**1. Introduction**

Establishing the initial model is one of the necessary and key steps of the modelbased inversion method, which has a great impact on the inversion effect. Traditionally, the method of generating the initial model is to extrapolate the inversion parameters observed from the wells or vertical seismic profile (VSP) along the interfaces picked from seismic profiles [1,2] or to extrapolate the seismic attributes [3–7]. In these methods, seismic horizons are necessary and manual interpretations are required. Horizon picking is one of the most time-consuming and labor-intensive steps [8]. Automatic horizon interpretation technology has been developing. Many geophysicists have proposed a variety of algorithms to improve the efficiency and accuracy of automatic horizons tracking [9–14], but these methods still need some prior information, such as manually setting up seed points or carrying out manual interpretation of large sets of strata. It is difficult to establish the initial model directly without manual interpretation. In addition, seismic inversion usually requires the initial model to cover the entire 3D region, but in practice, the automatic interpretation of the entire data volume is difficult to achieve. Therefore, there is a need for an efficient and accurate seismic extrapolation method to establish an initial model with the condition of lack of horizons.

Neidall et al. [15] presented the concept of velocity spectrum. The seismic gathers are automatically scanned with equal velocity intervals, and the velocity spectra are generated by stacking energy or similarity coefficient. Compared with artificial horizon interpretation, the velocity spectra are easier to generate. In conventional seismic data processing, stacking or migration velocity is obtained by velocity analysis on velocity spectra. The hyperbolas in the prestack seismic gathers reflect the characteristics of the interfaces, and the velocity

**Citation:** Sun, L.; Ding, L.; Wang, X. Research on Initial Model Construction of Seismic Inversion Based on Velocity Spectrum and Siamese Network. *Appl. Sci.* **2022**, *12*, 10593. https://doi.org/10.3390/ app122010593

Academic Editor: José A. Peláez

Received: 31 August 2022 Accepted: 19 October 2022 Published: 20 October 2022

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

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

spectrum contains the position and velocity information of each hyperbola in prestack gathers. Compared with the gathers, the features on velocity spectrum are more focused. The velocity spectrum is a 2D image, which can be used to identify the changes of the lateral characteristics of the formation.

Deep learning technology has shown much better performance than traditional neural network methods in speech and visual tasks, such as image classification [16], semantic segmentation [17], and image segmentation [18]. This is mainly due to the strong feature expression ability of deep networks such as convolutional neural network (CNN). In the field of exploration geophysics, deep learning has been successfully applied to seismic processing and interpretation [19,20]. However, most of the current applications need to manually make and label samples first, and then extract the corresponding type of feature information from the seismic images. For the establishment of the initial inversion model, it is still unable to get rid of the dependence on horizons. Deep learning has the potential to provide more global information. As an important part of computer vision, visual target tracking technology has been developed rapidly. The Siamese network uses the idea of similarity learning, describes the tracking problem as an object matching problem, and judges the position of objects by comparing the similarity of objects [21,22].

In this paper, we transformed the parameter extrapolation problem in constructing the initial inversion model into a 2D image tracking problem and proposed a method for constructing the initial model based on velocity spectrum and Siamese network. Firstly, the velocity spectra generated by CDP gathers were used to track the lateral variation of formation characteristics. Then, the target tracking results at different CDP positions were obtained by combining the similarity of the triple Siamese network analog velocity spectrum. Finally, the discrete inversion parameters were extrapolated along the tracking path to obtain the initial model. We proposed an improved triple Siamese structure, which adds an update branch to solve the lateral variation of velocity spectrum characteristics during target tracking. This improvement takes dynamic update into tracking. In order to verify the applicability of the method, we carried out tests on theoretical and practical data. The results show that it can automatically obtain the extrapolation paths and can be used to establish the initial model of seismic inversion.


*Review* **Insight on Electronic Travel Aids for Visually Impaired People: A Review on the Electromagnetic Technology**

#### **Emanuele Cardillo \* and Alina Caddemi**

Department of Engineering, University of Messina, Messina 98166, Italy<EMAIL_ADDRESS>**\*** Correspondence<EMAIL_ADDRESS>
Received: 15 October 2019; Accepted: 28 October 2019; Published: 4 November 2019

**Abstract:** This review deals with a comprehensive description of the available electromagnetic travel aids for visually impaired and blind people. This challenging task is considered as an outstanding research area due to the rapid growth in the number of people with visual impairments. For decades, different technologies have been employed for solving the crucial challenge of improving the mobility of visually impaired people, but a suitable solution has not yet been developed. Focusing this contribution on the electromagnetic technology, the state-of-the-art of available solutions is demonstrated. Electronic travel aids based on electromagnetic technology have been identified as an emerging technology due to their high level of achievable performance in terms of accuracy, flexibility, lightness, and cost-effectiveness.

**Keywords:** electronic travel aids; radar; electromagnetic; visual impairment; blind people; microwaves; mobility; antennas

#### **1. Introduction**

It is estimated that 188.5 million people worldwide have mild vision impairment, 217 million have moderate-to-severe vision impairment, and 36 million people are blind [1].

This is a continuously growing number that has fueled the interest of many in in the pursuit of improving the quality of life of those affected by blindness [2–6].

Usually, the mobility of visually impaired people is addressed by the use of travel supports, with the traditional white-cane being the most renowned and still the most popular. However, there are many electronic travel aids (ETAs) available to make mobility safer and more familiar for both inside and outside use.

The National Research Council published a list of the most important requirements of an ETA [7,8]:

These six points represent the landmark for designing an effective ETA. The first three requirements provide indications about the obstacles that an ETA should be able to detect. Points four to six suggest additional information is needed for easier and safer movement in a complex environment.

Since eighty years ago, when the first ETAs were introduced, the work of researchers has concentrated on the design of better aids, but as yet these have not materialized. [9]. To date, none of the available aids has fulfilled all the requirements to fully satisfy users' expectations.

The ETAs are usually divided into two categories: primary and secondary aids. Primary aids allow autonomous movement without other aids since they provide all the required information independently. Secondary aids only enhance the mobility and must be used in conjunction with other aids (e.g., primary aids). Moreover, ETAs can be classified as mobility and navigation aids, where the former help the user in detecting and avoiding obstacles in their path and the latter allow to the user to find the path itself from the starting point to the destination [10].

Different technologies have been used with the aim of creating better devices, the most significant being based on ultrasonic sensors and optical/vision systems [11–17].

Although technological advancement has reached an elevated level of maturity and a variety of devices are available on the market, ultrasonic systems have limitations [10]. To specify, the maximum detectable range is limited when smooth surfaces need to be detected, even worse if the angle of incidence of the ultrasonic beam is small. Furthermore, due to the relatively large radiation beam, it is difficult for small apertures to be accurately detected (e.g., the presence of doors). Nevertheless, this kind of aid is widely used due to the easy-to-access technology and the availability of low-cost ultrasonic sensors.

ETAs based on optical technologies are very robust in terms of object detection capabilities under normal operating conditions. They typically employ cameras, and the target recognition is provided by means of complex algorithms [18]. The main drawbacks are due to the high sensitivity to ambient light, which can seriously limit the system performance, especially in outdoor environments [10]. Additional issues are the strong dependence on the optical properties of obstacles and the bulky dimensions of the devices.

Due to the lack of a competitive and robust technology, ETA devices based on the electromagnetic technology have only been developed in the past decade; These devices usually achieve target detection by sending electromagnetic waves into the space and analyzing the reflected signal.

Most aids are radar-based systems, where the frequency-modulated continuous wave (FMCW) is the typical mode of operation [19–25]. In a FMCW radar, the information about the target is contained in the frequency of the received signal. The transmitted signal is modulated (e.g., using a linear frequency modulation or phase modulation), reflected back by the target, and conditioned by a homodyne receiver. Thereafter, it is possible to extract the range information from the intermediate frequency (IF) signal. For example, by considering a saw-tooth modulation, the range (*R*) of the target can be computed by using (1):

$$R = \frac{cf\_{IF}\pi}{2B},$$

where *B* and τ are the modulation bandwidth and the duration of the signal, respectively, *c* is the speed of light, and *fIF* is the frequency of the IF signals whose amplitudes overcome a certain threshold [26,27].

The advantages of this technology compared to ultrasonic and optical systems can be summarized as follows:

To the best knowledge of the authors, this is the first review article concerning ETAs based on electromagnetic technology. This review is necessary due to the previous lack of a paper that collects and illustrates the main results in this field.

In Section 2, the pertinent studies and prototypes based on the electromagnetic technology are reported. Particular attention is given to the illustration of both practical and technological aspects by clearly explaining the basic modes of operation of the considered aids.

In Section 3, the particular differences between the devices are analyzed providing, in the authors' opinion, the possible future developments of this technology. Finally, the conclusions are outlined in Section 4.

#### **2. Electromagnetic Aids for Visually Impaired and Blind People**

The idea of employing an electromagnetic system as an aid for visually impaired people is quite recent. In [30], a vector network analyzer and a double-ridge horn antenna were used for transmitting and receiving a sequence of pulses from 1 to 6 GHz. Then, an inverse Fourier transform was applied to the frequency-domain measurements in order to recreate a very short time-domain pulse. The measured delay of the received pulse corresponds to the round-trip time-of-flight (TOF) of the electromagnetic wave from the transmitting antenna to the obstacle. Thereafter, the range of the object could be easily computed [31]. The diagram of the system and the echo pulse reflected by a 25 × 25 cm2 metal plate at a distance of 2 m are shown in Figure 1.

**Figure 1.** Diagram of the system.

The proposed approach was compared with an ultrasonic system, making the two experimental set-ups as equivalent as possible. The proposed system showed better precision and the ability to detect obstacles that are not detectable by ultrasonic systems (e.g., a partially open door). Although the system is very bulky and expensive, its future miniaturization has been claimed.

An enhanced version of the system proposed in [30] has been reported in [32]. A portable vector network analyzer was employed for testing the effectiveness of the methodology in both indoor and outdoor scenarios. The maximum range was fixed to 3 m, whereas the operating frequencies from 4 to 6 GHz achieved a spatial resolution of 12 cm. The tests were carried out by a blind volunteer during different test conditions (i.e., obstacles located in different arrangements). The test set-up was arranged in order to have the obstacles aligned, shaped as a cage, and with an obstacle at chest level—as depicted in Figure 2. The system could correctly detect the presence of one or more obstacles even at the level of the head or chest (e.g., an open window or a tree branch).

**Figure 2.** *Cont*.

**F**

**Figure 2.** Test set-up with (**a**) aligned obstacles, (**b**) obstacles shaped as a cage, and (**c**) obstacles at chest level.

A peculiar application of an electromagnetic aid for visually impaired people is reported in [33], where a system capable of guiding a blind athlete during running and physical activities was designed, realized, and tested. This arose from the need to enhance the runners' freedom during competitions or training. As a matter of fact, athletes currently need to be guided by a non-extendible cable carried by another runner, thus limiting the possibility of free movement [34].

The proposed system is composed of: (a) a mobile unit preceding the runner that generates two "electromagnetic walls" (i.e., two electromagnetic waves radiated on the left and right sides of the athlete) and (b) a receiving unit worn by the runner equipped with vibro-tactile devices.

A representation of the guiding system is shown in Figure 3.

**Figure 3.** Representation of the guiding system [33].

The mobile unit generates a vibro-tactile warning when the athlete is leaving the area delineated by the electromagnetic walls, to guide them towards the center of the path.

In this circumstance, the use of electromagnetic waves ensures confinement of the wall to a narrow and well-defined area. The operating bandwidth of the system is inside the X-band, thus reducing the dimension and weight of the antenna in the receiving unit worn by the blind runner. To achieve this, the antenna was designed with lightweight and compact microstrip technology. The system was tested by a blind athlete using four different paths as reported in Figure 4, demonstrating the proper operation of the prototype. A video of the tests can also be watched online [34].

**Figure 4.** Paths traveled by the athlete in a rectangular room (20 × 17 m2): (**a**) square, (**b**) spiral, and (**c**,**d**) complex paths [33].

In [19], the challenge of avoiding collision with obstacles for visually impaired users was achieved by creating a radar-based navigation device. The system employs a frequency-modulated continuous-wave signal. Although the authors plan to have a main sensor working at the center frequency of 80 GHz with a bandwidth of 24 GHz, a bandwidth of 4 GHz—from 80 GHz to 84 GHz—was adopted for the tests. With this frequency bandwidth, a spatial resolution equal to 6 cm can be obtained. The radar sensor was attached on the head of the blind person, whose position was continuously tracked by an inertial sensor. This sensor scanned the surrounding environment and detected the position of obstacles up to 5 m. The intention of the authors is to scan the scenario using a beam-steering antenna. Its design is shown in [19], but since it is still under development, the rotation was mechanically achieved by a stepper motor. Due to the size of the setup, the radar was placed on a large mounting. A virtual map of the environment was created and mapped into a 3D audio signal. The authors assert that a long training process is not required.

A superior system performance compared to camera-based systems is claimed in terms of independence from light conditions, ability to detect transparent obstacles such as glass doors, and better visual range in rain or fog [35,36].

The challenge of tailoring a suitable electromagnetic aid is shown in [20], highlighting the effectiveness of the authors' solution. A clinical investigation was performed with 25 visually impaired people wearing a radar-based assistive device.

The working frequency of the system was 24 GHz, selected to facilitate a lightweight wearable device, because at this frequency the signal can penetrate textiles, making it possible to wear the device under clothing. The system automatically compensates small tilt errors or notifies the person if the incline is excessive. It is able to detect obstacles up to 3.5 m, within a horizontal sector of 25◦ and a vertical sector of 70◦.

The maximum usage time of the prototype was measured as between 4 and 5 h, but this can be improved in a future designs. The warning about the presence of obstacles is fed back to the user by means of acoustic or haptic feedback. In Figure 5, the operating principle of the system and a picture of the device is shown.

**Figure 5.** Operating principle of the system and picture of the device [20].

The clinical investigation was implemented during an observation period of two weeks, using the electromagnetic aid during everyday activities.

The results of an interview after usage of the device are shown in Figure 6a in response to the question "Does the assistive device help you to perceive your environment better?", whereas in Figure 6b the question was "Does the assistive device increase your confidence in independent walking?".

**Figure 6.** Results of the interview questions: (**a**) "Does the assistive device help you to perceive your environment better?" and (**b**) "Does the assistive device increase your confidence in independent walking?" The answers reported on the X-axes are: (1) not at all, (2) not much, (3) to some extent, (4) significantly, (5) very significantly [20].

For both graphs in Figure 6, the answers recorded on the X-axes are: (1) not at all, (2) not much, (3) to some extent, (4) significantly, (5) very significantly.

In the results, 92% of the participants stated that the perception of the surrounding environment was enhanced by the electromagnetic aid, whereas 80% noticed an increased confidence in independent mobility.

Solutions employing 24 GHz radars based on FMCW modulation are presented in [21–23], but different choices were made concerning the way the users employ the devices.

Despite the extensive range of available ETAs, the most widespread aid is still the traditional white-cane, due to its ease of use and cost effectiveness [37,38].

Therefore, the authors decided to create a device so small and lightweight that it can be integrated into a white-cane—a kind of "microwave cane". The authors based their design on feedback from users during interviews and the remarks of visually impaired and blind users, considering the positive reaction to a new device as the main task. Volunteers testing ETAs usually suggest the simultaneous use the white cane.

Since the self-confidence that the traditional white cane is reported to give users is absent in ETAs, the authors chose to go down this route.

Of course, this new device must overcome the limitations of the traditional cane (i.e., it does not provide any protection against obstacles at head or chest height and the range is confined to the length of the cane). At the same time, the system has to be cost effective and easy to use without extensive training.

In [21], great attention is paid to the design of a customized antenna. It was designed by employing microstrip technology in order to have a compact system that can be attached to the cane. They highlight the advantages of the system compared to ones based on ultrasonic or optical technology, overcoming the limitations of limited range and poor performance operating within a low incidence angle on smooth surfaces. The system was tested up to 5 m, with a spatial resolution of 60 cm (24.0–24.25 GHz).

In [22], a feasibility study was undertaken with the aim of accurately identifying the technical specification of the microwave cane. The behavior of the entire system was simulated using computer-aided design (CAD), with preliminary results obtained from newly designed transmitting and receiving antennas. The first experimental activity using the microwave cane is outlined in [23].

The authors obtained system spatial resolution close to 14 cm, reduced energy consumption, a maximum range of 5 m, and horizontal and vertical antenna patterns close to 12◦ and 40◦, respectively.

A picture of the prototype is shown in Figure 7.

**Figure 7.** Picture of the radar prototype [23].

The radar system was tested with different obstacles commonly used for daily tasks, that is, a wooden chair, a chest of drawers, and a human subject. The targets, characterized by different materials, dimensions, and shapes, were located at the range of 1.5 m and successfully detected, as shown in Figure 8.

**Figure 8.** Intermediate frequency (IF) signals obtained from obstacles at the range of 1.5 m. From the top to bottom, results are shown for a wooden chair, a chest of drawers, and a human target [23].

An interesting solution is illustrated in [24,25], where the radar technology was employed in conjunction with an RGB-D sensor. The radar operated from 77 to 81 GHz, implementing an FMCW signal. This 4 GHz bandwidth gives a spatial resolution of 6 cm. The purpose of the exercise was to enhance the features of object detection and localization. Whereas mm-wave radar is capable of providing speed information and a relatively high spatial resolution in the radial direction, the RGB-D sensor accomplishes the task of distinguishing two targets at the same distance. A block diagram of the system is shown in Figure 9.

**Figure 9.** Schematic diagram of the system in [24]. FMCW: frequency-modulated continuous wave.

Obstacles at the same distance are recognized using color information and the application of a single deep neural network or object instance segmentation [39,40].

In Figure 10, results relating to tests performed in an indoor environment are shown. In Figure 10(a1,a2) the mask R-CNN (Region convolutional neural network) was used for detecting and recognizing the targets, whereas in (b1,b2) the SSD (Single Shot MultiBox Detector ) network was used. In (c1,c2) the image results are shown with, detection results enclosed in a red box, whereas in (d1,d2) the data detected by the radar are illustrated. Finally, in (e1,e2), the data are fused using a particle filter.

The potential of this technology is also highlighted by the Integrated Smart Spatial Exploration System (INSPEX H2020 project), where the main goal is to take advantage of the automotive techniques concerning spatial exploration and obstacle detection in the design of portable sensors [41]. Potential applications employing radar-based sensors range from safer human navigation in reduced visibility to navigation for visually impaired people. The authors intend to integrate the INSPEX system into a smart white cane.

**Figure 10.** Tests performed in an indoor environment and related results [24]. (**a1**,**a2**) Detection performed by R-CNN, (**b1**,**b2**) detection performed by SSD network, (**c1**,**c2**) image results, (**d1**,**d2**) data detected by the radar, (**e1**,**e2**), data fused using a particle filter.

#### **3. Discussion**

In Section 2, the main contributions in the field were reported, showing properties of the proposed systems such as working principle, maximum range, cost, size, spatial resolution, operating frequency, and the existence of a final prototype. These aspects are organized in Table 1.

**Table 1.** Properties of the main electromagnetic aids for visually impaired people.

By analyzing the properties shown in Table 1, we can conclude that different devices could be market ready very quickly, which would provide useful products that could offer a good compromise between the capability of scanning the environment, cost-effectiveness, and size.

Therefore, it is legitimate to ask why there is still no valid electromagnetic aid available on the market. We believe that it can be explained by the fact that there is insufficient collaboration between research groups and industry, which would provide a more effective transition from laboratory prototypes to the final engineered product. Indeed, since the core technology is based on radar, the necessary components, used by the relevant advancement in the automotive field, are readily available for use Moreover, the miniaturization of the operating frequencies in a typical of microwave and millimeter waves could be utilized to minimize size and weight, affording easy integration in everyday activities.

Although the electromagnetic aids exhibit better performance than ultrasonic or camera-based solutions, recent years have seen these technologies more broadly diffused due to their wider availability in the market. Moreover, in published papers only limited attention has been given to finding the best solution for providing feedback to the user. A greater effort in this direction could be very beneficial for enhancing the usability of this category of travel aid.

#### **4. Conclusions**

In this review, the most recent developments relating to electromagnetic travel aids for visually impaired and blind people are presented. The main contributions in the field were analyzed, highlighting the working principle, the impact on the end user's life, and the main properties. The presented solutions mostly employ radar-based devices, with only one system exploiting a (vector network analyzer) VNA-based technology. Overall, the trend is towards employing ever-higher

operation frequencies in order to realize compact systems and to increase the resolution capabilities. The electromagnetic technology shows better performance than other types of electronic travel aid, as they have a more reliable signal processing technique, smaller dimensions, higher precision and resolution, and better immunity to noise. Although the "perfect" aid has not yet been realized, this contribution highlights that the technology is mature and different solutions can be adopted for fulfilling this challenging task.

**Author Contributions:** E.C. collected the required bibliography and wrote the review article. A.C. supervised the research activities and revised the paper.

**Funding:** This research received no external funding.

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

#### **References**

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**Appendix C**

In this appendix, the fields are formulated for TE*m*0 modes in a rectangular waveguide filled with anisotropic dielectrics. The outcome of this analysis is the attenuation constant and wave-impedance for the special case of anisotropic ENG and MNG dielectric fill.

Consider an anisotropic dielectric material whose permeability and permittivity tensors in rectangular coordinates are respectively:

$$
\mu = \begin{bmatrix}
\mu\_{xx} & 0 & 0 \\
0 & \mu\_{yy} & 0 \\
0 & 0 & \mu\_{zz}
\end{bmatrix} \tag{A6}
$$

$$
\boldsymbol{\varepsilon} = \begin{bmatrix}
\varepsilon\_{xx} & 0 & 0 \\
0 & \varepsilon\_{yy} & 0 \\
0 & 0 & \varepsilon\_{zz}
\end{bmatrix} \tag{A7}
$$

where *μxx*, *μyy* and *μzz* are the *x*, *y* and *z* directed permeability respectively, and *εxx*, *εyy* and *εzz* are the *x*, *y* and *z* directed permittivity respectively. It is therefore assumed that the material is non-bianisotropic, non-gyrotropic and non-chiral which give zero off-diagonal elements. This assumption is reasonable for the types of metamaterials considered in this work comprising either broad-side-coupled SRRs, or thin strips.

*Electronics* **2019**, *8*, 84

Consider a rectangular waveguide directed in the *z* direction and whose perfect-electric-conducting walls are located at *x* = 0, *x* = *a*, *y* = 0 and *y* = *b*. All field component phasors of a wave propagating in the z direction will contain the common factor exp(- *γz*) where *γ* is the complex valued propagation constant. If we restrict the waves to be TE (transverse electric) waves, and further restrict the wave to containing no *y* dependence (i.e., TEm0 modes), substitution of (A6) and (A7) into Maxwell's curl equations in phasor form, yields:

$$
\gamma E\_y = -j\omega\mu\_{xx}H\_x \tag{A8a}
$$

$$\frac{\partial E\_y}{\partial x} = -j\omega\mu\_{zz}H\_z \tag{A8b}$$

$$-\gamma H\_{\rm x} - \frac{\Im H\_z}{\partial \mathbf{x}} = j\omega \varepsilon\_{yy} E\_y \tag{A8C}$$

Equations (A8a)–(A8c) shows that only the tensor components *μxx*, *εyy* and *μzz* play a role in TE*m*0 mode behavior. Based upon the waveguide boundary conditions, the solution of the non-zero field phasors, that also satisfy (A8a) and (A8b) must take the form:

$$H\_{\mathbf{x}} = \frac{-\gamma A}{j\omega\mu\_{\text{xx}}} \sin k\_{\mathbf{x}} \mathbf{x} \tag{A9a}$$

$$E\_{\mathcal{Y}} = A \sin k\_x \mathfrak{x} \tag{A9b}$$

$$H\_z = \frac{-k\_x A}{j\omega\mu\_{zz}} \cos k\_x x \tag{A9c}$$

where the cut-off wavenumber *kx* is given by (A3), *A* is an amplitude (which could be complex valued), and *m* is a non-zero integer. Substituting (A9) into (A8c) yields the dispersion relation:

$$
\gamma^2 = \frac{\mu\_{xx}}{\mu\_{zz}} k\_x^{\;2} - \omega^2 \mu\_{xx} \varepsilon\_{yy} \tag{A10}
$$

For an evanescent mode, *γ* will be pure real and equal to the attenuation constant *α* which is positive valued. For a TE wave travelling (or decaying) in the *z* direction, the wave-impedance is −*Ey*/*Hx*. Hence from (A9a) and (A9b):

$$Z\_{TE} = \frac{\mathbf{j}\omega\mu\_{xx}}{\mathfrak{a}}\tag{A11}$$

Thus from (A11), *ZTE* will be pure imaginary whose is sign is that of *μxx*.

There are two types of anisotropic dielectric that are of interest here. They are MNG with negative permeability confined to the *x* direction, and ENG with negative permittivity confined to the *y* direction.

#### *C.1. Anisotropic MNG*

For the first case, *μxx* is negative valued, *μzz* equals *μ*0 the permeability of a vacuum, and *εyy* is positive valued. The permittivity *εyy* is greater than the permittivity of a free-space (*ε*0) due to the inherent electric polarizability of the constituent elements comprising the metamaterial, and the presence of insulating substrates to support such elements. Hence, from (A10):

$$
\alpha = \sqrt{\frac{-\mu\_{xx}}{\mu\_0} \left(\omega^2 \mu\_0 \varepsilon\_{yy} - k\_x^{-2}\right)}\tag{A12}
$$

and is real (i.e., evanescent mode) only if *ω*2*μ*0*εyy* > *kx* 2 which is denoted the "anti-cut-off" property [7]. Clearly from (A11), *ZTE* will be a capacitive reactance.

### *C.2. Anisotropic ENG*

For the second case, *μxx* = *μzz* = *μ*0, and *εyy* is negative valued. Hence, from (A10):

$$
\mathfrak{a} = \sqrt{k\_x^2 - \omega^2 \mu\_0 \varepsilon\_{yy}} \tag{A13}
$$

and is real for all frequencies provided *εyy* is negative valued. Clearly from (A11), *ZTE* will be an inductive reactance.

The foregoing indicates that pairing of the above anisotropic ENG and MNG filled waveguides can yield the required impedance tunneling condition. Namely, apart from the anti-cut-off property, the conclusions are similar to the isotropic ENG and MNG cases.

In practice, for SRR realizations of anisotropic MNG, and strip realization of ENG, *μxx* and *εyy* are frequency dependent and are only negative over a restricted frequency range; particularly for the SRR loaded waveguide. Therefore, the above theoretical analysis needs to be interpreted in that context.


**Author details**

Livija Šakić1,2\*, Kata Šakić2,3 and Šime Šakić4

1 Department of Anaesthesiology, Reanimatology and Intensive Medicine, University hospital "Sveti Duh", Zagreb, Croatia

2 Faculty of Dental Medicine and Health Osijek, University of Josip Juraj Strosmayer, Osijek, Croatia

3 School of Medicine, Croatian Catholic University, Zagreb, Croatia

4 Department of Orthopaedic Surgery, School of Medicine, University of Zagreb, Zagreb, Croatia

\*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.


3.2.2 Wheat germ agglutinin (WGA) conjugated solid lipid nanoparticles

Conjugation of bioadhesive ligand molecules with SLNs helps in improving drug absorption/bioavailability by increasing residence time in the GIT and reducing

dosing frequency. Lectins (a group of diverse proteins/glycoproteins) are the bioadhesive ligand and have stable structure and receptor binding ability. It offers resistance to enzymatic digestion/degradation, which helps in its in vivo survival for a prolonged time period [22]. Wheat germ agglutinin (WGA) is an example of nontoxic plant lectin which binds particularly to N-acetyl-D-glucosamine and sialic acid on cell surfaces. Conjugation of WGA with SLNs could be helpful for targeted delivery of antitubercular drugs to the lungs.

WGA conjugated SLNs loaded with rifampicin (WRSLNs) were successfully developed for lungs specific delivery of rifampicin (RIF). The conjugation of WGA to RIF loaded SLN was carried out by two-steps carbodiimide reaction reported by Ertl et al. with slight modification [23]. Even after conjugation with the SLNs, the WGA retained its bio-recognition activity and sugar-binding specificity [24]. From the in vivo study result, the interaction of WRSLNs with porcine mucin was confirmed. However, non-conjugated SLNs did not show any conjugation with the porcine mucin. The WRSLNs were stable at refrigerated temperature and also in presence of electrolytes up to 1.0 M Na2SO4 concentration. The WRSLNs were 10 times more stable than RIF-SLN which could be owing to the steric stabilization by lectin present on nanoparticle surface. Prepared WRSLNs had shown narrow size distribution, controlled release of a drug, retention of biorecognition activity and improved physical stability of drug against electrolyte induced flocculation [25].

#### 3.2.3 Mannose conjugated solid lipid nanoparticles

Studies on tuberculosis revealed overexpression of the mannose receptors specifically on alveolar macrophages (AM) surfaces [26]. Keeping it in mind attempts were taken to develop mannosylated SLN to deliver antitubercular drugs targeting to alveolar macrophages which have higher affinity for mannose. Rifabutin loaded mannosylated SLNs were successfully developed. Manosylation was done by ring opening reactions followed by reaction of aldehyde groups of mannose in 0.1 M sodium acetate buffer (pH 4.0) with the amino groups of lipid. This leads to formation of Schiff's base (–N=CH–), which may then get reduced to secondary amine (–NH–CH2–) and remain in equilibrium with Schiff's base at basic pH. In vivo studies were conducted to evaluate for their cytotoxicity, targeting potential, AM uptake, and hematological studies. It was reported that mannosylation improved uptake (up to 6-fold) of rifabutin by the AM. Moreover, mannosylated rifabutin-SLNs were less immunogenic and helped in sustained delivery of drug. Thus, the mannosylated SLNs may be employed for AM targeted delivery of rifabutin for effective management of TB [27].

carcinoma—25%, and lung metastases—30%) [29]. These receptors allow folate derivatives to bind preferentially that permits intracellular incorporation of folate

Folate-conjugated copolymer of polyethylene glycol (PEG) and N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) SLNs loaded with Paclitaxel (PTX) were successfully developed. The conjugation of folate- conjugated PEG and HTCC to RIF-SLN was carried out by carbodiimide mediated coupling chemistry. Pulmonary administration of the developed F-PEG-HTCC–SLNs selectively delivered the PTX to the lung's cancer cells with improved penetrability and prolonged lung residence. Moreover, the developed SLN significantly reduced the in vitro half-maximum inhibitory concentration of PTX in M109-HiFR cells [30]. List of SLNs and their different ligand conjugated forms for lungs targeting have

Drugs Target Model Comments Ref.

effect

by AMs.

lungs

to Lungs

RIF Mucin Porcine Controlled release of RIF [25]

RIF AM SD rats Improved drug uptake

DXM Lungs Mice Improved therapeutic

Improved hypoglycemic

efficacy targeting to

AM specific drug delivery. Enhanced therapeutic efficacy.

Alveolar cell site delivery of drug with enhanced antiproliferative efficacy.

 Brain targeted delivery of drugs is the most challenging task because of the presence of strongest physiological barrier, i.e.,

derivative by endocytosis.

Sl. no SLN (Type)

03 SLN SL,

04 Lf-SLNs

05 WGA-SLN

06 Msy-SLN

07 Msy-SLN

Table 1.

101

08 Ft-SLN CHO,

Lipids Preparation method

DOI: http://dx.doi.org/10.5772/intechopen.88268

double emulsion

Solid Lipid Based Nano-particulate Formulations in Drug Targeting

film hydration method

Ultrasonication with high pressure homogenization

SL Solvent injection method

> Single emulsification followed by solvent evaporation

TSN Modified solvent injection method

> Nano precipitation method

Modified solvent injection method

List of SLNs and their different ligand conjugated forms for lungs cells targeting.

01 SLN SA, PA Reverse micelle-

02 SLN SA, PA Modified lipid

GTS

SA, GMS

TSN, DSPE

GS

been summarized in Table 1.

4. Solid lipid nanoparticles for brain targeting

Overexpression of mannose receptors in case of lungs cancer was reported by numerous investigators. The mannosylated-distearoyl phosphatidyl-ethanolamine SLNs loaded with paclitaxel (PTX) was developed for lungs targeted delivery of PTX. Manosylation was done by ring opening reaction followed by reaction of an aldehyde group of mannose with the free amine group provided by stearylamine and DSPE in sodium acetate buffer (pH 4.0). The stability testing data indicated that SLNs formulations stored at 4 2°C were more stable than those stored at 27 2°C. It was revealed that mannosylated SLNs deliver significantly higher concentration of drug to the alveolar cell sites and showed improved antiproliferative efficacy as compared to PTX solution and PTX-SLNs [28].

#### 3.2.4 Folate conjugated solid lipid nanoparticles

The folate receptors (α- form) are overexpressed on the surface of lung tumor cells. The extents of the overexpression are different in different types of lung tumors (adenocarcinomas—72%, squamous cell carcinomas—51%, small cell

Table 1.

dosing frequency. Lectins (a group of diverse proteins/glycoproteins) are the bioadhesive ligand and have stable structure and receptor binding ability. It offers resistance to enzymatic digestion/degradation, which helps in its in vivo survival for a prolonged time period [22]. Wheat germ agglutinin (WGA) is an example of nontoxic plant lectin which binds particularly to N-acetyl-D-glucosamine and sialic acid on cell surfaces. Conjugation of WGA with SLNs could be helpful for targeted

WGA conjugated SLNs loaded with rifampicin (WRSLNs) were successfully developed for lungs specific delivery of rifampicin (RIF). The conjugation of WGA to RIF loaded SLN was carried out by two-steps carbodiimide reaction reported by Ertl et al. with slight modification [23]. Even after conjugation with the SLNs, the WGA retained its bio-recognition activity and sugar-binding specificity [24]. From the in vivo study result, the interaction of WRSLNs with porcine mucin was confirmed. However, non-conjugated SLNs did not show any conjugation with the porcine mucin. The WRSLNs were stable at refrigerated temperature and also in presence of electrolytes up to 1.0 M Na2SO4 concentration. The WRSLNs were 10 times more stable than RIF-SLN which could be owing to the steric stabilization by lectin present on nanoparticle surface. Prepared WRSLNs had shown narrow size distribution, controlled release of a drug, retention of biorecognition activity and improved physical stability of drug against electrolyte induced flocculation [25].

Studies on tuberculosis revealed overexpression of the mannose receptors specifically on alveolar macrophages (AM) surfaces [26]. Keeping it in mind attempts were taken to develop mannosylated SLN to deliver antitubercular drugs targeting to alveolar macrophages which have higher affinity for mannose. Rifabutin loaded mannosylated SLNs were successfully developed. Manosylation was done by ring opening reactions followed by reaction of aldehyde groups of mannose in 0.1 M sodium acetate buffer (pH 4.0) with the amino groups of lipid. This leads to formation of Schiff's base (–N=CH–), which may then get reduced to secondary amine (–NH–CH2–) and remain in equilibrium with Schiff's base at basic pH. In vivo studies were conducted to evaluate for their cytotoxicity, targeting potential, AM uptake, and hematological studies. It was reported that mannosylation improved uptake (up to 6-fold) of rifabutin by the AM. Moreover, mannosylated rifabutin-SLNs were less immunogenic and helped in sustained delivery of drug. Thus, the mannosylated SLNs may be employed for AM targeted delivery of rifabutin for

Overexpression of mannose receptors in case of lungs cancer was reported by numerous investigators. The mannosylated-distearoyl phosphatidyl-ethanolamine SLNs loaded with paclitaxel (PTX) was developed for lungs targeted delivery of PTX. Manosylation was done by ring opening reaction followed by reaction of an aldehyde group of mannose with the free amine group provided by stearylamine and DSPE in sodium acetate buffer (pH 4.0). The stability testing data indicated that SLNs formulations stored at 4 2°C were more stable than those stored at 27 2°C. It was revealed that mannosylated SLNs deliver significantly higher concentration of drug to the alveolar cell sites and showed improved antiproli-

The folate receptors (α- form) are overexpressed on the surface of lung tumor cells. The extents of the overexpression are different in different types of lung tumors (adenocarcinomas—72%, squamous cell carcinomas—51%, small cell

ferative efficacy as compared to PTX solution and PTX-SLNs [28].

3.2.4 Folate conjugated solid lipid nanoparticles

100

delivery of antitubercular drugs to the lungs.

Role of Novel Drug Delivery Vehicles in Nanobiomedicine

3.2.3 Mannose conjugated solid lipid nanoparticles

effective management of TB [27].

List of SLNs and their different ligand conjugated forms for lungs cells targeting.

carcinoma—25%, and lung metastases—30%) [29]. These receptors allow folate derivatives to bind preferentially that permits intracellular incorporation of folate derivative by endocytosis.

Folate-conjugated copolymer of polyethylene glycol (PEG) and N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) SLNs loaded with Paclitaxel (PTX) were successfully developed. The conjugation of folate- conjugated PEG and HTCC to RIF-SLN was carried out by carbodiimide mediated coupling chemistry. Pulmonary administration of the developed F-PEG-HTCC–SLNs selectively delivered the PTX to the lung's cancer cells with improved penetrability and prolonged lung residence. Moreover, the developed SLN significantly reduced the in vitro half-maximum inhibitory concentration of PTX in M109-HiFR cells [30].

List of SLNs and their different ligand conjugated forms for lungs targeting have been summarized in Table 1.


**1. Introduction**

The consequences of fire related incidents leave a serious impact on human life and its property as well. Frequently, these fires are ignited from the polymeric materials, including textiles we use in our daily life as in consumer goods, home furnishings, transportation, apparel and protective clothing, etc. The world fire statistics reveal that the textiles and upholstered furniture are the first item to be ignited by small flames such as cigarettes and candles, thus producing the most common fire [1, 2]. Meanwhile, the textile materials release smoke and toxic gases while going through the combustion process, which in turn limit the evacuation in fire disasters and worsen the fire scenario [3, 4]. In addition, these fires cause huge economical losses as well as deaths of human beings [5, 6]. Thus, the researcher is making continual efforts to investigate the flammability of textiles in order to improve their fire performance. On the other hand, the growing concern over the sustainability issues infer that fire retardants should leave a low impact on health and the environment during the entire life cycle including recycling and disposal. It also demands to come up

with significant researches with a focus on alternate flame retardant chemistries and methodologies, including the use of more environmentally benign raw materials and eco-friendly approaches in the synthesis and application of new flame retardants.

Nylon is the oldest man-made fiber (MMF) among the synthetic textiles, which remains as an important fiber in the synthetic fiber community till the date. Initially Nylon, also in the name of polyamide, is developed for a limited number of end uses; however, these days, the fibers belonging to the nylon/polyamide group share a big market, from regular apparels to technical textiles. For example, carpet is a significant application for nylon and accounts for 17.5 percent of total usage globally. Other applications of nylon include airbag, heavy-duty tires, intimate apparel, military apparel, sheer hosiery and swimwear, etc. [7]. Among, different types of polyamides, polyamide 66 (Nylon 66) and polyamide 6 (Nylon 6) represents one of the most used technical fibers. Both of them possess almost similar physical properties, namely high mechanical properties (tensile strength is higher than that of wool, silk, rayon or cotton), high chemical stability, high melting point, resistance to shrinkage and abrasion [8]. However, like other common textiles these fibers are also flammable due to their organic structure; alongside they also show serious dripping. Thus, the nylon textiles cannot meet industrial and civil requirements in many cases, which ultimately limit their uses in the mentioned sectors [9–11].


**3. Camelid intrabodies: a versatile research tool**

Over the years, nanobodies have earned their mark as a research tool. A variety of extracellular and intracellular applications using nanobodies exist, and the latter will be discussed here. Intrabodies are often used to unravel protein functions and to gain insight into their dynamics. The versatility of nanobodies and the ease by which they can be engineered allow researchers to use different lines of approach (**Figure 2**). Chromobodies, consisting of a nanobody fused with a fluorescent protein, allow researchers to recognize and trace endogenous proteins in living cells [70]. Since they are already well known, they will not be discussed in detail here.

and thus to confirm the in vivo functionality of nanobodies. Moreover, in this way, one can also disturb protein function by restricting free diffusion of the protein and limiting its availability at places where it is needed [71]. Considering that the paratope of the nanobody is located at its N-terminal end, it is safer to fuse the tag at the C-terminal end of the nanobody. Otherwise, a substantial risk at disturbing antigen binding exists [2], although there are examples where a long tag is added to the nanobody N-terminus without disturbing its functionality [72]. Beghein et al. elegantly demonstrated how effectively nanobodies can delocalize their target protein to a variety of subcellular organelles. A survivin Nb (Kd ~ 1 nM) was capable of guiding endogenous survivin in or out the nucleus (*nuclear localization sequence* tag and *nuclear export sequence* tag), capturing survivin on the outer membrane of mitochondria (*mitochondrial outer membrane* tag) and even at the intermembrane area (*mitofilin* tag) which probably required (partial) unfolding of the nanobody and possibly chaperone-assisted entry into mitochondria. This had not yet been investigated. Also, transport of survivin in the peroxisomes (PST-1 tag) was demonstrated [72]. Since interaction between the nanobody and survivin apparently did not perturb survivin functionality, the tagged nanobody is a perfect research tool for further elucidating survivin biology. This strategy also provided evidence that only actin-free gelsolin is able to migrate to the nucleus (in contrast to the actin-gelsolin complex) to potentially act as a nuclear cofactor for

specific protein-protein interaction, the co-localization is absent.

**Figure 2.** Schematic overview of nanobody-based applications in research. Upper panel. *Left*: Nanobodies can be equipped with a delocalization signal sequence, which allows the enrichment of a protein of interest (POI) at a specific subcellular localization, such as mitochondria. *Middle*: Nanobodies can exert a direct inhibitory effect which induces a protein knockout. *Right*: Nanobodies can be used to target a POI for proteasomal degradation. For this purpose, the nanobody is fused with a cullin-RING E3 ubiquitin ligase. Lower panel. *Left*: A chromobody, consisting of a fusion between a nanobody and a fluorescent protein (FP), allows the visualization and tracing of endogenous proteins in a background of contaminating proteins. *Right*: This nanobody-based application allows one to determine whether or not two proteins of interest interact. Both proteins are labeled with a different fluorophore. One protein, labeled with GFP, is recruited to a specific location via a GFP Nb coupled with a targeting signal. If the second protein, in this example labeled with RFP, is an interaction partner, both fluorescent signals will co-localize. When a compound interferes with a

Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

http://dx.doi.org/10.5772/intechopen.70495

217

#### **3.1. Pinpointing protein functions**

Nanobodies are an attractive tool for the determination of endogenous protein function. They not only complement well-known RNAi and CRISPR/Cas9 techniques but also allow a more detailed insight by pinpointing specific functions with "surgical precision" by targeting individual protein domains (rather than eliminating the entire protein altogether) and protein conformations, which cannot be achieved by expression modulation. In other words, nanobodies can be of inestimable value to deepen our knowledge of several biological pathways. Researchers have employed several strategies for assessing the functionality of proteins or protein domains, and the different options will be discussed here.

As stated earlier, nanobody cDNAs are available, and these are easily engineered. This implies that the addition of a delocalization tag is fairly straightforward. A variety of targeting sequences are available and can be used to induce the enrichment of both nanobody and its target at specific (ectopic) subcellular compartments. This strategy allows researchers to assess the interaction between the nanobody and its target in the strongly reducing intracellular environment Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs http://dx.doi.org/10.5772/intechopen.70495 217

[24]. Using adeno-associated virus as a vehicle, a bispecific nanobody was introduced in these mice that protects against both furin and MT1-MMP, yielding similar effects on muscle contraction speed [64, 65]. Inhibiting the enzymatic activity of furin could be an alternative strategy, and noncompetitive furin-inhibiting nanobodies have been identified although they have not been tested for treatment of gelsolin amyloidosis [66]. However, despite the involvement of furin in several pathological processes, some considerations have to be made regarding its use as a therapeutic target. Although a complete/partial cleavage redundancy of furin toward several substrates was observed in the liver of an interferon-inducible Mx-Cre/loxP, furin knockout mouse model and obvious adverse effect were absent; a complete knockout of furin in a mouse model resulted in embryonic lethality at day 11 [67, 68]. This observation probably precludes their use in chronic treatments because it is rather unlikely that the long-term inhibition of furin does not go hand in hand with severe adverse effects. Therefore, shielding mutant PG from aberrant cleavage seems to be the better strategy. Moreover, this approach is already successfully implemented in the treatment of early-stage familial amyloid polyneuropathy caused by amyloidogenic variants of transthyretin, thus highlighting its feasibility [69].

Over the years, nanobodies have earned their mark as a research tool. A variety of extracellular and intracellular applications using nanobodies exist, and the latter will be discussed here. Intrabodies are often used to unravel protein functions and to gain insight into their dynamics. The versatility of nanobodies and the ease by which they can be engineered allow researchers to use different lines of approach (**Figure 2**). Chromobodies, consisting of a nanobody fused with a fluorescent protein, allow researchers to recognize and trace endogenous proteins in living cells [70]. Since they are already well known, they will not be discussed in

Nanobodies are an attractive tool for the determination of endogenous protein function. They not only complement well-known RNAi and CRISPR/Cas9 techniques but also allow a more detailed insight by pinpointing specific functions with "surgical precision" by targeting individual protein domains (rather than eliminating the entire protein altogether) and protein conformations, which cannot be achieved by expression modulation. In other words, nanobodies can be of inestimable value to deepen our knowledge of several biological pathways. Researchers have employed several strategies for assessing the functionality of proteins or

As stated earlier, nanobody cDNAs are available, and these are easily engineered. This implies that the addition of a delocalization tag is fairly straightforward. A variety of targeting sequences are available and can be used to induce the enrichment of both nanobody and its target at specific (ectopic) subcellular compartments. This strategy allows researchers to assess the interaction between the nanobody and its target in the strongly reducing intracellular environment

**3. Camelid intrabodies: a versatile research tool**

protein domains, and the different options will be discussed here.

detail here.

216 Antibody Engineering

**3.1. Pinpointing protein functions**

**Figure 2.** Schematic overview of nanobody-based applications in research. Upper panel. *Left*: Nanobodies can be equipped with a delocalization signal sequence, which allows the enrichment of a protein of interest (POI) at a specific subcellular localization, such as mitochondria. *Middle*: Nanobodies can exert a direct inhibitory effect which induces a protein knockout. *Right*: Nanobodies can be used to target a POI for proteasomal degradation. For this purpose, the nanobody is fused with a cullin-RING E3 ubiquitin ligase. Lower panel. *Left*: A chromobody, consisting of a fusion between a nanobody and a fluorescent protein (FP), allows the visualization and tracing of endogenous proteins in a background of contaminating proteins. *Right*: This nanobody-based application allows one to determine whether or not two proteins of interest interact. Both proteins are labeled with a different fluorophore. One protein, labeled with GFP, is recruited to a specific location via a GFP Nb coupled with a targeting signal. If the second protein, in this example labeled with RFP, is an interaction partner, both fluorescent signals will co-localize. When a compound interferes with a specific protein-protein interaction, the co-localization is absent.

and thus to confirm the in vivo functionality of nanobodies. Moreover, in this way, one can also disturb protein function by restricting free diffusion of the protein and limiting its availability at places where it is needed [71]. Considering that the paratope of the nanobody is located at its N-terminal end, it is safer to fuse the tag at the C-terminal end of the nanobody. Otherwise, a substantial risk at disturbing antigen binding exists [2], although there are examples where a long tag is added to the nanobody N-terminus without disturbing its functionality [72]. Beghein et al. elegantly demonstrated how effectively nanobodies can delocalize their target protein to a variety of subcellular organelles. A survivin Nb (Kd ~ 1 nM) was capable of guiding endogenous survivin in or out the nucleus (*nuclear localization sequence* tag and *nuclear export sequence* tag), capturing survivin on the outer membrane of mitochondria (*mitochondrial outer membrane* tag) and even at the intermembrane area (*mitofilin* tag) which probably required (partial) unfolding of the nanobody and possibly chaperone-assisted entry into mitochondria. This had not yet been investigated. Also, transport of survivin in the peroxisomes (PST-1 tag) was demonstrated [72]. Since interaction between the nanobody and survivin apparently did not perturb survivin functionality, the tagged nanobody is a perfect research tool for further elucidating survivin biology. This strategy also provided evidence that only actin-free gelsolin is able to migrate to the nucleus (in contrast to the actin-gelsolin complex) to potentially act as a nuclear cofactor for the androgen receptor [73], that fascin plays a role in the formation of filopodia/cell spreading and is also involved in MMP9 secretion, and that the SH3 domain of cortactin directly regulates MMP secretion [74].

called DeGradFP, was expressed in mammalian cells or *D. melanogaster* embryos, certain GFPtagged proteins were depleted. DeGradFP was also capable of phenocopying specific loss of function mutations. In spite of these successful results, treatment with DeGradFP was not always followed by the degradation of the targeted protein (e.g., GFP) [81]. In addition to that,

Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

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219

Just like DeGradFP, the cullin-RING E3 ubiquitin ligases were used as the framework for synthetic E3 ligase design. In an attempt to enhance the E3 activity, however, the GFP Nb was fused directly to a truncated adaptor protein instead of the substrate recognition protein. The best results were obtained with Ab-SPOP, a synthetic version of the CLR3 E3 ligase complex, displaying a 10-fold stronger signal reduction of a GFP-tagged protein compared to DeGradFP (50-fold vs. 5-fold). Importantly, the construct degrades only nuclear proteins, and possibly in the future, similar constructs may become available that degrade cytoplasmic proteins. The in vivo effectiveness of Ab-SPOP was confirmed in zebra fish embryos. Ab-SPOP-induced depletion of Hmg2a-citrine, a protein responsible for the modulation of nucleosome and chromatin structure, resulted in various early developmental defects [83]. Fulcher et al. tailored the von Hippel-Lindau (VHL) protein as an affinity-directed protein missile, called AdPROM. Under normoxic conditions, this substrate recognition protein recruits the hypoxia-inducible factor (HIF1α) to the CLR2 E3 ligase. AdPROM is composed of a fusion between the VHL protein and a GFP Nb. It was of crucial importance that the GFP Nb was positioned at the C-terminus of the VHL protein in order to obtain a proper orientation of the target proteins to the CLR2 E3 ligase complex. Since the paratope of a nanobody is localized at the N-terminal end, one should definitely check for potential detrimental effects of this fusion on the binding capacity of the nanobody itself. However, the affinity-directed protein missile was competent in inducing the specific degradation of GFP-tagged VPS34 and PAWS1 proteins in human cell lines, which was further substantiated by the observation of functional effects. Interestingly, during these experiments the researchers observed the co-degradation of UVRAG which is a regulatory component of the VPS34 kinase complex. This suggests that AdPROM has the potential of destroying protein complexes although only individual proteins are targeted [82]. Targeted degradation of proteins of interest by the use of nanobodies holds great potential and might be the perfect complement to CRISPR/Cas systems or RNAi in the elucidation of protein function. The tunability of this system is a huge benefit. Future experiments should point out whether the GFP Nb can be replaced by highly selective nanobodies targeted against specific proteins. In this way, one could investigate the functions of the protein of interest in a more direct manner, without the requirement of protein tags.

Nanobodies can be utilized for the detection of protein-protein interactions in cell-based assays. There is a large supply of in vitro methods which can be used for the detection of protein-protein interactions. These methods are widely used and highly efficient for highthroughput screenings but are limited by the fact that they don't operate in intact mammalian cells. Screening for interaction between proteins in their native environment guarantees their proper folding and the presence of necessary cofactors or regulatory proteins. Both

a broader application of DeGradFP is still to be demonstrated.

**3.3. Detection of protein-protein interactions**

Some nanobodies exert a direct inhibitory effect, resulting in a functional knockout of the protein. These nanobodies can help researchers to define the biochemical activities of proteins. For example, mechanistic insights in podosome formation were revealed by two inhibitory nanobodies targeted against L-plastin (LPL). LPL Nb5 is capable of blocking the actin-bundling activity of L-plastin, and LPL-Nb9 locks LPL in an inactive conformation. Experiments involving these nanobodies revealed the participation of L-plastin (LPL) in podosome formation and stability [75]. Furthermore, L-plastin is a component of cancer cell invadopodia and contributes to matrix degradation and cancer cell invasion. These effects are mediated by the actin-bundling activity of L-plastin and its bundling independent role in MMP9 secretion and activity, as revealed by the differential effects observed in the presence of LPL Nb5 and LPL Nb9 [76]. One can also interfere with signaling pathways by specific inhibition of the transcriptional activity of proteins, like beta-catenin and p53 [77, 78]. These nanobodies can be used to elucidate the impact of cofactors and post-translational modifications on the targeted protein and allow us to broaden our understanding of the respective signaling pathways. Insight into pathological mechanisms, which might result in the identification of druggable targets, can also be obtained. For example, nanobodies were used to investigate the role of two enzymatic domains of TcdB, a toxin produced by *Clostridium difficile*. Using specific inhibition of the effector glycosyltransferase activity or the cysteine protease, it was, among other things, established that the TcdB-cytopathic effects are mainly mediated by the glycosyltransferase activity [79].

Finally, nanobodies are known to stabilize certain protein conformations and are often used as an aid in crystallization experiments [2]. This property also comes in handy when one wants to study the mechanisms by which cellular receptors translate extracellular cues into intracellular responses. Depending on which conformation the receptor adopts after ligand binding, certain downstream signaling events can be either activated or inhibited. Staus et al. have identified nanobodies that preferentially recognize and stabilize the β2 adrenergic receptor in its active or inactive conformation resulting in a variety of functional effects [80]. These experiments indicate that nanobodies, by acting as an allosteric modulator of receptors, can help us to understand receptor biology.

#### **3.2. Depleting endogenous proteins through proteasomal targeting**

An alternative way to determine the function of a protein of interest (POI) in an in vivo setting is to selectively induce their degradation and study the resulting knockout phenotype. To achieve this goal, three different groups have exploited a combination of nanobodies and the endogenous ubiquitin proteasome pathway, a system that is responsible for selective protein degradation in eukaryotes [81–83]. Caussinus et al. were the first to use the ubiquitin pathway for targeted degradation by making adaptations of an E3 ubiquitin ligase, more specifically the cullin-RING 1 (CLR1) E3 ligase complex. For this purpose, a fusion between the F-box domain of *Slmb* and a GFP Nb (VHH GFP4) was made. *Slmb* is part of an F-box protein, responsible for substrate recognition that is expressed in *Drosophila melanogaster*. When this construct, called DeGradFP, was expressed in mammalian cells or *D. melanogaster* embryos, certain GFPtagged proteins were depleted. DeGradFP was also capable of phenocopying specific loss of function mutations. In spite of these successful results, treatment with DeGradFP was not always followed by the degradation of the targeted protein (e.g., GFP) [81]. In addition to that, a broader application of DeGradFP is still to be demonstrated.

Just like DeGradFP, the cullin-RING E3 ubiquitin ligases were used as the framework for synthetic E3 ligase design. In an attempt to enhance the E3 activity, however, the GFP Nb was fused directly to a truncated adaptor protein instead of the substrate recognition protein. The best results were obtained with Ab-SPOP, a synthetic version of the CLR3 E3 ligase complex, displaying a 10-fold stronger signal reduction of a GFP-tagged protein compared to DeGradFP (50-fold vs. 5-fold). Importantly, the construct degrades only nuclear proteins, and possibly in the future, similar constructs may become available that degrade cytoplasmic proteins. The in vivo effectiveness of Ab-SPOP was confirmed in zebra fish embryos. Ab-SPOP-induced depletion of Hmg2a-citrine, a protein responsible for the modulation of nucleosome and chromatin structure, resulted in various early developmental defects [83]. Fulcher et al. tailored the von Hippel-Lindau (VHL) protein as an affinity-directed protein missile, called AdPROM. Under normoxic conditions, this substrate recognition protein recruits the hypoxia-inducible factor (HIF1α) to the CLR2 E3 ligase. AdPROM is composed of a fusion between the VHL protein and a GFP Nb. It was of crucial importance that the GFP Nb was positioned at the C-terminus of the VHL protein in order to obtain a proper orientation of the target proteins to the CLR2 E3 ligase complex. Since the paratope of a nanobody is localized at the N-terminal end, one should definitely check for potential detrimental effects of this fusion on the binding capacity of the nanobody itself. However, the affinity-directed protein missile was competent in inducing the specific degradation of GFP-tagged VPS34 and PAWS1 proteins in human cell lines, which was further substantiated by the observation of functional effects. Interestingly, during these experiments the researchers observed the co-degradation of UVRAG which is a regulatory component of the VPS34 kinase complex. This suggests that AdPROM has the potential of destroying protein complexes although only individual proteins are targeted [82]. Targeted degradation of proteins of interest by the use of nanobodies holds great potential and might be the perfect complement to CRISPR/Cas systems or RNAi in the elucidation of protein function. The tunability of this system is a huge benefit. Future experiments should point out whether the GFP Nb can be replaced by highly selective nanobodies targeted against specific proteins. In this way, one could investigate the functions of the protein of interest in a more direct manner, without the requirement of protein tags.

#### **3.3. Detection of protein-protein interactions**

the androgen receptor [73], that fascin plays a role in the formation of filopodia/cell spreading and is also involved in MMP9 secretion, and that the SH3 domain of cortactin directly regulates

Some nanobodies exert a direct inhibitory effect, resulting in a functional knockout of the protein. These nanobodies can help researchers to define the biochemical activities of proteins. For example, mechanistic insights in podosome formation were revealed by two inhibitory nanobodies targeted against L-plastin (LPL). LPL Nb5 is capable of blocking the actin-bundling activity of L-plastin, and LPL-Nb9 locks LPL in an inactive conformation. Experiments involving these nanobodies revealed the participation of L-plastin (LPL) in podosome formation and stability [75]. Furthermore, L-plastin is a component of cancer cell invadopodia and contributes to matrix degradation and cancer cell invasion. These effects are mediated by the actin-bundling activity of L-plastin and its bundling independent role in MMP9 secretion and activity, as revealed by the differential effects observed in the presence of LPL Nb5 and LPL Nb9 [76]. One can also interfere with signaling pathways by specific inhibition of the transcriptional activity of proteins, like beta-catenin and p53 [77, 78]. These nanobodies can be used to elucidate the impact of cofactors and post-translational modifications on the targeted protein and allow us to broaden our understanding of the respective signaling pathways. Insight into pathological mechanisms, which might result in the identification of druggable targets, can also be obtained. For example, nanobodies were used to investigate the role of two enzymatic domains of TcdB, a toxin produced by *Clostridium difficile*. Using specific inhibition of the effector glycosyltransferase activity or the cysteine protease, it was, among other things, established that the TcdB-cytopathic effects are mainly mediated by the glycosyltransferase activity [79]. Finally, nanobodies are known to stabilize certain protein conformations and are often used as an aid in crystallization experiments [2]. This property also comes in handy when one wants to study the mechanisms by which cellular receptors translate extracellular cues into intracellular responses. Depending on which conformation the receptor adopts after ligand binding, certain downstream signaling events can be either activated or inhibited. Staus et al. have identified nanobodies that preferentially recognize and stabilize the β2 adrenergic receptor in its active or inactive conformation resulting in a variety of functional effects [80]. These experiments indicate that nanobodies, by acting as an allosteric modulator of receptors,

MMP secretion [74].

218 Antibody Engineering

can help us to understand receptor biology.

**3.2. Depleting endogenous proteins through proteasomal targeting**

An alternative way to determine the function of a protein of interest (POI) in an in vivo setting is to selectively induce their degradation and study the resulting knockout phenotype. To achieve this goal, three different groups have exploited a combination of nanobodies and the endogenous ubiquitin proteasome pathway, a system that is responsible for selective protein degradation in eukaryotes [81–83]. Caussinus et al. were the first to use the ubiquitin pathway for targeted degradation by making adaptations of an E3 ubiquitin ligase, more specifically the cullin-RING 1 (CLR1) E3 ligase complex. For this purpose, a fusion between the F-box domain of *Slmb* and a GFP Nb (VHH GFP4) was made. *Slmb* is part of an F-box protein, responsible for substrate recognition that is expressed in *Drosophila melanogaster*. When this construct,

Nanobodies can be utilized for the detection of protein-protein interactions in cell-based assays. There is a large supply of in vitro methods which can be used for the detection of protein-protein interactions. These methods are widely used and highly efficient for highthroughput screenings but are limited by the fact that they don't operate in intact mammalian cells. Screening for interaction between proteins in their native environment guarantees their proper folding and the presence of necessary cofactors or regulatory proteins. Both nanobody-based methods rely on the interaction between a GFP Nb and a GFP-tagged protein. Herce et al. covalently linked a GFP Nb with a protein that accumulates at a specific subcellular location. In mammalian cells, this protein could be, for example, laminin B1 or centrin, which results in the delocalization of the GFP Nb to the nuclear lamina or the cytoplasmic centrioles, respectively. Subsequently, a GFP-tagged protein will be recruited to a specific location. If the second protein of interest, labeled with another fluorophore, interacts with the first protein of interest, the fluorophores will co-localize at a discrete spot. This interaction can be visualized by a single-fluorescence snapshot. Interestingly, this technique also allows screening for inhibitors of protein-protein interactions [84]. Another recently developed technique uses biocompatible engineered upconversion nanoparticles (UCNPs) conjugated with GFP Nbs. Visualization of the interaction between two proteins of interest is based on the lanthanide resonance energy transfer (LRET). As a proof of concept, they probed for the indirect interaction between the mitochondrial proteins TOM20 and TOM7. The latter was expressed as a fusion protein with EGFP and the former as a fusion protein with dsRed and a Halo tag. This Halo tag was subsequently labeled with tetramethylrhodamine (TMR), while the EGFP was recognized by the GFP Nb-labeled UCNPs. Co-localization of both proteins results in the detection of LRET-sensitized TMR emission. Remarkably, TOM7 and TOM20 are spatially separated by TOM40. The capacity of this technique for reporting indirect longdistance interactions might be of interest to unravel cellular protein complexes [85].

[86, 87]. Indeed, particularly polyclonal antibodies run out of stock at some point in the future, making experimental verification impossible. Because nanobody cDNAs are readily obtained and researchers all over the world can use exactly the same nanobody in their experiments, problems of reproducibility can be reduced. In the future, we hope to stimulate a closer consultation within the nanobody field and by doing so taking the research to

Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

http://dx.doi.org/10.5772/intechopen.70495

221

This work was supported by grants from the Research Foundation Flanders (Fonds Wetenschappelijk Onderzoek (FWO) Vlaanderen) and Ghent University (BOF13/GOA/010). AS and LB are supported by the Agency for Innovation by Science and Technology in Flanders

(IWT-Vlaanderen). We apologize to those researchers whose work could not be cited.

Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University,

[1] Hamerscasterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, et al. Naturally-occurring antibodies devoid of light-chains. Nature. 1993 Jun

[2] Muyldermans S. Nanobodies: Natural single-domain antibodies. In: Kornberg RD, edi-

[3] Famm K, Hansen L, Christ D, Winter G. Thermodynamically stable aggregationresistant antibody domains through directed evolution. Journal of Molecular Biology.

[4] Kim DY, To R, Kandalaft H, Ding W, van Faassen H, Luo Y, et al. Antibody light chain variable domains and their biophysically improved versions for human immunother-

[5] Ward ES, Gussow D, Griffiths AD, Jones PT, Winter G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from *Escherichia coli*. Nature.

3;**363**(6428):446-448 PubMed PMID: WOS:A1993LE93800057

tor. Annual Review of Biochemistry. 2013;**82**:775-797

Anneleen Steels, Laurence Bertier and Jan Gettemans\*

\*Address all correspondence to<EMAIL_ADDRESS>
the next level.

**Acknowledgements**

**Author details**

Belgium

**References**

2008;**376**(4):926-931

1989;**341**(6242):544-546

apy. MAbs. 2014/01/01;**6**(1):219-235


**1. Introduction**

Running is a sport that has been growing in popularity over the years [1]. Through multiple years of training, runners accomplish a variety of changes at different levels that include metabolic, neuromuscular, and biomechanical efficiency and ultimately result in improved running economy [2]. The changes in running economy play an important role in running performance, since a smaller energy expenditure at a given speed is beneficial, especially in disciplines that require running with submaximal speed for long distances. Therefore, not only cardiovascular and metabolic fitness (e.g., VO2max) but also biomechanical and neuromuscular efficiency are crucial for individual running economy [2]. Even though VO2max is typically used as a measure of running economy [3,4], studies that track the performance development of elite athletes for several years have reported improvements in performance without significant changes in VO2max [5,6]. This emphasizes the importance of considering the whole scope of variables that are associated with

**Citation:** Fadillioglu, C.; Möhler, F.; Reuter, M.; Stein, T. Changes in Key Biomechanical Parameters According to the Expertise Level in Runners at Different Running Speeds. *Bioengineering* **2022**, *9*, 616. https://doi.org/10.3390/ bioengineering9110616

Academic Editors: Christina Zong-Hao Ma, Zhengrong Li and Chen He

Received: 26 September 2022 Accepted: 23 October 2022 Published: 26 October 2022

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

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

running economy. Apart from factors such as age, gender, body temperature, and muscle fiber distribution, several biomechanical parameters as well as the running style have been shown to influence the running economy [7,8].

Positive correlations have been observed between isolated parameters and running economy. In particular, the vertical oscillation of the center of mass (osc\_CoM) [2,8–10], step length (SL) [2,10], and step frequency (SF) [3,10] were identified as factors that influence the running economy in various running-related studies. The findings of these studies have indicated that an increase in running economy is associated with a lower osc\_CoM [2,8,9,11] and a lower SF [3,10], whereas the results for SL have not been consistent across studies [10,12]. These varying results can be explained by the self-optimized SL/SF ratio [13,14]. On this basis, analyzing different running styles may be preferable to a comparison of isolated parameters when trying to understand the kinematic adaptations that occur as a result of training. However, the parameters used for the operationalization of the running style vary across the literature. Recently, in a study by van Overen et al. [13], it was suggested that the duty factor (DF), which is the ratio of stance to stride time, together with the SF normalized to the leg length (SF\_norm) would be suitable to describe the running style and can be used for comparisons between individuals.

The expertise level is a major factor that influences various biomechanical parameters, including spatio-temporal parameters. Therefore, in studies that attempt to distinguish between groups of runners in terms of their expertise level and define them, several terms are used, including "expert", "novice", "elite", "amateur", and "good". In this study, the terms expert (to refer to elite runners), good, and novice (to refer to amateur runners) will be used to avoid confusion between terminologies. Such studies have reported that SF was found to increase with increase in experience, stride time was found to be unaffected [15–18], and DF was found to decrease [19]. In addition, the effects of running experience on running economy have been reported in various studies. Importantly, experts were shown to have a more efficient running style than novices [9,20]. In studies that compared the performance of expert and good distance runners, it was found that expert runners have a slightly lower osc\_CoM and a better running economy than good runners [2].

Apart from the running experience, the running speed was also shown to influence the kinematic variables. Padulo et al. [21] showed that both for expert and for novice runners, SL and SF increased, while stance time decreased with an increase in running speed. A study by García-Pinillos et al. [22] investigated the alterations in spatiotemporal parameters in novice endurance runners and showed that stance time becomes shorter, while SL and flight time become longer, as speed increases. Furthermore, not only the mean values but also the variability in spatiotemporal parameters was found to change with an increasing speed. Jordan et al. [23] suggested that the variability in running gait is not random but manifests self-similarity depending on the gait speed and therefore, it may help to understand the control of human locomotion.

The term "movement variability" can refer to different aspects according to the context [24]. In sports, the main way to achieve the desired goal (e.g., hitting the basketball hoop) is to achieve consistency over multiple repetitions. On the other hand, a certain level of variability may increase the flexibility of the whole biological system and ultimately help to adapt to different conditions, such as fatigue and environmental changes, and may also help to reduce injuries [24]. It was suggested that the variability of a parameter, which is important as a movement goal, can be minimized over several movement repetitions, and thereby, variations in less important parameters may be allowed to a certain level so that their co-variation establishes a flexible but stable system [25–27]. However, in the case of sports such as running, it is difficult to definitively determine which parameters are prioritized by the central nervous system (CNS) in terms of control. Typically, runners aim to run as fast as possible for a given distance and, thereby, try to improve their running style to make it more energy-efficient [28]. Thereby, the goal is to ensure that the parameters that are crucial for this goal are kept stable, whereas the less important parameters may

vary on a larger scale. For the analysis of running in terms of the variability and stability of biomechanical parameters, different methods exist (e.g., uncontrolled manifold approach and tolerance noise covariation) and were applied in several studies [29–31]. Even though these methods are ideal for investigating the structure of motor variability in redundant solution spaces, they are not useful for distinguishing a target parameter [27,29,30]. Analyzing the variability characteristics of biomechanical parameters may help to understand which parameters are of high priority for the CNS [27,32]. Despite its relevancy, only a few studies have analyzed the variability in running kinematics [22,33–36].

To sum up, three key biomechanical parameters, i.e., vertical oscillation of CoM, stride frequency, and duty factor, have been shown to be closely related to running economy and running style. However, the influence of expertise level and running speed on the variability of these key biomechanical parameters has only been partially researched, even though they may reveal valuable information regarding the control of human locomotion. Therefore, the goal of this study was to investigate the effects of expertise level on vertical oscillation of CoM, stride frequency, and duty factor, as well as their variabilities at two different running speeds. It was hypothesized that expert runners would show lower mean values and lower variability of the considered parameters compared with novice runners, regardless of the running speed.

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

The data used in the present study were analyzed with the uncontrolled manifold approach as in a previous study [37].

#### *2.1. Participants*

The participants of this study were all male and comprised two groups: one group of 13 expert runners (EXP: age, 23.5 ± 3.6 years; height: 1.80 ± 0.06 m; weight, 66.8 ± 5.4 kg) and one group of 12 novice runners (NOV: age, 23.9 ± 3.8 years; height: 1.83 ± 0.07 m; weight, 72.2 ± 6.6 kg). EXP included runners with a 10 km personal best time below 35 min (32:59 ± 01:19 min) who had been members of a running club for at least 2 years (7.2 ± 3.2 years) and ran a minimum of 50 km per week (duration: 6.5 ± 1.7 h/week). NOV included runners who participated in a maximum of two sport sessions per week, including a maximum of one running session (0.2 ± 0.2 h/week). Importantly, this group only included runners who had never prepared for a running event or trained in a running club. It was important for all participants to be free of recent injuries or pain in the lower limbs. They were asked not to perform any intense workout on the day preceding the measurement. All participants provided their written informed consent prior to participation. This study was approved by the ethics committee of the Karlsruhe Institute of Technology.

#### *2.2. Protocol*

The study was performed on a motorized treadmill (h/p/cosmos Saturn; Nussdorf-Traunstein, Germany), with participants wearing a safety harness that was connected to an emergency off button. Before the measurements, a total of 22 anthropometric measurements were taken manually for each participant, and 41 markers were attached on their body according to the guidelines of the ALASKA (Advanced Lagrangian Solver in kinetic Analysis) modelling system [38]. After a standardized session of treadmill familiarization (6 min of walking and 6 min of running, [39,40], the speed of the treadmill was accelerated up to 15 km/h and held for 15 s in order for the participants to experience the running speed that they would run at during the measurements. After a 2 min break, the participants performed runs at speeds of 10 and 15 km/h in a counterbalanced order. The participants ran for approximately 1 min at each of the two speeds. For each speed, 3D marker data for the 41 markers were recorded during 20 consecutive gait cycles using 11 Vicon MX cameras (Vicon Motion Systems; Oxford Metrics Group, Oxford, UK) that were capable of recording at 200 Hz. The two running speeds for the measurements, 10 and 15 km/h, were

chosen based on previous comparable studies [16,22]. In the pre-tests for this experiment, the chosen running speeds were confirmed to be proper.

#### *2.3. Data Processing*

The marker data were post-processed using the Vicon Nexus software (V1.8.5) and filtered with a 10 Hz low-pass Butterworth filter using Matlab (The MathWorks, Natick, MA, USA). The gait cycles were segmented using the foot marker data [41]. The CoM was estimated with the ALASKA Dynamicus modelling system [38]. To calculate osc\_CoM, the difference between the minimum and the maximum height of the CoM (Equation (1)) was calculated for each of the 20 gait cycles. SF\_norm and DF were calculated using previously published formulae [13]. To calculate SF\_norm, SF was calculated as 60 divided by the sum of stance time and flight time, and then it was normalized to the leg length (Equation (2)). To calculate DF, the stance time was divided by twice the sum of the stance and flight time (Equation (3)). The variability of these parameters was calculated as the coefficient of variation (CV). Their mean values were included in the analysis to compare the results with those published in the existing literature.

$$\text{loss\\_CoM} = \text{CoM}\_{\text{Zmax}} - \text{CoM}\_{\text{Zmin}} \tag{1}$$

$$\text{SF\\_norm} = \frac{60}{\left(t\_{\text{state}} + t\_{flight}\right) \cdot \sqrt{\frac{I\_0}{g}}} \tag{2}$$

$$\text{DF} = \frac{t\_{\text{stance}}}{2 \cdot (t\_{\text{stance}} + t\_{flight})} \tag{3}$$

#### *2.4. Statistical Analysis*

Shapiro–Wilk tests were conducted to confirm the normality of data distribution. The dependent variables were osc\_CoM, SF\_norm, and DF, as well as their variabilities (operationalized by CV). For each of these dependent variables, a 2 × 2 repeated-measures ANOVA (rmANOVA) was calculated with the factors expertise level (EXP and NOV) and speed (10 and 15 km/h). In total, six rmANOVAs were performed. The Bonferroni–Holm method was used to correct the results for multiple comparisons [42]. The significance level was set a priori to *p* < 0.05. Partial eta-squared (small effect: *η* 2 *p* < 0.06; medium effect 0.06 ≤ *η* 2 *p* < 0.14; large effect: *η* 2 *p* ≥ 0.14) was calculated as a measure of the effect size for rmANOVA.

#### **3. Results**

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The results for the mean values and the variability are shown in Figure 1.

**Figure 1.** Mean value (**top row**) and coefficient of variation (CV) (**bottom row**) for the three parameters vertical oscillation of CoM (osc\_CoM), normalized stride frequency (SF\_norm), and duty factor (DF). The error bars show the standard deviation of the respective values. The values for the 10 km/h condition are shown in blue, and the values for the 15 km/h condition are shown in red; \* significant effect for the factor expertise level, # significant effect for the factor speed.

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*3.4. Utility*

Most of the sampled population (94.4%) found the exercise useful for real-life activities (M = 4.69, SD = 0.623). While 18.3% strongly and 19.7% mildly agreed that the exercise was useful for commanding officers, 29.6% remained neutral (M = 3.17, SD = 1.248). Some 42.3% either mildly or strongly agreed that the exercises were useful for ordinary operative staff. Here, 42.3% remained neutral (M = 3.40, SD = 0.900). Finally, under half (45.1%) agreed that their experiences of the exercises would affect their daily work, while 33.8% remained neutral (M = 3.40, SD = 1.102). The overall mean for the utility dimension was 3.68 (SD = 0.543).

#### *3.5. Bivariate and Multivariate Regression Analysis.*

#### **RQ 1:** *To what extent is there a relationship between participation in Norwegian wildland-fire collaboration exercises and perceived level of learning?*

*Bivariate analysis*. The most pronounced significance was found between learning and the item "Clear instructions of collaboration were presented" (r = 0.501, r2 = 0.239, F = 20,761, *p* ≤ 0.000). The item "The exercises were focused on collaboration" had the second highest pronounced significance (r = 0.451, r2 = 0.191, F = 16.349, *p* ≤ 0.000), followed by "Sufficient forms of discussions were provided" (r = 0.421, r2 = 0.164, F = 13.564, *p* ≤ 0.000). "My points of view were regarded" had an R-value of 0.398 (r2 = 0.145, F = 12.034, *p* ≤ 0.001), while "there were opportunities to improvise" had an R-value of 0.282 (r2 = 0.65, F = 5.508, *p* ≤ 0.022). "Collaboration was initiated immediately" had somewhat lower significance level (r = 0.255, r2 = 0.050, F = 4.439, *p* ≤ 0.039). The final item with a pronounced significant was "Personnel in need of exercise participated" with an *R*-value of 0.253 (r2 = 0.049, F = 4.291, *p* ≤ 0.042). The item "I performed well-known activities" was found insignificant (r = 0.06, r2 = −0.01, F = 0.30, *p* = 0.58) and was therefore excluded from the multiple regression analysis (Table 3).

> **Table 3.** Bivariate regression of the collaboration dimensions of learning.

*Note:* N = 71, sig = *p* ≤ 0.05.

*Multivariate analysis*. The joint collaborative characteristics predicted 29.3% (r2 = 0.293) of the learning variance, meaning that the remaining 70.7% of the predicted variance was unaccounted for. Still, the variables "There were opportunities to improvise," "Collaboration was initiated immediately," and "Personnel in need of exercise participated" were found to be significant (Table 4). The regression analysis indicated a 61% (r = 0.61) covariation between collaboration and learning, which is considered strong [49].

**Table 4.** Multiple regression of the collaboration dimensions of learning.

*Note*: N = 71, R = 0.611, *r*2 = 29.3 sig = *p* ≤ 0.05 **RQ 2: (2)** *To what extent is there a relationship between participation in Norwegian wildland-fire collaboration exercises and perceived level of usefulness?*

*Bivariate analysis* (Table 5). The most pronounced significance was found between usefulness and "I learned others concepts and abbreviations" (r = 0.436, r2 = 0.177, F = 14.755, *p* ≤ 0.000). This was followed by "I learned about other's organizational aspects" (r = 0.388, r2 = 0.137, F = 10,998, *p* ≤ 0.002) and "I learned new things during the exercise" (r = 0.343, r2 = 0.103, F = 8.259, *p* ≤ 0.006). The item "I learned about other's prioritizing of activities" received a R-value of 0.324 (r2 = 0.091, F = 7.290, *p* ≤ 0.009), and "I learned about other's communication patterns" a R-value of 0.323 (r2 = 0.090, F = 7.358, *p* ≤ 0.009).

**Table 5.** Bivariate regression of the learning dimension of usefulness.

*Note:* N = 71, sig = *p* ≤ 0.05.

*Multivariate analysis* (Table 6.). The perceived learning items predicted 17% (r2 = 0.170) of the usefulness variance, meaning that the remaining 83% of the predicted variance was unaccounted for. The only item that was found significant was "I learned about other's prioritizing of activities" (*p* = −0.049). Thus, these results indicate a medium to small [49] covariation between learning and utility.

> **Table 6.** Multiple regression of the learning dimensions of usefulness.

*Note*: N = 71, R = 0.488, *r*2 = 0.170, sig *p* ≤ 0.05


*Edited by Hojka Gregorič Kumperščak*

Attention Deficit Hyperactivity Disorder (ADHD) is the most prevalent neurodevelopmental disorder. Previously, it was considered a disorder that affected children only. Recently, however, there is no doubt that ADHD can affect adults as well, but with different clinical presentation. Thus, it is critical to understand how the clinical picture of the disorder changes with development. Traditional ADHD diagnostic procedures are broadening and incorporating new entities like endophenotypes. Comorbidity is a rule, especially if ADHD is not recognized and treated early. New genetic studies bring deeper and more concise knowledge about the disorder's etiology. This book addresses these aspects of ADHD to bring about more clarity and understanding of the disorder.

ISBN 978-1-83962-475-9

ADHD - From Etiology to Comorbidity

Published in London, UK © 2021 IntechOpen © AndreaObzerova / iStock

## ADHD From Etiology to Comorbidity

*Edited by Hojka Gregorič Kumperščak*