**1. Role of TCTP in Tumorigenesis**

TCTP (also known as histamine releasing factor, HRF; fortilin) is a highly conserved and multifunctional protein that participates in diverse biological and disease processes, including cancer [1–3]. Many studies have established the diverse roles of TCTP in tumorigenesis. The best characterized function of TCTP is its anti-apoptotic activity [2,4–6]. The anti-apoptotic role of TCTP was first identified in a study that showed the overexpression of TCTP in various cancer cells prevented etoposide-mediated apoptosis [7]. TCTP was shown to exert its anti-apoptotic activity by enhancing the stability of Mcl-1, a member of Bcl-2 family proteins [8]. Further, it was shown that depletion of TCTP in mice led to increased apoptosis during embryogenesis and that lethality supported its role in mediating apoptosis [9]. Crystallography studies revealed that the helical domain of TCTP displays similarity to helices H5-H6 of Bax which have been known to regulate mitochondrial membrane permeability during apoptosis. Based on this finding, a novel mechanism for the antiapoptotic activity of TCTP, which involves its localization to the mitochondria and inhibition of Bax dimerization was proposed [9]. TCTP also potentiates the antiapoptotic function of Bcl-xL through its BH3-like domain [10]. Moreover, genomic integrity is maintained by TCTP via its interaction with ataxia-telangiectasia mutated (ATM) and p53 [11,12]. Low-dose γ-irradiation induced localization of TCTP into the nucleus where it exists complexed with other proteins that participate in DNA damage sensing and repair [11].

TCTP is also implicated in metastasis, tumor invasion, and resistance to anticancer therapy [13–16]. Knockdown of TCTP in colon adenocarcinoma inhibited proliferation, migration and invasion of tumor cells both in vitro and in vivo [17]. Proteomic analysis revealed that the expression of proteins related to cytoskeleton biosynthesis was changed by TCTP knockdown [13]. In colorectal cancer, extracellular TCTP promoted disease progression and liver metastasis through Cdc42/JNK/MMP9 activation [14]. It was also suggested that TCTP regulates metastasis of colorectal cancer by regulation of the high mobility group 1

(HMGB1) and activation of the NF-KB pathway [17], or by regulating epithelial-mesenchymal transition (EMT) via mammalian target of the rapamycin complex 2 (mTORC2)/Akt/GSK3β/β-catenin pathway [15].

High expression of TCTP in a variety of cancers, including cholangiocarcinoma [18], ovarian cancer [19], and glioma [20], correlates with poor prognosis. Immunohistochemical analysis of normal and colorectal cancer tissues revealed that TCTP expression is significantly higher in the tumor tissues. Elevated TCTP was observed from the adenoma stage, indicating that TCTP is induced early in the disease, and further induction in later stages was not observed [21]. In a cohort of breast cancer patients, TCTP levels increased in later stages of cancer with the poor prognosis [22]. A significant association of TCTP in advanced stages of human hepatocellular carcinoma was observed. This study revealed transcriptional regulation mediated by the chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) [23].

TCTP plays multiple roles in the pathogenesis of cancer, including regulation of resistance to cancer therapy, especially including chemotherapy and radiation therapy [16,21,24]. Jung et al. showed that overexpression of TCTP in HeLa cells inhibited cell death by cytotoxic drugs through the inhibition of mitochondria-mediated apoptosis [16]. During the apoptosome formation induced by chemotherapeutic agents, C-terminally cleaved TCTP associates with Apaf-1 in the apoptosome, thus preventing the caspase activation cascade. In colorectal cancer cells, 5-FU and oxaliplatin treatment induced translational upregulation of TCTP via the mTORC1 pathway and decreased TCTP expression increased the sensitivity to the chemotherapeutic agents [21]. Moreover, breast and lung cancer cells with relatively high expression levels of TCTP were insensitive to radiation-induced cell death and response to radiation was influenced by TCTP levels [24].

Because of its anti-apoptotic properties, its role in the regulation of cancer metastasis, and its relevance to disease prognosis, TCTP has become a promising target for cancer therapy (Table 1). Mechanisms underlying how TCTP regulates cellular growth in cancer have been widely studied, but the question whether autophagy may be involved in how TCTP regulates cellular homeostasis has received relatively little attention. In this review, we discuss TCTP's role in cancer pathogenesis, especially in relation to its novel role in the regulation of autophagy and its potential as a target in cancer therapeutics.

**Table 1.** Roles of translationally controlled tumor protein (TCTP) in cancer pathogenesis.

### **2. Autophagy, a Machinery for Cellular Homeostasis**

Autophagy is the process that delivers cellular proteins and organelles to lysosomes for digestion by lysosomal hydrolases, thereby facilitating metabolic homeostasis [25–29]. By eliminating redundant proteins and damaged organelles and by providing building blocks and energy, autophagy serves as a recycling system for cell renewal. During metabolic stress, autophagy prevents the accumulation of toxic cellular components, enabling cells to adapt to environmental changes [29]. Autophagy occurs both in physiological stresses such as nutrient deprivation, ER stress, and hypoxia, as well as in pathological states, including infection, cancer, and neurodegeneration. Therefore, the modulation of autophagy as a therapeutic strategy has recently received increasing attention. But whether autophagy is the cause or result of disease is still an unsettled question. While it is an important catabolic survival mechanism, autophagy has also been classified as type II programmed cell death based on the observation that the cytoplasm of dying cells contains plenty of autophagic vacuolization [26]. However, whether autophagy results in cell death or merely accompanies cell death is still debated [30].

During the autophagic process, cells form double-membrane vesicles called autophagosomes. Lysosome then fuses with the autophagosomes to form autolysosomes. This results in the degradation of the cellular components originally captured by the autophagosomes and in the recycling of their building blocks. This process is divided into five steps: initiation, vesicle nucleation, vesicle elongation, vesicle fusion, and cargo degradation [31]. When cells are in a nutrient-rich environment, the mTORC1 complex inhibits autophagy by preventing the formation of the ULK1 complex (Figure 1) [32,33]. In the initiation step, the ULK1 complex dissociates from mTORC1 and activates the class III PI3K complex. In this complex, Beclin 1 works as a sca ffold protein to recruit VPS34, ATG15, UV radiation resistance-associated gene protein (UVRAG), and an activating molecule in BECN1-regulated autophagy protein 1 (AMBRA1). The class III PI3K complex is the main mediator of vesicle nucleation by localizing autophagic proteins into phagophores [33,34].

**Figure 1.** Negative regulation of autophagy by TCTP. In the initiation step of autophagy, TCTP may inhibit AMPK-induced ULK1 phosphorylation on Ser317 and Ser777, which activates the formation of the Beclin 1 complex. TCTP also enhances the mTORC1-induced phosphorylation of ULK1 on Ser757, which prevents ULK1 interaction with AMPK. TCTP also inhibits autophagosome formation by activating Bcl-2, which, in turn, inhibits the formation of the Beclin1 complex (see text for further details).

Once the phagophore, a double-membraned opened structure, has captured its cargo material, the autophagosome undergoes maturation by vesicle elongation process, which is regulated by two ubiquitin-like conjugation systems. The first system involves the incorporation of LC3II into a growing double-membrane, and it is regulated by the protease ATG4B and by the E1-like enzyme ATG7 [35]. The other system requires the formation of the ATG12-ATG5·ATG16L1 complex, which is mediated by ATG7 and by the E2-like enzyme ATG10 [36,37]. In the vesicle fusion step, the autophagosome fuses with the lysosome, facilitated by syntaxin 17 (STX17) [38], and the autophagosome contents are degraded due to the action of the lysosomal enzymes. Autophagy plays an indispensable role in these cellular metabolic events because absence or defective autophagy leads to metabolic, neurodegenerative, and infectious diseases [39]. Defective autophagy has also been reported to cause tumorigenesis, facilitated by several protein partners including TCTP. TCTP has been shown to be involved tumorigenesis, but its specific role in the regulation of autophagy, of which little is known, is discussed in Section 6 below.

### **3. Role of Autophagy in Tumorigenesis**

Autophagy plays a dual role in regulating tumorigenesis: (A) suppressing tumor growth and (B) maintaining the survival of cancer cells. Suppression of tumor growth by inhibiting the proliferation of cancer cells was initially considered as the primary role of autophagy [40]. Exogenous expression of Beclin 1 in human breast cancer carcinoma cells induced autophagy and this was associated with decreased cell proliferation and tumorigenesis in nude mice [41]. Studies in a mutant mouse model revealed BECN1 as a haploinsu fficient tumor suppressor gene [42] and provided evidence for negative regulation of tumorigenesis by Beclin-1 [43]. As a possible mechanism for tumor suppression by autophagy, it was proposed that autophagy prevents the accumulation of oxidative stress which would result in genomic instability [27]. During autophagy deficiency, accumulated p62, also known as sequestosome 1 (SQSTM-1), prevents Keap1-mediated ubiquitination of Nrf2, thereby activating an antioxidant defense pathway [44,45]. Another proposed mechanism suggests that autophagy suppresses tumorigenesis through inhibition of inflammation. When autophagy is deficient in cancer cells, necrotic cell death induces an inflammatory response and secretion of pro-inflammatory factors such as the HMGB1 protein, eventually leading to tumor growth [25]. Under chronic inflammation, a proliferation of cells occurs to compensate for the damage from inflammation-inducing cells that are susceptible to genetic mutations and oncogenic transformation [46]. Thus autophagy suppresses inflammation-induced tumorigenesis by mitigating cellular damage [47]. Autophagy also suppresses the proliferation of oncogene-expressing cancer cells by inducing senescence, which functions as a barrier to tumorigenesis [48]. Liu et al. showed that downregulation of ATG5 expression enhanced proliferation and prevented oncogene-induced senescence in melanocytes, thus confirming ATG5 as tumor suppressor gene [49]. Autophagic activity is also upregulated during oncogene-induced senescence and overexpression of ULK3, an autophagy-related gene, resulted in senescence [50]. Thus, autophagy was postulated to suppress tumorigenesis by forcing oncogene-expressing cells to exit the cell cycle.

Cancer cells with high metabolic demand due to increased proliferation depend on autophagy to maintain cancer cell survival [29]. Several studies have reported that autophagy supports cancer cell survival, proliferation, and metastasis-related behaviors. Ectopic expression of H-ras*v12* or K-ras*v12* in nontumorigenic epithelial cells showed increased basal autophagy, which supports the growth of RAS-driven tumors by maintaining functional mitochondria under the nutrient-limited condition [51]. Moreover, when autophagy is blocked by knockout of ATG5 or ATG7 in these cells, tumor growth in vivo is delayed [51]. Studies with a breast cancer mouse model also demonstrated the tumor-promoting role of autophagy [52]. In this study, conditional knockout of the protein, FIP200, essential for autophagy, inhibited breast cancer initiation, progression, and metastasis. Gene expression profiles of FIP200-knocked out mammary tumors revealed increased expression of immune response genes, suggesting that FIP200 deficiency induces anti-tumor immune surveillance that eventually suppresses tumor progression [52]. Moreover, autophagy inhibition by hydroxychloroquine (HCQ) decreased cellular proliferation and metastasis of dormant breast cancer cells. In this study, knockdown of ATG7 reduced the metastatic burden, while knockdown of BECN1 showed no e ffects, suggesting that dormant breast cancer cells are autophagic, which is dependent on ATG7 [44]. Additionally, autophagy serves

as a survival mechanism for cells under tumor-related conditions, such as hypoxia or metabolic stress. Under hypoxic conditions, hypoxia-inducible factor-1 α (HIF-1 α) induces mitochondrial hypoxia as an adaptive response to metabolic stress, preventing cell death [53]. With regard to cellular adaptation, autophagy provides cancer cells with a strategy to survive under metabolic stress conditions with limited nutrient and oxygen supply.

Overall, the e ffect of autophagy on tumorigenesis depends on cancer type, stage, and cancer environment [28,54]. Since genomic instability can initiate cancer [55], autophagy can delay tumorigenesis by maintaining genomic integrity. However, once tumors are formed and begin to proliferate, cancer cells themselves take advantage of autophagy to sustain their rapid proliferation.

### **4. Autophagy, a Potential Target for Cancer Treatment**

Probably due to the dual role of autophagy in cancer, both as an inhibitor and as an activator, there is an increasing interest to investigate the modulation of autophagy as a potential avenue for cancer therapy. The application of pharmacological modulators of autophagy has shown promise in some in vitro and in vivo studies, but not in others [56–58]. Several studies have reported that treatment with anti-cancer drugs resulted in the induction of autophagy in a range of cancer cell lines [59]. In human mammary carcinoma cells, tamoxifen treatment resulted in autophagic vacuole formation, and 3-methyladenine (3-MA), an inhibitor of autophagosome vacuoles, partially inhibited cell death caused by tamoxifen [60], suggesting that autophagy synergized with apoptosis to cause cell death under the tamoxifen treatment. However, more recent findings suggest that autophagy serves as a possible mechanism of tamoxifen resistance in breast cancer cells [61–63]. These studies imply that autophagy induced by anticancer therapy may serve as a cellular protection phenomenon.

Besides, autophagy is also related to radiation-induced cancer cell death. Human breast cancer cells, MCF7, are sensitive to standard doses of radiation. However, the accumulation of acidic vesicular organelles, a characteristic feature of autophagy, was observed in surviving cell population after the irradiation and a potent autophagy inhibitor bafilomycin A enhanced apoptosis-related cell death after the irradiation [64]. These findings suggest that autophagy induced by conventional cancer therapy could be a potential mechanism for the development of therapy resistance by providing cells with an alternative survival pathway to avoid apoptotic cell death. Thus, research on autophagy in the context of cancer therapy and considering autophagy inhibitors in combination with conventional cancer therapy could be a strategy to overcome therapy resistance.

Agents that inhibit autophagy have been suggested as anti-cancer agents, and most class III PI3K inhibitors are categorized to this type. Class III PI3K inhibitors mediate autophagosome formation, and agents such as 3-MA, wortmannin, and LY294002 have been shown to inhibit autophagy [65]. 3-MA, in particular, is distinguished from other PI3K inhibitors in that it exerts a dual role in autophagy. In nutrient-rich conditions, 3-MA promoted autophagy flux while still showing inhibitory e ffects on starvation-induced autophagy [66]. These di fferential e ffects on autophagy arise from the divergent effects of 3-MA on PI3K subunits. While blocking by 3-MA of class I PI3K is persistent, its inhibition of class III PI3K is transient. Under nutrient-rich conditions, prolonged treatment with 3-MA continuously blocks class I PI3K and the Akt-mTOR pathway, which inhibits autophagy while sparing class III PI3K, eventually activating autophagy [66]. Since class I PI3K has been known to be a negative regulator of autophagy [67], selecting PI3K inhibitors as autophagy-regulating anti-cancer agents should take into consideration of how they a ffect each subtype of PI3K.

Also, lysosomotropic agents such as chloroquine (CQ), HCQ, Lys0569, or monensin have been recommended for curing cancer by preventing acidification of lysosome and eventually inhibiting degradation of molecules in autophagosome [68,69]. Indeed CQ and HCQ are registered anti-malarial drugs and have shown anticancer e ffects when administered in combination with conventional chemotherapy and radiotherapy [70–72]. Furthermore, Bafilomycin A interferes with both lysosome acidification and autophagosome-lysosome fusion and this observation may have the potential to lead to the development of anti-cancer agents that overcome resistance to anticancer therapy [73,74]. A potent autophagy inhibitor spautin-1 [75] also has been reported to enhance the potency of imatinib in chronic myeloid leukemia [76].

On the other hand, inducing excessive autophagy is also suggested for treating cancer, especially in cancer cells that are resistant to apoptotic stimuli. The most commonly studied agent for inducing autophagy is rapamycin, which inhibits mTOR, thus activating autophagy by preventing the inactivating phosphorylation of ULK1. Although rapamycin is an FDA-approved drug for cancers, such as renal cancer, its clinical use has limitations because di fferent cancer cells respond di fferently to rapamycin [77]. Tyrosine kinase inhibitors and histone deacetylase inhibitors are also known to induce autophagy in cancer cells, but further investigations are needed to settle whether autophagy is induced by cells that are resistant to those inhibitors or as a readout of cell death. It is important to establish whether the induced autophagy results in drug resistance or cell death. Therefore, mechanistic studies about current cancer therapy and autophagy are still worthwhile for enhancing therapeutic e fficacy, although autophagy plays a complex role in tumorigenesis.


However, humans also sometimes negatively affect wild animals in ways that are less obvious and thus less well recognized: humans often cause *direct but unintended harm* to wild animals (Fraser 2012: 736). For example, agricultural practices, such as plowing felds, have a detrimental effect on many wild animals who live in the felds. A further example is road traffc: many animals are hit by cars, or their habitats are destroyed by the construction of new roads. Furthermore, considerable harm is caused to birds by communication towers and buildings with windows. Pollution (such as the routine discharge of machinery waste oil from ships) and the use of chemicals also negatively affect many wild animals. Yet despite their highly negative impacts on wildlife, these types of disturbances often go unnoticed (Fraser 2012: 736–737).

Wild animals who are more likely to be victims of both intended and unintended harms can be considered particularly vulnerable groups. The fact that some of these harms are unintended does not play a role in the assessment of whether these harms are morally problematic or not. Commensurate to their increased vulnerability, these animals should receive more attention. For example, when government agencies are constructing new buildings or roads, they should give additional care to the ways these endeavors may affect wild animals. As for agricultural practices, incentives could be given to subsidize farming practices which are less harmful to wild animals, or more research money could be devoted to studies on such topics.

A further important example of unintended harm caused to animals is anthropogenic climate change. Numerous publications have highlighted the disastrous impact of climate change on humans (one of the most recent being the Report of the Intergovernmental Panel on Climate Change IPCC 2021). However, the impact of climate change on animals has received less attention in the literature. This is problematic, as rising sea levels and weather extremes will negatively affect the lives of countless wild animals in the future. While some animal species may beneft from climate change, it has problematic effects on others. Yet—with a few notable exceptions (Palmer 2010; Pepper 2019; Rowlands 2021; Sebo 2021)—this topic is rather underdeveloped in the animal ethics literature.

I cannot develop here what compensation for climate-related harm to animals should look like in detail, as this would be a separate endeavor. A few words are nonetheless necessary. Moral agents have a duty not only to *mitigate* climate change (i.e., to take measures to address its causes), but also to *assist* animals who are victims of the effects of climate change. That is, to the extent that climate change jeopardizes animals' basic claims, we owe the affected animals assistance and aid. The specifc assistance depends on the kind of wrongs the animals are likely to incur along with the type of claims that are at stake. For example, animals' claims to live a selfdetermined life and to express normal behaviors are violated if their territory is fooded as a consequence of climate change. In this case, if it is a viable possibility, relocation of the animals concerned could be discussed. Of course, special attention will have to be paid to potential negative consequences of such resettlement on the animals affected and on other animals. Other strategic tools to facilitate adaptation of wild animals to climate change include land and water conservation, animal rehabilitation, habitat restoration, natural resource management, along with better legislation and regulation (Mawdsley et al. 2009; Palmer 2021).

There are two ways to move forward from here regarding the harm that climate change causes wild animals. First, we are under the moral obligation to do more research on how to both mitigate climate change and assist wild animals adapt to a changing climate. Second, "we can make practical progress by advocating for moral and political standing for wild animals, so that we will have the social and political capital necessary for taking the interests of wild animals into account as we recreate human societies in the face of climate change" (Sebo 2021: 61). To extend vulnerability language to wild animals and by describing them publicly as a particularly vulnerable group whose basic claims are likely to be overlooked may contribute to bringing about these changes.

Again, note that if I say that "we" are under a moral obligation, I have in mind those moral agents who have the power to positively infuence the course of wild animals' lives and who are best placed to fulfll such duties. In the case of climate change, many humans beneft from past or current emissions, and thus owe wild animals a duty to mitigate the effects of climate change as well as adaptive measures if it becomes clear that animals will be negatively impacted. This means that moral agents are minimally under the duty to publicly talk about the effects of climate change on wild animals, and to support organizations conducting research on how to mitigate its effects and assist animals in their adaptation process.

### 7.4 Wild Animals as a Particularly Vulnerable Group

In this chapter, I have argued that wild animals hold basic claims—no less so than animals destined for the food industry and for research. I showed that wild animals' claims are often not given the weight they are due, and that wild animals therefore often qualify as a vulnerable group. There are two reasons for the heightened vulnerability of wild animals. First, many wild animals are particularly vulnerable because they are at increased risk of having their claims disrespected by humans—be it intentionally or unintentionally. Second, when humans disrespect animals' basic claims, we frequently neglect to provide the compensation and reparations these animals are due. In sum, wild animals are frequently at increased risk of being denied what they are entitled to. This problem is particularly acute for those species who tend to be overlooked, such as small animals, animals who prematurely die in high numbers (r-strategists), and animals who are deemed unattractive by the public or who fulfll no key function in the ecosystem. All these animals' claims are often not given due weight when it comes to decisions about land use, the effects of climate change, and after disasters.

A further problem is that wild animals are often not seen as individuals with their own lives but rather as members of a specifc species, particularly those animals who are deemed "invasive species" and thus regarded as a nuisance. Often, lethal conservation practices are used to limit their numbers, overlooking the fact that these animals are sentient individuals (Abbate and Fischer 2019; Wallach et al. 2020). Publicly describing wild animals as particularly vulnerable groups may change the way we perceive these creatures. It draws our attention toward the fact that they are often at increased risk of not being given what they are due, and that more protection and attention is needed when we are dealing with them.

From what has been said in Chaps. 5, 6, and 7, it follows that many groups of animals are particularly vulnerable. We may then be tempted to wonder: if so many groups of animals (and humans, for this matter) are particularly vulnerable, does the concept of vulnerability lose its meaning? And if so many groups are vulnerable, where should we start to reduce increased vulnerability? Should those particularly vulnerable receive priority? I turn to these questions in the next chapter.

### References

from Predators. *Res Publica* 21 (3): 273–289. https://doi.org/10.1007/ s11158-015-9281-2.

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# Conclusion

This book set out to explore what vulnerability is for humans and for animals, and to establish why and when vulnerability matters from a moral point of view. The frst goal was to present a defnition of vulnerability for the domain of bioethics which would resolve the confict in the literature regarding the *scope* of vulnerability. In particular, I investigated whether vulnerability is, as some have suggested, a property belonging to everyone by their very nature ("universal vulnerability"), or whether it is a property restricted to some individuals and groups in certain contexts who are in need of special protection and additional attention ("situational vulnerability"). I suggested that a viable account of vulnerability should reconcile these two views.

This book has aimed to contribute a more nuanced and detailed understanding of vulnerability to contemporary debates in philosophy, bioethics, and the vulnerability literature in general—that is, an understanding of vulnerability that is free from the diffculties of former defnitions. I proposed that vulnerability is a matter of degree: all beings with interests can be considered generally vulnerable, yet some individuals, in certain situations, are more likely to have their interests unfairly considered and to consequently experience wrongful harms and mere wrongs. These individuals and groups should be regarded as *particularly* vulnerable in this specifc context and should be afforded additional attention and special protection to receive what they are due.

The second goal was to trace the ethical implications of situational vulnerability for the domain of (bio-)ethics in general and for the feld of animal ethics in particular. That is, a substantial part of the book was devoted to the question of whether animals can be regarded as *particularly* vulnerable in certain situations or contexts, and to outline the moral implications of situational vulnerability for animals. I thus aimed to fll an important gap in the literature, as animal vulnerability and its moral implications have yet to be examined in-depth and systematically.

I have argued that vulnerability discourse is already established with regard to humans, and fulflls a specifc role—that is, to orient our attention toward those individuals and groups in need of special protection and additional attention in specifc situations. The description of a group as "particularly vulnerable" in a specifc context thus has not only rhetorical force, but also normative weight: it shows that additional attention and potentially even special protection may be needed when dealing with certain groups, as the individuals of these groups run a higher risk of being denied what they are due.

My main suggestion has been that vulnerability discourse, so understood, can fulfll the same role for sentient animals. Indeed, the language of vulnerability can highlight those animals who are in need of additional attention and better protection because they are members of groups who are likely often denied what they are due. Extending the concept of vulnerability to animals, I argued, may positively infuence how we think about and treat these creatures, by refocusing our attention on their claims and needs. At the same time, though, we should remain humble about what vulnerability language can achieve. To be sure, vulnerability discourse cannot work miracles; nonetheless, this language can fulfll the same function for animals as for humans—no more and no less.

In the following, I summarize the main results of the book in greater detail, and assess whether the goals set out in the beginning of this book have been reached. In a second step, I discuss several objections one could make against the arguments presented in this book, and offer tentative responses.

### 8.1 Summary

Chapter 1 introduces the topic of vulnerability language and stresses its importance. Chapter 2 presented three scenarios involving what we would likely deem vulnerabilities: the Tuskegee syphilis study, the separation of refugees' children from their parents at the border between the U.S. and Mexico, and the BSE scandal in the U.K., which led to the (premature) killing of millions of cattle. The purpose of these case studies was to illustrate that we all probably have an intuitive understanding of what vulnerability means. However, when we attempt to properly defne it, we quickly run into challenges, one reason being that vulnerability seems to come and exist in various forms.

In Chaps. 2 and 3, I examined what vulnerability is and suggested how we ought to understand it. I began by presenting two conceptions of vulnerability in the literature: universal or ontological vulnerability on the one hand, and situational or circumstantial vulnerability on the other. Some philosophers and bioethicists assume that vulnerability is a shared property of all humans (and sometimes animals as well)—that is, we are all vulnerable by our very nature. Others understand vulnerability as situational: they stipulate that some individuals are vulnerable in certain situations or contexts and should accordingly be afforded special protection and additional attention. These two conceptions of vulnerability are in tension—at frst sight, they seem contradictory and irreconcilable. Indeed, the idea that vulnerability encompasses everyone *qua* their nature conficts with the view that vulnerability is a property of only some individuals or groups. Moreover, this confict has problematic consequences: it remains unclear whether vulnerability is a merely descriptive term, or whether it has normative pull. Not clarifying what vulnerability consists in may result in opposition to the idea that vulnerable beings should be afforded special protection and additional attention. After all, if all beings are vulnerable by their very nature, then how can special protection only for some be justifed?

I noted that any convincing account of vulnerability must reconcile these two conceptions: we need a defnition of vulnerability which encompasses universal vulnerability, but which can, at the same time, account for the situational vulnerability of some individuals or groups in specifc contexts. Furthermore, the defnition should be easily applicable to different cases and scenarios—that is, it should be formal and suffciently general in scope. Finally, it should be able to explain why some vulnerabilities, but not others, are morally relevant and require action.

To achieve this goal, I argued in favor of an explicative defnition—that is, a defnition that respects some central uses of ordinary language, yet is stipulative about others. In a word, the defnition of vulnerability presented in this book is *ameliorative*, insofar as it does not merely follow ordinary language but rather outlines how we *ought* to use and understand the concept of vulnerability. Thus, one aim has been to propose a defnition of vulnerability which *should* be used, not necessarily to describe one which currently *is* used in ordinary language. This ameliorative defnition, in turn, should facilitate the identifcation of those individuals who are situationally vulnerable and thus have a claim to special protection and additional attention. I referred to these individuals and groups as "particularly vulnerable" (or as "individuals with increased vulnerability") throughout the book.

To arrive at such an account, I closely analyzed the term "vulnerability" along with sets of phrases involving "X is vulnerable to Y." I concluded that vulnerability ascriptions express that some Y *may* happen to X. That is, vulnerability expresses that some state Y has a certain likelihood, under certain circumstances, of manifesting itself. I inferred from this that vulnerability is a *dispositional concept*, concluding that three aspects must be distinguished and accounted for when defning it: the *reasons* why an object is vulnerable; the *conditions* under which vulnerability may appear; and the various *manifestations* of vulnerability.

These distinctions led to the defnition of vulnerability presented in Chap. 3: *Vulnerable beings are individuals with either welfare interests or agency interests that may be frustrated by the individuals themselves, by external circumstances, or by other living beings.* The possession of welfare and agency interests is the *reason* why a being is vulnerable. These interests may be frustrated by individuals themselves, by external circumstances, or by other living beings—they represent *the conditions of manifestation*. Finally, the manifestations of vulnerability are justifed or unpreventable harm, unjustifed harm, and mere wrongs which do not entail any harm. I argued in favor of the view that manifestations of vulnerability are externally caused—that is, vulnerability does not inhere within things or individuals; rather, entities are caused to manifest a certain state by external circumstances or actions, in relation to and in interaction with their environment.

Some manifestations of vulnerability simply cannot be prevented, such as accidental injuries, harm resulting from natural catastrophes, or being attacked by someone who is not responsible for their actions. These incidents all diminish welfare or agency and may result in harm. However, if nobody had the *power* or *ability* to prevent these events, then they cannot be considered *wrongful* harms.

Some manifestations of vulnerability, though, could and *should* have been prevented. In such cases, individuals experience *wrongful harm* or *mere wrongs without any harm*. Regarding wrongs without harm, I described cases in which an individual's welfare or agency interests are unjustifably disregarded by another moral agent (e.g., due to prejudices and stereotypes), but where the individual is not actually made worse off. In such cases, the person is wronged although she does not experience any harm. Examples are breaches of confdentiality or disrespect for someone's autonomous decision-making in the healthcare sector which do not result in any negative consequences, or which may even improve the individual's welfare. From a moral point of view such actions are wrong, even though they do not necessarily result in harm or make someone worse off.

Furthermore, I can wrong someone without being in her vicinity. In many cases, I have the power to positively or negatively affect a person's interests even if she is located far away from me. In such cases, I still often have the capacity to infuence the course of a situation, for example, if I could assist an individual by providing money or time. Consequently, I may have a *duty* to justly consider the interests of some individual, even if I do not know her personally. Indeed, distance does not matter, from a moral perspective. If we have *duties* toward individuals, we are no longer merely talking about their *interests*—they have legitimate *claims*.

These considerations allowed me to specify who is *particularly vulnerable* in certain situations: some individuals are at comparatively higher risk of having their claims intentionally or unintentionally disregarded, overlooked or unfairly considered. As a consequence, they are more likely to incur wrongful harm or mere wrongs. The reasons for these unjustifed manifestations of vulnerability may include ignorance of the specifc needs of a population (e.g., due to language barriers or lack of interest in the specifc group), implicit or explicit biases and prejudices, discriminatory attitudes, conficts of interest, and so on. The individuals concerned are in need of additional attention and special protection in order to obtain what they are due.

To lower the risk of unjustifable manifestations of vulnerability, we need to identify those individuals in specifc situations who run a higher risk of not receiving what they are due. In practice, this means that we must think about the *groups* we tend to overlook, neglect, and forget, but also recognize which of their *claims* we tend to ignore. The question then becomes: When is vulnerability so acute that it warrants special protection and additional attention? Admittedly, this is often a matter of degree, and there may be a gray zone. Nonetheless, in many situations, there are some groups who may more often be victims of stereotypes or who tend to be ignored, forgotten, and overlooked. In these cases, steps should be taken to reduce their risk of incurring unjustifed harm and mere wrongs.

I concluded that there is only one type of vulnerability encompassing everyone who has interests. But, depending on the specifc situation and the groups involved, vulnerability has different likelihoods of manifestation: a change in setting may render some previously only generally vulnerable individuals particularly vulnerable. The controversy as to whether vulnerability is a property of all or of only some beings can thus be resolved. Any account of particularly vulnerable individuals in specifc situations who are in need of special protection and additional attention must be embedded within a broader conception of universal vulnerability.

After these considerations, we can come back to the examples with which I started out Chap. 2, and answer the question of what rendered these groups particularly vulnerable in their specifc contexts. The individuals concerned were all members of groups that were (and probably still are) comparatively more likely to be denied what they are due. In the case of the Tuskegee syphilis study, a poor and oppressed group—Black Americans—were enrolled in the study. Among other problems with the study, the participants were not given the necessary information about the study and its aims, nor were they informed about a treatment which became available during the study. The legitimate claims of the men enrolled in the study, as well as the claims of their family members, were unfairly considered due to racist prejudices and attitudes. As for the case of the children separated from their parents at the border between the U.S. and Mexico, the specifc interests and basic claims of the children of refugees and immigrants were ignored (e.g., the children were separated from their primary caregivers, and they were not given access to education). Finally, in the case of cattle during the BSE scandal in Great Britain, we can now see that these creatures were wronged in different ways. They had not been given the type of food they needed to thrive (their feed was complemented with meat-and-bone meal), and once the disease outbreak happened, they were summarily killed—isolation and medical treatment were not provided (or even considered), a situation we would fnd inacceptable in the case of humans. The cattle were treated this way precisely because they were animals: for many people, animals have a merely economic value; their lives do not count for their own sakes. Our treatment of animals is still determined, in many cases, by *speciesism*—a disadvantageous treatment or consideration of those beings who do not belong to a particular species (Horta 2010).

These considerations reveal an advantage of my account of vulnerability: it is *context-sensitive*. It is *formal* enough to be applied across different situations, groups, and even *species*. In Chaps. 4, 5, 6, and 7, I explored, in greater detail, the potential of my account of vulnerability for animals. I discussed whether the concept can be meaningfully extended and applied to animals and considered what it adds to debates in animal ethics. In Chap. 4, I inquired whether animals can fulfll the conditions of vulnerability ascriptions, contending that sentient animals do possess welfare interests, and that some animals also have agency interests (which can potentially be frustrated). Examples of the latter type of interests are caring for and protecting offspring, advancing in a social hierarchy, and the fact that some animals want to freely choose where they go and with whom they interact. Using the Five Freedoms of Animal Welfare as a basis and further adapting and complementing them, I developed a list of basic welfare and agency interests that most animals likely share

The question is then whether these interests have the status of *legitimate claims,* involving corresponding *duties* of moral agents. To this end, I assessed the extent to which animals count from a moral perspective. I defended the view that moral agents owe it *directly* to sentient animals to take their most fundamental interests into account—these duties are not, for example, owed to their human companions or to humanity in general. Moreover, I maintained that animals' fundamental interests should be considered in accordance with the principle of equal consideration by those holding power over the satisfaction of these interests: similar interests should have equal weight, regardless of their bearer's species. I thereby rejected speciesism, a form of discrimination based on species-membership.

In practice, these basic interests of animals have the status of *legitimate claims*, provided that a moral agent can be identifed who has some power over their satisfaction. Consequently, moral agents must take the legitimate claims of animals into consideration by fairly applying the principle of equal consideration—that is, weighting like claims alike. If they fail to do so, the animals concerned may incur unjustifed harm. Importantly, these basic claims of animals are not necessarily absolute, nor need they be satisfed in all cases. In some circumstances, they can be outweighed by more important claims. In other words, the claims listed here are *pro tanto*, rather than *pro toto* claims.

The remaining chapters applied my defnition of vulnerability to three groups of animals, specifcally: animals used for food (Chap. 5), research animals (Chap. 6), and wild animals (Chap. 7).

In Chap. 5, I discussed whether *using* and *killing* animals for food can be ethically justifed, or whether we should deem animals used for food a particularly vulnerable group. In a nutshell, I concluded that animal products which are harmfully produced are often—but not always—morally problematic. As for whether painlessly killing happy animals is morally permissible, I argued that killing animals for food is ethically problematic in developed Western societies which have plant-based alternatives to meat products readily available. Indeed, animals' claim to continued existence should not be overridden to satisfy a mere gustatory preference. One might think that these arguments result in a "principled veganism"—that is, the view that seeking a vegan lifestyle is morally obligatory. I deny this implication, showing that not all uses of animals are necessarily exploitative: consuming animal products can be compatible with respect for animals' basic claims, in some cases. Furthermore, I showed that farmed animals' dependency on humans does not necessarily imply that they are particularly vulnerable: ideally, moral agents are capable of respecting the claims of those who are dependent on them. Finally, I concluded that we should nonetheless recognize many groups of animals commonly used for food as particularly vulnerable, and increase their protection accordingly.

Chapter 6 examined whether research animals should be deemed a particularly vulnerable group, or whether the harm they incur during research should be considered ethically justifed. I showed that research animals are not suffciently protected by currently implemented research guidelines and principles. These research principles are sometimes not respected in practice, and remain insuffcient from a moral perspective. As a consequence, a large number of animals can be deemed particularly vulnerable in research settings nowadays: animals' legitimate claims are more likely to be unjustly considered merely because they are animals. In a word, current animal research practices are often speciesist. I then turned to the question of what animal research that respects animals' claims would look like. For animal research to be ethical, I proposed, it must respect animals' basic claims (e.g., bodily integrity and continued existence). This can be done by rendering animal research more similar to research with humans. In this vein, I explored how the requirements of social value, scientifc validity, independent review, fair subject selection, favorable risk-beneft ratio, informed consent, and respect for research subjects can be fruitfully applied to animal research.

Finally, in Chap. 7, I turned to the question of whether wild animals should be regarded as a particularly vulnerable group. This topic may have come as a surprise. After all, one may regard the manifold forms of harm encountered by wild animals in their daily lives as unpreventable and thus morally unproblematic, as their origin seems to lie with nature, not with human actions. However, I contended that naturally occurring suffering in the wild is not necessarily justifed from an ethical perspective. Wild animals can also hold legitimate claims against moral agents, I argued, although humans often fail to respect or even consider these claims. Many humans directly negatively affect wild animals, or cause them indirect harm without a second thought. In addition, while humans often have the ability to improve the lives of wild animals (e.g., by assisting or rescuing them), we often fail to do so. I supported the view that humans sometimes owe positive duties of assistance to wild animals. This does not necessarily imply that humans have to immediately intervene in nature to stop all forms of harm (e.g., predation), as such interventions would likely come at too high a cost for both humans and animals. Nonetheless, moral agents—individually and collectively—are not exempt from conducting research on how to improve wild animals' life and health or making efforts to respect wild animals' basic claims. Furthermore, I proposed that if moral agents violate wild animals' basic claims without an acceptable justifcation, then we owe the animals concerned some duty of compensation and reparation. For example, if humans pollute animals' habitat, then we have a duty to clean and restore it. But, in practice, this rarely happens. Consequently, wild animals can frequently be considered a particularly vulnerable group: their claims seldom receive the attention they are due. Humans are likely to ignore and overlook wild animals' fates, thinking that they fall outside our moral responsibility. On the contrary, humans must change the way we think about wild animals: if wild animals are considered a particularly vulnerable group, they can hopefully beneft from the special protection and additional attention they need to have their legitimate claims ultimately respected.

Importantly, the focused discussions of these three different groups of animals—animals used for food, research animals, and wild animals mostly served to illustrate how to apply my account of vulnerability. It is possible, even likely, that other animal groups are particularly vulnerable, in various contexts. Think, for example, about animals in captivity (e.g., in zoos), who cannot live out species-typical behavior due to a lack of space and who depend on humans for their survival. Now imagine that a disaster hits, such as a food or a war. In February 2022, Russia invaded Ukraine. This event was not only devastating to humans (it led to the largest refugee crisis in Europe since the Second World War), but it has also brought enormous suffering to millions of animals. While many Ukrainians fed their homes with their companion animals (such as cats and dogs), countless other animals had to be left behind. The situation is likely dire for animals in barns, research laboratories, and zoos. These animals depend on human care for their survival and well-being, yet buildings with animals were often bombarded, leaving animals buried in rubble or burned in fres. Some zoos ran out of food for their animals, while others tried to evacuate animals to neighboring countries. In war, not only are certain human groups particularly vulnerable (e.g., the elderly and the physically impaired who cannot easily fee), but also many groups of animals: their fates tend to be forgotten, overlooked, and denied due consideration. However, if animals count morally for their own sake, then they should also count in times of crisis and be considered for humanitarian rescue actions (Milburn and van Goozen 2021; Singer and Todorchuk 2022). By framing not only humans but also some groups of animals as particularly vulnerable in confict situations, we raise awareness about their plight and their legitimate moral claims.

Various steps can be taken to lower the risk of unjustifed manifestations of vulnerability. Recall the examples in the beginning of Chap. 1 regarding vulnerabilities during the COVID-19 pandemic: healthcare providers, children, and people who lost their income due to a lockdown. What we should do about these individuals' risk of incurring unjustifed harm or mere wrongs depends on their specifc situation and which of their claims are likely to be ignored or unfairly considered. Since vulnerability is context-dependent, so must responses to vulnerability be determined on a case-by-case basis. Special protection and additional attention can require different kinds of actions and measures, depending on the situation. In the case of healthcare providers, personal protective equipment must be organized to protect them against COVID-19. In the case of school children deprived of access to education because of the pandemic, online education must be provided. And in the case of people losing their income because they are obliged to stay at home, monetary compensation may be needed.

In the case of animals, a major reason for their being at risk of having their interests unjustly considered is the infuence of speciesist prejudices and attitudes. Therefore, a frst general step toward diminishing the manifold wrongs animals are likely to incur would be to educate people about speciesism and the precarious situations of many animals (whether under human care or in the wild). More attention should be paid to their claims, so that they can ultimately receive what they are due.

Following this overview of the book's main arguments and ideas, I can assess whether the account of vulnerability presented here fulflls the conditions outlined at the beginning. I submit that it does resolve the confict in the literature regarding the scope of vulnerability. Indeed, the defnition of particularly vulnerable individuals in need of special protection is embedded in a broader conception of vulnerability—that is, vulnerability as a property of all beings with certain types of interests. Furthermore, since my defnition is both formal in scope and context-sensitive, it can be applied to different domains, such as medical research, healthcare, and humanitarian crises. Moreover, as discussed, it can be extended to various groups of animals. Describing specifc animal groups as particularly vulnerable turns our attention toward their fates and reminds us that we should do something to help or protect them. Finally, by distinguishing among different manifestations of vulnerability, I showed why some vulnerabilities require actions (such as in the form of special protection and additional attention), while others do not: some types of harm are morally unproblematic or simply unpreventable; others could—and should—be prevented.

### 8.2 Objections and Outlook

Some questions may have remained unanswered so far, and some potential challenges and objections to my account of vulnerability may still be open. I address some of them here.

First, one might object that the defnition of vulnerability proposed in this book is eliminativist: insofar as it can be reduced to other concepts and terms, it is not substantive or explanatory in its own right. Be that as it may, this objection is not particularly problematic. Even if we reduce the notion of increased or particular vulnerability to other terms or expressions, such as "increased likelihood of having one's legitimate claims disregarded" and "being at higher risk of incurring unjustifed harm," the concept of vulnerability can still fulfll an important role and function namely, highlighting those individuals in need of more attention and special protection, in specifc contexts. The crucial function of vulnerability discourse is thus to "serve as a very useful means of marking something out for special attention" (Wrigley 2015: 485). This is a rather pragmatic function: vulnerability language serves as a type of warning or signal and points us toward those in need of special protection and additional attention. Moreover, the account of vulnerability proposed here does not need to cover all that we mean by vulnerability in everyday language. As outlined earlier, my aim has been to put forward an *ameliorative* account of vulnerability—that is, to suggest how we *ought* to understand vulnerability, rather to describe how the term is commonly used.

Another potential concern with my account of vulnerability is related to priority-setting. It is important to note that vulnerability does *not* necessarily imply priority. For instance, during the COVID-19 pandemic, there were often not enough hospital beds, medication, and healthcare professionals to treat all the patients. From an ethical viewpoint, we should prioritize those patients who are most likely to survive thanks to medical treatment and who are at the same time likely to die without treatment (Singer 2011: 205). However, this does not necessarily mean that the patients in question are *particularly vulnerable*. The particularly vulnerable beings in a specifc situation are those whose claims we tend to forget, overlook, or disregard. As a consequence, they should be afforded special protection and additional attention. Yet they do not necessarily need to be prioritized for medical treatment, for example.

As shown throughout Chaps. 5, 6, and 7, not only humans but also many animals can be deemed particularly vulnerable in specifc situations and contexts. The possibility of extending vulnerability discourse to animals in general might arouse skepticism. As Carol Levine et al. noted in the case of humans, "[i]f everyone is vulnerable, then the concept becomes too nebulous to be meaningful" (Levine et al. 2004: 46). If we extend the concept of vulnerability to animals, as suggested here, this problem becomes even more acute: Will the concept of vulnerability become void and meaningless if we extend it to animals by describing them as particularly vulnerable and in need of special protection and additional attention?

Two points are noteworthy here. First, who is particularly vulnerable in a specifc situation is context-dependent. Not all groups who, at some point, qualify as particularly vulnerable remain so *at the same time and in the very same situation*. This is a specifc advantage of my formal and context-sensitive account of vulnerability. Second, and more importantly, if many groups of humans and animals are particularly vulnerable, this does not indicate a weakness within the concept of vulnerability; rather, it points us to a *problem with how certain groups of humans and animals are likely to be treated*. Indeed, prejudices and stereotypes are deeply entrenched in many societies, and as a consequence, many groups are forgotten, overlooked, or willfully ignored.

Animals can suffer from the very same mechanism: speciesist prejudices are highly prevalent in most societies. Many humans are far from treating animals as they should be treated (with the exception of some domesticated animals in some societies). Therefore, most groups of animals are likely to not be given what they are due. The problem, then, is not that the concept of vulnerability is too broad—the problem is that widespread speciesist practices deprive so many animals of their due. The more we respect the basic claims of domesticated animals, research animals, and wild animals, the less vulnerable they will become. In turn, our attention will probably have to switch to other groups and species which remain particularly vulnerable in specifc situations: animals who are regarded as mere nuisances, disease-carriers, or pests and thus often incur many forms of morally problematic harms; animals who may be forgotten or ignored during disasters; and animals who will become victims of climate change.

What can we do to reduce vulnerability? The more we take concrete steps to diminish implicit and explicit biases and the more we carefully analyze certain situations to identify those individuals and groups who are at a higher risk of incurring unjustifed forms of harm and wrongs, the more we reduce the risk of unjust manifestations of vulnerability in the case of humans and animals.

While currently many, if not most, animal species can be considered particularly vulnerable, this situation will change as more animals are given their due. If we truly respect animals' basic claims without any speciesist prejudices and attitudes, fewer groups of animals will be particularly vulnerable. The concept of animal vulnerability can therefore unfold its true force and potential once we start to treat more animals as we should commensurately with what they deserve.

### References

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.


4.1. Photovoltaic panels generation forecasting

In order to forecast the electricity produced by photovoltaic panels, we used input data from one PV power plant of 7.6 kW located in Giurgiu City, Romania, installed at a prosumer side on his building facade. PV generates electricity for the consumption of the prosumer, and when the consumption is lower than the PV output, the electricity is sent to the grid. For our experiments, data was recorded at 10 minutes interval, from January to December 2015 and includes the following attributes: ambient temperature, humidity, solar radiation, wind direction, wind speed and PV output (generated power), having more than 50,000 records.

First, we applied the ARIMA models and we calculated the error distribution, MSE, MAPE and R correlation coefficient (Table 4).

From our observations, the correlation coefficient indicates a strong relationship between solar radiation and the PV power forecast. This close dependence showed that regression models are appropriate for this time series. The best results were obtained with ARIMA model where the accuracy is 96.5% that indicates that the model can be used in PV panels generation forecast.

We consider a second method based on feed-forward neural networks. Therefore, we trained and validated a set of ANN in Matlab using Levenberg-Marquardt (LM), Bayesian Regularization (BR) and Scaled Conjugate Gradient (SCG).

For ANN architecture, we analyzed various settings regarding the number of neurons per layer, number of hidden layers and training algorithms. After several tests, we chose the following architecture: the input layer with 5 neurons (ambient temperature, humidity, solar irradiation, wind direction and wind speed) 60 neurons on a hidden layer and a single output (the energy produced).

Table 4. Stochastic models results in case of PV generation forecasting.

For training, validating and testing: we have allocated 70% of the records for the training process, 15% for the validation process and 15% for the testing process. For training errors, we used the mean square error (MSE) by applying an error normalization process by configuring the normalization parameter to "standard". Thus, output parameter values were standardized, ranging from [�1, 1].

Taking into account the seasonal variations of the influence factors in Romania, we built artificial neural networks based on the three algorithms for each month and we compared the results in Table 5.

Comparing the ARIMA and ANN results, we consider that the most efficient approach is to use ANN on monthly data sets, which leads to excellent accuracy for every analyzed month. We also found that in almost 70% of cases, BR algorithm has a better generalization than LM or SCG algorithms. In 30% of cases, the highest accuracy was obtained with LM algorithm.


**2. Experimental**

Recycling process of kraft pulp fibres was observed on white kraft pulp sample prepared from hardwood mixture.

Pulp sample was processed into sheets of surface weight 800 – 900 g.m2 and brightness 82.7 % MgO.

Original sample after first processing by defibering, milling and drying represents zero recycling. Following pulp fibres processing simulated recycling. Pulp fibres undergo recycling for 8 times. Simulation could be considered as sufficient because usually pulp fibres are re-utilized for 4 to 5 times in practice.

Pulp was returned back into defibering, milling and drying process. The beating value was chosen as 29 °SR because of achieving sufficient strength of paper sheets without redundant fibre weakening and ensuring the possibility of next recycling.

Recycling of the Hardwood Kraft Pulp 271

Dimensional characteristics were observed by microscope until recently. Nowadays modern apparatus which can measure faster and are able to evaluate fibre dimensions are used.

Dimensional characteristics were monitored by Fiber Tester, which allows the evaluation of app. 20 000 fibres in pulp suspension containing 0.1 g of fibres in 100 ml of suspension in one measure (Karlsson, 2006). Double-dimensional view allows fibre length measuring separately and measuring of pulp fibre deformation during the running between two-glass

Following pulp fibre characteristics in recycling process were monitored during

• Local fibre deformation – angle of fibre deviation and fibre length between particular

Average values of chosen dimensional characteristics - length, width and final fibre shape

Dimensional characteristics of pulp fibres

sample 0,824 20,8 90,1

0th recycling 0,801 0,792 0,792 20,3 21,0 20,3 89,7 89,9 89,9 1st recycling 0,774 0,792 0,782 20,2 20,9 20,2 89,9 89,5 90,1 2nd recycling 0,760 0,784 0,789 20,0 20,6 19,8 90,0 89,2 91,2 3rd recycling 0,766 0,777 0,771 19,8 20,5 20,0 89,5 89,5 89,6 4st recycling 0,769 0,770 0,772 20,4 20,4 20,2 87,2 89,6 90,2 5st recycling 0,767 0,769 0,761 19,8 20,3 19,9 86,7 89,7 89,3 6st recycling 0,757 0,768 0,761 19,5 20,3 19,9 90,3 89,5 89,4 7st recycling 0,769 0,762 0,754 19,9 20,2 19,8 89,4 89,5 89,4 8st recycling 0,771 0,762 0,744 19,8 20,2 19,8 89,8 89,7 89,3

Average fibre width [μm]

Drying temperature [°C] 80 100 120 80 100 120 80 100 120

Fibre shape factor [%]

• Fibre length distribution – in 75 classed divided by 0.1 mm upto 7.5 mm • Fibre width distribution – in 50 classes divided by 2 µm upto 100 µm

• Percentual fibre distribution within configured length and width interval

factor from multiple measures of fibre suspension are stated in tab. 2.

Average fibre length [mm]

Table 2. Dimensional characteristics of pulp fibres.

plates.

dimensional analysis:

Pulp sample

Original

• Average fibre length • Average fibre width • Fibre shape factor

• Fine ratio – fibres up to length 0.2 mm

angles of fibre deviation.

The drying temperature influence on chosen pulp fibre properties was observed at 80 °C, 100 °C a 120 °C. These chosen temperatures cover the action of usually used temperature (100 °C) and extreme temperature values (80 °C and 120 °C) which were chosen for comparison.

Pulp samples taken from each recycling level were used for sheet preparation. Sheets surface weight was determined as relative value needed for calculation of chosen characteristics.

Standardly used procedures were applied for recycled pulp treatment (dry substance determination, wet laboratory defibering, laboratory beating in PFI mill, laboratory sheet preparation for physical tests, determination of dewatering ability according to Schopper-Rieglera, determination of surface weight).

Chosen properties were observed on the sheets prepared from original pulp, pulp after first processing (zero recycling) and pulp from particular recycling degrees:


**5. Conclusions**

The NR-TiO2 TENG for harvesting mechanical energy into electricity was successfully fabricated. The addition of rutile TiO2 nanoparticles at 0.5%wt of NR latex to form NR-TiO2 composite was found to enhance energy conversion efficiency of the TENG. The modification of TiO2 by the ball-milling technique for 24 h prior to mix with NR materials was found to effectively disintegrate TiO2 nanoparticles which consequently helped the dispersion of the nanoparticle fillers in the polymer matrix. Owing to the high dielectric constant of TiO2 fillers, the dielectric constant of the NR-TiO2-B24h film increased with increasing TiO2 concentration. The NR-TiO2-B24h film with improved dielectric constant attributed to the enhancement of TENG electrical output with the highest power density of 237 mW/m2. This work showed the potential applications of NR-TiO2 TENG as an environmentally friendly power source for portable electronic devices.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/polym13132213/s1, Video S1: The demonstrations of TENG to operate a portable calculator.

**Author Contributions:** Conceptualization, V.H.; Methodology, V.H. and W.B.; Performing experiment, W.B.; Validation, V.H., C.C., V.N.H.; Investigation, V.H. and W.B.; Writing—original draft preparation, V.H.; Writing—review and editing, V.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Basic Research Fund of Khon Kaen University, grant number 1500147, the Research Network NANOTEC (RNN) program of the National Nanotechnology Center (NANOTEC), NSTDA, Ministry of Higher Education, Science, Research and Innovation (MHESI) and Khon Kaen University, Thailand, the Thailand Center of Excellence in Physics (ThEP), and Research and Graduate Studies, Khon Kaen University.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the article.

**Acknowledgments:** This work was supported by the Research Network NANOTEC (RNN) program of the National Nanotechnology Center (NANOTEC), NSTDA, Ministry of Higher Education, Science, Research and Innovation (MHESI) and Khon Kaen University, Thailand, the Thailand Center of Excellence in Physics (ThEP), the Basic Research Fund of Khon Kaen University[Grant No. 1500147], and Research and Graduate Studies, Khon Kaen University. WB would like to thank for the support from Thailand Graduate Institute of Science and Technology (TGIST) (SCA-CO-2563-12216-TH).

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


**Hyaluronan Associated Inflammation and Microenvironment Remodelling Influences Breast Cancer Progression**

Caitlin Ward1, Catalina Vasquez1,2, Cornelia Tolg1,3, Patrick G. Telmer1 and Eva Turley1,4,5 *1London Regional Cancer Program, London Health Sciences Center, Victoria Hospital, London ON 2Dept. of Medical Biophysics University of Western Ontario 3The Hospital for Sick Children, Toronto ON 4Dept. of Oncology, University of Western Ontario, London ON 5Dept. of Biochemistry, University of Western Ontario, London ON Canada*