*Article* **The Pathogens Spillover and Incidence Correlation in Bumblebees and Honeybees in Slovenia**

**Metka Pislak Ocepek 1,\*, Ivan Toplak 2, Urška Zajc 3 and Danilo Bevk 4**

**Abstract:** Slovenia has a long tradition of beekeeping and a high density of honeybee colonies, but less is known about bumblebees and their pathogens. Therefore, a study was conducted to define the incidence and prevalence of pathogens in bumblebees and to determine whether there are links between infections in bumblebees and honeybees. In 2017 and 2018, clinically healthy workers of bumblebees (*Bombus* spp.) and honeybees (*Apis mellifera*) were collected on flowers at four different locations in Slovenia. In addition, bumblebee queens were also collected in 2018.  In bumblebee queens, only the presence of BQCV, *A. bombi* and *C. bombi* was detected with 73.3, 26.3 and 33.3% positive samples, respectively. This study confirmed that several pathogens are regularly detected in both bumblebees and honeybees. Further studies on the pathogen transmission routes are required.

**Keywords:** bumblebees; honeybees; viruses; *Nosema* spp.; *Crithidia bombi*; *Apicystis bombi*; *Lotmaria passim*; pathogens transmission


**Author details**

Bei-Yu Wu1 , Chun-Ting Liu1 , Yu-Chiang Hung1,2 and Wen-Long Hu1,3,4\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Chinese Medicine, Kaohsiung Chang Gung Memorial Hospital and School of Traditional Chinese Medicine, Chang Gung University College of Medicine, Kaohsiung, Taiwan

2 School of Chinese Medicine for Post Baccalaureate, I-Shou University, Kaohsiung, Taiwan

3 Kaohsiung Medical University College of Medicine, Kaohsiung, Taiwan

4 Fooyin University College of Nursing, Kaohsiung, Taiwan


**2. Materials and Methods**

#### *2.1. Specimens' Preparation*

The machining and finishing processes of the AISI 316L stainless steel used for this test (chemical composition shown in Table 1) are performed in a 3-axis CNC Machine Lathe "Pinacho SE 200 × 750 (Pinacho, Castejón del Puente, Spain).

The machining conditions prior to the burnishing were kept constant for all the specimens, being 60 m/min as cutting speed, 0.20 mm/rev as feed per turn, and a final diameter of 13 mm achieved in 3 passes. The insert used for this process was a rhombic 80◦ shape with a negative geometry CNMG120408-MM-YG213, made of carbide with a CVD TiCN+Al2O3+TiN coating, the clearance angle is set to 6◦, and the rake angle to −6◦ once is assembled on the PCLNR 2020K 12 shank tool. The average quality roughness prior to burnishing was set to 1.5 ± 0.2 μm in order to obtain significant differences between all burnishing conditions applied to the specimens.

#### *2.2. Ball Burnishing Equipment and Specimens*

The vibration-assisted ball burnishing system used for this investigation is composed of the burnishing tool (the union of a base armor, a pre-load unit force based on a calibrated spring, one ultrasonic actuation unit built by a resonant ceramic piezoelectric excited to 40 kHz of frequency and an operational head formed by one 10 mm diameter 100Cr6 chromium steel ball at the top) and an external 40 kHz wave generator both plugged by a UFH connector, characterized by Fernández-Osete et al. [25]. The amplitude of vibration obtained at the top of the tool by the resonance of the system is about 8 μm.

The definitive force applied onto the surface is a combination of multiple forces: the forces due to the irregularities of the material when the feed movement is performed, the force related to the vibration of the sonotrode when the assistance is applied, and the preadjusted force of the spring lodged. The final result of the process is shown in Figure 1.

**Figure 1.** Burnishing tool head and specimen manufactured.

#### *2.3. Experimental Campaign and Specification*

Based on the research group's previous experience with the process on other materials such as AISI 1045 [12] or AISI301LN [26], a screening design of experiments was planned with the following factors and their corresponding levels:

• Burnishing force Fb: three numerical levels, from 80 N to 160 N with a central point in 120 N. Attabi et al. [21] found a surface degradation at slim plates with 240 N and a 10 mm ball, so by applying the radius corrections (in the function of ball mechanical and geometrical properties of the specimen) it is decided to apply a maximum force of 160 N to avoid surface degradation.

The final experimental campaign resulted in a total of 12 specimens, and all combinations are shown in Table 2.

**Table 2.** Design of Experiments for the burnishing operation.

The tribological procedure performed is based on Velázquez-Corral et al. previous work [12] and the ASTM G132-96 standard [27], see Figure 2. The conditions used are the following:

**Figure 2.** Tribology setup used for the testing.

During the execution of each test, the friction and input force is tracked and the *cof* (Coefficient of friction) is calculated in real-time at 100 Hz sampling frequency. The final acquisition is filtered using a low-pass filter, using a minimum-order filter with a stopband attenuation of 60 dB and compensating the delay introduced by the filter itself, with a passband frequency of 20 Hz. After that, the total worn imprint is measured using the 3D non-contact profilometer Alicona with a 10 nm of resolution, obtaining a 3D surface and measuring the volume difference between this surface and the one prior to the tribology testing, which defines the wear of the test in volumetric units.


**5.2 Laparoscopic repair**

Parastomal hernia repair with prosthetic mesh is recommended, since recurrence rates are unsatisfactory high after suture repair or relocation of the stoma. Complications that can arise with mesh placement for parastomal hernia are mesh-infection, fistula formation and adhesion formation. Laparoscopic repair is effective in correction of these hernias and has the advantages of improved vision and definition of the fascial edges of the hernia.

Laparoscopic techniques for repair of parastomal hernia with prosthetic mesh can be divided in 'keyhole techniques' and modified 'Sugarbaker techniques'. All involve introduction of trocars, extensive adhesiolysis, and identifying and measuring the fascial

The reported failure following laparoscopic Nissen fundoplication for GERD and paraesophageal hernia is between 2-33%. Although failure of fundoplication is unusual when performed by an experienced surgeon, wrap herniation ('slipped Nissen') is the most common mechanism of failure. Other causes of failure are represented by disrupted fundoplication, slipped fundoplication, crural stenosis, too tight wrap, misplaced fundoplication or twisted fundoplication. In carefully selected patients who have recurrent or persistent symptoms (heartburn, dysphagia, chest pain, regurgitation, asthma, hoarsness or laryngitis) after laparoscopic or open fundoplication a laparoscopic redo fundoplication can be safely performed by an experienced surgeon. The overall conversion rate of redo laparoscopic fundoplication is 10%. Complications occur in approximately 15%, slightly increasing with multiple redos. After redo laparoscopic fundoplication 70% of patients is

Occurrence of parastomal herniation is a common complication after stoma formation. The reported incidence of parastomal hernias varies from 28% in ileostomies to 56% in colostomies (Carne et al., 2003; LeBlanc et al., 2005; Rieger et al., 2004). A parastomal hernia is more likely to occur when the stoma emerges through the semilunar line rather than the rectus sheath. Although most hernias become present within two years after stoma

Perstomal type: bowel prolapsing through a circumferential hernia sac enclosing the

Parastomal hernia repair with prosthetic mesh is recommended, since recurrence rates are unsatisfactory high after suture repair or relocation of the stoma. Complications that can arise with mesh placement for parastomal hernia are mesh-infection, fistula formation and adhesion formation. Laparoscopic repair is effective in correction of these hernias and has

Laparoscopic techniques for repair of parastomal hernia with prosthetic mesh can be divided in 'keyhole techniques' and modified 'Sugarbaker techniques'. All involve introduction of trocars, extensive adhesiolysis, and identifying and measuring the fascial

 Intrastomal: hernia sac between the intestinal wall and the everted intestinal layer Symptoms patients may experience are pain, poor fitting of stoma-material resulting in leakage of stomal contents, obstruction, incarceration and cosmetic disfigurement. Fortunately, most parastomal hernias can be treated conservatively and surgical intervention is only indicated in 15% of patients with parastomal hernias (Hansson et al., 2003). Recurrence rates after surgical repair are reported up to 76%, and can be explained by

the underlying defect in wound healing and collagen metabolism in most patients.

the advantages of improved vision and definition of the fascial edges of the hernia.

**4.3 Treatment of recurrence**

**5. Parastomal hernia**

**5.1 Classification**

stoma

**5.2 Laparoscopic repair**

GERD-related symptom free (Smith et al., 2005).

construction, the risk of herniation extends up to 20 years.

Interstitial type: hernia sac within the aponeurotic layers of the abdomen

Parastomal hernias can be classified in four types: Subcutaneous type: subcutaneous hernia sac

defect. A mesh should provide at least 5 cm of overlap of the fascial edges and should be secured with tacks or constructed with transfascial sutures.

Several different 'keyhole techniques' have been described, which have in common that a mesh is placed with a central hole or slit in the mesh to allow the bowel to pass through the mesh to the stoma site. One of the main drawbacks is shrinkage of the mesh that can result in obstruction or recurrent herniation by enlargement of the hole. In the modified Sugarbaker technique no hole is made in the mesh but the bowel to the stoma is lateralized and covered by the mesh (Berger & Bientzle, 2007; Mancini et al., 2007; Sugarbaker, 1985). The mesh is secured to the abdominal wall at the margin of the mesh at 5 cm intervals. A second row of tackers is placed at the margin of the hernia defect with additional tackers at each side of the colon. Both techniques are promising, however long term results are not yet available. Perhaps prevention of development of parastomal hernia by placement of a lightweight sublay mesh is the key (Janes et al., 2004).


**13. Pharmaceutical manufacturing and distribution: a clear health security concern**

In 1982, seven people died in the greater Chicago, Illinois metropolitan area after consuming acetaminophen that had been intentionally laced with potassium cyanide [257]. While the response by the drug manufacturers has since been widely recognized and praised as a textbook case in corporate crisis and public relations management, the greater concern was the recognition of how vulnerable pharmaceuticals are to potential tampering and bioterrorism [258, 259]. As a result, the United States Food and Drug Administration (US FDA) and Federal Government—working jointly with pharmaceutical manufacturers—established

harsh anti-tampering laws and guidelines to limit the risk of further contamination of the drug supply [260, 261].

However, it became clear that such "tampering" events—especially if intentional—can occur on a global scale [258, 259]. This is particularly true when one considers the extensive worldwide supply-chain manufacturing process that is involved in the production of consumer pharmaceuticals. Even slight changes in a drug's production—be it the initial recipes, mixing with drug stabilizers, buffers, or binders, all the way to packaging and distribution—can have catastrophic, if not fatal, consequences. The complexity of pharmaceutical development and distribution illustrates that there are numerous opportunities for sabotage, bioterrorism, neglect, or various forms of human error [262, 263]. Even local events can spark worldwide concerns and public fear. Any breakdown, intentional or otherwise, can compromise the safety of medications that millions depend on for their daily health. Potential observed effects may include avoidance of long-term maintenance medications, clinically significant manifestations of fear, and tremendous economic damage related to combined supply chain disruptions, in addition to increased downstream health-care costs.

While there are safeguards built into the entire process to assure pharmaceutical purity—including oversight by government agencies, such as the US FDA and European Union (EU) European Medicines Agency (EMA)—the system is not without potential risk. Many LMIR areas are challenged by the lack of appropriate regulatory oversight, government penalties, and even public (i.e., public company shareholders) accountability. In addition, pharmaceuticals that are no longer protected by intellectual property laws may be manufactured and distributed worldwide with lower regulatory or quality oversight [264, 265]. In fact, many wellestablished pharmaceuticals can vary significantly in their drug bio-availabilities and potencies depending on how and where they are manufactured [266, 267].

#### **14. Conclusions**

International health security is a complex and highly heterogeneous area of expertise. The American College of Academic International Medicine IHS Consensus Group (ACAIM-CG) developed a summative assessment highlighting the main issues that can impact IHS including emerging infectious diseases; chronic health conditions; bioterrorism; planetary changes (volcanic eruptions, earthquakes, wildfires, and climate change); nuclear incidents; information and cyber health; industrialization; globalization; pharmaceutical production; and communication platforms (social media). These concerns can directly and indirectly impact IHS both in the long and short term. When considering each IHS component, we aim to emphasize the utility of applying a predefined framework to effectively approach health security threats. This framework comprises of prevention, detection, assessment, reporting, response, addressing needs, and the ongoing repetition of this cycle (inclusive of appropriate mitigation measures). It is hoped that this collective work will provide a foundation for further research within the redefined, expanded scope of IHS.

#### **Acknowledgements**

The authors of this manuscript would like to thank all members of the ACAIM International Health Security Consensus Group, including non-author contributors, for their input and guidance throughout the process of drafting this important publication.

**29**

**Author details**

Tamara Worlton8

Thomas J. Papadimos5

and Stanislaw P. Stawicki17\*

, Manish Garg2

provided the original work is properly cited.

, Ricardo Izurieta3

2 Department of Emergency Medicine, New York Presbyterian Hospital, Cornell

© 2020 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,

Harry L. Anderson III12, Rebecca Jeanmonod13, Donald Jeanmonod13, Ijeoma Nnodim Opara14, Kristiana Kaufmann15, Juan A. Asensio16

, Bonnie Arquilla6

1 University of South Florida College of Medicine, Florida, USA

and Columbia University Schools of Medicine, New York, NY, USA

, Michael S. Firstenberg9

, Sona M. Garg4

, Andrew C. Miller7

,

, Sagar Galwankar10, Sunil Raina11,

, Abbas M. Khan7

,

Nicole K. Le1

*International Health Security: A Summative Assessment by ACAIM Consensus Group*

*DOI: http://dx.doi.org/10.5772/intechopen.93214*

CHC chronic health condition CHS cyber health security

HIR high-income regions

PC planetary changes PCC planetary climate change PHE public health emergency PHI protected health information

POC point-of-care

SM social media

EID emerging infectious diseases

IHS international health security LMIR low- and middle-income regions NGO nongovernmental organization

PTSD post-traumatic stress disorder SA summative assessment SDH social determinants of health

BT bioterrorism CG consensus Group

EOL end-of-life GH global health

**Glossary**

*International Health Security: A Summative Assessment by ACAIM Consensus Group DOI: http://dx.doi.org/10.5772/intechopen.93214*

## **Glossary**


**1. Introduction**

The ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated disease, Coronavirus Disease 2019 (COVID-19), have resulted in the generation of a tremendous amount of literature on various aspects of the disease and the virus. Of importance to this chapter is the literature on physical disinfection strategies for the virus, and infection prevention and control (IPAC) strategies for reducing potential transmission of the virus. In addition, physical inactivation approaches are used for rendering patient samples safe for handling in laboratories conducting diagnostic assays. Certain physical inactivation approaches also are used as barrier technologies for rendering human and animal raw materials safe for use in biologics manufacture. The literature specific to SARS-CoV-2 that has been published in the past 18 months is supplemented by previous literature on other relevant human and animal coronaviruses. These include human coronavirus 229E (HCoV-229E), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine hemagglutinating encephalomyelitis virus (PHEV), and canine, feline, and bovine coronaviruses (this list is not all-inclusive). For the present review chapter, the authors searched the literature for gamma irradiation, electron beam, high pressure, UVC, and heat (thermal) inactivation of coronaviruses in general, and in particular, the specific coronaviruses listed above. No limits were placed on date of publication, although, for obvious reasons, the data on SARS-CoV-2 were obtained from papers published since 2019.

Strategies for IPAC of SARS-CoV-2 include an impressive arsenal of pharmaceutical (vaccines, palliative therapies) and non-pharmaceutical interventions (face mask usage, social distancing, testing, contact tracing and quarantine), as well as chemical and physical approaches for liquid, surface, and air disinfection and for personal hygiene. In this chapter, we have attempted to review the physical inactivation efficacy data for SARS-CoV-2 and other coronaviruses. Our primary emphasis in this review is on IPAC, but other applications of physical inactivation approaches, such as rendering laboratory samples safe for handling within a biosafety I or II facility, and barrier treatments for inactivating potential contaminants in biologics animal-derived materials, are discussed.


**1. Introduction**

Melanoma is one of the most aggressive forms of malignant skin neoplasms and one of the main causes of cancer mortality. Although it represents only 4% of dermatological cancers, it is responsible for 80% of skin cancer deaths due to its high metastatic capacity and high refractoriness to chemotherapy [1–4]. The 5-year survival rate of patients with metastatic melanoma is less than 20% [5]. Dacarbazine, an alkylating agent, is the main treatment for advanced melanoma. However, serious side effects have been observed, and the therapeutic response rate is approximately 10% [5,6]. These results indicate that new therapies for melanoma are urgently required.

Oxidative stress plays a central role in the pathophysiology of melanoma since the generation of melanin leads to the generation of hydrogen peroxide and consumption of reduced glutathione (GSH) [7,8]. Consequently, melanoma maintains high baseline

**Citation:** Fontes, S.S.; Nogueira, M.L.; Dias, R.B.; Rocha, C.A.G.; Soares, M.B.P.; Vannier-Santos, M.A.; Bezerra, D.P. Combination Therapy of Curcumin and Disulfiram Synergistically Inhibits the Growth of B16-F10 Melanoma Cells by Inducing Oxidative Stress. *Biomolecules* **2022**, *12*, 1600. https://doi.org/10.3390/ biom12111600

Academic Editor: Vladimir N. Uversky

Received: 27 September 2022 Accepted: 16 October 2022 Published: 31 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/).

ROS levels compared to normal cells, which makes melanoma cells more susceptible to oxidative stress [7,9].

Curcumin (CUR) is a polyphenolic phytochemical isolated from turmeric, a food spice made from the rhizome of *Curcuma longa* L. [10]. Turmeric is traditionally used in many South Asian countries, both in traditional medicine and cooking, and has been used for over 2000 years as a medicine in China and India [11]. Curiously, many studies have reported that CUR causes cell death by stimulating reactive oxygen species (ROS) production in fibroblasts [12], leukemia [13], lymphoma [14], melanoma [15], and colon cancer [16].

Disulfiram (DSS) is a thiocarbamate drug approved by the United States Food and Drug Administration (US-FDA) for the clinical treatment of alcoholism and has been used for over 60 years [17]. DSS is an irreversible inhibitor of aldehyde dehydrogenase (ALDH). Interestingly, ALDH can decrease intracellular oxidative stress due to its ROS scavenger action. Consequently, DSS can act by inhibiting the intracellular antioxidant system [18,19].

Importantly, both DSS and CUR not only have antitumor activities but can also potentiate the action of anticancer chemotherapy drugs by blocking drug efflux pumps [20–26]. Furthermore, these compounds have been reported as agents capable of reducing the adverse reactions of highly toxic chemotherapeutic agents [27,28].

Therefore, we hypothesized that CUR, an oxidative stress inducer [29], combined with DSS, which acts as an antagonist of the intracellular antioxidant system [22], will have a synergistic effect, providing greater sensitization of cancer cells and resulting in cell death. Herein, we aimed to study the antitumor potential of the combination of CUR with DSS in B16-F10 melanoma cells using in vitro and in vivo models.


**5. The occurrence of the glutathione in the nucleus; active transport, de novo synthesis, diffusion or something else**

How GSH enters the nucleus and how it is regulated during the different phases of the cell cycle is still a matter of debate. The regulation of such interactions is also unclear. According to Smith and colleagues (Smith et al., 1996), the possible biochemical mechanisms responsible for the turnover of nuclear GSH are the following: 1.GSH may be taken up from the cytoplasm into the nuclei either passively or through energy-dependent processes 2.GSH may be synthesized *de novo* in the nucleus by the enzymes glutamate cysteine ligase and GSH synthetase 3.GSH may function to transport -glu-cys-cys.


**3. Results and Discussion**

*3.1. Characterization of WEEE Samples*

Samples of the three varieties of WEEE were submitted to SEM/EDS to identify the chemical distribution in the PCB through a semiquantitative approach. The detection mode for this study is related to Backscattered Electrons. In this context, Figure 3 presents such results for a sample of the Type 1 variety. It can be observed that as expected the metallic area of the PCB features a bright shade of grey, associated with a higher average atomic number, while the dark area is associated with the polymeric material. Some clouds of dark structures could also be observed over the bright area, possibly associated with oxidation of the metallic parts, possibly associated with nickel and copper. It is noteworthy to mention that the image and the respective EDS results are mainly associated with the surface area of the sample. In the dark area, a major presence of carbon, oxygen, and bromine can be observed, while in the bright area, gold stands as the major constituent, at least at the sample surface. These are interesting and expected findings as brominated compounds are used in this context as flame retardants, while gold layers on the surface of the metallic contacts are responsible for increasing efficient current transmission.

**Figure 3.** SEM/EDS characterization of Type 1 WEEE sample: (**a**) microanalysis of the dark area; (**b**) microanalysis of the bright area.

However, the amounts of other valuable metals, such as copper (in the inner contact) and aluminum (in the fiberglass) could only be qualitatively assessed by utilizing a chemical mapping that analyses the composition distribution deeper in the sample. Such analysis is presented in Figure 4.

**Figure 4.** Chemical mapping of the most relevant elements detected in the microanalysis of the dark and bright areas of the Type 1 WEEE sample.

Figure 5 presents the SEM/EDS analysis while Figure 6 shows the qualitative chemical mapping of the Type 2 variety of WEEE in the received sample. It can be observed that Type 2 follows Type 1 in terms of the overall chemical composition of the bright (metallic) and darker (polymeric) areas, with a cleaner surface regarding the presence of clouds potentially associated with metal oxidation. Comparing the two varieties, at a semiquantitative level, the composition in both areas in each sample is relatable.

**Figure 5.** SEM/EDS characterization of Type 2 WEEE sample: (**a**) microanalysis of the dark area; (**b**) microanalysis of the bright area.

**Figure 6.** Chemical mapping of the most relevant elements detected in the microanalysis of the dark and bright areas of the Type 2 WEEE sample.

On the other hand, the Type 3 variety, with a paler metallic hue, does not follow the other two in terms of composition and surface integrity, as shown in Figure 7. It was verified that the samples of this variety present a similar overall composition for the dark (polymeric) area as the one observed in the previous cases, but with a larger number of elements in the lower range of relevance. For the bright (metallic) area, the distinction is clear, with nickel as the major metallic constituent and with gold still present at an important level. The dark clouds were most present in this variety, which could be related to lower levels of Au in the surface.

**Figure 7.** SEM/EDS characterization of Type 3 WEEE sample: (**a**) microanalysis of the dark area; (**b**) microanalysis of the bright area.

Following the same approach, Figure 8 displays the chemical mapping of the Type 3 variety. The major qualitative difference between this variety and the others is related to the lower presence of gold and the higher distribution of oxygen, which corroborates the behavior associated with the more distinguished dark clouds observed in Figure 7.

**Figure 8.** Chemical mapping of the most relevant elements detected in the microanalysis of the dark and bright areas of the Type 3 WEEE sample.

In parallel to the SEM/EDS characterization, samples of each of one of the three varieties of WEEE were also submitted to non-isothermal TGA in oxidizing and chemical inert conditions, as presented in Figure 9. In praxis, the same thermal behavior as a function of temperature regardless of PCB type and reaction atmosphere was verified. The total weight variation indicates a mass loss of 30%. Regarding the chemical environment, at N2 atmosphere, carbon and hydrogen were volatilized as organic compounds, possibly carrying flame-retardant components, whereas in the oxidative experiment these elements were possibly being transported to the gas phase as oxidized compounds such as water, monoxide, and carbon dioxide [65]. The observed degradation temperature is in accordance with the presence of thermoplastic materials in the sample, at about 250 ◦C [27].

**Figure 9.** Thermal behavior characterization of WEEE sample: (**a**) in an inert atmosphere; (**b**) in an oxidizing atmosphere.

Both materials' characterization results indicate similarities between the three varieties, particularly regarding the polymeric fraction, and therefore to establish the chemical process more simply, the thermal processing of the received sample was considered without the variety distinction, to remove at least a fraction of the organic phase and to liberate constituents. Consequently, the thermochemical processing of the WEEE samples was carried-out for the material in the same condition as it was received from the local recycling center, without any classification, in a tubular furnace above 300 ◦C.

#### *3.2. Thermal Processing of WEEE Samples*

Table 1 shows the experimental results associated with the pyrometallurgical processing of 2.5 g of PCB connector at 300 and 400 ◦C, using compressed air (incineration) and argon (inert processing). As expected, the weight loss was again around 30%. However, at the inert gas atmosphere, the formation of some droplets of a black liquid at the far end of the tubular furnace were observed. This was interesting, and an indicative of the volatilization and condensation of the organic fraction, as reported previously by other authors [27,53].

**Table 1.** Observed WEEE samples' mass loss after thermal processing in a tubular furnace as a function of the temperature and the atmospheric chemical composition.

To produce more liquid and to generate more solid material, another thermal degradation experiment at 350 ◦C was carried out in argon using 54.5 g of the WEEE sample. The reaction time of 60 min was also applied to this test. A weight loss of 30.1% was observed, resulting in a solid weight of 38.1 g. In this context, Figure 10 presents the macroscopic aspect variation before and after thermal degradation. It is noteworthy that some substance has been removed from PCBs, exposing the metallic compounds, some copper sheets as well as the inner glass fibers, now covered with some dark material, possibly carbon black.

**Figure 10.** Macroscopic aspects of the WEEE samples: (**a**) before thermal processing; (**b**) after thermal processing.

The disassembling and sizing unit operation was applied to the solid product, and, in this context, Table 2 presents the size classification of the obtained material after these operations.

**Table 2.** Size classification of the thermal processing solid product after grinding operation using glass pebbles as friction agents for materials disassembling.

It can be observed that most of the size classification is associated with large particulate material. A total of 80.71% is associated with glass fibers (17.4 g), copper sheets (1.3 g) and non-liberated material (12.0 g). The fact that 31.58% of the sample is associated with non-liberated material indicates that the disassembling unit operation could be the subject of future developments, to optimize larger recovery of metals. Moreover, the non-liberated material could also be submitted to hydrometallurgical processes as most of the organic phase has been removed from it, in a roast–leach type of route. Material below 2.80 mm was submitted to magnetic separation. It was observed that 4.7 g was susceptible to the

effects of the magnetic field and recovered easily. Therefore, it can be said that 12.4% of the solid product is composed of magnetic-metal-containing materials. Figure 11 illustrates the macroscopic aspect of the most relevant materials recovered.

**Figure 11.** Macroscopic aspects of some relevant materials recovered in the process: (**a**) fiberglass; (**b**) copper sheets; (**c**) magnetic contacts; (**d**) condensed liquid.


*4.2.1 Debridement*

Debridement is one of the cornerstones of wound management, which could be surgical or non-surgical. It involves removal of necrotic tissue to aid wound healing. Debridement of open foot ulcers is required if unhealthy tissue is present, as this will aid in decreasing pressure points at callused sites on the foot [140, 141]. Removal of unhealthy tissue is linked to reducing colonizing a "clean" wound and allowing for examination of deep-lying tissues in the ulcer. Wound dressing is the cornerstone in diabetic foot care. As a convention, dressing changes and wound inspection and dressing should be done daily or as determined by the physician [142]. An ideal dressing should contribute to a moist wound environment, absorb excessive exudates, and not increase the risk for infections. Luckily, some non-surgical debriding agents have been incorporated with biologics such as transforming growth factor-β, vascular endothelial growth factor and epidermal growth factor [142–144]. Compared to conventional wound dressings, this modern wound dressing technique hastens the process of wound healing, as it stimulates angiogenesis, fibroblast proliferation, collagen synthesis and deposition, epithelization and remodeling of new extracellular matrix.


**1. Introduction**

#### **1.1 Playing and human learning**

The fundamentals of how learning occurs and its relationship to playful activity can be found in some of the concepts developed by the Russian-born psychologist and epistemologist Lev Semyonovich Vygotsky. This material has been popular in the West since the 1960s and, despite the passage of time, is still largely considered to be relevant. Therefore, the following paragraphs will highlight some of these concepts and present them as a necessary introduction to further developments.

Vygotsky [1] introduced the concept of the zone of proximal development, which refers to the distance (in the abstract) between the skills and competencies that a learner should have attained as a natural result of growth, maturation, and academic achievement at a given point in his or her life and those that he or she should attain at an immediately later point in his or her development. A zone of proximal development thus defines what a learner can achieve independently and what he/she can achieve with the help of a more competent adult or peer.

In this way, this approach emphasizes the importance of interaction and collaboration between learners, both to facilitate their own learning processes and to enhance the quality and breadth of their learning. In addition, when applied to a playful environment, this concept can be used to help each learner reach their full potential in collaboration with peers and teachers.

In this sense, we arrive at the concept of collaborative learning [1]. This author accentuated the importance of social interaction and cooperation in learning. According to Vygotsky, students learn best when they work together, support each other, and engage in joint activities. In an inclusive environment, this means encouraging participation and collaboration between them so that they can learn from each other and build knowledge together, mediated by gamification.

In the same vein, the concept of scaffolding is also introduced, which refers to the temporary and adapted help provided to students to support their learning. It is important to distinguish between the assistance itself and the apparatus designed and arranged to provide such assistance to students, which is how the concept could best be described. Thus, the structure of a class, the pedagogical strategies, the teaching aids, the order of participation, and other similar elements constitute the scaffolding that supports the growth that learning entails.

A crucial clarification is very important at this point. On the one hand, teachers should provide support structures, strategies, and concrete examples to help students understand new concepts and perform more complex tasks. However, it is important to stress that the help referred to in the previous paragraphs does not imply the provision of a specific solution to a specific problem. Rather, it is a somewhat paradoxical situation: on the one hand, elements are provided in the form of clues that lead the subject to find solutions on his or her own, although such a solution would not be possible without this assistance.

On the other hand, there is a large body of literature that clearly shows that the use of games has many benefits for learning, providing both structure and context for learning and for applying what has been learned. The recreation of game environments in relation to Vygotskian concepts and theoretical assumptions can facilitate the development of inclusive gamification as a very useful tool in educational contexts.

To conclude the Vygotskyan proposal, another relevant clarification relates to the conceptualization of the use of tools and mediation. In this sense, Vygotsky highlighted the importance of tools and cultural resources in learning and development. These tools can be physical, such as technology and gamification, or symbolic, such as language and other representational systems. In an inclusive environment, a variety of adapted tools and resources can be used to meet learners' individual needs and help them to participate fully in educational activities.

On the other hand, as a result of the constant evolution of educational systems, one of the areas of learning theory that has become more relevant is the focus on cooperative learning. It is therefore considered appropriate to look at other similar approaches to the subject, one of which is Johnson and Johnson's [2] theory of promoting learning through collaboration.

This methodology is based on the premise that students can learn more effectively and meaningfully when they work together in collaborative groups rather than in competition with each other. The basic principles are that students learn best when they work together to achieve common goals and that this collaboration promotes not only the acquisition of knowledge and skills but also the development of social and emotional skills.

In a cooperative environment, students depend on each other to achieve group success. This fosters a sense of shared responsibility and encourages real cooperation.

In addition, direct communication and exchange of ideas between students is essential for effective learning. Discussion and dialog allow students to develop their understanding through articulation and questioning. Each member of the group

#### *Gamification as a Tool for Inclusion DOI: http://dx.doi.org/10.5772/intechopen.113229*

has a specific role and responsibility to contribute to achieving the group goal. This prevents some students from taking advantage of each other's work and encourages mutual accountability, giving students the opportunity to develop communication, leadership, decision-making, and conflict resolution skills.

These skills are essential both in the educational context and in everyday life. Cooperative learning theory therefore has a profound impact on the educational environment. By implementing this approach, teachers can encourage student participation, promote critical thinking and problem-solving, and create a sense of community in the classroom. Pupils learn to listen to and understand different perspectives, to work in teams, and to value the contributions of their peers. In addition, collaboration rather than competition reduces the anxiety and stress associated with individual assessment.

While the benefits of cooperative learning theory are many, its successful implementation requires careful planning and consideration of certain challenges. Creating balanced groups, managing conflict, and ensuring the active participation of all students are aspects that require attention. In addition, some students may prefer to work individually, which requires flexibility on the part of the teacher.

Another approach refers to George Herbert Mead's theory of symbolic interactionism and inclusion in the classroom [3]. This perspective is based on the idea that education is a fundamental pillar of society and that its nature is as diverse as the individuals that make it up. In this context, the theory of symbolic interactionism emerges as a valuable lens for understanding how social interactions and symbols shape people's reality. When applied to the educational setting, this theory provides a solid basis for promoting inclusion in the classroom and ensuring that every student has the opportunity to develop fully, regardless of their differences [4].

Symbolic interactionism focuses on how individuals interact through shared symbols and signs and how these interactions influence the construction of meaning and reality. Mead argues that people see themselves through the eyes of others, what he calls the "generalized other". This implies that one's sense of self and place in society is shaped by social interactions and the interpretation of cultural symbols.

When this theory is applied to the educational context, it is evident that classroom inclusion is vital to establishing an environment that facilitates the cultivation of an inclusive and respectful "generalized other". Inclusion extends beyond the mere existence of diverse students in the classroom. It encompasses creating an atmosphere that values and respects all perspectives, with differences serving as opportunities for learning and enrichment [5].

A vital aspect of symbolic interactionism in education is cultivating empathy and mutual comprehension among learners. By recognizing that their own viewpoints are shaped by the interpretations of others, students can cultivate a heightened awareness of the experiences and opinions of their classmates. Such an approach encourages an educational setting in which diversity is valued and embraced, rather than viewed as a source of division [6].

Furthermore, Mead's theory underscores the significance of language and communication when it comes to the formation of meaning. Teachers can adopt this viewpoint to establish an environment that facilitates candid exchanges and efficient communication among students. By urging students to convey their opinions and emotions, they are presented with the chance to exchange their distinctive outlooks and assimilate with one another [7].

Inclusion in the classroom requires adjusting teaching methods and learning resources to cater to the varying needs of students. By applying the theory of symbolic interactionism, educators can develop exercises that enable learners to communicate their identities and experiences using symbols and signs. This not only provides students with a sense of empowerment but also fosters a feeling of togetherness and affiliation in the classroom [8].

These principles clarify why gamification is a valuable tool for learning when used appropriately, as it promotes collaborative learning, mediation, and interaction with others. Therefore, once the epistemic support has been established, it is pertinent to address the question: What is gamification and how does it aid inclusion?

Starting from the definitions of gamification, one of the noticeable premises is that it is not inherently designed for inclusion of diverse learners. It does not consider their abilities, special requirements, gender, or ethnicity, among other particularities. This can be confirmed by examining some of the definitions, as shown below:

While it is true that gamification can facilitate educational processes by promoting learning, fostering motivation, developing cognitive skills, maintaining concentration, increasing engagement, and providing rewards and incentives [9, 12–14], its use in promoting inclusion is not a common goal.

However, the games that are most likely to enhance inclusivity are serious games, distinct from educational games since they aim to encourage societal transformation. These games are formulated considering persuasion theories or studies, which aspire to behavioral transformation, particularly related to social connectivity, employing deliberately applied theoretical principles.

According to Ullauri et al. [15], numerous studies exhibit deficient commitment by designers toward incorporating accessibility features, resulting in a low percentage of accessible serious games. However, given the significant utilization of games in educational contexts, it is imperative to ensure accessibility to maximize usage by individuals.

Now, what do serious games contribute? The intervention's structure is based on observing the desired behavior through a character or other players. This is based on the Elaboration Likelihood Model (ELM) and incorporates a theory of persuasive communication [16]. Joining a persuasive narrative immerses the participant in an experience that can lead to a change in their behavior or, at the very least, an intention to do so.

According to Wendorf [17], serious games function by leveraging entertainment characteristics (hedonic processing) with cognitive and emotional engagement for

#### *Gamification as a Tool for Inclusion DOI: http://dx.doi.org/10.5772/intechopen.113229*

specific outcomes, through focusing on persuasion, formation and change of attitudes, increased/enhanced awareness, and overt behavioral changes.

From this perspective, it can be affirmed that these games are primarily aimed at convincing players to adopt, reinforce, or modify a certain viewpoint regarding a specific issue or situation, leading to a shift in their interpretation methods [18]. The realm of serious games encompasses persuasive games, health games, military training simulations, impactful social games, and educational games, with their deployment having experienced significant growth over the last few decades.

Serious games are purposive tools, whose impact emerges from their design and mechanics. They offer immersive experiences that support a deeper understanding of the problem situations or contexts they are intended to address. Consequently, they provide people with an embodied experience of complex social problems, enabling them to develop a stronger understanding [17].

Moreover, research has shown the effectiveness of games for prosocial change in diverse contexts [19] and areas, including cognitive, social, and behavioral aspects [20, 21]. For instance, certain games have been successful in targeting specific goals, such as reducing stigmatized beliefs and prejudices. In this manner, games can act as convincing tools (activators of cognitive processes) that aim to mold and alter attitudes and behaviors [22], not solely *via* their messages and content, but additionally through their mechanics and playful nature [23].

Kahne et al. [24] explored the use of video games to illustrate civic practices as a complement to formal education. The study found no clear correlation between video games and civic engagement. Nonetheless, young people who have experience with such games are more inclined to participate in social processes than those who do not. In this regard, research has shown that games that follow theoretical frameworks are more efficacious as intervention tools [19, 25, 26].

However, it is vital to comprehend that numerous factors play a role in developing and utilizing a serious game, including technological advancements that enhance interactivity, the efficacy of narratives and game structure, connectivity, and immediacy of feedback, among other characteristics [25, 27].

In terms of purpose, "serious games" are used for educational aims in diverse subject areas and contexts, such as in military schools [19, 28]. Although serious games have been applied to various contexts, there appears to be a specific interest in utilizing games as tools to promote health, coexistence, conflict management, and psychological, behavioral, or treatment changes at an individual level, among other subjects [29].

Regarding design, serious games are created to intentionally provide immersive experiences. As noted earlier, this facilitates a more thorough comprehension of situations and contexts, allowing individuals to obtain a stronger understanding. This is due to the fact that serious games offer an immersive experience that allows for both transportation and identification [17].

Researchers Romero et al. [30] suggest that gamification can act as a catalyst for social change. This is achieved by providing an interactive and transversal character and by fostering desired behaviors that promote moral development and active engagement in the community.

Additionally, serious games have been employed as strategies to promote psychological safety [31] by enabling participants to express conflicting views. The game's narrative structure and rules create a fictional world where disagreements are encouraged, limiting confrontational behavior. The aim is to facilitate "practice" scenarios where participants can acknowledge alternative viewpoints and diminish the potential negative impact in real-life situations, thus maintaining engagement [26, 32, 33].