**4. Conclusion**

OPG is rare and difficult to treat the tumor. Improvement in vision and/or checking further progression should be the aim of selecting a treatment modality. Results with advanced techniques of radiotherapy and newer chemotherapy regimens are promising.


**Table 1.** Stability constants of complexes of bispidine-based bis-azoles with Cu(II) with various stoichiometry. Stability constants were calculated based on the UV-vis measurements using the Bouguer-Lambert-Beer law and the mass action law (see Supplementary Information file).

\* the complexes of this stoichiometry did not form.

Importantly, λmax of the obtained complexes were in the visible wavelength range and closer to 633 nm laser (Table 1) compared to absorbance of CAs [43]. The measured stability constants demonstrated that more stable CuL complexes were formed between the Cu(II) cation and L3, as with the most nonpolar compound. For the most stable complex, CuL3, it was possible to obtain a solid sample and take an X-ray spectrum, confirming the 1:1 structure stoichiometry. For L1, a 1:1 complex could not be obtained, which was presumably due to the excessive polarity of the substituent. Based on the obtained data, a 1:1:1 complex was chosen for further determination of CA with various bispidine-based bis-azoles.

We first studied the intrinsic spectra of Cu(II) with the synthesized bispidine-based bis-azole ligands, which could cause a background in SERS spectra for CA detection. Thus, the optimal concentrations of the indicator system were selected by varying the Cu(II) and each of the considered ligands concentrations in the corresponding solvents from Table 1 in the range of 1–10 mM. The complex formation for L1 with copper ions on the SERS surface resulted in the significant interfering background signals (Figure 3a). This made determination of catecholamines in the corresponding ternary complexes challenging. Most likely, this was related to the additional interaction of –COOH groups of L1 with –NH2 groups of chitosan deposited onto silver nanoparticles. Thus, the indicator system with Cu(II) and L1 was not further considered for sensitive determination of CAs. For L3, its complex formation with copper ions also contributed to the additional spectral background (Figure 3b) due to the intrinsic intense characteristic vibrations of the phenyl groups in L3 structure. The SERS spectra shown in Figure 3 demonstrated that L2 caused the least background, and the ligands could be used at 1, 4, and 10 mM concentrations for L1, L3, and L2, respectively. L3 was advantageous as it showed higher SERS intensities, while L2 provided lower spectral background. Thus, L3 and L2 were chosen for further development of approach for CA detection.

#### *3.3. Complexes of Catecholamines with Cu(II) Ions and Bispidine-Based Bis-Azole Ligands*

To identify CA by SERS, the optimal concentration of the indicator system was selected by varying the Cu(II) and each of the considered ligands concentrations in the corresponding solvents from Table 1 in the range of 1–10 mM at a constant concentration of DA, as a model catecholamine. The SERS spectra were recorded with a 633 nm laser wavelength and an extinction time of 10 s. Raman spectra of CAs have been preliminary modeled with density functional theory (DFT) (see Supplementary Information file). Strong modes of CA agreed well with experimental SERS of CA complexes. The SERS spectra in Figure 4a showed that even at an indicator system concentration of 1 mM, intense signals were observed in the SERS spectra for the DA complex with L3 and Cu(II) ions. The indicator system with Cu(II) and L2 demonstrated stronger SERS signals of DA at 8 mM concentration based on the majority of Raman peaks (Figure 4b). The difference in the optimal concentrations might be connected to the differences in chemical structure of the studied ligands. From the SERS spectra shown in Figure 3, carboxyl and phenyl groups of L1 and L3, respectively, demonstrated more effective coordination to the AgNPs surface, compared to amino groups of L2.

**Figure 4.** Raman intensities of characteristic DA bands in the SERS spectra at different concentrations of Cu(II) and ligands for: (**a**) L3 and (**b**) L2. Error bars are shown for *n* = 20, *p* = 0.95.

With using identical experimental conditions, areas of the SERS-sensing surface, concentrations and volumes of solutions of the Cu(II):bispidine-based bis-azole components applied to the surface, and Raman microscope settings, we obtained the calibration curves for CAs (Figure 5). Notably, the L3 ligand with phenyl groups demonstrated sharper SERS peaks as well as higher sensitivity compared to the indicator system with amino groups in L2. This could be explained by NH2-groups in L2 structure also interacting with the

AgNP-based surface resulting in the presence of differently oriented DA:Cu(II):L2 complexes on the SERS substrate surface. Figure 5c shows the SERS spectra of DA obtained on the developed SERS-active surface at various concentrations of DA in the reaction mixture (the indicator system is L3:Cu(II) ions at 1 mM concentration). The linear trend for DA determination was observed at a 3.2 × 10−12–1 × 10−8 M concentration range. Normal values of catecholamines in blood plasma were reported to be 4 × 10−11–4.5 × 10−9 M [46], 4.5 × 10−10–2.49 × 10−9 M [47], and 2 × 10−11–4.67 × 10−10 M [47] for DA, NA, and AD, respectively. Thus, the proposed approach demonstrated promise for rapid multiplex determination of catecholamines: dopamine, adrenaline, and noradrenaline which could be applied for "point-of-care" analysis (Figure 6).

**Figure 5.** (**a**) Typical SERS spectra of the blank non-modified SERS substrate, DA (0.1 mM), L2 (8 mM), L3 (1 mM), the indicator system: Cu(II) ions with L2 (8 mM), the indicator system: Cu(II) ions with L3 (1 mM), DA (0.1 mM) applied onto the SERS substrate modified with Cu(II) ions and L2 (8 mM), and DA (0.1 mM) applied onto the SERS substrate modified with Cu(II) ions and L3 (1 mM). (**b**) Single-crystal structure of the bispidine-based bis-azole ligand L3. (**c**) SERS spectra of various DA concentrations applied onto the SERS substrate modified with Cu(II) ions and L3 (1 mM). All SERS spectra were measured on the silver nanostructured surface coated with the chitosan layer at the resonant 633 nm laser wavelength. (**d**) Calibration curve for DA determination with Cu(II) ions and L2 (8 mM) based on Raman intensity of 1597 cm−1 peak. Pale orange lines demonstrate confidence corridor with *p* = 0.95. (**e**) Calibration curve for DA determination with Cu(II) ions and L3 (1 mM) based on Raman intensity of 1612 cm–1 peak. Pale blue lines demonstrate confidence corridor with mboxemphp = 0.95.

**Figure 6.** SERS spectra of the DA (0.1 mM), NA (0.1 mM), and AD (0.1 mM) applied onto the SERS substrate modified with Cu(II) ions and L3 (1 mM) at 633 nm excitation laser wavelength.

#### **4. Conclusions**

Herein, we propose an approach for catecholamine detection based on trapping them into colored stable complexes on the SERS-active surface. The MIRRACLE (Molecular Immobilization and Resonant Raman Amplification by Complex-Loaded Enhancers) methodology allowed for sensitive determination of dopamine in 3.2 × 10−12–1 × 10−8 M concentration range. The determination of catecholamines in complexes with Cu(II) and bispidine-based bis-azole ligands on a nanostructured silver surface coated with a thin layer of chitosan by SERS allowed the selective determination of dopamine, adrenaline, and noradrenaline. Four synthesized bispidine-based bis-azole ligands served as pliers in the ternary complex with Cu(II) ions and catecholamines. The limits of detection of catecholamines using the developed method make it possible to determine them at the level of picomolar concentrations, which corresponds to the reference values in the blood plasma of healthy people. Therefore, the use of the proposed technique provides a potential opportunity to determine catecholamine neurotransmitters in biological fluids using SERS spectroscopy, specifically for diagnosis of neuroendocrine disorders. Further prospects in this area lie in the improvement and optimization of methods for the multiplexed determination of the largest possible set of molecules–markers of neurotransmitter metabolism

disorders, as well as the subsequent detection of the correlation of neuroendocrine and neurodegenerative diseases with concentrations and their ratios in biological fluids with the possibility of non-invasive diagnostics.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bios13010124/s1. Figure S1: UV-vis spectra of 5 mM solutions of 8a–AD, 8b–DA and their corresponding complexes with: 1–Cu(II), 2–Al(III), 3–Fe(II), 4–Co(II), 5–Fe(III), 6–Ni(II), 7–Mg(II). Figure S2: UV-vis spectra of DA and Cu(II) equimolar mixture in time 0–15 min. Table S1: Raman bands theoretically calculated, published in the literature, and experimentally observed on the AgNP-based substrate for [Cu(semiquinone)3] − (λex 632.8 nm). Figure S3: UV-vis spectra for spectrophotometric titration of L4 with Cu2+ solution in (a) 100% MeCN; (b) 50% MeCN–50% H2O. Figure S4: Spectrophotometric titration of L4 with Cu2+ solution in (a) 100% MeCN (λabs = 577 nm); (b) 50% MeCN–50% H2O (λabs = 583 nm). Figure S5: (a) UV-vis spectra for spectrophotometric titration of Cu2+ with L4 solution in 50% MeCN–50% H2O. (b) Spectrophotometric titration of Cu2+ with L4 solution in 50% MeCN–50% H2O (λabs = 583 nm). Figure S6: UV-vis spectra for spectrophotometric titration of L3 with Cu2+ solution in (a) 100% MeCN; (b) 50% MeCN–50% H2O. Spectrophotometric titration of L3 with Cu2+ solution in (c) 100% MeCN (λabs = 577 nm and λabs = 697 nm); (b) 50% MeCN–50% H2O (λabs = 583 nm). Figure S7: (a) UV-vis spectra for spectrophotometric titration of Cu2+ with L3 solution in 50% MeCN–50% H2O. (b) Spectrophotometric titration of Cu2+ with L3 solution in 50% MeCN–50% H2O (λabs = 583 nm). Figure S8: (a) UV-vis spectra for spectrophotometric titration of Cu2+ with L4 solution in 100% MeCN. Figure S9: Spectrophotometric titration of L1 with Cu2+ solution in H2O (λabs = 577 nm). Table S2: The calculated values of the constants of complex formation of copper with water-insoluble bispidine-type ligands. Table S3: The protonation constants for L1 in H2O (μ = 0.1 M) at 25 ◦C. Figure S10: (a) Potentiometric titration of L1 (1 mM) in H2O in the presence of HClO4 (4 mM), μ = 0.1 M KNO3 at 25 ◦C with NaOH solution (0.1 M). (b) Distribution of protonated and deprotonated forms of L1 (1 mM) in solution depending on pH. Table S4: The stability constants of complexes of L1 with cations Cu2+ and hydroxocomplexes Cu2+ in an aqueous solution (μ = 0.1 M) at 25 ◦C. Figure S11: (a) Potentiometric titration of L1 and L1 in the presence of Cu2+ (Cu2+ + L) in H2O (μ = 0.1 M KNO3) at 25 ◦C. (b) Distribution of different forms of L1 (1 mM) in the presence of Cu(II) perchlorate (1 mM) depending on pH. Figure S12: Typical SEM images of a proposed silver nanoparticle sensor surface (a) coated with a silver chitosan film of a silver nanoparticle surface (d) and its cross section (c); optical photograph of a silver nanostructured surface consisting of overlapping "coffee rings" (b). Figure S13: SERS spectra of various AD concentrations applied onto the SERS substrate modified with Cu(II) ions and L3 (1 mM). All SERS spectra were measured on the silver nanostructured surface coated with the chitosan layer at the resonant 633 nm laser wavelength. Figure S14: SERS spectra of various NA concentrations applied onto the SERS substrate modified with Cu(II) ions and L3 (1 mM). All SERS spectra were measured on the silver nanostructured surface coated with the chitosan layer at the resonant 633 nm laser wavelength. Figure S15: Optimized geometry for dopamine (DA) in "Q-Chem" with PBE0 method and 6-31G\* basis. Figure S16: Calculated normalized Raman spectrum for dopamine (DA) in "Q-Chem" with PBE0 method and 6-31G\* basis (Lorentzian). Table S5: Calculated normalized Raman shifts with intensities for dopamine (DA) in "Q-Chem" with PBE0 method and 6-31G\* basis (Lorentzian). Figure S17: Optimized geometry for noradrenaline (NA) in "Q-Chem" with PBE0 method and 6-31G\* basis. Figure S18: Calculated normalized Raman spectrum for noradrenaline (NA) in "Q-Chem" with PBE0 method and 6-31G\* basis (Lorentzian). Table S6: Calculated normalized Raman shifts with intensities for noradrenaline (NA) in "Q-Chem" with PBE0 method and 6-31G\* basis (Lorentzian). Figure S19: Optimized geometry for adrenaline (AD) in "Q-Chem" with PBE0 method and 6-31G\* basis. Figure S20: Calculated normalized Raman spectrum for adrenaline (AD) in "Q-Chem" with PBE0 method and 6-31G\* basis (Lorentzian). Table S7: Calculated normalized Raman shifts with intensities for adrenaline (AD) in "Q-Chem" with PBE0 method and 6-31G\* basis (Lorentzian).

**Author Contributions:** Conceptualization, O.E.E., T.N.S. and I.A.V.; methodology, O.E.E., O.O.K., A.V.M. and B.V.E.; software, O.E.E., A.S.Z. and A.V.M.; validation, O.E.E., O.O.K. and A.S.Z.; formal analysis, A.S.Z., A.V.M. and B.V.E.; investigation, O.E.E., O.O.K., A.S.Z., A.V.M. and B.V.E.; resources, S.Z.V., T.N.S. and I.A.V.; data curation, O.E.E., O.O.K., A.S.Z., A.V.M. and B.V.E.; writing—original draft preparation, O.E.E.; writing—review and editing, all authors; visualization, O.E.E. and A.S.Z.; supervision, O.E.E., I.A.V. and S.Z.V.; project administration, T.N.S. and I.A.V.; funding acquisition, T.N.S. and I.A.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Russian Science Foundation (grant No. 20-13-00330).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This research was supported by the Interdisciplinary Scientific and Educational School of Moscow University "Future Planet: Global Environmental Change." The research was carried out using the equipment of MSU Shared Research Equipment Center "Technologies for obtaining new nanostructured materials and their complex study" and purchased by MSU in the frame of the Equipment Renovation Program (National Project "Science") and in the frame of the MSU Program of Development.

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

#### **References**

on copper (II)-chitosan-modified SERS-active metallic nanostructured substrates for multiplex determination of dopamine, norepinephrine, and epinephrine. *Mikrochim. Acta* **2022**, *189*, 211. [CrossRef] [PubMed]

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*4.1. Animals*

Three-week-old, male, non-obese diabetic/severe combined immunodeficiency (NOD/Scid-NOD.CB17-Prkdscid/NCrHsd) mice (*n* = 10) (Harlan Laboratories S.r.l. Barcelona, Spain) were used in this study. These mice present immunological multidysfunction, including absence of mature T and B cells, reductions in macrophage function, complement-dependent hemolytic activity, and NK cell activity. [62] All mice were caged under standard light and temperature conditions with free access to food and water throughout the study. All e fforts were made to minimize su ffering and all animals were sacrificed at the end of the study.

All experimental procedures were approved and regulated by the Animal Experimentation Ethics Committee—University of Alcalá and University Hospital of Getafe (IRB: PROEX reference 237/15, 9 October, 2015), in accordance with the Royal Decree 53/2013 and the European Community Council Directive.

#### *4.2. PU model on Human Skin*

Ten NOD/Scid mice were engrafted under general anesthesia (Ohmeda, BOC Health Care) with female human skin. All human skin (*n* = 5) came from abdominoplasty procedures. Written informed consent was obtained from all patients. The skin was immediately stored after haverst in D-MEM (Dulbecco/Vogt modified Eagle's minimal essential medium) at a temperature of 4 ◦C. Skin grafting was performed during the first 24 h after extraction. Human full-thickness skin grafts (FTSGs) were placed onto a 4 × 3 cm wound. Mice skin was incised down to the muscle and removed, exposing muscular layer. FTSGs were sutured in place with 4/0 nylon. A tie-over bolster dressing was placed for the first 5 days. The same skin donor was used to graft two mice (in order to pair two mice with the same human skin donor). After 60 days, a compression device was applied to the human skin graft, as previously described. [59] The clip exerted a pressure of 150 mmHg, measured with a dynamometer. Three cycles of compression–release (8 h of clamping after 16 h of no compression) were applied to simulate a pressure ulcer, based on the method described by Stadler et al. [63].

#### *4.3. Recombinant Human Growth Hormone (rhGH)*

Genotonorm Kabipen 5.3 mg (Pfizer, Madrid, Spain), solution for injection in a pre-filled pen, was provided by the Department of Pharmacy of the University Hospital of Getafe and stored at a temperature between 2 and 8 ◦C. The use of the rhGH is restricted to the hospital setting, and its use was approved and regulated by the Animal Experimentation Ethics Committee University Hospital of Getafe (PROEX reference 237/15) in accordance with the Royal Decree 53/2013 and the European Community Council Directive.

#### *4.4. Local Treatment of the PU with rhGH*

PU formation was confirmed by visual assessment after the last cycle of compression–release. Paired mice were assigned randomly to the rhGH group (*n* = 5) or to the control group (*n* = 5). We designed a protocol for local administration of the hormone. The concentration of GH was determined based on previous studies. [55] In the rhGH group, the five mice were treated with four local rhGH intradermal injections, each of 0.15 mg (0.5 IU), applied to the PU edges on human skin (Figure 8). The injection protocol started 24 h after the last cycle of compression–release, and the administration of the hormone was repeated once per week for four weeks, always the same day of the week at the same hour. In the control group, the five mice received the same care but without the local administration of the rhGH.

**Figure 8.** Local administration of the rhGH. Note the intradermal injection of the rhGH applied to the pressure ulcer edges on human skin.


Under this approach, the obligation of civility towards the people you work alongside, live with as near neighbours, queue with in GP surgery waiting rooms, accidentally jostle as you board public transport, is to be replaced with mistrust and suspicion, right to the point of a continual questioning as to whether this person even has a right to be in the country. A mandate to adopt this attitude of mind is already imposed on employers, who need to check the immigration status of anyone to whom they are thinking of offering a job. The new legislation extended this obligation to landlords in the private rented sector, compelling them under pain of a financial civil penalty to be sure that a prospective tenant was not an 'illegal'. At GP clinics, receptionists flick through patient passports trying to determine whether the person in front of them is entitled to treatment on the NHS. Schools include details of the nationalities of the children they are teaching in reports delivered to the Department for Education, and these details can then be shared with Home Office immigration enforcement officials. The business of querying, questioning and checking the immigration bona fides of just about everyone with whom they come into contact as the providers of services is now conducted by staff at local bank branches as much as by police officers on the beat. At more and more points in which citizens and neighbours interact with one another, with easy-going conviviality lubricating these countless transactions, the surveillance of the state is revealing itself in pursuit of its wish to see a higher degree of hostility between people.

As a consequence of the 2014 Act, which was followed by the 2016 Immigration Act, much of civil society and the realm of public service has been configured as a series of tripwires over which some would stumble and identify themselves as undesirable interlopers. But the experience of being subjected to this scrutiny leaves its mark even on the 'legal' migrant, and indeed on the person of colour who is associated with immigration because it was something their parents or grandparents underwent. Decades of progress towards something that deserves to be called integration based on mutual respect is in danger of being undone as the logic of immigration control forces everyone into an awareness of where their physiognomy or accent places them on the scale of potential immigrant. Places where, in the past, people would feel they could walk free and easily are in danger of becoming recast as areas to steer clear of because of the risk of being subjected to challenge about their right to be there or to use that service.

What does this point to in the future? The discussions we have had today have pointed to the possibility of using convivial research methodologies to assess relationships of power that exist between individuals, and also the role of the state in local communities on issues of surveillance and control. If it is to play this role, it must be designed into the types of toolkits that migrant community organisations and their allies can use in their day-to-day practical work. Building trust between citizens and migrants back into relationships that the government is striving to strip out of civil society will only happen if it is part of a strategy that brings tangible benefits for the newcomers. An area where this is happening is the collaboration that is helping migrants to overcome the worst of the disadvantages associated with immigration statuses that refuse 'recourse to public funds' ('No Recourse to Public Funds', or NRPF). Migrant support groups have had a degree of success in forcing social services departments to provide measures of relief to families that are on the edge of absolute poverty because of this restriction. When this lobbying power is married to legal casework around human rights issues, then it is possible to get the NRPF condition lifted entirely.

Campaigns against NRPF have worked hard to incorporate the voice of migrant communities directly into advocacy work, using techniques such as theatre and drama to tell stories. As has been presented by contributors to our discussion today, this works best when the idea of performance goes alongside the building of confidence among groups of people who might otherwise be demoralised by their experiences. The dual outcome here is both a coherent and compelling re-enactment of the predicament of living without the support available to others, and also a group of individuals less afraid to speak out, and confident in the power of the account of life that they have to offer. This moves us to the point where we are tooling up to challenge the power of governments and state authorities who are imposing these hardships on migrant communities.

If we do this properly, we should be aiming to get beyond the tendency of many activists to evade the realities of politics in favour of a softer, apolitical, human rights approach. I do not necessarily mean just traditionally understood parliamentary party politics, but the sort that goes on around the power relations of art, of everyday life and of personal experience. These all have something to say about the type of society that Britain is becoming, the forces that are mobilising to bring it about, and the things that are happening at the level of grass-roots communities that constitute resistance to these developments. Emancipatory interruptions to the logic of a society steeped in attitudes of neocolonialism are the outcomes we are aiming for here, and consciousness about the role of conviviality seems to be a good thing to carry into the work we do.

My final point about it concerns one of the power issues that have been discussed today and that are very important for groups working in the migrant rights field – namely those that we have with the people who fund us. We continually find ourselves having to report on the things we have achieved during the time the grant covers, and how our client group has benefited from our work. Yet it often feels false to report on what we are asked to think of as progress during times when we know that so much in the area of policy and rights has been going backwards. Insofar as it has taken place, it has been in the form of partial victories that have scarcely interrupted the succession of crises, and the return of tension and having to go back to fight once again on issues we thought we had settled decades ago. If we present our work as an advance along a linear route that has 'victory' marked at the end of it, we are surely deluding ourselves. We need another way to think about what we are winning from the battles we are having to fight on a daily basis.

As an answer to this, I am very much intrigued by the idea of a 'Red Queen race', which features in the story of *Alice through the Looking Glass*, and which is used by evolutionary biologists to explain why some species win out in the race for survival, and others become extinct. Alice's Red Queen described her predicament as a ruler as one that involved so much work that one has to run very hard merely to stay in the place where one is. That is a pretty accurate description of where we are in the world of the fight for the rights of migrants at the moment. As hard as we campaign, it is unlikely that our work will see any significant victories in the near future, given all the evidence about the anti-immigrant moods that prevail over public opinion and mainstream politics.

What we can say is that we are not alone in seeing difficulties in abundance for the years that come. Any sober analysis of the options that are open to government must consider the immense problems that stand before policymakers as they attempt new systems of immigration control within an economic and political context that otherwise demands the openness of borders and the circulation of workforces. We are now at the end of twenty years of attempts at immigration management, which have seen governments return to the statute books time and time again, constantly redrafting volumes of complex legislation and reconfiguring the agencies that are supposed to be enforcing these controls. The result at this moment in time is a sense of crisis for the direction of policy that is just as intense as it has been at any earlier point. We need to understand that our adversary in this work is running just as hard as we are, and also reporting as little in the way of clear, indisputable progress as we are.

So, what then is the point of a Red Queen race? It is to stay in the competition and match your opponent, pace for pace, across time. Our job can be seen as the need to keep people – our clients – in the race and supported well enough to ensure that they endure. But if this sounds dismal we should at least be encouraged by the fact that there is an end to a Red Queen race, and that comes when you are still around years after it has started, and the people you are opposing have dropped with exhaustion as time has gone by.

That is what we have to project into the debate about the rights of migrants: not just in terms of their dealings with the Home Office and immigration enforcement, but in terms of their access to social justice right the way across the spectrum. Over recent years we have seen more and more political forces alongside us running exactly the same Red Queen race, whether it is UKIP, whether it is the anti-immigration lobby group Migration Watch UK, or whatever. Their work has not been easy because it is not long before the logic of their political position can be represented as being patently ridiculous and unworkable.

Perhaps the thing we are looking for in our work is something as simple as the capacity to survive and endure in conditions where we are supposed to buckle and give in. This will mean countless battles on countless fronts in order to sustain a social existence that will surely not easily give up on the ideal of conviviality. Being conscious of that fact, and having strategies that make conviviality a political weapon, is one of the most important things for which we should be working.

#### Note

1. This chapter is an edited transcript of the discussant comments given by the author at the workshop at which most chapters were originally presented.

### Reference

Gilroy, Paul. 2004. *After Empire: Melancholia or Convivial Culture?* London: Routledge.

## 11 Breaking down barriers to co-production between research teams and civil society organisations

Karin Woodley and Charlotte Gilsenan

The work of civil society organisations has a value that is distinct from that of other sectors of society. Civil society organisations comprise independent, non-governmental and non-profit-making organisations, and include charities, voluntary and community groups, and social enterprises. They are predominantly 'value-driven' and work to achieve a broad range of social aims, including (but not limited to): extending social justice and promoting equality; poverty reduction; and dismantling barriers to societal cohesion, health, economic and environmental well-being.

In addition to their well-known function in undertaking direct interventions in social and economic life, civil society organisations have a long history of facilitating varying types of self-help, which, as a form of collective efficacy, often fill gaps in statutory services and respond directly to changing local need. Characterised by participatory decision-making and/or community ownership, civil society organisations have specialist knowledge of the needs of communities. The best of these do their work in partnership with a broad range of community members and engage with the most marginalised and vulnerable. This means they can be ideally placed to understand the changing needs of communities and to deliver innovative and solution-focused responses, particularly where state interventions are failing.

The needs of communities are varied and interlinked; many civil society organisations have therefore evolved to be multipurpose so that they can respond to people's needs holistically. These organisations have a unique capacity to identify relationships and interdependencies between issues that are not captured or addressed by more siloed statutory services.

Increasing political emphasis on devolution and localism requires the generation and collation of evidence at a local level, much closer to the complexities and dynamics of whole communities, which are so often hidden from national view, with more accessible opportunities for evidence sharing. This provides opportunities for place-based civil society organisations to demonstrate what delivers social and economic benefits in neighbourhoods, as well as evidence of what activities successfully support vulnerable people to take control of their lives and their environment, and build the resilience they need to thrive.

## The need for co-production between research teams and civil society organisations

Increasing economic and social vulnerability, as well as increasing ethnic and religious conflict, drives us to identify and share effective solutions to poverty and social injustice. However, although these are the foremost concerns of civil society, what works in civil society has either not been captured or not been organised into a cumulative and coherent body of knowledge. As a result, the effectiveness (or lack of effectiveness) of civil society in helping to ensure society is economically and socially accountable, responsive, inclusive and effective can be obscured and/or misunderstood.

Decentralisation means that previously state-delivered services are increasingly contracted to civil society organisations operating on local and neighbourhood levels. This means it is increasingly important that we improve our understanding of what works and what does not. Effective co-production between research teams and civil society organisations is therefore needed to:

## The barriers to co-production

Locally driven and participatory civil society organisations have a rich history of gathering compelling evidence, often in alliance with academic leads, that can have a groundbreaking effect on national policy and practice. Work undertaken by the network of settlement organisations in the first half of the twentieth century, for example, ensured that evidence generated and owned at a community level played a significant role in the development of the welfare state. However, the types of insight generated by locally driven organisations no longer have the status they once did. This is due, in part, to the rise and subsequent dominance of the social sciences in the production and distribution of knowledge regarding social and economic life in the UK. Another factor has been the acquisition of greater research and development budgets by national charities to address government's preoccupation with knowledge production and evidence gathering that can support scalable initiatives.

Partnership working between locally driven organisations and research bodies can and does produce useful and relevant insights. However, there are significant limitations to the structures, mechanisms and outputs that characterise current partnership working. For example, the predominance of civil society–research partnerships restricted to impact measurement means that outputs produced by such activities are of value to practitioners and, to a limited extent, policymakers, but they offer limited usefulness to more complex analyses of social and economic life.

When research partnerships set out to produce more ambitious outputs, traditional research practices often fail to engage effectively with the stakeholders of civil society organisations or to achieve mutually and reciprocally identified aims. This is, in part, due to structural and environmental conditions. It is often the case that civil society organisations have insufficient resources and expertise to independently and autonomously pursue effective partnerships. Asymmetries in resources mean that, when partnerships are created, hierarchical relationships emerge between researchers and civil society, with researchers as the dominant 'partner'. These asymmetries limit the ability of civil society organisations (and their stakeholders) to influence and shape research design and to take ownership of research outputs. This presents a significant obstacle to the development of an analytical research framework that can fully exploit the potentialities within civil society–research partnership.

Such structural limitations to effective engagement in research partnerships are compounded by divergences in certain key assumptions underpinning research design, methods and objectives. On the whole, civil society organisations are driven to produce knowledge with, rather than for or about, research subjects. They tend to opt for participatory and/or problem–solution-oriented research methodologies, such as action research and peer-to-peer approaches.

Although there are good academic practitioners in these fields, in the main, such methods challenge conventional academic models, ethics and doctoral training by questioning and disrupting the traditional relationship between researchers and their research subjects.

## Breaking down barriers to co-production

## Conviviality

For researchers to capture and analyse the realities of community members' lives, methodologies must give communities a voice and role in the research process beyond providing access to research participants. A one-way process of 'tell us, tell us, tell us' lacks reciprocity and, by failing to respect and value the life experiences and creativity of community members, undermines the quality of evidence gathered.

To ensure that communities are treated like partners as opposed to information portals:

Research bodies need to:

Co-production opens up opportunities to:

## Cambridge House

Cambridge House is a social action centre tackling poverty and social injustice. We deliver intensive front-line services for people facing multiple disadvantage and complex, interrelated needs from our own building in Southwark, south London.

Working regionally and nationally, we remain firmly rooted in our local neighbourhood as a community asset and hub. We deliver services to more than a hundred thousand people each year, and use this experience to challenge public perception and stigma, shape research and knowledge-exchange activities, and promote systemic change.

Founded in 1889 by the University of Cambridge, Cambridge House is one the founders of the Settlement Movement and played a key role in the establishment of the welfare state in Britain. A century later, we continue to pursue our vision of a society without poverty where all people are valued, treated equally and lead fulfilling and productive lives.

Our front-line services include a law centre, professional advocacy services for children and adults with learning difficulties and mental health conditions, youth empowerment activities, projects for children and adults with disabilities, and office and conference spaces for 16 resident charities and community groups.

## 12 Afterword: Giving multiculture a name

Les Back

The idea of conviviality, as many of the contributions to this important book demonstrate, provides us with an alternative way to give a name to multicultural coexistence (Gilroy 2004). As Nowicka and Vertovec (2014, 1) point out, the idea of conviviality is based on the Latin root for 'with' and 'living'. In Gilroy's formulation – inspired by Ivan Ilich and Theodor Adorno – conviviality is always proximate to its negation. For Gilroy, the shadow of racism, imperial melancholia, 'anti-terrorist' securitisation and war is cast over the impulse to live differently. Understanding how these tensions are lived out involves writing what he calls 'counter histories of cultural relations'. 'This negative work', Gilroy (2004, 161) argues in his book *After Empire*, 'can discover and explore some of the emancipatory possibilities that are implicitly at stake in convivial culture but do not announce themselves, preferring to remain hidden and unpredictable'. Gilroy himself leaves the notion of conviviality under-explicated, in part because it acts like a fugitive hinterland in the context of racism and melancholic nationalism.

For Gilroy (2006, 27), a 'habitable multiculture depends upon working through the legacies of departed empire'. This means reckoning with the legacy of racisms both old and new that remain socially alive within worlds of coexistence. 'Even if today's unwanted newcomers – from Brazil or Eastern Europe – are not actually postcolonials, they may still carry all the ambivalence of the vanished empire within them' (Gilroy 2006, 31). Gilroy offers a way of understanding conviviality as an unruly, spontaneous social pattern produced by metropolitan social groups living in close proximity with each other. Here, racial differences become ordinary, banal, unremarkable and sometimes mundane to the point of boredom. In Gilroy's conception, the forms of comprehension and communication that result produce 'everyday virtues' that enrich city life. Looser social bonds result, in which 'a degree of differentiation can be combined with a large measure of overlapping' (Gilroy 2006, 40). What the contributors to this book argue is that this prosaic form of multicultural 'good sense' is best understood by focusing on the sensibilities – as practical tools – that make convivial culture.

What the chapters in this book also do is embody in their practice a kind of convivial methodology, or what I would call a sociable form of research craft that traverses traditional disciplinary boundaries in which the relationships between the participants and the researcher or writer are reformulated. This method is attentive to the minor miracles or routine wonders that are so often looked past and unremarked upon. Taken together, the chapters of the book are inventive both in terms of form and in terms of practice that is deeply attentive to the embodied nature of thinking and learning, whether it be through walking along a street or being still and attentively present in one spot.

Gilroy (2006, 40) insists that 'recognising conviviality should not signify the absence of racism'. What the notion of conviviality does offer is an alternative understanding of culture that focuses on what people do every day, rather than always reducing them to their cultural origins. As Gilroy (2006, 40) concludes, 'Culture is misunderstood and oversimplified through being conceived as ethnic property to be owned and held under copyright. The vital alternative comprehends unruly, convivial multiculture as a sort of "Open-Source" co-production.' Gilroy offers a series of analytical instruments that enable us to understand the coexistence of both racism and convivial culture.

Conviviality, then, should not be a byword for saccharine diversity fantasies. Gill Valentine (2008, 334) points out rightly that it is foolhardy to replicate the mistaken logic of Gordon Allport's (1954) famous 'contact hypothesis', and assume that conviviality is the simple consequence of proximity to difference, although many of Valentine's findings merely restate observations that have been long-established in the field (see Hewitt 1986). Gilroy (2004) – recalling Adorno – stresses the 'negative dialectics of convivial culture' because he insists on avoiding a simplistic account of affirmative multiculture. While there are certainly shifts and forms of cultural bridging, there is no positive resolution of the dialectical tension between racism and multiculture. This involves having to live with countervailing conditions, or 'metropolitan paradoxes', both in places and in people, that do not hold out the possibility of synthesis (Back 1996).

Rather, conviviality here is a capability recalling philosopher Martha Nussbaum's (2006, 74) focus on human capabilities as a set of minimum core entitlements for 'the dignity of a human being, and of a life worthy of that dignity'. In Nussbaum's treatment, capabilities are minimum measures of justice that should be afforded to all to ensure 'human flourishing'. Her parameters are much wider than the concerns of the authors of this book, but they include relevantly: 'being able to live with and towards others, to recognize and show concern for other human beings, to engage in various forms of social interaction; to be able to imagine the situation of another' (Nussbaum 2006, 77). Racism damages that capability. Unlike Nussbaum, I am not making a normative or universal argument, and I think this scepticism is shared across this wonderful collection. Rather, we want to foreground how people who come into profound contact with each other use specific tools to make living in overlapping worlds and urban contact zones workable.

Central to Ivan Illich's initial formulation of conviviality is the emphasis on *tools*, and this has been returned to as an incitement in the book to think about how convivial multicultural worlds are made. This offers a way out of either reducing conviviality to a sense of 'identity' or claiming that a kind of underlying 'cultural ecology' structures and therefore explains convivial life. By paying close attention to the experience of how people live multicultural coexistence, we can find glimpses of the tools and resources that might enable us to make a different kind of society, one that is less disposed to melancholic hatred. In a time of populism and emboldened racist voices, this seems to me to be an urgent political task. Illich's (2009, 11) concerns were very different – he was seeking to find alternatives to the damaging nature of industrial society – and he notes, 'individuals need tools to move and dwell'. These tools include a refusal of hatred, as well as an attentiveness to, and curiosity for, what is both familiar and strange in the street. Using these tools affords the capacity to build and make sense out of the tangle of local circumstances and global connections. Interestingly, Illich (2009, 15) focuses on the 'structure of the tools, not on the character structure of the users'. In the same vein, I do not read this book as the search to identify the character structure of nice 'convivial people'. Rather, I read its chapters as in various ways trying to identify the toolbox of convivial capabilities that people in their ordinary circumstances of life use to navigate their way through a world shaped not only by hatred but also through coexistence with their neighbours, their friends and even their foes.

## References

Allport, Gordon W. 1954. *The Nature of Prejudice*. Reading, MA: Addison-Wesley.

## Index

absolutism, ethnic 2 academic knowledge distrust of 4; production of 29–30 academic researchers collaboration with 10; evaluating the output of 124, 132, 135–8, 169 action research 18, 123; *see also* participatory action research activism 175 Adorno, Theodor 100, 102–3, 192–3 advocacy for family members 77 agency, collaborative 7 Ahmed, Nazneen 8; *co-author of Chapter 4* Allport, Gordon 193 Amin, Ash 26, 61, 76, 78, 135 Amit, Vered 127 analytical conviviality 21–3, 53 antisocial behaviour 128 area-based social policy initiatives 127, 138 Arendt, Hannah 135, 137 Arizpe, Lourdes 20 arts-based research methods 99–100 arts-based workshops 77–92, 104 arts in social life 97, 100 Askins, Kye 62, 71 asylum seekers 99–102, 106, 147, 177 Attia, Mariam 28 authoritarianism 128 Back, Les 10, 22, 39–40, 98, 117, 124, 129, 132, 155; *author of Chapter 12* Basch, Linda 161 Bauman, Zygmunt 127, 138 Beckett, Samuel 123–5, 141–2 Beinart, Katy 8, 59, 64–71; *co-author of Chapter 4* Benjamin, Jessica 100, 102 Berg, Mette Louise 162–3, 171; *co-author of Chapter 1 and co-editor* Bergold, Jarg 5 Bermondsey 129 Big Society programme 130 Birmingham 129, 144–52 Blanc, Cristina Szanton 161 Boddy, Janet 47 Boetti, Alighiero 64 Borland, Katherine 50 Bosnian community in Britain 96–7, 103–4 Brubaker, Rogers 170 Butler, Judith 39

Caillé, Alain 20 Callahan, Manuel 39 Camberwell 128 Cambridge House 10, 190–1 Cameron, David 130 'cannibalistic *gemeinschaft*' (Keith) 131, 134, 137 capabilities 10, 194 Center for Convivial Research and Autonomy (CERA) 38 Centrala 144–51 Centre on Migration, Policy and Society (COMPAS) 124 Centre for Urban and Community Research (CUCR) 124 changed understandings of social life 41 Chicago 129 Chicago School 160 child language brokers 76–82, 87, 90, 92 children's homes 43–4 civil society and civil society organisations 181–3, 186–7 civility 39–40, 182 climate change 49 collaborative research 124, 127, 136, 138, 143, 155 community, concepts of 129 community arts 102 consensus 52 'contact hypothesis' (Allport) 193 contention, value of 137–8, 169 'Conversio' project 150–1 *Convivencia* 20 convivial culture 21, 193 convivial economies 20 convivial encounters 17, 28–9, 66 convivial findings from research 43 convivial interpretations of research 40 convivial living together 1, 8, 76, 124, 194 convivial research 2–10, 18–19, 26, 29–30, 36, 38, 45, 53, 96, 99, 104, 118, 124, 131, 143, 156, 182; arts practice seen as 60; components of 40 convivial sensibility 87, 96, 103 convivial situations 25–8 convivial space 92, 117, 144 'convivial turn' in the social sciences 2–3, 61, 76, 159–60 convivialism, definition of 20

conviviality aspects of 3; challenge of 29; concept of 2–4, 17–18, 37, 76, 181, 192–3; consciousness of the role of 183; critiques of the theory of 61; as a desired state 20; definition of 99; development of 70–1; 'doing' of 9; hallmarks of 137; helping or hindrance of 9–10; institutional arrangements for 9; lack of 46; literature on 61; manifestations of 17; mediators of 9; multifaceted nature of 37, 53; *normative* or *analytical* 8, 18–23; as an outcome of urban complexity 22; as a political weapon 185; in the research process 36–7, 41–53; seen as a capability 194; seen from different perspectives 10, 46–7, 49; as a skill and capacity 7, 10; temporal aspects of 154; understanding of 39; uses of the term 17, 38 Cooke, Bill 138 co-production 29, 132; barriers to 188–90; between research teams civil society organisations 187; opening up opportunities 190 cosmopolitanism 3, 29 counter-hegemonic knowledge 103 craft skills in research 132–4 Crafter, Sarah 8–9; *co-author of Chapter 5* cultural contact zones 9, 79–80, 90 cultural ecology 194 culture, misconception of 193

Dawidek, Małgorzata 150 Deptford 131 Disch, Lisa Jane 134–5 diversity 4, 20, 137, 159; *see also* super-diversity 'diversity turn' in ethnicity and migration studies 123–4 Dwyer, Claire 8; *co-author of Chapter 4*

Ealing 60, 64, 67–8 economic migrants 176 Edge, Julian 28 Elias, Norbert 128–9 elicitation techniques *see* music-elicitation interviews; photo elicitation Elliott, Jane 42 emotions aroused by research 40; importance and power of 98 empathy 27–8, 103 environmental issues 47–9 essentialism 26–7 ethical reflection 29 ethics 4, 103, 170; analytical 50 ethno-mimesis 102–4, 117 *ethnographic sensibility* 136 ethnographic studies 5, 18, 98, 101–4, 117, 130–1, 135–6, 160 European Union (EU) 132, 176 Evans, James 47 everyday situations and encounters 29–30, 61, 76–9, 133, 135, 159, 164, 181, 193 Ex-votos 81–2 *exclusive* communities 128 Exiled Writers Ink 105

Fabric of Faith project 59–63, 66–7, 70–1 facilitation of conviviality 9, 27–8, 143 failing in research 62, 123–35, 138; better ways of 123–4, 127–32, 135, 141–3, 156, 169 faith backgrounds 70–1 faith communities 59–64 Fals Borda, Orlando 100 family life 47–9 Family Lives and the Environment, study of 47 Fanon, Frantz 99 'feel-good' element in research 39–40 Feltz, Vanessa 42–3 feminine sociability 64–6 feminism and feminist scholarship 6, 18–19, 24, 27, 30 first encounters with data 51 Fitzgerald, F. Scott 142 Flynn, Don 10, 39; *author of Chapter 10* focus groups 45, 151–3 Fraser, Karen 106 freedom of movement 176 friendships, women's 98 funding cycles 132 Galipo, Adele 3, 9–10; *author of Chapter 9* gentrification 164–5 Giaquinto, Bea 9, 96–8, 105–9, 113–15; *co-author of Chapter 6* Gidley, Ben 9, 76, 169; *author of Chapter 7* Gilroy, Paul 21, 26, 39, 61, 79, 159, 181, 192–3 Gilseman, Charlotte 10; *co-author of Chapter 11* Given, Michael 4 Glick Schiller, Nina 161–2 globalisation 176 Grenfell Tower 166 Griffin, Chris 44–5 group analysis 51–2 'groupism' (Brubaker) 170 gypsies and travellers 133–4 Hackney 137–8, 164 Hackney, Fiona 66 Halberstam, Judith 142, 149, 154 Hall, Stuart 61, 99 Hasedžić, Fahica 9, 96–8, 105–17; *co-author of Chapter 6* Hatoum, Mona 64 Haylett, Chris 125 Heil, Tilmann 40, 130–1, 143, 161 Heynen, Hilde 100 Higgins, Kate 4

hipsters 137–8 Holloway, Wendy 50 Holmes, Douglas R. 5–6 hope 142–3, 156 'human rights' approaches 183 Humphry, Debbie 133 identities, study of 47

Illich, Ivan 6–7, 10, 19, 38, 53, 77, 91, 99, 192, 194 Illouz, Éva 20 immigration control 176–85

impact of research on participants and researchers 40 India 47–8 Ingold, Tim 62 injunction to participate 125–6, 130, 138 innovative research methods 1–2, 36–9, 46–7, 53 integration 182 intellectual humility 134–6, 169 interactions 2, 4, 7, 10, 21–4, 28, 51, 62, 66, 78, 80, 170 Internet resources 19 interpretive phenomenological analysis (IPA) 49–50 interviews for purposes of research 24, 40, 47 Iqbal, Humera 8–9; *co-author of Chapter 5* Islam 1 Jefferson, Tony 50 Johnson, Shanell 40 joint analysis 50–3 Jones, Phil 47 Kaczmarek, Alicja 9, 124, 133; *co-author of Chapter 8* Keith, Michael 124, 131, 134 knowledge production 24, 27, 29, 39, 49–50, 53, 98, 123–4, 130–1, 143, 169, 189; positivistic model of 131 Kothari, Uma 138 Lapiņa, Linda 37, 53 Laurier, Eric 160–1 'learning by doing' 62 Lee, Emily 28 Lelliot, Sue 133 Lisiak, Agata 9, 124, 133; *co-author of Chapter 8* Local Knowledge for Local Solutions project 127–8 'looked after' children 43–4 Luton 137 Maiter, Sarah 26 Making Suburban Faith project 59–60 Malsbary, Christine B. 79 Marcus, George E. 5–6 marriage 23 meaning, production of 24 mediation, cultural 77–8 'methodological neighbourhoodism' (Berg *et al*.) 162–3, 171 micropolitics 135, 161 micropublics 76–7, 90–1 Mignolo, Walter D. 30 Migrants' Rights Network (MRN) 175–9 migration 18, 42–6, 50; forced 100; processes driven by 18 migration studies, shortcomings of 19 Mill, John Stuart 135–6 mimesis in art 100; *see also* ethno-mimesis mind maps 84 mixed-methods research 5, 48 mixed-parentage young people 41–2 mixing of people from different cultures, religions and places 20

Mouffe, Chantal 20 Mountz, Alison 154 'multicultural drift' 61, 129 multiculture and multiculturalism 21, 192–3 mundane conviviality 39, 124, 161 music-elicitation interviews 47 Muslim communities 1, 106 *My Life is but a Weaving* (installation) 60, 67–9 Myers, Martin 133 Neal, Sarah 61, 76 'neighbourhood', definition of 10 neighbourhood analysis 161 neighbourhoods divided from one another 129 New Deal for Communities programme 137 New Labour 126, 130 Newtown Neighbourhood Project 133, 137

'no recourse to public funds' (NRPF) 183 Noble, Greg 53 nostalgia 129 Nottingham Castle 107, 112–13, 115; City Arts 105; Market Square 111–12; Theatre Royal 110–11; Trent University 108–9 Nowicka, Magdalena 3, 8, 28, 38, 40, 79, 90, 103, 130–1, 143, 147, 161, 192; *author of Chapter 2, co-author of Chapter 1 and co-editor* Nussbaum, Martha 194

Oakley, Ann 24 O'Connor, Pat 98 Office for Standards in Education (Ofsted) 44 Öhberg, Emilia 126–7 O'Neill, Maggie 9, 96–8, 102, 106–11; *co-author of Chapter 6* Ortega y Gasset, José 100 outsourcing 1 Overing, Joanna 8, 61 Owen, Charlie 43

Pahl, Ray 98 Pain, Rachel 62, 71 Paradise, Annie 40 participation obstacles to 127; political economy of 130–2; *see also* injunction to participate participatory action research (PAR) 26, 100–4, 116–17, 126, 178 participatory arts (PA) 101–4, 116–17 participatory research 6, 8, 18, 36–9, 53, 62, 96–104, 123–4, 127–8, 131–2, 135–6, 189; *see also* research participants 'participatory turn' in the social sciences 2–3, 5, 36, 87, 123–6, 130 partnership working 188–9 Passes, Alan 8, 61 Pastore, Ferruccio 170 *performative* social science 80 Pettit, Jethro 39 phenomenological analysis 4, 49–50 Philo, Chris 160–1 Phoenix, Ann 3, 8–9, 50–2; *author of Chapter 3* Phoenix Art Gallery, Brighton 70 photo elicitation 151–3 'pikeys' 133 Pitkin, Hannah 135

INDEX 199

placement of children by local authorities 44 policy outcomes from research 43–4 Polish Expats Association (PEA) 143–4, 147–51, 155 political correctness 129, 137 political nature of convivial research 30 'politics of feeling' 104 Ponzo, Irene 170 popular culture 181 populism 1, 4, 10, 194 positivism 131, 134–5, 138 poverty, study of 42 power relations of art, of everyday life and of personal experience 183; in contentious spaces 79; in family life 49; histories and geographies of 30; in the research process 2, 5, 8, 37–8, 41, 45, 50–2 Pratt, Mary 79 psychosocial methods of research 46 public art 71 public service cuts 1 qualitative research 123, 131

racial identities 22 racism 22, 27, 178, 192–4 Rancière, Jacques 137 *rapport* 45 'Red Queen race' 184–5 reflexivity 27–8 refugees 99–102, 106, 176 reifying community 127, 132, 138 representation and representativeness, different concepts of 130, 135 research encounters 25–7 research participants assumptions and expectations about 24–5; compared with researchers 25; contributions made by 44; inventing new tools 38–9; marginalisation of 36; relations with 9, 36, 39–40, 170; remuneration of 155; skills needed by 27 Roberts, Brian 80 Rooke, Alison 133 Rose, Gillian 27 Roy, Alastair 116

Salverson, Julie 100 schools as research sites 92 scientific neutrality 138 segregation 129, 137 Sennett, Richard 127, 129, 131 separation from parents 42–3 sexism 27 'shadowing' 166 shared-ness, sense of 71 Shepherd's Bush 159–68 Shire, George 98 Sigona, Nando 163 *Simon Had a Little Overcoat* 141 Singh, Jaspal Naveel 50 Sinha, Shamser 155 'slower' forms of science and scholarship 6, 124–5, 134–5, 154, 169 Smith, Anne Marie 99 *sociable* sociology 98

'social capacity' 79 social exclusion 126 social interaction and dialogue 4, 80 social justice 103–4, 185 social theory 23 socialisation 7, 18–19, 155 sociality and sociability 19, 64–6, 71 Socratic mode 136 Solnit, Rebecca 142–3 Somali migrants 164, 167–8 Southwark 127 Spencer, Liz 98 Springgay, Stephanie 80 super-diversity 1–4, 22, 124, 136, 159, 163–4, 169 Sure Start programme 131–2, 135 sustainability and sustainable development 20–1 Taback, Simms 141 Taussig, Michael 103 Thomas, Stefan 5 togetherness 22, 138 tools *convivial* or *industrial* 38; for conviviality 77, 90–2, 99, 130, 155, 175, 194; cultural 91; for research 7, 10, 38–9 Townsend, Peter 42 TRANSFORmIG project 144–6, 151 'transmigrants' 161 transnationalism 159–70 *transparent* analysis 49, 52–3 triangulation 49–50 trust, building of 175

University College London (UCL) 70 urban areas 20–3, 170

Valentine, Gill 61, 193 Vandenberghe, Frédéric 20 Vertovec, Steven 38, 79, 90, 147, 192 'voice' for less powerful groups 41, 125 Vygotsky, Lev 91

Wainwright, Hilary 136 walking as a research method 47, 96–7, 106–9, 112, 116–18 Webster, Mark 101 Welsh, Irvine 125–6 Wessendorf, Susanne 137–8, 164 White, Sarah 39 'white backlash' 129 Wiles, Rose 36, 39, 46 Wilson, Helen 61 Wise, Amanda 53 'with-ness' 29 Woodley, Karin 10; *co-author of Chapter 11* workshops, difficulty in securing engagement in 149

xenophobia 178

Young, Iris Marion 127 young mothers, research on 41


**1. Introduction**

Lignocellulosic biomass (LB) is a key resource for the sustainable manufacture of bio-based chemicals and fuels. LB can be processed according to the biorefinery concept, which involves consecutive treatment stages to achieve an integral benefit of the feedstock, with minimal or no waste generation [1]. The success of biorefineries also depends on the right application of technology methodologies for obtaining multi-products [2]. The contribution of biorefineries to a future bio-economy inspires business opportunities based on product diversification while improving environmental performance [3].

In the field of biomass valorization, the biorefinery acts as a platform for chemicals and energy production through the inclusion of diverse conversion technologies [3,4].

The complexity of biomass utilization lies in both the polymeric nature of its main constituents (cellulose, hemicelluloses, and lignin) and their different chemical reactivities. In the last few decades, several fractionation methods have been developed, as summarized in recent literature reviews [2,5–8]. Galbe and Wallberg [6] classified the conventional methods according to their mode of action:

mechanical treatments (e.g., milling or grinding) reduce the particle size of biomass, increasing the surface area of the particles and improving the enzyme accessibility; dilute acid pretreatments (with H2SO4, H3PO4 or other strong acids), and hydrothermal pretreatments (with hot, compressed water or steam) promote the hydrolysis of hemicelluloses; whereas alkaline and organosolv processing enable the extraction of lignin.

Hydrothermal pretreatments (autohydrolysis, performed with hot, compressed water) causes the selective solubilization of hemicelluloses by depolymerization into soluble compounds of lower molecular weight. Soluble, low molecular weight polymers from hemicelluloses show potential for applications in the manufacture of barrier films or hydrogels [6]; oligosaccharides show prebiotic properties with potential in the pharmaceutical and food markets [9,10]; and monosaccharides can be transformed into value-added chemicals such as xylitol and furfural [11]. Additionally, autohydrolysis increases the biomass surface area and decreases the crystallinity of the cellulosic fraction remaining in the solid phase, facilitating its further hydrolysis.

Solutions of alkalis (such as NaOH, KOH, or ammonia) are suitable for removing lignin by saponification of intermolecular ester bonds, and for increasing the digestibility of cellulose [5]. Alternatively, delignification can be achieved by organosolv treatments with organic solvents (e.g., ethanol) or solvent-water mixtures, in the presence or absence of catalysts. From an environmental point of view, the separation of lignin from the organosolv media by precipitation with water facilitates the wastewater treatment. From an economic perspective, organosolv lignin is a valuable product with potential applications in diverse fields, including the manufacture of adhesives, fibers, films, biodegradable polymers [12], and natural antioxidants [13].

The solids from delignification treatments, with increased cellulose contents, may show increased susceptibility to enzymatic saccharification [1,14,15], or serve as substrates for manufacturing cellulose nanocrystals [16]. In the first case, glucose can be transformed in value-added compounds (e.g., bioethanol or lactic acid) [15,17]; whereas in the second one, cellulose nanocrystals are used in a number of fields, including biological applications.

"Conventional" pretreatments (or their combinations) are expected to play an important role in the future full-scale biorefineries [6]. Combined pretreatments may allow process designs enabling an improved fractionation and an efficient processing into a spectrum of multi-products [8]. For example, consecutive stages of autohydrolysis and delignification allow the separate recovery of hemicellulose-derived saccharides, lignin, and cellulose [18], enabling the integral fractionation of biomass, an aspect of crucial importance for a successful biorefinery approach [19].

Hazelnut (*Corylus avellana* L.) is a commercial crop, whose fruit is widely used in food industries [20]. Turkey is the main producer and exporter of hazelnut, followed by Italy, Azerbaijan, USA, China, Georgia, Iran, France, Chile, and Spain. According to the Food and Agriculture Organization (FAO), 863,888 tons of hazelnuts (with shell) were produced worldwide in 2018 [21]. Hazelnut shells (HS) are the most important byproduct of the hazelnut processing industry, representing more than half of the total nut weight. HS are a low–cost byproduct, usually burned, but could be valorized on the basis of their lignocellulosic nature. Recently, getting value from selected biochemicals in HS has become a motivation for research, due to the compelling economic benefits [3,22]. In terms of composition, HS are mainly made up of lignin: Perez-Armada et al. [23] reported 40.1 wt% of lignin, whereas Demirbas [24] and Surek and Buyukkileci [25] found around 46%, and Ho¸sgün and Bozan [20] near 50%. HS hemicelluloses are mainly made up of xylan, acetyl, and uronic moieties, which accounted jointly for 25–32.5% of the dry weight, whereas the reported cellulose contents are about 26–27% [10,23,26].

Based on the above ideas, this work deals with the development of HS fractionation methods suitable for a multi-product biorefinery. HS and solids resulting from HS autohydrolysis (denoted AS) were subjected to diverse delignification treatments to assess their efficiency. The soluble hemicellulose-derived products were identified, and the solids from selected delignification stages were assayed as substrates for enzymatic hydrolysis. In summary, this work provides an experimental assessment on biorefinery schemes enabling an integral valorization of HS.


**3.2.4 Immobilization of living cells on islet surfaces**

Endothelial cells on the islets surfaces have a good tolerance towards blood presence and can provide a protection against IBMIR. Introduction of such protective cells around islets can be used as an effective protection strategy. The human aortic endothelial cells were introduced onto isolated human islets of Langerhans by mixing of both cell types and incubation for several hours (Johansson et al., 2005). The clotting can be significantly reduced with the 90%-cell-coating present on islet surfaces. The consistency of the cell amounts in such coating can be improved using the PEG-phospholipid-based approach (Teramura & Iwata, 2009; Teramura & Iwata, 2008a). In that case, islets and human endoderm kidney cells were first separately biotinylated through biotin-PEG-lipid anchors to cell membranes with further streptavidin coating on the kidney cells (Fig. 5). Immobilization of the endothelium cells around islets was then made via streptavidin-biotin reaction. The cell enclosure was stable on the islet surfaces within 3-5 days *in vitro*. To overcome the streptavidin immunogenicity, the cell deposition was also made via the PolyDNA-PEG-lipid conjugate (Teramura & Chen, 2010; Teramura & Minch, 2010). Human endoderm kidney cells were able to rapidly proliferate forming a cell multilayer on the islets surfaces protecting the encapsulated islets from the host immune response. However, the cell oxygen consumption can result in lowered oxygen available for the encased islets. Thus, additional studies are necessary to clarify the short- and long-term effects of the cell presence on islets surfaces.

## **3.3 Conformal coating of islets**

### **3.3.1 Layer-by-layer (LbL) approach**

The layer-by-layer (LBL) assembly of polymers based on sequential adsorption of oppositely charged components is one of the established methods for the preparation of thin polyelectrolyte multilayer films with controlled properties. The LBL represents a universal surface modification approach that allows for producing surface-attached films with controlled thickness, permeability, mechanical properties and surface chemistry. The technique has been recently applied to modify islet surfaces (Krol et al., 2006; Wilson et al., 2008). The LbL modification of islet surfaces is based on alternating deposition of water

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 19

To promote a multilayer film formation on the cell surfaces, the negatively charged cell surface is treated with a cationic polymer solution and the cell surface is further exposed to an anionic polymer solution to form an electrostatically-paired polyelectrolyte complex film (Fig. 3, A4). Effect of molecular weight of polyelectrolytes and the charge of outermost layer was demonstrated in case of the LBL encapsulation of human islets into poly(allylamine hydrochloride)/poly(styrenesulfonate sodium salt) (PAH)/(PSS) and poly- (diallyldimethylammonium chloride) (PDADMAC)/PSS layers. Islets encapsulated into PAH/PSS and PDADMAC/PSS multilayers using a higher polycation molecular weight demonstrated a limited insulin release due to a lowered permeability of insulin through the polyelectrolyte membrane. A decrease in a polycation molecular weight resulted in larger pores of the polyelectrolyte membrane and restored responsive relationship between

Most cationic polymers widely used in the LbL modification of surfaces such as poly(Llysine) (PLL) and poly(ethylene imine) (PEI) are extremely cytotoxic and cells treated with the polycations can be severely damaged. Their cytotoxic effect though has been observed to be dependent on polycation concentration and exposure time (De Koker et al., 2007). The overall cytotoxicity of the polyelectrolytes originates from positive charge of polycations which can induce pore formation within the cell membrane causing its damage and, eventually, cell death (Bieber et al., 2002; Godbey et al., 1999). The high toxicity of the PAH/PSS LbL film was confirmed by Wilson et al (Wilson et al., 2008). They demonstrated that coating the murine islets with only 3 layers of PAH/PSS/PAH led to the reduction of islet viability by 70%. Similar effect was found for islets coated with 3 layers of PLL/alginate LbL film. Even 15 minutes of islets incubation with low concentration of PLL results in ~60% decrease in cell viability. Menger et al showed that PLL was able to pass through the lipid bilayer if it was previously allowed to form complex with anionic lipids (Menger et al., 2003). PEI was found extremely toxic to the islets. This polycation destroys the cell membrane immediately after its interactions with the membrane surface (Teramura et al., 2008b). The overall charge arrangement of a polycation and its interaction with the cell membrane strongly depends on the three-dimensional structure and flexibility of the polymer chains. It has been shown that polymers with highly flexible chains and a high cationic density will exert tremendous cytotoxicity. Thus, the polycations with globular structures demonstrated good biocompatibility, whereas polymers with more linear and flexible structure such as PLL and PEI showed higher cytotoxicity (Teramura et al., 2008b). Since the polycations toxicity partially depends on the polymer charge density, it can be attenuated by conjugating neutral molecules, such as PEG, to the critical number of amino groups along the polycation backbones. PEGylation of PLL is carried out through grafting of N-hydroxysuccinimide-PEG (NHS-PEG) chains to amino groups on PLL backbone to produce PLL-g-PEG. The grafted PEGs are unbranched, hydrophilic, discrete-length molecules in the form of Methyl-PEGn-NHS ester, where the subscript "n" denotes a number of the ethylene glycol units. The NHS ester end group is spontaneously reactive with primary amines, providing for efficient PEGylation of amine-containing molecules or surfaces. The methoxy(ethylene glycol) grafts were conjugated to PLL backbone through a covalent attachment to lysine residues (Wilson et al., 2009). Forty percent of PEG substitutes on the PLL chain allowed for attenuation of the PLL positive charges without any

glucose stimulation and insulin response of the coated islets (Krol et al., 2006).

**3.3.2 The ionic LbL assembly**

Fig. 5. (a) Chemical structure of biotin–PEG-conjugate (biotin–PEG–lipid). (b) Schematic illustration of the interaction between streptavidin and biotin–PEG–lipid at the lipid bilayer cell membrane. Biotin–PEG–lipid has hydrophobic acyl chains and is incorporated into the cell surface by anchoring into the lipid bilayer. Streptavidin is immobilized on the cell surface by anchoring to biotin–PEG–lipid. (c) Scheme for the immobilization of streptavidin-immobilized HEK293 cells on the surface of biotin–PEG–lipid-modified islets. After mixing streptavidinimmobilized HEK293 cells and biotin–PEG–lipid-modified islets, they were cultured in medium at 37°C on a culture dish. During culture, HEK293 cells were spread and grown on the cell surface to cover the whole surface. (d) Hamster islets modified with biotin–PEG–lipid and immobilized with streptavidin-immobilized HEK293 cells. The HEK293 cells were labeled with CellTracker. Reprinted from Teramura & Iwata, 2009 with permission from Elsevier.

soluble polymers on surfaces from aqueous solutions which results in nano-thin coatings of controllable thickness and composition (Decher & Schlenoff, 2002; Kharlampieva & Sukhishvili, 2006; Tang et al., 2006). The ultrathin conformal coating affords a faster response to stimulation and the possibility to bind factors or protective molecules to the protective ultrathin shell with the later slow triggered release of these molecules (Chluba et al., 2001). By selecting specific polyelectrolytes, a defined cutoff of the coating (Kozlovskaya & Sukhishvili, 2006) is possible, as is inhibitor binding to prevent graft rejection, microphage attacks, or antibody recognition (Kim & Park, 2006). Modification of the last coating layer can be used to support functionality of islets and reduce the immune response from a host system. The cutoff of the polyelectrolyte multilayer (PEM) is defined by polyelectrolytes used in coating formation (Krol et al., 2006).


**4. Staging**

The cervix was the first organ for which The International Federation of Gynecology and Obstetrics (FIGO) established a system for only clinical staging in 1958. Thereafter, to document the presence or absence of nodes and distant metastases, a pathological TNM staging system was developed and implemented. The FIGO Committee of Gynecological Oncology updated the staging system in 2018 so that clinical, radiological, or pathological evidence may be used to designate the stage (**Table 3**).

Clinical examination and physical evaluation initiate staging. FIGO 2018 staging allows ultrasonography, CT, MRI, and PET to offer further information about tumor size, nodal status, and local or systemic metastasis [18]. MRI is helpful for primary

*a Imaging and pathology can be used, where available, to supplement clinical findings with respect to tumor size and extent, in all stages. Pathological findings supersede imaging and clinical findings.b The involvement of vascular/ lymphatic spaces should not change the staging. The lateral extent of the lesion is no longer considered.c Isolated tumor cells do not change the stage but their presence should be recorded.d Adding notation of r (imaging) and p (pathology) to indicate the findings that are used to allocate the case to Stage IIIC. For example, if imaging indicates pelvic lymph node metastasis, the stage allocation would be Stage IIIC1r; if confirmed by pathological findings, it would be Stage IIIC1p. The type of imaging modality or pathology technique used should always be documented. When in doubt, the lower staging should be assigned.*

#### **Table 3.**

*FIGO staging of cancer of the cervix uteri (2018).*

cancers beyond 10 mm. In experienced hands, ultrasonography provides high diagnostic preciseness. For future evaluation, note the staging modality. Imaging can provide new prognostic indicators to assist in choosing the best therapy. PET-CT is

more accurate than CT and MRI (4–15% false-negative results) at detecting nodal metastases above 10 mm.


**Abstract**

Nasopharyngeal carcinoma is a unique disease entity among head and neck cancers due to its epidemiology and clinical behavior. Non-keratinizing or undifferentiated carcinoma is the most common histological type in endemic areas. Radiotherapy is the treatment for early-stage disease. With the widespread use of IMRT, loco-regional control has improved significantly in locally advanced diseases. But distant metastasis continues to be the most common pattern of failure. To address this issue, chemotherapy has been incorporated into radiotherapy in various settings; as concurrent, induction, and adjuvant. The initial trials of concurrent chemotherapy incorporated adjuvant chemotherapy also and the magnitude of benefit contributed by each treatment was not clear. Later trials proved that adjuvant chemotherapy was not beneficial. Induction chemotherapy when added to concurrent chemoradiation resulted in improvement in Failure Free Survival, Overall Survival, and Distant Metastasis Free Survival. Thus, induction chemotherapy followed by concurrent chemoradiation became the standard of care for locally advanced disease (stage III and IVA). The role of chemotherapy in stage II disease is still evolving. Metastatic nasopharyngeal carcinoma is treated by platinum doublet chemotherapy, Cisplatin-gemcitabine is the standard regimen.

**Keywords:** Nasopharyngeal carcinoma, locally advanced, metastatic, concurrent chemotherapy, induction chemotherapy, adjuvant chemotherapy

## **1. Introduction**

Nasopharyngeal carcinoma (NPC) is unique from other head and neck cancers due to its difference in epidemiology, etiology and propensity for distant metastasis. It is endemic in Southern China, South East Asia, North Africa, and Artic region. Non-keratinizing or undifferentiated carcinoma is the most common histological type in endemic areas. Radiotherapy is the backbone of treatment owing to the complex anatomical location and high radiosensitivity. Higher local control and survival are reported for early-stage disease with radiotherapy alone [1]. But around 70% of patients present with locoregionally advanced disease and outcomes with radiotherapy alone are poor [2]. Many strategies have been tried to improve outcomes in locoregionally advanced NPC; the incorporation of chemotherapy and the use of modern radiotherapy techniques. IMRT when compared with two-dimensional

radiotherapy showed significantly better locoregional control and survival with a lower incidence of radiation- induced toxicities [3, 4]. IMRT is mainly aimed at reducing the toxicities in the early stages whereas it improves loco-regional control (LRC) in advanced stages. After the widespread use of IMRT, distant metastasis remains the predominant pattern of failure. Hence chemotherapy was added to radiotherapy in various settings; as concurrent, induction, and adjuvant. Patients with metastatic disease are treated by palliative chemotherapy or palliative radiotherapy.


**5. Final considerations**

According to the available evidence, ageing constitutes a vulnerable stage for cell fate mainly because of the delicate balance of several molecular conditions. Under these conditions, any additional challenge to any homeostatic system can trigger the breakdown of such equilibrium, leading to the manifestation of pathological processes, such as neurodegenerative disorders. AD and PD, the first and second most prevalent age-related diseases, can be favoured by a spontaneous imbalance in the homeostatic system and, once initiated, further contribute to increasing the homeostatic imbalance. A complex network of molecular alterations, organelle dysfunction and cellular signalling, among others, increases the difficulty of properly addressing the cloudy edge between healthy ageing, pathological ageing and age-related disorders. However, neuroinflammation and the perpetuation of a chronic pro-inflammatory status have emerged as a central axis connecting all these conditions. Although our knowledge regarding the inflammatory process has increased during years, the intricate molecular network that drives the final inflammatory response is still incomplete. In this regard, Wnt signalling, which has been demonstrated to be a relevant player in ageing and age-related disorders, such as AD and PD, should also be considered among the potentially relevant molecular pathways that could be involved in the modulation of the inflammatory process. Moreover, the still limited information regarding the Wnt-inflammatory response crosstalk already suggests interesting potentialities of an anti-inflammatory intervention based on the modulation of Wnt signalling. On the other hand, based on our research and considering the age-related changes in Wnt activity, it is possible to suggest that Wnt signalling can also be an interesting target to support physiological ageing.

**References**

[1] Alzheimer's Disease International. World Alzheimer Report. 2016. Available from:

Molecular Basis of Neurodegeneration: Lessons from Alzheimer's and Parkinson's Diseases

http://dx.doi.org/10.5772/intechopen.81270

59

[2] Ashapkin VV, Kutueva LI, Vanyushin BF. Aging as an epigenetic phenomenon. Current

[3] Masser DR, Hadad N, Porter HL, Mangold CA, Unnikrishnan A, Ford MM, et al. Sexually divergent DNA methylation patterns with hippocampal aging. Aging Cell.

[4] Hadad N, Unnikrishnan A, Jackson JA, Masser DR, Otalora L, Stanford DR, et al. Caloric restriction mitigates age-associated hippocampal differential CG and non-CG methylation. Neurobiology of Aging. 2018;**67**:53-66. DOI: 10.1016/j.neurobiolaging.2018.03.009 [5] Wezyk M, Spólnicka M, Pośpiech E, Pepłońska B, Zbieć-Piekarska R, Ilkowski J, et al. Hypermethylation of TRIM59 and KLF14 influences cell death signaling in familial Alzheimer's disease. Oxidative Medicine and Cellular Longevity. 2018;**2018**:6918797.

[6] Lardenoije R, van den Hove DLA, Havermans M, van Casteren A, Le KX, Palmour R, et al. Age-related epigenetic changes in hippocampal subregions of four animal models of Alzheimer's disease. Molecular and Cellular Neurosciences. 2018;**86**:1-15. DOI:

[7] Berson A, Nativio R, Berger SL, Bonini NM. Epigenetic regulation in neurodegenerative diseases. Trends in Neurosciences. 2018;**S0166-2236**:30133-30134. DOI: 10.1016/j.

[8] Bhattacharjee P, Sanyal T, Bhattacharjee S, Bhattacharjee P. Epigenetic alteration of mismatch repair genes in the population chronically exposed to arsenic in West Bengal, India. Environmental Research. 2018;**163**:289-296. DOI: 10.1016/j.envres.2018.01.002 [9] Skinner AM, Turker MS. Oxidative mutagenesis, mismatch repair, and aging. Science of Aging Knowledge Environment. 2005;**2005**:re3. DOI: 10.1126/sageke.2005.9.re3

[10] Conde-Pérezprina JC, León-Galván MÁ, Konigsberg M. DNA mismatch repair system: Repercussions in cellular homeostasis and relationship with aging. Oxidative Medicine

[11] Harman D. Aging: A theory based on free radical and radiation chemistry. Journal of

[12] Lushchak VI. Free radicals, reactive oxygen species, oxidative stress and its classification. Chemico-Biological Interactions. 2014;**224**:164-175. DOI: 10.1016/j.cbi.2014.10.016

[13] Garaschuk O, Semchyshyn HM, Lushchak VI. Healthy brain aging: Interplay between reactive species, inflammation and energy supply. Ageing Research Reviews. 2018;**43**:26-

and Cellular Longevity. 2012;**2012**:728430. DOI: 10.1155/2012/728430

https://www.alz.co.uk/research/world-report [Accessed: 2017-11-20]

Genomics. 2017;**18**:385-407. DOI: 10.2174/1389202918666170412112130

2017;**16**:1342-1352. DOI: 10.1111/acel.12681

DOI: 10.1155/2018/6918797

10.1016/j.mcn.2017.11.002

Gerontology. 1956;**11**:298-300

45. DOI: 10.1016/j.arr.2018.02.003

tins.2018.05.005


**1. Introduction**

262 Biochemistry

Wolterink-van Loo, S., Levisson, M., Cabrières, M.C., Franssen, M.C.R., & van der Oost, J.

Work, E. (1962). Diaminopimelic racemase. In: *Methods in Enzymology,* Colowick, S. P. & Kaplan, N. O., pp. 858-864, Academic Press, ISBN 0076-6879, New York Yugari, Y., & Gilvarg C. (1965). The condensation step in diaminopimelate synthesis*. J. Biol. Chem.*, Vol.240, No.12, (December 1965), pp. 4710-4716, ISSN 0021-9258

469, ISSN 1431-0651

(2008). Characterization of a thermostable dihydrodipicolinate synthase from *Thermoanaerobacter tengcongensis. Extremophiles*, Vol.12, No.3, (May 2008), pp. 461-

> Flavonoids comprise a group of plant polyphenols with a broad spectrum of biological activities. They have been shown to exert beneficial effects on human health and play an important role in prevention and/or treatment of several serious diseases, such as cancer, inflammation and cardiovascular disease (Middleton et al., 2000; Rice-Evans, 2001). Flavonoids are important beneficial components of food, pharmaceuticals, cosmetics and various commodity preparations due to their antimutagenic, hepatoprotective (Stefani et al., 1999), antiallergic (Berg & Daniel, 1988), antiviral (Middleton & Chithan, 1993) and antibacterial activity (Tarle & Dvorzak, 1990; Tereschuk et al., 1997; Singh & Nath, 1999; Quarenghi et al., 2000; Rauha et al., 2000). They are known to inhibit nucleic acid synthesis (Plaper et al., 2003; Cushnie & Lamb, 2006), cause disturbance in membranes (Stepanovic et al., 2003; Stapleton et al., 2004; Cushnie & Lamb, 2005) and affect energy metabolism (Haraguchi et al., 1998). But the most studied activity is their antioxidant action since they can readily eliminate reactive oxygen and nitrogen species or degradation products of lipid peroxidation and are thus effective inhibitors of oxidation (Ross & Kasum, 2002).

> However, their commercial applications are limited due to low solubility in lipophilic environment and low availability for a living organism. Although aglycons, prenylated and methoxylated flavonoid derivatives may be implemented into such systems, they are rarely found in nature and are often unstable. In some plant species, the last step in the flavonoid biosynthesis is terminated by acylation which is known to increase solubility and stability of glycosylated flavonoids in lipophilic systems. Selectively acylated flavonoids with different aliphatic or aromatic acids may not only improve physicochemical properties of these molecules (Ishihara & Nakajima, 2003) but also introduce various beneficial properqties to the maternal compound. These include penetration through the cell membrane (Suda et al., 2002; Kodelia et al., 1994) enhanced antioxidant activity (Viskupicova et al., 2010; Katsoura et al., 2006; Mellou et al., 2005), antimicrobial (Mellou et al., 2005), anti-proliferative (Mellou et al., 2006) and cytogenic (Kodelia et al., 1994) effect and improvement of thermostability and light-resistivity of certain flavonoids.

In nature, flavonoid acylation is catalyzed by various acyltransferases which are responsible for the transfer of aromatic or aliphatic acyl groups from a CoA-donor molecule to hydroxyl residues of flavonoid sugar moieties (Davies & Schwinn, 2006). Acylation is widespread especially among anthocyanins; more than 65% are reported to be acylated (Andersen & Jordheim, 2006). While the exact role of plant acylation is not yet fully understood, it is known that these modifications modulate the physiological activity of the resulting flavonoid ester by altering solubility, stability, reactivity and interaction with cellular targets (Ferrer et al., 2008). Acylation might be a prerequisite molecular tag for efficient vacuolar uptake of flavonoids (Kitamura, 2006; Nakayama et al., 2003). Some acylated flavonoids have been found to be involved in plant-insect interactions; they act as phytoalexins, oviposition stimulants, pollinator attractants (Iwashina, 2003), and insect antifeedants (Harborne & Williams, 1998). With respect to novel biological activities, acylation of flavonoids can result in changes in pigmentation (Bloor, 2001), insect antifeedant activity (Harborne & Williams, 1998) and antioxidant properties (Alluis & Dangles, 1999).

Over the past 15 years, there has been a substantial effort to take advantage of this naturally occurring phenomenon and to implement acylation methods into laboratories. However, the use of acyltransferases as modifying agents is rather inconvenient, as they require corresponding acylcoenzyme A, which must be either in stoichiometric amounts or regenerated *in situ*. Natural acyltransferases and cell extracts from *Ipomoea batatas* and *Perilla frutescens* containing acyltransferases were applied for selective flavonoid modification with aromatic acids (Tab.1) (Nakajima et al., 2000; Fujiwara et al., 1998).

Table 1. Acyltransferase catalysis of flavonoid acylation and their nature sources.

To solve this problem, the chemical approach was first investigated. It possessed a low degree of regioselectivity of esterification and drastic reaction conditions had to be applied (Patti et al., 2000). Later on, hydrolytic enzymes (lipases, esterases and proteases) have been recognized as useful agents due to their large availability, low cost, chemo-, regio- and enantioselectivity, mild condition processing and no need of cofactors (Collins & Kennedy, 1999; Nagasawa & Yamada, 1995).

Since the enzymatic preparation of flavonoid derivatives is a matter of several years, commercial applications have just been emerging. There are several patented inventions available to date, oriented on the flavonoid ester production and their use for the manufacture of pharmaceutical, dermopharmaceutical, cosmetic, nutritional or agri-foodstuff compositions (Fukami et al., 2007; Moussou et al., 2004, 2007; Ghoul et al., 2006; Bok et al., 2001; Perrier et al., 2001; Otto et al., 2001; Nicolosi et al., 1999; Sakai et al., 1994).

This review presents available information on enzyme-mediated flavonoid acylation *in vitro*, emphasizing reaction parameters which influence performance and regioselectivity of the enzymatic reaction. In the second part, the paper focuses on biological effects of synthesized flavonoid esters as well as of those isolated from nature. Finally, the paper ends with application prospects of acylated flavonoids in the food, pharmaceutical and cosmetic industry.


*Edited by Xingshun Qi and Sam Koruth*

The book focuses on the recent advances in the digestive system. It is composed of 3 sections: gastrointestinal duct, liver, and biliary system. There are 9 chapters: peptic ulcers; mid-gastrointestinal bleeding; gastrointestinal manifestations of IgA vasculitis; biomechanics of intestinal contractions; evaluation of serum sodium change after terlipressin in cirrhosis; history and background of biliary system; gallbladder carcinoma; ERCP for cholecysto-choledocholithiasis; and cystic artery variations and associated vascular complications in laparoscopic cholecystectomy. The knowledge presented in this book should be valuable for family physicians, internists, gastroenterologists, hepatologists, endoscopists, radiologists, and pathologists who are interested in digestive diseases to guide the clinical practice and management. This book should be also useful for patients and their relatives to better understand the digestive system.

ISBN 978-1-78985-139-7

Digestive System - Recent Advances

Published in London, UK © 2020 IntechOpen © Dr\_Microbe / iStock


**Abstract**

This work analyses the distributed-ledger technology as a digital framework for the representation and exercise of tokenized rights, as well as the private-law characterization and EU regimen applicable to non-fungible tokens (NFTs). After a brief introduction to permissioning in blockchain and to permissioned distributed ledgers (PDLs), we reflect on its relevance to NFT contracting and markets, focusing on the legal and economic consequences of NFT trading in PDLs, with particular emphasis on public permissioned networks (PPDLs). We then discuss key challenges about law-compliant trading for NFTs, such as proper legal qualification of their nature, determination of optimal conditions for the legal trading of NFT underlying assets and investing applicable regime, including specific primary-market and essential public surveillance and intellectual-property (IP) issues.

**Keywords:** permissioned blockchain networks, non-fungible tokens, public-permissioned DLT networks, primary-market obligations, IP protection


**2.1. Historical background**

In the abundance of biomass in the world's nature, traditional utilisation of biomass in order to survive has long been established. Primitively, when man knows how to create fire, biomass utilisation evolved from domestic usage to even larger application such as building houses, clothes production, paper making, etc. Yet the biomass utilisation seems endless and continues to produce more relevant products.

Henry Ford was the first to design a model car that runs using ethanol, and it was also reported that Rudolph Diesel intended on using vegetable oil to power his car engine [8]. However, at the time of World War II, fossil fuels were more practical based on supply, price and efficiency which caused biofuel to be unpopular. In contrast, in the late 1990s, high intensity of research and investment were made to produce biofuel from biomass due to reverse effect on supply and price of fossil fuel which is unpredictable.

The first biomass used was in solid form—wood—which was used to produce fire and oil derived from plants and animals that has been turned to a useful medium to light up lamp [9, 10]. In the present day, biofuel is derived from food crops like sugar beets, grains and vegetable oils or, namely, first-generation biomass [11]. However, the impact on the global food supply and sustainability of land led to non-food stock for energy sources and the second generation of energy sources such as cellulosic biomass, inedible oil and algae-derived oil [11, 12]. Microalgae can reproduce in a mass rapidly per day and have been found enriched of high level of oil in total dry biomass [13]. However, regardless the high growth rates or high oil content, algae-derived oil production faces challenges that needed attention such as strain isolation, nutrient sourcing or availability, on-site management in production and handling and residual biomass or waste management [14].

Meanwhile, biobutanol, another type of fuel, has becoming popular in recent years that surpass bioethanol. Biobutanol is referred as the next generation of biofuels that offers many advantages. It can be produced directly with few combinations of physical, chemical and biological processes from food crops such as cereal crops, sugar beets and sugar cane. The first synthesis of biobutanol was achieved during 1912–1914 by acetone-butanol-ethanol (ABE) fermentation using Clostridium species as catalysts [15].


*2.4. Analysis*

To prepare data for processing by the CNNs, all images were resized (Figure 2C). The dimensions of each image were rescaled to the correct input shape for the training algorithm (224 × 224 pixels for the bespoke CNN, VGG-16, and ResNet-50 and 229 × 229 for InceptionV3). Resizing images in this manner also ensures that the magnification, resolution, or quality of the photos available when using the tool do not affect classification.

Before transfer learning was undertaken, a benchmark was established using a bespoke handmade CNN in TensorFlow. The architecture used for this CNN comprised a ReLU activated 3 × 3 input layer with 16 nodes, a (1, 1) stride, and 'same' padding (so that output size was equal to input size). This input layer then fed into three 3 × 3 ReLU activated hidden layers, with the same stride and padding as the input layer and whose number of nodes doubled from the previous layer (e.g., 16, 32, 64, and 128). Each convolutional layer fed into a 2 × 2 pooling layer, with a (2, 2) stride, to prevent overfitting. The final hidden layer was used as the input into a binary densely connected softmax output layer to capture either fertile (0) or infertile (1). As the model was a binary classifier, it was compiled using the 'Sparse Categorical Cross-Entropy' cost method, 'Root-Mean-Squared Propagation' optimiser, and 'Binary Accuracy' metric [41]. The model was trained against two training sets and used to generate two classifiers. The first classifier was trained against the original, pre-data augmentation, training set (i.e., 367 images) and the second full training set including data augmentation (i.e., 6973 images). During fitting, experimentation found that five epochs and a batch size of 32 produced the optimal performance. These models provided two benchmarks showing the impact of both data augmentation and transfer learning.

Once a benchmark was established, transfer learning was undertaken. The VGG-16 [26], ResNet-50 [27], and InceptionV3 [29] architectures with parameters pretrained against the ImageNet dataset were repurposed using the full training set (i.e., 6973 images). Although the architectures' layers were frozen, to maintain their ImageNet weighting, each was slightly altered for its new purpose. The output layer of each architecture was replaced with a densely connected softmax layer with two outputs, so as to accommodate the binary classification of 'fertile' or 'infertile'. These altered models were then compiled and fit to the training set. As each model is a deep net classifying a binary problem, all three were compiled using the 'Sparse Categorical Cross-Entropy' cost method, 'adam' optimiser, and 'Binary Accuracy' metric [41]. The training data were then used to improve the target predictive function of the architectures to detect and classify fertility status. When fitting, manual fine-tuning of the models' hyperparameters found that five epochs and a batch size of 32 maximised performance.

#### *2.5. Resources and Requirements*

Image pre-processing, data augmentation, and analysis were performed using the TensorFlow 2.4.1 library in Python through a Jupyter notebook created for this project by the lead author. All analysis found here was performed on an Intel 2.20 GHz 10 Core Xeon Silver 4114 CPU equipped on a desktop computer with 25.8 GB of RAM.