**1. Introduction**

Artists believe that art is central to human life. Unfortunately, many people with visual impairments (PVI) do not have access to the world's visual culture or the opportunity to experience the life-enhancing power of visual art. PVI must have access to the world's visual culture if they are to participate fully in their communities and the world at large. Such access will improve the quality of their lives and help them gain skills crucial to their education and employment opportunities [1]. Findings from participant observations in touch tours for blind and visually impaired people at the Metropolitan Museum of Art were discussed by Hayhoe [2]. Touch Graphics Inc. exhibits a reproduced painting called "Talking Tactile" at the San Diego Museum of Art [3]. Recently, "BlindTouch" [4,5] has been introduced to enhance PVI's artwork experience by reproducing painting masterpieces as 3D-printed models that contain touch recognition sensors (based on conductive painting) and provide relevant audio descriptions and sound e ffects (such as the sound of the wind or a flying butterfly) while the user explores the di fferent features of the artwork with their fingers. The touch interface was evaluated by PVI and found to be e ffective. The audio feedback can be activated by tapping the fingers. However, PVI still find it di fficult to e ffectively experience color when appreciating a museum's artwork. Tactile color pictograms (TCPs), which are embossed on a surface that PVI can touch to perceive color information, are used to allow easy accessibility to color information. TCPs have two advantages as an assistive tool when used in conjunction with audio descriptions. First, they allow immediate access to color information through color patterns, just as a sighted person sees colors

immediately.

However, for color recognition, tactile patterns coding colors (TPCs) alone might not provide a good user experience for PVI (especially those with congenital blindness or complex disabilities) because tactile interactions tend to be slow. Congenitally blind people understand colors through physical and abstract associations. Color audition means the reaction of feeling color in one sound. Gauguin tried to pursue a symbol of innerness as a color. He said, "Color which, like music, is a matter of vibrations, reaches what is most general and therefore most indefinable in nature: its inner power" [7].

Therefore, it might be desirable to provide color images for PVI that convert the color being touched into a corresponding musical sound, such as orchestral or classical music, that codes the color. In other words, to provide better color perception for PVI, a multisensory user experience that combines tactile and musical stimuli might be e ffective. A combination of TPCs and sound coding colors (SCCs) could also enable PVI to interpret the overall color composition of a piece of artwork. Therefore, we here explore the association of sound tones (timbre, pitch, and intensity) with the color properties (hue, value, and chroma) of the Munsell color system. Based on the observations and series of user tests performed in this research, we here introduce two SCC sets that represent 18 colors in 6 hues.

### *1.1. Review of the Color System*

The Munsell color system is a color space that specifies colors based on the three properties of hue (basic color), value (lightness), and chroma (color intensity) [8]. In this system, the higher the lightness value, the closer the color is to white, and the lower the value, the closer it is to black. Chroma is the vividness (clearness) of a color. Colors with the highest chroma are pure colors (saturated colors), meaning that they are not mixed with other colors, and colors with the lowest chroma are achromatic (white, gray, black). Palmer et al. described 37 colors that reflect the color preferences of US college students [9]. Munsell's hue/value/chroma data from those 37 colors can be found in [9]. The SCC sets proposed in this paper can express six unique hues (red (R), orange (O), yellow (Y), green (G), blue (B), and purple (P)) using three color dimensions—saturated (S), light (L), and dark (D)—for each hue, as shown in Figure 1.

**Figure 1.** Red (R), orange (O), yellow (Y), green (G), blue (B), and purple (P) using three color dimensions—saturated (S), light (L), and dark (D).

For example, in Figure 2, the color marked "Light" has a value of seven and chroma of eight. The color marked "Dark" has a value of three and a chroma of eight. The color marked "Saturated" has a value of five and a chroma of 15. The colors with the lowest chroma (0) are achromatic.

**Figure 2.** Saturated (S), light (L), and dark (D) for red in [9].

### *1.2. Review of the Sound Representations of Colors*

The perception of a sound has several aspects:

Pitch: the frequency of a sound (high or low).

Tone: the timbre of a pitch.

Key: is it a major or minor scale?

Timbre: the characteristic sound of an instrument (e.g., a flute and an oboe playing the same note sound differently).

Chords: two or more notes played at the same time.

Melody: sequentially arranged notes.

Volume: loudness.

#### Velocity (the musical term): **the force with which a note is played, and it is vitally important in making MIDI performances sound human.**

Synesthesia, a mixing of the senses, occurs in some individuals. People with strong synesthesia, synesthetes, experience a perception in one sense when a stimulus for another sense is presented (e.g., seeing a color when hearing a sound). This is what Martino and Marks [10] call "strong synesthesia" as opposed to "weak synesthesia," which describes the simple cross-sensory correspondences that most people experience, e.g., being able to match a tone of a certain pitch to a light of a certain brightness [11]. One of the most common types of synesthesia is seeing color while listening to music or musical notes (e.g., [12–15]). Gaboski and Odebert [12] showed some tendency to associate color with short musical selections. Slow music is associated with blue, high tones are light, and low tones are dark. Fast and cheerful notes are reminiscent of bright and intense warm colors.

As a synesthete, Kandinsky saw colors corresponding to the sounds of different instruments and used those correspondences in his paintings [13]. He attributed musicality to his paintings and said that painting could generate energy like music, "drawing is rhyming the form with color and showing the moving power through color." He believed that just as musicians express their feelings in musical forms such as rhythm, timbre, and melody, artists can express their inner experiences of fear, sadness, and joy using various arrangements of color and form. He associated colors and forms with specific emotions, connected them with elements of music, and then sought to write those subjective and emotional experiences into logical and universal laws. "Yellow is stimulus, red is energy, blue is infinite sensibility, and green is calm," said Kandinsky.

People without synesthesia also report correspondences between music and color. For example, [14,16] found some evidence that music–color correspondences result from an emotional

link. Brighter colors such as yellow, red, green, and blue were usually assigned to happy songs, and gray was usually assigned to sad songs [17]. A study by Palmer [9] also suggested that fast notes in a major key are yellow or orange and slow notes in a major key are blue and gray. He found cross-modal correlations based on the associative sensibility between color and music with the help of music that involves emotion.

Newton's *Opticks* [18] showed that the colors of the spectrum and the pitches of musical scales are similar (for example, "red" and "C"; "green" and "A*b*"). Maryon [19] also explored the similarity between the ratio of each tone to the wavelength of each color to connect them. This method of associating the pitch frequency of the scale with color can be a way of substituting colors and notes for one another [20]. However, the various sensibilities that can be obtained through color are limited by simply substituting colors into the musical scale. Alber Lavigna [21] found that the technique of a composer in organizing an orchestra seems very similar to the technique of a painter applying colors. In other words, a musician's palette is a list of orchestral instruments.

A comprehensive survey of associations between color and sound can be found in [22], including how di fferent color properties such as value and hue are mapped onto acoustic properties such as pitch and loudness. Using an implicit associations test, those researchers [22] confirmed the following cross-modal correspondences between visual and acoustic features. Pitch was associated with color lightness, whereas loudness mapped onto greater visual saliency. The associations between vowels and colors are mediated by di fferences in the overall balance of low- and high-frequency energy in the spectrum rather than by vowel identity as such. The hue of colors with the same luminance and saturation was not associated with any of the tested acoustic features, except for a weak preference to match higher pitch with blue (vs. yellow). In other research, high loudness was associated with orange/yellow rather than blue, and high pitch was associated with yellow rather than blue [23].

Chroma has a relationship with sound intensity [23,24]. When the intensity of a sound is strong and loud, its color is close, intense, and deep. However, when the sound intensity is weak, the color feels pale, faint, and far away. Higher value is associated with higher pitch [17,25]. Children of all ages and adults matched pitch to value and loudness to chroma. The value (i.e., lightness) is high and heavy dependent on the light and dark levels of the color. Using the same concept in music, sound is divided into light and heavy feelings according to the high and low octaves of a scale. When the intensity of the sound is strong, the color sensed is close and sharp, whereas when the intensity of the sound is weak, the color becomes distant and muted [26].

Another way to match color and sound is to associate an instrument's tone with color, as in Kandinsky [14].  As shown in Table 1, it is possible to compare the sensibility felt in each instrument tone with the sensibility felt in color.

In "SeeColor" [27], when you touch a relief-shaped, embossed outline area, the color associated with that area is transmitted to an instrument's sound.

In color sonification [28], the color hue, chroma, and value attributes of pixels are mapped onto sound parameters as the f0 (which is related to a sound's perceived pitch), the spectral envelope of the sound (which influences the perception of timbre), and the intensity (which is related to the sound's perceived loudness), respectively. The sound output of a hue value is thus a sinusoid whose frequency depends on that value. The lower frequency colors, such as red and orange, give sensations of strength and power, so they relate well with higher pitched sounds.

The future work from [28] is to deal with more complex sounds and rhythmic patterns. However, there was no assessment provided from these methods to confirm the sound-code literacy of PVI.

In this paper, based on the aforementioned observations, we introduce two SCC sets, VIVALDI and CLASSIC, produced with rhythmic instrumental sounds of classical melody. Table 1 shows the previously proposed color–instrument matchings and the two SCC sets proposed by us.

### **Table 1.** Existing SCCs with instruments and ours.

### **2. Materials, Methods**

### *2.1. Chord Coding Colors (CCC)*

The purpose of this study was to create sounds by which the colors in an image are expressed. Our first SCC set is a so-called chord coding colors (CCC) set as shown in Table 2. Each color's chroma and value are represented by a unique musical chord (e.g., the G chord is made up of the notes G, B, and D. The Gm chord is comprised of G, Bb, and D) that sounds as a single note. In the CCC set, the colors red, orange, yellow, blue, green, indigo, and purple are represented by the instruments violin, viola, trumpet, oboe, cello, horn, and saxophone, respectively. The color–instrument assignment mostly stems from Kandinsky in Table 1. High chroma (saturated) maps to a sound intensity denoted by "ˆ" (velocity = 127), and the medium chroma light and dark map to sound intensity (velocity = 60). A high value (light) maps to a high pitch, denoted by "H" (C4 to C6), and a low value (dark) maps to a low pitch, denoted by "L" (C2 to C4). The codes between colors are all the same, so there is no difficulty to understanding saturated, light, and dark.

**Table 2.** *Cont.*

To listen to a wav file marked with (In Supplementary Materials), left-click the wav audio file and drag it to the computer screen; then right-click to enter the program menu and launch Windows Media Player.

### *2.2. VIVALDI SCC*

The user feedback on the initial pilot test motivated us to develop our two melody-based SCC sets (Tables 3–5). Our VIVALDI SCC was inspired by Vivaldi's The Four Seasons, which musically represents elements of the four seasons of the year. We extracted some of Vivaldi's melodies from spring, autumn, and summer that matched the characteristics of saturated, light, and dark colors, respectively. The most important part in matching instruments and colors is avoiding similar tones so that the sounds (colors) are clearly distinguishable. Therefore, to express each color, we used two strings, two wind instruments, and two percussion instruments for easy identification. In addition, the composition of the excerpted part was changed from original sound, and the velocity and pitch were adjusted to clarify the distinction between value and chroma.


**3.2 GEFS+ and SMEI**

194 Novel Aspects on Epilepsy

On the other hand, some of the disorders display locus heterogeneity where mutations in distinct genes result in the same syndrome. This indicates that other factors beside the primary mutation influence the clinical manifestation of the epilepsy, e.g. environmental factors, developmental events, or differences in inheritance of genetic susceptibility alleles. The latter is supported by mouse models where differences between the genetic backgrounds of two mouse strains influence the severity of a disease caused by the same

Unfortunately, discovery of the responsible gene for an epilepsy syndrome have not led to a prompt understanding of the pathogenesis of the disease. Many of the mutated channels have been characterized in expression systems, but only in some cases have this led to a better understanding of the disease. In other cases this have led to more confusion, as some mutations in a particular channel are found to enhance channel function while others appear to cause a loss of function, even though the clinical manifestation are similar. There are also large discrepancies between results depending on the expression system used to characterize the channels. The mutated channel can e.g. show enhanced function when expressed in *Xenopus laevis* oocytes, while the opposite is shown when expressed in mammalian cells (Meadows et al., 2002). To make it even more difficult, it has been demonstrated that depending on the type of neuron in which a mutated channel is expressed, it can have strikingly different effects on the excitability of the cell (Waxman, 2007). While a mutation can make one type of neuron hyperexcitable, the same mutation can make another neuron hypoexcitable. So changes in neuronal fuction are not necessarily predictable solely from the change in the behaviour of the mutated channel itself, but have to be considered in the cell background in which the mutated channel is expressed. Further, depending on whether a mutated channel mainly is expressed in excitatory or inhibitory neurons, it can have completely opposite effects on the excitability status of the neuronal

The ion channel mutations are bound to cause relative subtle changes in neuronal function. Mutations that cause dramatic changes would likely result in a more severe phenotypes or lethality. The mutations apparently allow normal behaviour under most circumstances, but disturb the equilibrium between excitatory and inhibitory neuronal networks, so that small external perturbations such as fever are sufficient to break the homeostasis and induce

Voltage-gated sodium channels play an essential role in the initiation and propagation of action potentials. These channels open as the membrane depolarizes and inactivate within a few milliseconds of opening. As the membrane polarizes again, the inactivation is removed

Sodium channels are large, multimeric complexes composed of an α subunit and one or more auxiliary β subunits. The α subunit has four homologous domains, each consisting of six transmembrane helices. The β subunit has one transmembrane segment and an extracellular domain with an immunoglobulin-like fold and belongs to the Ig superfamily of cell adhesion molecules (CAMs) (Catterall, 2000). The association with β subunits modulate cell surface expression and localization, voltage-dependence and kinetics of activation and inactivation, as well as cell adhesion and association with signalling and cytoskeletal

sodium channel mutation (Bergren et al., 2005).

**3. Mutations in sodium channel subunit genes**

and a second depolarizing stimulus is able to reopen the channel.

**3.1 Voltage-gated sodium channels**

network (Yu et al., 2006).

seizures.

Febrile seizures, i. e. seizures induced by elevated body temperature, affect approximately 3% of children under 6 years of age and are by far the most common seizure disorder. Generalized Epilepsy with Febrile Seizures Plus (GEFS+) is an autosomal dominant epileptic syndrome where the febrile seizures may persist beyond 6 years of age and which may be associated with afebrile generalized seizures (Scheffer and Berkovic, 1997). The disease has a penetrance of approximately 60%. In 1998, GEFS+ was linked to mutation in SCN1B, the voltage-gated sodium channel β1 subunit gene (Wallace et al., 1998). GEFS+ can also result from mutations in the sodium channel α subunit genes SCN1A (Escayg et al., 2000) and SCN2A (Sugawara et al., 2001), and from mutations in the GABRG2 gene which encodes the γ2 subunit of the GABAA receptor (Baulac et al., 2001). Heterozygous mutations in SCN1A can also result in Severe Myoclonic Epilepsy of Infancy (SMEI), also known as Dravet syndrome (Claes et al., 2001). This rare form of epilepsy is characterized by generalized tonic, clonic, and tonic-clonic seizures that are initially induced by fever, light, sound, or physical activity and typically begin around 6-9 months of age. Later, SMEI patients also manifest other seizure types including absence, myoclonic, and simple and complex partial seizures. Psychomotor development stagnates around the second year of life and the patients often respond poorly to antiepileptic drugs. The disorder usually occurs in isolated patients as a result of *de novo* mutations (Claes et al., 2003; Ohmori et al., 2002).

#### **3.3 How mutations in sodium channels can cause seizures**

As sodium channels are responsible for the upstroke of the action potential one might expect that epilepsy-causing mutations in sodium channel genes increase the activity of the channel, thereby allowing increased influx of sodium ions and consequently neuronal hyperexcitability. Indeed, biophysical analyses of the mutant channels have shown that several of the mutations are gain-of-function mutations that increase sodium currents, e. g. by impairing inactivation or by causing a hyperpolarizing shift in the voltage-dependence of the channel (Lossin et al., 2002; Spampanato et al., 2003; Spampanato et al., 2004). The first identified GEFS+ mutation, a C121W missense mutation that disrupts a conserved disulphide bridge in the extracellular Ig domain of the β1 subunit, causes subtle changes in modulation of sodium channel function and alter the ability of β1 to mediate protein-protein interactions that are critical for channel localization (Meadows et al., 2002; Wallace et al., 1998). Electrophysiological and biochemical studies on the mutant C121W β1 subunit coexpressed with NaV1.2 or NaV1.3 have shown that the C121W mutation causes a reduction in current rundown during high-frequency channel activation and increases the fraction of sodium channels that are available to open at subthreshold membrane potentials (Meadows et al., 2002). The mutation is therefore thought to enhance sodium channel function, thereby increasing neuronal excitability and predisposing to seizures.

On the other hand, many of the characterized sodium channel mutations are found to cause attenuation of sodium current (Barela et al., 2006; Lossin et al., 2003; Sugawara et al., 2001).

Epileptic Channelopathies and Dysfunctional

specifically susceptible to febrile seizures.

**4. Mutations in GABAA receptor subunit genes**

by GABAA receptors (Chebib and Johnston, 1999).

(Oakley et al., 2009).

**4.1 GABA receptors**

**4.2 GEFS+ and ADJME**

some patients.

Excitability - From Gene Mutations to Novel Treatments 197

only led to a moderate increase in the respiration rate and did not cause respiratory alkalosis and seizures (Schuchmann et al., 2006). Fever and the accompanying elevated pH and enhanced neuronal activity seem therefore to be the drop that makes the barrel overflow and induce the seizures. As several ion channels are sensitive to changes in pH (Jensen et al., 2005; Prole et al., 2003), it will be interesting to see whether some mutations in sodium channel genes render the channels pH-sensitive, which could make the affected individuals

SMEI patients are normal until their first seizure that typically occurs around 6-9 months of age. This age-specificity may to be related to the time-specific expression of sodium channels. NaV1.1 is undetectable during prenatal and early postnatal development, a stage where NaV1.3 is preferentially expressed. NaV1.3 expression declines at the expression of NaV1.1 increases. An animal model of SMEI has shown that loss of inhibition and seizure onset correlates in time with an increase in NaV1.1 levels and decline in NaV1.3 levels

GABA is the major inhibitory neurotransmitter in the central nervous system. There are three types of GABA receptors: GABAA, GABAB, and GABAC. GABAA and GABAC receptors are ionotropic while GABAB receptors are G-protein coupled and often act by activating potassium channels. Most of the cortical inhibitory effects of GABA are mediated

The GABAA receptors are pentameric chloride channels formed by various combinations of different types of α (α1 to α6), β (β1 to β3), γ (γ1 to γ3), δ, ε, π, θ, and ρ (ρ1 to ρ3) subunits, that each have four transmembrane segments, M1 to M4 (Benarroch, 2007). The most prevalent subunit combination consists of α1β2γ2 (McKernan and Whiting, 1996). The subunit composition determines the functional and pharmacological characteristics of the receptors (Meldrum and Rogawski, 2007; Sieghart and Sperk, 2002). Binding of GABA to the receptor triggers opening of the chloride channel, allowing rapid influx of chloride that hyperpolarizes

As mentioned, GEFS+ can also result from mutation in the GABRG2 gene encoding the γ2 subunit of the GABAA receptor (Baulac et al., 2001). Mutations in the α1 subunit gene (GABRA1) have been linked to Autosomal Dominant Juvenile Myoclonic Epilepsy (ADJME) (Cossette et al., 2002), an idiopathic epilepsy that is not associated with febrile seizures. This disorder typically manifests itself between the ages of 12 and 18 with myoclonic seizures occurring early in the morning and with additional tonic-clonic and absence seizures in

It has been shown that mutations in the γ2 subunit of the GABAA receptor cause retention of the receptor in the endoplasmatic reticulum (ER) (Harkin et al., 2002; Kang and Macdonald, 2004). Similarly, the A322D mutation in the α1 subunit causes rapid ER associated degradation of the subunit through the ubiquitin-proteasome system (Gallagher et al., 2007).

the neuron and thereby decreases the probability of generation of an action potential.

**4.3 How mutant GABAA receptor subunits can cause seizures**

While it seems like the mild phenotype of GEFS+ mostly is associated with missense mutations that alter the biophysical properties of the channels, the more severe SMEI phenotype is usually caused by nonsense or frameshift mutations that prevent production of functional channels (Claes et al., 2003; Claes et al., 2001; Nabbout et al., 2003; Ohmori et al., 2002). But how can loss-of-function mutations in a sodium channel cause epilepsy when reduced sodium current should lead to hypoexcitability rather than hyperexcitability? The answer seems to be related to the expression pattern of the channels. NaV1.1 is predominantly found in inhibitory interneurons and is thought to conduct most of the sodium current in these cells, whereas excitatory pyramidal neurons express only negligible levels of NaV1.1 (Ogiwara et al., 2007). Catterall and co-workers showed that haploinsufficiency of NaV1.1 channels in heterozygous knock-out mice led to a phenotype resembling that of SMEI (Oakley et al., 2009; Yu et al., 2006). In these mice, sodium currents in GABAergic interneurons in the hippocampus were substantially reduced, whilst the effect in pyramidal cells was much less severe. Loss of one SCN1A copy led to a reduction in action potential number, frequency and amplitude in the interneurons (Yu et al., 2006). Similarly, studies in several animal models carrying nonsense or missense mutations in SCN1A show impaired interneuron function (Martin et al., 2010; Mashimo et al., 2010; Ogiwara et al., 2007; Tang et al., 2009). These studies indicate that functional loss of one copy of SCN1A reduces the inhibitory function of GABAergic interneurons and enhances the excitability of downstream synaptic targets, thereby predisposing to epileptic seizures.

But if this is true, how does the predicted changed NaV1.1 function in many of the patients lead to hyperexcitability when the consequence should be increased GABA action? One possibility is that enhanced sodium current in the interneurons causes too much inhibition, and that this leads to synchronization of the downstream synaptic targets, as has been suggested in the pathogenesis of autosomal dominant nocturnal frontal lobe epilepsy (ADNFL) (Klaassen et al., 2006) (discussed later). Another possibility is that the functional consequences of the mutations in vivo are different from that predicted after in vitro characterization of the mutant channels, and that all of the mutations actually cause a reduction of sodium current in inhibitory neurons. This is supported by studies on knockout mice lacking the β1 subunit (Chen et al., 2004). These mice show downregulated NaV1.1 expression, indicating that β1 function might be necessary for normal expression of NaV1.1. As the inhibitory interneurons seem to be most affected by a reduction in NaV1.1, the consequences of the β1 mutations might be reduced sodium current in interneurons rather than, or in addition to, increased NaV1.2 and NaV1.3 function.

As mutations in SCN1A most often are associated with febrile seizures the mutations seem not to be sufficient to cause spontaneous seizure themselves. Why are the seizures triggered by fever? Why are the seizures most prevalent in young children? And what is the reason for the age-specific onset of SMEI? It is known that an increase in body temperature leads to an increase in the rate of respiration, especially in young children (Gadomski et al., 1994). This increased respiration can cause respiratory alkalosis in the immature brain, and alkalosis of brain tissue can lead to enhanced neuronal activity and to epileptoform activity (Lee et al., 1996). Studies on rat pups showed that seizure activity induced by hyperthermia had a well-defined pH threshold and that a rise in brain pH to the threshold level by injection of bicarbonate could provoke seizures (Schuchmann et al., 2006). By suppressing the alkalosis with a moderate elevation of ambient CO2 to 5%, seizures could be abolished within 20 seconds without affecting body temperature. Bicarbonate-induced pH changes and seizures could also be blocked by elevation of ambient CO2. In older rats, hyperthermia only led to a moderate increase in the respiration rate and did not cause respiratory alkalosis and seizures (Schuchmann et al., 2006). Fever and the accompanying elevated pH and enhanced neuronal activity seem therefore to be the drop that makes the barrel overflow and induce the seizures. As several ion channels are sensitive to changes in pH (Jensen et al., 2005; Prole et al., 2003), it will be interesting to see whether some mutations in sodium channel genes render the channels pH-sensitive, which could make the affected individuals specifically susceptible to febrile seizures.

SMEI patients are normal until their first seizure that typically occurs around 6-9 months of age. This age-specificity may to be related to the time-specific expression of sodium channels. NaV1.1 is undetectable during prenatal and early postnatal development, a stage where NaV1.3 is preferentially expressed. NaV1.3 expression declines at the expression of NaV1.1 increases. An animal model of SMEI has shown that loss of inhibition and seizure onset correlates in time with an increase in NaV1.1 levels and decline in NaV1.3 levels (Oakley et al., 2009).


**2. Materials for ANG storage**

The selection of a suitable adsorbent is one of the most important criteria for the utilization of the technology for successful commercial application. An adsorbent with high surface area, large pore volume, narrow micropore distribution, the pore size of 1–1.2 nm, and high density is required to achieve the ambitious ANG storage target for vehicular application set by the US DOE. The porous materials studied in recent literature for adsorption based natural gas storage at relatively low pressure compared to that of the CNG and ambient temperature conditions are resins, zeolites [5], xerogels [6], aerogels, carbon-based materials [7] such as carbon nanotubes and fibers, metal organic frameworks (MOF) and covalent organic frameworks (COF). The methane adsorption efficiency is known to be linearly dependent on the surface area of the adsorbent (**Figure 1**) [8]. Earlier studies have revealed that Zeolites exhibiting adsorption capacity up to 100 V/V storage capacity are not suitable to reach the target set by the DOE and the interest has shifted to the carbon-based materials and 3D frameworks [4].

*Adsorbed Natural Gas Storage for Vehicular Applications DOI: http://dx.doi.org/10.5772/intechopen.101216*

**Figure 1.**

*Schematics showing the effect of the surface of the carbon-based adsorbents on the methane uptake capacity, the red squares represent the carbon in granular powder form and the green circles represent the coal samples.*

The volumetric storage capacity of the adsorbed natural gas may be calculated in a simple manner by following the equation given below;

$$\mathbf{n}\_{\text{stg}} = \mathbf{n}\_{\text{exc}} + \mathfrak{p}\_{\text{gas}} \left[ \mathbf{1} - \left( \mathfrak{p}\_{\text{pack}} / \mathfrak{p}\_{\text{He}} \right) \right] \tag{1}$$

where, nstg is the volumetric storage capacity, nexc is the excess amount of adsorbate per volume of adsorbent, ρgas, ρpack, and ρHe is the gas, packing and helium density respectively.

### **2.1 Porous carbon materials**

Porous carbon materials are one of the widely studied systems for said application. In general carbon-based materials have exhibited higher adsorption capacity compared to that of the other studied porous materials possessing similar surface area due to their slit-shaped pore structure [9]. The packing density of activated carbon-based materials is also known to exhibit a linear dependence on the applied pressure and the value reaches up to 0.8 g/cm3 on applying pressure till 980 MPa [10]. Furthermore, the activated carbon-based materials can be easily synthesized from readily available low-cost starting materials such as wooden materials, corns, different fossil fuels, and polymer and are cost-effective. These materials can be easily physically activated using steam or CO2 and chemically activated in an industrial scale using acids and bases under high-temperature conditions [11]. Earlier studies on carbon-based adsorbent for the natural gas storage application was based on the single-walled carbon nanotubes (SWCNT). The Monte Carlo simulation-based theoretical studies predicted that the SWCNT bundles may be suitable for methane storage under moderate pressure conditions [12]. However, experimental studies afterward suggested that the adsorption capacity is limited to 0.11 g/g in these systems at 60 bar and 30°C [13]. The activated carbon-based materials have been successful in achieving excess gravimetric methane uptake till 0.2 g/g at 35 bar and ambient temperature conditions and in this case the delivery capacity was 170 V/V at 65 bar pressure [14]. Further increase in pressure to 100 bar improved the adsorption capacity to 263 V/V, which surpassed the target set by DOE. However, the deliverable capacity of carbon-based materials is reported to be inferior, which limits the utilization of these materials for storage applications. The carbon nanotube (CNT) though has shown promise to achieve high adsorption capacity, the experimental value is limited to 160 V/V at 35 bar and 25°C [15].

One of the advantages of these carbon-based materials is that these can be synthesized from renewable, polymeric, and cost-effective sources and activated via multiple routes. For example, olive stones were utilized to prepare carbon-based materials, which on carbonization at 500°C under inert atmosphere produced the carbon material. The sample was activated using KOH at a high temperature of 800°C [16]. The sample exhibited 3551 m2 /g BET surface area and 215 V/V methane uptake capacity at 100 bar with a working capacity of 135 V/V. In another instance, polyacrylonitrile beads were pyrolyzed at 600°C to synthesize carbon flowers. The carbon flowers were then activated with KOH by heating the mixture at 800°C. The flowers were also activated by heating these under CO2 flow at 850°C. The resulting carbon materials exhibited a BET surface area of 1077 m2 /g and methane adsorption capacity of 196 V/V at 65 bar [17]. Other biomass precursors for the synthesis of porous carbon materials include coconut shells [18], corn straws [19], banana peels [20], and soya [21]. Xiao and coworkers published a summary on the use of various biomass for the synthesis of porous carbon materials and the surface area of the resulting materials [22]. The review also summarized the different activation methods utilized to activate the carbon materials such as strong inorganic bases [23], lewis acid [24], and H3PO4 [25] based procedures to optimize the pore structure further and enhance their adsorption performance. Templating is another useful technique utilized in literature to achieve highly ordered and large surface area carbon materials. Both soft and hard templating can be used for this purpose. Hard templates could be a various nanoparticles, silica, and molecular sieves. For example, mesoporous silica sieve hard templates in presence of 1,10-phenanthroline ligand were utilized to synthesize Co immobilized nitrogen-doped porous carbon materials for catalytic applications. The calcination for the material synthesis was carried out at 800°C [26]. Typically, in the case of the soft templating method, the polymers or surfactant self-assemble into a particular ordered shape, which is then immobilized into the mesoporous material to be synthesized. Subsequently, the templates are removed to expose the pores and obtain the porous material. Ionic liquid and self-assembled block polymers have been utilized as the soft templates for this purpose [27, 28]. Direct carbonization of ordered nanostructures is also becoming another attractive option to generate porous carbon materials. For example, the MOF can be utilized as precursors to directly synthesize porous carbon materials via pyrolysis under an inert environment [29]. The presence of various organic ligands may also serve as the carbon source for the above synthesis, whereas the metal nodes often help to control the pore structure and control the physical properties of the carbon material. For example, Yamaguchi and coworkers utilized a Zn and Co-based bimetallic MOF to synthesize nanoporous carbon by pyrolyzing the precursor under an inert atmosphere at 900°C [30]. The shape of the precursor was replicated in the carbon material produced as can be shown in **Figure 2** below. Metal such as Zn evaporates at a high temperature allowing the formation of pores in the resulting carbon product. In some cases, the metal ions convert to the corresponding nanoparticles and serve as a catalytic site for further applications. The type of metal, the metal content, and ligand type play important role in developing nanoporous carbon structures.


**5.2 Falling syndrome**

Muscle weakness syndrome, which involves motor incoordination and transient loss of standing and balance, all encompassed under the common term of "falling syndrome," has been worrying different authors for almost a century [76]. The frequency with which this problem occurs in the arena had not become worrisome until the beginning of the last century, from the being of 1930 when the manifestation of the problem became general and the falls were more frequent and alarming [77], reaching incidence percentages in the most critical decades close to 99% [78] or 98% [26] of the sampled animals. It affects both males and females and specimens of all ages: bulls, calves, and cows [79, 80]. It is observed in individuals of different livestock farms, regardless of their weight, the category of the arena where they fought, the distance from its farm of origin [77], and, additionally, within the same livestock, the incidence of this problem can be very diverse.

Despite recent research work done in this regard, the falling syndrome of the brave bull is an issue in which consensus is not yet perceived. The theories that have come to light in order to explain the etiology of the syndrome have been very numerous and varied, without any of them providing definitive conclusions to date. The simplest attributes the problem to physical reasons such as transport trauma and intentional fraud, while others, more complex, consider that the origin of the syndrome is genetic, due to the inheritance of a gene that determines the fall [81]. However, given the appearance of the problem in cattle ranches whose original genetic distance is very wide, it is logical to assume that the appearance of this syndrome must be influenced by the action of the environment, within which food management, in addition to other factors, such as the health status of the livestock itself would play a very important role.

**123**

drome [69].

capacity [89, 90].

first moments of fight.

mately 3 km, accompanied by horsemen.

*The Lidia Breed: Management and Medicine DOI: http://dx.doi.org/10.5772/intechopen.92008*

metabolic, endocrine, genetic, or ethological causes [76].

mean a lack of physical condition for the show.

enclosure where they normally live.

Nowadays, in view of the different studies carried out, it is possible to think that the falling syndrome is a multicausal problem, where we can observe some predisposing causes, such as the genetic endowment, the characteristics of the transport, the physical demands of the fight, the effect of the *puya* and the *banderillas*, the lack of functional gymnastics, nutritional deficiencies, and other more determinants, such as the possible pathological, circulatory, nervous,

On the other hand, the bull is by nature a sedentary animal. In the last year of life, he is transferred to small enclosures where his chances of exercising naturally are limited and the energetic components in his diet are increased. Although cattle are not considered an athletic species, the bull is subjected to tremendous exercise in the arena, lasting approximately 20 min, maintaining a physical and metabolic effort of great intensity to which it is not accustomed [82]. These circumstances

This muscle weakness, manifested in the falling syndrome, is projected in various acute muscle injuries associated with intense physical exercise and in chronic muscle injuries that may result from nutrient deficiencies of selenium and vitamin E [83]. On the other hand, Aceña et al. [84] demostrated the existence of a reduction in glycogen stored and very high concentrations of lactic acid in the muscles at the end of the fight, results that indicate the existence of muscle fatigue due to physical exercise in an anaerobic situationss. Similarly, a high correlation has been observed between the main parameters indicative of metabolic acidosis (HCO3

lactate, and low blood pH) and respiratory acidosis (PCO2) with the falling syn-

Therefore, it is essential to subject the animals to a physical preparation and adaptation to the fight. In fact, in recent years, the number of farmers who seek to achieve adequate physical condition in their animals has increased, through an empirical training program along a running track or by moving them in the same

There are few studies on the effect of training on the physiology of the bull [85–87]; however, we can state that training potentially increases athletic performance, as can be deduced from muscular and blood metabolic adaptations [88, 89]. It has been observed that training favors the β-oxidative metabolic pathway of fatty acids (oxidative metabolism) prevailing over the glycolytic pathway, requiring a protocol of at least 6 months to increase its antioxidant

In addition, this training would increase the muscle mass of the animal favoring physical performance [91]. To train, and for the result to be effective, great care of the diet should be taken into account since, in the finishing phase of the bulls, it is intended that the animal's body weight increases and that the training will serve to increase muscles and adapt the cardiovascular system to an aerobic exercise. With this training management, it is being pursued that the bull endures the fight better, increasing its mobility while achieving greater lung capacity and, therefore, a greater chance of recovery, after efforts made in the

With training, physical capacity is enhanced, stimulating the body's level of work above normal. These animals have a great capacity for adaptation and although at the beginning of the training they show signs of fatigue and body loss, this is followed by a phase of recovery/adaptation and maintenance of body weight. A basic training program would consist of three sessions per week, within a total period of 5–6 months, depending on the date scheduled for the fight. A group of animals, with a variable number of bulls, around 12, are forced to move for approxi-

−,

#### *The Lidia Breed: Management and Medicine DOI: http://dx.doi.org/10.5772/intechopen.92008*

*Animal Reproduction in Veterinary Medicine*

*Small format "unifeed" mixer truck, adapted to Lidia cattle (BIGA).*

mucosa [27].

**Figure 7.**

**5.2 Falling syndrome**

fed through traditional feeding management. In addition, the feeding time generates a negative effect on the severity of the lesions, with the animals fed for more than 6 months being the ones with the greatest lesions at the level of the rumen

Another strategy to prevent RA is the use of additives both chemical and microbial. Among the first are buffer substances such as bicarbonate, alkalizing agents such as magnesium oxide, or adjuvants such as bentonite, which can help fight RA because it absorbs part of the volatile fatty acids at the ruminal level [5, 65, 73, 74]. The most commonly used microbial additives to combat RA are yeast extracts and live yeasts. These microorganisms help maintain ruminal pH by stimulating the growth of cellulolytic bacteria and lactic acid users, preventing their accumulation in the rumen [75].

Muscle weakness syndrome, which involves motor incoordination and transient loss of standing and balance, all encompassed under the common term of "falling syndrome," has been worrying different authors for almost a century [76]. The frequency with which this problem occurs in the arena had not become worrisome until the beginning of the last century, from the being of 1930 when the manifestation of the problem became general and the falls were more frequent and alarming [77], reaching incidence percentages in the most critical decades close to 99% [78] or 98% [26] of the sampled animals. It affects both males and females and specimens of all ages: bulls, calves, and cows [79, 80]. It is observed in individuals of different livestock farms, regardless of their weight, the category of the arena where they fought, the distance from its farm of origin [77], and, additionally, within the

Despite recent research work done in this regard, the falling syndrome of the brave bull is an issue in which consensus is not yet perceived. The theories that have come to light in order to explain the etiology of the syndrome have been very numerous and varied, without any of them providing definitive conclusions to date. The simplest attributes the problem to physical reasons such as transport trauma and intentional fraud, while others, more complex, consider that the origin of the syndrome is genetic, due to the inheritance of a gene that determines the fall [81]. However, given the appearance of the problem in cattle ranches whose original genetic distance is very wide, it is logical to assume that the appearance of this syndrome must be influenced by the action of the environment, within which food management, in addition to other factors, such as the health status of the livestock

same livestock, the incidence of this problem can be very diverse.

**122**

itself would play a very important role.

Nowadays, in view of the different studies carried out, it is possible to think that the falling syndrome is a multicausal problem, where we can observe some predisposing causes, such as the genetic endowment, the characteristics of the transport, the physical demands of the fight, the effect of the *puya* and the *banderillas*, the lack of functional gymnastics, nutritional deficiencies, and other more determinants, such as the possible pathological, circulatory, nervous, metabolic, endocrine, genetic, or ethological causes [76].

On the other hand, the bull is by nature a sedentary animal. In the last year of life, he is transferred to small enclosures where his chances of exercising naturally are limited and the energetic components in his diet are increased. Although cattle are not considered an athletic species, the bull is subjected to tremendous exercise in the arena, lasting approximately 20 min, maintaining a physical and metabolic effort of great intensity to which it is not accustomed [82]. These circumstances mean a lack of physical condition for the show.

This muscle weakness, manifested in the falling syndrome, is projected in various acute muscle injuries associated with intense physical exercise and in chronic muscle injuries that may result from nutrient deficiencies of selenium and vitamin E [83]. On the other hand, Aceña et al. [84] demostrated the existence of a reduction in glycogen stored and very high concentrations of lactic acid in the muscles at the end of the fight, results that indicate the existence of muscle fatigue due to physical exercise in an anaerobic situationss. Similarly, a high correlation has been observed between the main parameters indicative of metabolic acidosis (HCO3 −, lactate, and low blood pH) and respiratory acidosis (PCO2) with the falling syndrome [69].

Therefore, it is essential to subject the animals to a physical preparation and adaptation to the fight. In fact, in recent years, the number of farmers who seek to achieve adequate physical condition in their animals has increased, through an empirical training program along a running track or by moving them in the same enclosure where they normally live.

There are few studies on the effect of training on the physiology of the bull [85–87]; however, we can state that training potentially increases athletic performance, as can be deduced from muscular and blood metabolic adaptations [88, 89]. It has been observed that training favors the β-oxidative metabolic pathway of fatty acids (oxidative metabolism) prevailing over the glycolytic pathway, requiring a protocol of at least 6 months to increase its antioxidant capacity [89, 90].

In addition, this training would increase the muscle mass of the animal favoring physical performance [91]. To train, and for the result to be effective, great care of the diet should be taken into account since, in the finishing phase of the bulls, it is intended that the animal's body weight increases and that the training will serve to increase muscles and adapt the cardiovascular system to an aerobic exercise. With this training management, it is being pursued that the bull endures the fight better, increasing its mobility while achieving greater lung capacity and, therefore, a greater chance of recovery, after efforts made in the first moments of fight.

With training, physical capacity is enhanced, stimulating the body's level of work above normal. These animals have a great capacity for adaptation and although at the beginning of the training they show signs of fatigue and body loss, this is followed by a phase of recovery/adaptation and maintenance of body weight.

A basic training program would consist of three sessions per week, within a total period of 5–6 months, depending on the date scheduled for the fight. A group of animals, with a variable number of bulls, around 12, are forced to move for approximately 3 km, accompanied by horsemen.

It usually begins with a weekly session, increasing the pace until reaching 3 sessions/week in the second month. The intensity is progressive, each session begins with the first minute to the step, to warm the animals, increasing the pace until they are trotted or lightly galloped, to return to the initial point in a progressive cooling. The orography of the land is usually flat, but there are farmers who prefer to exercise the cattle on sloping terrain to increase the intensity of the session. This training is interrupted approximately 15 days before the fight [92].

Each breeder has been carrying out a particular training protocol, adapted to their availability of time and cowboys, the number of animals they intend to prepare, and the date of their fight. Generally, a more intense preparation is usually carried out with bulls whose destiny is first or second category arena. In turn, the orographic characteristics of the farm, its distribution of fenced areas, and its extension will have an important influence on the programmed exercise.

#### **5.3 Health management**

Considering the high economic value of the Lidia breed animals, the number of farmers who establish a health management program in their livestock as a control system against infectious or parasitic diseases, and to increase fertility and pregnancy rates as well as to decrease mortality rates in new-born calves, is rising in recent years.

Problems related to infectious and contagious diseases represent the main source of economic losses. The pathogens that have tropism for the reproductive, respiratory, or digestive system stand out. Therefore, reproductive and respiratory alterations and neonatal diarrhea are the main problems we find in these cattle [93].

Currently, there are several emerging diseases that could affect these animals during the last decade such as blue tongue, foot and mouth disease, or bovine spongiform encephalopathy, which have joined those that already have an eradication program in our country (brucellosis and tuberculosis), which require periodic official livestock checking on farms (Order DES/6/2011). On many occasions, the health problem itself is linked to a cumbersome legislation that hinders the transit of animals through the various communities of the national territory and between intracommunity countries such as Spain, France, and Portugal (Royal Decree 186/2011).

The official campaigns of eradication of brucellosis and tuberculosis are based on hard controls of the herds and on the application of a legal regulation on these aspects that makes, in certain cases, the movement of animals from the infected cattle ranches, including sales for bullfighting, impossible [94]. It is essential to consider the peculiar factors of this cattle production system. One of them is the level of consanguinity within some farms with a very small number of individuals, which works against disease resistance. It is also necessary to consider the complexity of handling these animals, which coexist in extensive systems with species of different sanitary categories (hunting and/or wild) that could be reservoir for numerous diseases.

In addition, cross-reactions with paratuberculosis (a widespread disease in the Spanish countryside) compromise the reliability of diagnostic analytical tests, posing serious problems when addressing eradication plans [95]. The fight against diseases, both endemic (tuberculosis and brucellosis) and emerging (bluetongue), to achieve eradication and control, will be one of the workhorses for the Lidia sector. This should not entail, in any case, any risk to the maintenance of the diversity of *encastes* and genetic lines that characterize this breed. Important and unique farms for their genealogy are being decimated by this cause, to the point of endangering the survival of certain *encastes*.

**125**

**7. Conclusions**

*Lidia bull with protected horns.*

**Figure 8.**

*The Lidia Breed: Management and Medicine DOI: http://dx.doi.org/10.5772/intechopen.92008*

One of the most valued and delicate body parts of the bull is its horns. They suffer a risk of deterioration, mainly in the last year of life, as a result of potential fights, friction, contacts, or blows with the ground, with trees, fencing, feeders, or the walls of the handling facilities [96]. Therefore, to protect the horns during the last year of animal live, a fiberglass bandage is placed on the horns, easy to handle, porous and that hardens quickly by polymerization with water, providing good consistency (**Figure 8**). The technique consists of immobilizing the animal in the restraining facilities and wrapping the horn with this bandage to protect it from any aggression or friction. The distal part of the horn is reinforced in many cases with metal tubes or similar hard materials, in order to reduce the wear of the apical zone

The horn is increased in thickness by the sheath, and the end of the horn is blunt, which decreases the effect of the lesions of horns between animals by 90% and, in addition, improves their handling for vaccinations, deworming, and other treatments, due to the risks of deterioration of the defenses when the animals pass through the handling facilities minimized [99]. In spite of the obvious advantages of the sheathing mentioned above, and the answer to many questions about the influence of this management practice on the structure and corneal anatomy and the ethological performance of the animal in the arena provided by Alonso et al.

Lidia cattle production presents unique characteristic that requires farmer and veterinary knowledge about the particularities of these animals and its management. The Lidia production sector, from its origins, has been adapting to the new times making use of the most current technological advances. In this way, the feeding system, selection criteria, and reproductive techniques have been modified, driving the need for a modernization of the medical and management practices.

[100], there is still some controversy about its usefulness.

**6. Sheathed of horns**

[97, 98].


**2.10 Soil erosion prevention**

The major environmental problem of soil erosion. It is necessary to take action to solve this issue. Some strategies for preventing soil erosion include the ones listed below:

1.To stop soil erosion, add mulch and rocks to the grass and plants below.

2.Plant trees to prevent soil erosion on bare land.


*6.3.1 Hand shaking or non hand shaking of lipid based film by hydration*

This method was first described by [1] and which is simple for liposomes formation with one limitation of having low drug loading. Phospholipids and cholesterol are to be dispersed in organic solvent and then evaporated by using rotary evaporator at low pressure and vacuum. When solvent is evaporated then a dry film will be formed on the wall of rota flask and that should be hydrated by using aqueous phase buffer. The lipids in film spontaneously gets swell when hydrated to form heterogeneous multilamellar liposomes (MLVs).

#### *6.3.2 Micro-emulsification*

Micro fluidizer helps in preparing small MLVs from liposomal dispersion [23]. Micro fluidizer pumps liposomal fluid at pressure of 10,000 psi through 5 μm orifice and by micro channels that directs two pathways of dispersion to colloid with high velocity. The large MLVs liposomal dispersion or organic medium containing lipids can also be passed through fluidizer. The dispersion collected is replaced through the micro fluidizer until vesicles with spherical dimensions obtained.

## *6.3.3 Sonication method*

When the liposomal vesicles sizes are above 1 μm then sonication is done to reduce size to form SUVs or extrusion done with polycarbonate filters for producing smaller and uniform sized vesicles. Size reduction by ultrasonication for aqueous dispersion can be done mainly by bath or probe sonicators [24].

## *6.3.4 French pressure cell*

The French pressure press breaks cells with appropriate conditions compared with ultrasound techniques [25]. French pressure press is advantage because sonication procedure degrades lipids, proteins and sensitive compounds. This cell can be used for dispersions with low volume of less than 40 ml and not applicable for high volume production batches. Hence, a scale-up-based strategy was established by using micro fluidization technique.

#### *6.3.5 Membrane extrusion*

Another method for liposomes downsizing is extrusion. The vesicles with force are passed through membranes with a lower pressure than french press. Extrusion studies using polycarbonate filters were done and performed on extrusion behavior and membrane properties [26, 27]. Lipex Biomembranes Inc., now called Northern Lipids Inc., invented extrusion vessel from milliliter to several liters. This Lipex extruder allows higher temperatures with jacketed mode. An alternative to this lipex extruder is Maximator device, which is continuous pumping system which was introduced by Schneider et al., 1994. The Maximator has glass vessel which is thermostable and connected directly to pneumatic piston pump. This method consists of preparing liposomes followed by freeze–thaw and finally extrusion. This long process and disadvantage because of high product loss.

## **6.4 Methods on fusion of preformed vesicle**


*<IP_ADDRESS> Sciatic notch index*

The sciatic notch index is given by dividing the hundred times width of sciatic notch with its depth.

ð Þ Width of the sacrum*=*diameter of sacrum ∗ 100 (5)

acetabular height (vertical diameter) and indices 1 (total pelvic height/acetabular height), 2 (midpubic width/acetabular height) and 3 (pubic length/acetabular height). Pelvic brim depth, minimum width of ischiopubic ramus and indices 4 (pelvic brim chord \* pelvic brim depth) and 5 (pubic length \* 100/ischial length) were also good discriminators of sex. The remaining parameters used in the study were not significant as they showed a lot of overlap between the male and female categories. The results indicated that one exclusive criterion for sexing was index 3

The sacrum is a large flattened triangular bone formed by the fusion of five sacral vertebrae and forming the posterosuperior part of bony pelvis. It articulates on either side with the corresponding innominate or hip bone forming sacroiliac joint. Morphological and metric differences of sex determination are given in the

The sacral index [30] is given by dividing the hundred times length of anterior superior breadth of the sacrum at the first sacral vertebrae with anterior length of sacrum. The anterior length was measured along the midline from the anterosuperior margin of the promontory to the middle of antero-inferior margin of the last sacral vertebra. The anterior superior breadth was measured between the

¼ ð100 ∗ Anterior superior breadth of sacrum*=*Anterior length of the sacrumÞ

The study [30] also calculated the demarcating point (DP) which increases the accuracy by 100%. The range of sacral index in male is 80.7–106.4 and in females is 93.1–108.8 and DP for sacral index in males is less than 90.29 and in females is

In a study [31] done on 150 fully ossified dry human sacrums (59 male and 91 females), it was observed that the mean straight length of sacrum in the male and in the female was 104.27�5.76 mm and 92.82�7.59 mm respectively. The mean width of sacrum in the male and the female was 99.51�5.80 mm and 102.98�6.69 mm respectively. The mean sacral indices were 95.42�3.14 and 111.27�7.66 in males and

2 Curvature More evenly distributed Curvature not seen in the upper

**Sr. No Trait Male Females** 1 Size and shape Longer, Narrower Shorter, wider

3 Sacral Promontry Well marked Less marked 4 Body of first sacral vertbra Larger Smaller

vertebrae

5 Sacroiliac articulation Large, Extends to 2.5to 3

*Showing difference in human sacrum with respect to sex.*

(7)

half, lower half curves suddenly

Small, Extends to 2 to 2.5 vertebrae

(pubic length/acetabular height).

*Forensic Osteology and Identification*

*DOI: http://dx.doi.org/10.5772/intechopen.99358*

lateralmost points of the ala of the sacrum.

**4.2 Sacrum**

**Table 7**.

*4.2.1 Sacral index*

Sacral Index

greater than 112.43.

females respectively.

**Table 7.**

**115**

In adult males: 145; in adult females: 166. In the male fetus: 4–5; in the female fetus: 5–6.

### *<IP_ADDRESS> Chilotic line index*

The chilotic line index is obtained by dividing the hundred times length of the sacral part of the pectineal line with the pubic part of pectineal line.

ð Þ Sacral part of pectineal line*=*pubic part of pectineal line ∗ 100 (6)

In males: the CLI is greater than 100, In females: the CLI is less than 100.

These indexes are not used routinely. Nowadays, discriminant function analysis is used by anthropologists. This was first used by Howells [28]. He worked on Gaillard's skeletal collection (75 males, 69 females) and took four parameters, ischial and pubic lengths and the index obtained from it, he took four measurements of the greater sciatic notch and acetabular region. These included sciatic height, cotylosciatic length (shortest distance from acetabular rim to greater sciatic notch), cotylopubic length (from acetabular rim to pubic symphysis) and the difference between SS-SA, in which SS is the distance between the anterior superior iliac spine and the closest point on the greater sciatic notch, and SA is the distance between the anterior superior iliac spine and the closest point on the auricular surface (**Table 6**).

In another study, Dixit [29] observed twelve measurements and five indices from 100 human hip bones of unknown sex of Indian origin. Each of the hip bones was classified as male, female and intermediate on the basis of morphological characters. Afterwards discriminant function analysis was done and it was observed that sex can be accessed with greater accuracy from parameters such as the

#### **Table 6.**

*Showing discriminant function coefficients for determining sex from the Os Coxa.*

#### *Forensic Osteology and Identification DOI: http://dx.doi.org/10.5772/intechopen.99358*

acetabular height (vertical diameter) and indices 1 (total pelvic height/acetabular height), 2 (midpubic width/acetabular height) and 3 (pubic length/acetabular height). Pelvic brim depth, minimum width of ischiopubic ramus and indices 4 (pelvic brim chord \* pelvic brim depth) and 5 (pubic length \* 100/ischial length) were also good discriminators of sex. The remaining parameters used in the study were not significant as they showed a lot of overlap between the male and female categories. The results indicated that one exclusive criterion for sexing was index 3 (pubic length/acetabular height).


**2.3 Adverse production-load situations (summer-winter)**

As gas is used – at least in middle Europe and Germany - for heating to a high percentage there will be a big difference in consumption figures between winter and summer (see figure 2).

Typical locations of biogas plants and the major cities the corresponding gas transportation

and distribution network are shown in figure 1.

Fig. 1. Biogas plant locations in Germany 2011 (source: DENA)

**2.3 Adverse production-load situations (summer-winter)**

Typical biogas plants have preferred locations in rural areas, where the renewable material grows and the transportation is short and easy. In opposition to the rural places the areas of higher gas demand or actual consumption are located in or near urban areas. So, in most cases the biogas must be transported in pipelines over a longer distance spreading also over a wider network area before the biogas is consumed totally. Depending on the network structure and the total consumption figures there exist mixing areas of biogas and natural

As gas is used – at least in middle Europe and Germany - for heating to a high percentage there will be a big difference in consumption figures between winter and summer (see

**2.2 The location paradox**

gas or pure biogas.

figure 2).

Fig. 2. Total load/feed-in (blue), industrial (RLM) and small consumer load (red) vs. time (hours) of a big city

In certain areas the biogas feed-in in the network is in wintertime only a small percentage and the area of influence is therefore small, too; but it is large in summertime. This fact principally leads to problems in pipeline connection, operation, constant gas quality delivery and fair billing (see below, Operational Aspects). In the future – when the number of plants and/or biogas production will increase - we will expect a considerable higher impact on network operation and surveillance tasks.


**5. Conclusions**

Our studies showed for the first time that anticancer drugs ixazomib, oprozomib, and delanzomib had a critical role in upregulating OAT3 transport activity and expression, suggesting their potential impact on the OAT3-mediated drug disposition and clinical drug–drug interactions during combination therapies of proteasome inhibitor drugs and other types of drugs (Figure 11).

**Author Contributions:** Conceptualization, Y.F. and G.Y.; methodology, Y.F.; software, Y.F.; validation, Y.F.; formal analysis, Y.F.; investigation, Y.F., Z.L., J.Z.; resources, Y.F.; data curation, Y.F.; writing—original draft preparation, Y.F.; writing—review and editing, G.Y.; supervision, G.Y.; project administration, G.Y.; funding acquisition, G.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by grants (to Guofeng You) from National Institute of General Medical Sciences (R01-GM079123 and R01-GM127788).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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


**2.  Instead of the usual 'disc'-shaped eyes normally found in birds, owls have developed large 'tubular'-shaped eyes (**Figure 2**) that are held tight within the orbit and protected by the *scleral ossicles*, or 'sclerotic ring', composed of a series of small interlocking bones that form a bony ring within the sclera. These 'tubular' eyes are so large that in some small species of owl they can take up to 50% of the skull area. Some eagle species have developed 'tubular' eyes, but these are much shorter in length than an owl's, while the majority of diurnal raptors have much smaller 'globose' eyes (**Figure 3**) [2].

The owl's large, forward-facing eyes allow for considerable binocular vision, giving an excellent, but a fairly narrow field of view of 110 degrees, with an overlap of

*Designed for Darkness: The Unique Physiology and Anatomy of Owls DOI: http://dx.doi.org/10.5772/intechopen.102397*

#### **Figure 2.** *Diagram of an owl's eye. Image credit: owlpages.com*

approximately 70 degrees (man, by comparison, has a field of view of 180 degrees with an overlap of 140 degrees) [3]. With such a narrow field of view, many species resort to that very distinctive behaviour of owls, head bobbing, to accurately judge distance and position.

The eyes have extremely large cornea (the transparent outer coating of the eye) and pupil (the opening at the centre of the eye). A larger cornea allows for a larger pupil size, which in turn serves to increase the number of photons that reach the retina (light-sensitive tissue on which the image is formed), thereby improving visual sensitivity [4]. The pupil's size is controlled by the iris (the coloured membrane suspended between the cornea and lens). When the pupil is larger, more light passes through the lens and onto the large retina. Light sensitive retinal cells act as receptors and form images. These receptors are made up of two types of cells, rods and cones; so named for their shapes. Cones distinguish colours, function in bright light, and are needed for sharp resolution, while rods function in low light or at night and

#### **Figure 3.**

*Eye orbit comparison nocturnal-diurnal. L—Little owl (*Athene noctua*). R—Kestrel (Falco tinnuculus). Photo: Alan Sieradzki.*

#### **Figure 4.**

*The nictitating membrane of the Eurasian eagle owl (*Bubo bubo*). Photo: Bruce Marcot.*

are sensitive to movement. Primarily a nocturnal predator, an owl's eyes are packed with rods, giving owls excellent nocturnal vision without the need of the *tapetum*, a reflecting layer at the back of the eye found in most nocturnal animals, including those other nocturnal birds, the Caprimulgiformes. While the ambient light on a cloudy, moonless night rarely drops below an illumination level of 0.004-foot candles, experiments have shown that some species, such as Long-eared Owl (*Asio otus*), Tawny Owl (*Strix aluco*), and Barn Owl (*Tyto alba*), can spot and approach prey from a distance of 6 feet or more under illumination as low as 0.000,00073 foot candles [5]. A recent study's findings indicate that owls may have independently evolved a DNA packaging mechanism in the retina that enhances light channelling in photoreceptors, a feature that has not been observed in any other bird species to date [1].

But an owl's eyes also contain enough cones to enable it to see perfectly in daylight; owls are by no means blind in the daylight. In fact, with its wide range of pupil adjustment, an owl's ability to see sharply is as developed as in any diurnal raptor and has allowed many owl species, such as the Burrowing Owl (*Athene cunicularia*), the Snowy Owl (*Bubo scandiacus*) and the Short-eared Owl (*Asio flammeus*), to become highly successful diurnal hunters. To protect their eyes, owls are equipped with three eyelids. They have a normal upper and lower eyelid, the upper closing when the owl blinks (as in humans—the only bird to do so), and the lower closing up for sleep. The third eyelid is called a *nictitating membrane*, which is a thin layer of tissue that closes diagonally across the eye, from the inside to the outside and cleans and protects the surface of the eye (**Figure 4**).


**2. Molecular and cellular structure of the extracellular matrix**

In skeletal muscles, the ECM is involved in a variety of processes during development, contractile maturation, fibre regeneration following injury, physiological adaptations to changed functional demands and the natural aging process [8]. As outlined in **Figure 1**, the ECM is a highly dynamic non-cellular system that undergoes frequent cycles of modifications, degradation and reassembly. The muscle ECM functions on the one hand as an embedding and stabilizing structural support and on the other hand as a cellular interaction and signalling medium. The molecular lattice of collagens and proteoglycans with its associated matricellular proteins, enzyme systems and adhesion receptors mediates various physiological and biochemical mechanisms, including (i) the overall maintenance of muscle tissue stability and elasticity, (ii) the mechanical transduction of force from the contractile fibres to their anchoring tissues, (iii) cytoskeletal coupling to enable the efficient execution of frequent excitation– contraction–relaxation cycles, (iv) the provision of signalling pathways at the fibre periphery, (v) the preservation of neuromuscular homeostasis, and (vi) the physical scaffold and embed‐ ding medium of cellular constituents, such as contractile fibres, capillaries, motor neurons and satellite cell pools. Cell–matrix interactions are of central importance during cell adhesion and cell migration and thus essential during both embryonic and adult myogenesis.

issues related to high energy demand, elevated levels of cellular stress and enormous physi‐ cal strains during excitation–contraction–relaxation cycles [1]. The survival of skeletal muscle fibres therefore depends heavily on (i) a high degree of physiological adaptability, (ii) a unique level of tissue plasticity, (iii) efficient molecular chaperoning to prevent proteotoxic insults and (iv) a sophisticated repair machinery that can counter-act frequent cellular injuries [2]. A crucial stabilizing element that is intrinsically involved in this continuous maintenance of contractile tissues is the extracellular matrix (ECM) [3]. On the one hand, the complex layers of the muscle ECM provide the physical structure for force transmission between contracting fibres and their surrounding tissue environment [4], and on the other hand the ECM functions as an embed‐ ding medium for essential supportive components of muscles such as capillaries and motor

70 Composition and Function of the Extracellular Matrix in the Human Body

The composition and organization of the ECM adapts considerably in response to changed functional or structural demands during myogenesis, fibre maturation and exercise-induced changes [6]. During the natural aging process and in association with a variety of muscular disorders, a hyperactive connective tissue may trigger myofibrosis with a detrimental impact on muscle elasticity and fibre contractility [7]. Physiological or pathological changes in the muscle ECM frequently mirror the different phases of altered muscle structure and function [8]. The main component of the ECM is represented by collagen, which exits in a large number of isoforms that connect with proteoglycans, matricellular proteins and adhesion receptors to

This chapter provides an overview of the molecular components of the ECM from skeletal muscle and describes the proteomic concept of the ECM complexome. The formation, matu‐ ration and flexibility within the various layers of the ECM in developing, maturing and adapting skeletal muscles is outlined, as well as the crucial role of myofibrosis in neuromus‐

In skeletal muscles, the ECM is involved in a variety of processes during development, contractile maturation, fibre regeneration following injury, physiological adaptations to changed functional demands and the natural aging process [8]. As outlined in **Figure 1**, the ECM is a highly dynamic non-cellular system that undergoes frequent cycles of modifications, degradation and reassembly. The muscle ECM functions on the one hand as an embedding and stabilizing structural support and on the other hand as a cellular interaction and signalling medium. The molecular lattice of collagens and proteoglycans with its associated matricellular proteins, enzyme systems and adhesion receptors mediates various physiological and biochemical mechanisms, including (i) the overall maintenance of muscle tissue stability and elasticity, (ii) the mechanical transduction of force from the contractile fibres to their anchoring tissues, (iii) cytoskeletal coupling to enable the efficient execution of frequent excitation– contraction–relaxation cycles, (iv) the provision of signalling pathways at the fibre periphery, (v) the preservation of neuromuscular homeostasis, and (vi) the physical scaffold and embed‐

form an elaborate extracellular network and tight cell–matrix interactions [9].

**2. Molecular and cellular structure of the extracellular matrix**

neurons [5].

cular pathology.

**Figure 1.** Overview of the complex functions and interactions of the extracellular matrix from skeletal muscles.

The distinct layers of the ECM that surround muscle fibres, muscle fascicles and the entire skeletal muscle are formed by the endomysium, perimysium and epimysium, respectively [3]. **Figure 2** shows diagrammatically the arrangements of the ECM from skeletal muscles and lists the main molecular constituents, including various isoforms of collagen, proteoglycans, matricellular proteins, crosslinking proteins and matrix metalloproteinases. Crucial adhesion systems that maintain sarcolemma–matrix interactions are marked. They include the collagenlaminin-α/β-dystroglycan-dystrophin/utrophin axis and the collagen–fibronectin–integrin axis that link the basal lamina via the plasmalemma to the underlying membrane cytoskeleton [10].

The stabilizing linkage between the outside and inside of muscle cells, provided by the ECM– sarcolemma–cytoskeleton axis, is of critical importance for maintaining normal contractile functions [8]. Primary or secondary abnormalities in individual binding partners of these surface complexes may result in severe neuromuscular disorders, as discussed in below section on the role of the ECM in skeletal muscle pathology and myofibrosis.

Collagens form tight helical structures and function as the main structural protein species in the extracellular space. Muscle-associated collagens are highly abundant in the interstitial matrix, ECM microfibrils and the basal lamina [11]. Collagen isoform COL I, the most abundant protein in the mammalian body [12], is the primary collagen in the perimysium and tendon. The interstitial matrix contains mostly collagen COL I, COL III and COL V. Minor types of

**Figure 2** Diagrammatic presentation of the various layers of the extracellular matrix (ECM) from skeletal muscle and an overview of the molecular components that form the interstitial matrix, microfibrils and the basal lamina. Abbrevia‐ tions: COL, collagen; DG, dystroglycan; FN, fibronectin; LAM, laminin; INT, integrin; MMP, matrix metalloproteinase; NMJ, neuromuscular junction; PG, proteoglycan; PRELP, proline-arginine-rich end leucine-rich repeat protein; SLRP, small leucine-rich repeat proteoglycans; TIMP, tissue inhibitors of matrix metalloproteinases.

collagens in this extracellular region are COL XI, XII, XIV, XV and XVIII and are mostly expressed during muscle development [3]. The main structural constituent in ECM microfibrils is collagen isoform COL VI. Microfibrils provide structural support during the physical strains of continuing contraction–relaxation cycles. Directly overlaying the sarcolemma membrane is the basal lamina consisting of the main non-fibrillar collagen isoform COL IV and a few minor constituents, including COL VI, XV and XVIII. The collagen network of the basement mem‐ brane interacts with two crucial plasmalemma adhesion complexes, the integrin complex and the dystrophin–glycoprotein complex [10]. These sarcolemma-bridging protein assemblies form the laminin–dystroglycan axis [13] and the fibronectin-integrin (α7β1) axis [14], whereby the ECM glycoprotein fibronectin mediates the connection between laminin-211 (α2β1γ1 merosin) and collagen COL IV [15].

A variety of regulatory ECM proteins are involved in matrix assembly and the modulation of cell–matrix interactions, such as dermatopontin, nidogen/entactin, periostin (osteoblastspecific factor OSF-2) and osteopontin [16–18]. Matricellular proteins represent non-architec‐ tural ECM components and are crucial factors during muscle development and fibre repair. Matrix metalloproteinases are an important class of ECM-associated enzymes that regulate the degradation of ECM proteins and support tissue integrity during phases of collagen deposition and muscle regeneration [19]. The main isoforms present in skeletal muscles are matrix metalloproteinases MMP-1, MMP-2, MMP-9, MMP-10 and MMP-13 [20,21]. The tissue inhibitors of matrix metalloproteinases, named TIMP, are endogenous regulatory factors involved in the formation, adaptation and controlled degradation of the ECM [22]. TIMP molecules play a crucial role in the migration and differentiation of muscle stem cells during regeneration following cellular injury [19].

A large variety of proteoglycans fill the gaps between collagen molecules and thereby form an integral part of the highly complex ECM structure. Proteoglycan molecules are highly glycosylated with glycosaminoglycans at multiple sites along the peptide backbone [23]. Skeletal muscles contain small leucine-rich repeat proteoglycans (SLRP), heparan sulfate proteoglycans and chondroitin sulphate proteoglycans. Muscle-associated proteoglycans of the type SLRP are asporin, biglycan, decorin, mimecan (osteoglycin), fibromodulin and lumican. Asporin is mostly found in the cartilage matrix. Biglycan is a small SLRP-type proteoglycan that interacts with α-sarcoglycan and γ-sarcoglycan of the dystrophin-glyco‐ protein complex [24]. Decorin is the primary proteoglycan molecule of the perimysium and tendon structures [25]. Fibromodulin is involved in collagen fibril formation, which is illustrated by the biomedical fact that fibromodulin-deficient tendons exhibit abnormal collagen fibrils [26]. The heparan sulfate proteoglycan syndecan is transiently up-regulated during tissue differentiation and is involved in stem cell maintenance and muscle regeneration [27]. Perlecan is located to the basement membrane and its expression is also transiently increased during muscle differentiation [28]. The chondroitin sulphate proteoglycan named aggrecan forms large aggregates in cartilage [29]. A proline-arginine-rich end leucine-rich repeat protein (PRELP) is presented by prolargin of the basal lamina. At the highly differen‐ tiated neuromuscular junction region, the large proteoglycan molecule agrin is present and forms via α-dystroglycan a tight linkage to the utrophin-glycoprotein complex. Agrin is essential for the normal development of the neuromuscular junction and agrin-induced clustering processes are crucial for the anchoring of the acetylcholine receptor complex in the junctional folds [30].


**4. Conclusions**

The second-order polynomial model provided adequate mathematical description of the UAE of phenolic compounds from black locust flowers. ANOVA analysis confirmed that extraction time has the highest impact on the TPC, followed by the extraction temperature and ethanol concentration. The optimal extraction conditions were 60% (v/v) ethanol, 59 ◦C, and 30 min at the liquid-to-solid ratio of 10 cm3 g−1. Under these conditions, the TPC was found to be 3.12 gGAE 100 g−1 d.p.m. Compared to the maceration and Soxhlet extraction, the UAE has a higher yield of phenolic compounds for shorter extraction time and better antioxidant activity. Therefore, the UAE represents an effective, reliable, and feasible technique for preparing extracts enriched with phenolic compounds, such as rutin quantified by HPLC analysis. The structural changes in the flowers obtained by the different techniques were evaluated using SEM analysis. The micrographs indicate that the different action mechanisms are characteristic for the used extraction techniques. Further studies will be directed to more detailed identification and quantification of other phenolic compounds available in the extracts obtained under optimal conditions and to investigate their biological activity.

**Author Contributions:** I.S. and I.S.G., conceived, designed, and performed the experiments. D.G., S.Ž., I.B., and I.M., participated in the data analysis and contributed intellectually to the manuscript.

**Funding:** This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia under the project TR-34012. Ivana Boskov is the recipient of a scholarship from the Ministry of Education, Science and Technological Development of the Republic of Serbia.

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