**Abstract**

This chapter will review the altered brain structure and function associated to drug addiction, with a focus on brain regions involved in learning and motivated behavior. As evidenced by both clinical and preclinical studies, repeated drug exposure affects whole brain neuroplasticity including the mesolimbic system which is a main locus for reward, an action-control center such as the dorsal striatum, and limbic brain regions such as the prefrontal cortex, the hippocampus, and the amygdala that are involved in behavioral control, memory, and mood. In this way, the drug-seeking actions that were initially intentional responses become involuntary habits governed by the dorsal striatum. Drug addiction may also curse with a reduced ability to experience rewards that are unrelated to drugs and emotional dysregulation, while the impairment on limbic regions contributes to generate cognitive symptoms. These entail persistent memories for previous experiences with the drug contrasting with a global cognitive decline that may hamper the acquisition of new, adaptive learnings. Overall, these features promote a desire for the drug, leading to relapse in drug use. Further drug exposure, in turn, aggravates its consequences on the brain and behavior, creating the harmful "addiction cycle."

**Keywords:** substance use disorders, habits, motivation, memory, mood, accumbens, striatum, limbic regions


**Author details**

Nancy S. Landínez-Parra *Group of Mathematical Modeling and Numerical Methods GNUM-UN, Faculty of Engineering, National University of Colombia, Colombia*

*Human Corporal Movement Department, Faculty of Medicine, National University of Colombia, Colombia*

Diego A. Garzón-Alvarado and Juan Carlos Vanegas-Acosta *Group of Mathematical Modeling and Numerical Methods GNUM-UN, Faculty of Engineering, National University of Colombia, Colombia*


*2.1.1. Examples*

and pressure conditions. Sometimes, surfactants are also added to control the growth and

Internal pressure is set up by the amount of temperature and solution used. This process is mainly used for the preparation of small-sized particles for achieving enhanced surface area.

morphology of the particles can be varied by changing crystallization temperature, time and concentration of etching chemicals. **Table 2** shows the various routes by which hierarchical

(TT)

K2 TiO2 (C2 O4 )2 and

Anatase 36.93 15–25μm Ti foil Flower-like

(DEG)

Titanium tetrabutoxide

diethylene glycol

and titanium isopropoxide

microspheres

dichloride

Anatase 64.8 1–1.5μm Titanium powder Radial nanoflakes Gas sensing

(TBT) and acetic

acid

(Ti(SO4 )2 ) and urea

nanostructures produced by various hydrothermal routes.

Anatase 168.3 2–4μm Titanium powder Nanospheres

(CO(NH2 )2 ) from amorphous one. The

DSSCs

DSSCs

DSSCs

DSSCs

Photocatalysis

Photocatalysis

Gas sensing

Photocatalysis

Lithium sulfur battery

**Precursor materials Morphology Used in application**

1D/3D nanorods DSSCs

Flower-like structures

Nanowire trunk on which nanorods are grown

Nanoparticle-based

HNs

Hierarchical microspheres

structure formed from nanobelts

Flower-like shapes formed from nanosheets

Overlapped subunits of nanoflakes

Nanothorn-like hierarchical structures

composed of nanosheets

Basically, this synthesis is used for preparation of crystalline TiO2

structures have been prepared by hydrothermal route.

**Particle size (diameter)**

Rutile 67 1–1.5 μm Tetrabutyl titanate

length) 150 nm diameter

95 nm, NR 5 nm

Anatase — 300–600 nm Dodecylamine

Anatase 27.4 4–6μm Primary Titania

Anatase 170 350 nm Titanocene

Anatase 116.6 3–4μm Tetrabutyl titanate

Anatase — 20–50 nm Titanium sulfate

morphology of target materials.

8 Titanium Dioxide - Material for a Sustainable Environment

**Reference Phase Surface**

**area (m2 g−1)**

Rutile 75.53 (4.4 μm

Anatase — NW trunk

TiO2

Lin et al. [56]

Wang et al. [50]

Qiang et al. [3]

Xiang et al. [57]

Zheng et al. [60]

Shao et al. [58]

Zhu et al. [29]

Wang et al. [32]

Gao et al. [2]

Yang et al. [59]

Min et al. [59]

**Table 2.** Hierarchical TiO2

Lin et al. have reported rutile TiO2 hierarchical flower-like structures via hydrothermal synthesis without using any surfactant. Precursor tetrabutyltitanate (TT) is first mixed with HCl for acidification and is subsequently hydrolyzed using distilled water. To ensure complete hydrolysis, the reaction mixture is stirred for about one and a half hour. Next, the mixture is transferred to a Teflon-based autoclave and is placed in an electric oven for 5 h at 150°C for crystallization. Hierarchical nanoflower-like structures are obtained as an end product. By increasing the HCl concentration, the etching rate of TiO2 structures is enhanced and symmetric flower-like structures are produced [56] (**Figure 4**). These structures are employed for DSSCs and 8.6% of conversion efficiency is achieved.

Wang et al. have prepared rutile Titania 1D/3D structures via hydrothermal treatment. The precursor Titanium tetrabutoxide (0.9 m) is acidified using HCl (16 ml) and then hydrolyzed using DI water (16 ml). The reaction mixture is subsequently heated to 150°C and kept at this temperature for 10 h for crystallization. The position of FTO substrates is varied to obtain different HNSs. 1D/3D HNSs are produced while the FTO substrate lied flat on the bottom of the reactor with the conductive side facing up. 3D nanorods are produced when the conductive side of substrate is placed downwards.

Rutile Titania 3D flower-like nanorods are grown on 1D nanorods with length in microns [50] (**Figure 5**). These structures are employed as a photo anode material in DSSCs and significant improvement in device performance is seen. 1D structure provides directed pathway for electron percolation and 3D morphology provides large surface area for light scattering and dyeloading. Also, the structures exhibit long life time due to less electron-hole recombination.

Qiang et al. have obtained hierarchical anatase TiO2 nanowire trunks with short nanorod branch HNSs by facile one-way hydrothermal synthesis on FTO glass without using any surfactant/stabilizing agent. The solution is prepared using precursor K2 TiO2 (C2 O4 )2 (0.002 mol)

**Figure 4.** FESEM images of TiO2 structures. Change in morphology is produced from changing HCl conc. at (a) 1M (b) 2M (c) 3M (d) 4M (e) 5M (f) 6M (f) 7M (g) 8M [56].

mixed with ethanol (360 ml) and DI water (120 ml) for hydrolysis under vigorous stirring

mixture and white powder of anatase Titania is obtained, which is then washed with water

in different ratios. The solution is transferred to Teflon-lined autoclave, kept at 250°C for 12 h

HNSs made up of symmetrically arranged interconnected spherical nanoparticles are obtained as a result of these syntheses [57]. These structures are employed as a photoanode material in this article. The large spheres can provide maximum scattering of sunlight for light-driven

Teflon-lined autoclave at an inclined angle in 5 M NaOH solution for its reduction. Sodium titanate is formed as a result of this reaction. The temperature is maintained at 220°C for 24 h

with water and ethanol to remove all the acidic content and to maintain its pH at neutral. The

nanoparticles with NaOH results in the formation of Na2

TiOx + NaOH ⟶ Na2 TiO3 + H2 O (1)

Na2 TiO3 + H+ /H2 O ⟶ H2 TiO3 + NaOH (2)

**Figure 7.** TEM images of hierarchical TiO2 spherical structures composed of nanoparticles (a) Agglomerated TiO2

ions can be replaced with H+ ions by washing them with deionized water or acid.

ion. After calcination, nanobelts (**Figure 8**) forming nanoflower-like structures

formed during reaction is removed from the reaction

Hierarchical Nanostructures of Titanium Dioxide: Synthesis and Applications

flower-like structures on Ti foil by placing it in

nanostructures. The sample is then washed

ions of sodium titanate would get

TiO3

, which is a

is added in reaction mixture

11

http://dx.doi.org/10.5772/intechopen.74525

at ambient room temperature. HNO3

reactions like photocatalysis (**Figure 7**).

nanoparticles (b) Dispered TiO2 nanoparticles [57].

Shao et al. have prepared hierarchical TiO2

for complete reaction of converting Ti foil to TiO2

sample is then immersed in HCl solution so that all Na+

nanoporous structure. The reaction that takes place is as follows.

to dope Ta particles with TiO2

replaced by H+

are formed [58].

Reaction of TiO2

It can be shown as:

These Na+

and ethanol to maintain the pH of particles at 7. After that, TaCl5

.

**Figure 5.** (a,d) SEM images of 1D/3D nanorod structures (b) 1D nanorods (c) 3D nanoflower-like structures [50].

**Figure 6.** SEM images of hierarchical anatase nanowire trunk covered by short nanorod branches (a) Nanorods covering the nanotrunks (b) Enlarged image of nanorods (c) Nanotrunks covered by nanorods [3].

and diethylene glycol (DEG) (30 ml) as Titania precursor. About 10 ml H2 O is used for hydrolysis. The solution is then spin coated on FTO substrate for seeding of TiO2 structures. The spin-coated substrate is immersed in Teflon-based autoclave, which is kept at 180°C for 1–12 h for crystallization of Titania structures. **Figure 6** shows nanotrunks produced having nanowire-like structures grown on them [3].

These structures are employed as a photo anode material in DSSCs and impressive power conversion efficiency of 7.34% is achieved. Hierarchical morphology aids in efficient electron transfer but these structures provide additional recombination sites so results are inferior to bare TiO2 nanowires.

Xiang et al. have reported Ta-doped and -undoped hierarchical TiO2 nanostructures. Dodecylamine (8 g) and titanium isopropoxide (TIP, 8 g) are used as precursor materials and are mixed with ethanol (360 ml) and DI water (120 ml) for hydrolysis under vigorous stirring at ambient room temperature. HNO3 formed during reaction is removed from the reaction mixture and white powder of anatase Titania is obtained, which is then washed with water and ethanol to maintain the pH of particles at 7. After that, TaCl5 is added in reaction mixture in different ratios. The solution is transferred to Teflon-lined autoclave, kept at 250°C for 12 h to dope Ta particles with TiO2 .

HNSs made up of symmetrically arranged interconnected spherical nanoparticles are obtained as a result of these syntheses [57]. These structures are employed as a photoanode material in this article. The large spheres can provide maximum scattering of sunlight for light-driven reactions like photocatalysis (**Figure 7**).

Shao et al. have prepared hierarchical TiO2 flower-like structures on Ti foil by placing it in Teflon-lined autoclave at an inclined angle in 5 M NaOH solution for its reduction. Sodium titanate is formed as a result of this reaction. The temperature is maintained at 220°C for 24 h for complete reaction of converting Ti foil to TiO2 nanostructures. The sample is then washed with water and ethanol to remove all the acidic content and to maintain its pH at neutral. The sample is then immersed in HCl solution so that all Na+ ions of sodium titanate would get replaced by H+ ion. After calcination, nanobelts (**Figure 8**) forming nanoflower-like structures are formed [58].

Reaction of TiO2 nanoparticles with NaOH results in the formation of Na2 TiO3 , which is a nanoporous structure. The reaction that takes place is as follows.

$$\text{TiO}\_{\text{x}} + \text{NaOH} \longrightarrow \text{Na}\_{2}\text{TiO}\_{3} + \text{H}\_{2}\text{O} \tag{1}$$

These Na+ ions can be replaced with H+ ions by washing them with deionized water or acid. It can be shown as:

$$\text{Na}\_2\text{TiO}\_3 + \text{H}^+/\text{H}\_2\text{O} \longrightarrow \text{H}\_2\text{TiO}\_3 + \text{NaOH} \tag{2}$$

and diethylene glycol (DEG) (30 ml) as Titania precursor. About 10 ml H2

the nanotrunks (b) Enlarged image of nanorods (c) Nanotrunks covered by nanorods [3].

Xiang et al. have reported Ta-doped and -undoped hierarchical TiO2

ire-like structures grown on them [3].

10 Titanium Dioxide - Material for a Sustainable Environment

nanowires.

bare TiO2

lysis. The solution is then spin coated on FTO substrate for seeding of TiO2

spin-coated substrate is immersed in Teflon-based autoclave, which is kept at 180°C for 1–12 h for crystallization of Titania structures. **Figure 6** shows nanotrunks produced having nanow-

**Figure 6.** SEM images of hierarchical anatase nanowire trunk covered by short nanorod branches (a) Nanorods covering

**Figure 5.** (a,d) SEM images of 1D/3D nanorod structures (b) 1D nanorods (c) 3D nanoflower-like structures [50].

These structures are employed as a photo anode material in DSSCs and impressive power conversion efficiency of 7.34% is achieved. Hierarchical morphology aids in efficient electron transfer but these structures provide additional recombination sites so results are inferior to

lamine (8 g) and titanium isopropoxide (TIP, 8 g) are used as precursor materials and are

O is used for hydro-

nanostructures. Dodecy-

structures. The

**Figure 7.** TEM images of hierarchical TiO2 spherical structures composed of nanoparticles (a) Agglomerated TiO2 nanoparticles (b) Dispered TiO2 nanoparticles [57].

Yang et al. prepared hierarchical Titania structures by hydrothermal synthesis. Titanium sul-

tion was complete and white powder was obtained, which was then moved in Teflon-lined autoclave at 180°C for 10 h for its complete crystallization. The powder formed was washed with DI water and ethanol to maintain pH at neutral and annealed to recrystallize. Reactions

+ 3H2 O ⟶ 2NH4

[Ti(H2 O)(edta)] ⟶ Ti4+ + EDTA + H2 O (4)

Mesoporous nanothorn-like structures are produced as a result of this process [59] shown in **Figure 10**. These structures are employed for gas sensing application of acetone. This hierarchical morphology provides much higher sensitivity and fast response time with minimum

Min et al. prepared hierarchical Titania structures by hydrothermal synthesis. About 1 g of Titanium powder was being dissolved in (0.5 M) 30 ml HF for its oxidation as it is a power-

Teflon-lined autoclave at 150°C for crystallization for 5 h. The powder obtained was washed via centrifugation and was then dried at 80°C. Nanosheets (**Figure 11**) are being produced as

These structures are employed as a photocatalyst material for photodegradation of organic dye. Methylene blue is used as a model dye in this article. Complete photodegradation of

acetic acid (EDTA) disodium salt was added in them as the chelating agent for TiO2

(3 mmol), urea (24 mmol) and EDTA (3 mmol) were mixed and NH4

) were used as precursor materials. Ethylene diaminetetra

Hierarchical Nanostructures of Titanium Dioxide: Synthesis and Applications

TiO3

O)(edta)] with F−1 ions. The reaction mixture was

formation.

13

F (9 mmol) was

. After stirring for 3 h, the reac-

http://dx.doi.org/10.5772/intechopen.74525

+ + 2OH− + CO2 (3)

.H2 O + 8H+ (5)

. This solution was moved to

.H2 O ⟶ 2TiO2 + 2H2 O (6)

fate (Ti(SO4

Ti(SO4 )2 )2

that took place were:

recovery speed.

a result of this process [52].

organic compound is observed in just 60 min.

) and urea (CO(NH2

added in the mixture for attaching [Ti(H2

O〓C (NH2)

H2 Ti2 O5

) 2

2

dissolved into 60 ml of deionized water for formation of H2

2Ti4+ + 6H2 O ⟶ H2 Ti2 O5

ful oxidizing agent. The solution was then mixed with 3 ml NH3

**Figure 10.** FESEM images of hydrothermal synthesis at (a) 5 min, (b) 1 h, (c) 3 h and (d) 5 h [59].

**Figure 8.** SEM images of nanoflower-like hierarchical TiO2 structures produced after annealing at 500°C [58].

**Figure 9.** SEM images of hierarchical TiO2 prepared by hydrothermal heating: (A) after 1 h, (B) after 2 h and (C) after 12 h. (D, E) Annealed powder of sample B [29].

By reduction of nanoparticles and increasing time duration of crystallization, nanoflower-like structures can be grown.

Zhu et al. have prepared HNSs of TiO2 using titanocene dichloride (Ti(Cp)2 Cl2 ) (20 mg) as precursor. DI water (10 ml) is added for hydrolysis, and ethylene diamine (EDA) (2 drops) acts as chelating agent. This results in the production of TiO2 nanocrystals. The mixture then after sonication is placed in an autoclave at 120°C for 1–12 h. The powder obtained is then washed with water and ethanol and is annealed at 400°C for 2 h. Flower-like HNSs (**Figure 9**) are formed in this process [29].

By increasing the duration of hydrothermal synthesis, the nanoparticles have attained more flower-like mesoporous morphology due to increased time provided for etching. These structures have proved to be better photocatalytic agents as compared to nanocrystals of TiO2 as they can provide maximum enhanced surface area, and due to their mesoporosity, maximum dye can be loaded on them. Hence, these structures can be used with perspective of various solar cells and photocatalysis for efficient light-driven reactions.

Yang et al. prepared hierarchical Titania structures by hydrothermal synthesis. Titanium sulfate (Ti(SO4 ) 2 ) and urea (CO(NH2 )2 ) were used as precursor materials. Ethylene diaminetetra acetic acid (EDTA) disodium salt was added in them as the chelating agent for TiO2 formation. Ti(SO4 )2 (3 mmol), urea (24 mmol) and EDTA (3 mmol) were mixed and NH4 F (9 mmol) was added in the mixture for attaching [Ti(H2 O)(edta)] with F−1 ions. The reaction mixture was dissolved into 60 ml of deionized water for formation of H2 TiO3 . After stirring for 3 h, the reaction was complete and white powder was obtained, which was then moved in Teflon-lined autoclave at 180°C for 10 h for its complete crystallization. The powder formed was washed with DI water and ethanol to maintain pH at neutral and annealed to recrystallize. Reactions that took place were:

$$\text{O=C (NH}\_2\text{)}\_2 + 3\text{H}\_2\text{O} \longrightarrow 2\text{NH}\_4^+ + 2\text{OH}^- + \text{CO}\_2 \tag{3}$$

$$\text{[Ti(H}\_2\text{O)(edta)]} \longrightarrow \text{Ti}^{4+} + \text{EDTA} + \text{H}\_2\text{O} \tag{4}$$

$$2\text{Ti}^{\ast} \star \text{6H}\_{2}\text{O} \longrightarrow \text{H}\_{2}\text{Ti}\_{2}\text{O}\_{3}\text{H}\_{2}\text{O} + 8\text{H}^{\ast}\tag{5}$$

$$\text{H}\_{\text{z}}\text{Ti}\_{\text{z}}\text{O}\_{\text{y}}\text{H}\_{\text{z}}\text{O} \longrightarrow 2\text{TiO}\_{\text{z}} + 2\text{H}\_{\text{z}}\text{O} \tag{6}$$

Mesoporous nanothorn-like structures are produced as a result of this process [59] shown in **Figure 10**. These structures are employed for gas sensing application of acetone. This hierarchical morphology provides much higher sensitivity and fast response time with minimum recovery speed.

Min et al. prepared hierarchical Titania structures by hydrothermal synthesis. About 1 g of Titanium powder was being dissolved in (0.5 M) 30 ml HF for its oxidation as it is a powerful oxidizing agent. The solution was then mixed with 3 ml NH3 . This solution was moved to Teflon-lined autoclave at 150°C for crystallization for 5 h. The powder obtained was washed via centrifugation and was then dried at 80°C. Nanosheets (**Figure 11**) are being produced as a result of this process [52].

These structures are employed as a photocatalyst material for photodegradation of organic dye. Methylene blue is used as a model dye in this article. Complete photodegradation of organic compound is observed in just 60 min.

**Figure 10.** FESEM images of hydrothermal synthesis at (a) 5 min, (b) 1 h, (c) 3 h and (d) 5 h [59].

By reduction of nanoparticles and increasing time duration of crystallization, nanoflower-like

precursor. DI water (10 ml) is added for hydrolysis, and ethylene diamine (EDA) (2 drops)

after sonication is placed in an autoclave at 120°C for 1–12 h. The powder obtained is then washed with water and ethanol and is annealed at 400°C for 2 h. Flower-like HNSs (**Figure 9**)

By increasing the duration of hydrothermal synthesis, the nanoparticles have attained more flower-like mesoporous morphology due to increased time provided for etching. These structures have proved to be better photocatalytic agents as compared to nanocrystals of TiO2

they can provide maximum enhanced surface area, and due to their mesoporosity, maximum dye can be loaded on them. Hence, these structures can be used with perspective of various

using titanocene dichloride (Ti(Cp)2

prepared by hydrothermal heating: (A) after 1 h, (B) after 2 h and (C) after 12 h.

structures produced after annealing at 500°C [58].

Cl2

nanocrystals. The mixture then

) (20 mg) as

as

structures can be grown.

**Figure 9.** SEM images of hierarchical TiO2

(D, E) Annealed powder of sample B [29].

are formed in this process [29].

Zhu et al. have prepared HNSs of TiO2

**Figure 8.** SEM images of nanoflower-like hierarchical TiO2

12 Titanium Dioxide - Material for a Sustainable Environment

acts as chelating agent. This results in the production of TiO2

solar cells and photocatalysis for efficient light-driven reactions.

morphology ranges from hierarchical arrangement of nanoparticles to nanorods producing

by increasing duration of hydrothermal synthesis, better etched morphology was obtained. **Table 2** summarizes reported examples of hydrothermal synthesis of HNSs with respect to precursor used for the synthesis and various properties of obtained HNSs together with the application studied. The HNSs obtained from this method varies in size from 20 nm to 4.5

exploited in various applications including solar cells, photocatalysis and sensors, etc. and

In this synthesis, the conditions are the same as for a hydrothermal method but non-aqueous solvent is used instead of water. The process takes place in an autoclave and temperature can be increased because of high boiling of certain organic solvents as compared to water. This method results in uniform particle size distribution and high purity products. Also by changing the temperature, morphology of the grown crystals can be varied. In addition to that, different morphologies result due to differences in steric hindrance offered by different functional groups in various organic solvents. **Table 3** shows various routes adopted to synthesize

vent is added to this solution followed by heating in a 50 ml Teflon-lined autoclave at 150°C for 0.5, 1, 6 and 12 h. The resultant white precipitates are then dried in air. Nanoflower-like structures are grown over the nanorod structures with average crystallite size of 4 nm (**Figure 12**) [10]. These structures are then employed for photocatalytic degradation of methylene blue dye. Complete degradation of organic dye has been achieved in 120 min by these structures. Hence, these HNSs can be used for determining photocatalytic behavior and applications like

> **Precursor materials**

HNSs by solvothermal synthesis technique. Their method

Hierarchical Nanostructures of Titanium Dioxide: Synthesis and Applications

 fibers 3D nanoflowers grown on nanofibers

> like structure composed of nanoneedles

TiCl4 3D urchin-

fibers. Then, 15 ml ethanol sol-

**Morphology Application**

Photocatalysis

Photocatalysis

have improved respective performance owing to their unique structural properties.

particles, and

15

g−1. These structures are

http://dx.doi.org/10.5772/intechopen.74525

flower-like structures. Increasing temperature increases crystallization of TiO2

μm. The surface area of the resultant HNSs ranges from 27 to 170 m2

**2.2. Solvothermal synthesis**

HNSs via solvothermal route.

Ochanda et al. have presented TiO2

**Reference Phase Surface**

**area (m2 g−1)**

Anatase 37 2.5–3.0μm

involves mixing of 15 ml of 4 M NaOH solution with 0.5 g TiO2

**Particle size (diameter)**

(microspheres), 20–40 nm (nanorods)

nanostructures produced via solvothermal method.

Anatase 94.01 50–70 nm TiO2

TiO2

*2.2.1. Examples*

solar cells.

Ochanda et al. [10]

Xiang et al. [35]

**Table 3.** Hierarchical TiO2

**Figure 11.** FESEM images of Titania hierarchical nanospheres composed of nanosheet-like structures [52].


**10.3 Surface modified anodes**

The electrode surface has a tremendous role in the total anode's efficiency. Currently, several reports have stated that surface alteration is advantageous in actuating increased bacterial adhesion and better biocompatibility that favors electron transfer kinetics. The surface alterations using TiO2-carbon fabric-based nanofiber usually attain the highest current density of 7.99 A/m2 , whereas a changed surface with carbon nanotubes and coated with conductive polymer had a maximal power density of 1573 mW/m2 . The two broad surface treatments that are most generally used are silicone coating and graphite or carbon surface application. Each of these surface alteration forms is discussed in subsequent subsections.


*Article*

## **Comparison of the Clinical Efficacy and Adverse Events between Intravesical Injections of Platelet-Rich Plasma and Botulinum Toxin A for the Treatment of Interstitial Cystitis Refractory to Conventional Treatment**

**Jia-Fong Jhang, Wan-Ru Yu and Hann-Chorng Kuo \***

> Department of Urology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Tzu Chi University, Hualien 970, Taiwan

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Background: Intravesical injection of Botulinum toxin A (BoNT-A) and platelet-rich plasma (PRP) have been reported to alleviate bladder pain and decrease nocturia in patients with refractory interstitial cystitis/bladder pain syndrome (IC/BPS). Both treatments are novel and there has no comparison between them. This study compared the therapeutic effects and adverse events between IC/BPS patients receiving PRP or BoNT-A injections. Materials and Methods: This study retrospectively analyzed female patients with IC/BPS who were refractory to conventional treatment and received BoNT-A (*n* = 26) or PRP (*n* = 30) injections within the previous two years. Patients were arbitrarily treated with four monthly injections of PRP or a single injection of 100 U of BoNT-A. All injections were followed by cystoscopic hydrodistention. The primary endpoint was the global response assessment (GRA), and secondary endpoints were changes in the O'Leary-Sant IC symptom score, visual analog score (VAS) of bladder pain, voiding diary, and uroflow measures from baseline to six months after the first injection day. Results: The baseline demographics revealed no significant difference between groups. The GRA at one, three, and six months was similar between groups. A significant improvement in IC symptom scores was noted in both groups. Although VAS was significantly improved in overall patients, no significant difference was noted between the PRP and BoNT-A groups at 6 months. Only half of the study cohort had a GRA ≥2 at six months. An increase in the post-void residual was noted one month after the BoNT-A injection, but there was no difference between groups at three and six months. More patients reported dysuria (19.2% vs. 3.3%, *p* = 0.086) and urinary tract infection (UTI, 15.4% vs. 0%, *p* = 0.041) after BoNT-A injection than after the PRP injections. The time from the first injection to receiving alternative treatment was similar between groups. Conclusion: Both intravesical PRP and BoNT-A injections have similar efficacy in IC symptom improvement. However, only half of the study cohort had a GRA of ≥2 at the six-month follow-up BoNT-A injection carries a potential risk of UTI after treatment.

**Keywords:** botox; platelet-rich plasma; interstitial cystitis; intravesical injection

**Key Contribution:** Intravesical injections of either PRP or BoNT-A are safe and effective for improving IC/BPS symptoms without significantly increasing the PVR. However, only half of patients remained effective at 6 months; repeat treatment was necessary for both groups.


*4.3. A Simulation Model for Rebar Fabrication's Optimal Production Plans*

Figure 4 shows the models for SHD10 and SHD13 and the corresponding verification processes. There are seven components to the simulation of rebar plant processing: (1) raw steel import, (2) transportation, (3) priority and rebar type setting, (4) cutting, (5) rebar reuse, (6) bending, and (7) machine schedule setting.

First, in the simulation model, a rebar with a particular diameter is put up as an agent to represent the raw steel imported into the rebar fabrication plant. Second, the rebar is transported and assigned to a cutter. It is believed that the hoist's maximum capacity can be gathered and transported all at once (for a hoist, the transport time typically varies with the transport distance). Third, different sites from which to collect various rebar types are taken into consideration before processing. Each agent is assigned a priority, which determines the order in which they move through the cutting process. By identifying various sitespecific combinations in this step, the simulation can ultimately choose the optimum site-specific combination when prioritizing the agent. Fourth, the rebar is cut. Similar to

the case of hoists, for which the capacity is set considering simultaneous transportation, the number of simultaneous cuts is set for the cutters.

**Figure 4.** Simulation modeling that reflects processes within a rebar processing plant.

The cutting machine is employed, and the required time is input to represent the number of times the imported rebar can be cut (with the maximum amount) and the time needed to perform one cut, which adds time to the process. The length of the raw steel and the needed length are taken into consideration while defining the cut steel bars, which are then repeatedly moved to the cutting process. Fifth, we considered the reuse of the cut rebar. The following factors determine the loss rate: the cutting procedure is repeated to reuse the rebar if the original steel is cut and the residual rebar is longer than the type of rebar to be processed, and vice versa. Sixth, the rebars go through bending procedures. The long-term steel without the need for bending is designed to finish the rebar process, and the rebar bending process takes as much time as the number of bends to the specific rebar type. Seventh, the machine schedule employed in the process of fabricating rebar is taken into account. The machine operation in the plant under consideration begins at 8:00 a.m. and ends at 9:30 p.m., with three intervals in between. The suggested model takes into account the machinery turning on and off to match the actual state of a rebar production plant.

By simulating the combination of several locations and the ratio of raw steel by length, we were able to build an optimal production plan for the rebar fabrication plant by calculating the time required for rebar fabrication and the loss rate.

#### **5. Results**

#### *5.1. Optimization*

The situation of importing raw steel with a length ratio of 2:1 (8 m:10 m) corresponds to the largest reduction in the loss rate when taking into account both SHD10 and SHD13 scenarios. The loss accumulation pattern is comparable for length ratios of 1:1, 1:2, and 1:3, as shown in Table 5. Moreover, the loss accumulation patterns are similar for length ratios of 2:1 and 3:1.

To optimize the site combination, the loss rate was analyzed for different combinations of multiple sites. In the considered case, 16 rebar types were selected, and the maximum loss rate was observed when the length of the imported raw steel was only 8 m. A combination of the two sites in the rebar fabrication process corresponded to the optimal production plan.

**Table 5.** Analysis results of four site combinations.

#### *5.2. Model Verification*

The data of the rebar fabrication plant were collected, and a model was built by reflecting the actual environment of the plant. The model was evaluated, and minor system errors were corrected. The model was additionally validated using information on the status of the rebars made at the actual rebar fabrication plant.

The simulation model was noted to exhibit a reasonable performance. According to Figures 5 and 6, the cutting process and flow of the processed rebars into the bending well were stopped as the machinery was shut off for one hour during lunch, 30 min during break time, and 30 min during dinner.

**Figure 5.** Model verification: hourly fabrication flow of D10 rebar.

**Figure 6.** Model verification: hourly fabrication flow of D13 rebar.


**8. Pathology and resilience**

Both NET and PCT were found to be safe and efficacious treatments reducing PTSD symptoms in older adults suffering from PTSD. Compared with NET, the PCT-group showed significantly stronger short-term reductions in PTSD pathology, whereas this group did not show significantly improved resilience, neither comparatively, nor longitudinally. Nevertheless, the study findings suggest that posttraumatic recovery in later life extends beyond reductions in re-experiencing, avoiding and arousal symptoms, adding nuance to the current centrality of PTSD symptoms [53]. One might say that the current concept of PTSD in DSM-5 (including depression symptoms in the diagnostic criteria of PTSD), already reflects this nuance. These findings may inspire further research on resilience factors, both in individual and community contexts.


**5. Final comments**

In the last 400.000 years there were variations in the molluskan paleocommunities of north Patagonia (southern BAP and north of Río Negro Province). The marine malacofauna of this area is composed by two associations, the first one is formed by Holocene sites with abundance of *Heleobia australis* and *Corbula patagonica*, and the other, mainly by Pleistocene sites and modern beaches with *Amiantis purpurata* as the most abundant, together with *Buccinanops globulosus*, *Bostrycapulus odites* and *Tegula patagonica*. The record of *Crassostrea rhizophorae*, *Anomalocardia brasiliana* and *Tegula atra*, in north Patagonia suggests that the interglacials MIS 7, MIS 5e and MIS 1 were slightly warmer than today. These latter species, except for *Tegula atra* live today in lower latitudes. However, the associations determined for the analyzed interglacials did not change concerning the faunal composition as a whole, and, except for the latitudinal changes of the three species mentioned above, and the record of temperate to cold water associations since the Interglacial MIS 1 in north Río Negro Province (ecotone), the composition remained similar, showing only changes in abundance of species.

*Update on Malacology*


**2.3 Injection machine and molded part description**

As illustrated in **Figure 1(a)**, a horizontal injection molding machine "FCSHN 125SV" was used to elaborate the PC/ABS parts. Their maximum injection pressure and clamping force were 217 MPa and 125 kN, respectively. Its screw's diameter was 34 mm.

The injection mold forms three imprints presented in **Figure 1(b)**. Imprint A was the tensile specimen. Imprint B was the dynamic tensile specimen. Imprint C was the torsion specimen [17].

This study is an alternate to investigate the impact of the injection process on the mechanical properties of injected PC/ABS parts.

**Figure 1.** *(a) Injection molding machine, (b) obtained specimens [17].*


**Branched-Chain Fatty Acids as Mediators of the Activation of Hepatic Peroxisome Proliferator-Activated Receptor Alpha by a Fungal Lipid Extract**

#### **Garima Maheshwari 1,2, Robert Ringseis 1,\*, Gaiping Wen 1, Denise K. Gessner 1, Johanna Rost 2, Marco A. Fraatz 2, Holger Zorn 2,3 and Klaus Eder 1**

Received: 8 June 2020; Accepted: 28 August 2020; Published: 31 August 2020

**Abstract:** The study aimed to test the hypothesis that monomethyl branched-chain fatty acids (BCFAs) and a lipid extract of *Conidiobolus heterosporus* (CHLE), rich in monomethyl BCFAs, are able to activate the nuclear transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha). Rat Fao cells were incubated with the monomethyl BCFAs 12-methyltridecanoic acid (MTriA), 12-methyltetradecanoic acid (MTA), isopalmitic acid (IPA) and 14-methylhexadecanoic acid (MHD), and the direct activation of PPARalpha was evaluated by reporter gene assay using a PPARalpha responsive reporter gene. Furthermore, Fao cells were incubated with different concentrations of the CHLE and PPARalpha activation was also evaluated by using the reporter gene assay, and by determining the mRNA concentrations of selected PPARalpha target genes by real-time RT-PCR. The reporter gene assay revealed that IPA and the CHLE, but not MTriA, MHD and MTA, activate the PPARalpha responsive reporter gene. CHLE dose-dependently increased mRNA concentrations of the PPARalpha target genes acyl-CoA oxidase (ACOX1), cytochrome P450 4A1 (CYP4A1), carnitine palmitoyltransferase 1A (CPT1A) and solute carrier family 22 (organic cation/carnitine transporter), member 5 (SLC22A5). In conclusion, the monomethyl BCFA IPA is a potent PPARalpha activator. CHLE activates PPARalpha-dependent gene expression in Fao cells, an effect that is possibly mediated by IPA.

**Keywords:** branched-chain fatty acids; *Conidiobolus heterosporus*; peroxisome proliferator-activated receptor α; lipid metabolism; fatty acid oxidation; hepatocyte

#### **1. Introduction**

The peroxisome proliferator-activated receptor alpha (PPARalpha) is a transcription factor that belongs to the superfamily of nuclear hormone receptors and is predominantly expressed in tissues with high rates of fatty acid oxidation. PPARalpha can be activated by micromolar concentrations of a variety of peroxisome proliferators including synthetic agonists, such as fibrates, a class of hypolipidemic drugs, and natural agonists, such as fatty acids (e.g., dietary ω-3 polyunsaturated fatty acids (PUFAs), like arachidonic acid (AA), linoleic acid (LA) or α-linolenic acid (ALA)), eicosanoids

and their derivatives [1–3]. Upon activation, PPARalpha upregulates the expression of a large set of target genes involved in fatty acid uptake, transport and oxidation through binding to a specific sequence, called peroxisome proliferator response element (PPRE), in the regulatory region of these genes [4]. In addition, PPARalpha inhibits several proinflammatory genes through a negative crosstalk with the key regulator of inflammation, nuclear factor-kappa B, especially in the vasculature [5]. Owing to this, PPARalpha activators exhibit lipid-lowering and atheroprotective effects, thereby reducing the risk of developing cardiovascular diseases (CVDs) [6].

Apart from the abovementioned PPARalpha activators, the tetramethyl branched-chain, isoprenoid-derived fatty acids phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) and pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) as well as their CoA-thioesters were shown to be high-affinity natural ligands and potent activators of PPARalpha [7–10]. In addition, the feeding of phytol, which is metabolized to phytanic acid following absorption from the intestine, was found to activate PPARalpha in tissues of mice [11]. Branched-chain fatty acids (BCFAs) are primarily saturated fatty acids, both even-carbon- and odd-carbon-numbered, with one or more methyl branches, accordingly called mono- or multimethyl BCFAs, respectively. The branching is mainly at the penultimate (*iso*) or next to the penultimate carbon atom (*anteiso*). BCFAs belong to the minor fatty acids in food and are found in ruminant products (dairy and beef) due to synthesis by ruminal microorganisms [12,13]; they are also present at significant levels in fermented foods of animal (shrimp paste, fish sauce) and nonanimal origin (sauerkraut) [13,14]. In addition, BCFAs occur naturally in bacterial lipids and in several fungi [15,16]. As early as 1967, Tyrrell described the composition of fatty acids in various species of the fungal genus *Conidiobolus* and reported proportions of BCFAs of up to 73% of total fatty acids [17]. Among these *Conidiobolus* species, the *Conidiobolus heterosporus* Drechsler 1953, first described by Tyrrell in 1971, belonging to the phylum *Zygomycota*, contains 53% of the total fatty acids as BCFAs, with the main BCFA being 12-methyltridecanoic acid (MTriA, *iso*-C14:0) (33% of total fatty acids), 12-methyltetradecanoic acid (MTA, *anteiso*-C15:0) (13%), 14-methylpentadecanoic acid (=isopalmitic acid, IPA, *iso*-C16:0) (6%) and 14-methylhexadecanoic acid (MHD, *anteiso*-C17:0) (1%) [18]. In contrast to the multimethyl BCFAs phytanic acid or pristanic acid, the BCFA species contained in *Conidiobolus heterosporus*, like MTriA, MTA, IPA and MHD, are monomethyl BCFAs. Whether these monomethyl BCFAs are also able to activate PPARalpha, however, is currently unknown.

Therefore, the present study aimed to test the following two hypotheses: First, based on the structural similarity between multimethyl BCFAs and monomethyl BCFAs, we tested the hypothesis that monomethyl BCFAs are potent PPARalpha activators. Second, due to the high amount of monomethyl BCFAs in *Conidiobolus heterosporus* lipids, we tested the hypothesis that a lipid extract of *Conidiobolus heterosporus* (CHLE) also activates PPARalpha and thereby induces the expression of PPARalpha target genes. To test these two hypotheses, we treated rat Fao hepatoma cells, a frequently used cell line to investigate the ability of different substances to activate the PPARalpha pathway, with the monomethyl BCFAs MTriA, MTA, IPA and MHD, in isolated form and with various concentrations of the CHLE and tested their ability to activate PPARalpha by determining PPARalpha transactivation and induction of PPARalpha target genes. As a positive control, we used WY-14643, a synthetic agonist of PPARalpha, and two PUFAs, LA and ALA, both of which are naturally occurring PPARalpha agonists [2].

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

#### *2.1. Submerged Cultivation of Conidiobolus heterosporus, Fungal Lipid Extraction and Preparation of Fatty Acid Methyl Esters*

*Conidiobolus heterosporus* Drechsler 1953, which belongs to the phylum Zygomycota, order Entomophtorales, family Ancylistaceae and the genus *Conidiobolus*, was obtained from Centraalbureau voor Schimmelcultures (CBS 138.57; Utrecht, Netherlands), and grown in submerged cultures following a previously described method [19]. For the precultures, 100 mL of yeast extract (3 g/L) and malt extract (30 g/L) (YM) medium (Merck KGaA, Darmstadt, Germany) in 250 mL Erlenmeyer flasks was used. After 3 days, the precultures were homogenized for 30 s at 9800 rpm using an Ultra Turrax homogenizer (IKA, Staufen, Germany). For the main cultures, 20 mL of the homogenized suspension was used to inoculate 200 mL of YM medium in 500 mL Erlenmeyer flasks. The cultivations were carried out in an Ecotron incubation shaker (25 mm shaking diameter, 150 rpm, 24 ◦C) (Infors GmbH, Einsbach, Germany) for 5 days. For harvesting, the fungal mycelium was separated from the culture broth by vacuum filtration with a Buchner funnel (110 mm) and a DP 595 cellulose filter paper (Albet LabScience, Dassel, Germany).

Total lipids were extracted by the Soxhlet method. After saponification of 150 mg CHLE with 4 mL 0.5 M NaOH in methanol (80 ◦C, 10 min), free fatty acids were converted into their corresponding fatty acid methyl esters (FAMEs) by addition of 3.5 mL boron trifluoride-methanol solution (20%) and heating to 80 ◦C for 5 min as described previously [19]. After addition of 1 mL n-hexane and incubation at 80 ◦C for 1 min, 3 mL saturated NaCl solution was added. The organic phase was dried over anhydrous sodium sulfate overnight.

#### *2.2. Analysis of Fatty Acid Composition*

The fatty acid composition of the fungal lipid extract was determined by using an Agilent 7890A gas chromatograph (Agilent Technologies, Waldbronn, Germany), equipped with a model 5975C mass spectrometry detector (Agilent Technologies), under the following conditions: carrier gas, helium (5.0); constant flow rate, 1.2 mL/min; inlet temperature, 250 ◦C; split ratio, 10:1; septum purge flow rate, 3 mL/min; 30 m × 0.25 mm i.d., 0.25 μm BP21 (FFAP) column (SGE Europe Ltd, Milton Keynes, UK); temperature program, 40 ◦C (3 min) and 5 ◦C/min to 240◦C (12 min); scan mode, TIC; scan range, m/z 33–400; electron ionization energy, 70 eV; source temperature, 230 ◦C; quadrupole temperature, 150 ◦C; transfer line temperature, 250 ◦C. The identification of the fatty acids was carried out by the comparison of retention indices with the reference standard Supelco 37 component FAME mix (Sigma-Aldrich, Taufkirchen, Germany). To determine the fatty acid composition, all FAME peak areas were summed up, set to 100%, and the results were expressed as a relative percentage for each fatty acid. The fatty acid composition of the CHLE is listed in Table 1.

#### *2.3. Chemical Reagents*

Ham's F12 medium, fetal calf serum (FCS), gentamycin and Trizol were purchased from Invitrogen (Karlsruhe, Germany). WY-14,643; the isolated fatty acids LA (≥99% pure), ALA (≥99% pure), IPA (≥98% pure), MTriA (≥98% pure), MHD (≥98% pure) and MTA (≥98% pure); and the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; Thiazole Blue) stock solution were obtained from Sigma-Aldrich (Steinheim, Germany).

#### *2.4. Cell Culture*

The rat hepatoma Fao cell line [20] was obtained from the European Collection of Cell Cultures (ECACC Cat. No. 89042701; Salisbury, UK) and grown in Ham's F12 medium supplemented with 10% FCS and 0.5% gentamycin at 37 ◦C and humidified atmosphere of 95% air and 5% CO2. Fao cells were seeded either in 24-well culture plates (Greiner Bio-One, Frickenhausen, Germany) at a cell density of 2.1 × 105 per well for cell viability assay and qPCR analysis or in 96-well culture plates at a cell density of 17 × 104 per well.

#### *2.5. Cell Treatments*

After reaching 70–80% confluency, cells were treated with either CHLE, WY-14,643 or various isolated fatty acids (LA, ALA, MTriA, MTA, IPA or MHD) for 24 h at the concentrations indicated. Incubation media containing isolated fatty acids were prepared by diluting the fatty acid stock solutions (100 mM in ethanol) with low-serum Ham's F12 medium (0.5% FCS) as described previously [21]. Prior to adding CHLE into the incubation media, an aliquot of the stock solution (100 mM in ethanol, based on the molecular weight of an average triglyceride of 850) was evaporated under a nitrogen stream and dissolved in 0.03 N NaOH at room temperature to saponify triglycerides and bidistilled water. Afterwards, the saponified CHLE was diluted in low-serum Ham's F12 medium (0.5% FCS) (pH 7-8). WY-14,643 was added to the low-serum medium from a 100 mM stock solution dissolved in DMSO. Cells treated with the vehicle alone (DMSO for WY-14,643, ethanol for isolated fatty acids, low-serum medium for CHLE) were used as controls and contained the same vehicle (ethanol) concentration (ethanol: 0.5 % (*v*/*v*); DMSO: 0.05% (*v*/*v*)). After addition of either CHLE, isolated fatty acids or WY-14,643 to the medium, the medium was gently mixed at RT to ensure complete solubility of the added substances. No signs of precipitation could be observed.

**Table 1.** Fatty acid composition of the *Conidiobolus heterosporus* lipid extract.

1 Only fatty acid methyl esters in quantities greater 0.1% were considered.

#### *2.6. Cell Viability Assay*

Cell viability after treatment with either CHLE or isolated fatty acids was assessed by the MTT assay [22]. Briefly, after removing the incubation media, 5 mg/mL MTT stock solution dissolved in PBS (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.4) was added to each well, and cells were incubated at 37 ◦C for 4 h. Subsequently, the MTT/PBS solution was removed, and the formazan crystals generated during the incubation period were dissolved by adding isopropanol in 0.04 N HCl. After the crystals were completely dissolved, the solution was transferred into a 24-well plate, and its absorbance was measured at the wavelength of 540 nm using an Infinite 200 M microplate

reader (Tecan, Männedorf, Switzerland). Cell viabilities are expressed as percentage of control cells, which was set to 100%.

#### *2.7. Transient Transfection and Dual Luciferase Reporter Assay*

After reaching 70–80% confluency, cells were transiently transfected with 50 ng of a 3X ACO-PPRE vector (containing three copies of consensus PPRE from the ACO promoter in front of a luciferase reporter gene) and pGL4.10 vector as negative control using FuGENE 6 transfection reagent (Roche diagnostics, Mannheim, Germany) for 12 h according to the manufacturer's protocol. Cells were also co-transfected with 5 ng of pGL4.74 Renilla luciferase (encoding the Renilla luciferase reporter gene; Promega, Mannheim, Germany), which was used as internal control reporter vector to normalize for differences in transfection efficiency (Promega). After transfection, cells were treated with either WY-14643; the isolated fatty acids LA, ALA, MTriA, MTA, IPA or MHD; or the CHLE at the concentrations indicated or vehicle alone for 24 h. Afterwards, cells were washed with PBS and lysed with lysis buffer (Promega). Luciferase activities were determined with Beetle-Juice and Renilla-Juice Kits from PJK (Kleinblittersdorf, Germany) in a Mithras LB940 luminometer (Berthold Technologies, Bad Wildbad, Germany). For control of background luminescence, Firefly and Renilla luciferase activities were also determined in the lysates of nontransfected control cells and subtracted from luminescence of transfected cells. Data were normalized for transfection efficiency by dividing Firefly luciferase activity of the 3X ACO-PPRE plasmid by that of Renilla luciferase activity of the co-transfected pGL4.74 Renilla luciferase plasmid. Results represent normalized luciferase activities and are shown relative to cells transfected with pGL4.10 vector and treated with the vehicle only, which was set to 1.

#### *2.8. Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction (qPCR)*

After treatment, the media were discarded, the cells were washed once with PBS and total RNA was isolated using Trizol reagent according to the manufacturer's protocol. Total RNA (1.2 μg) was reverse-transcribed using 60 U M-MuLV reverse transcriptase in a Biometra Thermal Cycler (Whatman Biometra, Göttingen, Germany). The cDNA was stored in aliquots at −20 ◦C. Subsequent real-time RT-PCR analysis of selected PPARalpha target genes and reference genes and calculation of gene expression data was performed as described recently in detail [23]. The normalization factor was calculated as the geometric mean of expression data of the three most stable out of six tested potential reference genes (CANX, MDH1, ACTB, RPL13, TOP1 and ATP5B). Means and SD were calculated from normalized expression data for cells of the same treatment group. The mean of the vehicle control cells was set to 1, and the mean and SD of the treated cells were scaled proportionally. Features of gene-specific primer pairs are listed in Supplementary Table S1.

#### *2.9. Statistics*

Statistical analysis was performed using the Minitab statistical software (Release 13, Minitab Inc., State College, PA, USA). Normal distribution of variables was tested with the Anderson–Darling test. Since all data showed normal distribution, the effects of different concentrations of CHLE, isolated fatty acids and WY-14643 in comparison to control treatment were analyzed by Student's *t*-test. Post-hoc analysis was performed using Fisher's multiple comparison test. Means were considered significantly different from control at *p* < 0.05. Data are means ± SD calculated from three independent experiments. In each independent experiment, all treatments were represented in 4 (qPCR) and 8 (reporter assay, MTT assay) wells (representing the numbers of technical replicates per treatment).

#### **3. Results**

#### *3.1. E*ff*ects of Isolated Fatty Acids and CHLE on Cell Viability of Rat Fao Cells*

Incubation with either isolated BCFAs or straight-chain fatty acids for 24 h did not impair cell viability up to a concentration of about 200 μM, with the exception of MTriA, as demonstrated by the MTT assay (Figure 1A). Cell viability of Fao cells was, however, not reduced by 24 h incubation with the monomethyl BCFA MHD up a concentration 500 μM, the highest concentration tested (Figure 1A). Cell viability of Fao cells was not reduced by 24 h incubation with CHLE up to a concentration of 500 μM (Figure 1B)

**Figure 1.** Effects of isolated fatty acids (**A**) and *Conidiobolus heterosporus* lipid extract (CHLE) (**B**) on cell viability of Fao cells. Fao cells were treated either without (vehicle) or with different concentrations of linoleic acid (LA), α-linolenic acid (ALA), isopalmitic acid (IPA), 12-methyltridecanoic acid (MTriA), 14-methylhexadecanoic acid (MHD), 12-methyltetradecanoic acid (MTA) or CHLE for 24 h, and cell viability was measured by the MTT assay. Bars represent means ± SD of three independent experiments. (**A**) The dashed line indicates the vehicle control (=100%). \* *p* < 0.05 compared with vehicle control.

#### *3.2. E*ff*ects of Isolated Fatty Acids and CHLE on PPARalpha Transactivation*

Using a PPARalpha-responsive reporter gene, we studied the effect of CHLE and isolated fatty acids contained in the CHLE on PPARalpha transactivation. As shown in Figure 2, the synthetic PPARalpha agonist WY-14,643 markedly increased the luciferase activity of the PPARalpha-responsive reporter gene about 12-fold compared to vehicle control (*p* < 0.05). IPA, one of the main monomethyl BCFAs of the CHLE, increased the luciferase activity of the reporter gene about 3.5-fold (*p* < 0.05), while the other monomethyl BCFAs, namely MTriA, MHD and MTA, did not (Figure 2). Treatment of cells with CHLE increased the luciferase activity of the PPARalpha-responsive reporter gene about 4-fold compared to vehicle control (*p* < 0.05). The straight-chain fatty acids LA and ALA increased the luciferase activity of the reporter gene about 4- and 3-fold, respectively, compared to vehicle control (*p* < 0.05, Figure 2).

#### *3.3. E*ff*ect of CHLE on the mRNA Concentration of PPARalpha Target Genes*

Treatment with CHLE increased the mRNA concentrations of the PPARalpha target genes solute carrier family 22 (organic cation/carnitine transporter), member 5 (SLC22A5), acyl-CoA oxidase (ACOX1), carnitine palmitoyltransferase 1A (CPT1A) and cytochrome P450 4A1 (CYP4A1) in a dose-dependent manner (*p* < 0.05, Figure 3). The strongest increase was observed for CYP4A1, which was increased 6-fold by 500 μM CHLE compared to control (*p* < 0.05, Figure 3). As expected, the synthetic PPARalpha agonist WY-14,643 caused a markedly stronger induction of all PPARalpha target genes investigated when compared to CHLE (*p* < 0.05, Figure 3).

**Figure 2.** Effects of isolated fatty acids, *Conidiobolus heterosporus* lipid extract (CHLE) and synthetic PPARalpha agonist WY-14,643 on PPARalpha transactivation in Fao cells. Fao cells were transiently transfected with a 3X ACO-PPRE vector and a Renilla luciferase expression vector for normalization using FuGENE6. After transfection, cells were treated either without (vehicle) or with different isolated fatty acids (50 μM each), CHLE (500 μM) or WY-14,643 (50 μM) for 24 h. Afterwards, cells were lysed, and luciferase activities were determined by dual luciferase assay. Bars represent means ± SD of three independent experiments. \* *p* < 0.05 compared with vehicle control.

**Figure 3.** Effects of *Conidiobolus heterosporus* lipid extract (CHLE) on relative mRNA levels of the PPARalpha target genes SLC22A5, ACOX1, CPT1A and Cyp4A1 in Fao cells. Fao cells were treated either without (vehicle) or with CHLE at two different concentrations (200 and 500 μM) or WY-14,643 (50 μM) for 24 h, and mRNA levels were determined by qPCR. Bars represent means ± SD of three independent experiments. \* *p* < 0.05 compared with vehicle control.

#### **4. Discussion**

In the present study, we tested the hypothesis that both isolated monomethyl BCFAs and CHLE, which is rich in monomethyl BCFAs, activate the nuclear transcription factor PPARalpha and CHLE induces the expression of PPARalpha target genes in rat Fao cells. Our data from the reporter gene assay clearly show that the monomethyl BCFA IPA and the CHLE are potent activators of PPARalpha. Moreover, we found that CHLE induces the expression of known PPARalpha target genes involved

in fatty acid metabolism, like ACOX1, CPT1A, CYP4A1 and SLC22A5, in a dose-dependent manner. Compared with WY-14,643 the extent of activation of PPARalpha by IPA and CHLE was lower, but this is not surprising given that WY-14,643 is a high-affinity synthetic ligand of PPARalpha and such ligands typically cause a stronger stimulation of PPARalpha transactivation than naturally occurring ligands [2].

Several studies are available reporting that tetramethyl BCFAs, like phytanic acid, pristanic acid and their CoA-thioesters, as well as the synthetically produced short-chain BCFA valproic acid, cause PPARalpha activation [9,24,25]. However, to our knowledge, there is no study available exploring the effect of the monomethyl BCFAs MTriA, MTA, IPA and MHD on PPARalpha activation in isolated form. Thus, our study shows for the first time that the monomethyl BCFA IPA, which makes up about 6% of total fatty acids of CHLE, significantly stimulates PPARalpha transactivation, indicating that IPA is responsible for the effect of the CHLE. A few studies are available investigating the effect of a mixture of different BCFAs in laboratory animals. For instance, Shirouchi et al. [26] showed that feeding 5% porpoise oil, which contains 15.5% BCFAs (including the monomethyl BCFA IPA), to obese rats alleviates hepatic triglyceride accumulation and increases serum adiponectin levels, effects that are known to be mediated by PPARalpha activation [27]. The decrease of hepatic triglyceride accumulation in response to PPARalpha activation is mechanistically explained by the physiological function of the proteins encoded by many PPARalpha target genes, including those considered in the present study as indirect markers of PPARalpha activation. The enzymes encoded by ACOX1, CPT1A and CYP4A1 are directly involved in peroxisomal, mitochondrial and microsomal fatty oxidation pathways, respectively, and induction of these genes causes a decrease of hepatic triglyceride levels due to an increased oxidation of fatty acids [4]. SLC22A5 encodes the novel organic cation transporter OCTN2, which mediates carnitine uptake into tissues. Because carnitine is essential for the transport of long-chain fatty acids into the mitochondrial matrix, where mitochondrial fatty acid oxidation takes place, genetic defects of this carnitine transporter result in decreased intracellular carnitine levels and an impaired fatty acid oxidation [28]. A limitation of the abovementioned study [26] is that porpoise oil also contains ω-3 PUFAs, which are also known to be activators of PPARalpha. Thus, the contribution of BCFAs to the effect of porpoise oil in this study remains unclear. In another study, it was reported that rat pups receiving milk substituted with BCFAs (including IPA) showed an increased intestinal expression of the anti-inflammatory interleukin-10 (IL-10) compared with pups receiving no BCFAs and a reduced incidence of necrotizing enterocolitis [29], which is indicative of an anti-inflammatory action of BCFAs in the intestine. This effect might be also mediated by activation of PPARalpha by the BCFAs, because upregulation of IL-10 has been reported to occur in response to administration of WY-14,643 [30]. In addition, the anti-inflammatory effect of BCFAs in the neonatal rat intestine might be also explained by the well-known negative crosstalk of PPARalpha with critical inflammatory signaling pathways such as nuclear factor-kappa B [5].

In summary, the observation of the present in vitro study that the monomethyl BCFA IPA is an activator of PPARalpha may provide an explanation for the PPARalpha-mediated effects of BCFA-rich diets in in vivo studies [26,29]. Nevertheless, other fatty acids, particularly PUFAs like LA, AA and ALA (all of which were present in the CHLE), are naturally occurring PPARalpha activators [1,2]. Thus, not only LA and ALA, used as further positive controls in the reporter gene assay, but also AA may have also contributed to the PPARalpha-dependent effects of CHLE or BCFA-rich diets. In any case, the results of the present study suggest that dietary supplementation of CHLE might be a useful approach to induce the beneficial effects associated with PPARalpha activation like lipid lowering and reduction of CVD risk.

Considerations with regard to the use of BCFA-rich sources as a dietary supplement have to include the induction of possible adverse effects. In this context, it should be noted that various feeding studies in rats, lambs and steers did not observe any toxic or adverse effects after treatment with different BCFAs [31–33]. In contrast, adverse effects including teratogenic, hepatotoxic, neurotoxic and proapoptotic effects have been reported for valproic acid, which is also used as an antiepileptic drug, but only at extremely high concentrations [34,35]. Such effects might be also, at least partially, explained by the activation of PPARalpha, because administration of synthetic PPARalpha activators to rodents was found to cause hepatic peroxisome proliferation, hypertrophy, hyperplasia and even hepatocarcinogenesis [36], with this last condition being attributed to induction of oxidative stress and an imbalance between apoptosis and cell proliferation [37]. Apart from this, clinical observations in patients with peroxisomal disorders like Zellweger syndrome or Refsum's disease and studies employing corresponding mouse models have shown that elevated concentrations of BCFAs are associated with a high level of toxicity [38,39]. However, it has to be considered that in such cases the BCFA concentrations in blood markedly exceed those achieved by dietary supplementation. Proapoptotic effects were also found for naturally occurring BCFAs, like MTA [40,41]. Such proapoptotic effects however were found to occur in tumor cells, an effect that is probably beneficial. Collectively, it can be stated that more studies are required for the evaluation of safety aspects of BCFA-rich sources as food supplements.

#### **5. Conclusions**

This study shows for the first time that the monomethyl BCFA IPA and the CHLE are able to induce PPARalpha activation in cultured hepatocytes. These findings indicate that lipid extracts from fungi, such as CHLE, are a source of PPARalpha-activating biomolecules which might be considered as dietary supplements to induce beneficial effects associated with PPARalpha activation, like lipid lowering and reduction of CVD risk, provided that no safety concerns exist. In light of recent evidence showing that administration of BCFAs in neonatal rats increases the expression of anti-inflammatory cytokines and reduces the incidence of necrotizing enterocolitis, an anti-inflammatory action of BCFAs in the intestine might be postulated, which is likely mediated by the well-established negative crosstalk of PPARalpha activation with critical inflammatory signaling pathways. Regarding adverse effects reported from the use of synthetic PPARalpha activators in rodents, potential safety issues associated with BCFA-rich sources as food supplements have to be thoroughly evaluated.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-273X/10/9/1259/s1, Table S1: Characteristics of gene-specific primers used for qPCR analysis.

**Author Contributions:** Conceptualization, R.R., K.E. and H.Z.; formal analysis, G.M., G.W., D.K.G., J.R. and M.A.F.; writing—original draft preparation, R.R.; writing—review and editing, R.R. and K.E.; supervision, R.R.; project administration, K.E.; All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by the Hessen State Ministry of Higher Education, Research and the Arts (HMWK) via the LOEWE research center "Insect Biotechnology and Bioresources". G.M. was funded by the German Academic Exchange Service (DAAD; funding ID: 57243780).

**Acknowledgments:** The authors are grateful for the provision of the 3X ACO-PPRE vector by Prof. Dr. Sander Kersten, Department of Agrotechnology and Food Sciences, Chair of Nutrition, Metabolism and Genomics, University of Wageningen, The Netherlands.

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

#### **References**

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**2.2 Roadside component**

The roadside component includes variable message signs, traffic lights and other units which are equipped with communication capabilities. This way, the roadside component can communicate with other vehicles by sending them information or acting as a relay station supporting multi-hop communication. Moreover, this component can communicate with other roadside units and the central system and therefore forward information received from vehicles. The roadside component can be also connected to the Internet.

#### **2.3 Personal component**

The personal component is actually a nomadic device, that is a personal navigator or a smartphone, which can host a variety of ITS applications. These devices can also support cooperative ITS applications based on communication with other road users or the road infrastructure.


*4.1. Agglomeration*

A simple pharmaceutical process of agglomerating blends with two different concentrations of the active pharmaceutical ingredient (API) was simulated. The process structure is illustrated in Figure 4. The solid material is described using two interdependent distributed parameters:

**Figure 4.** Flowsheet structure of the agglomeration process.

The initial blend entering the agglomerator is a mixture of materials from two feeders (Figure 5):

**Figure 5.** Input distribution for the agglomeration process.2 To improve readability, in all the distributions shown here and further, values of mass fractions less than 10−5 were cut off.

Both particle sizes and API concentrations are given in terms of the Gaussian normal distribution with mean values μ and standard deviations σ shown in Table 1. Feeders continuously supply the material at a rate of 0.25 kg/s each. The final distribution after their mixing is shown in Figure 5. The agglomerator is initially filled with 200 kg of the same blend. All model parameters used to simulate the agglomeration process are listed in Table 1.

Figures 6 and 7 show the distribution of the product stream in steady-state, which was reached after 25 min of process time. Clearly visible individual peaks, which were formed as a result of the agglomeration of particles with different sizes and different API concentrations. Since the initial blend is supplied continuously at a constant rate during the entire agglomeration process, the initial distributions *A* and *B* (Figure 7) are still distinguishable in the final mixture. Agglomeration of material *B* with itself gives *F*, which has a doubled mean size and the same API concentration as *B*, as expected. In turn, agglomeration of *B* with *F* (having the same concentrations) gives a peak at *M*, which does not alter API content and has a particle size equal to (*B* + *F)*. The same is true for the material from feeder 1, agglomerating with itself according to the schema: (*A* + *A*) = *C*, (*A* + *C*) = *E*, and (*A* + *E*) + (*C* + *C*) = *H*.

**Figure 7.** Agglomeration diagram.

More complex behaviour is observed if particles with different API concentrations agglomerate. In this case, the particle sizes are added up and the concentration is averaged according to Equation (59). For example, agglomeration of particles from *A* and *B* gives *D*. Further, G is formed as a mixture of (*A* + *D*) and (*B* + *C*). The result of the agglomeration of *B* and *C* is shifted below the concentration value of 0.05 due to the mutual influence of two factors. First, the introduction of the size-dependent growth rate β∗ (Equation (57)) leads to faster growth of smaller particles. Second, averaging by the second distribution takes into account the actual amount of material being agglomerated in selected classes (see Equations (59) and (60)).

The complete scheme of material transitions during the agglomeration, shown in Figure 7, is represented in Table 2.

**Table 2.** Material transitions during agglomeration.

Thus, information on the change in particle size during the agglomeration process, obtained from the generated transformation matrix, made it possible to take into account and correctly process the dependent distributed parameters of the agglomerated material.


Abstract

The study of the solar wind-Earth's magnetosphere and atmosphere interaction is a topic of great interest. The solar wind energy is transferred to the Earth's environment also through ultralow frequency (ULF, 1 mHz–5 Hz) waves of the geomagnetic field, with higher efficiency at high latitudes where magnetic reconnection processes occur, making the polar cap an important laboratory for these investigations. Several studies suggest that the atmosphere responds to the geomagnetic activity driven by the solar wind, although the interaction processes are not yet completely understood. In this context, the results of recent investigations, showing the coupling on timescales of 1–2 days between geomagnetic ULF activity and the middle-low (h < 50 km) atmosphere in the polar cap, are summarized, based on geomagnetic measurements at Terra Nova Bay, in Antarctica (λ 80°S) and atmospheric parameters from the reanalysis dataset.

Keywords: ULF waves, solar wind, high-latitude atmosphere, polar cap electrodynamics, energetic particle precipitation, atmosphere processes, cloud microphysics


**Aleksander Lisiecki**

Silesian University of Technology, Faculty of Mechanical Engineering, Department of Welding Engineering, Konarskiego 18A street, 44-100 Gliwice, Poland<EMAIL_ADDRESS>
Received: 9 August 2019; Accepted: 20 September 2019; Published: 24 September 2019

**Abstract:** This study measured optical properties, such as specular, diffuse, and total reflection for 808 nm wavelength, characteristic for high power diode lasers radiation, from the surface of titanium alloy Ti6Al4V at delivery conditions, polished, and oxidized. Moreover, the optical properties of surface layers produced by high power direct diode laser (HPDDL) melting and nitriding were determined. Additionally, a methodology for determining the value of absorption for 808 nm wavelength of the HPDDL radiation on the surface of a melt pool during laser surface melting and nitriding of titanium alloy was proposed. The results show that the distinct differences in absorption affect the heat transfer, thermal conditions of laser heating and thereby the penetration depth during laser melting and nitriding of the titanium alloy.

**Keywords:** titanium nitrides; reflectivity; absorption; high power diode laser


*2.2. Experimental Design*

The field experiment was arranged as a two-factorial design. The first factor was the nitrogen fertilization system (NFS), related to the type of N fertilizer applied: (i) ammonium nitrate, 34-0-0, (acronym M-NFS), (ii) organic N: digestate (O-NFS), (iii) mixed: 2/3 of digestate + 1/3 of ammonium nitrate (AN) (OM-NFS). The rate of applied N was: 0 (N control), 60, 120, 180, and 240 kg ha−1. Each year, digestate was applied at the end of November, and AN on the 1st of March of the following year. The applied digestate on average contained: DM: 72, N-NH4: 7.2, P2O5: 5.45, K2O: 2.85, MgO: 1.88, S: 1.99 kg m−3. Winter barley was a fore-crop for WOSR. A hybrid WOSR variety *Impression Cl,* characterized by a high yielding potential for medium fertile soils, was used as a test crop. It was sown at the rate of 3.0 kg ha−1 at the end of August and harvested at the end of July from an area of 12 m2. The seeds were harvested at maturity when the moisture content was 8% dry weight.

**Table 2.** Main characteristics of meteorological conditions during the winter oilseed rape (WOSR) growing season on the background of the long-term averages 1.

meteorological station at Szczecin.


**Preface to "Genetics, Genomics and Biotechnology of Plant Cytoplasmic Organelles"**

Chloroplasts and mitochondria are essential plant organelles involved in many fundamental processes (photosynthesis, energy production, metabolism, cell homeostasis, etc.). They are semi-autonomous organelles; hence, their function is dependent upon cross-talk between nuclear and organelle genetic systems. The Special Issue Book entitled "Genetics, Genomics and Biotechnology of Plant Cytoplasmic Organelles" provides new advances in the sequencing of both mitochondria and chloroplasts' genomes using Next-Generation Sequencing technology in plant species and algae including important crop and tree species, in vitro culture protocol, and identification of a core module of genes involved in plastid development. In particular, the published studies focus on the description of adaptive evolution, elucidate mitochondrial mRNA processing, highlight the effect of domestication process on plastome variability and report the development of molecular markers. A meta-analysis of recently published genome-wide expression studies allowed the identification of novel nuclear genes, involved in the complex and still unrevealed mechanisms at the basis of communication between chloroplast and nucleus (retrograde signalling) during plastid development (biogenic control). Finally, an optimized regeneration protocol useful in plastid transformation of recalcitrant species, such as sugarcane, has been reported. We would like to thank to all the authors and their contributions as well as the reviewers for their constructive suggestions. Further, the Plants MDPI team and, particularly, Hinata Fang deserve our thanks for their support.

The editors declare no conflict of interest.

**Nunzia Scotti, Rachele Tamburino** *Editors*


1.1 Dusty plasmas

Dusty plasmas are also known as nonideal complex plasmas that contain particulates of condensed matter. The dust particles may have sizes ranging from nanometers to micrometers, and typically much more massive than that of plasma ions, electrons, and neutrons. When the dust particles immersed in the plasma, they attain a high electric charge (negative charge) which makes the dusty plasmas interesting and technological important in the area of applied plasma physics. The dynamical behavior of these massive dust charge particles is much complex and occurs on considerably slower time scales, because their charge-to-mass ratio is in orders of magnitude smaller than that of the corresponding charge-to-mass ratio of either the electrons or ions. Dust particles are found in the large abundant in planetary plasmas, cosmic plasmas, plasmas in the laboratory, and plasmas near the earth. As a matter of fact, one may cogitate that except in the hottest regions of fusion plasmas, where the dust particles would not survive, most are known as dusty plasmas in the sense that some dust particles might be present. To understand this fact, one recognizes two cases in which: (1) there are just few secluded (noninteracting) dust particles, with the goal that they have nearly nothing if any impact on the plasma, and (2) there are countless number of dust particles in the plasma so that their existence really changes the properties and behavior of the plasma. In the event (1), the dust particles are charged by their interactions with the plasma yet do not change the plasma in any noticeable way. Then, again case (2) agrees to the situation in which the charge dust is a component of the plasma, subject to the collective interactions that recognize an ionized gas from a neutral gas. Case (2) is what is ordinary characterized as "dusty plasmas." At a significantly bigger scale, it is outstanding that comets for the most part have two tails. One tail is expected to the comet's dust particles, the other is because of ionized gas comet coma. These tails are not separate near the coma but overlap forming dusty plasma [3].


4.4 Influence of free water and strain-rate on the confined strength of ordinary concrete and high-strength concrete

strength of OC in saturated condition whereas both concretes present similar

OC and HSC concretes. (d) Dynamic deviatoric behavior of saturated OC and HSC concretes.

Quasi-oedometric compression tests applied to ordinary concrete (R30A7) and high-strength concrete in dry and saturated conditions [34]. (a) Quasi-static deviatoric behavior of dry OC and HSC concretes. (b) Dynamic deviatoric behavior of dry OC and HSC concretes. (c) Quasi-static deviatoric behavior of saturated

Investigation of the Quasi-Static and Dynamic Confined Strength of Concretes by Means…

DOI: http://dx.doi.org/10.5772/intechopen.89660

The quasi-oedometric compression testing technique constitutes one of the most convenient and efficient testing methods to characterize the quasi-static and dynamic confined behavior of concrete and rock-like materials. However, precautionary measures need to be considered in the mounting procedure, to fill the gap between ring and the confining cell, and in the data processing to take into account for the barreling deformation of the cell, the shortening of the sample and, when necessary, for the plastic behavior of the cell, the possible effects of friction at each interface and the axial dissymmetry between the sample and the confining cell. Thus, it is possible to determine the hydrostatic and deviatoric behaviors of these materials under pressure ranging from few tens to a thousand of MPa in quasi-static loading conditions with large capacity hydraulic press or at high strain-rates with a Split-Hopkinson Pressure Bar apparatus. The main obtained results illustrate the beneficial effect of the presence of strong particles added in mortars, a strong influence of free water content on both hydrostatic and deviatoric behaviors of concretes especially in the case of microconcrete and ordinary concrete. Finally, it is concluded that concrete composition and water content have a strong influence on the concrete behavior

strengths in dry condition.

5. Conclusion

83

Figure 12.

The experimental results of quasi-static and dynamic QOC tests performed on dried and saturated ordinary concrete (OC, in this case, R30A7 concrete) and high-strength concrete (HSC) were compared in [34]. The main results obtained regarding their quasi-static and dynamic deviatoric responses are reported in Figure 12. First, under dry conditions, both OC and HSC concretes exhibit similar strength with a slightly higher strength of HSC at low pressure but with a more reduced increase of strength with pressure as compared to OC (Figure 12(a, b)). In addition, it can be noted that the dynamic strength of dried OC and HSC is significantly higher than their quasi-static strength whatever the considered level of pressure. Finally, the influence of strain-rate on the confined strength of dried concretes cannot be neglected.

On the other hand, under saturated conditions, both concretes exhibit completely different strength. In quasi-static conditions, a saturation of strength around 70 MPa was observed with OC, whereas the strength of HSC goes up to 270 MPa. In dynamic loading conditions, higher strengths are observed with both concretes compared with their quasi-static strengths and the influence of strain-rate cannot be neglected (Figure 12(c, d)). As in quasi-static loading, the dynamic confined strength of saturated HSC is much higher than that of saturated OC. Now, if dried and saturated concretes are compared, it can be concluded that the confined strength of dried samples is higher than that of saturated samples for both concretes. As concluded with microconcrete, it is supposed that interstitial waterpressure inside saturated concretes reduces their confined strength. In addition, this effect is even more pronounced in OC compared with HSC due to the higher level of porosity (11.8%) in OC as compared to HSC (8.8%), which explains the much lower Investigation of the Quasi-Static and Dynamic Confined Strength of Concretes by Means… DOI: http://dx.doi.org/10.5772/intechopen.89660

#### Figure 12.

cement paste. In addition, a correlation was noted between the large porosities of millimeter class and the compaction of the concretes under high level of pressure. Finally, it was demonstrated that the experimental data provided by QOC experiments could be used to simulate numerically impact tests involving high levels of

4.3 Influence of free water and strain-rate on the confined behavior of

microconcretes in [16]. The dynamic tests exhibited an important dissimilarity between dry and saturated specimens concerning both deviatoric and hydrostatic behaviors. First, dried microconcretes exhibited a continuous compaction whereas saturated specimens showed a non-linear (hardening) hydrostatic behavior (Figure 11(b)). Moreover, a strong and continuous increase of the strength with pressure was noted with dry samples whereas water-saturated specimens exhibited an almost-perfect saturation of the strength (Figure 11(a)). The quasi-static results allowed highlighting the reason of this dissimilarity (Figure 11(c, d)). On the one hand, dried specimens behave similarly in dynamic tests and no strain rate effect is observed. On the other hand, the behavior of saturated specimens gradually tends to that of dried specimens when the loading rate is decreased, and an expulsion of water during slow quasi-static tests was observed. Finally, it was concluded that, waterpressure inside saturated microconcrete plays a major role on their fast-quasi-static or

dynamic confined behavior by reducing drastically their shear strength.

concrete and high-strength concrete

concretes cannot be neglected.

82

4.4 Influence of free water and strain-rate on the confined strength of ordinary

The experimental results of quasi-static and dynamic QOC tests performed on dried and saturated ordinary concrete (OC, in this case, R30A7 concrete) and high-strength concrete (HSC) were compared in [34]. The main results obtained regarding their quasi-static and dynamic deviatoric responses are reported in Figure 12. First, under dry conditions, both OC and HSC concretes exhibit similar strength with a slightly higher strength of HSC at low pressure but with a more reduced increase of strength with pressure as compared to OC (Figure 12(a, b)). In addition, it can be noted that the dynamic strength of dried OC and HSC is significantly higher than their quasi-static strength whatever the considered level of pressure. Finally, the influence of strain-rate on the confined strength of dried

On the other hand, under saturated conditions, both concretes exhibit completely different strength. In quasi-static conditions, a saturation of strength around 70 MPa was observed with OC, whereas the strength of HSC goes up to 270 MPa. In dynamic loading conditions, higher strengths are observed with both concretes compared with their quasi-static strengths and the influence of strain-rate cannot be neglected (Figure 12(c, d)). As in quasi-static loading, the dynamic confined strength of saturated HSC is much higher than that of saturated OC. Now, if dried and saturated concretes are compared, it can be concluded that the confined strength of dried samples is higher than that of saturated samples for both concretes. As concluded with microconcrete, it is supposed that interstitial waterpressure inside saturated concretes reduces their confined strength. In addition, this effect is even more pronounced in OC compared with HSC due to the higher level of porosity (11.8%) in OC as compared to HSC (8.8%), which explains the much lower

Quasi-static and dynamic QOC tests were performed on dry and water-saturated

confining pressure in front of the projectile head [41].

microconcrete

Compressive Strength of Concrete

Quasi-oedometric compression tests applied to ordinary concrete (R30A7) and high-strength concrete in dry and saturated conditions [34]. (a) Quasi-static deviatoric behavior of dry OC and HSC concretes. (b) Dynamic deviatoric behavior of dry OC and HSC concretes. (c) Quasi-static deviatoric behavior of saturated OC and HSC concretes. (d) Dynamic deviatoric behavior of saturated OC and HSC concretes.

strength of OC in saturated condition whereas both concretes present similar strengths in dry condition.

### 5. Conclusion

The quasi-oedometric compression testing technique constitutes one of the most convenient and efficient testing methods to characterize the quasi-static and dynamic confined behavior of concrete and rock-like materials. However, precautionary measures need to be considered in the mounting procedure, to fill the gap between ring and the confining cell, and in the data processing to take into account for the barreling deformation of the cell, the shortening of the sample and, when necessary, for the plastic behavior of the cell, the possible effects of friction at each interface and the axial dissymmetry between the sample and the confining cell. Thus, it is possible to determine the hydrostatic and deviatoric behaviors of these materials under pressure ranging from few tens to a thousand of MPa in quasi-static loading conditions with large capacity hydraulic press or at high strain-rates with a Split-Hopkinson Pressure Bar apparatus. The main obtained results illustrate the beneficial effect of the presence of strong particles added in mortars, a strong influence of free water content on both hydrostatic and deviatoric behaviors of concretes especially in the case of microconcrete and ordinary concrete. Finally, it is concluded that concrete composition and water content have a strong influence on the concrete behavior

under high confinement and the strain-rate cannot be neglected whatever the concrete types and the water-saturation conditions.

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