**2. Materials and methods**

#### **2.1. Materials**

of cyclic nucleotides in the normoxic newborn pulmonary circulation assist in the downregulation of postnatal PAFR-mediated responses and that under hypoxic conditions, increasing the levels of cyclic nucleotides will abrogate PAF-mediated vasoconstriction thereby ameliorating PAF-induced persistent pulmonary hyperten‐

Platelet-activating factor (PAF) is an endogenous phospholipid which evokes a wide range of biological activities, such as vasoconstriction and systemic hypotension [1], mainly under pathophysiological conditions. The discovery of PAF, its cellular origin, and biological actions were first reported by Benveniste and Associates [2, 3]. Following these reports, investigations of the physiological effects of PAF involved its roles in fetal lung maturation and lung function [4-9], and its role in reproduction where it is involved in implantation of embryos, among other effects [10, 11]. PAF produces a myriad pathological effects in vivo, including platelet aggre‐ gation [12-14], mediation of immune response and bronchoconstriction [15, 16], and smooth muscle contraction [17-21], which hinges on its role as an inflammatory mediator and vaso‐ constrictor [17]. In the fetus, PAF plays an important physiological role in maintaining a high level of vasomotor tone in the pulmonary circulation [22]. Therefore, the high PAF receptor (PAFR) binding in fetal lamb lungs supports the existence of a high level of pulmonary vasomotor tone in utero [23]. On the other hand, in lungs of the newborn lamb, PAFR binding and receptor mRNA expression are low, suggesting a down regulation of PAFR-mediated

PAF acts by binding to its Gq protein isoform of G protein-coupled receptors, which is a seven transmembrane receptor [24]. Activation of G protein-coupled receptors by an agonist results in activation of signal transduction pathways [25], which may involve recruitment of intra‐ cellular second messengers such as cAMP, cGMP, inositol 1,4,5-triphosphate (IP3), and calcium [26, 27]. cAMP and cGMP act via their endogenous receptors, cAMP-dependent protein kinase (cAMP/PKA) and cGMP-dependent protein kinase (cGMP/PKG), respectively, to elicit relaxation of smooth muscle, and cAMP and cGMP mediate relaxation of pulmonary vessels, but cGMP has been shown to be more effective than cAMP in producing relaxation of perinatal ovine pulmonary vessels [28, 29]. Acute hypoxia upregulates PAFR-mediated intracellular signaling in fetal ovine pulmonary vascular smooth muscle [30]. Chronic hypoxia in the perinatal period may result in abnormal upregulation of PAFR protein expression, PAFR binding, and PAFR-mediated cell signaling, leading to increased pulmonary vasomotor tone and vascular remodeling, a key event in the onset of clinical disorders such as persistent

We are interested in understanding the mechanisms of pulmonary vascular relaxation at birth. Our primary hypothesis is that with oxygenation at birth and the increased production of

**Keywords:** Pulmonary artery, PAFR binding, cyclic nucleotides, siRNA

sion of the newborn.

**1. Introduction**

460 Muscle Cell and Tissue

effects in vivo [22, 23].

pulmonary hypertension of the newborn (PPHN) [31].

The study was approved by the Institutional Animal Care and Use Committee of Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Pregnant ewes (146-148 d gestation, term being 150 d) were purchased from Nebekar Farms (Santa Monica, CA). Authentic standards of PAF (C16-PAF) as well as 8-Br-cAMP, Rp-cAMPS, 8-Br-cGMP, Rp-8 pCPT-cGMPS were purchased from Biomol, Plymouth Meeting, PA. Radiolabeled PAF standards and substrates: hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, 1-O-[ace‐ tyl-3 H-(N)]-, (3 H-acetyl-C16-PAF), 21.5 Ci/mmol (370 GBq/mmol), and 3 H-thymidine were purchased from Perkin Elmer Life Sciences (Boston, MA). siRNA to PKA-Cα and its control were purchased from Cell Signaling Technologies (Carlsbad, CA). Phenylmethysulfonyl fluoride, leupeptin, pepstatin, as well as bovine serum albumin, were purchased from Sigma Chemical Company (St. Louis, MO). Antibody to PKA and PKG were purchased from Cell Signaling, while PAFR antibody was purchased from Cayman Chemical Company (Ann Arbor, MI). Studies were done with freshly made reagents. Ecolite(+) liquid scintillation cocktail was purchased from MP Biochemicals (Irvine, CA).

### **2.2. Methods**

Arterial intrapulmonary vessels were isolated from freshly killed term fetal lambs and then smooth muscle cells were harvested from the excised arteries and veins under sterile condi‐ tions as previously reported [20, 32]. Cells were used at the 3rd to 10th passage. Cell phenotype did not change from 1st to 10th passage as determined by the expression of α-smooth muscle actin and myosin light-chain kinase proteins.

#### **2.3. Study conditions**

Studies were done with adherent cells in normoxia and in hypoxia.

*Normoxia:* Cells were studied in humidified incubator at 37 °C aerated with 5% CO2 in air.

*Hypoxia:* An incubator set at 37 °C was first equilibrated for at least 1 h with a gas mixture of 2% O2, 10% CO2, and balance N2 to maintain incubator culture media pO2 < 40 Torr, and to mimic fetal lung environment, and monitored with TED 60T percent oxygen sensor, Teledyne Analytical Instruments (City of Industry, CA). Cells were then placed in this equilibrated incubator and continuously aerated with the hypoxia gas mixture throughout the duration of the study.

#### **2.4. Study of PAFR binding**

### *2.4.1. General protocol*

Receptor-binding assays were performed during hypoxia and normoxia as we previously reported [30]. Briefly, after incubation in normoxia or hypoxia, unbound 3 H-PAF was washed off with ice-cold phosphate buffer saline, and then incubated on ice for 30 to 45 min in saline/ EDTA mixture containing 154 mM saline and 5 mM EDTA. Receptor bound 3 H-PAF was extracted on Whatman GF/C membrane filters using inline vacuum system. Then culture flasks or dishes were washed with calcium-free 0.25% bovine serum albumin-containing Tyrodes buffer, pH 6.4. Cell-bound PAF radioactivity was quantified by scintillation spectrometry (Beckman Instruments, Fullerton, CA). In studies probing the interaction of PAF with its receptors in the presence of other agonist or antagonists, cells were pre-incubated with the agent before the addition of 3 H-PAF, and then incubated further according to the specific experimental protocol.

#### *2.4.2. Specific protocols*

*Effect of cAMP/cGMP on PAFR binding:* Cells were pre-incubated for 30 min, in normoxia or hypoxia, with buffer for controls or 10 µM each of cell permeable cyclic nucleotide analogs, 8- Br-cAMP or 8-Br-cGMP; the cAMP/PKA receptor inhibitor Rp-cAMPS or the cGMP/PKG receptor inhibitor Rp-8-pCPT-cGMPS. Then 1.0nM 3 H-PAF was added and incubated for 30 min more.

*Effect of cAMP/cGMP on PAFR protein expression:* Cells were pre-incubated for 3 hr min, in hypoxia or normoxia, with buffer for controls or 10 µM each of cell-permeable cyclic nucleotide analogs, 8-Br-cAMP or 8-Br-cGMP. Membrane proteins were isolated from cultured cells and probed for PAFR protein expression by Western blotting.

*Proliferation assay:* Sub-confluent cells were serum starved by culturing in 0.1% fetal bovine serum (FBS) for 72 hr, then cells were cultured in 10% FBS with or without the test agents in the presence of 5µCi/well of 3 H-thymidine and incubated for 24 hr more in the oxygen condition [32]. Cell DNA that were labeled with 3 H-thymidine were extracted with 0.5N NaOH and quantified on LKB 6500 scintillation counter (Beckman Coulter, Fullerton, CA).

*Transient cell transfection:* Cells were seeded in 6-well culture plates at 5 × 104 cells per well in an antibiotic-free growth media and allowed to stabilize for 24 h. Then they were treated with 1.5µg/ml of each plasmid in lipofectamine transfection reagent with 50nM of PKA-Cα siRNA according to the vendor's protocol (Cell Signaling) and incubated for 48 hr after which the transfection medium was replaced with fresh 10% FBS culture medium, which was also used to study cell proliferation or to study PAFR binding. Transfection efficiency was between 25% and 35% within 24 hr of transfection as judged by the pGFP fluorescence [32]. The proliferative phenotype of transfected cells was compared to that of untransfected cells. In studying cell proliferation or PAFR binding, transfected cells were incubated for 24 hr for proliferation study or 30 min for PAFR binding, with and without 10nM PAF in 10% FBS. Control for cell proliferation is 10% FBS alone while cells transfected with scrambled siRNA (sham siRNA) were used as control for PKA-Cα siRNA effect.

#### **2.5. Western blotting**

mimic fetal lung environment, and monitored with TED 60T percent oxygen sensor, Teledyne Analytical Instruments (City of Industry, CA). Cells were then placed in this equilibrated incubator and continuously aerated with the hypoxia gas mixture throughout the duration of

Receptor-binding assays were performed during hypoxia and normoxia as we previously

off with ice-cold phosphate buffer saline, and then incubated on ice for 30 to 45 min in saline/

extracted on Whatman GF/C membrane filters using inline vacuum system. Then culture flasks or dishes were washed with calcium-free 0.25% bovine serum albumin-containing Tyrodes buffer, pH 6.4. Cell-bound PAF radioactivity was quantified by scintillation spectrometry (Beckman Instruments, Fullerton, CA). In studies probing the interaction of PAF with its receptors in the presence of other agonist or antagonists, cells were pre-incubated with the

*Effect of cAMP/cGMP on PAFR binding:* Cells were pre-incubated for 30 min, in normoxia or hypoxia, with buffer for controls or 10 µM each of cell permeable cyclic nucleotide analogs, 8- Br-cAMP or 8-Br-cGMP; the cAMP/PKA receptor inhibitor Rp-cAMPS or the cGMP/PKG

*Effect of cAMP/cGMP on PAFR protein expression:* Cells were pre-incubated for 3 hr min, in hypoxia or normoxia, with buffer for controls or 10 µM each of cell-permeable cyclic nucleotide analogs, 8-Br-cAMP or 8-Br-cGMP. Membrane proteins were isolated from cultured cells and

*Proliferation assay:* Sub-confluent cells were serum starved by culturing in 0.1% fetal bovine serum (FBS) for 72 hr, then cells were cultured in 10% FBS with or without the test agents in

*Transient cell transfection:* Cells were seeded in 6-well culture plates at 5 × 104 cells per well in an antibiotic-free growth media and allowed to stabilize for 24 h. Then they were treated with 1.5µg/ml of each plasmid in lipofectamine transfection reagent with 50nM of PKA-Cα siRNA according to the vendor's protocol (Cell Signaling) and incubated for 48 hr after which the transfection medium was replaced with fresh 10% FBS culture medium, which was also used to study cell proliferation or to study PAFR binding. Transfection efficiency was between 25%

and quantified on LKB 6500 scintillation counter (Beckman Coulter, Fullerton, CA).

H-PAF, and then incubated further according to the specific

H-thymidine and incubated for 24 hr more in the oxygen

H-PAF was added and incubated for 30

H-thymidine were extracted with 0.5N NaOH

H-PAF was washed

H-PAF was

reported [30]. Briefly, after incubation in normoxia or hypoxia, unbound 3

EDTA mixture containing 154 mM saline and 5 mM EDTA. Receptor bound 3

the study.

462 Muscle Cell and Tissue

**2.4. Study of PAFR binding**

agent before the addition of 3

the presence of 5µCi/well of 3

receptor inhibitor Rp-8-pCPT-cGMPS. Then 1.0nM 3

probed for PAFR protein expression by Western blotting.

condition [32]. Cell DNA that were labeled with 3

experimental protocol.

*2.4.2. Specific protocols*

min more.

*2.4.1. General protocol*

*Preparation of proteins for Western analysis:* Proteins were prepared from stimulated and unstimulated cells that were studied in normoxia or hypoxia according to our previous reports. Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Western blotting for PAFR quantification following our published methods [20, 30, 32, 33]. After x-ray exposure, band corresponding to PAFR protein was quantified and normalized to β-actin density.

#### **2.6. Data analysis**

All numerical data are mean ± SEM. In all instances where radioisotope was used, background radioactivity was subtracted before quantifying radioactivity. Data were analyzed with twotailed *t*-test followed with ANOVA (GraphPad Prism 6, San Diego, CA). Results were consid‐ ered significant at *p* < 0.05.


**3.3 Summary**

As mentioned above, the usefulness of the circuit that can be designed by arbitrarily determining the three matching frequencies using the LC-ladder type configuration was examined. It was analytically and experimentally shown that a dual-band power divider with an absolute constant bandwidth in the UHF/SHF (sub6) band can be realized by closing the two matching frequencies to each other on the lowfrequency side. The design method in this study is considered to be very useful in the situation where the use of the SHF band becomes more active due to social factors such as the spread of 5G. In the future, we plan to conduct an experimental study on the design of power dividers in the higher frequency range, such as the X-Band.


*2.2.2 Objective 2: identification and ranking of different threats to BAT conservation and their causes*

Different threats to BAT conservation in its natural ecosystems and their causes, Mt. Elgon areas (Site 1) were identified in each zone and classified under three major threat categories, namely human-use threat, livestock threat and habitat destruction threat.

In each study zone, the identified threats and their causes were arranged and ranked as; very high, high, medium, low and very low when the threat in question is capable of destroying at least 80%, 70–80%, 50–69%, 30–49%, and below 30% of in-situ BAT species, respectively [13]. The threats are ranked as Very High: when a very large area of conservation target species and site is likely to be destroyed and High: when many locations of the target conservation site and the number is likely to be destroyed. Medium thread occurs when it's localized in its scope with a moderate degradation level and affects limited locations of the target conservation zone. Low and very low threats occur when the threat only impairs small and insignificant portions of the target conservation area and species; respectively [13].


*10.4.1 Scanning electron microscopy (SEM)*

The protein morphology of the extracted collagen can be studied using SEM. The morphology of the extracted collagen can be compared to the standard bovine

#### **Figure 8.**

*SEM images of extracted collagen, with 1) acid-soluble collagen of Catla fish (a), pepsin-soluble collagen of Catla fish (B), acid-soluble collagen of Rohu fish (C) and pepsin-soluble collagen of Rohu fish (D) [139], (2) being from buffalo skin [14] and 3) being SEM image of porcine skin collagen [142] (used with permission).*

collagen available in the market. The expected microstructure of collagen from SEM images would be to observe collagen sheets which would be a combination of collagen fibrils and fibres that are bundled together to form a fibril network and dense sheet-like structure.

**References**

p. 929–958.

[1] Shoulders, M.D. and R.T. Raines, *Collagen structure and stability.* Annual review of biochemistry, 2009. **78**:

*DOI: http://dx.doi.org/10.5772/intechopen.94266*

*Collagen: From Waste to Gold*

[10] Schmidt, M., et al., *Collagen extraction process.* International Food Research Journal, 2016. **23**(3).

[11] The European IPPC Bureau, *Best available techiques (BAT) reference document for the tanning of hides and*

[12] Wallace Corporation Limited. *Wallace Corporation Limited*. 2015 [cited 2015 5/05/2015]; Available from: http://

[13] Palacios, M.J.E.L., Maria Angeles Perez; Aran, paqui, *From leather waste to functional leather* 2016, European Commission through the LIFE+ Programme Elda, Spain.

[14] Rizk, M.A. and N.Y. Mostafa, *Extraction and Characterization of Collagen from Buffalo Skin for Biomedical*

*Applications.* Oriental Journal of Chemistry, 2016. **32**(3): p. 1601–1609.

[15] Maffia, G., et al., *Collagen processing.* Journal of the American Leather Chemists Association, 2004. **99**(4): p. 164–169.

[16] SigmaAldrich. *Calf Skin collagen* 2018; Available from: http://www.sig maaldrich.com/catalog/product/sigma/

[17] Yang, L., *mechanical properties of collagen fibrils and elastic fibres explored by AFM*. 2008, University of Twente

[18] Stryer, L., *Biochemistry*. 1975, New York: W. H. Freeman and Company.

[19] Neuman, R.E. and M.A. Logan,*The determination of collagen and elastin in tissues.* Journal of Biological Chemistry,

[20] Naked Nutrition. *Collagen synthesis*. 2020 10/02/20]; Available from: https://

c3511?lang=en&region=NZ.

Enschede The Netherlands

1950. **186**(2): p. 549–556.

*skins*. 2013: Seville.

www.wallace.co.nz/.

[2] Gelse, K., E. Pöschl, and T. Aigner, *Collagens—structure, function, and biosynthesis.* Advanced drug delivery reviews, 2003. **55**(12): p. 1531–1546.

[3] Peng, Y., et al., *Evaluation for collagen*

*Mechanical Properties of Heat-cured Whey Protein-based Edible Films Compared with*

*Manufacturing Conditions.* Journal of food science, 2005. **70**(2): p. E131-E134.

*products for cosmetic application.* International Journal of Cosmetic

[4] Simelane, S. and Z. Ustunol,

*Collagen Casings under Sausage*

[5] MBG, P.M.C., et al., *An oral supplementation based on hydrolyzed collagen and vitamins improves skin elasticity and dermis echogenicity: a clinical placebo-controlled study.* Clin Pharmacol Biopharm, 2015. **4**(142): p. 2.

[6] O'Sullivan, O., et al., *Extraction of Collagen from Fish Skins and Its Use in the*

Aquatic Food Product Technology 2006.

*Manufacture of Biopolymer Films.*

[7] Meena, C., S. Mengi, and S. Deshpande. *Biomedical and industrial applications of collagen*. in *Proceedings of the Indian Academy of Sciences-Chemical*

[8] Pereira, R.F. and P.J. Bártolo, *3D bioprinting of photocrosslinkable hydrogel constructs.* Journal of Applied Polymer

[9] Aberoumand, A., *Comparative study between different methods of collagen extraction from fish and its properties.* World Applied Sciences Journal, 2012.

*Sciences*. 1999. Springer.

Science, 2015. **132**(48).

**16**(3): p. 316–319.

**231**

**15**(3): p. 21–32.

Science **26**(6): p. 313.

Ramanathan et al. [140] used SEM to observe the surface morphologies of freeze-dried acid-solubilised fish skin collagen. The images showed a smooth surface texture, in two of the images, a layer-by-layer structure was observed (no definite fibres), and this was related to the intertwining of collagen fibres. Similarly, Rizk et al. [14], Tziveleka et al. [141], Rodrigues et al. [142], Pal et al. [139] all carried out SEM to assess the surface morphology of extracted collagen and all showed SEM images to have smooth or slightly wrinkled surfaces or sheet-like structures (**Figure 8**).

#### *10.4.2 Transmission electron microscopy (TEM)*

Transmission electron microscopy is usually carried out to observe collagen fibril structure and it is uniformity in a much deeper level. SEM only provides limited information on collagen morphology. **Figure 8** is showing an electron transmission image of mammalian lung tissue collagen at a magnification of 50 nm, while is showing a TEM image of collagen fibrils and fibres.

The preparative steps of collagen TEM are very specific and usually requires a technician to carry out each step carefully in order to observe the fibrillar structure of collagen. The Karnovsky fixative is mostly used as a preparative method prior to taking TEM images.


**3. Simulation of rising vapour bubble in convective boiling conditions using the ISM method**

Here, two examples of rising vapour bubble simulations due to the convective boiling conditions are illustrated. Firstly, vapour bubble growth in superheated water; secondly, bubble condensation in sub-cooled boiling conditions.


**4.3 X-ray diffraction analysis (XRD)**

X-ray diffraction patterns of all studied samples are shown in **Figure 3**.

In order to provide a more effective visualization of involved samples, as well as their behavior in the present study, components were separated into individual graphs, according to **Figures 4-7**, as follows.

#### **Table 3.**

*Use of Gamma Radiation Techniques in Peaceful Applications*

**(ATR-FTIR)**

4000–650 cm−1

range.

**3.6 Tensile and elongation at break**

relative humidity, for 24 h, prior to testing.

lizing from melt, are shown in **Figure 1**.

**4.1 Differential scanning calorimetric analyses (DSC)**

*Melting and crystallization curves for PLA, PBAT, and PLA/PBAT (50/50).*

**4. Results and discussion**

**3.5 Attenuated total reflection Fourier-transform infrared spectroscopy**

FTIR is a sensible method for identifying chemical modification in a material and, so, is capable to detect chemical modifications in a polymeric material. This method detects vibrational movements imparted from chemical bonds for the material that is being analyzed. As each chemical group absorbs vibrational energy at a given value, it is possible to differentiate them via infrared spectrum. Spectra were obtained from a PerkinElmer, universal ATR sampling accessory spectrum 100 FTIR spectrometer. Setup collection sample was adjusted for 64 scans, within a

Tensile and elongation at break essay are relevant instruments for evaluating loss of properties and evolution of degradative process of the polymer. Parameters that contribute for mechanical behavior of polymers are chemical structure, crystallinity degree, molar mass, moisture, and reinforcing agent present, among others. All these properties are modified during degradation processes. In case of reinforcing agents, the concentration is not changed; nevertheless, their interaction can be modified in consequence of chemical modifications suffered by the polymer. Tensile and elongation at break tests were accomplished at 25 ± 5°C, in an EMIC

dance with ASTM D 638-14. Specimens were conditioned at 25 ± 5°C and 50 ± 5%

DSC heating curves of PLA, PBAT, and PLA/PBAT (50/50) blends, after crystal-

PLA was primarily amorphous when it was cooled from melt, and this result

suggests that PLA was not able to crystallize within the cooling time frame.

, in accor-

model DL 300 universal essay machine, 20 kN load cell, 50 mm min−1

**144**

**Figure 1.**

*Thermal properties of materials studied.*

**Figure 2.** *TG and DTG curves for PLA, PBAT, and PBAT/PLA (50/50).*

**Figure 4.**

*DRX diffractograms of basic components: PBAT, PLA, CaCO3, and PLA gamma-radiated at 150 kGy.*

In general, the sample is composed by crystals and amorphous phases: the sharp peaks are related to crystallite diffraction, and larger peaks are related to amorphous phases. In **Figure 4**, pure PBAT and PLA exhibited four peaks in 17.5, 20.5, 22.5, and 24.5°, in which 22.5° 2θ was the most intense. Bio-CaCO3 exhibited the most intense peak at 30.0 2θ, among other crystalline ones. PLA gamma-irradiated at 150 kGy exhibited two peaks at 20.0 and 22.0, 2θ, proving the efficacy of gamma irradiation treatment.

PBAT/PLA blends, 82/18 and 65/35, corresponding to **Figures 5** and **6**, respectively, as well as their composites, exhibited two intense peaks at 22.5 and 30.0 2θ, emphasizing that composites showed a higher intensity for peaks than based blends.

PBAT/PLA blend (50/50) and its composite showed just one intense peak at 30.0 2θ, much more intense for corresponding composite.

#### **4.4 Scanning electron microscopy (SEM)**

The cell morphology of all formulations processed using SEM is shown in **Figures 8** and **9**. Images were taken in a 100 × magnification, confirming structural foam nature [45].

**147**

**Figure 6.**

*gamma-radiated at 150 kGy.*

**Figure 5.**

*gamma-radiated at 150 kGy.*

mens are shown.

*Study of Bio-Based Foams Prepared from PBAT/PLA Reinforced with Bio-Calcium Carbonate…*

*DRX diffractograms of PBAT/PLA (82/18) and their compositions with 10 phr of CaCO3 and 5 phr of PLA*

The higher PBAT concentration in PLA/PBAT blends, the easier will be the miscibility between both PBAT and PLA, as shown in **Figure 8**; in **Figure 9** pure

*DRX diffractograms of PBAT/PLA (65/35) and their compositions with 10 phr of CaCO3 and 5 phr of PLA*

Addition of PLA gamma-irradiated at 150 kGy contributed for an effective distribution of bio-calcium carbonate 125 μm reinforcement in PBAT/PLA compo-

In **Figure 11** foamed samples obtained from 4 mm die extruder and final speci-

*sea-phase* type morphology, as can be observed in **Figure 9a** and **b**.

sitions and buildup of structural foams, as can be seen in **Figure 10**.

PLA shows an irregular dispersion and PBAT a continuous phase in blends; that is, PLA has a typical and irregular morphology *island-phase* type and the PBAT a

PLA and PBAT micrographs are presented.

*DOI: http://dx.doi.org/10.5772/intechopen.85462*

*Study of Bio-Based Foams Prepared from PBAT/PLA Reinforced with Bio-Calcium Carbonate… DOI: http://dx.doi.org/10.5772/intechopen.85462*

#### **Figure 5.**

*Use of Gamma Radiation Techniques in Peaceful Applications*

In general, the sample is composed by crystals and amorphous phases: the sharp

PBAT/PLA blends, 82/18 and 65/35, corresponding to **Figures 5** and **6**, respectively, as well as their composites, exhibited two intense peaks at 22.5 and 30.0 2θ, emphasizing that composites showed a higher intensity for peaks than based blends. PBAT/PLA blend (50/50) and its composite showed just one intense peak at 30.0

The cell morphology of all formulations processed using SEM is shown in **Figures 8** and **9**. Images were taken in a 100 × magnification, confirming structural

peaks are related to crystallite diffraction, and larger peaks are related to amorphous phases. In **Figure 4**, pure PBAT and PLA exhibited four peaks in 17.5, 20.5, 22.5, and 24.5°, in which 22.5° 2θ was the most intense. Bio-CaCO3 exhibited the most intense peak at 30.0 2θ, among other crystalline ones. PLA gamma-irradiated at 150 kGy exhibited two peaks at 20.0 and 22.0, 2θ, proving the efficacy of gamma

*DRX diffractograms of basic components: PBAT, PLA, CaCO3, and PLA gamma-radiated at 150 kGy.*

**146**

foam nature [45].

irradiation treatment.

**Figure 4.**

**Figure 3.**

*DRX diffractograms of all studied samples.*

2θ, much more intense for corresponding composite.

**4.4 Scanning electron microscopy (SEM)**

*DRX diffractograms of PBAT/PLA (82/18) and their compositions with 10 phr of CaCO3 and 5 phr of PLA gamma-radiated at 150 kGy.*

#### **Figure 6.**

*DRX diffractograms of PBAT/PLA (65/35) and their compositions with 10 phr of CaCO3 and 5 phr of PLA gamma-radiated at 150 kGy.*

The higher PBAT concentration in PLA/PBAT blends, the easier will be the miscibility between both PBAT and PLA, as shown in **Figure 8**; in **Figure 9** pure PLA and PBAT micrographs are presented.

PLA shows an irregular dispersion and PBAT a continuous phase in blends; that is, PLA has a typical and irregular morphology *island-phase* type and the PBAT a *sea-phase* type morphology, as can be observed in **Figure 9a** and **b**.

Addition of PLA gamma-irradiated at 150 kGy contributed for an effective distribution of bio-calcium carbonate 125 μm reinforcement in PBAT/PLA compositions and buildup of structural foams, as can be seen in **Figure 10**.

In **Figure 11** foamed samples obtained from 4 mm die extruder and final specimens are shown.

**Figure 7.**

*DRX diffractograms of PBAT/PLA (50/50) and their compositions with 10 phr of CaCO3 and 5 phr of PLA gamma-radiated at 150 kGy.*

#### **Figure 8.**

*SEM micrographs of PLA/PBAT blends, 100 X magnification: (a) PBAT/PLA, 82/18; (b) PBAT/PLA, 65/35; (c) PBAT/PLA, 50/50.*


**1. Introduction**

Gamification, as detailed throughout this publication, is a diverse and multifaceted process, which can be applied to many if not all areas of society and industry. The focus here is on the field of the Arts, and more specifically in cultural spaces, such as Art Galleries and Museums. This context offers much to a prospective scholar of gamification; willing audiences, compelling objects and spaces to unveil and hidden narratives to expose [1, 2]. This book chapter explores the development and delivery of a gamified, augmented reality smartphone application called TNAR (Temple Newsam Augmented Reality). The application, being educational in focus and site-specific in nature, was situated within Temple Newsam, a stately home on the outskirts of Leeds (UK), often referred to as the Hampton Court of the north. Here,

the intention was to develop gamified mechanics to not only motivate and maintain user engagement, but also to be the vehicle for the delivery of the educational content, thus being at once the medium and the message.

In order to develop and deliver the TNAR application, and subsequently to assess its efficacy, a number of areas are considered. Initially, a consideration of gamification as a means to motivate engagement and the characteristics of gamification that are perceived to foster this motivation will be undertaken, reflecting upon the dichotomy between the mechanistic and experiential perspectives [3] (Sections 2). Following this, an exploration of the relationship between pleasure and motivation when game playing will be undertaken, with a focus on individual play, in which one participant interacts with a gamified system, this being the most common model applied to cultural spaces [4] (Sections 3 and 4). This exploration of the relationship between play and pleasure supports the identification of three distinct characteristics of individual play: challenge, completion and creation. These characteristics are used to reflect upon existing examples of gamified systems placed in cultural spaces, the capacity of each to elicit these characteristics of play, and the impact this had on the experience of the participant (Section 5). This then is the rationale used in the design and development of the TNAR project (Section 6). A description of project implementation and a reflection on methodology applied to capture participant play is provided in Section 7. Finally, a reflection on the results obtained through observing participants engage with the TNAR project is presented in Section 8, with overarching conclusions and closing remarks contained in Section 9.


**2. Properties and ion transport mechanism in PEO**

PEO has chemical structure H▬(O▬CH2▬CH2)n▬OH which is a polyether compound. Depending on the molecular weight of polymer, it is also known as polyethylene glycol (PEG). Generally, polymer having molecular weight above 20,000 g/ mol is called PEO while, below 20,000 g/mol is known as PEG [21]. PEO is a low toxic compound; therefore, it is widely used in many applications such as chemical, industrial, medical, biological etc. PEO has ethylene oxide unit which provides flexibility and high donor number for Li+ ions and promotes the ion transport. Also, it has high dielectric constant and Li+ ion solvating ability [22]. The ion transport mechanism in PEO occurs by the coordination of Li+ ions with the ether oxygen atom of PEO chain. This process occurs by formation and dissociation of Li▬O bond by local segmental motion of polymer chain (**Figure 2**) [23]. The ion transportation in polymer

electrolytes mainly depends on polymer chain segmental motion which becomes faster in amorphous region or above the glass transition temperature (Tg) of polymer (as shown in **Figure 2(a)**). Hence, ideally polymer should have lower Tg to remain in rubbery state or to promote the Li+ ion conductivity at RT [17]. In order to enhance Li+ ion conductivity of polymer electrolytes different strategies such as addition of nano fillers, use of plasticizers or gel polymer electrolytes has been proposed. On this basis, polymer electrolytes are classified into different categories as discussed below.


3.2.1. Impacts on Traffic in Barcelona

Upon changing the respective speed limit parameters within TransCAD®, the model was reapplied to assess how the traffic flows within the city have changed. Figure 14 shows speed difference maps calculated from the TransCAD® model runs for a 1 in 100 year event for the Baseline and Climate Change scenarios. Overall appearance shows a similar spatial distribution of speed reductions with slight increases (higher negative values) in reductions for the Climate Change Scenario.

**Figure 14.** Comparison of speed differences for 1 in 100-year event for Baseline and Climate Change Scenarios.

Figure 15 shows a comparison of the derived Exceedance Probability (EP) Curves for the Baseline and Future Climate Change prediction scenarios based on summation of all the link cost values. This curve was derived via plotting the total accumulated losses for a pluvial flood event across the network with respect to the probability of occurrence (1/Return Period) of the event. Here, we see that under future climate change conditions the predicted monetary losses/impacts when traffic disruption increases with respect to the severity of the flood event. Table 7 shows a comparison of the loss values with an average increase of monetary losses due to the vehicular speed in the network was reduced by 10% or more under future climate change scenarios.

**Figure 15.** Flood Impact on Traffic EP Curves for Barcelona.

**Table 7.** Monetary losses as a result of traffic speed disruption.


*2.2. Materials*

In the presented study, the Corine Land Cover (CLC) database from 2018, Chief Geodesy and Cartography Office (GUGiK), and OpenGeodata of Nordrhein-West walen data were used. The analyses were conducted using the MapInfo Pro 17.0.

It must be emphasized that studies relying on CLC data have certain limitations, such as the detailed nature of the input data and a high degree of generalization. The CLC is a far more useful resource for small-scale studies, but it is a less reliable tool for analyses conducted on a larger scale [47].

The CLC has its own nomenclature that is consistent over the databases from different years and for the whole area. The nomenclature consists of 3 hierarchical levels, which are called standard levels. There are five main classes of land cover (level 1 and 2):

Different land-use types within each of the above groups are specified at the second and third levels of the inventory. In the presented study, level 3 was chosen (see Table 2 in Section 2.3). The CLC uses a Minimum Mapping Unit (MMU) of 25 hectares (ha) for areal phenomena and a minimum width of 100 m for linear phenomena. The CLC vector layers were downloaded from the Pan-European datasets of Copernicus Land Monitoring Services.

**Table 2.** The values of the degree of belonging of land cover to the urban landscape.


**4.2 Algorithm of MALESAbrain**

To trace the algorithm, we suggest readers browsing the definitions first. Then reading the instructions, comments in the algorithm and checking definitions when tracing. Afterwards compare the algorithm with the example in previous section.

MALESAbrain(*kq*,*kr* ,*sm*,*sd*,,,due-date )

> SET *kq* / \* the minimum requirement of an AK-cell to join the competition for promotion to a higher discussion position - called *qualification threshold* (Definition 1-5)\*/

> SET *kr*, / \* the worthless AK-cells will be deleted whenever lower than the *rejection threshold* (Definition 1-5) \*/

SET *sm* / \* a consensus of the learning group agrees the solution is able to solve the problem whenever a solution reached the *solution maturity threshold* (Definition 1-5)\*/

SET *sd* / \* the worthless solutions will be deleted whenever lower than the *solution disagreement threshold* (Definition 1-5) \*/

SET / \* *growth-factor* limits the posted number of AK-cells at each level, which converts the architecture of knowledge base from linear structure into a hierarchical structure of branch tree in the forum. (Definition 1-6) \*/

SET / \* *convergent-factor* is used to decide whether the percentage of mature AK-cells has achieved the educator's training target (Definition 1-7) \*/

SET due-date / \* *due-date* is an expected date to end the discussion \*/

#### REPEAT

/\* *Critical thinking* \*/

*COMPARE personal viewpoint "x" with retained knowledge pieces "ki" in MALESAbrain K* = {*ki*  MALESAbrain} /\* Definition 1-4 \*/

Proposing Two Algorithms to Acquire Learning

**4.3 A calculation example**

therefore is illegal.

(*w1* = 3.1)

(

*s2.1,2*

*i=*

∴*w2.1,1* = 0.9 + 0.5 + (-0.7) = 0.7…①

*jj sw*

,1.2,1.2 || =

= *w2.1,1* · |*s2.1,1*| + *w2.1,2* · |*s2.1,2*|

= 0.7·(1) + (1.4) · (1) = 2.1 (∵

Example 1. The calculation of knowledge weight(*w2.1*).

∴*w2.1,2* = 0.6 + 0.8= 1.4…②

*m*

*w2.1* =

*j*

1

*2.1* = (*p2.1*,

*j*

(*s2.1,2* , *w2.1,2*=1.4)

By Definition 1-3 *wi,j* is the learners' judgment scores towards *si,j*

solutions is two, therefore *m* = 2), so the knowledge-weight is:

= 0.7· |*s2.1,1*| + (1.4)· |*s2.1,2*| (∵ ① and ②)

*K2.1*

a *knowledge-content*

*X:*

*K1*

*K2*

Knowledge in Problem-Based Learning Environment 149

In this subsection, we use a knowledge retained snapshot and a calculation example to look

Discussion topic "How to Fix an Illegal Operation" - An operation requested to be performed by either the Operating System or CPU, which is not understood and

Running a source with a dirty CD or diskettes can cause data to be read

Corrupt, bad or missing files can cause illegal operations.

*s2.1,1* Finding program bugs in the program and fix them. (*s2.1,1* , *w2.1,1*=0.7)

Fig. 12. A snapshot of the retained knowledge pieces in MALESAbrain's knowledge base. Fig. 12 shows a snapshot of knowledge pieces retained in the knowledge base. The knowledge posted by the learners has been organized in a tree-like manner according to the respective weights of individual nodes. In this example shown, the AK-cell *Ki = K2.1* includes

the knowledge-weight *wi=w2.1* bonds with solution *si,j=s2.1,1* and solution *si,j=s2.1,2*. Let us assume three visited learners have given their preferences on solution *s2.1,1* as 0.9, 0.5 and -

0.7; and two visitors have given their preferences on solution *s2.1,2* as 0.6 and 0.8.

*2.1* includes the problem *p2.1* and two solution *s2.1,1* and *s2.1,2* (because the number of

,1.2,1.2 ||

*jj sw*

 )(1 )(0 || *xwhen*

*xwhen x* see Definition 1-3)

∵ *s2.1,1* has three visited learners' judgment scores (0.9, 0.5 and -0.7)

∵ *s2.1,2* has two visited learners' judgment scores (0.6 and 0.8)

*j*

 2 1

It is recommended that you attempt to uninstall and or reinstall the program causing the illegal operation to verify that any corrupt, bad or missing files are replaced or repaired during the reinstallation.

*2.1* and a *knowledge-weight wi=w2.1*. For illustration the calculation on

Running a software or game when memory shortage can cause illegal operations.

into the internal storage structure of the knowledge base of MALESAbrain.

improperly causing illegal operations. (*w2* = 2.9)

 *s2.1,j*), *w2.1* = 2.1)

Learners INPUT their *Pref*:( *learnerk* , *si,j* ) *→ agreementk,i,j* (-1 ~ +1) /\* learners' show their judgment score to others' problem-solutions while browse the forum (Definition 1-2) \*/

IF (*Pref* between –1 ~ +1 but not nil) THEN allow ENTER chat-room or MOVE to next pieces of knowledge /\* Learners must give their preference scores prior to moving on to chat room or next pieces of knowledge for discussion (Definition 1-2) \*/

MALESAbrain COMPUTE the *knowledge-weight m j i jiji sww* 1 ,, || /\* Definition 1-3 \*/

MALESAbrain DISPLAY (or POST) *learners' discussion issues based on "growth-factor "* /\* Definition 1-6 \*/

```
/* Attention */
```
IF *wi kq* THEN /\* the AK-cell is a qualified AK-cell (Definition 1-5,7)\*/ INCREMENT a "qualified-mark" AK-cell *ki* to knowledge base *K* = {*ki*  MALESAbrain} IF *wi* > parent(*wi* ) THEN /\* higher weight AK-cell should be in the front level \*/ SWAP(*ki* ,parent(*ki* ) ) END IF

END IF

IF *wi* < *kr* THEN /\* the AK-cell is a worthless knowledge-piece (Definition 1-5)\*/ DELETE *ki*

END IF

IF *wi,j sm* THEN /\* the AK-cell becomes an matured AK-cell in the knowledge base (Definition 1-5,7)\*/

DISPLAY a "mature-mark" on solution *si,j*

INCREMENT a "mature-mark" AK-cell *ki* to *M* = {*ki* | *ki*  MALESAbrain *si,*j where *wi,j sm*}

END IF

IF *wi,j* < *sd* THEN /\* the solution is worthless solution (Definition 1-5)\*/ DELETE *si,j*

END IF

IF (dd:mm:yy = due-date) THEN

IF ( || || K M ) THEN /\* if learning-rate || || K Mis greater or equal to convergent-factor

```
"" then end discussion (Definition 1-7) */
```
PRINT (MALESAbrain meeting with || || KM% learning-rate)

endDiscussion = TRUE /\* stop the meeting \*/

ELSE

endDiscussion = FALSE

CALL MALESAbrain(*kq*,*kr* ,*sm*,*sd*,,,due-date ) /\* re-calibrate the learning thresholds and start another session of discussion whenever the learning-rate lower than when time is due \*/

END IF

END IF

UNTIL (endDiscussion = TRUE) /\* *learning-rate* \*/ END MALESAbrain


**6.4 Eclampsia**

It is defined as the presence of seizures or coma in a patient with preeclampsia, and that cannot be explained by another underlying cause.

## **6.5 Chronic hypertension**

BP > 140/90 mm Hg before pregnancy or the same obtained values on two separate occasions prior to week 20 of gestation or persistent hypertension after the sixth week postpartum.

### **6.6 Chronic hypertension and pregnancy-induced hypertension**

It is defined as an increase in SAP greater than 30 mm Hg or an increase greater than 15 mm Hg in DAP, on two separate occasions, prior to week 20 of gestation, initial proteinuria, and generalized edema.

## **6.7 Transient or late arterial hypertension**

This is hypertension developing in the postpartum without previous preeclampsia; values return to baseline after the tenth day postpartum.