC, Inc., using GLP/GMP guidelines, as previously described [1]. **1** is very stable in the solid state under ambient temperature and was adminis‐ tered in various vehicle media for the animal studies. For the rat studies, **1** was formulated as a buffered emulsion of soybean oil (20%), egg yolk lecithin (10%), glycerin (3%), histidine (3.1%) and water emulsion (containing 2-7 mg/mL of **1**) [IND 86,876]. For the mouse and dog IV studies, **1** was formulated as a 0.3% Klucel+0.3-3.3% Tween® 80, saline suspension and for the oral mouse study an emulsion of **1** in a 8% Tween-80®/Neobee®-1053 (Squibb) solution was used.

#### **2.2. High pressure liquid chromatography (HPLC) analysis of 1 and a metabolite, 2**

HPLC analysis was performed using an Agilent Technologies (New Castle, DE) 1200 model HPLC fitted with a diode array UV detector set at a wavelength of 244 nm (λmax for **1**). A Rheodyne Model 7725 injection port (Cotati, CA, USA) with a 20µL sample loop was used to inject the samples. Chromatograms were recorded with an Agilent Technologies integrator. Samples were chromatographed using an Alltech 150 x 4.6 mµ column that contains *Luna* C8 (2) 100A packing (diameter of particles=5 mµ).

The mobile phase for **1** consisted of 80% THF: 20% water and for **2**, was 45% THF: 47% water, which was degassed, filtered through a 0.45 µm Rainin filter (Woburn, MA, USA) and delivered at a flow rate of 1.0 mL/min.

Plasma and erythrocyte samples were stored at-74o C until analyzed. Standard solutions were prepared by dissolving 6 mg of **1** or **2** in 20 mL THF. Internal standards were 20 mg of cholesteryl benzoate (**ChB**, Sigma Aldrich Co) or phenol (**P**, Sigma Aldrich Co) each in 20 mL THF. All solutions were stable for at least 2 months at 5o C. Standard assays for **1** and **2** consisted of 0.25 mL of plasma, 25 µL of **1** or **2** and internal standard (**ChB** or **P** – 2 µL) and 2 mL dichloromethane. The samples were vortexed for 10 min and frozen to separate the layers. The bottom organic layer was removed with a 25 gauge needle and glass syringe, filtered through a 0.45 mm Acrodisc syringe filter and evaporated to dryness under vacuum, reconstituted with 100 µL of THF and analyzed as below.

Control dog and rat whole blood and plasma samples were spiked with **1** in the concentration range 5-1000 ng/mL, plus 20 µL of the above **ChB**. Similar controls were prepared for **2** (ng/mL) and its int. std. – **P**. Peak-area ratios of **1**/**ChB** or (**2**/**P**) *vs*. the concentration of **1** or **2** (ng/mL) were subjected to linear regression analysis. Retention times for **1** and **ChB** were 6.41 and 4.63 min., respectively and for **2** and **P** were 6.6 and 18.5 min., respectively.

Verification of the HPLC assays included calibration curves derived from the assay of five (5) erythrocyte and eleven (11) plasma standards in duplicate prepared each with **1** and **2** (0.5 ng/ mL-600 µg/mL). Plasma and erythrocyte samples were obtained from healthy rats and dogs and spiked with **1** or **2** and their respective internal standards. Drug concentrations in all samples were calculated using the results of linear regression analysis. Reproducibility was higher than 85%. Limit of quantitation for **1** and **2** was 0.2 ng/mL.

All mice, rat and dog studies were conducted at MPI Research (Mattawan, MI) under GLP regulations as described in the Guide for the Care and Use of Laboratory Animals, Office for

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243

For mouse toxicity studies, **1** was administered IV bolus *via* the tail vein or per oral gavage; for the dog studies, **1** was administered as an IV bolus *via* the femoral vein and for rats, administration was during a 3 h IV infusion via indwelling femoral vein catheters. The observation period for all studies was 14-days – followed with euthanasia.  For the mouse

For the acute behavioral studies, rats received drugs and controls to identify/verify gross behavioral patterns employing a Morris modified water maze (4' x 3' x 1.5') with a single layer of white polyethylene peanuts that floated (in 6'' of water) and covered a single mounting stage [13, 14]. Adult female rats (Hsd:SD, 175-225 g.) were grouped 3-6 animals per drug arm. The test agents were dissolved in or suspended in 5% aqueous TweenR 80-hydroxypropyl cellulose in 5% saline and administered intraperitoneally (IP). Controls received the vehicle only. Swimming trials began – 1, 2, 3 and 20 hours post-dosing. For each time period postdosing, the rats were challenged on six (6)-back-to-back swim trial events to find the platform. The daily swimming times and ranges were compared to vehicle controls *vs*. chemotherapeu‐ tics alone in rats. The data (latency to find the platform) was analyzed by variance (ANOVA). Body weights and water temperature-prior to each dosing and during each FOB assessment

Groups of rats (5/sex) were administered 100, 200, or 300 mg/kg of **1** as a 3 h timed infusion and samples of blood collected (in EDTA tubes) at various time points IPEOI:-15,+10,+45 min, 1.5, 3, 6, 8, 12, 24 h and 14-days. Each animal possessed an indwelling femoral catheter for ease of the study. The catheters were flushed after each blood draw. The plasma and erythrocytes

Adult beagle dogs (8 M) were administered **1** as a slow bolus injection once through a femoral vein in doses of 10, 20, and 30 mg/kg. A 20-gauge venous catheter was inserted into a saphenous vein for bleeding and samples were withdrawn at 0, 5, 15, and 30 min. and 1, 1.5, 2, 4, 8, 12 and 24 h into EDTA containing tubes. Plasma was separated and stored at-70o C until assayed.

Model parameters were estimated using Micropharm software and nonlinear least squares regression was performed using Simplex and Gauss Newton algorithms []. An open twocompartment model provided the best fit. Clearance, volume of distribution and half-lives

C until analyzed.

Laboratory Animal Welfare, NIH, Bethesda, MD.

were monitored.

**2.5. Pharmacokinetic studies**

were separated and stored separately at -74o

Animals were anesthetized with ketamine.

**2.6. Animal pharmacokinetic data analysis**

were derived from estimates of the model parameters.

studies physical/gross necropsy examinations were conducted.

#### **2.3. P-glycoprotein (P-gp) transport studies**

**Cell lines**-three (3) human cancer cell lines-A549: *lung*; MCF7: *breast*; and HeLa: *ovarian* were obtained from ATCC, Manassas, MD, and maintained in culture in a temperature controlled (36 o C), 5% carbon dioxide, and humidity controlled incubator system. All culture transfers were in a laminar flow hood under sterile conditions. Tissue culture medium used was RPMI 1640 containing 10% FBS and penicillin/streptomycin/fungizone (1%) – all purchased from InVitrogen.

**Material preparation-**Rho was prepared in distilled water as a stock solution (1 mg/mL), stored frozen at-22o C. Rho (0.2µg/mL) was added to the culture medium in the presence or absence of Vpml and/or **1**. Vpml (2-23 µg/10 mL) was prepared in PBS solution and **1** (0.5-73 µg/10 mL) in PBS+5% DMSO.

**A typical assay**-contained: **1**-7.3 µg/mL, Vpml-2.5 µg/mL and Rho-0.2 µg/mL. Cells per test system were 0.8-1.2 x 106 /mL. Incubations occurred in the above incubator conditions for the times and schedules discussed below. Experiments were conducted in triplicate and at times as specified in the Results. Reaction incubations were 15-60 minutes in length as discussed in the Results section.

**Post incubation**-After treatment, cells were rapidly trypsinized (~2 min), washed with cold PBS and kept on ice until analyzed. Assay conducted with a Becton-Dickinson FACS-ARIA II flow cytometer with CELLQUEST software. The Rho fluorescence was measured at laser excitation 488 nm with emission at 530 nm and is expressed in arbitrary units compared to control cells (untreated). Measurements were conducted on 10,000 cells per assay.

#### **2.4. Animals**

Adult Sprague-Dawley mice [Crl: CD1(ICR) BR] (males 20-25 g and female 18-25 g) and rats [Crl: CD1(ICR) BR] (males 300-350 g and female 225-250 g) were obtained from Harlan Industries (Indianapolis, IN), housed in groups of three-five per cage in light-controlled (12 h/ day) and temperature-controlled (24o C) animal isolators with filtered vents and exhausts. They were fed a diet of Purina Laboratory Chow (Purina Feed) and received tap water *ad libitum.* The rats were fasted and 3-4 mL of blood was drawn *via* the jugular vein. The order of bleeding was alternated (one animal from each dose group, then repeating) to reduce handling and time biases.

Adult male and female beagle dogs (6.5-7.5 mo. of age, 6.5-9.49 kg) were raised and maintained at MPI Research (Mattawan, MI). They were fed a diet of Purina Dog Chow (Purina Feed), received tap water *ad libitum* and exercised per IACUC protocol. Mouse, rat and dog were euthanized with phenobarbital/ketamine anesthesia and/or carbon dioxide inhalation. The chest cavities were exposed to insure death. Institutional animal care and use committees (IACUC) reviewed and approved all the studies.

All mice, rat and dog studies were conducted at MPI Research (Mattawan, MI) under GLP regulations as described in the Guide for the Care and Use of Laboratory Animals, Office for Laboratory Animal Welfare, NIH, Bethesda, MD.

For mouse toxicity studies, **1** was administered IV bolus *via* the tail vein or per oral gavage; for the dog studies, **1** was administered as an IV bolus *via* the femoral vein and for rats, administration was during a 3 h IV infusion via indwelling femoral vein catheters. The observation period for all studies was 14-days – followed with euthanasia.  For the mouse studies physical/gross necropsy examinations were conducted.

For the acute behavioral studies, rats received drugs and controls to identify/verify gross behavioral patterns employing a Morris modified water maze (4' x 3' x 1.5') with a single layer of white polyethylene peanuts that floated (in 6'' of water) and covered a single mounting stage [13, 14]. Adult female rats (Hsd:SD, 175-225 g.) were grouped 3-6 animals per drug arm. The test agents were dissolved in or suspended in 5% aqueous TweenR 80-hydroxypropyl cellulose in 5% saline and administered intraperitoneally (IP). Controls received the vehicle only. Swimming trials began – 1, 2, 3 and 20 hours post-dosing. For each time period postdosing, the rats were challenged on six (6)-back-to-back swim trial events to find the platform. The daily swimming times and ranges were compared to vehicle controls *vs*. chemotherapeu‐ tics alone in rats. The data (latency to find the platform) was analyzed by variance (ANOVA). Body weights and water temperature-prior to each dosing and during each FOB assessment were monitored.

#### **2.5. Pharmacokinetic studies**

samples were calculated using the results of linear regression analysis. Reproducibility was

**Cell lines**-three (3) human cancer cell lines-A549: *lung*; MCF7: *breast*; and HeLa: *ovarian* were obtained from ATCC, Manassas, MD, and maintained in culture in a temperature controlled

**Material preparation-**Rho was prepared in distilled water as a stock solution (1 mg/mL), stored frozen at-22o C. Rho (0.2µg/mL) was added to the culture medium in the presence or absence of Vpml and/or **1**. Vpml (2-23 µg/10 mL) was prepared in PBS solution and **1** (0.5-73 µg/10 mL)

**A typical assay**-contained: **1**-7.3 µg/mL, Vpml-2.5 µg/mL and Rho-0.2 µg/mL. Cells per test

times and schedules discussed below. Experiments were conducted in triplicate and at times as specified in the Results. Reaction incubations were 15-60 minutes in length as discussed in

**Post incubation**-After treatment, cells were rapidly trypsinized (~2 min), washed with cold PBS and kept on ice until analyzed. Assay conducted with a Becton-Dickinson FACS-ARIA II flow cytometer with CELLQUEST software. The Rho fluorescence was measured at laser excitation 488 nm with emission at 530 nm and is expressed in arbitrary units compared to

Adult Sprague-Dawley mice [Crl: CD1(ICR) BR] (males 20-25 g and female 18-25 g) and rats [Crl: CD1(ICR) BR] (males 300-350 g and female 225-250 g) were obtained from Harlan Industries (Indianapolis, IN), housed in groups of three-five per cage in light-controlled (12 h/

were fed a diet of Purina Laboratory Chow (Purina Feed) and received tap water *ad libitum.* The rats were fasted and 3-4 mL of blood was drawn *via* the jugular vein. The order of bleeding was alternated (one animal from each dose group, then repeating) to reduce handling and time

Adult male and female beagle dogs (6.5-7.5 mo. of age, 6.5-9.49 kg) were raised and maintained at MPI Research (Mattawan, MI). They were fed a diet of Purina Dog Chow (Purina Feed), received tap water *ad libitum* and exercised per IACUC protocol. Mouse, rat and dog were euthanized with phenobarbital/ketamine anesthesia and/or carbon dioxide inhalation. The chest cavities were exposed to insure death. Institutional animal care and use committees

control cells (untreated). Measurements were conducted on 10,000 cells per assay.

/mL. Incubations occurred in the above incubator conditions for the

C) animal isolators with filtered vents and exhausts. They

C), 5% carbon dioxide, and humidity controlled incubator system. All culture transfers were in a laminar flow hood under sterile conditions. Tissue culture medium used was RPMI 1640 containing 10% FBS and penicillin/streptomycin/fungizone (1%) – all purchased from

higher than 85%. Limit of quantitation for **1** and **2** was 0.2 ng/mL.

**2.3. P-glycoprotein (P-gp) transport studies**

242 Tumors of the Central Nervous System – Primary and Secondary

(36 o

InVitrogen.

in PBS+5% DMSO.

the Results section.

**2.4. Animals**

biases.

day) and temperature-controlled (24o

(IACUC) reviewed and approved all the studies.

system were 0.8-1.2 x 106

Groups of rats (5/sex) were administered 100, 200, or 300 mg/kg of **1** as a 3 h timed infusion and samples of blood collected (in EDTA tubes) at various time points IPEOI:-15,+10,+45 min, 1.5, 3, 6, 8, 12, 24 h and 14-days. Each animal possessed an indwelling femoral catheter for ease of the study. The catheters were flushed after each blood draw. The plasma and erythrocytes were separated and stored separately at -74o C until analyzed.

Adult beagle dogs (8 M) were administered **1** as a slow bolus injection once through a femoral vein in doses of 10, 20, and 30 mg/kg. A 20-gauge venous catheter was inserted into a saphenous vein for bleeding and samples were withdrawn at 0, 5, 15, and 30 min. and 1, 1.5, 2, 4, 8, 12 and 24 h into EDTA containing tubes. Plasma was separated and stored at-70o C until assayed. Animals were anesthetized with ketamine.

#### **2.6. Animal pharmacokinetic data analysis**

Model parameters were estimated using Micropharm software and nonlinear least squares regression was performed using Simplex and Gauss Newton algorithms []. An open twocompartment model provided the best fit. Clearance, volume of distribution and half-lives were derived from estimates of the model parameters.

#### **2.7. Data analysis**

Data analysis was performed on all plasma and *in vitro* studies and analyzed via non-linear regression using a non-weighed quasi-Newtonian/simplex fitting algorithm (Statistical software available from Stat soft, Tulsa, OK).

cold to touch. No seizures or loss of coordination was noted. The deaths at 400 and 600 were of a very immediate nature, occurring within minutes or less post-dose, with no clinical signs exhibited prior to death. While transient incidences of rapid breathing were also noted in a couple of control animals, a definitive relationship to the vehicle was unclear. No definitively clear treatment-related body effects were noted in those mice surviving the 14-day observation period when compared with controls. No macroscopic findings were noted in any animal at

**Method LD10/50 (mg/kg) Time\* (Days) Comments**

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cholesterol – elevated, but

36 M 50-600 IV 132/385 2-14 days Deaths were erratic

30 F Gavage 5d – no deaths No deaths

acceptable 73 F 50-300 Infusion (LD10) 15 days

8 F 10-30 Bolus >30.0 10 days No deaths

4 F 10-30 Bolus >30.0 10 days No deaths

Based on the conditions and findings of this study, the intravenous LD10 for **1** was calculated to be 136 mg/kg (95% confidence limits could not be calculated) in mice (combined sexes),

A study was conducted in groups of 10 male/10 female mice per dose. The study evaluated **1** administered daily for five days at doses of 0, 800, 1000, 1200, 1500 and 2000 mg/kg per gavage to mice. Only one death occurred at 800 mg/kg on day 2 after dosing. All animals demonstrated some degree of lethargy and unkempt appearance. Similar body appearances were noted with

while the intravenous LD50 was calculated to be 385 mg/kg (95% confidence limits).

73 M IV 100 1-Rat died @ 100 mg/kg;

Neither aplastic bone marrow nor splenic depletion of lymphocytes was noted.

30 M 800-2000 Oral – 0.8 – 2.0 g/d x 21 days

36 F Bolus (LD10/50)

necropsy.

Mice

Mice

Rat

Dog

Dog

\*The time of the last death.

**Table 1.** Medial dose summary for **1**

**Species No/ Sex**

**Doses (mg/kg)**

8 M IV

4 M IV

**3.4. Sub-chronic oral mouse toxicity (Table 1)**

the controls. No seizures were noted.


**4. The effect of mosses on the hydraulic media**

In conditions with minimal moss, vegetable growth looks very optimal, with indications of wider leaves and dense stem conditions. On the other hand, in conditions where there is quite a lot of moss growth, the growth of kale plants looks not optimal, with indications of thinner leaves and poor stem conditions. Solid (**Figure 2**). The appearance of moss in a hydroponic system will be very difficult to prevent. Farmers cannot avoid the factors that lead to the appearance of moss, even at the beginning of growing seedlings.

The reason moss is not good for hydroponic plants

**Figure 2.** *Moss in the pipe of Hidrophonic instrument.*

**Figure 3.** *The death of the vegetable that grows together with the moss.*


**Author details**

Afam I.O. Jideani\* , Henry Silungwe, Thakhani Takalani, Tonna A. Anyasi, Henry Udeh and Adewale Omolola

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Food Science and Technology, School of Agriculture, University of Venda, Thohoyandou, South Africa


*1.1.4 Dielectric layer deposition and patterning*

The following process steps were followed for the deposition and patterning of dielectric Silicon Nitride (Si3N4) on the central signal line of the CPW layer.

a. **Deposition of Si3N4:** This layer provides the dc isolation between the signal line and the ground line when the switch is actuated to the down-state position. A thinner layer of Si3N4 will result in a higher capacitance in the downstate but would lead to pinhole problems which occur in thin dielectric layers. Also, the thin dielectric layer must be able to withstand the actuation voltage without breakdown.

Oxford Instruments Plasma technology Plasma Enhanced Chemical Vapor Deposition (PECVD) system is used for deposition of Si3N4 as shown in **Figure 7**. PECVD is a process by which thin films are deposited from the conversion of

*RF MEMS Switch Fabrication and Packaging DOI: http://dx.doi.org/10.5772/intechopen.95003*

**Figure 6.** *Optical microscope image.*

**Figure 7.** *Oxford PECVD for Si3N4 deposition at CeNSE.*

gaseous materials into solid state, due to a chemical reaction occurring in the presence of plasma. PECVD uses electrical energy to generate the plasma. Due to the presence of plasma, the gas mixture is transformed into highly reactive ions and molecules, which leads to low temperature requirements as compared to CVD processes. PECVD processes results in high quality films which have good adhesion, uniformity and good step coverage [18].

Silane (SiH4) is usually supplied along with an inert gas like Nitrogen, Argon or Helium. Silane reacts with Ammonia (NH3) to produce Si3N4 and a byproduct Hydrogen. This reaction is as depicted by the chemical reaction as given below.

3*SiH*4 þ 4 *NH*3 ! *Si*3*N*4 þ 12 *H*2

b. **Lithography for Si3N4:** The patterning of Si3N4 is achieved by first depositing a positive photoresist AZ4562 by placing it on a spin coater. The spin coater rotates at 4000 rpm for 40 sec. After soft baking at 110°C for 1 minute, the PR is exposed to UV rays through a mask aligner at proximity of

etch and 10 to 20 sec for Cr etch. **Figures 5** and **6** represent the mask for

The following process steps were followed for the deposition and patterning of

a. **Deposition of Si3N4:** This layer provides the dc isolation between the signal line and the ground line when the switch is actuated to the down-state position. A thinner layer of Si3N4 will result in a higher capacitance in the downstate but would lead to pinhole problems which occur in thin dielectric layers. Also, the thin dielectric layer must be able to withstand the actuation

Oxford Instruments Plasma technology Plasma Enhanced Chemical Vapor Deposition (PECVD) system is used for deposition of Si3N4 as shown in **Figure 7**. PECVD is a process by which thin films are deposited from the conversion of

dielectric Silicon Nitride (Si3N4) on the central signal line of the CPW layer.

patterning and the resulting CPW layer respectively.

*1.1.4 Dielectric layer deposition and patterning*

**Figure 4.**

**Figure 5.**

**258**

*Mask 1 for CPW layer patterning.*

*EVG mask aligner at CeNSE, IISc.*

*Nanofibers - Synthesis, Properties and Applications*

voltage without breakdown.

30 μm and energy of 110 mJ. The PR is then developed using the developer AZ 351B for 45–60 seconds. Next, the wafer is hard baked on an oven at 110°C for 3 minutes.

using Isopropyl Alchohol (IPA). This is to prevent the re-deposition of stripped photoresist on the substrate since Acetone has high vapor pressure. This is followed by cleaning with Ultrsonicate Acetone for 3 minutes.

The sacrificial layer is the layer which will be etched out to release the top metal layer. The topography and planarity of the top membrane is defined by the sacrificial layer planarity. Several materials like metals, dielectrics and photoresists have been used as the sacrificial layer. The choice of the sacrificial layer is based on the processing steps that follow the deposition of this layer, the temperature range, the required planarity and profile of surface. Here, a positive Photoresist (PR) S1813 is used as the sacrificial layer. This PR has to be deposited with utmost accuracy in order to define the gap between the top electrode and bottom electrode of the RF MEMS switch. The complete process of sacrificial layer deposition and patterning

**Sacrificial layer Optimization**: The PR S1813 is a positive photoresist which has excellent adhesion, excellent coating uniformity with effective broadband exposure. This PR is used for a wide variety of process flow requirements such as lift-off, dry etch, wet etch, the thickness of the PR to name a few. The plot in **Figure 11** gives the resist thickness versus spin for the Shipley family of PRs. Thick PR layers can be achieved in one step, however they have the disadvantage of being nonuniform over the wafer surface. In order to achieve uniform and thick PR coating, the coating process is performed in three steps. In the first step, the spin coater is run at low speeds of 500 rpm for 30 sec. This low spin speed and reduced spin time

**Figure 10** shows the patterned silicon nitride layer.

*1.1.5 Sacrificial layer deposition and patterning*

*RF MEMS Switch Fabrication and Packaging DOI: http://dx.doi.org/10.5772/intechopen.95003*

can be explained by the following steps:

*Optical microscope image of silicon nitride layer formed.*

**Figure 10.**

**Figure 11.**

**261**

*PR deposition using multiple step method.*

c. **Etching of Si3N4:** The etching of Si3N4 is performed using a dry etch process called Reactive Ion Etch (RIE). Reactive Ion etching is a process wherein the reactive species react with the material to be etched only when the surfaces of the material are activated by the collision of incident ions from the plasma. The etching characteristics like etch rate, etch profile, etch uniformity, etch selectivity can be controlled very precisely by selecting the right combination of recipes of chamber pressure, flow rate of gases, applied RF power and electrode bias. The etch rates are slow typically about 10 nm/ min up to 50 nm/min.

The RIE-F equipment used at CeNSe, IISc is as shown in **Figure 8**. For etching of Si3N4 the chamber pressure is set at 10 mTorr, RF power at 50 W with the main power at 2000 W. The flow rate of Sulfur Hexa Flouride (SF6) is set at 45 scc/m with the temperature at 5°C. For etching out 100 nm of Si3N4 the required time was 12 seconds. The mask used for the patterning of the Si3N4 layer is as shown in **Figure 9**.

d. **Photoresist strip:** This is followed by the wet etching of the photoresist by dipping the wafer in acetone for 5 minutes followed by immediate cleaning

**Figure 9.** *Mask 2 for silicon nitride.*

#### *RF MEMS Switch Fabrication and Packaging DOI: http://dx.doi.org/10.5772/intechopen.95003*

30 μm and energy of 110 mJ. The PR is then developed using the developer AZ 351B for 45–60 seconds. Next, the wafer is hard baked on an oven at 110°C

c. **Etching of Si3N4:** The etching of Si3N4 is performed using a dry etch process called Reactive Ion Etch (RIE). Reactive Ion etching is a process wherein the reactive species react with the material to be etched only when the surfaces of the material are activated by the collision of incident ions from the plasma. The

etching characteristics like etch rate, etch profile, etch uniformity, etch

selectivity can be controlled very precisely by selecting the right combination of recipes of chamber pressure, flow rate of gases, applied RF power and electrode bias. The etch rates are slow typically about 10 nm/ min up to 50 nm/min.

The RIE-F equipment used at CeNSe, IISc is as shown in **Figure 8**. For etching of Si3N4 the chamber pressure is set at 10 mTorr, RF power at 50 W with the main power at 2000 W. The flow rate of Sulfur Hexa Flouride (SF6) is set at 45 scc/m with the temperature at 5°C. For etching out 100 nm of Si3N4 the required time was 12 seconds. The mask used for the patterning of the Si3N4

d. **Photoresist strip:** This is followed by the wet etching of the photoresist by dipping the wafer in acetone for 5 minutes followed by immediate cleaning

for 3 minutes.

*Nanofibers - Synthesis, Properties and Applications*

layer is as shown in **Figure 9**.

**Figure 8.** *RIE F CeNSe, IISc.*

**Figure 9.**

**260**

*Mask 2 for silicon nitride.*

using Isopropyl Alchohol (IPA). This is to prevent the re-deposition of stripped photoresist on the substrate since Acetone has high vapor pressure. This is followed by cleaning with Ultrsonicate Acetone for 3 minutes. **Figure 10** shows the patterned silicon nitride layer.

## *1.1.5 Sacrificial layer deposition and patterning*

The sacrificial layer is the layer which will be etched out to release the top metal layer. The topography and planarity of the top membrane is defined by the sacrificial layer planarity. Several materials like metals, dielectrics and photoresists have been used as the sacrificial layer. The choice of the sacrificial layer is based on the processing steps that follow the deposition of this layer, the temperature range, the required planarity and profile of surface. Here, a positive Photoresist (PR) S1813 is used as the sacrificial layer. This PR has to be deposited with utmost accuracy in order to define the gap between the top electrode and bottom electrode of the RF MEMS switch. The complete process of sacrificial layer deposition and patterning can be explained by the following steps:

**Sacrificial layer Optimization**: The PR S1813 is a positive photoresist which has excellent adhesion, excellent coating uniformity with effective broadband exposure. This PR is used for a wide variety of process flow requirements such as lift-off, dry etch, wet etch, the thickness of the PR to name a few. The plot in **Figure 11** gives the resist thickness versus spin for the Shipley family of PRs. Thick PR layers can be achieved in one step, however they have the disadvantage of being nonuniform over the wafer surface. In order to achieve uniform and thick PR coating, the coating process is performed in three steps. In the first step, the spin coater is run at low speeds of 500 rpm for 30 sec. This low spin speed and reduced spin time

**Figure 10.** *Optical microscope image of silicon nitride layer formed.*

**Figure 11.** *PR deposition using multiple step method.*

will result in uniform coating of thick resist on the wafer. In the second step the speed is ramped upto 1000 rpm within a time of 30 sec. A solid film of the photoresist is formed with the complete evaporation of the solvent. This step decides the thickness and uniformity of the photoresist. The third step consists of the spin coater speed set at 2000 rpm for 40 sec. This last step ensures that any leftover solvent is completely evaporated. The complete cycle of spin coating is as shown in **Figure 11**. Using a Dektak optical profiler the thickness of this layer was confirmed to be 3 μm.

pressure of 6.5x103 Torr, target to substrate distance set at 7.5 cm, with the Argon flow rate at 250 sccm. The DC Power was set at 25 W with a pre sputtering time of 30 seconds followed by a deposition time of 1100 seconds

b. **Gold layer patterning:** The four switch designs chosen for the top Gold layer are shown as four respective masks in **Figure 13**. The lithography involved the use of AZ5412E positive PR. This was spin coated at 4000 rpm for 40 sec. The wafer was then soft baked aligner at a proximity of 10 μm and energy of 50 mJ The PR is then developed using the developer MF 26A with the wafer dipped in the developer of 20–140 sec. Next, the wafer is hard baked on an oven at 90°C for 30 minutes. For Gold etch, freshly made Potassium Iodide and Iodine (KI/I2) solution in a ratio of KI:I2:H2O = 4 g:1 g:40 ml is used. The unwanted Chrome deposition on the bottom layer is also etched out using a

A/sec.

The release of the top switch membrane is the most crucial step in the whole fabrication process. There are many methods to release the top layer without deformation and stiction. The first step in the top layer release is to etch the sacrificial layer. This could be achieved by using dry etching or wet etching. In wet etching, conventional liquid solvents are used to completely remove the sacrificial layer followed by drying. The drying could be through the process of air drying or

*Four top layer designs for RF MEMS switch. (a) Fixed-fixed beam switch. (b) Fixed-fixed Flexure switch.*

*(c) Fixed-Fixed Single Flexure switch. (d) Crab leg Flexure switch.*

with the deposition rate set at 50

*RF MEMS Switch Fabrication and Packaging DOI: http://dx.doi.org/10.5772/intechopen.95003*

Chrome etchant for 5 to 10 seconds.

*1.1.7 Top layer release*

through critical point drying.

**Figure 13.**

**263**

**Sacrificial layer patterning**: The patterning of the sacrificial layer photoresist is processes by first depositing one more layer of positive PR S1813 on this layer. This was achieved by the spin coater speed set to 500 rpm for 30 seconds, followed by a ramp up of 1000 rpm for 30 sec and 200 rpm for 40 sec. After soft baking the PR is exposed to UV rays through a mask aligner at a proximity of 30 μm and energy of 75 mJ. The mask used for generating the pattern for this layer is as shown in **Figure 12**.

The PR is then developed using the developer AZ 351B for 30–60 seconds. Next, the wafer is hard baked on an oven at 90°C for 30 minutes. The PR layer thickness shrunk from 3 μm to 2.09 μm after development and baking.

#### *1.1.6 Top layer deposition and patterning*

The top layer or beam formation defines the performance of the RF MEMS switch. The top layer designs were simulated using Coventorware™. These designs have been chosen due to their lower pull-in voltages. Gold is the choice for the top layer due to its favorable characteristics such as, its high conductivity, non-tarnishing property, high Young's Modulus and compatibility with micromachining processes. The top metal layer deposition and patterning is described in the following sections.

a. **Gold layer deposition:** The deposition of this layer was carried out using the TECPORT sputtering equipment. It may be recalled that the bottom layer has the composition of Cr/Au/Cr. This composition would lead to excellent adhesion of the top layer anchors with the previously deposited Chrome layer. Several Iterations were carried out in order to sputter the top Gold layer without residual stress. Several parameters such as temperature, rate of deposition were optimized in order to arrive at top layers without buckling after release process.

Finally, with the optimized parameters setting temperature and rate of deposition a stress free top layer was arrived at. The stress free top layer is of critical importance for reduction in actuation voltage. The process parameters of the sputter coater were set at a base pressure of 5x106 Torr, deposition

**Figure 12.** *Mask 3 for PR layer.*

pressure of 6.5x103 Torr, target to substrate distance set at 7.5 cm, with the Argon flow rate at 250 sccm. The DC Power was set at 25 W with a pre sputtering time of 30 seconds followed by a deposition time of 1100 seconds with the deposition rate set at 50 A/sec.

b. **Gold layer patterning:** The four switch designs chosen for the top Gold layer are shown as four respective masks in **Figure 13**. The lithography involved the use of AZ5412E positive PR. This was spin coated at 4000 rpm for 40 sec. The wafer was then soft baked aligner at a proximity of 10 μm and energy of 50 mJ The PR is then developed using the developer MF 26A with the wafer dipped in the developer of 20–140 sec. Next, the wafer is hard baked on an oven at 90°C for 30 minutes. For Gold etch, freshly made Potassium Iodide and Iodine (KI/I2) solution in a ratio of KI:I2:H2O = 4 g:1 g:40 ml is used. The unwanted Chrome deposition on the bottom layer is also etched out using a Chrome etchant for 5 to 10 seconds.


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**83**

**Chapter 6**

**Abstract**

affordable housing

**1. Introduction**

Solutions

Socioeconomic Influences on

*Deborah J. Bowen and Lisa Quintiliani*

Affordable Housing Residents:

Problem Definition and Possible

Socioeconomic status (SES) is a powerful social determinant of health. Often, affordable housing is an important step in promoting reliable economic and social health among individuals living in poverty. However, we argue that we must go further to improve the long-term health outcomes of these individuals and families. First, we use survey data and geographical analysis to identify the socioeconomic status of neighborhoods and residents of affordable housing in a major urban center. SES levels are certainly lower among affordable housing residents, and SES was significantly lower in public housing development neighborhoods than other neighborhoods. We offer solutions from our own and other research experiences that identify potential changes to affordable housing to promote and maximize health of residents. These data have implications for multilevel intervention.

**Keywords:** built environment, neighborhood, socioeconomic status,

between socioeconomic status (SES) and health outcomes [3–5].

ences and calls for research into them.

Public housing residents have a much higher burden of chronic disease and disease risk than other urban dwellers, even when controlling for income [1]. One possible driver of these differences may be the environment in which public housing developments are located [2]. There is growing evidence that the physical surroundings and resources available to individuals can have an impact on the relationship

Early research led to several large-scale interventions that provide alternatives to living in densely populated, very low SES neighborhoods and buildings [6]. The Moving to Opportunity study used a randomized design to demonstrate that health outcomes, including obesity, could be improved when moving from high- to low-poverty areas. Many of these interventions and changes required physical changes to public and affordable housing that take years to create and are expensive and labor intensive. In contrast, we believe that with simple changes, we can create affordable housing experiences that promote health, rather than reduce the likelihood of health promotion efforts. This chapter articulates those potential experi-

## **Chapter 6**

*Different Strategies of Housing Design*

regulation and the credit crunch. London, New York: Routledge; 2008

York: Routledge; 2018

[37] Spinozzi P, Mazzanti M. Cultures of sustainability and wellbeing theories. Histories and Policies. London, New

**82**


**1. Introduction**

In suspension bridges, cable clamps are key nodes connecting the main cables and hangers. With the progress and development of suspension bridges, cable clamps have gradually evolved from eyebars and pin-type hinge structures to riding straddle and pintype structures. The choice is closely related to the type of hanger used: riding straddle cable clamps are commonly used for wire rope hangers, whereas pin-type cable clamps are used for parallel wire hangers. In the U.S. and Japan, preference is given to riding straddle cable clamps, whereas pin-type cable clamps are more common in China and Europe [1]. Figure 1 shows the different types of cable clips.

During the construction and operation, the cable clamp will no longer be firmly anchored because of the loosening of the fastening bolts, the main cable has a thinner section under long-term loading, and the linear expansion coefficient is inconsistent under the influence of temperature. These factors are important inducements that cause slippage of the suspension bridge cable clamps. Therefore, the cable clamp slippage is a common phenomenon that is detrimental to suspension bridges. In the inspection manual for suspension bridges developed by the Honshu–Shikoku Bridge Construction Corporation, Japan, cable clamp slippage is listed as a key inspection item [2]. In the United States, the cable inspection and strength assessment guidelines provide detailed inspection requirements for hangers and cable clamps [3]. There are strict regulations on cable clamp slippage in the Chinese Highway Bridge Technical Condition Assessment Standard [4].

**Citation:** Li, H.; Liu, Y.; Li, C.; Hu, H.; Sun, Q. Force Analysis of Self-Anchored Suspension Bridges after Cable Clamp Slippage. *Symmetry* **2021**, *13*, 1514. https:// doi.org/10.3390/sym13081514

Academic Editors: Yang Yang, Ying Lei, Xiaolin Meng and Jun Li

Received: 22 July 2021 Accepted: 16 August 2021 Published: 18 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Different types of cable clips: (**a**) abridged general view of a riding straddle cable clip; (**b**) abridged general view of a pin-type cable clamp.

Cable clamps with poor anti-sliding performance tend to slide along the main cable, as shown in Figure 2, which directly causes a loss of stress in the entire structure and major safety hazards in the structural system. Insufficient anti-skid performance of the cable clamp will cause the cable clamp to slide down the main cable, which will lead to the redistribution of the internal force of the principal force-bearing components such as the main cable, hanger, and main beam. The internal force deviates from the design value and the safety reserve of the structure is cut. In practice, engineering problems due to the poor anti-sliding performance of cable clamps may force a project to be terminated or even rebuilt. Hence, scholars have extensively studied the problem of cable clamp slippage.

**Figure 2.** Actual images of a cable clip after slipping.

In 2000, Ji et al. analyzed the additional force due to the hanger pull acting on the screws in the hanger, with an objective to improve the cable clamp design of the Kurima Bridge, Japan. The Liaison Bridge Corporation proposed the application of pin-type cable clamps for the first time [5]. Since 2009, scholars have started paying attention to cable clamp slippage in suspension bridges. Ren et al. analyzed the anti-skid friction coefficient of a cable clamp and the main cable void ratio by conducting an anti-sliding test on the main cable and cable clamp [6]. Huang et al. studied the variations in the lateral displacement of the main cable and the lateral deflection angle of the cable clamp under the effect of hanger tension in the main cable [7]. In 2013, Li et al. determined the upper limit of the transverse elastic modulus based on the macroscopic force of an ideally arranged main cable [8]. In 2014, Ruan et al. studied and analyzed the spatial effect and stress distribution characteristics of a cable clamp [9]. Ma et al. conducted jack push tests on the main cable of a suspension bridge to analyze the friction coefficient of the cable clamp against sliding and the internal and external void ratios of the cable clamp [10]. Zhuge et al. studied the friction factor of a carbon fiber reinforced plastic (CFRP) cable–cable clamp interface [11]. Li et al. proposed the Tsai–Hill failure criterion for composite materials [12]. In 2015, Zhou et al. calculated the anti-sliding friction coefficient and the internal and external void ratios of a steel wire main cable clamp coated with a zinc–aluminum alloy [13]. In 2016, Sun et al. derived a simplified calculation formula to determine the increase in the elevation of the main cable mid-span control point and the average increment in the hanger cable force [14]. In 2018, Shen et al. analyzed the change laws of the ultimate anti-sliding friction resistance of a cable clamp under the action of the cable force, and the contact force between the main cable and the cable clamp surface [15,16]. In 2019, Ruan et al. proposed a theoretical model considering transversely isotropic materials based on the generalized Hooke's law. Through anti-slipping performance tests, they determined the actual ultimate sliding resistance and comprehensive friction coefficient of a cable clamp [17]. In 2020, Miao et al. proposed a new slippage-based failure criterion based on an analytical model [18] and briefly revised the original slippage resistance formula using the Coulomb friction law.

In summary, most studies have been based on the interface performance of the main cable and the cable clamp, focusing on the analysis of the effects of the clamping force of the cable clamp and the friction coefficient on the anti-slipping performance. Studies on the total force acting on suspension bridges are lacking. The integrity of the overall structure of a full bridge following cable clamp slippage has rarely been studied. In practice, although cable clamp slippage is minor, it is a key factor causing a change in the forces acting on the entire bridge. Therefore, this study mainly analyzed the overall force acting on suspension bridges following cable clamp slippage.

In this paper, the influence of pin-type cable clamp slippage on a self-anchored suspension bridge is reported. Considering the geometric nonlinear influence of the bridge, a finite element simulation was conducted. First, we selected a large-span self-anchored bridge as an example to describe the geometry of self-anchored suspension bridges and the magnitude of the cable clamp slippage in detail. Subsequently, a method for simulating the cable clamp slippage was proposed and verified through examples. Additionally, the total force acting on the suspension bridge before and after cable clamp slippage was analyzed, and recommendations for operation and maintenance was provided.