**3. Changes associated with normal aging, which might influence swallowing**

A holistic approach is required while studying swallowing in the elderly. Swallowing cannot be regarded as an isolated action; one has to take into account the age-related functional decline occurring in several body-functions and its repercussions on swallowing (figure 1).

First of all there is an age-related decrease of sense of smell and taste, which are important for the pleasure we enjoy while eating [35-37].

Also, some anatomical differences arise in the older person such as a smaller cross-sectional area of masticatory muscles (masseter and medial pterygoid) and an increased lingual atrophy [38]. Next to the anatomical changes also, functional alterations occur in the muscle activity of the masseter, orbicularis oris, the supra- and infra-hyoidal muscles [39], and the thyroaryte‐ noid muscle [39].

The respiratory system undergoes some changes as well; there is a decreased cough reflex, a diminished ciliary clearing, and a weakening of the respiratory muscles. These changes in combination with a deterioration of the immune system make the elderly more prone to developing an aspiration pneumonia.

As far as the digestive system is concerned, a delayed gastric emptying may lead to an earlier feeling of fullness at mealtime. Recent studies have shown a decreased sensibility and an increased stiffness of the esophagus in old age [40].

Another important issue is fatigue [41]. Fatigue, being a very common complaint in the elderly, is often associated with functional decline and may, as well as sleeping disturbances and depression, lead to a reduced food intake [42-44]. The elderly also often experience a declined perception of thirst and subsequently they have a low fluid intake. Tongue strength and endurance decline during a meal and this in combination with a diminished reserve may negatively influence deglutition especially in already weakened elderly [45].

As people get older, the slower swallowing act may actually also be a benefit as it can allow greater time to recruit the necessary number of muscle fibers to generate the necessary pressures for adequate bolus propulsion through the oropharynx. Hence, speeding up an elderly patient's swallow may induce contradictory results, as it may lead to insufficient swallow pressures and therefore may be contraindicated as a therapy technique.

Cognitive changes are also considered to be part of the normal aging process and cognitive processes such as concentration, attention, and double-tasking are influenced by age. A decline in concentration and attention together with a reduced reserve may lead to aspiration. Moreover as eating is a social event, people tend to talk during mealtime further increasing the risk of penetration and aspiration.

Staying physically active is associated with healthy aging, therefore elderly who are bedridden are additionally exposed to a number of important risk factors due to the sedative life style such as a diminished lung capacity and a weaker cough, a greater risk to develop a pneumonia, muscle weakness, and a loss of appetite [46].

Finally, medication may also negatively influence deglutition [47]. Drugs with an anticholi‐ nergic effect may cause xerostomia while some may lead to a diminished (e.g., allopurinol, carbamazepine, and penicillamine) or an altered (e.g., captopril lithium) taste perception. Sedatives can reduce the level of alertness and neuroleptics may mimic the swallowing problems encountered in Parkinson's disease. Nitrates are relatively contraindicated in gastroesophageal, reflux disease as they lower the pressure in the lower gastro-esophageal sphincter and steroids can not only induce a Candida infection orally but they can also provoke a steroid myopathy. Moreover, 40% of already weakened elderly take at least one medication that is completely superfluous [48].

**Figure 1.** The relationship between presbyphagia and dysphagia


**2. Epidemiology of burns**

According to the statistics of the World Health Organization, 180,000 deaths every year are caused by burns [2]. Majority of them occur in low- and middle-income countries. It is estimated that almost two thirds occur in the African and South-East Asia regions. The highest fire-related mortality rates are in Southeast Asia, 11.6 deaths per 100,000 population per year; in the Eastern Mediterranean, 6.4 deaths per 100,000 population per year; and in Africa, 6.1 deaths per 100,000 population per year [3].

Among pediatric population the rate of deaths from burns is over seven times higher in lowand middle-income countries than in high-income countries. Lowest mortality rates due to burns in high-income countries are the successful result of preventive interventions of many kinds, such as promotion of the use of smoke detectors, the lowering of temperatures of hot water heaters, the installation of sprinkler systems, the promotion of flame-retardant children's sleepwear, and the development of safer buildings and household fuels [3].

The epidemiology of burns differs in different age groups and depends on sex [3]. For example, in low- and middle-income countries, fire-related burns are the sixth leading cause of death among 5–14-year-old victims and the eighth leading cause death among 15–29-year-old victims [3].

In low- and middle-income countries, women (especially young) are at higher risk of burns [3]. However in high-income countries, men are at higher risk of burns.

In pediatric population, it is estimated that worldwide approximately 1% of all children sustain a burn injury each year [4].

The results of our studies conducted in Lower Silesia (the region of Poland) among children with burns indicate that boys are at higher risk of burn injury [5, 6].

The analysis of mechanism of trauma revealed that the most common type of injury in children treated in ambulatory conditions by general practitioners (GP) and requiring hospitalization due to burns in Lower Silesia was thermal burns [5, 6]. The second reason for hospitalization and ambulatory treatment were chemical burns [5]. In the studied population, there were no cases of burns caused by radiation [5, 6].

Among the pediatric patients hospitalized due to thermal injury in Lower Silesia, 2010–2012, burns were usually located in the upper limbs [5]. Trunk and lower limbs were also frequently affected [5].

The detailed analysis of the thermal burns of upper limbs revealed that the hand and wrist were more often affected than forearm, arm, and shoulder. However, the detailed analysis of the thermal burns of lower limbs revealed that the injury affected more often the hip, tight, knee, and lower leg than ankle and feet [5].

Most of patients under 18 years old treated by GP due to burns in Lower Silesia of children were under 2 years old [6]. Moreover, in the studied population, the percentage of children hospitalized due to burns in Lower Silesia in 2010 according to individual age groups in age group under 2 years old was 0.5%, in children from 3 to 6 years old was 0.12%, in children from 7 to 12 years old was 0.06%, in children from 13 to 15 years old was 0.07%, and in children from 16 to 18 years old was 0.06% [5]. Similarly in the studied population, percentage of children hospitalized due to burns in Lower Silesia in 2011 according to individual age groups in age group under 2 years old was 0.54%, in children from 3 to 6 years old was 0.13%, in children from 7 to 12 years old was 0.06%, in children from 13 to 15 years old was 0.07%, and in children from 16 to 18 years old was 0.08% [5].

The chemical injuries were less common in pediatric population of Lower Silesia in the analyzed period of time. However, it was noticed that this type of burns more frequently affects the upper gastrointestinal tract than thermal burns [5, 6].

The obtained results are coherent with other studies realized in Polish pediatric population [7].


**1. Introduction**

Although children with acquired brain injury (pediatric acquired brain injury—pABI) may suffer long-lasting physical, cognitive, behavioral and social symptoms as well as disturbances in psychological adaptation [1–3], few randomized controlled trials have

**Citation:** Holthe, I.L.;

Rohrer-Baumgartner, N.; Svendsen, E.J.; Hauger, S.L.; Forslund, M.V.; Borgen, I.M.H.; Øra, H.P.; Kleffelgård, I.; Strand-Saugnes, A.P.; Egeland, J.; et al. Feasibility and Acceptability of a Complex Telerehabilitation Intervention for Pediatric Acquired Brain Injury: The Child in Context Intervention (CICI). *J. Clin. Med.* **2022**, *11*, 2564. https://doi.org/10.3390/ jcm11092564

Academic Editor: Massimiliano Valeriani

Received: 29 March 2022 Accepted: 30 April 2022 Published: 3 May 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/).

explored the effectiveness of complex interventions targeting these children, their families and their everyday context at home and in school. This may be due to the considerable complexity of designing and conducting such studies where one must take a range of factors into account. These include the child's age and developmental level, the heterogeneity of symptoms that may follow a pABI, family factors that may influence the ability and willingness to participate in an intervention, and the heterogeneity in factors related to the child's everyday life in the community, such as school and other areas of participation. The possible pitfalls are many. Exploring the feasibility of a complex intervention is therefore important prior to large-scale RCTs. This paper describes the investigation of the feasibility of a complex, individualized and community-based intervention targeting children with pABI and their families.

Brain damage acquired after birth can be caused by traumatic (TBI) or non-traumatic injuries such as stroke (i.e., brain hemorrhage or infarction), tumor, inflammation, infection or hypoxia. pABI may cause a wide range of disruptions in the child's developmental course [1–3]. Deficits may be persistent, leading to reduced participation in school and social life [4–6], and reduced quality of life [2,7,8]. Prior studies have identified needs for long-term follow-up after ABI; psychological support for parents, siblings and the affected child; as well as support in the return to school [9]. Interestingly, reported needs may change over time, from primarily physical concerns in the first months post injury (79%), to cognitive (47%) and socioemotional needs (68%) later on [10]. Importantly, rehabilitation needs after pABI are described as largely unmet [11].

As family needs after a pediatric brain injury are complex and heterogeneous, rehabilitation services and follow-up care should be individualized to each patient's needs. In line with this, rehabilitation should be multidisciplinary and take place within a biopsychosocial framework [12]. In addition, rehabilitation needs to be flexible, monitoring the development and goals of the patient, which may change over time [12]. A recent study mapped treatment goals set by young people with ABI to the domains in the International Classification of Functioning, Disability and Health (ICF) [13], in which 52% were related to activities and participation, and 20% to environmental factors [14]. This highlights the need to include community-based rehabilitation that is individualized and contextsensitive [14]. The complexity of pediatric ABI rehabilitation may explain the small body of evidence-based recommendations. However, existing knowledge provides evidence for family/caregiver-focused interventions and the use of technology in rehabilitation [15]. In addition, direct interventions related to cognitive functions such as attention, memory, executive function and emotional/behavioral functioning have given positive results [15]. However, a critique against the direct training approach in cognitive rehabilitation (i.e., interventions directed toward retraining the child's abilities) is that generalization to everyday functioning is often unknown or disputed [16]. Therefore, indirect approaches such as behavioral compensation and modification, environmental modification and supports, educational supports and instructional strategies are recommended, including education for parents and caregiver involvement in handling executive dysfunction [17,18]. The effects of parenting/family interventions after pABI have been explored in randomized controlled trials (RCTs) by Wade and colleagues with promising results for online problem-solving training, improving behavior problems, executive functioning, and family functioning [15,19–22]. The use of technology in rehabilitation is relatively new, but telerehabilitation has shown to be a reliable alternative to in-person meetings [23,24] with promising results in cognitive rehabilitation [25], speech and language therapy [26] and family therapy [27,28]. Recent reviews on technology-based or -assisted rehabilitation programs for children with ABI have found that in addition to the mentioned evidence for online problem-solving, there is promising evidence for training of cognitive, social and behavioral skills [15,22,29,30]. The findings are characterized by heterogeneity and small samples, but the involvement of a clinician in addition to the technology-based intervention may be of importance for a positive result [30]. Although most of this research has been conducted in the USA, some of the technology-based intervention studies presented in the

literature reviews have also been performed in Europe. In addition, goal-oriented rehabilitation is considered a key ingredient in pABI rehabilitation [14,31], and goal attainment scaling can be a sensitive and meaningful way to measure rehabilitation outcomes [32]. Children's self-identified goals have been found to be as achievable as their parents' goals in a sample of children with a mean age of 9 years [33], pointing toward the importance of including the children in defining rehabilitation goals.

To our knowledge, no single intervention has included all these important perspectives simultaneously in a pABI-population. However, an intervention targeting individualized, everyday rehabilitation needs in a home setting has been carried out for the adult TBIpopulation in the US [34] and is currently ongoing in Norway [35]. This intervention defined individualized rehabilitation goals in collaboration with the patient and family member when available and worked on strategies to reach these goals over a period of four months. To meet the demands of holistic, person-centered and goal-oriented interventions for the pABI-population, this adult intervention was adapted to the pediatric population. In the Child in Context Intervention (CICI), the family and the child's school are included, and collaboration is established with the Norwegian Service for Special Needs Education ("Statped"). The intervention has largely been adapted to a telerehabilitation format to facilitate access to rehabilitation services, and to ease participation for families and schools.

The Medical Research Council [36] has pointed out the need to carry out feasibility studies as part of the development of complex interventions. This is especially important as the complexity of such studies is influenced not only by the intervention components, but also by contextual factors, in addition to the interactional processes occurring between the intervention components and the context [37]. In pediatric rehabilitation, one also needs to consider a range of transactional processes: between child and parent; between the child, family and school environment; and in the community and with peers, as children rely on their environment even more than adults [38]. In the CICI study, the complex interactions that may take place between children, parents, schools and the intervention components is hard to foresee before the intervention has been tried out. Some key uncertainties include whether the complex logistics of the study are feasible, whether the families are able to maintain participation in an intensive intervention (seven sessions over 4–5 months), whether recruitment is feasible, whether it will be possible to maintain a sustainable working alliance with both children and parents through the telerehabilitation format and whether the goal-oriented approach is feasible when working with an entire family that may not have the same priorities or needs. In addition, as there have been no previous studies carried out including schools in a similar rehabilitation program, it is also of importance to assess the feasibility of this intervention component. Conducting a feasibility study prior to the future definitive RCT is therefore principal in order to evaluate the intervention manual, identify possible obstacles and be able to adjust the protocol before the RCT.

#### *Objectives*

The objectives of the CICI feasibility study were to assess the feasibility of (1) the recruitment procedures and (2) the contents and structure of the intervention, including the feasibility of treatment delivery through a videoconference solution. We also wished to evaluate the acceptability of (3) the intervention for children, parents and therapists, (4) the baseline (T1) and outcome (T2) assessment methods and finally (5) the quality of treatment delivery. In order to inform the final decisions on outcome measures in the RCT, considering preliminary indications regarding the usefulness of outcome measures was also addressed.

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

This article adheres to the CONSORT extension for the Pilot and Feasibility Trials Checklist [39].

#### *2.1. Trial Design*

This feasibility trial applied a one-group pre−post design, with a baseline (T1) and a follow-up assessment immediately after the intervention period of 4-5 months (T2). The future definitive RCT will include a two-group RCT-design. For the RCT, outcome assessments will also be performed about 9 months after baseline (T3). The T3-assessment was not included in the feasibility trial to minimize time expenditure as T3 largely matches T2.

#### *2.2. Participants and Recruitment Procedures*

The inclusion criteria were (1) school-aged children (6–16 years at inclusion) with a radiologically verified diagnosis of ABI, or loss of consciousness post-insult and verified neurological symptoms in cases where radiology could not be administered; (2) time since insult at least 1 year; (3) self- or parent-reported persistent ABI-related cognitive, emotional or behavioral challenges influencing participation in everyday life related to family, friends, school or local community, assessed through a telephone interview; (4) children attending school regularly; and (5) the family is able to participate actively in a goal-oriented study for the next 4–5 months.

Exclusion criteria were (1) severe pre- or comorbid neurological or neuropsychological disorders that would confound assessment and/or outcome measurements; (2) children with brain tumors in active treatment or at great risk of relapse; (3) children with severe psychiatric illness or with injuries so severe that they were currently in institutionalized care; (4) parental severe psychiatric illness, drug abuse or indications of a history of or risk of domestic violence; and (5) not fluent in Norwegian language, although exceptions could be made for English-speaking parents who understand and read Norwegian.

Participants were identified from the medical charts at Sunnaas Rehabilitation Hospital. The families were invited to participate through written age-appropriate information for the children and information to parents. Parents and teenagers from 16 years of age provided written informed consent. A scripted telephone interview was used to screen for inclusion and exclusion criteria and willingness to participate. Eligible and consenting families performed a baseline screening (T1) at Sunnaas Rehabilitation Hospital.

After inclusion, the children's teachers and principals at their schools received written information about the study and consent form for the teachers. They were thereafter contacted by telephone by the CICI Statped collaborator.

#### *2.3. Assessments*

The outcome measures to be evaluated in the feasibility study are listed in Table 1. Neuropsychological assessment was included at baseline to provide descriptive data regarding the children with ABI and to inform the goal-setting process. Primary outcomes were post-concussive symptom burden (HBI), parenting self-efficacy (TOPSE) and quality of life (PedsQL). Questionnaires were completed at home and returned by mail.

As part of the baseline assessments, the families were asked to name the current three most challenging areas related to the child's brain injury.  Parents agreed on three areas but scaled them separately. The child was similarly asked to name and rate his or her own three most troublesome pABI-related problem areas.

#### *2.4. Intervention*

A detailed intervention manual was developed. It was based on the manual developed for an adult population by Borgen and colleagues [35] and adapted to the pediatric and family context by experienced rehabilitation therapists (authors M.L., S.L.H., I.M.H.B., M.V.F., N. R.-B., I.L.H.). In addition to descriptions of the content of each session, the manual contained general information on the study's rationale and aims as well as detailed guidance on how to establish SMART-goals and strategies, and references to evidence-based strategies and "tools" to handle different challenges. The manual also included detailed guidelines on how to manage therapy and communication through videoconference.

**Table 1.** Measures included in the feasibility study.

The intervention included seven family sessions, four school-meetings interspersed with the family sessions (starting after the first family-session) and a one-day parent group seminar (occurring about halfway through the family sessions for all participants) over the treatment period of four to five months. Figure 1 gives an overview of the intervention components. The family sessions and the school sessions were delivered through an encrypted videoconferencing solution. The feasibility study was performed from August to December 2020. See Adaptations due to COVID-19 below.

**Figure 1.** Overview of the intervention.

Three therapists, two clinical neuropsychologists and one experienced pediatric nurse (authors N.R.-B., I.L.H. and E.J.S., respectively) with extensive rehabilitation experience delivered the intervention. Therapists received training from the therapists of the adult community-based TBI-study [35,53] concerning the goal-oriented approach and strategies related to different commonly reported ABI-related problem areas. This was discussed in repeated meetings throughout the feasibility study. Therapists delivered nearly all the family sessions in pairs to ensure adherence to the study manual and a uniform delivery across therapists. The school sessions were performed by an experienced special education counselor from Statped (author A.P.S.-S.) in close collaboration with the three therapists. The therapists conducted the parent group seminar. Weekly meetings between the therapists, the Statped counselor and the study Principal Investigator (author M.L.) were held to discuss ongoing therapies and to ensure protocol adherence. The therapists also received training in the technical solutions of telerehabilitation as well as education on important therapeutic aspects of using this format, by a researcher with relevant experience (author H.P.Ø.), and the telerehabilitation team at Sunnaas Rehabilitation Hospital.


**Meet the editor**

Prof. Dr. Jolita Vveinhardt is a PhD of Social Sciences in the field of Management Science. She works at two universities in Lithuania in the position of a professor and senior scientist. In the scientist role, she heads three scientific groups: "Neuro-Relationships" (Lithuanian Sports University), "Managerial Solutions to Violence in Sport" (Lithuanian Sports University), "The Group of

Interdisciplinary Research on Working Environment" (Vytautas Magnus University). She successfully supervised and implemented three scientific research projects. Currently she is the head of an international scientific research project. Prof. Dr. Jolita Vveinhardt is the author and co-author of three monographs, two science studies, one textbook, and five educational books. She has published more than 200 scientific articles, 80 of which were published in peer-reviewed journals of Web of Science Core Collection (Clarivate Analytics) database and read more than 50 papers in national and international scientific conferences. She is a member of editorial boards of 14 scientific periodicals. Prof. Dr. Jolita Vveinhardt is a member of the International Baltic Management Development Association, Lithuanian Educational Research Association, Lithuanian Association for Quality Management and Innovations, Lithuanian Trauma Psychology Association, European Society for Traumatic Stress Studies, Lithuanian Neuroscience Association, Lithuanian Ergonomics Association, Lithuanian Project Management Association and Federation of European Neuroscience Societies. She teaches the following subjects for Master's degree students: Contemporary Organization Theories and Novelties of Management Science. Prof. Dr. Jolita Vveinhardt supervised 4 successfully defended doctoral dissertations, she is currently supervising three doctoral students. Her main research interests are destructive relationships among employees (mobbing, bullying, nepotism, favoritism, social loafing, social ostracism, organizational cynicism, cronyism, protectionism), business ethics, organizational culture, management culture, organizational climate, personal and organizational values, value congruence, corporate social responsibility, decision-making, neuromanagement, etc.

Contents

**Preface VII**

Jolita Vveinhardt

**Analysis 35**

**Industry 61**

Ivana Šandrk Nukić

Andrey Morozenko

**Organization 123**

Riaz Ahmed

Saniye Çelik

Chapter 2 **Model of Culture for Innovation 13**

Chapter 1 **Introductory Chapter: Organizational Culture - How Much Underused Potential Does Science Have? 1**

Chapter 3 **The Role of National Cultures in Shaping the Corporate**

Mohammad Ayub Khan and Laurie Smith Law

Luong Hai Nguyen and Tsunemi Watanabe

Chapter 6 **Reflex-Adaptive Organizational Structure in the**

Chapter 7 **Leadership Competencies Affecting Projects in**

Chapter 4 **Project Organizational Culture Framework in Construction**

Chapter 5 **Organizational Culture as a Determinant of Construction**

**Implementation of Large-Scale Projects 107**

Chapter 8 **Transformational Leadership and Organizational Culture: Keys to Binding Employees to the Dutch Public Sector 145**

**Companies' Competitiveness: Case Study of Croatia 85**

Julia C. Naranjo-Valencia and Gregorio Calderon-Hernández

**Management Cultures: A Three-Country Theoretical**


**Energy Transfer in Alternative Vehicles**

Editor

**Wojciech Cieslik**

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*Editor* Wojciech Cieslik Division of Alternative Powertrains Poznan University of Technology Poznan Poland

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This is a reprint of articles from the Special Issue published online in the open access journal *Energies* (ISSN 1996-1073) (available at: www.mdpi.com/journal/energies/special issues/Energy Transfer Alternative Vehicles).

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*Anthocyanins: Dietary Sources, Bioavailability, Human Metabolic Pathways, and Potential… DOI: http://dx.doi.org/10.5772/intechopen.99927*

example, anthocyanins in vegetables and cereals are chemically more complex in comparison with fruits, but berries are the major source of these compounds. Anthocyanin bioavailability has been reported to be very low, with recovery of less than 1% of the ingested anthocyanin dose. However, nowadays much greater bioavailability values have reported taking into account not only the phase I and phase II metabolites but also the microbiota catabolites. One of the peculiarities of anthocyanin metabolism is their capacity of interconversion between them. For example, dehydroxylation reaction can arise pelargonidin from cyanidin and methylation reactions can convert delphinidin into petunidin and malvidin. For this reason, metabolism data after anthocyanin ingestion is more straight forward to interpret. Regarding metabolism, cyanidin is the most studied anthocyanin due to ubiquitous character in the nature. However, more studies are necessary to better understand the similarities and differences with the other less studied anthocyanins. Even though several papers have reported the potential anti-neuroinflammatory effect of rich anthocyanin extracts, anthocyanins or their metabolites, the number of papers are very scarce. The most important limitation to study the activity of anthocyanin metabolites is the lack of commercial phase II and microbiota catabolites compounds. Thus, the chemical synthesis is the most employed technique to obtain standards although more developments are requires in order to obtain greater quantities. Moreover, little is known about the molecular mechanisms implicated in the observed effects. Furthermore, the majority of works are based on the study of the microglia M1 phenotype, so more studies are necessary to know if anthocyanins and their metabolites are able to induce an anti-inflammatory phenotype. To sum up, more research is necessary to stablish if anthocyanins and their metabolites are efficacious in slowing the progression of brain aging or of neurodegenerative diseases with an inflammatory component.


**4. Two "special topics" in benthic BEF studies**

As mentioned in the former sections, most BEF studies assess ecosystem functioning in terms of biomass production, effects on community composition or element cycling. However, there are other possible effects with important implications for the whole ecosystem. We choose two examples to illustrate this: trophic cascades and invasion success. We chose these two special topics because the former shows that slightly different levels of intraguild diversity can lead to completely opposed effects, whereas the latter illustrates that although experimental approaches meet hypotheses based on theory, they sometimes do not reflect what is actually happening in nature.

## **4.1 Trophic cascades**

Trophic species-level cascades occur when a change in predator abundance induces changes in the biomass of primary producers, due to a control of the abundance of grazers, thereby releasing lower trophic levels from grazing pressure (Polis et al., 2000). Predator diversity may reduce trophic cascades and may therefore be important for the population development of lower trophic levels and primary production. The importance of such intraguild diversity was studied by manipulating predator species richness (1, 2 or 3 species). The predator species pool consisted of several invertebrates such as hunting and web-building spiders preying on an arthropod (planthopper) assemblage inhabiting *Spartina* cordgrass (Finke & Denno, 2005). The occurrence of trophic cascades was

Integrating Different Organizational Levels in Benthic

availability may be the underlying mechanism.

should be interpreted with caution.

**approach**

Biodiversity – Ecosystem Functioning (BEF) Studies 101

processes. To investigate the relationship between native species diversity and invasion success of exotic species, experiments were conducted on marine sessile epibenthic invertebrates of the southern rocky coasts of New England, exposed to the invasion of the colonial ascidian *Botrylloides diegensis*, native of the Pacific Ocean (Stachowicz et al., 1999, 2002). Experimental communities were composed of zero to four native species. Species richness was manipulated by transplanting juveniles or by allowing larvae to settle on 2x2 cm tiles that were (after the successful colonization) combined in a 5x5 grid (i.e., a complete plate consisted of 25 interchangeable subunits). During the course of the experiment all but the five target species were removed regularly. As a result, survival of the invasive species decreased with higher species richness, and it was hypothesized that reduced resource

Contrary to the previous example, two observational studies on natural assemblages evidenced the opposite to what had been demonstrated in controlled experiments before. A long-term (11 months) survey of sessile invertebrates on a rocky jetty on the Tasmanian coast revealed a positive relationship between colonization rates of both native and introduced species and natural species richness (Dunstan & Johnson, 2004). Similarly, in a natural riparian assemblage, the most diverse assemblages were the most invaded by exotic plants; the same patterns were evidenced by an *in situ* experiment manipulating local diversity (Levine, 2000). A possible explanation therefore could be the different niche opportunities across varying environmental gradients (Shea & Chesson, 2002). Therefore at a larger observational scale, invasion success will be higher, given that more suitable niches are available, be it for native or exotic species, whereas invasion success would negatively relate to species richness at smaller, i.e. local scales (Shea & Chesson, 2002). On a more individual basis, the specific characteristics and functions of the resident species as well as the interactions among them and with the invasive species, rather than species richness *per se* may be the determinant for the invasion success (Dunstan & Johnson, 2004). These examples show that isolated experiments can sometimes be misleading in their outcomes. Therefore a combination of field observations, and field as well as laboratory experiments would certainly be the best approach to avoid such inherent biases. As this extensive approach is often not feasible, isolated laboratory experiments

**5. Integrating species and genetic diversity in one experiment: an alternative**

The preceding sections provided an overview of benthic experimental studies that alter either species or genotypic diversity separately. It has been argued that genetic diversity is only important in ecosystems that rely on or are dominated by one or a few key or habitat providing species, such as *Zostera marina* ecosystems (Hughes et al., 2008). However, all living organisms are hierarchically organized: genes make up genotypes, genotypes define populations and populations collectively constitute a species (Reusch & Hughes, 2006). If there is variation and heritability, as well as selection in ecologically important traits such as, e.g., growth rate or resistance to parasites, diversity at any level can have important ecological effects (Hughes et al., 2008). Moreover, it has been shown in terrestrial plant communities that genetic diversity can have direct consequences on species diversity: in a long-term experiment, genetically diverse communities reduced the rate at which species diversity declined (Booth & Grime, 2003), and genetic and species diversity maintained each

dependent on predator species richness, however the magnitude of the effect depended on whether the predators were a mixture of strict predators only, or of strict and intraguild predators combined. Higher intraguild predator richness led to antagonistic interactions among predators and dampened trophic cascades. As a consequence, it diminished herbivore suppression and therefore reduced primary productivity, indicating that predator diversity indirectly affects primary biomass production via trophic topdown control (Finke & Denno, 2005).

A similar outcome resulted from an experiment containing five species of predators, namely four carnivores (crabs, shrimp, blennies and killifish) and one omnivore (pinfish), preying on a herbivore assemblage dominated by amphipods and isopods, which in turn grazed on five species of macroalgae (Bruno & O'Connor, 2005). In an outdoor experiment, one, three or five predators were randomly assigned to mesocosms containing algae and herbivores. Predator diversity and identity had strong effects on the strength of the trophic cascade: when a generalist carnivore was present, grazers were significantly limited and algal biomass increased, whereas algal biomass decreased with increasing predator diversity, especially when the omnivorous fish was included.

Analogous to the previous examples, field observations in kelp forest ecosystems have revealed that predator diversity is negatively correlated with herbivore abundance and positively correlated with kelp density (Byrnes et al., 2006). To confirm the causality of this observation, predator richness was manipulated experimentally in kelp mesocosms. Accordingly, decreasing predator richness stimulated herbivore grazing leading to a decrease in giant kelp biomass. The underlying mechanism was not antagonistic behaviour among multiple predator species as observed in Finke & Denno (2005); instead, the herbivores changed their behaviour towards the different predators and therefore spent less time grazing.

Duffy et al. (2005) tested the effect of food chain length and trophic cascades on biomass of primary producers and consumers. In outdoor mesocosm tanks, experimental eelgrass communities were assembled in treatments of two (plants, grazers) and three (plants, grazers and predator) trophic levels. Grazer diversity was altered in the different treatments, and one omnivore predator (blue crab) was in- or excluded in order to modify the length of the trophic chain (Duffy et al., 2005). Results showed that the crabs had a strong effect on the grazers, resulting in higher algal biomass.

The results of these studies show that slight changes in predator diversity (e.g., one *versus* two species) can cascade to lower trophic levels. However, the exact outcome depends on the intraguild interactions at the predator's trophic level on one hand, and on the predatorprey response on the other. This implies that the loss of certain species at the top of the food chain can have unprecedented effects inducing fundamental changes to the whole system. In the case of seagrass and macroalgal systems, the loss of predators may induce the loss of key habitat-providing species with harmful consequences for the whole community (e.g. Estes et al., 1998).

#### **4.2 Invasion success**

Marine biological invasions are a major issue at regional and global scales. One of the major causes is the large number of transport vectors, like cargo vessels, but it has also been hypothesized that a depauperate native flora and fauna may facilitate the invasion success of exotic species (Elton, 1958). Non-native species can be a threat to regional biota due to competitive advantage, with uncontrollable consequences for the functioning of ecological

dependent on predator species richness, however the magnitude of the effect depended on whether the predators were a mixture of strict predators only, or of strict and intraguild predators combined. Higher intraguild predator richness led to antagonistic interactions among predators and dampened trophic cascades. As a consequence, it diminished herbivore suppression and therefore reduced primary productivity, indicating that predator diversity indirectly affects primary biomass production via trophic top-

A similar outcome resulted from an experiment containing five species of predators, namely four carnivores (crabs, shrimp, blennies and killifish) and one omnivore (pinfish), preying on a herbivore assemblage dominated by amphipods and isopods, which in turn grazed on five species of macroalgae (Bruno & O'Connor, 2005). In an outdoor experiment, one, three or five predators were randomly assigned to mesocosms containing algae and herbivores. Predator diversity and identity had strong effects on the strength of the trophic cascade: when a generalist carnivore was present, grazers were significantly limited and algal biomass increased, whereas algal biomass decreased with increasing predator diversity,

Analogous to the previous examples, field observations in kelp forest ecosystems have revealed that predator diversity is negatively correlated with herbivore abundance and positively correlated with kelp density (Byrnes et al., 2006). To confirm the causality of this observation, predator richness was manipulated experimentally in kelp mesocosms. Accordingly, decreasing predator richness stimulated herbivore grazing leading to a decrease in giant kelp biomass. The underlying mechanism was not antagonistic behaviour among multiple predator species as observed in Finke & Denno (2005); instead, the herbivores changed their behaviour towards the different predators and therefore

Duffy et al. (2005) tested the effect of food chain length and trophic cascades on biomass of primary producers and consumers. In outdoor mesocosm tanks, experimental eelgrass communities were assembled in treatments of two (plants, grazers) and three (plants, grazers and predator) trophic levels. Grazer diversity was altered in the different treatments, and one omnivore predator (blue crab) was in- or excluded in order to modify the length of the trophic chain (Duffy et al., 2005). Results showed that the crabs had a

The results of these studies show that slight changes in predator diversity (e.g., one *versus* two species) can cascade to lower trophic levels. However, the exact outcome depends on the intraguild interactions at the predator's trophic level on one hand, and on the predatorprey response on the other. This implies that the loss of certain species at the top of the food chain can have unprecedented effects inducing fundamental changes to the whole system. In the case of seagrass and macroalgal systems, the loss of predators may induce the loss of key habitat-providing species with harmful consequences for the whole community (e.g.

Marine biological invasions are a major issue at regional and global scales. One of the major causes is the large number of transport vectors, like cargo vessels, but it has also been hypothesized that a depauperate native flora and fauna may facilitate the invasion success of exotic species (Elton, 1958). Non-native species can be a threat to regional biota due to competitive advantage, with uncontrollable consequences for the functioning of ecological

down control (Finke & Denno, 2005).

spent less time grazing.

Estes et al., 1998).

**4.2 Invasion success**

especially when the omnivorous fish was included.

strong effect on the grazers, resulting in higher algal biomass.

processes. To investigate the relationship between native species diversity and invasion success of exotic species, experiments were conducted on marine sessile epibenthic invertebrates of the southern rocky coasts of New England, exposed to the invasion of the colonial ascidian *Botrylloides diegensis*, native of the Pacific Ocean (Stachowicz et al., 1999, 2002). Experimental communities were composed of zero to four native species. Species richness was manipulated by transplanting juveniles or by allowing larvae to settle on 2x2 cm tiles that were (after the successful colonization) combined in a 5x5 grid (i.e., a complete plate consisted of 25 interchangeable subunits). During the course of the experiment all but the five target species were removed regularly. As a result, survival of the invasive species decreased with higher species richness, and it was hypothesized that reduced resource availability may be the underlying mechanism.

Contrary to the previous example, two observational studies on natural assemblages evidenced the opposite to what had been demonstrated in controlled experiments before. A long-term (11 months) survey of sessile invertebrates on a rocky jetty on the Tasmanian coast revealed a positive relationship between colonization rates of both native and introduced species and natural species richness (Dunstan & Johnson, 2004). Similarly, in a natural riparian assemblage, the most diverse assemblages were the most invaded by exotic plants; the same patterns were evidenced by an *in situ* experiment manipulating local diversity (Levine, 2000). A possible explanation therefore could be the different niche opportunities across varying environmental gradients (Shea & Chesson, 2002). Therefore at a larger observational scale, invasion success will be higher, given that more suitable niches are available, be it for native or exotic species, whereas invasion success would negatively relate to species richness at smaller, i.e. local scales (Shea & Chesson, 2002). On a more individual basis, the specific characteristics and functions of the resident species as well as the interactions among them and with the invasive species, rather than species richness *per se* may be the determinant for the invasion success (Dunstan & Johnson, 2004). These examples show that isolated experiments can sometimes be misleading in their outcomes. Therefore a combination of field observations, and field as well as laboratory experiments would certainly be the best approach to avoid such inherent biases. As this extensive approach is often not feasible, isolated laboratory experiments should be interpreted with caution.


*2.6. HPLC Analysis of Organic Acids*

The predominant organic acids found in kombucha tea were determined by high performance liquid chromatography (HPLC). Six organic acids including glucuronic acid (Sigma-Aldich, Darmstadt, Germany), gluconic acid (Merck, Darmstadt, Germany), d-saccharic acid 1,4-lactone (Sigma-Aldich, Germany), acetic acid (Merck, Germany), ascorbic acid (Merck, Germany), and succinic acid (Merck, Germany) in kombucha tea were optimized for detection by isocratic HPLC systems with a conventional C18 column. The kombucha tea samples were filtered through a 0.22 μm sterile microfilter and 50 μL of the filtrate was injected into the HPLC system (Agilent technologies 1200 series, Santa Clara, CA, USA). The C-18 column (4.6 × 150 mm, 5 μm; GL Sciences, Tokyo, Japan) employing a UV detector (210 nm) was used for the analysis. Moreover, the HPLC system was controlled with a flow rate of 0.8 mL per minute and a running time of 40 min at 25 ◦C. Six organic acids in kombucha tea were separated by 20 mM KH2PO4 elution buffer at pH 2.4 and the standards of organic acids were used for comparison with kombucha tea. The peak area of each organic acid was calculated by Agilent ChemStation level-5 program, and then standard graphs of each organic acid were generated. Thus, the content of each organic acid in kombucha tea was calculated from the standard graph of each organic acid.

#### *2.7. DPPH Radical Scavenging Assay of Kombucha Tea*

The radical scavenging activity of kombucha tea was determined against DPPH free radical content [15]. The kombucha tea was diluted with methanol by 10-fold serial dilution. Each concentration of the kombucha tea (0.5 mL) was incubated with 1.5 mL of 0.1 mM DPPH solution (Sigma-Aldich, Germany). Absorbance at 517 nm was measured by spectrophotometer (Genesys 20, Thermo Scientific, Dreieich, Germany) after the solution mixture was kept in the dark at room temperature for 20 min. Methanol was used as a blank solution, while DPPH without kombucha tea was used as a control. The absorbance of the DPPH solution A1, and the absorbance of kombucha tea mixed with the DPPH solution A2, were measured. The percentage of DPPH free radical inhibition was calculated as follows: percentage inhibition = {(A1–A2)/A1} × 100.

The antioxidant activity of kombucha tea was assessed by comparing the samples to standard gallic acid and was expressed as milligrams of gallic acid per milliliter of kombucha tea (mg GAE/mL kombucha tea).


2.2.7. Enzymes Antioxidant Activity

Figure 8 illustrates the effects of the EP application (vapor and dipping) on the enzymatic activity of tomato fruits. During the seventh day of vapor application of 0.8% EP, CAT activity was increased (12.78 units mg−1 protein) compared to control and 0.4% EP (7.55 and 7.01 units mg−1 protein, respectively), while 0.8% EP also increased CAT activity on the last day of storage in comparison to chlorine treated fruits (Figure 8A). On the other hand, dipping in 0.4% EP decreased CAT activity on the 14th day compared to control (Figure 8B). Interestingly, both EP application methods did not significantly affect SOD activity of tomato fruits (Figure 8C,D). POD activity increased with the EP vapor application (0.4 and 0.8%) compared to control on the seventh day of storage, while 0.8% EP led to decreased enzyme activity on the last day of storage compared to chlorine (2.52 and 3.59 units mg−1 protein, respectively) (Figure 8E). In contrast, dipping in 0.4% EP resulted in decreased POD activity on the seventh day of storage compared to chlorine, whilst the same treatment increased enzymes' activity on the last day of storage in comparison to control (Figure 8F).

**Figure 8.** Effects of vapor (**A**,**C**,**E**) or dipping (**B**,**D**,**F**) application with eco-product (EP) at different concentrations (0%, 0.4% and 0.8%), chlorine (0.02%) or control (application of water) on enzyme activity (catalase-CAT, superoxide dismutase-SOD and peroxidase-POD), expressed as units of enzyme per mg of protein, stored up to 14 days at 11 ◦C. In each day, means (±SE) followed by different Latin letters significantly differ according to Duncan's MRT (*p* = 0.05). ns: not significant.

#### 2.2.8. Sensory Evaluation

The effects of the EP's application (via vapor and dipping) on tomato's sensory attributes (marketability, aroma, appearance) are illustrated in Figure 9. Tomato's marketability was not affected during vapor application (Figure 9A), while dipping on 0.4% EP and chlorine decreased marketability (lower scoring values) on the seventh day of storage compared to control (Figure 9B). Vapor treatment with chlorine decreased aroma scoring on the seventh day of storage compared to control, while on the last day of storage all vapor treatments were able to decrease aroma values (Figure 9C). Moreover, dipping in chlorine resulted in lower aroma values on the seventh day compared to other treatments (Figure 9D). During vapor application, all treatments resulted in lower appearance values compared to control on the last day of storage, while dipping method did not affect tomato's appearance (Figure 9E,F).

**Figure 9.** Effects of vapor (**A**,**C**,**E**) or dipping (**B**,**D**,**F**) application with eco-product (EP) at different concentrations (0%, 0.4% and 0.8%), chlorine (0.02%) or control (application of water) on marketability (scale 1–10), aroma (scale 1–10) and appearance (scale 1–10) of tomato fruits stored up to 14 days at 11 ◦C. In each day, means (±SE) followed by different Latin letters significantly differ according to Duncan's MRT (*p* = 0.05). ns: not significant.


**4. Conclusion**

diffuse and focal perivascular infiltrates and proliferates to a small or virtually absent cellu‐ lar inflammation is associated with the virulence of the virus strains and to a large extent with the immunological reactivity of the organism to infection. In assessing the nature of the pathological process, we stand in solidarity with Yerman, concluding that the immunopa‐ thology of tick-borne encephalitis includes both immediate type hypersevsitivity, and de‐ layed-type hypersevsitivity, a different combination of which creates the impression of

Cerebral symptoms +++ +/++ - Focal neurological symptoms ++ +++ -

The current of disease heavy heavy / moderate

The during the onset of death, week the first week 2-3-4 weeks Till one week

Alteration of neurons +++ ++/+++ +/± Gemocirculating disorders +++ ++/+++ ±/-

neutrophils ++ + ±/ lymphocytes/ histiocytes +/++ ++/+++ ++

focal ++ ++/+++ +/± diffuse ++ ++/+++ - Reactions of ITH +++ ±/+ - Reactions of DTH ±/+ ++/+++ +/±

Note: + + + pronounced, + + moderate severity, + weak expression, ± low intensity, - the lack of symptom/sign.

**Table 3.** Clinical and morphological variants of tick-borne encephalitis

**Clinical and morphological variants of the disease The first The second The third**

> 2 weeks. -56.6% 3 weeks. - 13.4% 4 weeks. - 30%

light, with or without clinical picture

no

different types of inflammatory reaction.

Mortality 100 %

**Indexes**

136 Encephalitis

*The clinical picture*

*The morphological picture*

Infiltration due to:

Proliferation of glia:

On the basis of their own and literature data is given the modern interpretation of the patho‐ morphology of tick-borne encephalitis (TBE) in terms of immunopathological nature of the inflammation. It is shown that the pathological picture of central nervous system reflects the hyperergic nature of inflammation with TBE. In the morphogenesis of the pathological proc‐ ess traced the consistent development of immediate type hypersensitivity reactions, initiated by the damaging effect of the virus in brain tissue, and delayed type hypersensitivity reac‐ tions to ensure the formation of local (tissue) immunity. For a comprehensive assessment of pathology of tick-borne encephalitis in humans and experimental animals set the variability of its manifestations, this depends on the properties of the infecting virus strain, such as the virus-induced immune response and the stage of disease morphogenesis. On this basis, div‐ ided into three clinical and morphological variant manifestations of infection with TBEV.