**Author details**

Kazuaki Yamada Faculty of Science and Engineering, Department of Machine Engineering, Toyo University, Kujirai, Kawagoe-shi, Saitama, Japan

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


2. Definitions: parametric curves

autonomous cars on the roads, as there are both technical and legal problems involved [2, 3]. The intelligent system is composed of three different groups and subgroups: acquisition and

Although the vast majority of the literature often depicted the problems by focusing mainly on these groups or subgroups of processes, functionality in intelligent vehicles, or in mobile robots in general, cannot be conceived as composed of separate blocks, and therefore, a

This chapter is devoted to the use of parametric curves in the field of robotics. Parametric curves are mainly used in the decision block when the path is defined. However, they are also employed in other blocks, and some of their properties are beneficial for other processes.

Focusing on the decision-making block, the "path planning" or the design of the path to follow has been the subject of study in the last decades, where many authors divide the problem into global and local path planning. On the one hand, the global path planning generates an overall path composed of a set of points to be followed, covering large distances and considering static obstacles in the environment. On the other hand, the local path planning constructs a short path with much more precision, even in continuous form, taking into account unexpected

In general, path planning techniques can be grouped into four large groups: graph search,

• Graph search-based planners search a grid for the optimal way to go from a start point to a goal point. Algorithms, such as Dijkstra, A-Start (A \*) and its variants Dynamic A\* (D\*),

• Sampling-based planners try to solve the search problem restricting the computational time. The idea is to randomly explore/sample the configuration space, looking for connections between source and destination. The main problem is that the solution is suboptimal. The most common techniques are the probabilistic roadmap method (PRM) and the rapidly

• Interpolating curve planners try to insert a new group of data within the previously defined data group. In other words, both graph search and sampling-based planners are global planners that provide a rough approximation of the solution. In this case, it is a matter of interpolating this group of points. At this stage, the design of the trajectory is when the properties of continuity, smoothness and geometrical restrictions of the vehicle, among others, intervene. Computer-aided geometric design (CAGD) techniques are generally used to smooth the gross path provided by the global planner. The use of lines and circles is usually employed as a first solution, with Dubin's curves defined when the vehicle moves forward and Reed and Sheep's curves when the vehicle moves backward. The clothoid appears as a solution to the discontinuity in curvature between the line and the circle since, by definition, it has a constant relationship between the length of the arc and its curvature. The polynomial curves are another alternative to the previous ones. The modification of its coefficients allows taking into account, among others, the adjustment of positions, curvature restrictions, etc.

field D\*, Theta\*, etc., have been extensively studied in the literature.

sampling, interpolating and numerical optimization, see [3]:

sufficiently efficient system can only be achieved if all the systems work in unison.

perception, decision and actuation-control.

126 Advanced Path Planning for Mobile Entities

obstacles that may appear.

exploring random tree (RRT).

Curves in both space and plane are a part of the geometry necessary to represent certain shapes in different areas. Curves arise in many applications, such as art, industrial design, mathematics, architecture, engineering, etc.

#### 2.1. Different ways of defining a curve. Advantages and disadvantages

There are different ways of defining a curve: implicit, explicit, and parametric.

#### 2.1.1. Implicit and explicit expression of a curve

The coordinates (x, y) of the points of an implicitly defined plane curve verify that:

$$F(\mathbf{x}, \mathbf{y}) = \mathbf{0} \tag{1}$$

for some function F. If the curve is in R3 , then the curve must satisfy these two conditions simultaneously:

$$F(\mathbf{x}, \mathbf{y}, z) = \mathbf{0} \text{ and } G(\mathbf{x}, \mathbf{y}, z) = \mathbf{0} \tag{2}$$

The explicit representation of a curve clears one of the variables as a function of the other. In the plane, the coordinates (x,y) of the points in the curve explicitly defined satisfy either.

$$\mathbf{y} = f(\mathbf{x}) \text{ or } \mathbf{x} = \mathbf{g}(\mathbf{y}) \tag{3}$$

In the case of a curve in the space, its explicit form could be defined as:

$$\infty = f(z) \text{ and } y = \mathbf{g}(z) \tag{4}$$

A Bézier curve of degree n is specified by a sequence of (n + 1) control points, and its explicit expression is (6). The polygon that joins the control points is called the control polygon, and

The dimension of the vector containing the control points is related to the dimension of the

A conic is a curve obtained as the intersection of a plane with the surface of a double cone. There are three types of irreducible conics: hyperbola, parabola, and ellipse. Parabolas can be parameterized by polynomial functions, but hyperbolas and ellipses need rational functions such as RBC. The explicit definition of an RBC is (8), where Pi are the control points, Bi,n(u) are the Bernstein bases, and ωi are weights associated with each control point. These weights allow

ð6Þ

129

Path Planning Based on Parametric Curves http://dx.doi.org/10.5772/intechopen.72574

ð7Þ

ð8Þ

the functions or bases used are the Bernstein polynomials Bi,n(u), defined in (7).

space where the curve is represented.

Figure 1. Classification of the most important curves in CAGD.

a new way of modifying the curve.

2.2.2. Rational Bézier curves

#### 2.1.2. Parametric expression of a curve

In this case, the coordinates of a parametric curve are expressed as a function of a parameter, for example, u. The definition of a curve defined in Rn could be done as in (5), where functions αi are the coordinate functions or component functions. The image of α(u) is called the trace of α and α(u) is the parametrization of α.

$$\alpha: [a, b] \to \mathbb{R}^n \land \alpha(u) = (\alpha\_1(u), \dots, \alpha\_n(u)); u \in [a, b] \tag{5}$$

Parametric curves are the most used in computer graphics and geometric modeling because the curve points are calculated in a simple way. In contrast, the calculation of the points through the implicit expression is much more complex.

Within the parametric curves, it is possible to differentiate between polynomial curves and rational curves. Polynomial curves are those whose component functions are polynomials, and rational curves are those expressed as the quotient of polynomials. The representation in the form of parametric curves allows a great variety of curves, some known, some strange, some complex and others surprising for their symmetry and beauty.

The advantageous properties of the parametric curves that make them widely used are intuitivity, flexibility, affine-invariant, fast computation, and numerical stability.

In order to model complicated shapes or surfaces, it is necessary to introduce a way of representing curves based on a polygon. From this idea, the most used parametric curves arise in computer-aided geometric design (CAGD): Bézier, B-splines, RBC, and NURBS. Figure 1 shows a schematic of the most important curves in CAGD. It can be seen how NURBS are the most general curves, and Bézier are the most particular ones. Among them, Bézier is the simplest, possessing properties that make them be the most extensively used.

## 2.2. Most common parametric curves: Bézier, B-spline, NURBS, and RBC

#### 2.2.1. Bézier curves

Bézier curves arose as a result of the car modeling in both Renault and Citroën companies, by the engineers Pierre Bézier and Casteljau. The simplicity in the manipulation of these curves makes their use and application widespread.

The popularity of the Bézier curves is due to their numerous mathematical properties that facilitate their manipulation and analysis. Moreover, their use does not require great mathematical knowledge, which is very interesting for designers who shape objects.

Figure 1. Classification of the most important curves in CAGD.

A Bézier curve of degree n is specified by a sequence of (n + 1) control points, and its explicit expression is (6). The polygon that joins the control points is called the control polygon, and the functions or bases used are the Bernstein polynomials Bi,n(u), defined in (7).

$$\alpha\left(u\right) = \sum\_{\iota=0}^{n} \mathbf{P}\_{\iota} B\_{\iota,\iota}\left(u\right); u \in \left[0, 1\right] \tag{6}$$

$$B\_{i,n}\left(u\right) = \binom{n}{i} \mu'\left(1-u\right)^{n-i}; i = 0, \square \quad , n \tag{7}$$

The dimension of the vector containing the control points is related to the dimension of the space where the curve is represented.

#### 2.2.2. Rational Bézier curves

ð3Þ

ð4Þ

ð5Þ

In the case of a curve in the space, its explicit form could be defined as:

In this case, the coordinates of a parametric curve are expressed as a function of a parameter, for example, u. The definition of a curve defined in Rn could be done as in (5), where functions αi are the coordinate functions or component functions. The image of α(u) is called the trace of

Parametric curves are the most used in computer graphics and geometric modeling because the curve points are calculated in a simple way. In contrast, the calculation of the points

Within the parametric curves, it is possible to differentiate between polynomial curves and rational curves. Polynomial curves are those whose component functions are polynomials, and rational curves are those expressed as the quotient of polynomials. The representation in the form of parametric curves allows a great variety of curves, some known, some strange, some

The advantageous properties of the parametric curves that make them widely used are

In order to model complicated shapes or surfaces, it is necessary to introduce a way of representing curves based on a polygon. From this idea, the most used parametric curves arise in computer-aided geometric design (CAGD): Bézier, B-splines, RBC, and NURBS. Figure 1 shows a schematic of the most important curves in CAGD. It can be seen how NURBS are the most general curves, and Bézier are the most particular ones. Among them, Bézier is the

Bézier curves arose as a result of the car modeling in both Renault and Citroën companies, by the engineers Pierre Bézier and Casteljau. The simplicity in the manipulation of these curves

The popularity of the Bézier curves is due to their numerous mathematical properties that facilitate their manipulation and analysis. Moreover, their use does not require great mathe-

intuitivity, flexibility, affine-invariant, fast computation, and numerical stability.

simplest, possessing properties that make them be the most extensively used.

2.2. Most common parametric curves: Bézier, B-spline, NURBS, and RBC

matical knowledge, which is very interesting for designers who shape objects.

2.1.2. Parametric expression of a curve

128 Advanced Path Planning for Mobile Entities

α and α(u) is the parametrization of α.

2.2.1. Bézier curves

makes their use and application widespread.

through the implicit expression is much more complex.

complex and others surprising for their symmetry and beauty.

A conic is a curve obtained as the intersection of a plane with the surface of a double cone. There are three types of irreducible conics: hyperbola, parabola, and ellipse. Parabolas can be parameterized by polynomial functions, but hyperbolas and ellipses need rational functions such as RBC. The explicit definition of an RBC is (8), where Pi are the control points, Bi,n(u) are the Bernstein bases, and ωi are weights associated with each control point. These weights allow a new way of modifying the curve.

$$\alpha(u) = \frac{\sum\_{i=0}^{n} \alpha\_i \mathbf{P}\_i B\_{i,n}(u)}{\sum\_{i=0}^{n} \alpha\_i B\_{i,n}(u)}; u \in [0, 1] \tag{8}$$

#### 2.2.3. B-spline curves

B-splines are polynomial curves defined in pieces, continuously differentiable up to a prescribed order. The name spline is a word that means "elastic slats". These slats were used by craftsmen to create curves describing the surfaces to be built, such as boat hulls and aircraft fuselages. Constrained by weights, these elastic slats or splines assume a shape that minimizes their elastic energy.

B-spline curves were developed to overcome the limitations of Bézier curves: the need for a local control of the curve, the difficulty in imposing C2 continuity and the fact that a number of control points of a Bézier curve imposes its degree.

Analogous to the definition of a Bézier curve, a B-spline curve of degree k (or k + 1 order) is expressed in (9) as an affine combination of certain control points Pi, where Ni,k are polynomial functions by pieces with finite support of order k (degree k-1, meaning that they are zero out of a finite interval) that satisfy certain conditions of continuity. Each of these functions can be calculated using the Cox-de-Boor recursive formulas.

$$\alpha(u) = \sum\_{\iota=0}^{n} \mathbf{P}\_{\iota} N\_{\iota,k} \left( u \right) \tag{9}$$

B-splines can be defined by a recurrence relationship; simplicity is considered a double infinite sequence of simple nodes such that for all i. B-splines are then defined through the following recurrence relationship.

For the sake of simplicity, a double infinite sequence of simple nodes ai is considered such that ai < ai+1 for all i. Then, the B-splines Ni,k are then defined through the recurrence relationships (10) and (11).

$$\mathcal{N}\_{\iota,0}\left(u\right) = \begin{cases} 1 \text{ if } u \in \left[a\_{\iota}, a\_{\iota+1}\right] \\ 0 \text{ otherwise} \end{cases} \tag{10}$$

2.3. Comparison of properties

Table 1. Comparison of the properties of parametric curves.

When dealing with curves, their representation is important, but their shape manipulation is a key factor in their usability. The object to be modeled will determine the type of parametric curve chosen, depending on the properties required. In Table 1, we can see a comparison of the properties of the parametric curves in CAGD. In the following section, some of the most

Path Planning Based on Parametric Curves http://dx.doi.org/10.5772/intechopen.72574 131

Predicting the movement of a robot is important as it implies the computation of a proper path that meets the kinematic and dynamic properties of the robot. Simply moving a mobile robot from an initial position (xi,yi,θi) to a final position (xg,yg,θg) safely implicates many research

relevant works in mobile robots using parametric curves are described.

3. Use of parametric curves in robotics: state of the art

3.1. Generation of trajectories of mobile robots through parametric curves

fields, which are involved in the generation of efficient path planning algorithms.

$$N\_{i,k}\left(u\right) = \frac{t - a\_l}{a\_{i \leftrightarrow k-l} - a\_l} N\_{i,k-l}\left(u\right) + \frac{a\_{l \leftrightarrow k-l}}{a\_{l \leftrightarrow k} - a\_{l \leftrightarrow l}} N\_{i \leftrightarrow l,k-l}\left(u\right) \tag{11}$$

#### 2.2.4. Non-uniform rational B-spline curves

Rational B-spline curves are obtained in a similar way as the RBCs from Bézier curves. The definition of a NURBS curve is:

$$\alpha\left(u\right) = \frac{\sum\_{i=0}^{n} \text{P}\_i \text{o}\_i \text{N}\_{\iota,k}\left(u\right)}{\sum\_{i=0}^{n} \text{o}\_i \text{N}\_{\iota,k}\left(u\right)}\tag{12}$$

Table 1. Comparison of the properties of parametric curves.

### 2.3. Comparison of properties

2.2.3. B-spline curves

130 Advanced Path Planning for Mobile Entities

their elastic energy.

recurrence relationship.

2.2.4. Non-uniform rational B-spline curves

definition of a NURBS curve is:

(10) and (11).

control points of a Bézier curve imposes its degree.

calculated using the Cox-de-Boor recursive formulas.

B-splines are polynomial curves defined in pieces, continuously differentiable up to a prescribed order. The name spline is a word that means "elastic slats". These slats were used by craftsmen to create curves describing the surfaces to be built, such as boat hulls and aircraft fuselages. Constrained by weights, these elastic slats or splines assume a shape that minimizes

B-spline curves were developed to overcome the limitations of Bézier curves: the need for a local control of the curve, the difficulty in imposing C2 continuity and the fact that a number of

Analogous to the definition of a Bézier curve, a B-spline curve of degree k (or k + 1 order) is expressed in (9) as an affine combination of certain control points Pi, where Ni,k are polynomial functions by pieces with finite support of order k (degree k-1, meaning that they are zero out of a finite interval) that satisfy certain conditions of continuity. Each of these functions can be

B-splines can be defined by a recurrence relationship; simplicity is considered a double infinite sequence of simple nodes such that for all i. B-splines are then defined through the following

For the sake of simplicity, a double infinite sequence of simple nodes ai is considered such that ai < ai+1 for all i. Then, the B-splines Ni,k are then defined through the recurrence relationships

Rational B-spline curves are obtained in a similar way as the RBCs from Bézier curves. The

ð9Þ

ð10Þ

ð11Þ

ð12Þ

When dealing with curves, their representation is important, but their shape manipulation is a key factor in their usability. The object to be modeled will determine the type of parametric curve chosen, depending on the properties required. In Table 1, we can see a comparison of the properties of the parametric curves in CAGD. In the following section, some of the most relevant works in mobile robots using parametric curves are described.


*<IP_ADDRESS> Halocin C8 (HalC8)*

Halocin C8 is produced by *Natrinema* sp. AS7092, a strain isolated from the large Chaidan Salt Lake, China (7.44 kDa, 76 amino acids) [28]. It is a unique polypeptide with an isoelectric point of 4.4 [33]. Its activity is retained after desalting, boiling and frozing [33]. Halocin C8 has a very broad spectrum of activity against several species and genera of *Halobacteriales* members including *Natronobacterium gregoryi*, *Nbt*. comb. nov and *Natronomonas pharaonis* [28]. The *halC8* gene encodes both halocin C8 and its immunity protein HalI.

**87**

*Halocins, Bacteriocin-Like Antimicrobials Produced by the Archaeal Domain: Occurrence…*

Halocin A4, also called also halocin U1, is produced by an uncharacterized haloarchaea strain isolated from a Tunisian saltern [34]. Its molecular weight is 7.435 Da, as determined by the spectrometric mass, and is both acidic (pH = 4.14) and hydrophobic (eluent at ~85% acetonitrile) [26]. Halocin A4 has been reported to inhibit the growth of crenarchaeal *Sulfolobus* sp. strains [26]. G*ene* encoding HalA4 is *located* on a 300 kpb megaplasmid, pHM300 (NC\_017943) [29].

Halocin H6 is produced by *Haloferax gibbonsii* Ma 2.39 species [27]. Its activity is resistant to trypsin. Stabilities of this peptide were studied and have shown that HalH6 can be desalted and it retained its activity after heat treatment up to 10 min at 100°C [27]. Halocin H6 is considered as a bactericidal substance which causes cell

Halocin Sech7a was excreted by the extremely halophilic haloarchaeon Sech7a, isolated from brine samples of Secovlje solar salterns crystallizers in Slovenia [36]. Sech7a is about 11 kDa. It is stable over a wide pH range and is heat labile at temperatures above 80°C. Its optimal activity was observed in the early exponential phase growth at 45°C. It loses activity under low salt conditions, but its activity can

Halocin SH10 is produced by *Natrinema* sp. BTSH10, a strain isolated from the Kanyakumari salt marsh, Tamil Nadu, India [37]. The optimal production of halocin SH10 is at 42°C, pH 8.0 and 3 M NaCl at the stationary phase. In this context, it was reported that the activity is lost under acidic conditions [37]. Production of SH10 is influenced by the carbon source composition of the medium — *Natrinema*

This class comprises halocins composed of proteins greater than 10 kDa in size. Currently, there are two characterized protein halocins, HalH1 and HalH4, in the

Halocin H4, produced by *Haloferax mediterranei* R4 (ATCC 33500) was isolated from a Spanish solar salt pond in Alicante. It is the first halocin that was studied [20]. The optimal activity was detected at the midpoint between exponential and stationary phases [20]. Halocin H4 is sensitive to proteases, high temperature and desalting. HalH4 has an antimicrobial activity against other haloarchaeons. It Interacts with the membrane of the target cells where it causes permeability changes that result in an ionic imbalance leading to cell lysis and death [21, 38]. The *halH4* gene, encoding halocin activity, is located on the pHM300 megaplasmid, a single

sp. BTSH10 could produce maximal halocin in the presence of beef [37].

lysis and the specific target of HalH6 is the Na+/H+ antiport [27, 35].

be restored after dialysis against initial saline conditions [36].

*DOI: http://dx.doi.org/10.5772/intechopen.94765*

*<IP_ADDRESS> Halocin A4 (HalU1)*

*<IP_ADDRESS> Halocin H6 (HalH6)*

*<IP_ADDRESS> Halocin Sech7a*

*<IP_ADDRESS> Halocin SH10*

*3.1.2 Protein halocins*

*<IP_ADDRESS> Halocin H4*

range of 30 to 35 kDa [28].

polypeptide of 34.9 kDa.

*Halocins, Bacteriocin-Like Antimicrobials Produced by the Archaeal Domain: Occurrence… DOI: http://dx.doi.org/10.5772/intechopen.94765*

### *<IP_ADDRESS> Halocin A4 (HalU1)*

*Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological...*

of them have been characterized and purified.

Halocins, bacteriocins-like peptides and proteins produced by extremely halophilic archaea, were first discovered in 1982 by F. Rodriguez Valera [24, 25]. They are classified according to their size into two major classes: high molecular mass (protein, > 10 kDa) and low molecular mass (peptide, ≤10 kDa) called microhalocins [26, 27]. It has been shown that halocins are effective against *Haloarchaea* and *Crenarchaea* such as *Sulfolobus* spp. and *Methanosarcina thermopila*, and thus act across the main subdivision of the archaeal domain. These compounds represent a general class of antiarchaeal toxins and there is no confirmation about the inhibition of bacteria [26, 27]. Production of halocin is a practically universal feature of archaeal halophilic rods [27]. Although several halocins were identified, only some

These halocins are composed of a peptide with size below or in the range of 10 kDa. Seven halocins have been characterized including HalS8, HalR1, HalC8, HalU1, HalH6, Sech7a and Sech10. They are hydrophobic and retain their activity in the absence of salt and can be stored at 4°C. They are relatively insensitive to heat

HalS8 is the first characterized microhalocin with 36 amino acids (3580 Da), it is synthesized by the uncharacterized S8a haloarchaea [29]. Halocin S8 showed a narrow inhibitory spectrum and can only inhibit *Halobacterium salinarum* NRC817, *Halobacterium* GRB and *Haloferax gibbonsii* [29]. It can be desalted and it is heat resistant. Its activity is resistant to trypsin but sensitive to proteinase K and is undetectable in the transition to the stationary phase [29, 30]. The *halS8 gene* is *encoded*

Halocin R1, the second characterized microhalocin, is produced by

*Halobacterium salinarum* GN101, a strain isolated from solar salt marsh in Mexico [31]. Initially HalR1 was described with a molecular weight of 6.2 kDa [32] and later on, it was shown that the HalR1 peptide is composed of 38 amino acids [24, 29]. Like HalS8, the activity is not affected by desalting and is resistant to acids, bases, organic solvents DNase and RNase, and against some proteases such as papain, trypsin or thermolysin, but it is sensitive to proteinase K, pronase P and

Halocin C8 is produced by *Natrinema* sp. AS7092, a strain isolated from the large Chaidan Salt Lake, China (7.44 kDa, 76 amino acids) [28]. It is a unique polypeptide with an isoelectric point of 4.4 [33]. Its activity is retained after desalting, boiling and frozing [33]. Halocin C8 has a very broad spectrum of activity against several species and genera of *Halobacteriales* members including *Natronobacterium gregoryi*, *Nbt*. comb. nov and *Natronomonas pharaonis* [28]. The *halC8* gene encodes both

**3.1 Halocins**

*3.1.1 Microhalocins*

and organic solvents [28].

*<IP_ADDRESS> Halocin S8 (HalS8)*

on a ∼200-kbp megaplasmid [29].

*<IP_ADDRESS> Halocin HalR1 (HalR1)*

elastase [20, 32].

*<IP_ADDRESS> Halocin C8 (HalC8)*

halocin C8 and its immunity protein HalI.

**86**

Halocin A4, also called also halocin U1, is produced by an uncharacterized haloarchaea strain isolated from a Tunisian saltern [34]. Its molecular weight is 7.435 Da, as determined by the spectrometric mass, and is both acidic (pH = 4.14) and hydrophobic (eluent at ~85% acetonitrile) [26]. Halocin A4 has been reported to inhibit the growth of crenarchaeal *Sulfolobus* sp. strains [26]. G*ene* encoding HalA4 is *located* on a 300 kpb megaplasmid, pHM300 (NC\_017943) [29].

### *<IP_ADDRESS> Halocin H6 (HalH6)*

Halocin H6 is produced by *Haloferax gibbonsii* Ma 2.39 species [27]. Its activity is resistant to trypsin. Stabilities of this peptide were studied and have shown that HalH6 can be desalted and it retained its activity after heat treatment up to 10 min at 100°C [27]. Halocin H6 is considered as a bactericidal substance which causes cell lysis and the specific target of HalH6 is the Na+/H+ antiport [27, 35].

### *<IP_ADDRESS> Halocin Sech7a*

Halocin Sech7a was excreted by the extremely halophilic haloarchaeon Sech7a, isolated from brine samples of Secovlje solar salterns crystallizers in Slovenia [36]. Sech7a is about 11 kDa. It is stable over a wide pH range and is heat labile at temperatures above 80°C. Its optimal activity was observed in the early exponential phase growth at 45°C. It loses activity under low salt conditions, but its activity can be restored after dialysis against initial saline conditions [36].

### *<IP_ADDRESS> Halocin SH10*

Halocin SH10 is produced by *Natrinema* sp. BTSH10, a strain isolated from the Kanyakumari salt marsh, Tamil Nadu, India [37]. The optimal production of halocin SH10 is at 42°C, pH 8.0 and 3 M NaCl at the stationary phase. In this context, it was reported that the activity is lost under acidic conditions [37]. Production of SH10 is influenced by the carbon source composition of the medium — *Natrinema* sp. BTSH10 could produce maximal halocin in the presence of beef [37].

#### *3.1.2 Protein halocins*

This class comprises halocins composed of proteins greater than 10 kDa in size. Currently, there are two characterized protein halocins, HalH1 and HalH4, in the range of 30 to 35 kDa [28].

## *<IP_ADDRESS> Halocin H4*

Halocin H4, produced by *Haloferax mediterranei* R4 (ATCC 33500) was isolated from a Spanish solar salt pond in Alicante. It is the first halocin that was studied [20]. The optimal activity was detected at the midpoint between exponential and stationary phases [20]. Halocin H4 is sensitive to proteases, high temperature and desalting. HalH4 has an antimicrobial activity against other haloarchaeons. It Interacts with the membrane of the target cells where it causes permeability changes that result in an ionic imbalance leading to cell lysis and death [21, 38]. The *halH4* gene, encoding halocin activity, is located on the pHM300 megaplasmid, a single polypeptide of 34.9 kDa.

#### *<IP_ADDRESS> Halocin H1*

Halocin H1 is produced by *Haloferax mediterranei* M2a (previously known as *H*. *mediterranei* Xia3) isolated from salt ponds in Santa Pola (Alicante, Spain) [20]. Halocin H1 is a single 31 kDa polypeptide characterized by a broad inhibitory spectrum among *Halobacteriales* members. HalH1 activity is temperature and salt dependant. It is stable at 50°C only and requires a salt concentration of 1.5 M to maintain its activity [38, 39]. Optimum activity was observed at mid-exponential phase. The sensitivity to proteases and the gene encoding activity were not determined yet.

### **3.2 Applications of halocins**

Some studies reported the role of halocins in a variety of environmental, industrial and biotechnological applications \*\*\*(REFERENCES?). However, this topic is poorly documented and somewhat controversial. One of these applications is the use of halocin producing strains in the textile industry during the tanning process characterized by high salinity concentration, halocins could inhibit the growth of pathogenic microbes affecting the quality of products. [7, 10]. Moreover, some halocins have also been reported for biomedical and therapeutic uses, for example, Halocin H7 has been shown to inhibit the Na+/H+ antiport in *Haloarchaea*, can be used as a treatment to reduce the injuries caused when ischemic organ transplantation is re-infused [35]. The therapeutic potential of halocins needs more research on their physical structures and their modes of action. On the other hand, halocins are known also to have a potential application in food industry as preservative agents by controlling the growth of haloarchaea in salted food products [40].


van der Wal, Jenneke, 95, 96, 103, 106, 107, 109, 110, 119, 120 van Koppen, Marjo, 188 van Riemsdijk, Henk C.,126,128–130, 135, 137, 142 van Urk, Coppe, 290, 291, 313–315 Varlokosta, Spyridoula, 326–329, 337 Verbeke, Saartje, 25, 34 Verdenius, Andries A., 186, 191, 211, 213 Vergnaud, Jean-Roger, 260, 342, 343 Vilkuna, Maria, 164 Vinokurova, Nadya, 52, 53, 58, 63 Viotti, Evani, 99, 378 Visser, Fredericus Theodorus, 290 Vogel, Ralf, 127, 133 Warner, Anthony, 352 Watters, David E., 38 Wilder, Chris, 130 Willems, Klaas, 25, 34 Williams, Edwin, 129, 202, 287, 289, 290 Wiltschko, Martina, 210, 315 Woods, Rebecca, 156 Woolford, Ellen, 44, 420 Wurmbrand, Susi, 319, 342, 345, 346, 348, 352

### Xu, Zheng, 8, 18

Zaenen, Annie, 344 Zamparelli, Roberto, 226 Zanuttini, Raffaella, 55, 59, 60 Zdrojewski, Pablo, 406 Zeijlstra, Hedde, 69, 244 Zombolou, Katerina, 8, 13, 15 Zwart, Jan-Wouter, 27, 39, 45, 209, 210

# **Language index**

Abaza, 41 Afrikaans, 163, 21419 Ainu, 226 Albanian, 3–5, 5 2 , 5 4 , 6 5 , 8,11–13,1314 , 18 Ancient Greek, 127 Bantu, 264 , 76, 77, 82, 86, 95, 96, 99, 100,1003 ,1004 ,1005 ,101–103, 106, 110, 119, 120 Belhare, 341, 3421 , 345, 347–349, 3507 , 351, 352, 3528 , 353 Brazilian Portuguese, 96–99,101–105, 1057 , 106–109, 1098 , 110, 111, 114,115,117,119,120, 359, 361– 364, 3658 , 367–372, 37215 , 373, 375–378, 37819 , 37820 , 379, 37922 , 380, 381, 38124 , 383–385, 38527 , 386–388, 38828 , 389, 390, 402, 404, 405, 409, 410, 4105 , 411, 4127 , 4128 , 413–415, 41510 , 416, 41610 , 417–419, 421 Bulgarian, 326 Burushaski, 75–77, 81, 82, 86 Capeverdian, 359 Chamorro, 3110 Chichewa, 101 Chukchi, 75, 226 Coast Tsimshian, 24, 25, 27, 3110 , 32, 3212 , 35–37, 39, 44 Cuzco Quechua, 79

Dominican Spanish, 37215 Dutch, 6 5 , 132, 137, 163, 185–191, 1915 , 192–200, 2019 , 202, 203, 205– 210, 21015 , 211–214, 21419 , 313, 314, 316–319, 359, 387 English, 1314 , 17, 20, 55, 57, 61, 62, 86– 89, 104, 108, 1098 , 131, 145, 146, 1461 , 147, 148, 1482 , 149, 153,156,15714 ,15715 ,158,159, 15917 , 160–168, 180–182, 190, 212, 226, 233, 251, 260, 261, 2619 , 263, 265, 26512 , 266, 272, 273, 289, 290, 308, 330, 3305 , 334, 342, 3528 , 3593 , 361, 371, 376, 380, 386–390, 408, 414 European Portuguese, 96–98,103–108, 110, 113, 114, 11713 , 324, 361, 362, 371, 373, 375, 376, 386, 387, 389, 401 Evenki, 63 Finnish,145–147,149–151,153,161–167, 359, 361, 382–384, 389 French, 7, 220, 221, 237, 246, 251, 252, 255, 260–266, 272, 273, 277, 278, 2783 , 279–281, 283, 324, 3252 , 358, 359, 363, 365, 368, 369, 372, 380, 383, 386, 409 Frisian, 21116 , 21318 Georgian, 3110

Danish, 15715

### Language index

German, 7, 14, 1415 , 16–18, 24, 26, 27, 32, 3210 , 127, 130, 132, 133, 140,163,1926 ,1988 , 289, 303, 313–315, 318, 319, 3192 , 320, 324, 358, 359, 387 Germanic, 3, 5, 9 7 , 13, 14, 17, 20, 2267 , 362, 3624 Gitksan, 41 Gothic, 1273 Greek, 3, 4, 6 5 , 8, 109 , 1110 , 12, 13, 15, 1517 ,16–18, 69, 71, 77–80, 805 , 81–89,126,176, 239, 260, 288, 292–296, 298–305, 3054 , 307, 308, 326–331, 335–337, 3377 , 350, 359 Greenlandic, 75 Haitian, 359 Hebrew, 17, 359, 361 Hindi, 44, 77 Hungarian,145–147,151–153,160,163, 165–168, 2257 , 23413 , 242, 24219 243, 244, 24521 Hunzib, 3110 , 3721 Icelandic, 83, 1538 , 164, 324, 344, 359, 361, 387, 388 Indo-Aryan, 25 Indo-European, 31, 3210 , 77, 95, 100, 110, 120 Ingush, 75, 81, 87 Irish, 16523 Italian, 6, 7, 14, 16, 18, 15714 , 16523 , 171, 173,174,178–180,182–184, 266, 275–277, 302, 3054 , 324, 325, 358, 359, 371, 372 Itelmen, 345, 347, 350, 352 Jamaican, 359

,

Japanese, 260, 2619 , 359, 378–381, 387, 401 Kashmiri, 3416 Kham, 38, 39 Kimbundu, 101, 102 Kinande, 76, 100 Kiranti, 341 KiRimi, 420 Kirundi, 110 Korean, 79 Kutchi Gujarati, 3416 Lak, 3110 Latin, 3–5, 5 2 , 6 5 , 7, 8, 109 , 12, 1312 , 18, 102, 303, 350, 372 Lubukusu, 102 Makhuwa, 120 Malimiut Iñupiaq, 35, 36 Mapudungun, 226, 227, 229, 23614 Marathi, 3110 , 33, 34, 3416 , 37, 4024 , 44, 359, 361 Marwari, 3416 Matengo, 120 Mayali, 229–231, 23112 Mohawk, 229, 420 Nahuatl, 226 Nez Perce, 3210 , 34, 39, 3923 Ngiyambaa, 3110 Nias, 3620 Niuean, 225, 2257 , 23413 , 24521 Norwegian, 88, 89, 163, 16321 , 2267 , 290 Old French, 362 Old Portuguese, 362 Oromo, 75, 87 Otjiherero, 100

Language index

Paumarí, 28–30, 3110 , 32, 33, 35, 37, 39, 40, 42 Pitjantjatjara, 3110 Polish, 1275 , 1538 Quechua, 79 Romance, 3, 5, 7, 9 7 , 14, 17, 18, 95–99, 103, 104, 108–111, 16523 , 183, 220, 238, 331, 335, 345, 3624 , 368, 372, 410, 421 Romanian, 85, 857 , 86, 323, 326, 327, 330–337, 410, 4106 , 415, 416 Russian, 324, 359 Sakha, 51–57, 573 , 58–63, 638 , 65, 66, 71–74, 774 , 87 Selayarese, 41 Shipibo, 3210 , 75, 79 Slavic, 183 Sotho, 420 Southern Tiwa, 229 Spanish, 27, 276 , 3210 , 40, 16523 , 171, 172,174,176,178–182,184, 239, 23916 , 260, 3054 , 345, 386, 399, 400, 405, 406, 408–410, 4104 , 4106 , 411, 4127 , 413, 414, 416, 418, 420, 421 Suena, 3110 Swahili, 26, 264 , 27, 102 Swedish,145–149,1494 ,1495 ,153,154, 15510 , 15511 , 156, 157, 15714 , 15715 , 158, 159, 163, 166, 167 Tagalog, 3421 Tamil, 75, 81 Thai, 359 Tongan, 41, 42 Tsez, 36, 3721 , 40, 44, 4427 , 75, 81, 87, 343, 344, 352

Turkish, 53, 573 , 595 , 77, 359, 381 Wambaya, 275 , 3210 , 33, 35, 36, 39, 42, 43 Warlpiri, 3444 Welsh, 16523 West Greenlandic, 3110 , 40–42 Wichita, 229 Yiddish, 387 Yidiny, 3210 Yucatec Mayan, 235 Yup'ik, 3210

absolutive alignment, 343, 346, 347, 349–352 absolutive case, 75, 225, 343 accusative case, 38, 44, 45, 51–57, 616 , 63, 638 , 65, 77, 79, 88, 126, 128, 1482 , 22810 , 235 adjunction, 2213 , 223, 224, 227, 229– 233, 235, 237–240 adnominal pronoun construction,146– 148, 1482 , 149, 1495 , 150–153, 1538 ,162,163,16321 ,165–167 Agree, 70–73, 75, 77, 78, 80–89, 23413 , 289, 291, 306, 307, 325 agreement long-distance agreement, 75, 84, 345 object agreement, 264 , 27, 275 , 33, 34, 3416 , 3518 , 345, 408, 420 parasitic agreement,185–187,193, 198, 199, 201, 203–205, 208, 209, 211, 215 Spec–head agreement, 201, 204, 209, 211, 213, 214 subject agreement, 264 , 34, 3518 , 72, 86, 1057 , 118, 16523 topic agreement, 264 , 103, 384 alignment, 23–29, 299 , 30, 37, 3721 , 38–40, 42–46, 341–343, 350, 351 animacy,15,16, 32, 38, 3922 , 367, 399– 401, 405, 407–413, 416, 419– 421

APC,*see* adnominal pronoun construction auxiliaries, 275 , 33, 75, 420 avoid pronoun principle, 363, 373 bare phrase structure, 20413 binding, 253, 254, 2542 , 2605 , 2606 , 263, 264, 26511 , 289, 298, 299, 301, 313, 316, 318, 319, 3192 ,

320, 328

case

abstract Case, 106, 110, 119 case features, 63, 112, 128, 136 case filter, 225, 23413 , 235 case mismatches, 126, 13311 , 134, 140, 141 inherent case, 51, 52, 54, 277 morphological case, 24, 125, 150, 153, 162, 165, 23413 syntactic Case, 95, 120 classifiers, 24017 , 241, 242 clefts, 274 clitic climbing, 331, 333, 334 clitic doubling, 5 4 , 15, 16, 16523 , 220, 239, 3053 clitic left dislocation,16, 252, 253, 257, 258, 262, 265, 266, 3053 clitic right dislocation, 257, 265, 266, 3053 clitics,1005 ,108,119,15714 ,16523 , 220, 238, 241, 242, 246, 251, 258,

261, 266, 304, 345, 371, 372, 402, 408, 420 complementizers, 107, 272, 2721 , 273, 276, 279 compounding, 2224 , 223, 232 control, 62, 77, 846 , 107, 183, 323–325, 3252 , 326–330, 3305 , 331–335, 337, 341, 342, 3421 , 343–348, 3486 , 349–353, 367, 375, 376, 380 exhaustive control, 325–327, 3284 , 330, 332, 334, 337 implicit control, 288, 290–292, 297, 313–316, 3203 partial control, 295, 323, 3231 , 324, 325, 3252 , 326–328, 3284 , 329, 330, 3305 , 331, 334, 336, 337 coordination,1251 ,145,156,157,15713 , 15714 , 15715 , 158, 159, 15917 , 166, 167 copulas, 190, 211 copy deletion, 162, 167 core grammar, 357, 361, 37012 , 387, 390 criterial freezing, 274, 275, 280 criterial positions, 275, 282 dative case, 5, 11, 1516 , 55, 58, 712 , 83, 1005 , 105, 115, 11711 , 126, 127, 151, 152, 165, 251, 2511 , 258, 261, 26410 , 266, 2772 , 293, 314, 316, 344, 3443 , 365, 386, 408, 420, 421 defective goal, 220, 2202 , 221–223, 227– 229, 235, 23615 , 237, 238, 241– 246 degree words, 185–189, 191–196, 199, 200, 202, 203, 205–209, 211, 213, 215 δ-features, 381, 384

dependent case, 26, 43, 44, 4428 , 45, 54, 55, 616 , 69–71, 75, 77, 79, 80, 805 , 88 deponence, 3421 deponent verbs, 3, 4, 4 1 , 5, 5 2 , 7–9, 9 8 , 1313 differential object marking, 264 , 53, 399, 400, 405, 406, 4063 , 407– 410, 4104 , 4106 , 411, 4127 , 414, 415, 41610 , 418, 421 Distributed Morphology, 8 DOC, *see* double object construction double object construction,1098 , 251, 2553 , 260, 261, 2618 , 2619 , 262– 265, 26512 , 266, 420 E-language, 361, 371 ellipsis, 399, 401, 4012 , 402–404, 411, 413, 41510 , 417 empty category principle, 271–274, 278, 279, 2793 , 280–283 ergative alignment, 24, 38, 40, 42, 45, 343, 344, 346, 350, 351 ergative case, 3415 , 3518 , 42, 43, 45, 4530 , 343, 344, 418 ergativity, 25, 28, 30, 3212 , 33, 40, 42, 45 morphological ergativity, 42 syntactic ergativity, 40–42 exceptional case marking, 77, 774 , 81, 84 exhaustive control, *see* control expletives, 17, 18, 11610 , 291, 358, 359, 368, 371, 379, 37921 , 37922 , 382– 384, 387, 389 expressive emphasis, 209 extended projection principle,103,105, 106,110,112,117,120,160,162, 164, 228, 412

, 37921

, 381,

movement, 104–106, 108, 113, 114, 161,

279, 281, 282, 3679

164, 252, 253, 255, 265, 275,

focalisation, 3679 focus, 152, 275, 362, 381 functional items, 8, 52, 62, 15713 , 166, 171, 220, 236, 241, 244, 245, 24521 , 246, 274, 291, 313, 360, 399, 402, 413 genitive case, 52, 70, 72, 79, 86, 1005 , 126, 145–148, 150, 151, 1538 , 15511 ,156,15612 ,162,163,16321 , 164, 165, 167, 2019 , 256, 257, 2607 , 298, 303, 304 grammatical functions, 23, 25, 30, 37, 3721 , 38, 44, 418 grammaticalization, 308, 365, 3658 , 372 head movement, 161, 219, 220, 244, 279 hyper-raising, 97, 102, 104, 108, 110, 114, 120 I-language, 370 idioms, 132 imperatives, 51–56, 59–61, 616 , 62, 63 implicational relations, 4325 implicit arguments, 288, 289, 2891 , 291, 292, 294, 297–302, 304, 305, 3054 , 306–308, 315, 318 intensifiers,186,189,191,193,197, 201, 202, 212, 21217 , 213, 21318 , 214 islands, 272, 401 labelling, 171, 173–175, 177–180, 182– 184, 273, 275 language acquisition, 357, 360, 361, 370, 371, 418, 419 left dislocation, 385 mapping hypothesis, 56, 58 Merge, 19, 45

399, 401, 403, 406, 411, 413, 414, 418, 420–422 multiple spell-out, 209 nanosyntax, 236 nominative case, 11, 24, 277 , 43, 51– 53, 55, 69–71, 712 , 72, 73, 733 , 74, 75, 77, 79–86, 88, 89, 98, 102,105,106,111,112,115–117, 11713 ,119,120,128,133,13311 , 134, 141, 1461 , 148, 150, 151, 153, 15511 , 165, 314, 316, 328, 329, 342, 344, 3444 , 364, 365, 369, 382, 383, 418 noun incorporation, 219, 220, 222– 227, 229–234, 236–238, 244, 247 null objects,108, 37012 , 399–401, 4012 , 402–405, 409, 410, 4105 , 411, 4127 , 41510 , 416–419 null subject languages, 273, 276, 3054 , 358, 361, 362, 373, 386, 387, 389 partial null subject languages, 359, 361, 382, 38225 , 384, 389 radical null subject languages, 359, 361, 362, 378, 386, 387, 389, 390 semi null subject languages, 389 null subjects, 87,115,116,160, 293, 294, 301, 308, 346, 418 number features, 171, 174, 179, 182– 184, 407 parameter hierarchies, 359, 360 parameters, 358–360, 370, 37011

parasitic gaps, 185, 186, 20110 , 215 partial control, *see* control partitive case, 51–53, 531 , 54–57, 573 , 58, 59, 595 , 60, 61, 616 , 62, 63, 638 , 65, 659 passive, 4, 7, 9 8 , 17, 18, 61, 73, 287– 290, 292, 294, 298–302, 305, 3054 , 307, 308, 314–317, 3192 , 320, 3528 PCC, *see* person case constraint PDC,*see* prepositional dative construction person case constraint, 412, 420, 421 person features, 59, 60, 171, 174, 179, 182–184, 305, 308, 399, 402, 411–413, 4139 , 418, 421 phase impenetrability condition, 300 phases, 52, 60, 63, 638 , 74, 75, 80, 300 φ-features, 69, 75, 84, 86–88, 103, 104, 106, 110, 112–114, 11713 , 118– 120, 145, 160–162, 164, 166, 167, 237, 315, 320, 335, 381, 38123 , 384 PIC, *see* phase impenetrability condition possessor raising, 99, 101, 113 prepositional dative construction, 251, 255, 260–262, 264–266 raising, 253 , 69, 80, 81, 83, 85, 857 , 88, 275, 331, 335, 343, 3455 , 346, 348, 349, 378–381, 389 reanalysis, 9, 10 relative clauses, 72, 73, 125–127, 1275 , 128 raising analysis, 128 relative pronouns, 126, 128 structure-dependence, 186, 208, 215

topic, 264 , 87, 252, 253, 266, 275, 2772 , 282, 381 topicalization, 24521 , 3053 unaccusativity, 8, 12–14, 1516 , 16, 25, 55, 58, 59, 617 , 228, 22810 , 229, 230, 288, 290, 295, 299, 300 unagreement, 179, 180 unergativity, 18, 58 Universal Grammar, 63, 23413 , 273, 360, 370 V2 word order, 362, 3624 , 363 weak crossover, 2605 , 2606 wh-extraction, 108 wh-words, 141

# Syntactic architecture and its consequences III

This volume collects novel contributions to comparative generative linguistics that "rethink" existing approaches to an extensive range of phenomena, domains, and architectural questions in linguistic theory. At the heart of the contributions is the tension between descriptive and explanatory adequacy which has long animated generative linguistics and which continues to grow thanks to the increasing amount and diversity of data available to us.

The chapters develop novel insights into a number of core syntactic phenomena, such as the structure of and variation in diathesis, alignment types, case and agreement splits, and the syntax of null elements. Many of these contributions show the influence of research by Ian Roberts and collaborators and they provide varied perspectives on current research in synchronic and diachronic comparative syntax.

This book is complemented by two other volumes.


**Preface XIII**

Sajjad Abedi, Gholam Hossein Riahy, Seyed Hossein Hosseinian and Mehdi Farhadkhani

**Section 7 Fuel Cell 329**

**Section 8 Integrated System 363**

Agbossou

**Power Plant 389**

Chapter 15 **Wind Diesel Hybrid Power System with Hydrogen Storage 365**

Chapter 14 **Methodology of Designing Power Converters for Fuel Cell Based Systems: A Resonant Approach 331** Maria Teresa Outeiro and Adriano Carvalho

Chapter 16 **Sustainable Power Generation Through Co-Combustion of Agricultural Residues with Coal in Existing Coal**

Wan Azlina Wan Ab Karim Ghani and Azil Bahari Alias

Mamadou Lamine Doumbia, Karim Belmokhtar and Kodjo

Contents **VII**

	- **Section 4 Biomass 237**


*2.6. Chlorophyll Quantification*

Chlorophyll was extracted and quantified according to a previous method [18]. The chlorophyll and anthocyanin extractions were made using leaves different from the ones used for the terpene quantifications. The leaf position (e.g., lighting) alters the chlorophyll contents. Therefore, to minimize the errors among the analyses, we carefully harvested and chose the second and third leaves with similar sizes for terpene or chlorophyll measurement. Briefly, weighed leaf samples were placed into 2 mL of pure N,N-dimethylformamide (DMF; FUJIFILM Wako Chemicals, Osaka, Japan) at 4 ◦C in darkness overnight. The absorbance values at 647 and 664 nm of the aliquot of the extracted solution were measured using a spectrophotometer (U-5100, Hitachi, Tokyo, Japan). DMF was used as the blank control. The total amount of chlorophyll was calculated based on the following equation [18]: Chl a + b = 17.67 ∗ A647 + 7.12 ∗ A664.


**2.1 The Mediterranean region**

This area is represented by mountain ranges extending from Irbid in the north near to the south around Ra's Al Naqb. This region is characterized by the presence of several types of forests (The Phoenicean juniper, *Juniperus phoenicea*, Mt. Atlas mastic tree, *Pistacia atlantica*, Aleppo pine, *Pinus halepensis*, Palestine oak, *Quercus calliprinos*, kermes oak, *Quercus coccifera* and Mount Tabor oak, *Quercus ithaburensis*). Non-forested areas are characterized by a dense cover of the Thorny Burnet, *Sarcopoterium spinosum*. Altitude ranges from 700 to 1500 m asl, with 400–600 mm average annual rainfall and snowfall during winter. Several types of soil are found, including calcareous, *terra rosa*, sandy, and sandy-loamy caused by erosion of the Nubian sandstone that is common in the south [20].


Human Endogenous Retroviruses in Autism Spectrum Disorders: Recent Advances and New Perspectives at the Gene-Environment Interface

*Emanuela Balestrieri, Chiara Cipriani, Enrico Garaci, Claudia Matteucci and Paola Sinibaldi-Vallebona*

## **Abstract**

Human endogenous retroviruses (HERVs) are genetic elements, derived from their exogenous retroviral counterpart by a process of germline infection and proliferation within the human genome, and their integration as proviruses led to the fixation and the vertical transmission, following Mendelian laws. HERVs currently make up ~8% of the genetic material, and some of them have been cooped for physiological functions. Otherwise, their activation in response to environmental factors has been associated with human pathological conditions. In the setting of neurodevelopmental disorders, HERVs have been proposed as contributing factors involved in Autism Spectrum Disorders (ASD), spanning the bridge between genetic susceptibility, environmental risk factors and immune response. We described a distinct expression profile of some HERV families and cytokines in lymphocytes from autistic children and in their mothers suggesting a close mother-child association in ASD. Moreover, *in vitro* treatment with an antiretroviral drug was able to restore the expression level of HERVs and cytokines providing new insights into the potential role of HERVs as biomarkers of ASD and raising the possibility of using HERVs expression as a therapeutic target for a tailored approach to patient care.

**Keywords:** human endogenous retroviruses; HERVs, biomarker, mother-child association, gene expression, aetiology, antiretrovirals

### **1. Introduction**

In 1943, the child psychiatrist Leo Kanner described children preferring loneliness with repetitive patterns of behaviour. Similar symptoms were reported by Hans Asperger, an Austrian paediatrician, in 1944 mainly in people of high intelligence [1]. Kanner, first spoke about 'childhood or early-onset schizophrenia', and later he called this condition 'infantile autism', and concerning the aetiology of autism, he attributed autism to a lack of maternal warmth and attachment. Following this hypothesis, Bruno Bettelheim with his book 'The Empty Fortress' popularized the theory of 'refrigerator mother' by stating that 'the infant that misreads the mother's actions or feelings, or correctly assesses her negative feelings, may retreat from her and the world'. This view was widely criticized and nowadays represents an obsolete thought [2]. From this time, many hypotheses and models emerged to explain this complex condition focusing on symptomatology, phenotype and pathogenesis [3, 4]. However, despite many promising hypotheses, the current literature is made up of controversial findings and lacking of definitive proof about the mechanism underlying the complex aethiopathogenesis of Autism Spectrum Disorder (ASD). ASD is currently referred to as a pervasive neurodevelopmental disorder with an impact on emotional and social behaviour that persists throughout life [5]. The clinical presentation is very heterogeneous, and its incidence is continuously increasing [6]. Despite the consolidated evidence that the main contribution to the increase in the incidence of autism comes from the improvement of the diagnostic process, it has also been hypothesized that at the basis of the onset of autism, there is not a single cause but a set of risk factors acting together to produce the phenotype. Decades of studies have indeed shown that autism is a complex pathology influenced by the combination of genetic, environmental and epigenetic factors, mainly acting during prenatal and/or perinatal phases [7–9]. The concordance rate of ASD in monozygotic twins much higher than in dizygotic twins seemed to indicate that genetic factors were more likely to contribute to ASD than environmental factors [10]. More than a thousand ASD-associated genes known to be involved in brain development have been identified to date [11], and many genomic copy number variants have been associated with neurodevelopmental disorders including ASD [12, 13]. Several epidemiological studies indicate that potential risk factors for ASD also include various determinants [14, 15], such as the age of the pregnant woman, advancing paternal age [14, 16, 17] and prematurity [18]. However, an ever increasing important role has been attributed to risk factors related to the early foetal environment, including toxicants, diet, air pollution, smoking or chemicals exposure, which have been suggested to induce a prenatal and/or perinatal brain insult able to contribute to the development of autism in genetically predisposed individuals [19]. These environmental insults share in common the activation of the maternal immune system (MIA), which has therefore been recognized as an additional risk factor for ASD [20]. MIA is an inflammatory response triggered by pathogenic infection and autoimmune diseases in the mother. It is known that several microorganisms, vertically transmitted to the foetus, affect its development resulting in severe complications such as miscarriage and malformations [21]. However, even non-vertically transmitted infections during pregnancy can cause harm to the offspring by producing inflammatory cytokines, which directly damage the foetal brain by crossing the placental and blood-brain barrier [22, 23].

Preclinical studies, using mouse model of MIA induced by prenatal exposure to polyinosinic:polycytidylic acid (Poly I:C), a synthetic double-stranded RNA molecule targeting TLR-3, mimicking viral maternal infection, demonstrated that the exposure to a prenatal insult induced derailed neurodevelopment in offspring. Particularly, in the mothers, the Poly I:C injection leads to the production of interleukin-17 that reaches the foetal brain *via* the placenta inducing cell death and decreasing synaptic density and expression levels of synapse formation-associated proteins and resulting in ASD-like behavioural and morphological brain abnormalities, also described in the pathophysiology of human ASD [24–26]. In line with the hypothesis that maternal immune response could impact on neurodevelopment in the newborn,

#### *Human Endogenous Retroviruses in Autism Spectrum Disorders: Recent Advances… DOI: http://dx.doi.org/10.5772/intechopen.108671*

epidemiological studies have reported that MIA, caused by autoimmune diseases, also increases the risk of ASD [27]. Altogether these studies suggest that MIA-induced inflammation and cytokines can impair placental function and lead to the disruption of its barrier function, resulting in exposure of the foetus to toxic substances. Accumulated evidence also shows an important role of epigenetic factors, such as DNA methylation, histone modification and noncoding RNA in predisposition to disease development. Epigenetic mechanisms regulate chromatin structure and gene expression without altering the DNA sequence. In consideration of this last characteristic, in the past it was believed to have no role in the growth and development of the individual [28] while the study of their interaction with environmental conditions has highlighted their important role in the development of genes related to brain development. In recent years, there have been rapid advances in the understanding of epigenetic mechanisms that ultimately regulate gene activity and expression during development and differentiation or in response to environmental stimuli. Instead, it is now known that the main function of epigenetic factors is to regulate development through cell differentiation processes, tissue specification and maintenance of cell lineages [29]. Therefore, environmental stimuli can alter the epigenome and consequently gene transcription, changing the phenotype [30].

Within this interplay among genetic susceptibility, MIA, epigenetic and environmental factors are placed the human endogenous retroviruses (HERVs), which we proposed as novel contributing factors involved in ASD.

#### **2. Human endogenous retroviruses and their co-evolution with the host**

In contrast to the prevailing early twentieth century conception of genetic material as fixed, in the 1940s, the Nobel Prize Barbara McClintock discovered in maize the 'mutable loci' which were capable to move between chromosomes. This pioneering study paved the way for future research into the role of these 'jumping genes' or transposable elements (TEs) in both health and disease conditions [31]. Indeed, it was later discovered that about 46% of the human genome consists of TEs [32]. They consist of repetitive sequences that are able to insert copies of themselves elsewhere in the genome [33]. They are divided, according to their size and functionally related structures, into short interspersed elements (SINEs), long interspersed elements (LINEs), long terminal repetition retrotransposons (LTR) and DNA transposons [34]. The major subset of LTR retrotransposons is represented by HERVs, which together with their derivative sequences comprise at least 8% of the human genome [32, 35].

These elements have their origin in the numerous environmental events that shaped the human genome during evolution, including the occasional infection of germ cells of our ancestors by exogenous retroviruses and the insertion of their RNA genome as proviruses into the cell's chromosomal DNA [36]. Hence, HERVs are transmitted in a Mendelian manner to all subsequent generations (**Figure 1a**). Retroviral proviruses share the canonical structure of retroviruses consisting of an internal region of four essential viral genes (gag, pro, pol and env), flanked at either side by long terminal repeats sequences (the 5′ and 3′ LTRs) that are identical at the time of integration and contain promoter, enhancer and polyadenylation signals that shape the cellular transcriptome (**Figure 1a**) [37].

A non-coding sequence containing a tRNA-specifc primer-binding site (PBS) is usually present between the end of the 5′ LTR and the first codon of the gag gene,

**Figure 1.** *HERV origin (a) and their activation in response to environmental stimuli (b) (created by BioRender).*

and HERVs are classified into families on the basis of the tRNA that binds to the viral primer-binding site to prime reverse transcription.

During human evolution, HERVs invaded the human genome undergoing amplification, retrotransposition and/or reinfection events, and resulting in the presence of multiple copies fixed in the DNA of all nucleated cells [36]. While a majority of these sequences have accumulated mutations and/or deletions and are mostly defective, several HERVs preserve the properties of the ancient viruses and, still being transcriptionally active and competent to produce some retroviral proteins, have been co-opted for physiological functions [36, 38, 39] (**Figure 1a**). In the light of the current knowledge, what Weiss stated in 2016 'If Charles Darwin reappeared today, he might be surprised to learn that humans are descended from viruses as well as from apes' [40] is still current.

#### **3. The role of human endogenous retroviruses in physiological conditions**

For a long time, HERVs have been considered as junk DNA with no impact on the host. However, during the last decades, the great efforts of the scientific community highlighted that some 'well preserved' ERV sequences influence different physiological properties being involved in a variety of biological pathways [41, 42]. The most intriguingly example of the co-option of HERVs during the host evolution comes from the well-studied properties of the syncytins, the products of the Envelope (Env) genes of HERV-W-1 and HERV-FRD. Specifically, Syncytin-1, encoded by the HERV-W-1

#### *Human Endogenous Retroviruses in Autism Spectrum Disorders: Recent Advances… DOI: http://dx.doi.org/10.5772/intechopen.108671*

gene, is the first retroviral protein found to have a defined physiological function mediating the cell-cell fusion as the terminal differentiation of the trophoblast lineage [43]. As emerging from different reports, the decrease of syncytin expression and the consequential fusion deficiency could contribute to placental anomalies including pre-eclampsia disorder [44]. Moreover, other studies suggested that syncytin-1 also possesses non-fusogenic activities, as the regulation of trophoblast proliferation and apoptosis and indicated a widely expression in different cell types such as granulocytes, T lymphocytes, monocytes, glial cell of the brain and cancer cells [45, 46]. In human, syncytin-1 interacts with the type D mammalian retrovirus receptor ASCT-1/ASCT-2 (sodium-dependent neutral amino acid transporter type 1/2) on cell membranes. Syncytin-2, encoded by the HERV-FRD gene, is also expressed in trophoblasts and able of mediating cell fusion by interacting with a different receptor known as MFSD2 (major facilitator superfamily domain containing 2) [47, 48]. Moreover, Syncytin-2 is involved in maternal immune tolerance towards the semi-allogenic foetus [49]. A similar functional domestication emerged for ERV-encoded GAG gene, involved in the memory consolidation in the mammalian brain, including long-term potentiation and depression [50]. Given the abundance of HERVs in the human genome, they represent an important source of genomic variability, also providing potential coding and regulatory elements for the acquisition of new cellular functions [51, 52]. In line, growing evidence has been obtained regarding the general expression of HERVs in normal tissues [53, 54], and in this context, we demonstrated an age-related transcriptional activity of HERV-H, HERV-K and HERV-W in peripheral blood mononuclear cells (PBMCs) from a large cohort of healthy human subjects aged between 1 and 80 years, reinforcing the hypothesis of a physiological correlation between HERV activity and the different stages of life in human [55]. Among the proposed mechanisms by which HERVs could contribute to the human physiology, it is recognized that various sequences, concentrated in the LTRs, are involved in the regulation of the expression of neighbouring genes acting as promoters, enhancers, polyadenylation signals, regulators of chromatin folding and binding sites for transcriptional factors [56, 57]. In the genome, most HERVs reside as solo-LTRs, resulting from homologous recombination between the LTRs of a full-length HERV [58] able to act as alternative tissue-specific promoters to drive the expression of host genes [59–61]. During embryo development, some HERV sequences are also engaged by the host for the regulation of gene expression [62]. In particular, non-coding RNA expressed by the HERV-H group and the recruitment of specific cellular transcriptional factors on HERV-H LTRs seem to be involved in the conservation of stem cell identity [57, 63]. Of note, the HERV-H loci seem to be more preserved in a full-length state than other HERV families, suggesting that the full-length elements rather than solo-LTRs are useful to the host and that the internal regions of HERV-H may be involved in the process of exaptation [64]. Similarly, an ancestral env gene named HEMO [human endogenous MER34 (medium reiteration-frequency-family-34) ORF] has been found highly expressed in embryos, already in the early stages of development and in all subsequent differentiation periods as well as in the placenta and in the blood of pregnant women [65]. Finally, in the regulation of stem cell function, HERV-K ENV was highly expressed in the cell membrane of pluripotent stem cells and signals *via* direct binding to CD98HC, leading to activation of signalling pathways that regulate stem cell function [66]. Moreover, the expression of HERVs has a direct key role for the maintenance of human embryonic stem cells and induced pluripotent stem cells (iPSCs), and their activation could be considered a marker of pluripotency [67].

To conclude, the more profound insight into the mechanisms explaining the roles of HERVs in various biological/physiological contexts will help to clarify the contribution of HERVs in pathological conditions.

## **4. The activation of human endogenous retroviruses**

In addition to their physiological role, HERVs have been also proposed as possible cofactors in the aetiology of several human diseases. Proviruses are known to remain dormant for long periods of time within the host, but they may occasionally be triggered by factors present in the environment. Indeed, one of the peculiar features of HERVs is their intrinsic responsiveness to microenvironmental stress and various stimuli likely *via* epigenetic mechanisms [68, 69]. Epigenetics represents a fine mechanism to control HERV activity to ensure genomic stability and integrity and, on the other side, it represents one of the mechanisms by which HERVs could modulate the gene expression. HERV sequences are epigenetically silenced by DNA methylation and histone modifications in addition to being located in chromosomal regions with heterochromatic chromatin architecture leading to a low transcriptional degree in most cell types [70, 71]. It is known that epigenetics is a driver of the embryonic development, contributing to global remodeling, cell commitment and tissue specification [72] and that ERVs are highly active during early embryogenesis and germline development [62, 73]. Indeed, they are involved in the pre-implantation transcription network although the mechanisms are still unclear [62, 74]. Thus, during a sensitive phase, as the embryogenesis, any environmental insults could have an impact on the development, and epigenetic modifications could directly link the environmental stimuli and the molecular regulatory pathways, explaining some aspects of complex pathologies including HERV activation. A multitude of environmental factors and xenobiotics can activate HERV expression causing DNA rearrangements, HERV reinsertions and HERV copy number variation, resulting in abnormal HERV activity that could, in turn, potentially affect crucial pathway such as inflammation [69, 75, 76]. Microorganisms, cytokines, hormones, vitamins, nutrients and drugs could represent triggers leading to HERV transactivation (**Figure 1b**). Of particular relevance is the role played by the interaction with microbes, including viruses, exogenous retroviruses, intestinal microbiota and protozoan. Among viruses, the herpesviruses, Hepatitis B virus, the human immunodeficiency virus-1 (HIV-1), Influenza A virus and more recently SARS-CoV-2 have been found able to deregulate HERV activity and although so far, an unequivocal pathogenic cause-effect relationship has not been established, their contribution to the development of viral diseases, including virusassociated tumors, has been suggested [77–83]. Moreover, several *in vitro* studies demonstrated that cells express HERVs at high levels in response to stimulation by using lipopolysaccharide or interferon-γ (IFN-γ), cytokines as Interleukine-1β (IL-1β) and tumor necrosis factor-α (TNF-α) or mitogens, such as phytohemagglutinin [84–86]. These observations could be due to the fact that HERVs showed various regulatory sequences that have been linked to the transcriptional modulation systems [87]. More recently, the hormonal regulation of HERVs has been investigated, and specifically, cross talks among the female sex hormones and HERVs in contributing to breast cancer tumorigenesis and proliferation have been elucidated providing useful knowledge for the development of novel cancer therapies. Specifically, the effect of progesterone on HERV-K expression is at least partly mediated by OCT4 known to be involved in

*Human Endogenous Retroviruses in Autism Spectrum Disorders: Recent Advances… DOI: http://dx.doi.org/10.5772/intechopen.108671*

embryogenesis and expressed in diverse cancer types as well [88]. These findings were in line with the peculiar expression of HERVs in peripheral leukocytes during the menstrual cycle suggesting a well-coordinated hormonal regulation of HERV activity [89]. Also, drugs are able to modulate HERV expression, both *in vitro* and *in vivo* with different proposed mechanisms, mainly linked to the epigenetic one. In particular, neuroleptics and antidepressants influence HERV activity in human brain cell lines and in post-mortem brain samples of patients with mental disorders in therapy during their lifetime [90].

Thus, HERVs have been found particularly responsive to environmental stimuli that can determine their dysregulation at transcriptional levels and/or encoded protein expression that could influence the onset of complex diseases.


**1. Introduction**

Quantum mechanics is at the basis of all modern physics and fundamental for the understanding of the world that we live in. As a general theory, quantum mechanics should apply also to the measurement process. From the general experience of non-destructive measurements, we draw conclusions about the interaction between the observed system and the measurement apparatus and how this can be described within quantum mechanics.

We thus consider a quantum system *μ*, interacting with a measurement device. For simplicity we assume that *μ* is a two-level system that is not destroyed in the process. Then after the measurement, *μ* ends up in one of the eigenstates of the measured observable. If *μ* is prepared in one of these eigenstates, it remains in that state after the measurement. If *μ* is initially in a superposition of the two eigenstates, it still ends up in one of the eigenstates and the measurement result is the corresponding eigenvalue. The probability for a certain outcome is the squared modulus of the corresponding state component in the superposition (Born's rule).

An essential question is whether the probabilistic nature of quantum measurement with Born's rule is an inherent feature of quantum mechanics or whether it can be shown to hold as a result of a quantum-mechanical treatment of the measurement process combined with a statistical analysis. In the latter case, a single measurement would be a quantum-mechanical process in which the state of the measurement apparatus (possibly including its surroundings) determines the result. Born's rule would then emerge from the statistics of the ensemble that describes the measurement apparatus in interaction with the system subject to measurement.

The requirement that *μ*, if initially in an eigenstate of the observable, remains in that eigenstate after interacting with the apparatus, is usually considered to lead to a well-known dilemma: If applying the (linear) quantum mechanics of the 1930s to *μ* in an initial superposition of those eigenstates, the result of the process appears to be a superposition of the two possible resulting states for *μ* and the apparatus without any change in the proportions between the channels. This has been referred to as von Neumann's dilemma [1], and it has led to paradoxical conclusions such as Schrödinger's cat.

Attempts to get around this problem include Everett's relative-state formulation [2] and its continuation in DeWitt's many-worlds interpretation [3] as well as non-linear modifications of quantum mechanics [4–8]. In the non-linear modifications, one gets the bifurcation of the measurement process but the non-linear character of the basic theory introduces new conceptual difficulties. Mathematically our treatment can be seen to be very close to quantum diffusion [5]; we have chosen to follow the same conventions in handling the statistics of stochastic variables as in Ref. [5]. The ambition to understand quantum measurement as a deterministic process we share with the De Broglie-Bohm theory [9], with the difference that we look for how details in the measurement device influence the process.

Bell pointed out that the Everett-DeWitt theory does not properly reflect the fact that the presence of inverse processes and interference are inherent features of quantum mechanics [10]:

Thus, DeWitt seems to share our idea that the fundamental concepts of the theory should be meaningful on a microscopic level and not only on some ill-defined macroscopic level. However, at the microscopic level there is no such asymmetry in time as would be indicated by the existence of branching and the non-existence of debranching. [...] [I]t even seems reasonable to regard the coalescence of previously different branches, and the resulting interference phenomena, as the characteristic feature of quantum mechanics. In this respect an accurate picture, which does not have any tree-like character, is the 'sum over all possible paths' of Feynman.

Therefore, as suggested by Bell, we investigate work of Feynman for a correct theory. We choose the scattering theory of quantum field theory, including Feynman diagrams, as a basis for our description of the measurement process. This theory contains inverse processes that result in a non-linear dependence on the initial state which removes the von Neumann dilemma.

In the field of investigation of the measurement process, a strong belief has been established that microscopic details of the measurement interaction cannot lead to the bifurcation determining the result of measurement (see, e.g., [11,12]). This belief is based on von Neumann's way of handling linear quantum mechanics. In this situation, many have abandoned the ambition to understand the mechanism of a single measurement and concentrated on the full ensemble of measurements. There one has studied the irreversible decoherence process that takes the initial ensemble, with *μ* in a pure state, into a mixed state for the final ensemble after measurement. Since 1970, the year of DeWitt's many-worlds theory [3] and Zeh's paper on decoherence [11], a new tradition has developed that includes different views on how to interpret quantum mechanics, see for instance Refs. [13,14]. Epistemological aspects play an important role in these interpretations.

Our idea is that the microscopic details of the measurement apparatus affect the process so that it takes *μ* into either of the eigenstates of the measured observable and initiates a recording of the corresponding measurement result. This is possible to see in a more developed form of linear deterministic quantum mechanics, namely the scattering theory of quantum field theory.

The development of relativistic quantum mechanics led to quantum field theory. For a situation where a quantum system *μ* meets a part *A* of a measurement device, interacts with it and then leaves it, scattering theory can be an adequate description. As we have pointed out already, the scattering theory of quantum field theory has the reversibility that was requested by Bell. These are our reasons for the choice of studying measurement in the scattering theory of quantum field theory.

In our approach, measurement is part of the physics studied, rather than a subject for epistemological analysis. We show how non-linearities can be generated within quantum theory. Our statistical study of measurement processes then shows that those states of the apparatus which are competitive in leading to a final state, also take *μ* into *one* of the eigenstates of the measured observable. Moreover, this bifurcation, leading to one of the two possible final states for *μ*, occurs with the frequencies given by Born's rule.

In the following sections, we shall first give our scattering theory description and then make all possible processes subject to a statistical analysis.