*Article* **Measuring the Regional Availability of Forest Biomass for Biofuels and the Potential of GHG Reduction**

### **Fengli Zhang 1, Dana M. Johnson 2, Jinjiang Wang 1,\*, Shuhai Liu 1,\* and Shimin Zhang 1**

Received: 7 November 2017; Accepted: 8 January 2018; Published: 15 January 2018

**Abstract:** Forest biomass is an important resource for producing bioenergy and reducing greenhouse gas (GHG) emissions. The State of Michigan in the United States (U.S.) is one region recognized for its high potential of supplying forest biomass; however, the long-term availability of timber harvests and the associated harvest residues from this area has not been fully explored. In this study time trend analyses was employed for long term timber assessment and developed mathematical models for harvest residue estimation, as well as the implications of use for ethanol. The GHG savings potential of ethanol over gasoline was also modeled. The methods were applied in Michigan under scenarios of different harvest solutions, harvest types, transportation distances, conversion technologies, and higher heating values over a 50-year period. Our results indicate that the study region has the potential to supply 0.75–1.4 Megatonnes (Mt) dry timber annually and less than 0.05 Mt of dry residue produced from these harvests. This amount of forest biomass could generate 0.15–1.01 Mt of ethanol, which contains 0.68–17.32 GJ of energy. The substitution of ethanol for gasoline as transportation fuel has potential to reduce emissions by 0.043–1.09 Mt CO2eq annually. The developed method is generalizable in other similar regions of different countries for bioenergy related analyses.

**Keywords:** timber; harvest residues; ethanol; GHG savings; Michigan


*Article* **Potency of Urea-Treated Halloysite Nanotubes for the Simultaneous Boosting of Mechanical Properties and Crystallization of Epoxidized Natural Rubber Composites**

**Indra Surya 1 , Kamaruddin Waesateh 2 , Sitisaiyidah Saiwari 3,4 , Hanafi Ismail 5 , Nadras Othman 5 and Nabil Hayeemasae 3,4,\***

**Abstract:** Halloysite nanotubes (HNTs) are naturally occurring tubular clay made of aluminosilicate sheets rolled several times. HNT has been used to reinforce many rubbers. However, the narrow diameter of this configuration causes HNT to have poor interfacial contact with the rubber matrix. Therefore, increasing the distance between layers could improve interfacial contact with the matrix. In this work, Epoxidized Natural Rubber (ENR)/HNT was the focus. The HNT layer distance was successfully increased by a urea-mechanochemical process. Attachment of urea onto HNT was verified by FTIR, where new peaks appeared around 3505 cm−1 and 3396 cm−1 , corresponding to urea's functionalities. The intercalation of urea to the distance gallery of HNT was revealed by XRD. It was also found that the use of urea-treated HNT improved the modulus, tensile strength, and tear strength of the composites. This was clearly responsible for interactions between ENR and urea-treated HNT. It was further verified by observing the Payne effect. The value of the Payne effect was found to be reduced at 62.38% after using urea for treatment. As for the strain-induced crystallization (SIC) of the composites, the stress–strain curves correlated well with the results from synchrotron wide-angle X-ray scattering.

**Keywords:** epoxidized natural rubber; halloysite nanotubes; urea; tensile properties; wide-angle X-ray scattering


**6. Effect of reloading on the skeletal muscle**

dissolution and endothelial degradation, a marked reduction in the number of mitochondria, the accumulation of the connective tissue, the elimination of apoptotic myonuclei and a decrease in capillary density [85–87]. Selective susceptibility of fibre types to immobilization seems to exist, while the red muscle fibres show the greatest atrophy. The decreased synthesis of protein and increased protein degradation are characteristic features to muscle atrophy. At least half of the total muscle protein is myofibrillar protein, and this fraction is lost at a faster rate than other muscle proteins during atrophy [84]. Three major proteolytic systems to skeletal mass protein loss are the cytosolic calcium-dependent calpain system, the lysosomal proteases and the ATP-dependent ubiquitin-proteasome system, which work as partners during muscle proteolysis rather than one system being used exclusively during atrophy [88]. Recent advances in cellular biology show the oxidative stress to be an important regulator of pathways leading to muscle atrophy during periods of disuse, increasing the expression of the key components of the proteasome proteolytic system. This proteolytic system is a prominent contributor to protein breakdown in skeletal muscle during periods of inactivity [37,84,86].

Prolonged periods of time spent with a diminished or no-weight bearing have a deleterious effect on skeletal muscle with the decreased protein synthesis, the loss of muscle mass and alterations of biochemical parameters [74,77]. The main findings confirmed that the proportion of slow MyHC isoforms decreased and the proportion of MyHC fast isoforms increased in

Clinical observations show that the atrophy of the skeletal muscle occurs as a result of immobilization and is caused by the changed functional conditions in the muscular system. Skeletal muscle function depends on the intact proprioceptive activity, motor innervation, mechanical load, and joint mobility. If one of these factors is altered, the muscle will undergo adaptation. As an increased muscular activity leads to the enhancement of the structures involved in contraction, inactivity or disuse is followed by the reduction of the muscle mass

Alterations of biochemical parameters and changes at the ultrastructural level of the contractile apparatus are considered to be characteristic of atrophied muscles. The effect of disuse on the skeletal muscle depends on the fibre type composition of the muscle. The degenerative changes in disused muscles at the ultrastructural level have been shown to be most severe in slow oxidative muscle fibres. It is suggested that the most vulnerable muscles were antigravity

The properties of muscle contraction which depend on the MyHC isoform composition decrease in atrophied skeletal muscle [46,89,91]. Contractile activity can induce differential expression of myosin protein isoforms in skeletal muscle. MyHC composition has an important regulatory role in myosin ATPase activity and muscle fibre shortening velocity [92]. A prolonged activity causes alterations in the MyHC composition. A decrease in the mechanical load stimulates the conversion of slow myosin in muscles of mixed fibre type composition, whereas a decrease in the weight-bearing load results in a decrease in slow myosin content

**5.6. Effect of unloading on the skeletal muscle MyHC composition**

consequence of altered functional conditions [46,64,89].

58 Composition and Function of the Extracellular Matrix in the Human Body

[8,77].

[46,92].

muscles crossing a single joint [90].

The plasticity of muscle is apparent in the phenomena such as muscle atrophy caused by inactivity, and recovery from atrophy. When atrophic muscles once again become active, the muscle mass and the volume reportedly increase in a relatively short period of time, but the recovery of the muscle strength takes much longer [93]. The recovery of motor activity after the hindlimb suspension is as fast as the recovery of the muscle strength. It is probably related with the regeneration of the muscle structures from disuse atrophy [89]. The fact that the increases in the muscular strength lag behind those in the muscular mass suggests the presence of functionally immature muscle fibres during the recovery process following disuse atrophy [89]. Several studies have shown that the increases in the muscle mass soon after reloading are attributable to oedema and do not actually represent recovery [89]. The recovery of the muscle mechanical properties depends also on the metabolism of the skeletal muscle. The two-week reloading period has shown that the Sol muscle metabolism can be restored [94]. Full recovery of slow-twitch muscle function via cross-sectional area and myonuclear domain size has been shown to need more time for restoration of neural and mechanical properties of muscle [81].

Disuse muscle atrophy can be experimentally induced by suspending animals by their tails [89,95], immobilizing joints, severing tendons or conducting muscle denervation [96]. Muscle atrophy in tail suspension is caused by hindlimb unloading, conserving the functions of nerves and joints. Reloading is thus possible after tail suspension and is suitable for investigating the recovery process following disuse muscle atrophy caused by sports injuries [89].

The reloading after hindlimb suspension shows that collagen III mRNA level at the end of the second week is higher than in control group. It has been found that in response to reload, the skeletal muscle expression of collagen I and III was markedly induced from the second day of reloading [4].

It has been shown that non-fibrillar type IV collagen mRNA level is decreasing in both, slowtwitch and fast-twitch muscles during the three weeks of hindlimb suspension, but two weeks of reloading period is obviously not enough to restore the metabolic states of this collagen in the basal lamina of the muscle fibre [26]. It was demonstrated that the reorganization of the basement membrane compounds needs certain time [97]. As type IV collagen plays a role in the regenerative process on ECM, including the matrix-associated receptors that underline muscle fibre-matrix interactions, it shows how complicated is the evaluation of the functional significance of type IV collagen metabolism [5].

The recovery of the collagen degradation markers during reloading period is different in slowand fast-twitch skeletal muscles. MMP-2 level increased in slow-twitch soleus muscle during two weeks of reloading and in fast-twitch gastrocnemius muscle after one week reloading. The mRNA level of TIMP-1 increased in fast-twitch GM muscle after two weeks of reloading. A significant increase in mRNA level for MMP-2 was registered in Sol muscle during the reloading, showing that the reaction of MMP-2 on the pretranslational level is not fast in all muscles [52].

Concomitant to atrophy, numerous molecular events testify of a slow-to-fast transition of muscle properties [47,74]. The recovery of muscle properties effectively occurs on return to normal load [98]. It is also known that muscle fibre damage occurs during reloading, likely due to the inability of the muscle fibres to bear eccentric contractions and the consequent inflammation process [94,99]. Natural recovery seems to be most effective after reloading while several investigations show the delayed recovery of rats during running exercise [100].

The muscle tissue response to unloading seems to more pronounced than the connective tissue response. The connective structures are protected from rapid changes in tissue mass, while muscle, which is known to act as a protein store for the organism, is subject to substantial and fast changes in tissue mass. However, it should be considered that important changes occur in the connective tissue structures during unloading despite the small changes in tissue mass.


*Review* **Insights into the Role of Matrix Metalloproteinases in Precancerous Conditions and in Colorectal Cancer**

**Zahra Pezeshkian 1, Stefania Nobili 2,3, Noshad Peyravian 1, Bahador Shojaee 1, Haniye Nazari 4, Hiva Soleimani 5, Hamid Asadzadeh-Aghdaei 1, Maziar Ashrafian Bonab 6, Ehsan Nazemalhosseini-Mojarad 7,\* and Enrico Mini 8,9,\***

**Simple Summary:** Colorectal cancer (CRC) is one of the most common cancer worldwide. CRC is derived from polyps and many factors, such as Matrix Metalloproteinases (MMPs) can gain the progression of colorectal carcinogenesis. Many investigations have indicated the role of MMPs in CRC development while there is not enough knowledge about the function of MMPs in precancerous conditions. This review summarizes the current information about the role of MMPs in polyps and CRC progression.

**Abstract:** Colorectal cancer (CRC) is the third and second cancer for incidence and mortality worldwide, respectively, and is becoming prevalent in developing countries. Most CRCs derive from polyps, especially adenomatous polyps, which can gradually transform into CRC. The family of Matrix Metalloproteinases (MMPs) plays a critical role in the initiation and progression of CRC. Prominent MMPs, including MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, MMP-14, and MMP-21, have been detected in CRC patients, and the expression of most of them correlates with a poor prognosis. Moreover, many studies have explored the inhibition of MMPs and targeted therapy for CRC, but there is not enough information about the role of MMPs in polyp malignancy. In this review, we discuss the role of MMPs in colorectal cancer and its pathogenesis

**Keywords:** Matrix Metalloproteinases (MMPs); polyp; colorectal cancer; TIMPs; MMP polymorphisms; MMP targeting

### **1. Introduction**

At approximately 11% of all diagnosed cancer cases, CRC is the third most common cancer and the second most lethal cancer worldwide [1,2]. It is today well known that several factors contribute to the CRC pathogenesis, driving complex genetic and epigenetic processes that, ultimately, transform normal colonic mucosa to cancerous tissue [3]. CRC

**Citation:** Pezeshkian, Z.; Nobili, S.; Peyravian, N.; Shojaee, B.; Nazari, H.; Soleimani, H.; Asadzadeh-Aghdaei, H.; Ashrafian Bonab, M.; Nazemalhosseini-Mojarad, E.; Mini, E. Insights into the Role of Matrix Metalloproteinases in Precancerous Conditions and in Colorectal Cancer. *Cancers* **2021**, *13*, 6226. https:// doi.org/10.3390/cancers13246226

Academic Editor: Heike Allgayer

Received: 14 November 2021 Accepted: 7 December 2021 Published: 10 December 2021

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

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

may initiate from benign polyps with the mucosal origin and can develop into carcinoma. Colorectal polyps, especially adenomas, are proliferative lesions that have been defined as the precursor of CRC. Therefore, the early detection and removal of these polyps can interrupt the progression of the adenoma-carcinoma sequence [4,5].

Many molecular signaling pathways are involved in CRC initiation and progression, such as ERK/MAPK, TGF-β, PI3K/Akt, Src/FAK, and β-catenin pathways. These pathways can promote the hallmarks of cancer such as inflammation, angiogenesis, metastasis, and invasion, also via the activation and overexpression of MMPs [6,7]. Thus, MMPs have been suggested as potential prognostic factors for the malignancy risk of colorectal polyps. MMPs are proteolytic enzymes implicated in the degradation of stromal connective tissues and of the extracellular matrix (ECM), a complex network that plays a key role in sustaining signaling transduction and thus cancer development and progression [8]. As such, MMPs have key roles in tumor initiation, progression, and metastasis and can affect tumor cell behavior by cleaving proapoptotic agents and producing an aggressive phenotype [9]. Because of these roles, MMPs have been detected as biomarkers in CRC progression [10]. A new challenge in CRC treatment is finding an effective pharmacological and therapeutic method for suppression of MMPs and targeted therapy of CRC [11]. This review will deal with the role of MMPs in colorectal carcinogenesis from colorectal polyps to CRC.

### **2. CRC Pathogenesis and Molecular Classification**

Colorectal polyps result from atypical cell proliferation in the colorectal tissue. Based on histological and morphological features, colorectal polyps are divided into neoplastic (adenoma) and non-neoplastic (hyperplastic, hamartomatous, and inflammatory) types [5,12]. Neoplastic polyps, also known as adenomatous polyps, are subclassified by their histological characteristics as tubular, villous, or tubulovillous adenomas. Previous investigations demonstrated that approximately 5–10% of neoplastic polyps are villous adenomas and most of them show dysplasia. Approximately 10–15% of neoplastic polyps show morphological features of both villous and tubular types [13]. Adenomas are not usually transformed to carcinoma, but there is evidence that the adenoma-carcinoma sequence originates from adenomatous polyps [14]. Also, hyperplastic polyps may possess malignancy potential [15]. CRC is caused by the misregulation of some oncogenes such as *KRAS* and *c-MYC* and tumor suppressor genes such as *P53* and APC, which control cellular signal transduction [16–18].

### *2.1. Molecular Mechanism of CRC*

Specific features characterize CRC and its pathogenesis based on genetic, epigenetic, and transcriptomic factors. Three main molecular abnormalities are involved in CRC carcinogenesis:

### *2.2. Molecular Classification Based on Transcriptomic Analysis*

Based on gene expression profiles, CRC has been classified into subgroups with distinct molecular and clinical features [24].

	- CMS1 (14%) indicates MSI, CIMP, and *BRAF* mutation and immune activation.
	- CMS2 (37%) shows Wingless-Type MMTR integration site family member (WNT), MYC signaling activation, and epithelial involvement.
	- CMS3 (13%) demonstrates MSI, CIMP, and *KRAS* mutations and metabolic involvement.
	- CMS4 (23%) includes invasion, metastatic situations, and TGF-β signaling coactivation and angiogenesis. Also, epithelial-mesenchymal transition (EMT) is a crucial event in colorectal carcinogenesis and is involved in CMS4 status. EMT can result in advanced-stage CRC, poor patient survival, and worst clinical features [26,27] and CMS4 subgroup shows the most unfavorable prognosis.
	- CRIS-A is enriched for *BRAF*-mutated MSI tumors and *KRAS*-mutated MSS tumors that are without targeted therapeutic options.
	- CRIS-B is related to invasive tumors with poor prognosis and high TGF-ß signaling. CRIS-B is unconnected to the CMS4 mesenchymal subtype, which also indicates aggressive tumors with TGF-ß pathway activation.
	- CRIS-C is dependent on EGFR signals and is sensitive to anti-EGFR monoclonal antibody treatment.
	- CRIS-D shows IGF2 overexpression. This occurrence has been involved in desensitization to the EGFR blockade in patients with *KRAS* wild-type tumors.
	- CRIS-E indicates *KRAS*-mutated, Paneth cell-like CIN tumors refractory to anti-EGFR antibody treatment [28].

### **3.  The propeptide domain is highly conserved and can regulate the sequence that interacts with Zn2+. Also, cystine within this area permits the MMPs to be in the active or inactive status [30]. The catalytic domain possesses a conserved zinc-binding motif which, in the active condition, will disconnect from the propeptide domain. Movement between the catalytic and PEX domain is done via hinge regions [29]. According to their structural domains, MMPs have been categorized into collagenase, gelatinase, stromelysin, matrilysin, and membrane-bound MMPs (MT-MMPs) [31,32].

MMPs play a crucial role in the remodeling of the ECM by digestion of ECM components, stimulation of cell surface proteins. Also, they can control the activity of other proteinases, growth factors, chemokines, and cell receptors, and moderate many biological functions [33]. MMPs can regulate cellular growth, migration, survival, and adhesion in biological and pathological statuses (Table 1, Figure 1). Due to the MMP's key roles, the dysregulation of their expression levels and their activation lead cancerous cells to proliferation, angiogenesis, survival, invasion, malignant transitions, and immune dysregulation [34–36]. Also, the tissue inhibitors of metalloproteinase (TIMPs) control the activation of MMPs and have a critical action in precancerous conditions, CRC progression, and metastasis (Table 2, Figure 2) [11,37].

**Table 1.** Matrix Metallopeptidases Features in Humans.

**Figure 1.** Summary of the prominent *MMP* genes in CRC. MMPs play different functions in CRC.

**Table 2.** Summary of Investigations about the Roles of MMP Genes and Proteins in Colorectal Polyps and Cancer.

**Table 2.** *Cont.*

**Table 2.** *Cont.*

**Table 2.** *Cont.*

**Figure 2.** The diagram indicates the role of *MMPs* genes in adenoma development, colorectal adenoma-carcinoma sequence, and tumor progression. *MMP-1*, *MMP-3*, *MMP-7*, *MMP-9*, and *MMP-13* are involved in adenoma development. *MMP-1*, *MMP-2*, *MMP-3*, *MMP-7*, *MMP-8*, *MMP-9*, *MMP-12*, *MMP-13*, *MMP-14*, and *MMP-21* participate in adenoma-carcinoma sequence and tumor progression.

### **4. The Function of MMPs in Colorectal Polyps and Cancer**

*4.1. MMP-1, MMP-13, and MMP-8 (Collagenases)*

The specific targets for MMP-1 and MMP-13 are in the intestine. MMP-1 can digest type I, II, III, VII, VIII, X collagen, and gelatin. Upregulation of *MMP-1* gene was detected in CRC patients compared to normal tissue [6,59]. Eiro et al., found overexpression of *MMP-1* gene in serrated, villous, and tubulovillous adenomas (i.e., polyps with high potential for transformation to CRC) [44]. Previous investigations demonstrated the correlation between *MMP-1* gene expression and CRC progression: high expression levels of *MMP-1* were associated with invasion, advanced stage metastasis, LNM, and shorter overall survival [60,61]. Wang, et al. investigated the role of MMP-1 in the development of CRC. They found that the downregulation of MMP-1 expression inhibited the progression of CRC in vitro and in vivo by suppressing the PI3K/Akt/c-myc signaling pathway and the EMT [6].

MMP-13, another member of the collagenase category, could degenerate type III collagen. According to the strength of the association between pathologic stage and immunoreactivity scoring (IRS) of MMP-13, in high-grade adenomas and CRC, MMP-13 was observed with a moderate and strong staining intensity, respectively [46]. This result indicated that MMP-13 could help to predict metastatic behavior and prognosis of earlystage cancerous and precancerous colorectal adenoma [46,62]. The study of the association

between grade dysplasia and MMP-13 expression in 137 biopsies from patients with cancerous and non-cancerous colorectal adenomas showed that the high expression level of MMP-13 IRS could be helpful to predict metastatic state, prognosis, and recrudescence at an early stage of cancerous and precancerous colorectal adenoma. Moreover, the upregulation of MMP-13 IRS from low to high-grade adenoma was considered an early predictive cancer biomarker [46]. Meanwhile, several studies confirmed that upregulation of MMP-13 was related to advanced CRC and liver metastasis [62–64]. Also, the expression of MMP-13 on the primary tumor cell surface is increased in inflammatory bowel disease. The expression of MMP-13 is closely related to the progression, early relapse, and high mortality of CRC [63,65].

Another member of collagenase enzymes is MMP-8 which is frequently expressed by neutrophils. MMP-8 cleaves many substrates, such as type I, II, and III collagen. This MMP is mainly considered to play a protective role against cancer. However, more recent findings also suggest an oncogenic function of *MMP-8* gene [66,67].

Sirnio et al., found that enhanced-serum MMP-8 level in CRC patients was significantly related to advanced-stage CRC, distant metastasis, lack of MMR, and poor survival. Thus" they evidenced that MMP-8 is correlated with inflammation and CRC progression [68].

### *4.2. MMP-2 and MMP-9 (Gelatinase)*

MMP-2 and MMP-9, known as gelatinases, can digest type IV collagen and gelatin [69]. Murname et al. showed that MMP-2 protein activity in adenomas with high-grade dysplasia (HGD) was different from adenomas with low-grade dysplasia (LGD). They suggested that the active *MMP-2* gene could predict CRC malignancy risk in patients with adenomatous polyps [70]. Some studies also indicated high expression levels of MMP-9 protein in adenomas with HGD compared to adenomas with LGD and normal tissue. As such, researchers speculated that upregulation of MMP-9 is a primary event in the CRC adenoma-carcinoma sequence [41,71]. High expression levels of MMP-2 protein in CRC tumors compared to normal mucosa have also been reported [41,72]. In addition, a statistically significant relationship between upregulation of *MMP-2* gene with advanced-stage CRC or CRC progression has been observed [41,73–75]. On this basis, *MMP-2* has been suggested as a potential biomarker to detect CRC progression and predict patient survival. Furthermore, overexpression of the *MMP-2* gene was associated with metastasis of lymph nodes and a decrease of cell adhesion in tumors [73].

Finally, also the upregulation of *MMP-9* gene was associated with the advanced stage of CRC and suggested as a biomarker predictive of poor overall survival [41,76]. Chen et al. indicated that the overexpression of *MMP-9* gene promoted CRC metastasis through the MKK-3/p38/NF-κB pro-oncogenic pathway. Furthermore, they suggested *MMP-9* gene as a potential molecular target for targeted therapy to treat metastatic CRC patients [76].

On the contrary, some investigations reported that *MMP-9* gene has a protective role in CRC by stimulating Notch activation resulting in the activation of p21WAF1/Cip1 leading to the suppression of β-catenin [77,78]. In a recent study, although in colitisassociated colon cancer, Walter et al. confirmed this observation by revealing that MMP-9 protein expression was associated with reduced ROS levels, decreased DNA damage, and stimulated mismatch repair pathway [79].

In an interesting study, Wei et al., by analyzing microbiota in tumors obtained by patients with different prognoses, found that the expression of some inflammatory genes, including *MMP-9*, was associated with the abundance of specific bacteria. High levels of *MMP-9* expression were significantly correlated with the high abundance of *B. fragilis* and *F. nucleatum* whereas a high level of F*. prausnitzii* was associated with downregulation of *MMP-9* [80].

### *4.3. MMP-3, MMP-10 (Stromelysin)*

Another member of MMPs family is MMP-3, or stromelysin-1, which degrades collagen (types II, III, IV, IX, and X), proteoglycans, fibronectin, laminin, and elastin in ECM.

Sipos et al., found a positive association between MMP-3 protein expression and the adenoma–dysplasia–carcinoma sequence. In particular, they reported that high-grade dysplastic sessile adenomatous-stage and early-stage CRC conditions can be differentiated based on the stroma expression of MMP3 [81]. Meaningful positive associations between the protein expression level of MMP-3, invasion, lymph node metastasis, histological type of CRC, and poorly differentiated tumor were reported by Islekel et al. [82]. MMP-3 can activate other MMPs, such as MMP-1, MMP-7, and MMP-9, to promote the progression of tumor initiation [83,84].

MMP-10 also belongs to the stromelysin family. It can digest collagen types II, III, IV, IX, X, proteoglycans, fibronectin, laminin, and elastin. Also, MMP-10 enhances cell growth and invasion in CRC, and its upregulation was found to be associated with poor survival [49,85].

### *4.4. MMP-7 (Matrilysin)*

MMP-7, or matrilysin, digests fibronectin, laminin, type I collagen, and gelatin. It can provide the right condition for vascularization via cleavage of ECM [86]. A major ratio of MMP-7 expression in tumor cells has been reported. Qasim et al., found MMP-7 protein overexpression in villous adenomatous polyps compared to other types of polyps and demonstrated that MMP-7 protein overexpression is an initial event in CRC carcinogenesis that could lead adenomas to CRC [53]. In our laboratory, we observed high expression levels of *MMP-7* and *VEGF-A* mRNA in adenomatous polyps compared to normal tissue. We found that the expression levels of *MMP-7* and *VEGF-A* genes were higher in villous adenoma than in other types of adenomas. Thus, we concluded that the *MMP-7* gene overexpression has a critical role in colorectal adenoma angiogenesis and could be a primary event in the adenoma-carcinoma sequence [45].

*MMP-7* gene can enhance tumor growth and metastasis [87]. Also, MMP-7 activates other MMPs, such as proMMP9 and proMMP2 [88] In addition. MMP-7 exerts a wide spectrum of activities not only as an enzyme but also as a signaling molecule. In fact, it has been shown that MMP-7 trans-activates EGFR by releasing the heparin-binding epidermal growth factor (HB-EGF) in CRC cells, with consequent cell proliferation and apoptosis regulation [89,90].

### *4.5. MMP-12 (Metalloelastase)*

MMP-12, or metalloelastase, can digest different substrates. Several studies considered *MMP-12* gene as an anti-metastatic agent [91,92]. Also, it could inhibit angiogenesis by downregulation of *VEGF* and enhancement of the endogenous angiogenesis inhibitor angiostatin. Overall, the role of *MMP-12* in tumor suppression and increase in overall survival has been widely recognized [93–95].

Importantly, Klupp et al., found higher levels of MMP-12 protein expression in sera of CRC patients compared with those of healthy individuals. Also, they suggested an association between MMP-12 protein expression levels and CRC advanced disease and vascular invasion. Furthermore, a significant correlation between the upregulation of MMP-12 expression and poor survival was shown [49].

### *4.6. MMP-21 (XMMP)*

MMP-21 (XMMP) can degenerate aggrecan (cartilage-specific proteoglycan core protein) in the internal region of ECM [96]. Overexpression of MMP-21 protein in CRC compared with normal tissue was shown in many studies [97,98]. Furthermore, significant associations between MMP-21 protein expression and CRC tumor invasion, lymph node metastasis, and distant metastasis were found [97,99]. Wu et al., showed that MMP-21 not only affected CRC progression but also was an independent prognostic biomarker in patients with stage II and stage III CRC cancer. Taken together, these facts led them to conclude that MMP-21 could be used for targeted therapy in CRC [97]. Huang et al., demonstrated that the upregulation of MMP-21 protein was related to shorter overall survival in patients with CRC [98].

### *4.7. MMP-14 (MT1-MMP)*

MMP-14, called MT1-MMP, acts on matrix substrates, such as collagens I, II, III, and gelatin. The *MMP-14* gene plays a crucial role in many biological and pathological conditions and activation of proMMP2 [92,100]. The role of *MMP-14* in angiogenesis and cancer invasion has been identified by previous investigations [101–103]. Cui et al., observed statistically significant associations between the overexpression of *MMP-14* gene in CRC compared to normal mucosa. Their analysis indicated that high expression levels of *MMP-14* were associated with advanced-stage CRC, lymph node metastasis, and poor overall survival. They concluded that the *MMP-14* gene is an oncogene and may represent a potential prognostic biomarker in CRC [104].

Yang et al., showed in an in vivo CRC model that the STAT3 phosphorylation activity and the overexpression of MMP14 protein were enhanced by the overexpression of Hes1 gene. Also, they suggested that Hes1 promoted the invasion of colorectal cancerous cells via the STAT3-MMP14 pathway [103]. It was reported that the overexpression of MMP-14 protein was associated with Prox1 gene. When Prox1 gene was deleted, MMP14 protein was increased, and the mice showed slow-growing, matrix-rich, chemotherapy-resistance, and cancerous cells with malignant stromal features, including activation of fibroblasts, blood vessels dysfunction, and lack of cytotoxic T cells [105].

### **5. The Effects of Polymorphisms of** *MMP* **Genes on Colorectal Carcinogenesis**

Single-nucleotide polymorphisms (SNPs) are a common genetic variation involving a single base pair in DNA. SNPs are mostly located in the gene promoter region and may have an impact on gene and protein expression levels. The effects of MMP polymorphisms have been observed in many cancers such as CRC and hepatocellular carcinoma [106,107].

In a Japanese population, the *MMP-1* 1G/2G polymorphism was detected and associated with the development of CRC [108]. In the Iranian population, Kouhkan et al., demonstrated that *MMP-1* 2G/2G genotype polymorphism was correlated with invasion risk of CRC, especially in smoker men [109]. In the Netherlands, *MMP-2*-1306C>T SNP was detected in CRC patients, and the T/T genotype was found to be associated with poor overall survival whereas C/C and C/T genotypes showed better outcomes. No difference in overall survival was instead observed among patients with different genotypes of the *MMP-9*-1562C>T SNP [110]. Also, in a cohort study of Taiwanese CRC patients, Ting et al. indicated that patients carrying the A/A genotype of the *MMP-2*-1575G>A SNP had a higher risk to develop distant metastasis compared with patients carrying the T/T genotype [111]. In a Polish population with CRC, individuals with the G/G variant genotype of *MMP-7*-181A>G SNP had a higher risk of lymph node involvement and advanced tumor infiltration than patients carrying the A/A genotype [112]. A Chinese study showed that the *MMP-9* R279Q SNP relative to the R/R genotype was correlated with a higher risk of CRC compared with the QQ genotype. Also, the allele frequency of the *MMP-1* 16071G/2G and *MMP-7* 181 A/G polymorphisms were not associated with CRC [113]. In a Korean population, the homozygous *MMP-9*-1562C/C genotype was significantly more frequent in CRC cases than in the control group [114]. In Sweden, researchers found that the A/A genotype of *MMP-12*-82A>G increased the risk of disseminated malignancy in CRC patients while the A/A genotype of *MMP-13*-82A>G was not correlated to invasion [115].

Lièvre et al., investigated *MMP-3*, *MMP-7*, and *MMP-1* genes promoter polymorphisms in 295 patients with large adenomas and 302 patients with small adenomas. The analysis revealed a significant association between *MMP-3*-1612 ins/del A, *MMP-1*-1607 ins/del G polymorphism, and small adenomas; also, adenomas were associated with the combined genotype 2G/2G-6A/6A. However, no significant association between *MMP-7* polymorphism and the development of adenomas was found. The authors suggested that only the study *MMP-3* and *MMP-1* gene promoter polymorphisms had potential roles in

the development of adenomas from normal colon epithelial cells or in the earliest steps of CRC [57].

Tai et al., showed that *MMP-8* rs11225395 related to the risk of CRC and worst outcomes in a subpopulation of the Han Chinese population. On this basis, they suggested *MMP-8* rs11225395 polymorphism as a potential biomarker predictive of CRC susceptibility [116].

### **6. Targeting MMPs in CRC Treatment**

*6.1. Pharmacological Inhibition*

Several pharmacological inhibitors of MMPs (MMPIs) have been studied and tested in phase I-III clinical trials, but to date, none of these drugs has been approved for the treatment of cancer, including CRC. Overall, the late stages of the clinical experimentation failed because of the substantial toxicity and weak selectivity of MMPIs [117]. Mainly, candidate MMPIs are represented by small molecules, peptides, and antibodies [118]. Currently, only one broad-spectrum MMPI has been approved by FDA but it has not indication in cancer (i.e., the small molecule periostat) [117,119]. Other MMPIs, such as the small molecule prinomastat, selective for MMP-1, MMP-2, and MMP-9 [120–123] and the GA-5745/andecaliximab, a selective anti-body against MMP-9, have reached the phase III [124,125]. However, none of these trials includes CRC.

### *6.2. Inhibition of MMPs by TIMPs*

Since MMPs are naturally inhibited by TIMPs, these proteins have also been widely investigated mainly to exploit their ability to discover potential strategies for MMP inhibition [126]. The TIMP family consists of four members of proteins (TIMP1-4) that form a 1:1 complex with MMPs. Dysregulation of this complex due to the increased expression of MMPs or a decreased control by TIMPs has been observed in several diseases, including cancer. TIMPs control the activity of MMPs via binding to them (Figure 3) [126–128].

**Figure 3.** MMPs inhibition by TIMPs. TIMP-1 inhibits MMP-1, 3, 7, 9. TIMP-2 can suppress MMP-2 and 9, and TIMP-4 blocks MMP-2. These inhibitions result in the primary tumor transitioning to advanced CRC. Moreover, TIMP-3 has a protective effect on CRC cases and could bind to several MMPs [126–128].

TIMP-1 inhibits MMP-1, 3, 7, 9 and affects angiogenesis [37,129]. Previous investigations considered a dual activity for the *TIMP-1* gene: in particular, *TIMP-1* was associated with tumor growth at the early stages of colon cancer, and decreased activity of *TIMP-1* could lead to tumor invasion [130,131].

TIMP-2 can suppress MMP-2, MMP-9, and microvascularization [129,132]. Also, downregulation of TIMP-2 is related to invasive CRC [133]. Wang et al., reported that downregulation of *TIMP-2* in CRC tumor tissues was meaningfully correlated with the depth of invasion, lymph node metastasis, tumor stage, and poor survival [134].

*TIMP-3* is known as a tumor suppressor gene and inhibits several MMPs. *TIMP-3* downregulation is associated with advanced CRC [135]. Lin et al., represented that, adenovirus-mediated *TIMP-3* transduction in CT26 colon cancer cell line suppressed cell growth and stimulated apoptosis. Also, *TIMP-3* transduction inhibited migration and invasion. In vivo data indicated that *TIMP-3* prevented in vivo tumor growth and liver metastasis [136].

TIMP-4 protein suppresses MMP-2, and one study showed that overexpression of TIMP-4 increased the survival rate of rectal cancer [128].

Currently, no drug mimicking the TIMP activity has been obtained as well as no gene therapeutic approach able to modulate the activity of TIMPs is available.

### *6.3. MMPs Regulation by microRNA*

MicroRNAs, a class of small, endogenous RNAs of 21–25 nucleotides in length, control gene and protein regulation via binding and digesting target mRNA (Table 3). Suppression of MMPs by microRNAs is a suggested way for CRC treatment. Some evidence has been provided. In particular, microRNA-34 (miR-34a) plays a role as a tumor suppressor, and its overexpression could suppress *MMP-1*, *MMP-9*, and tumor cell proliferation, migration, and invasion via acetylation of *P53* in CRC [137–139]. The upregulation of miR-139 reduces proliferation, migration, and invasion by suppression of the IGF-IR/MEK/ERK signaling and *MMP-2* gene in CRC patients [140]. Upregulation of miR-29a increases CRC metastasis via suppression of KLF4 (Kruppel-like factor 4), transcription factor, and upregulation of *MMP-2* gene [141]. Also, miR-29b suppresses CRC metastasis, reduces angiogenesis and EMT by targeting the *MMP-2* gene [142]. Overexpression of miR-143 can suppress the *MMP-7* gene directly and prevent colorectal tumor cell proliferation and invasion [143].

**Table 3.** MMPs are Regulated by microRNAs in CRC.

### *6.4. MMPs Regulation by Long Non-Coding RNAs*

Long non-coding RNAs (lncRNAs) can regulate gene expression and have key roles in cell proliferation, migration, invasion, apoptosis, metastasis, and EMT in CRC. In this regard, lncRNA-targeted therapy is today considered a potential promising strategy for CRC treatment [144]. In fact, based on mechanistic studies investigating the complex lncRNAmediated sponge interactions in CRC, potential therapeutic targets for the treatment of this cancer may be identified. Among the available findings, Tian et al., demonstrated that the suppression of *TUG1* by shRNA prevented *MMP-14* expression, proliferation, invasion, and EMT in colon cancer [145]. Sun et al., found a significant association between XIST inhibition and suppression of *c-Myc*, *cyclinD1,* and *MMP-7* expression through inactivation of Wnt/β-catenin signaling pathway [146]. A recent investigation showed a meaningful correlation between the overexpression of LINC00963 and the upregulation of *MMP-2* and *MMP-9*, proliferation, migration, and invasion of CRC cells [147]. Duan et al., revealed that the inhibition of the CCEPR lncRNA reduced the expression levels of *MMP-2* and *MMP-9*, and prevented EMT in CRC cells [148]. Pan et al., realized that the expression level of MMP-2 protein was notably decreased when PCA3 was knocked out. In addition, suppression of PCA3 inhibited colon cancer cell invasion and migration [149].

### **7. Conclusions**

In summary, MMPs genes and proteins, through complex mechanisms involving the induction of many molecular signaling pathways and the EMT process, play a relevant role in the transition from pre-cancerous lesions and polyps to advanced CRC. However, further investigation is needed to understand how MMPs exactly work. This would improve the selectivity of MMPIs that could be exploited in a dual-mode: to treat CRC alone or in combination with targeted agents and/or chemotherapy and to prevent CRC development.

**Author Contributions:** Conceptualization, investigation, writing—original draft, designed tables and figures Z.P.; editing, validation, and revise, S.N.; writing, design table, and investigation, N.P.; revise, B.S.; investigation, H.N. and H.S.; validation, H.A.-A. and E.M.; supervision, validation, and revise E.N.-M. and M.A.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Fondo Beneficenza Intesa Sanpaolo S.p.A. (Milan, Italy) and Associazione Giacomo Onlus (Castiglioncello, Italy) to E.M.

**Acknowledgments:** The authors would like to thank all the staff of the Department of Cancer at the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

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

### **References**


*1.1. Related Work*

Multi-agent systems have been used intensively in recent year in the energy sector. In [4], the impact of the variation of charging prices on the behavior of electric vehicle users was investigated, comparing benefits when using variable and fixed charging prices. With the goal of generating a realistic population of customers, an EV usage simulator was developed that models the effects of city size. Offering variable prices showed good results and could be a promising approach where EV users have different prices when deciding the location of their next charging spot.

Xydas et al. [6] built a MAS for charging controllers and EVs. The objective of the controller was to operate the CS in order to perform peak shaving, valley filling, maximizing income, and for the EV to minimize costs in two different scenarios, one where the agent is reacting to variations of electricity prices, and one for charging processes irrespective of price signals. The aim of the research presented in [7] was to measure user satisfaction and their adaptability to new vehicle technologies (specifically, full electric vehicles). The work in [8] presented a simulation framework for electric vehicles in terms of energy consumption. In [9], vehicle behavior was simulated by an agent-based transportation simulation tool with detailed routes on a map of Zurich for the simulation of vehicle behavior with realistic vehicle energy consumption profiles. The approach uses multi-agent transportation simulation (MATSim). Sweda et al. [10] looked at the strategic investment in new charging infrastructure considering private EV usage and driving patterns. The study was done using an MAS to simulate user behaviors.

## *1.2. Multi-Agent Simulation Systems*

In the literature, different MAS frameworks have been employed. In [11], the authors used an MAS for investigating the issue of avoiding grid congestion in scenarios with a large number of EVs. The simulation was based on the JACK framework in conjunction with MATLAB/Simulink.

In [12], the authors briefly summarized multi-agent simulation tools and specifically evaluated MASON and NetLOGO via a test implementation for human modeling (We briefly cite one of their main conclusions: "NetLOGO has proved its reputation as an ABS platform where the simulation models can be implemented quickly and straightforwardly. . . . Had we simply looked for a handy standalone agent-based simulation tool for a limited number of agents, NetLOGO easily could have been our choice. . . "). NetLOGO was also employed in [13] to simulate human behavior.

Another comparison study on MAS tools as [14]. They also came to the conclusion that NetLOGO was surprisingly efficient for the task ("Perhaps because NetLOGO is clearly designed for one type of model (Section 3.1) and uses a simplified language, scientists tend to assume it is too limited for serious ABMs. We originally intended to exclude NetLOGO as too limited for full treatment in this paper, but found we could implement all our test models (Section 2.1) in NetLOGO, with far less effort than for other platforms.").


*1.1. PI3K-AKT Pathway*

Viruses activate metabolic pathways in order to meet their needs for production of appropriate macromolecules. Once such pathway is the multifunctional phosphatidylinositol 3-kinases (PI3K)-AKT pathway. Due to its central importance in metabolism but also other cellular functions, this pathway is a common target for viruses [1–4]. In this review, we describe the activation of the PI3K-AKT pathway by alphaviruses and the consequent cellular effects.

PI3Ks are a large family of kinases that is divided in three classes: class 1 (1A and 1B), class II, and class III. The PI3K-AKT pathway involves class 1A PI3Ks. These PI3Ks are heterodimers with a catalytic subunit (p110) and a regulatory subunit (p85). Class 1A PI3Ks are activated by receptor tyrosine kinases (RTK) or G protein-coupled receptors (GPCR) after binding of growth factors, either directly or indirectly via activation of the small GTPase RAS [5] (Figure 1). Once the regulatory action of p85 has been relieved, active class 1A PI3Ks phosphorylate phosphatidylinositol-4,5-biphosphate (PIP2, or PtdIns(4,5)P2) to phosphatidylinositol-3,4,5-triphosphate (PIP3, or PtdIns(3,4,5)P3) at the plasma membrane (PM). PIP3-enriched membranes are a docking site for the PI3K-dependent kinase-1 (PDK1) and mTORC2 (or PDK2). Furthermore, the serine/threonine kinase AKT (or protein kinase B, PKB) is relocated to membrane sites with PIP3, where it can be activated via phosphorylation by PDK1 and mTORC2 [6,7]. A number of downstream targets of AKT have been identified, several of which are multifunctional nodes, integrating AKT signalling with signalling through other pathways [7]. One of these is the serine/threonine kinase mammalian target of rapamycin (mTOR) in the multi-protein complex mTORC1. AKT signalling leads to the activation of mTORC1, which promotes cell growth by

inducing lipid biogenesis through activation of the transcription factors SREBP1 and PPARγ and by promoting protein synthesis by activating the S6 kinase (S6K) and by inactivating the translational inhibitor 4E-BP1. mTORC1 also inhibits autophagy by blocking ULK1 [6]. The serine/threonine kinase glycogen synthase kinase 3 (GSK3) is another multifunctional target of AKT. Through phosphorylation, active GSK3 inhibits most of its substrates. Upon phosphorylation by AKT, GSK3 itself is inhibited and thereby the downstream targets are positively regulated. These targets include the prosurvival BCL-2 family member MCL-1 and the transcription factor c-Myc, which is required for expression of many genes involved in proliferation. Other GSK3-targets, such as glycogen synthase, are involved in (regulation of) cellular metabolism [7]. The third multifunctional target of AKT are the forkhead box O (FoxO) transcription factors. Phosphorylation of FoxO transcription factors by AKT leads to acute translocation out of the nucleus. Thus, AKT signalling suppresses the expression of FoxO targets. These include targets involved in the induction of apoptosis, cell-cycle arrest, catabolic metabolism and growth inhibition. Thus, PI3K-AKT signalling via these multifunctional targets promotes cell survival, growth and proliferation and steers cellular metabolism towards anabolism.

**Figure 1.** Activation of PI3K-AKT pathway by external signals via G-protein coupled receptors (GPCR) or receptor tyrosine kinases (RTK) leads to dissociation of the p85 regulatory subunit from the active p110 PI3K subunit. The active subunit catalyses the conversion of PIP2 to PIP3 at the plasma membrane, leading to the recruitment and activation of the AKT kinase. Via multiple downstream effector pathways, cellular states of growth, proliferation, heightened metabolic activity and survival are promoted. For more details, see text.

The PI3K-AKT pathway is regulated in many ways. The RAF-MEK-ERK pathway also promotes cell survival and growth and the two pathways have co-regulated proteins and negatively regulate each other. For example, MEK promotes membrane localisation of the phosphatase and tensin homologue (PTEN), where it dephosphorylates PIP3 and inhibits AKT activation [6]. Also, post-translational modifications (including (de)phosphorylation and acetylation) of AKT play important roles in regulation. An important immediate negative feedback loop is provided by mTORC1. Through a variety of downstream targets of mTORC1, AKT signalling is inhibited [7].


**6. Summary and outlook**

In spite of several long-term efforts to employ biomaterials to improve the physiological relevance of stem cell-derived spinal motor neurons *in vitro*, the resulting cells are still far from representing accurate reflections of their native counterparts in many physiological respects *in vivo*. We believe the core problem lies in the fact that we do not currently have the necessary tools to enable us to recapitulate the dynamic flux of morphogens continuously occurring near the neural tube during the developmental process. One attractive candidate for helping to achieve this goal is micro- and nanotechnology that generates 3D scaffold with high cavity of reservoir for target morphogen loading with a controllable release rate and concentration for longer period of time *in vitro*. By virtue of having established production protocols for those biomaterials with highly tunable porous structure and surface properties, we believe such materials have great potential to function as a morphogen-releasing source that facilitates the establishment of *in vitro* environments corresponding more closely with those of the notochord and paraxial mesoderm.

In conclusion, the use of biocompatible materials has great promise to address a current roadblock in iPSC-neuron research, namely, the accurate recapitulation of native spinal neuron development in culture. Conversion into a 3D culture environment that incorporates materials of similar mechanical and physical properties to those of native extracellular matrices holds great potential to improve current neurodevelopment modeling. More importantly, future efforts to recapitulate the unique morphogen supply schemes present at different stages of spinal cord development in human embryos would help overcome one of the most challenging issues in producing reliable neural tissue models from iPSC-derived spinal neurons.

### **Acknowledgments**

This work was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (HI19C1095), and by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (RS-2023- 00209822), Republic of Korea.

*Exploring the Potential for Biomaterials to Improve the Development of Spinal Motor Neurons… DOI: http://dx.doi.org/10.5772/intechopen.113275*


**3. Conventional etiological mechanisms of neural proteins as antibodies**

The target antigens that play a critical role in neuronal transmission and in plasticity include the N-methyl-D-aspartate (NMDA) receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), the gamma-aminobutyric acid receptor (GABA), the glioma-inactivating leucine-rich protein (LGI1) and the contacting-associated protein 2 (CASPR2), a protein that plays a key role in the normal function of voltage-dependent potassium channels [21].

The structure of NMDA receptors (R-NMDA) are formed by combinations of different subunits: NMDAR1 (NR1), NMDAR2 (NR2), and NMDAR3 (NR3); which form a Ca++ permeable ion channel. A single gene encodes the NR1 subunit; however, transcription can generate at least eight isoforms, whereas for NR2-type subunits there are four different genes encoding NR2A, NR2B, NR2C, and NR2D7 subunits. Functional NMDA receptors are composed of heterotetramers, and formed by two dimers twisted by the subunits NR1-NR2, where in the NR1 subunit it possesses a glycine binding site and each in the NR2 subunit, a glutamate binding site, with two binding sites for glycine (S1) and two for glutamate (S2) in each receptor. The NR1-NR2 dimmer is considered the basic functional structure at each receptor, where different physiological and pharmacological binding sites are found for different ligands [22, 23]. Each ionotropic receptor subunit has similar molecular structure, which is organized into four functional domains, which are: an extracellular domain with the amino (N) terminal (DNT), a ligand binding domain (DBL), a region (M1–M4), where the M2 segment that partially enters the membrane forms the ion channel, and finally, a carboxyl domain (C) in the intracellular region (DCT) (**Figure 1(A)**) [24, 25].

In NMDARAS, IgG antibodies are directed to the N-terminal extracellular domain of the GluN1 subunit of the NMDA receptor (**Figure 1**), specifically an epitope region at GluN1 aa369 [26–28]; the cultures of dissociated rat hippocampal neurons and antibody-containing cerebrospinal fluid (CSF) from patients with NMDARAS have been used to study the molecular mechanism by which IgG antibodies cause hypo function of the NMDAR [29]; antibodies decrease the levels of synaptic NMDA receptor and disrupt NMDA receptor currents in cultured neurons. In addition, antibodies disrupt the interaction between NMDAR and the ephrin B2 receptor (EphB2R), a major stabilizer of NMDARs at postsynaptic sites, facilitating the displacement of NMDARs from the synapse [29]. The antibody does not act as a receptor antagonist, by modulating the physiological receptor binding domain, but causes capping and internalization of the receptor [30]. Antibody-mediated internalization is independent of NMDAR activity and does not occur as a compensatory response to the agonism of the receptor, suggesting that the mechanism of internalization is primarily NMDAR cross-linking by patient antibodies [29].

Besides, anti-AMPA-GluR3B antibodies have been associated with many pathological effects: they activate glutamate, AMPA receptors, and are involved in the processes of "excitotoxicity." The phenomenon is associated with various pathological states of the CNS including: epilepsy, hypoxia/ischemia, and trauma. In animal and *in vitro* models, anti-NMDA-NR1 antibodies may be highly pathogenic, as they may cause a decrease in surface NMDA receptors expressed in hippocampal neurons, and also decrease the density and synaptic localization of receptors NMDA. The expression of these NR1a subunits correlates with the distribution of high-affinity NMDA receptors by agonists. Anti-NMDA-NR1 antibodies induces reduction in expression through cross-linking and internalization of NMDA receptors. Such changes may impair glutamate signaling through NMDA receptors and lead to various abnormal neuronal

Autoimmune Epilepsy: New Development and Future Directions

http://dx.doi.org/10.5772/intechopen.70686

19

Nevertheless, anti-AMPA-GluR3B antibodies induce many pathological effects that activate glutamate/AMPA receptors,which are involved in excitotoxic damage, the complement activation is modulated by regulatory proteins in which the activation plays a central role in the pathogenesis of brain damage and induces behavior and motor impairments. It has been observed in animal and *in vitro* models that anti-NMDA-NR1 antibodies can be highly pathogenic, as they may cause a decrease in surface NMDA receptors expressed in hippocampal neurons, and the density and synaptic localization of NMDA, probably by the internalization of receptors, which can impair glutamate signaling through NMDA receptors and lead to various neuronal/behavior/cognitive and psychiatric alterations. Knock-out mice to the GluR2 gene show reduced scanning and motor coordination. In these animals, the AMPA receptor-mediated synaptic transmission is reduced, but the long-term potentiation is better [34]. Knock-out mice to the GluR2 gene also exhibit increased cell death, possibly due to the excitotoxicity related to the greater insertion of the compensating homomeric GluR1 protein

**4. Clinical features of epilepsy-associated autoimmune encephalitis**

appears to represent approximately 40% of all cases [27, 37, 38].

Each of the currently known neuronal cell surface or synaptic autoantibody associates with a specific syndrome or limited set off symptoms (**Table 1**). NMDAR antibody-associated encephalitis is a recently described disorder in which infrequent seizures are associated with the presence of autoantibodies directed against the extracellular domain of the NR1 subunit of the NMDAR. This disorder was first described as a clinical entity in 2005, in one in four young women who developed acute psychiatric symptoms, seizures, memory deficit, in association with the presence of an ovarian teratoma. In a study of 100 patients, it was shown that although the majority are young women (mean age 23 years), the disorder could occur in men and in children. This fact has allowed the number of pediatric cases to grow steadily and

Symptoms of anti-NMDA receptor encephalitis develop and resolve in a multi-stage process; most patients experience a prodromal similar to a viral picture, which is followed by a pattern

/behavioral/cognitive/psychiatric disorders.

in AMPA receptors [35, 36].

**4.1. NMDA receptor**

**Figure 1.** (A) Structure of AMPA receptor subunits. The transmembrane topology is shown, along with the flip/flop alternatively spliced exon, and the two ligand-binding domains (S1 and S2). Glycosylation sites are shown as trees in the N-terminal region; this region is associated with immune response. (B) Flow cytometry demonstrates the presence of T lymphocytes of the CD8+ class with greater activation, as well as B lymphocytes; here it can be known that the immune process has extravasated to the cerebral parenchyma, (C) and (D) the tissue based assay. Mouse brain tissue sections, such as hippocampus are stained with the patient's serum or CSF by indirect immunoperoxidase technique. (C) Shows CSF immunoreaction at the hippocampus level of the cytoplasmic and a neuronal surface in D (anti-human IgG-Px, Abcamab97225). (E) TBA in F, shown a reaction at neuronal surface level that colocalizes with GAD65 / 67 (Alexafluor 546, Invitrogen Molecular probes). (G) Immunoblot, CSF recognizes 100 and 50 Kd proteins(Anti-human IgG-Px, Abcam- ab97225).

Encephalitis associated with antibodies against GABAB1 receptor is generally presented as limbic encephalitis, as well as drug-refractory seizures. In a series of 15 patients, the mean age of presentation was 62 years (range 24–75) and both sexes were similarly affected. About half of the patients had an associated tumor, either a small cell lung carcinoma or a neuroendocrine lung tumor. These patients usually have antibodies to various non-neuronal proteins of uncertain significance, which suggests a susceptibility to autoimmunity [30].

In the knockout mice to the GABAB1 receptor, a variety of neurological and behavioral alterations are found, including spontaneous seizures, increased anxiety, hyperactivity, hyperalgesia and memory impairment, suggesting a dysfunction of the limbic system [31, 32].

In contrast, patients present with limbic encephalitis in conjunction with antibodies to the AMPAR were not present with seizures as frequently: only 3/10 had seizures as presenting feature with one other patient having seizures after a relapse [33].

Besides, anti-AMPA-GluR3B antibodies have been associated with many pathological effects: they activate glutamate, AMPA receptors, and are involved in the processes of "excitotoxicity." The phenomenon is associated with various pathological states of the CNS including: epilepsy, hypoxia/ischemia, and trauma. In animal and *in vitro* models, anti-NMDA-NR1 antibodies may be highly pathogenic, as they may cause a decrease in surface NMDA receptors expressed in hippocampal neurons, and also decrease the density and synaptic localization of receptors NMDA. The expression of these NR1a subunits correlates with the distribution of high-affinity NMDA receptors by agonists. Anti-NMDA-NR1 antibodies induces reduction in expression through cross-linking and internalization of NMDA receptors. Such changes may impair glutamate signaling through NMDA receptors and lead to various abnormal neuronal /behavioral/cognitive/psychiatric disorders.

Nevertheless, anti-AMPA-GluR3B antibodies induce many pathological effects that activate glutamate/AMPA receptors,which are involved in excitotoxic damage, the complement activation is modulated by regulatory proteins in which the activation plays a central role in the pathogenesis of brain damage and induces behavior and motor impairments. It has been observed in animal and *in vitro* models that anti-NMDA-NR1 antibodies can be highly pathogenic, as they may cause a decrease in surface NMDA receptors expressed in hippocampal neurons, and the density and synaptic localization of NMDA, probably by the internalization of receptors, which can impair glutamate signaling through NMDA receptors and lead to various neuronal/behavior/cognitive and psychiatric alterations. Knock-out mice to the GluR2 gene show reduced scanning and motor coordination. In these animals, the AMPA receptor-mediated synaptic transmission is reduced, but the long-term potentiation is better [34]. Knock-out mice to the GluR2 gene also exhibit increased cell death, possibly due to the excitotoxicity related to the greater insertion of the compensating homomeric GluR1 protein in AMPA receptors [35, 36].


**1. Introduction**

In the Northern Hemisphere, a seasonal snowpack can cover over 50% of the land area with the snow surface often the interface between the atmosphere and the earth [1]. The roughness of a snow surface is an important control on air-snow heat transfer [2], and changes in the snow surface can have substantial effects on the energy balance at this interface. Snow is a complicated surface with rapidly evolving physical roughness characteristics due to changing atmospheric conditions, the metamorphism of snow crystals, melting and freezing processes and redistribution by wind, especially in open areas [3]. Roughness characteristics also influence the air-surface momentum transfer on the snowpack due to wind [4]. The changes in wind momentum can reduce the energy budget, influence the formation of roughness features, and affect the aeolian movement of snow [4]. Heat flux modeling has typically used the aerodynamic roughness length (*z*0) as a static parameter, in hydrologic, snowpack, and climate models [5,6], with *z*0 only varying as a function of land cover type. For example, the Community Land Model version 4.0 (CLM4; http://www.cesm.ucar.edu/models/ccsm4.0/clm/) applies a single *z*0 value of 2.4 × 10−3 m to all snow-covered surfaces. However, *z*0 varies both spatially [7] and temporally [8], which may result in variable estimates of turbulent heat fluxes not captured by most models [9]. Wind velocity profile measurements are often used to calculate *z*0 estimates [6], but there are a limited number of sites that measure the wind profile over a snowpack surface, making the spatio-temporal representation of *z*0 challenging.

Millimeter-scale variations in snow-surface roughness features have been estimated from a black plate pushed partially into the snow [10–12], using two-dimensional photography, digital processing, and automated post-processing software [13–16]. Snow surface elevation data are now available over large areas at the resolution (±80 mm) of airborne light detection and ranging (lidar) [17–19], terrestrial laser scanner (TLS) (resolution of ±5 mm) [20–26], and photogrammetry [25]. Although most lidar and photogrammetry efforts have only focused on snow depth [26], only a few datasets have been used to evaluate snow surface roughness at the meter-scale or sub-meter scale [27,28]. However, few of these datasets have been applied to interpolate *z*0 and create a digital elevation model of the snowpack surface for evaluating surface roughness [27]. Aerodynamic roughness length (*z*0) has been estimated from the geometry of the snow surface [2,7,29–31]. However, this method is time consuming and typically only applicable over smaller scales [13]. Also, Fassnacht et al. [27] have identified potential errors with the different methods of computing *z*0 from the geometry of the surface that result in values varying over 1–3 orders of magnitude and have suggested these methods need to be evaluated for varying scales, resolutions, and environments.

This study used TLS-derived surface geometry and vertical wind profile measurements to compare concurrent *z*0 estimates for changing snow surface features of shallow snowpacks. Here, we asked the following questions: (1) How does the aerodynamic roughness length (*z*0) vary spatially and temporally for a shallow snow environment? (2) How does *z*0 estimated from geometric measurements (*z*0*G*) compare to *z*0 estimated from anemometric measurements (*z*0*A*), and (3) How does *z*0 vary with snow-covered area based on the underlying terrain?

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

The capability of a rough surface to absorb momentum from a turbulent boundary layer can be quantified by *z*0, which is a measure of the vertical turbulence that occurs when a horizontal wind flows over a rough surface [32]. In general, *z*0 is a quantity that is computed from the Reynolds number and the roughness geometry of the surface [29]. For rough, turbulent regimes occurring in the atmospheric boundary layer, dependence on the Reynolds number vanishes and *z*0 is only a function of roughness geometry [33]. Various relations have been found to relate the geometry of roughness elements with *z*0 [2,29]. For example, the dependence of *z*0 on the size, shape, density, and distribution of surface elements has been studied using wind tunnels, analytical investigations, numerical modeling, and field observations [34,35]. Smith [36] provides a comprehensive review of the different approaches and models developed to analyze surface roughness and highlights that almost all models were developed for simplistic natural surfaces (i.e., regular arrays of roughness elements).The lack of a clear method for calculating *z*0 as a function of surface roughness is due to the complexity of surfaces that exists in nature and the direction, spatial, and temporal dependence.

The most robust approach for estimating *z*0 is from the anemometric method used to generate a logarithmic wind profile and solve for *z*0 [32]. The anemometric method can be used for any surface with any arrangement of roughness elements but requires a meteorological tower of at least two vertically spaced wind, temperature, and humidity measurements that can be used to approximate the respective gradients. The measurements integrate over a footprint area rather than the single-point location of the sensors based on the distance from measurement source, elevation of sensor, meteorological conditions, turbulent boundary layer, and atmospheric stability. All of these factors can potentially create turbulent fluctuations affecting the downwind measurements of the wind profile [37,38]. The anemometric method is also very sensitive to the wind measurement heights; Munro [2] found that adding 0.1 m to any of the heights can alter *z*0 by an order of magnitude. In contrast, the geometric method uses algorithms relating *z*0 to characteristics of surface roughness elements and thus does not require tower instrumentation but only a measure of the geometry of the surface [29].

Anemometric data are used to estimate *z*0 from the logarithmic wind profile through an empirical relation that describes the vertical distribution of horizontal wind speeds within the lowest portion of the planetary boundary layer [39]. The wind speed (*Uz* in m/s) at height *z* (in m) above a surface is given by:

$$\mathcal{U}z = \frac{\mathcal{U}^\*}{k} \ln\left[\frac{z}{z\_0} + \psi\left(\mathbf{z}, \frac{z}{L}, L\right)\right] \tag{1}$$

where *U*\* is the friction velocity (m/s), k is the Von Kármán constant (~0.40), and *ψ* is a stability term, and *L* is the Monin-Obukhov stability parameter. This equation is only valid through the hypothesis of stationarity and horizontal homogeneity. Under neutral stability conditions, *z/L* tends towards zero, and *ψ* can be neglected.

The most common geometric method for estimating *z*0 is simply a function of the height of the elements:

$$z\_0 = f\_0 z\_h \tag{2}$$

where *zh* is the mean height of roughness elements in meters, and *f* 0 is an empirical coefficient derived from observation [28]. The frontal area index, which combines mean height and breadth (all in meters), and density of the roughness elements, is defined as roughness area density given by:

$$(\lambda F) = \text{ l.y } z\_h \text{ } \rho el \tag{3}$$

where *Ly* is the mean breadth of the roughness elements perpendicular to the wind direction, and *ρel* is the density or number (*n*) of roughness elements per unit area [40]. Lettau [29] developed a formula for *z*0 based on the geometry of the surface for irregular arrays of reasonably homogenous elements:

$$z\_0 = 0.5 \ z\_h \,\lambda \,\text{F} \tag{4}$$

In the Lettau formula, the coefficient 0.5 represents an average drag coefficient for the roughness elements, which was determined experimentally. Other geometric methods have been developed, especially to consider more regularly-shaped and distributed roughness elements, such as buildings in an urban setting [41,42]. The Counihan equation provides a geometric estimate of *z*0 as:

$$z\_0 = \ z\_h (1.8 \frac{A\_f}{A\_d} - 0.08) \tag{5}$$

where *Af* is the total area in square meters silhouetted by the roughness elements, and *Ad* is the total area covered by roughness elements.

A meteorological tower was erected at Colorado State University Agricultural Research, Development and Education Center (ARDEC) South (http://aes-ardec.agsci.colostate.edu/), (40.629680, −104.99699) on a flat field that had no obstructions at least 100 m in the prevailing wind direction. The fetch was 40 m wide with the tower placed in the middle, leaving 100 unobstructed, homogenous meters upwind. Ten anemometers and five temperature and relative humidity sensors were placed vertically at different heights on the tower. The accuracy of the air temperature and relative humidity sensors (METER VP-3) was variable across a range of ±0.25–0.50 ◦C and ±4%, respectively (see http://manuals.decagon.com/Manuals/14053\_VP-3\_Web.pdf for more information). The METER Davis Cup Anemometers have a wind direction accuracy of ±7◦ and a speed accuracy within ±5% (see http://manuals.decagon.com/Manuals/). Data were collected from February 2014 through March 2015. In mid-March 2014, the flat field was plowed to create additional underlying roughness, specifically furrows and troughs were formed perpendicular to the dominant wind direction at an approximate spacing of two meters. The approximate amplitude of the troughs and furrows was 25 cm deep and 50 cm wide.

Meteorological data were recorded every five minutes based on the average of one-minute observations. Anemometric data were evaluated for 153 instances when wind speeds were faster than 4 m/s to ensure neutral stability [8] and when the log-linear fit had an r2 greater than 0.95. The height of the instruments was calculated based on the depth of snow, which did not exceed 10 cm.

This study estimated *z*0 values from anemometric measurements and used them as a reference to evaluate concurrent geometric methods. The *z*0*A* values were calculated using Equation (1) from logarithmic anemometer wind profile data. Surface elevation was measured using a FARO Focus3D X 130 model TLS (https://www.faro.com/products/). This lidar tool generates a point cloud scan of a given area with an error of ±2 mm and a resolution of approximately 7.5 mm. The TLS was set up in 2–3 locations around the area of interest with 6 reference spheres to match the images using the FARO Scene Software. The data were generated into a point cloud and interpolated to a solid surface with 10 mm resolution with the kriging method using the Golden Software's Surfer 8 (https://www.goldensoftware.com/products/surfer). The gridded data were de-trended in the x-y plane to remove the bias in slope of the field or the angle of the lidar. Gaps in the scans tended to be small (<100 mm), and the kriging interpolation eliminated them. Individual roughness elements were identified and for each element the silhouette lot area and obstacle height were determined using a MATLAB code (https://www.mathworks.com/products/matlab.html). This was required to compute the Lettau formula (Equation (4)). The 1000-m2 area around the tower was scanned on 12 occasions when the concurrent anemometric and geometric measurements were acquired. One concurrent measurement set was made with no snow cover for each of the unplowed and plowed scenarios; seven concurrent measurement sets were made with partial snow-covered area (SCA) and three with complete snow cover. SCA was determined from digital photos taken from the TLS unit.

Both the Counihan and Lettau methods were used to calculate *z*0*G* (Equations (4) and (5), respectively). The Counihan method was appropriate for this study because the roughness elements (furrows) in this study site were semi-regular. During each concurrent anemometric and geometric measurement set, the percentage of the area covered in snow, or SCA, was estimated from photographs.


2. The legal defence of women's rights (1945–1970)

Ever since it was created in 1945, the objective of the United Nations has been to guarantee equality amongst all human beings and, more specifically, between men and women. Thus, in the Universal Declaration of Human Rights (1948), the organisation reiterated its commitment to promoting equality amongst people without distinction of their gender. These proclamations sparked intergovernmental actions taken in defence of women.

The Commission on the Status of Women created in 1946 was the first body in the United Nations system devoted to defending the status of women. The purpose of this Commission is to draw up recommendations and reports aimed at guaranteeing the rights of women under equal conditions with men in the political, economic, social, and educational spheres.

However, even before the creation of the UN, at the founding summit of the League of Nations (1919), women met at what could be called the "first parallel forum of NGOs". Over the next two decades, contacts between international non-governmental organisations for women and the League of Nations increased, placing pressure on the government delegations present at several summits on essential issues for women such as health, education, peace-keeping and disarmament, although an adequate legal and institutional framework was lacking [7].

It can be asserted that the earliest participation by women in the non-governmental sphere in drawing up international resolutions, conventions and declarations was in the area of labour rights, through the International Labour Organisation (ILO). The two objectives pursued by the ILO since its creation, in relation to female workers, were to protect their role as mothers and to promote equality between men and women in terms of employment. Both the contents of the conventions and the resolutions issued by the ILO have evolved from mere protection from a health and reproduction perspective, progressing towards the adoption of measures focusing on equal conditions and opportunities in employment [8].

The following conventions were issued during the years spanning from 1919 to 1970: Maternity Protection Convention (1919), Night Work (Women) Convention (1919), Underground Work (Women) Convention (1935), Equal Remuneration Convention (1951), Discrimination (Employment and Occupation) Convention (1958), Vocational Training Convention (1962), Employment Policy Convention (1964) and Workers with Family Responsibilities Convention (1965).

During the course of 1966, two International Human Rights Covenants applicable to both men and women were approved. These were the International Covenant on Civil and Political Rights and the International Covenant on Economic, Social and Cultural Rights. These covenants bolstered the Universal Declaration by developing the rights contained therein, turning them into legal obligations for the governments of the countries that ratified or acceded to them.

There is a greater emphasis on the principle of equality between men and women in these two International Covenants than there is in the Declaration of Human Rights. While gender-based discrimination is mentioned in the Declaration of Human Rights as one of several types of distinctions, such as race or religion, in the International Covenants on Civil and Political Rights and on Economic, Social and Cultural Rights, an entire article is devoted to stressing that men and women are equally entitled to these rights. Amongst the rights proclaimed in the first of these covenants, some highlights are the right to marriage above a minimum age, consent by husband and wife and equal rights and responsibilities by both spouses in relation to the marriage, within marriage and in the event of dissolution thereof. The right to vote and to hold public office established in the 1951 Convention on the Political Rights of Women was also reiterated.

The United Nations gradually recognised and expanded the rights of women in the civil, political, economic, educational and labour arenas. The status of women was seen essentially as a legal concern by the UN in its early years. The demands were mainly related to access to education, health services, food, work in decent health and wage conditions, etc. [9]. However, during this first period, the UN failed to consider the causes of the inequality that half the population continued to bear merely because they were women. At that time, their marginalisation in decision-making, lack of power and the established roles for men and women did not find their way into the agenda of the United Nations.

In all fairness, we must mention that some early displays of a different perception of the feminine issue were also seen during this first period, in which women were no longer passive beneficiaries of development or vulnerable individuals with rights to be protected, to become actors in the economic and social development of the nations [10]. Starting in the late 1960s, the international community began to make the substantial change that would be seen in its strategy on issues related to promotion of women, based on their integration in the development processes.


**3. Research development and methodology**

 In developing the research methodology an interview survey was devised, comprising a semi-structured interview. According to [31] there are two types of interviews relevant for subjective surveys. One of them is exploratory or in-depth interviews normally used for surveys. Retrofitting of LCBs was targeted for the survey and adopted for this study. The focus of this study was developed from part of a doctoral study on energy management in the reuse of LCBs. A set of questions was formulated in accordance with the objective of the study and a draft questionnaire was prepared and piloted. Comments were received back from the piloted questionnaire after which a number of redrafts of the questionnaire were undertaken. The final version of the questionnaire contained 85 questions which covered an extensive range of operational issues in retrofitting projects. The questionnaire was designed to address different areas of investigation categorised into six different parts namely: building characteristics, energy using equipment/systems, how the equipment is used, energy used, energy management strategies and user behaviour.

### **3.1 Sampling and selection process**

 Due to the qualitative approach of this study, a non-probability sampling technique was used. Specifically, purposive sampling technique was selected for the retrofitting projects. Zikmund ([32], p. 382) defines purposive sampling as "a non-probability sampling technique in which a researcher selects the sample based on his/her judgment about some appropriate characteristics required of the sample members". Thus, through a process of purposive sampling ([33], pp. 381–385) the researcher selected five case study buildings from the categories of retrofitting projects involving LCBs. The selected projects for this study were chosen from LCB retrofitting projects in the East of England. However, unlike statistical sampling, the sample is not a representative of the entire population of LCBs in England. Although the selected projects have various types of ownership, however, they are used for similar purposes.

#### **3.2 Data collection process**

 The data were collected through phone interviews, site interviews and case study buildings. The researcher conducted some interviews with the building tenants to collect information on how energy is used in the building. Following the phone interview, the researcher conducted six on-site interviews with the building managers/operators to learn more about the management practices being implemented in their building. The purpose was to build on the phone interviews to obtain a more in-depth view of the operational performance of the building and to gain a deeper understanding of the best practices in operating the building. Energy use data collection formed the main focus of the data collection of the selected

*Improving Environmental Sustainability in Reuse of Some of England's Churches: Challenges… DOI: http://dx.doi.org/10.5772/intechopen.81222*

buildings. According to [34] annual energy use can be estimated either by using top-down approach or a bottom-up approach. The bottom-up approach involves the use of the calculation methods while the top-down approach involves an analysis of measured energy consumption and appropriating it to the elements responsible for energy consumption. The bottom-up approach is mainly founded on theory, the calculated loads, and the rated capacity of energy-using equipment. The limitations of the bottom-up approach lies in the weakness of the calculated results rarely agree with metered data. This leads to overestimating energy consumption and in masking individual elements of energy use. Thus, the top-down approach was preferred for this study because of its advantage of providing a greater degree of accuracy as it is based on factual metered data [35]. In the top-down approach, the actual measured data are obtained from utility companies such as monthly utility bills and meter prints outs and are critically examined. The rational for this is to estimate the annual energy consumption and to determine how energy is being used for the activity within the building. Utility data from the buildings were collected for 12 months and the figures were converted to kg of CO2 and ranked in order of absolute energy consumption.

### **3.3 Data analysis method**

The data analysis method for this study comprise of two approaches. Firstly, benchmarking was adopted as an energy performance tracking strategy. It is a strategy most often use in normalising energy consumption-based metrics such as weather or square footage to promote realistic comparisons with other similar buildings [36]. Benchmarking as used in the analysis of the data in this study is the most prevalent performance tracking approach found in the literature capable of providing a high level picture of energy use. CIBSE TM46 [37] energy benchmarks were adopted to benchmark the performance of the investigated buildings (**Table 2**).

Secondly, the ranking was adopted to categorise higher performing buildings from the lower performing ones. Although the review of literature indicates that numerous ranking and scoring systems have been developed, however, there is no scientific consensus method [38]. The use of ranking will enable the building owners and the facilities managers to be able to compare their building performance to similar building's size and similar pattern of use in order to be adequately informed on the actions to be taken to boost the performance of their buildings. Energy use of the surveyed buildings was converted into CO2 emission using DEFRA [35] CO2 emission conversion factors. It assumes CO2 emission factors of 0.184 kg of CO2/kWh for gas and 0.542 kg of CO2/kWh for electricity. The carbon emissions of the buildings were calculated to determine both 'absolute' and 'relative' terms. The absolute emissions indicate the total footprint while relative emissions refer to the absolute figure indexed to a unit of this per m2 per performance also referred to as 'intensity indicators'. During the analysis of data, the interpretation and the presentation of results; ethical issues were taken into consideration

by intentional coding the surveyed buildings using an alphabet (**Table 4**) to keep the building's identities and location hidden. This is in line with the suggestion of ([39], p. 89) that the process of data collection should not put participants at risks and that the vulnerable population should be respected by the researcher.