**3. Rodent models of gliomas**

Animal models are often used when researching a disease and trying to understand how a particular pathological process occurs, as well as a means of studying the efficacy of potential new therapies. This review will provide examples of commonly used and new experimental animal models for gliomas, which make up a large portion of primary brain tumors. The majority of models involve intracerebral implantation of rodent (rat or mouse) or human glioma cells into synergetic rats or mice, or immunocompromised rodents (e.g. nude or athymic rats or mice). There are also a limited number of transgenic mouse models for gliomas. One approach to better simulate a human tumor is to obtain human glioma neurospheres from patients during tumor resection, and then culture the cells prior to intracerebral implantation into immunocompromised rodents. Another recent approach is to implant non-replicating viruses that can stimulate neuronal stem cells to turn into glioma cells which develop into diffuse tumors similar to those found in high-grade or malignant gliomas called, glioblastoma multiforme (or GBM).

### **3.1 Intracerebral cell implantation models**

252 Advances in the Biology, Imaging and Therapies for Glioblastoma

information regarding alterations in tissue structure associated with glioma tumors. An extension of the diffusion-weighted imaging technique is diffusion tensor imaging (DTI), which can provide information on white matter neuronal fiber tractography. MR spectroscopy can be used to assess alterations in tumor metabolites associated with glucose, bioenergetics, amino acid or lipid metabolism, for example. Many of these advanced MR techniques are used in a clinical setting. More recently, molecular MRI (mMRI) which incorporates a MRI contrast agent as a signaling molecule and an affinity component that targets specific tumor markers associated with tumor growth, angiogenesis, cell invasion, inflammation, or apoptosis, can be used to characterize *in vivo* molecular events associated

This review will focus on various glioma models that have been studied *in vivo*, with particular emphasis on MR image and/or spectroscopy evaluation of these models, and the use of MR image criteria (morphological, biophysical, molecular and metabolic) to evaluate therapeutic treatments. The aims are to: (1) provide an overview on current rodent glioma models being studied; (2) provide an overview regarding currently used MR methods, including advanced MR techniques (e.g. MRA, DWI, PWI, MRS and mMRI), relevant to glioma research; and (3) summarize studies that have used MR methods to evaluate

Gliomas represent 40% of all primary central nervous system (CNS) tumors diagnosed. Among them, glioblastomas (GBM) are the most malignant, with a very poor survival time of about 15 months for most patients diagnosed with this grade IV brain tumor (CBTRUS 2011). High grade gliomas are the most common primary brain tumors in adults, and their malignant nature ranks them as the fourth largest cause of cancer death (Niclou *et al.*, 2010). There are four tumor grades for gliomas: Grade I which is a non-malignant, fairly circumscribed astrocytoma that is rare and appears in young adults; Grade II which are a more common diffusely infiltrating astrocytoma; Grade III which is an anaplastic astrocytoma; and Grade IV which is a glioblastoma multiforme (Niclou *et al.*, 2010). Grades II-IV gliomas generally are found in the adult population, and often recur following current treatment options (including surgical resection, radiotherapy, and chemotherapy) (Niclou *et*

Grading and identification criteria that can be used to provide information regarding tumor behavior are cell proliferation (cellularity and mitotic activity), nuclear atypia, neovascularization and the presence of necrosis and/or apoptotic regions (Gudinaviciene *et al*., 2004). Grade II gliomas (also referred to as diffuse astrocytomas) and grade III gliomas (also referred to as anaplastic astrocytomas) only differ based on their mitotic activity, and this difference accounts for a substantial decrease in the 5-year survival for patients, from 47% to 29% (from grade II to III, respectively) (CBTRUS 2011). Grade IV gliomas (GBM) are often characterized by the presence of large necrotic areas (Gudinaviciene *et al*., 2004) and

Animal models are often used when researching a disease and trying to understand how a particular pathological process occurs, as well as a means of studying the efficacy of

therapeutic response in pre-clinical models for gliomas.

generally have a 3% 5-year survival (CBTRUS 2011).

**3. Rodent models of gliomas**

with gliomas.

**2. Human gliomas**

*al.*, 2010).

Glioma cells (rat, mouse or human origin) are injected into the cerebral cortex of rats or mice (synergetic if cells are transplanted into the same species and strain that they were obtained from, or immune-compromised rats or mice if human cells are used) using a stereotaxic device for precise implantation into a brain region. As tumors grow over a period of 1-2 months, this model is considered a short-term model. Different cell lines varying in their degree of malignancy, such as rat C6, 9L/LacZ, F98 and RG2 cells, mouse GL261 cells and human U87 cells, provide a range of gliomas from moderately aggressive to GBM-like.

Many of these models have some characteristics associated with human gliomas, such as aggressive tumor growth, angiogenesis, and tumor necrosis (in a few models), however the diffuse nature of high-grade gliomas, glioblastoma multiforme (GBM), is not well represented. In many instances the intracerebrally-implanted rodent tumors have defined tumor boundaries, which do not represent the infiltrative nature of GBMs well. A comprehensive review that discusses the advantages and disadvantages of rat brain tumor models, most of them involving intracerebral implantation of rat glioma cells, is discussed in a paper by Barth and Kaur (2009).

The rat C6 cell line produces diffusively invasive astrocytomas (Barth, 1998; Barth and Kaur, 2009), which have been found to be similar to human glioma cells regarding the expression of genes mainly involved in tumor progression (Sibenaller *et al*., 2005). C6 gliomas were induced in an outbred Wistar rat strain repeatedly injected with methylnitrosourea (MNU), which makes it non-syngeneic in inbred strains, and increases its potential to evoke an alloimmune response (Barth and Kaur, 2009). As a result of some genetic similarities to human gliomas, the C6 model has been widely used as a GBM model for a number of years (Grobben *et al*., 2002; Barth and Kaur, 2009). The 9L/LacZ-derived tumors are aggressive and infiltrative, and are angiogenic (Plate *et al*., 1993), which are some of the characteristics associated with human GBM (Weizsaecker *et al*., 1981). Although the aggressive 9L/LacZ gliomas are highly invasive (Szatmori *et al*., 2006) and have extensive neovascularization (Plate *et al*., 1993), due to their pronounced immunogenicity (Barth, 1998) and the fact that they are classified as gliosarcomas (Sibenaller *et al*., 2005), makes these cells a poor choice for glioma studies. F98 gliomas are classified as anaplastic malignant tumors, which have an infiltrative pattern of growth, and also have attributes associated with human GBM (Barth, 1998; Barth and Kaur, 2009). The aggressive and invasive nature (Barth, 1998; Barth and Kaur, 2009) of RG2 tumors (Groothuis *et al*., 1983), as well as the highly tumorigenic human glioblastoma U87 MG cell line *(Martens et al*., 2006; Cheng *et al*., 1996), both mimics human

Assessment of Rodent Glioma Models Using Magnetic Resonance Imaging Techniques 255

Hupp, 2011; Muller et al., 2011). PTEN, which stands for phosphatase and tensin homolog, is a tumor suppressor gene also involved in the regulation of the cell cycle (Natsume et al., 2011; Alexiou and Voulgaris, 2010). Rb/p53 mice developed malignant tumors in approximately 9 months (Jacques *et al*., 2010). PTEN/Rb/p53 tumors had an appearance that was similar to the Rb/p53 tumors (Jacques *et al*., 2010). Deletion of Rb/p53 or Rb/p53/PTEN resulted in the formation of primitive neuroectodermal tumors (PNET), which alludes to the role of an initial Rb loss involved in driving the PNET phenotype (Jacques *et al*., 2010). It was found that targeted deletion of PTEN and p53 in subventricular zone (SVZ) stem cells resulted in glioma formation with a latency period of approximately 7-8 months (Jacques *et al*., 2010). The tumors from the recombination of PTEN/p53 were histologically infiltrative, diffuse, necrotic and had signs of micro-vascular proliferation,

Another successful transgenic mouse model involves the deletion of the *TP53* (tumor protein 53) gene (*Trp53 null* background), and over-expressing human PDGF (plateletderived growth factor) under the control of the GFAP (glial fibrillary acidic protein) promoter, which developed tumors with human glioblastoma-like features and with the integrated development of PDGFRα+ tumor cells and PDGFRβ+ Nestin+ vasculature in 2-6 months (Hede *et al.*, 2009). The tumor suppressor gene *TP53* is either lost or commonly mutated in astrocytic brain tumors, and these *TP53* alterations are often combined with excessive growth factor signaling via the PDGF/PDGFRα complex (Hede *et al*., 2009). PDGF is one of many growth factors that regulate cell growth and division, and has been found to be widely associated with malignant gliomas (Calzolari and Malatesta, 2010; Shih and

GBM cancer-initiating cells have been found to mediate resistance to chemotherapy and radiation treatment, both used as follow-up therapies following surgical resection of the main tumor mass (Wei *et al*., 2010). Cells isolated from GBM that possess the capacity for self-renewal following radiation and chemotherapy, can form neurospheres when cultured *in vitro* (Wei *et al*., 2010). The glioma-associated cancer-initiating cells were found to express MHC-I (major histocompatibility I) but not MHC-II, CD-40 or CD80, which induces T-cell immune deficiency, and express the costimulatory inhibitory molecule, B7-H1, which plays a role in mediating immune resistance in gliomas and induces T-cell apoptosis (Wei *et al*., 2010). These neurospheres can be intracerebrally implanted into immune-compromised rodents to develop tumors *in vivo,* and therefore provide an experimental model that more closely resembles recurrent human GBM (radiation and chemotherapeutic resistant and induce immunosuppression) to evaluate new therapies. Another approach that takes into consideration the role of tumor-initiating stem cells, is to orthotopically implant tiny fragments of surgically-resected tumors, containing brain tumor stem cells within the glioblastoma tissue, into immunocompromised mice (xenograft model) brains with the use

Glial progenitor cells in the white matter and subventricular zone within the central nervous system were recently found to be the likely candidates for glioma-initiating cells (Assanah *et*

which are all characteristics of human high-grade gliomas (Jacques *et al*., 2010).

Holland, 2006).

**4. Human glioma neurospheres**

of a trocar system (Fei *et al*., 2010).

**4.1 Viral-induced glioma models**

high-grade gliomas via inducing vascular alterations. U87 pcDNA3 and U87 IRE1 DN human glioma cells were selected as malignant glioma models that form highly versus poorly vascularized tumors, respectively (Drogat *et al*., 2007; Wehbe *et al*., 2010). GL261 cells give rise to quickly growing, and diffusively invasive intracranial tumors in C57BL/6 mice (Szatmori *et al*., 2006). RG2 and F98 glioma cell lines were both obtained from chemical induction as a result of administering ethylnitrosourea (ENU) to pregnant rats, where the progeny developed brain tumors that were isolated, and propagated and cloned in cell culture (Barth and Kaur, 2009). Human U87 cells are of high interest for angiogenesis studies (Cheng *et al*., 1996). The immunogenicity issue of the 9L/Lacz model can be resolved by using non-immunogenic models (e.g. RG2).

Xenograft models, induced by orthotopic (into native tumor sites) injection of primary tumor cells or tumor cell lines, represent the most frequently used *in vivo* cancer model systems for glioma research (Waerzeggers *et al*., 2010). Both cell culture and xenograft model systems lack the stepwise genetic alterations that are thought to occur during tumor progression, and often do not represent the genetic and cellular heterogeneity of primary tumors, as well as the complex tumor-stroma interaction (Waerzeggers *et al*., 2010). Genetically engineered mouse models (discussed below in the "Transgenic Mouse Models" section) better represent the causal genetic events and subsequent *in situ* molecular evolution, the tumor-stroma interactions, and consist of cellular subpopulations such as cancer stem cells (discussed further in the "Human Glioma Neurospheres" and "Viral-Induced Glioma Models" sections below), that occur in native tumors (Waerzeggers *et al*., 2010).

#### **3.2 Chemical-induced model**

Slow-growing, low- and high-grade, spontaneous gliomas can be generated with a chemically-induced model from the administration of ENU (Kish *et al*., 2001; Koestner, 1990). Transplacental ENU exposure of a pregnant female a day before gestation, results in the formation of low-grade oligodendrogliomas and mixed gliomas, with a tumor incidence approaching 100%, in rat pups at approximately 3-6 months of age (Koestner, 1990). In addition to oligodendrogliomas and mixed gliomas, unfortunately the ENU-induced model also results in the formation of meningiomas (Koestner *et al*., 1971), spinal cord tumors (Koestner *et al*., 1971) and other primitive neuroectodermal tumors (Vaquero *et al*., 1992), decreasing its potential as a reproducible model. In addition to the isolation of RG2 and F98 rat glioma cells from ENU induction, A15A5 neoplastic astrocytes have also been cloned (Davaki and Lantos, 1980).

#### **3.3 Transgenic mouse models**

As we are beginning to understand the genetic mutations associated with gliomas, it is possible to generate transgenic mouse models that have these genetic mutations. Recent findings suggest that brain tumors originate from neural stem or progenitor cells. Some examples of transgenic mutations include deletions of gene combinations, such as Rb/p53, Rb/p53/PTEN or PTEN/p53 (Jacques *et al.*, 2010). pRb is a retinoblastoma protein, which is a tumor suppressor protein that is dysfunctional in many cancers. Rb controls excessive cell growth by inhibiting cell cycle progression until the cell is ready to divide (Chinnam and Goodrich, 2011; Lohmann, 2010). p53 which is also known as protein 53 is a tumor suppressor protein responsible for regulating the cell cycle (Kim et al., 2011; Maclaine and Hupp, 2011; Muller et al., 2011). PTEN, which stands for phosphatase and tensin homolog, is a tumor suppressor gene also involved in the regulation of the cell cycle (Natsume et al., 2011; Alexiou and Voulgaris, 2010). Rb/p53 mice developed malignant tumors in approximately 9 months (Jacques *et al*., 2010). PTEN/Rb/p53 tumors had an appearance that was similar to the Rb/p53 tumors (Jacques *et al*., 2010). Deletion of Rb/p53 or Rb/p53/PTEN resulted in the formation of primitive neuroectodermal tumors (PNET), which alludes to the role of an initial Rb loss involved in driving the PNET phenotype (Jacques *et al*., 2010). It was found that targeted deletion of PTEN and p53 in subventricular zone (SVZ) stem cells resulted in glioma formation with a latency period of approximately 7-8 months (Jacques *et al*., 2010). The tumors from the recombination of PTEN/p53 were histologically infiltrative, diffuse, necrotic and had signs of micro-vascular proliferation, which are all characteristics of human high-grade gliomas (Jacques *et al*., 2010).

Another successful transgenic mouse model involves the deletion of the *TP53* (tumor protein 53) gene (*Trp53 null* background), and over-expressing human PDGF (plateletderived growth factor) under the control of the GFAP (glial fibrillary acidic protein) promoter, which developed tumors with human glioblastoma-like features and with the integrated development of PDGFRα+ tumor cells and PDGFRβ+ Nestin+ vasculature in 2-6 months (Hede *et al.*, 2009). The tumor suppressor gene *TP53* is either lost or commonly mutated in astrocytic brain tumors, and these *TP53* alterations are often combined with excessive growth factor signaling via the PDGF/PDGFRα complex (Hede *et al*., 2009). PDGF is one of many growth factors that regulate cell growth and division, and has been found to be widely associated with malignant gliomas (Calzolari and Malatesta, 2010; Shih and Holland, 2006).


**Author details**

Helen D. Donoghue1 \*, G. Michael Taylor2 , Tom A. Mendum2 , Graham R. Stewart2 , Leen Rigouts3 , Oona Y-C. Lee4 , Houdini H.T. Wu4 , Gurdyal S. Besra4 and David E. Minnikin4 [9] Truman RW, Singh P, Sharma R, Busso P, Rougemont J, Paniz-Mondolfi A, Kapopoulou A, Brisse S, Scollard DM, Gillis GP, Cole ST. Probable zoonotic leprosy in the southern

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[12] Avanzi C, del-Pozo J, Benjak A, Stevenson K, Simpson VR, Busso P, McLuckie J, Loiseau C, Lawton C, Schoening J, Shaw DJ, Piton J, Vera-Cabrera L, Velarde-Felix JS, McDermott F, Gordon SV, Cole SY, Meredith AL. Red squirrels in the British Isles are infected with

[13] Besra GS, McNeil M, Minnikin DE, Portaels F, Ridell M, Brennan PJ. Structural elucidation and antigenicity of a novel phenolic glycolipid antigen from *Mycobacterium hae-*

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[16] Tufariello JM, Kerantzas CA, Vilchèze C, Calder RB, Nordberg EK, Fischer JA, Hartman TE, Yang E, Driscoll T, Cole LE, Sebra R, Maqbool SB, Wattam AR, Jacobs WR Jr. The complete genome sequence of the emerging pathogen *Mycobacterium haemophilum* explains its unique culture requirements. MBio. 2015;**6**:e01313-15. DOI: 10.1128/mBio.01313-15

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[21] Taylor GM, Tucker K, Butler R, Pike AWG, Lewis J, Roffey S, Marter P, Lee OY-C, HHT W, Minnikin DE, Besra GS, Singh P, Cole ST, Stewart GR. Detection and strain typing of ancient *Mycobacterium leprae* from a medieval leprosy hospital. PLoS ONE. 2013;**8**:e62406.

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DOI: 10.1371/journal.pone.0062406

\*Address all correspondence to<EMAIL_ADDRESS>
1 Centre for Clinical Microbiology, University College London, London, UK

2 Department of Microbial Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK

3 Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium

4 Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK


**Larval Settlement on Marine Surfaces: The Role of Physico-Chemical Interactions**

Editor

**Francesca Cima**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editor* Francesca Cima Department of Biology University of Padova Padova Italy

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This is a reprint of articles from the Special Issue published online in the open access journal *Journal of Marine Science and Engineering* (ISSN 2077-1312) (available at: www.mdpi.com/journal/ jmse/special issues/larval surfaces interactions).

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*3.1. Theoretical Research*

The theoretical aspect of the paper is articulated towards a better understanding of the role of traditional open public space as the dominant generator of a city's physical structure and the need to re-identify traditional street spaces according to the global needs of society. Further theoretical research treats the morphogenesis of the subject area in the context of di fferent temporal sequences and identifies the concept of creative street design as the method for sustainable regeneration in the case study. By applying the concept of creative urban design, it is possible to articulate the socio-spatial sustainability of public spaces, which operate on the intersecting line between the preservation of the local values of a place and the global uniformity that tends to shape contemporary urban spaces.

The public open spaces of cities can become new generators and interpreters of cultural values and technological opportunities. The wealth of information in a global context can be transposed through the creative regeneration of physical and material spaces, which will promote the sustainability, and more strongly encourage various forms of connections, from the local to global level.

### *3.2. Analysis of the Current State of Mirkova Varoš Street*

Field surveys carried out during the exploration phase of the analysis of the current spatial and physical condition of Mirkova Varoš streets included the detection of the following parameters: the length, position, building types, number of floors, tra ffic rating, greenery, and urban furniture (Table 2). The analysis of the existing physical conditions of the case study streets reflects the direct objective data obtained from the characteristics of the determinants in the space, including the tra ffic rating, which refers to the primarily objective urbanistic criteria for the assessment of the buildings in terms of construction preservation and general fit within their existing context (object location, size, shape, structure of the dimensions of the building, and architectural heritage).

Field research has also identified the following issues in the case study streets: functional issues (insu fficient number of parking spaces, inadequate parking of cars on the street and on the sidewalks, and lack of urban furniture on the streets), economic issues (unprofitable hospitality and trade facilities due to the small number of users in the centre), ecological issues (pollution from tra ffic congestion at the centre, a lack of greenery, impaired bioclimatic comfort, and noise), visual issues (inadequate facades of buildings, illegal upgrades, disappearance of genius loci), and psychological problems (unpleasant atmosphere (a consequence of an "empty" space) and the experience of a monotonous and unattractive ambience)) [70,71].

### **Table 2.** Physical characteristics of Mirkova Varoš streets. Current state analysis.

Building types: Co—commercial; R—residential; C—culture; A—administration; Floors: P—ground level; P + n—ground level and number of floors; Pk—attic. Building conditions: C—construction rating; A—ambient rating; poor, mediocre, high; Greenery: + over 75% street length covered with greenery; + - between 25% and 75%; - less than 25%. Urban furniture: + street with high quality urban furniture; + - street with insufficient or inadequate urban furniture; - street with no urban furniture.

### *3.3. Perception of the Streets of Mirkova Varoš*

### 3.3.1. Visual, Tactile, Auditory Perception

Urban perception as a result of the human-environment interaction in this study has served as the basis for the evaluation and proposal for physical regeneration of the streets in Mirkova Varoš in Podgorica.

The importance of the visual representation of a public space and its impact on the observer has been recognized by various researchers and repeatedly confirmed [23,37,38,62,63,72]. Visual perception is predominant in the interaction between man and space, whereby the complexity and nature of visual elements create the feeling of satisfaction in a particular area or a particularly negative experience.

Tactile stimuli play a significant role in the formation of the overall perceptual image of an urban space and can contribute to di fferent experiences and influence quality of life [61,73,74]. This is especially pronounced for people with visual disabilities [75]. However, despite the importance of tactile senses and their "universality and diversity," this form of perception is not su fficiently present in the evaluation of public spaces [61].

Auditory interaction in a physical space is very complex and contributes to di fferent experiences. The important role of auditory perception in the context of the sustainable development of urban open public spaces has been indicated by numerous measurements of sound perception, acoustic comfort evaluation in urban public spaces, analyses of factors that influence perception, experimental studies [76–80], analyses of aspects in creating "sound space" [81], and the understanding of urban spaces and design through auditory perception [82].

The Semantic Di fferential Scales for the perceptions of users and architecture students (with arithmetic means) for the case study of the Mirkova Varoš streets are shown in Table 3.

The results obtained by surveying the area users and architecture students indicate di fferent approaches in the evaluation of space, and in visual, tactile and auditory terms, within various categories of the respondents. There are di fferences in the perception of space by architects (in this case, students) and non-architects (area users) i.e., di fferences in their reactions to the impulses from public spaces, as has been indicated by various authors [83–86]. Architecture students had high criteria in their evaluation of the aesthetic components of the area (applied materials and colours), while other users of the public space were more critical toward the functional and infrastructural deficiencies in the public space, as well as acoustic comfort. On a semantic di fferential scale, the majority of respondents reacted positively to historically valuable objects and cultural content, while negative reactions were provoked by issues of tra ffic frequency, noise, pollution, and inappropriate parking. The survey found that streets with fewer users received lower ratings on the semantic scale of visual criteria. This is especially pronounced in the category of non-architect respondents. Area users generally used a rating of 0 for criteria without stimuli, either positive or negative.

Finally, it is possible to conclude that critical judgment and reasoning was more pronounced among students of architecture compared to area users. The respondent group, comprising randomly selected area users, has less ability to independently evaluate interpretive visual factors in relation to auditory "impressions" and the subjective experience of images in relation to the auditory display is also inseparable.

Figure 7 shows the qualitative results of the street evaluation in Mirkova Varoš, Podgorica.

**Table 3.** Semantic Di fferential Scales of perception of the Mirkova Varoš streets in Podgorica.

**Table 3.** *Cont.*

**Figure 7.** Qualitative results of the street evaluation in Mirkova Varoš, Podgorica.

## 3.3.2. Physical Street Regeneration Directions

The results achieved by the survey measuring the subjective perception of architecture students and other users of the space formed a qualitative basis (input data) for the physical street regeneration of Mirkova Varoš. By analyzing the results (the average values) achieved at the scales of the semantic di fferential, the characteristic positive and negative aspects of the 12 analyzed streets can be seen.

All streets were evaluated most positively for their morphological characteristics (street width) and natural sun conditions (100%). In addition to these criteria for the visual perception of streets, positive results were obtained for a large number of streets for their dynamic street fronts and the presence of greenery (75%). The highest percentage of negatively assessed streets in visual terms relates to the contrasting pairs disharmonized–harmonized (75% negative, 25% positive), colourless–colourful (83% negative, 17% positive), and disarranged–arranged (83% negative mean).

In terms of tactile perception criteria, the obtained mean values (close to neutral zero point) are fairly uniform in most streets (over 80%). In Hercegovaˇcka, Bokeška, Vuˇcedolska and Vuka Karadži´ca streets, there are greater fluctuations in the evaluation of criteria, in terms of the contrasting pairs artificial–natural, unpleasant–pleasant, and simple–complex.

Regarding auditory perceptual factors, a mostly negative mean was obtained for road tra ffic noise low–high level criteria (83%), unenjoyable–enjoyable (75%), as a result of the irritating–pleasing criteria (50%). This shows that, despite the negative e ffect of tra ffic noise, which is predominantly represented in Mirkova Varoš, and other types of noise, users do not record dissatisfaction (age structure defines the tolerance threshold for sound perception).

The structure of the obtained results indicates the dissatisfaction of the users of the space and students, with regard to the level of organization of the public space, as well as the lack of content and activities that could increase the number of users in the "abandoned" traditional city centre. In most cases, street comfort depends on the frequency of tra ffic through the street, except in streets where these deficiencies are o ffset by the aesthetic and ambient values of the street itself, but also by a satisfactory level of public facilities (cultural and creative capacities).

Based on the street space perception results obtained by surveying users and architecture students, several general directions for the physical regeneration of the case study streets were articulated.

### *3.4. Creative Street Regeneration in Mirkova Varoš*

Solutions for the physical regeneration of the 12 case study streets, made by architecture students, are presented in Table 4.

Creative regeneration, through the prism of the urban paradigms of the 21st century, has the task of preserving and improving the traditional values of a space (authenticity, recognisability) [8], to create a field of active creative interaction and enable economic prosperity with benefits to all stakeholders. Emphasis is placed on encouraging the creativity of all who are involved in the process, not just the creative class [12].

Street regeneration interventions in the context of socio-spatial sustainability in the case study streets have been specifically implemented in three key directions:

1. *Functional*: infrastructural (program dynamism and multifunctionality (hospitality, culture, trade, entertainment, and recreation), reducing road tra ffic and encouraging the dominance of pedestrians (underground car parking, new routes, integration with a narrower and wider context, and a network of micro public spaces), information points, digital street installations, etc.;

In a wider context, the creative urban street regeneration of Mirkova Varos used in this study is intended to highlight the important role of a combined, unconventional method of physical space regeneration, with a strong intention to promote a rational and realistic physical redefinition of the current image of traditional public open spaces and reactivate their role in 21st Century cities. In a narrower sense, this study suggests that it is possible to regenerate concrete traditional streets by respecting tradition and at the same time developing new spatial scenarios. These scenarios are intended to promote a dynamic relationship between man and space—a humane, cultural, creative, ecological, informational dimension—and interact with the local capabilities and needs of global society. Generating new forms of creative activities (by regenerating existing content and introducing new content, new forms of integration) reactivates the socio–spatial interaction in (1) the user–place system, (2) the place–city system, and (3) local processes–global flows.

**Table 4.** Proposals for the physical regeneration of the 12 streets in Mirkova Varoš in Podgorica.

**Table 4.** *Cont.*

The following students have participated in the proposals for the physical regeneration of streets: Danica Cvorovi´ c, Milan Rahovi´c, Bojana Raspopovi´c, Branka Vujovi´c, Aida Kujovi´c, Ana Nenezi´c, Vlado Jegdi´c, Stefan Mušiki´c, Ivana Kapetanovi´c, Marija Petriˇcevi´c, Andjela Markovi´c, Nikola Popovi´c, Nina Simovi´c, Jovana Juškovi´c, and Anja Lješkovi´c.

ˇ


Meet the editor

Andrzej Klimczuk, Ph.D., is a sociologist, public policy expert, and assistant professor in the Department of Public Policy of the Collegium of Socio-Economics, SGH Warsaw School of Economics, Poland. He worked as an external expert for institutions such as the European Commission, Interreg CENTRAL EUROPE Programme, and Fondazione Cariplo. Dr. Klimczuk has authored many scientific papers in the fields of gerontology,

labor economics, public management, and social policy. His recent monographs include *Economic Foundations for Creative Ageing Policy*, *Generations, Intergenerational Relationships, Generational Policy*, and *Perspectives and Theories of Social Innovation for Ageing Population*.

Contents

**Section 1**

*by Andrzej Klimczuk*

Change in Australia

**Section 2**

to Ivory Towers

*by Justin Dansou*

**Preface XI**

Selected Basics of Demographic Analysis **1**

**Chapter 1 3**

**Chapter 2 7**

**Chapter 3 23**

Demographic Analysis of Societies or Groups Defined by Various Criteria **35**

**Chapter 4 37**

**Chapter 5 55**

**Chapter 6 81**

**Chapter 7 113**

Demographic Analysis and the Decomposition of Social Change *by Parfait Eloundou-Enyegue, Sarah Giroux and Michel Tenikue*

Geographies of Ageing: A Visuospatial Approach to Demographic

Demographic Transition in Sub-Saharan Africa: From Grassroots

Variations in First Union Dissolution Patterns among the Lebou, Peuhl,

The Age-Sex Structure of Religion as a Determinant of the Social Inclusion

Age at First Marriage of Women in Bangladesh: Levels, Trends

*by Mohammad Salim Zahangir and Mosammat Zamilun Nahar*

Introductory Chapter: Demographic Analysis

*by Hamish Robertson and Nick Nicholas*

*by Kelvin Melkizedeck Leshabari*

of Internal Migrants in Maroua

*by Nanche Billa Robert*

and Determinants

Sereer, Toucouleur, and Wolof in Senegal