**The SUMOylation Pathway as a Potential Therapeutic Target in Multiple Myeloma**

James J. Driscoll *Division of Hematology and Oncology, Department of Internal Medicine University of Cincinnati Medical Center and Barrett Cancer Center Cincinnati, OH Medical Oncology Branch, National Cancer Institute, National Institutes of Health Bethesda, MD USA*

#### **1. Introduction**

128 Multiple Myeloma – An Overview

[290] He, H., et al., *Genistein down-regulates the constitutive activation of nuclear factor-κB of bone*

[291] Hideshima, T., et al., *MLN120B, a novel IκB kinase β inhibitor, blocks multiple myeloma cell growth in vitro and in vivo.* Clinical Cancer Research, 2006. 12(19): p. 5887-5894. [292] Kong, F., et al., *Inhibitory effects of parthenolide on the angiogenesis induced by human*

[293] Hossain, Z., et al., *Chitosan and marine phospholipids reduce matrix metalloproteinase*

[294] Ritchie, J.P., et al., *SST0001, a chemically modified heparin, inhibits myeloma growth and*

[295] Mori, Y., et al., *Anti-α4 integrin antibody suppresses the development of multiple myeloma*

[296] Olson, D.L., et al., *Anti-α4 integrin monoclonal antibody inhibits multiple myeloma growth in*

[297] Wang, X., Z. Zhang, and C. Yao, *Targeting integrin-linked kinase increases apoptosis and*

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*a murine model.* Molecular cancer therapeutics, 2005. 4(1): p. 91-99.

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*BMSCs.* Medical Oncology, 2010: p. 1-5.

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*marrow stromal cells in multiple myeloma, leading to suppression of gene expression and*

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*angiogenesis via disruption of the heparanase/syndecan-1 axis.* Clinical Cancer Research,

*decreases invasion of myeloma cell lines and inhibits IL-6 and VEGF secretion from*

The Ubiquitin+Proteasome System (UPS) represents a successful anti-cancer strategy that has translated from preclinical studies to clinical development and significantly improved the survival of Multiple Myeloma (MM) patients (1). The proteasome is high molecular weight structure that contains multiple proteolytic sites and functions as the catalytic core of the UPS to degrade Ub~conjugated proteins (2-4). Inhibition of the proteasome's chymotryptic-like peptidase activity blocks the entire UPS and leads to selective tumor cell death. Bortezomib is the first proteasome inhibitor used in human clinical trials and treatment leads to a reduction in tumor burden in MM patients (5,6). However, cellular resistance to proteasome-based therapy generally results and reduces the efficacy. Resistance to proteasome inhibitors may result from altered expression of either proteasome or non-proteasome encoding genes. SUMOylation is a post-translational modification that attaches a **S**mall **U**biquitin-like **MO**difier to target proteins and, similar to Ub, may target proteins for proteasomal degradation. Gene expression profiling of MM patient samples indicated that bortezomib resistance may be achieved through induction of non-proteasome components. Importantly, certain effectors of the SUMOylation pathway were induced in the samples of MM patients that did not respond to bortezomib. A molecular-based, biologically-relevant supervised approach was used to identify a compendium of genes within the SUMO+UPS that were induced in MM patients and correlated with a decreased response to bortezomib. This molecular approach is relevant to a heterogeneous disease like MM where patients with similar clinical and pathologic features have vastly different genetic mutations responsible for disease and varying responses to therapies and clinical outcomes. The potential role of the SUMOylation pathway is only beginning to emerge, however, evidence indicates that SUMOylation is induced in myelomagenesis and also in bortezomib-resistant cells.

An aggresome is a proteinaceous inclusion body that forms when the cellular protein degradation machinery is impaired or overwhelmed and leads to an accumulation of proteins for disposal (7,8). Aggresome formation is thought to represent a cellular protective

The SUMOylation Pathway as a Potential Therapeutic Target in Multiple Myeloma 131

highly effective synergistic drug combinations that have the potential to achieve a higher frequency of durable responses in a greater number of MM patients. Molecular, genomic and proteomic studies have further increased understanding of the biology of myelomagenesis, provided the basis for better prognostic classification in the context of uniform therapies and generated a sound rationale for combining targeted therapies in

The 26S proteasome is a high-molecular weight, multi-subunit complex that consists of a 20S catalytic core and a 19S regulatory particle. Proteasomes are found in eukaryotes, archaebacteria and some bacteria. The 20S proteasome functions as the catalytic core of the ubiquitin-proteasome system to hydrolyze proteins into short polypeptide and is capable of hydrolyzing peptides C-terminal to chymotryptic, tryptic or acidic residues. 20S proteasomes are cylindrical structures composed of four stacked rings with each ring composed of seven individual subunits. The two inner rings are made of seven *β subunits* that contain the various active sites and the two outer rings are each composed of seven *α subunits*. The two outer rings comprised of α subunits are then bound at either pole by the

Proteasome inhibition is a promising therapeutic strategy for the treatment of an increasing number of B cell malignancies, especially MM. Bortezomib is a low molecular weight boronbased agent that inhibits the chymotryptic-like activity of the proteasome. Inhibition of the chymotryptic-like activity inhibits all proteasome activity and acts as a blockade of the entire ubiquitin+proteasome pathway. The result is the intracellular accumulation of Ub~protein conjugates, activation of cellular stress sensors and eventually cell death. The selective cytotoxic effect of proteasome inhibition on tumor cells further validated protein degradation as a therapeutic target in oncology and has been translated in the treatment of certain malignancies. Bortezomib was US Food and Drug Administration (FDA) approved for the treatment of newly diagnosed, relapsed and refractory MM. The introduction of bortezomib into the treatment of MM resulted in a paradigm shift and significantly improved patient survival. The concurrent targeting of both the tumor cell as well as the surrounding bone marrow microenvironment may promote tumor cell death and efficacy. The FDA has recently approved a more effective three drug combination of bortezomib, lenalidomide and dexamethasone for the treatment of MM that may act on the malignant

plasma cell and also to counteract the protective effect of the bone marrow milieu.

Many MM patients, however, exhibit an intrinsic (primary) resistance and do not initially respond to bortezomib. Furthermore, in those patients that do respond, clinical efficacy is dampened by the inevitable emergence of acquired drug-resistance that eventually develops through unidentified mechanisms. While bortezomib has significantly transformed the management of MM, the mechanism(s) of action and bases of individual patient susceptibility remain unclear. These recent advances in mechanistic understanding and treatment modalities have extended median survival to >6 years and 10% of patients now survive >10 years. However, the vast majority of MM remains incurable even with these

Though the catabolism of ubiquitinated substrates has been targeted therapeutically with significantly improved prognosis, patient response to bortezomib remains highly variable and cannot be predicted accurately. E3 Ub ligases are the specific components of the UPS

current and emerging clinical development.

19S regulatory particles.

strategies.

response to the presence of abnormal, misfolded or damaged proteins within the cytoplasm that cannot be eliminated by the UPS. Aggresomes form adjacent to the microtubuleorganizing center (MTOC) and neighboring the centrosome. The microtubule-based motor protein dynein, heat shock proteins and the histone deacetylase 6 (HDAC6) have been reported as aggresome components (9). HDAC6 specifically functions as an adaptor protein between the dynein motor protein and accumulating polyubiquitinated substrate proteins. The protein aggregates that reside in an aggresome are sequestered in a cage that consists largely of intermediate filaments. Aggresome formation may serve as an alternative mechanism to eliminate the cytotoxic effect of accumulated Ub~protein conjugates. Recent evidence indicates that SUMOylation effectors and SUMO-modified proteins are related to aggresome formation. Inhibition of SUMOylation could preclude aggresome formation and consequently overcome bortezomib resistance.

 The formation of the aggresome functions as a mechanism not only to sequester potentially cytotoxic aggregates but also to serve as a platform for the eventual autophagic clearance of these aggregates (10, 11). Autophagy is a catabolic process that involves the degradation of the cell's own constituents through the lysosomal machinery (12, 13). It is a tightly-regulated process that plays a normal part in cell growth, development, and homeostasis and maintains the balance between the synthesis, degradation and subsequent recycling of cellular products. It is a major mechanism by which a starving cell reallocates nutrients from unnecessary processes to more-essential processes. A variety of autophagic processes exist, all having in common the degradation of intracellular components through the lysosome. The most well-known mechanism of autophagy involves the formation of a membrane around a targeted region of the cell, separating the contents from the rest of the cytoplasm. The resultant vesicle then fuses with a lysosome and subsequently degrades the contents.

Importantly, new evidence indicates upon proteasome inhibition, the accumulated Ub~protein conjugates are bound by autophagy cargo receptors for clearance (14, 15). Thus, it appears that aggresome formation is a prerequisite for autophagic clearance of Ub~protein conjugates (16). In addition, selective autophagy may serve to eliminate these aggregates and overcome proteasome inhibition and yield drug resistance. It remains to be determined whether SUMOylated proteins are also cleared by autophagic clearance. However, inhibition of the SUMOylation pathwya may prevent aggresome formation, impede their autophagic clearance of Ub~protein conjugates and overcome bortezomib resistance. Future studies will address these questions and may yield new therapeutic targets, novel anti-cancer agents and lead to improved strategies for the treatment of MM and other malignancies.

#### **2. Clinical success of proteasome inhibitor-based therapy in multiple myeloma**

Multiple myeloma (MM) is a hematologic neoplasm of B-cell origin characterized by the clonal proliferation and progressive accumulation of malignant plasma cells within the bone marrow. MM accounts for greater than 10% of all hematologic cancers and in spite of conventional high-dose chemotherapies, remains uniformly fatal, in part, because of either intrinsic (primary) or acquired (secondary) drug resistance. Recent advances in translational medicine have led to the development of molecularly-based targeted therapies as well as

response to the presence of abnormal, misfolded or damaged proteins within the cytoplasm that cannot be eliminated by the UPS. Aggresomes form adjacent to the microtubuleorganizing center (MTOC) and neighboring the centrosome. The microtubule-based motor protein dynein, heat shock proteins and the histone deacetylase 6 (HDAC6) have been reported as aggresome components (9). HDAC6 specifically functions as an adaptor protein between the dynein motor protein and accumulating polyubiquitinated substrate proteins. The protein aggregates that reside in an aggresome are sequestered in a cage that consists largely of intermediate filaments. Aggresome formation may serve as an alternative mechanism to eliminate the cytotoxic effect of accumulated Ub~protein conjugates. Recent evidence indicates that SUMOylation effectors and SUMO-modified proteins are related to aggresome formation. Inhibition of SUMOylation could preclude aggresome formation and

 The formation of the aggresome functions as a mechanism not only to sequester potentially cytotoxic aggregates but also to serve as a platform for the eventual autophagic clearance of these aggregates (10, 11). Autophagy is a catabolic process that involves the degradation of the cell's own constituents through the lysosomal machinery (12, 13). It is a tightly-regulated process that plays a normal part in cell growth, development, and homeostasis and maintains the balance between the synthesis, degradation and subsequent recycling of cellular products. It is a major mechanism by which a starving cell reallocates nutrients from unnecessary processes to more-essential processes. A variety of autophagic processes exist, all having in common the degradation of intracellular components through the lysosome. The most well-known mechanism of autophagy involves the formation of a membrane around a targeted region of the cell, separating the contents from the rest of the cytoplasm. The resultant vesicle then fuses with a lysosome and subsequently degrades the contents.

Importantly, new evidence indicates upon proteasome inhibition, the accumulated Ub~protein conjugates are bound by autophagy cargo receptors for clearance (14, 15). Thus, it appears that aggresome formation is a prerequisite for autophagic clearance of Ub~protein conjugates (16). In addition, selective autophagy may serve to eliminate these aggregates and overcome proteasome inhibition and yield drug resistance. It remains to be determined whether SUMOylated proteins are also cleared by autophagic clearance. However, inhibition of the SUMOylation pathwya may prevent aggresome formation, impede their autophagic clearance of Ub~protein conjugates and overcome bortezomib resistance. Future studies will address these questions and may yield new therapeutic targets, novel anti-cancer agents and lead to improved strategies for the treatment of MM

**2. Clinical success of proteasome inhibitor-based therapy in multiple**

Multiple myeloma (MM) is a hematologic neoplasm of B-cell origin characterized by the clonal proliferation and progressive accumulation of malignant plasma cells within the bone marrow. MM accounts for greater than 10% of all hematologic cancers and in spite of conventional high-dose chemotherapies, remains uniformly fatal, in part, because of either intrinsic (primary) or acquired (secondary) drug resistance. Recent advances in translational medicine have led to the development of molecularly-based targeted therapies as well as

consequently overcome bortezomib resistance.

and other malignancies.

**myeloma**

highly effective synergistic drug combinations that have the potential to achieve a higher frequency of durable responses in a greater number of MM patients. Molecular, genomic and proteomic studies have further increased understanding of the biology of myelomagenesis, provided the basis for better prognostic classification in the context of uniform therapies and generated a sound rationale for combining targeted therapies in current and emerging clinical development.

The 26S proteasome is a high-molecular weight, multi-subunit complex that consists of a 20S catalytic core and a 19S regulatory particle. Proteasomes are found in eukaryotes, archaebacteria and some bacteria. The 20S proteasome functions as the catalytic core of the ubiquitin-proteasome system to hydrolyze proteins into short polypeptide and is capable of hydrolyzing peptides C-terminal to chymotryptic, tryptic or acidic residues. 20S proteasomes are cylindrical structures composed of four stacked rings with each ring composed of seven individual subunits. The two inner rings are made of seven *β subunits* that contain the various active sites and the two outer rings are each composed of seven *α subunits*. The two outer rings comprised of α subunits are then bound at either pole by the 19S regulatory particles.

Proteasome inhibition is a promising therapeutic strategy for the treatment of an increasing number of B cell malignancies, especially MM. Bortezomib is a low molecular weight boronbased agent that inhibits the chymotryptic-like activity of the proteasome. Inhibition of the chymotryptic-like activity inhibits all proteasome activity and acts as a blockade of the entire ubiquitin+proteasome pathway. The result is the intracellular accumulation of Ub~protein conjugates, activation of cellular stress sensors and eventually cell death. The selective cytotoxic effect of proteasome inhibition on tumor cells further validated protein degradation as a therapeutic target in oncology and has been translated in the treatment of certain malignancies. Bortezomib was US Food and Drug Administration (FDA) approved for the treatment of newly diagnosed, relapsed and refractory MM. The introduction of bortezomib into the treatment of MM resulted in a paradigm shift and significantly improved patient survival. The concurrent targeting of both the tumor cell as well as the surrounding bone marrow microenvironment may promote tumor cell death and efficacy. The FDA has recently approved a more effective three drug combination of bortezomib, lenalidomide and dexamethasone for the treatment of MM that may act on the malignant plasma cell and also to counteract the protective effect of the bone marrow milieu.

Many MM patients, however, exhibit an intrinsic (primary) resistance and do not initially respond to bortezomib. Furthermore, in those patients that do respond, clinical efficacy is dampened by the inevitable emergence of acquired drug-resistance that eventually develops through unidentified mechanisms. While bortezomib has significantly transformed the management of MM, the mechanism(s) of action and bases of individual patient susceptibility remain unclear. These recent advances in mechanistic understanding and treatment modalities have extended median survival to >6 years and 10% of patients now survive >10 years. However, the vast majority of MM remains incurable even with these strategies.

Though the catabolism of ubiquitinated substrates has been targeted therapeutically with significantly improved prognosis, patient response to bortezomib remains highly variable and cannot be predicted accurately. E3 Ub ligases are the specific components of the UPS

The SUMOylation Pathway as a Potential Therapeutic Target in Multiple Myeloma 133

humans. E3 Ub ligases are engaged in the recognition of biologically relevant proteins that are degraded by the proteasome. While proteasome inhibition generates the accumulation of Ub~conjugated proteins and ultimately cell death, the selectivity of this process and the role of individual E3 ligases has not been previously addressed. We investigated whether UPS effectors and specifically, E3 ligases were differentially expressed in MM patients and whether expression correlated with resistance to bortezomib-based therapy. Recently, we demonstrated that genes encoding SUMOylation pathway effectors were induced during

Aggresomes are novel cellular structures that employ HDAC6 to sequester and transport protein aggregates to the autophagy-lysosome system for efficient disposal. These poorly defined structures have been shown to consist of HDAC6, microtubules, heat shock proteins and ubiquitin. Aggresome formation is induced by a number of cellular and genotoxic stressors, including proteasomal inhibition. These structures may represent a potential compensatory mechanism to for cells to eliminate Ub~protein conjugates that accumulate upon proteasomal inhibiton. The result may be resistance to proteasome inhibitors and promote tumor survival. Similar to Ub, the small Ub-like MOdifier (SUMO) is attached to target certain proteins for proteasomal degradation but an association with aggresomes has not been established. The proteasome is limited in its capacity to degrade certain proteins, such as membrane-associated, oligomeric and protein aggregates. SUMOylation, like ubiquitination, may be mechanistically and biologically linked to aggresomes to promote the removal of toxic proteins that accumulate upon proteasomal inhibition. SUMOylation may also be required for the formation of aggresomes. Genetic or pharmacologic targeting of SUMOylation pathway effectors may impair functioning and formation of aggresomes to

overcome drug resistance and enhance the cytotoxicity of proteasome inhibitors.

can be selective and mediated through specialized Ub-binding cargo receptors.

Removal of harmful protein aggregates is mediated by autophagy, a mechanism that sequesters cytosolic cargo and delivers it to the lysosome (17-20). Autophagy is of great importance for cellular homeostasis and survival, while its deregulation has been linked to pathological conditions, e.g., certain neurodegenerative diseases and cancer (21-23). The process of autophagy for many years has been thought of as a random degradative process to eliminate long-lived cell constituents. However, recent evidence indicates that autophagy

The involvement of Ub as a specificity factor for selective autophagy has resulted in a rapid emergence of studies that have demonstrated active communication between the UPS and the aggresome-autophagy-lysosomal system (25). The identification and characterization of autophagy receptors, such as p62/SQSTM1, NBR1, p97/ VCP which can simultaneously bind both Ub and autophagy-specific Ub-like modifiers, e.g., LC3/GABARAP, and possible SUMO has provided a molecular link between the UPS and autophagy (26-28). Since the UPS regulates the selective degradation of certain short-lived, mutant and misfolded proteins, autophagy may remove not only aggregates which are comprised of long-lived proteins but also UPS substrates. Selective autophagy may protect cells from oxidative and

myelomagenesis and correlated with decreased patient survival.

**4. Aggresomes and proteasome-based therapy**

**5. Autophagy**

that recognize substrates for degradation and confer specificity on target selection for the UPS. Therefore, the expression of individual E3's was analyzed using a microarray dataset obtained from MM patient tumor samples and found a striking variability in the expression level of individual E3 Ub ligases between normal plasma cells and patients MM cells. Specifically, RNF4, an E3 specific for poly-sumoylated proteins, was induced in MM patients and correlated with decreased patient response to the proteasome inhibitor bortezomib. Expression profiling of pretreatment tumor samples obtained from MM patients in independent clinical trials were used to generate a signature that correlated expression of SUMO+Ub+Proteasome pathway components with clinical outcome to predict patient response to bortezomib.

#### **3. The Ub+Proteasome System (UPS) and the Ub-like SUMOylation pathway**

The UPS regulates numerous critical cellular processes by maintaining the appropriate intracellular level of key proteins. Whereas *de novo* protein synthesis is a comparatively slow process, proteins are rapidly degraded at a rate compatible with the control of cell cycle transitions and cell death induction. The critical role played by Ub-mediated protein turnover in cell cycle regulation makes this process a high-value target for oncogenic mutations. SUMO proteins are similar to ubiquitin, and SUMOylation is directed by an three-step enzymatic cascade analogous to that involved in ubiquitination. SUMOylation is a highly dynamic, reversible, post-translational protein modification, similar to ubiquitination that attaches a family of polypeptides to target proteins to modify their function, subcellular localization or stability. A role for SUMOylation in the UPS has been established since SUMO attachment signals the recruitment of specific E3 Ub ligases to target proteins, e.g., PML, PEA, PARP-1 and HIF1-alpha, for ubiquitination and proteasomal degradation. Induction of individual SUMOylation and UPS effectors has been demonstrated in MM cell lines and patient samples and SUMO pathway induction has been correlated with resistance to bortezomib-based therapy. Similarly, proteasome components are induced in bortezomib-resistant cells ad ndecrease the toxic accumulation of Ub~conjugates. The mechanistic link of SUMOylation to proteasomal resistance is uncertain. An important unresolved issue is the clinical significance of sumoylation during proteasomal degradation and proteasome inhibitor-based therapy. SUMOylation was recently shown to have a role in proteasomal degradation since SUMO-2/3 attachment can signal Ub-dependent degradation. Novel RING family Ub ligases, e.g., RNF4, bear SUMO interaction motifs and have been implicated in the proteasomal degradation of SUMOmodified target protein PML. Similarly, the E3 Ub ligase von Hippel–Lindau (VHL) has been reported to control the levels of sumoylated HIF-1α while VHL and RNF4 control the levels of HIF-2� to indicate that sumoylated HIF-2α is degraded through SUMO-targeted ubiquitination. SUMO modification as a signal for degradation is conserved in eukaryotes and Ub ligases that specifically recognize SUMO-modified proteins have been discovered from yeasts to humans. proteasome components to further link the pathways. Individual components of the sumoylation pathway serve as biomarkers to predict clinical response to bortezomib and provide evidence for targeting SUMO pathway to improve therapeutic outcome in myeloma in general and specifically in combination with proteasome inhibitors.

SUMO modification as a signal for degradation is conserved in eukaryotes and Ub ligases that specifically recognize SUMO-modified proteins have been discovered from yeasts to humans. E3 Ub ligases are engaged in the recognition of biologically relevant proteins that are degraded by the proteasome. While proteasome inhibition generates the accumulation of Ub~conjugated proteins and ultimately cell death, the selectivity of this process and the role of individual E3 ligases has not been previously addressed. We investigated whether UPS effectors and specifically, E3 ligases were differentially expressed in MM patients and whether expression correlated with resistance to bortezomib-based therapy. Recently, we demonstrated that genes encoding SUMOylation pathway effectors were induced during myelomagenesis and correlated with decreased patient survival.

#### **4. Aggresomes and proteasome-based therapy**

Aggresomes are novel cellular structures that employ HDAC6 to sequester and transport protein aggregates to the autophagy-lysosome system for efficient disposal. These poorly defined structures have been shown to consist of HDAC6, microtubules, heat shock proteins and ubiquitin. Aggresome formation is induced by a number of cellular and genotoxic stressors, including proteasomal inhibition. These structures may represent a potential compensatory mechanism to for cells to eliminate Ub~protein conjugates that accumulate upon proteasomal inhibiton. The result may be resistance to proteasome inhibitors and promote tumor survival. Similar to Ub, the small Ub-like MOdifier (SUMO) is attached to target certain proteins for proteasomal degradation but an association with aggresomes has not been established. The proteasome is limited in its capacity to degrade certain proteins, such as membrane-associated, oligomeric and protein aggregates. SUMOylation, like ubiquitination, may be mechanistically and biologically linked to aggresomes to promote the removal of toxic proteins that accumulate upon proteasomal inhibition. SUMOylation may also be required for the formation of aggresomes. Genetic or pharmacologic targeting of SUMOylation pathway effectors may impair functioning and formation of aggresomes to overcome drug resistance and enhance the cytotoxicity of proteasome inhibitors.

#### **5. Autophagy**

132 Multiple Myeloma – An Overview

that recognize substrates for degradation and confer specificity on target selection for the UPS. Therefore, the expression of individual E3's was analyzed using a microarray dataset obtained from MM patient tumor samples and found a striking variability in the expression level of individual E3 Ub ligases between normal plasma cells and patients MM cells. Specifically, RNF4, an E3 specific for poly-sumoylated proteins, was induced in MM patients and correlated with decreased patient response to the proteasome inhibitor bortezomib. Expression profiling of pretreatment tumor samples obtained from MM patients in independent clinical trials were used to generate a signature that correlated expression of SUMO+Ub+Proteasome pathway components with clinical outcome to predict

**3. The Ub+Proteasome System (UPS) and the Ub-like SUMOylation pathway**  The UPS regulates numerous critical cellular processes by maintaining the appropriate intracellular level of key proteins. Whereas *de novo* protein synthesis is a comparatively slow process, proteins are rapidly degraded at a rate compatible with the control of cell cycle transitions and cell death induction. The critical role played by Ub-mediated protein turnover in cell cycle regulation makes this process a high-value target for oncogenic mutations. SUMO proteins are similar to ubiquitin, and SUMOylation is directed by an three-step enzymatic cascade analogous to that involved in ubiquitination. SUMOylation is a highly dynamic, reversible, post-translational protein modification, similar to ubiquitination that attaches a family of polypeptides to target proteins to modify their function, subcellular localization or stability. A role for SUMOylation in the UPS has been established since SUMO attachment signals the recruitment of specific E3 Ub ligases to target proteins, e.g., PML, PEA, PARP-1 and HIF1-alpha, for ubiquitination and proteasomal degradation. Induction of individual SUMOylation and UPS effectors has been demonstrated in MM cell lines and patient samples and SUMO pathway induction has been correlated with resistance to bortezomib-based therapy. Similarly, proteasome components are induced in bortezomib-resistant cells ad ndecrease the toxic accumulation of Ub~conjugates. The mechanistic link of SUMOylation to proteasomal resistance is uncertain. An important unresolved issue is the clinical significance of sumoylation during proteasomal degradation and proteasome inhibitor-based therapy. SUMOylation was recently shown to have a role in proteasomal degradation since SUMO-2/3 attachment can signal Ub-dependent degradation. Novel RING family Ub ligases, e.g., RNF4, bear SUMO interaction motifs and have been implicated in the proteasomal degradation of SUMOmodified target protein PML. Similarly, the E3 Ub ligase von Hippel–Lindau (VHL) has been reported to control the levels of sumoylated HIF-1α while VHL and RNF4 control the levels of HIF-2� to indicate that sumoylated HIF-2α is degraded through SUMO-targeted ubiquitination. SUMO modification as a signal for degradation is conserved in eukaryotes and Ub ligases that specifically recognize SUMO-modified proteins have been discovered from yeasts to humans. proteasome components to further link the pathways. Individual components of the sumoylation pathway serve as biomarkers to predict clinical response to bortezomib and provide evidence for targeting SUMO pathway to improve therapeutic outcome in myeloma in general and specifically in combination with proteasome inhibitors. SUMO modification as a signal for degradation is conserved in eukaryotes and Ub ligases that specifically recognize SUMO-modified proteins have been discovered from yeasts to

patient response to bortezomib.

Removal of harmful protein aggregates is mediated by autophagy, a mechanism that sequesters cytosolic cargo and delivers it to the lysosome (17-20). Autophagy is of great importance for cellular homeostasis and survival, while its deregulation has been linked to pathological conditions, e.g., certain neurodegenerative diseases and cancer (21-23). The process of autophagy for many years has been thought of as a random degradative process to eliminate long-lived cell constituents. However, recent evidence indicates that autophagy can be selective and mediated through specialized Ub-binding cargo receptors.

The involvement of Ub as a specificity factor for selective autophagy has resulted in a rapid emergence of studies that have demonstrated active communication between the UPS and the aggresome-autophagy-lysosomal system (25). The identification and characterization of autophagy receptors, such as p62/SQSTM1, NBR1, p97/ VCP which can simultaneously bind both Ub and autophagy-specific Ub-like modifiers, e.g., LC3/GABARAP, and possible SUMO has provided a molecular link between the UPS and autophagy (26-28). Since the UPS regulates the selective degradation of certain short-lived, mutant and misfolded proteins, autophagy may remove not only aggregates which are comprised of long-lived proteins but also UPS substrates. Selective autophagy may protect cells from oxidative and

The SUMOylation Pathway as a Potential Therapeutic Target in Multiple Myeloma 135

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genotoxic stresses. In addition, this process may remove Ub~ and SUMO~protein conjugates that accumulate upon proteasome inhibition and overcome the cytotoxic effect of bortezomib. Induction of selective autophagy may be induced in tumor cells to provide a mechanism of cell survival and drug resistance (29, 30). Selective autophagy represents a promising new pathway to investigate as means to overcome resistance to proteasome inhibitors and further advance the treatment of MM.

#### **6. Conclusions**

Multiple myeloma accounts for a substantial amount of all hematologic malignancies seen worldwide. In the US and Europe, the incidence of MM exceeds 40,000 cases annual. MM has assumed a position of prominence as a model system for preclinical studies, drug development and clinical trials. Unlike other malignancies, in which surgery and radiation are important treatment modalities, MM is nearly exclusively treated with stem cell transplantation (SCT) or synergistic chemotherapeutic combinations. SCT has produced statistically significant survival benefit for MM patients. However, a number of MM patients are not eligible for SCT either because of age, toxicities and many patients can relapse. Novel therapeutic agents such as the immunomodulatory agents and bortezomib have revolutionized therapy and improved overall survival. However, despite these advances, MM remains largely incurable as disease relapse is inevitable. Therefore, there is an urgent need for new, tolerable agents that demonstrate the ability to either overcome or prevent drug resistance. The role of the SUMOylation pathway in Multiple Myeloma is only beginning to be understood and appears to be a novel therapeutic target that can be used not only to treat MM but also to overcome drug resistance. Future studies will address the role of this emerging pathway in myelomagenesis.

#### **7. References**

genotoxic stresses. In addition, this process may remove Ub~ and SUMO~protein conjugates that accumulate upon proteasome inhibition and overcome the cytotoxic effect of bortezomib. Induction of selective autophagy may be induced in tumor cells to provide a mechanism of cell survival and drug resistance (29, 30). Selective autophagy represents a promising new pathway to investigate as means to overcome resistance to proteasome

Multiple myeloma accounts for a substantial amount of all hematologic malignancies seen worldwide. In the US and Europe, the incidence of MM exceeds 40,000 cases annual. MM has assumed a position of prominence as a model system for preclinical studies, drug development and clinical trials. Unlike other malignancies, in which surgery and radiation are important treatment modalities, MM is nearly exclusively treated with stem cell transplantation (SCT) or synergistic chemotherapeutic combinations. SCT has produced statistically significant survival benefit for MM patients. However, a number of MM patients are not eligible for SCT either because of age, toxicities and many patients can relapse. Novel therapeutic agents such as the immunomodulatory agents and bortezomib have revolutionized therapy and improved overall survival. However, despite these advances, MM remains largely incurable as disease relapse is inevitable. Therefore, there is an urgent need for new, tolerable agents that demonstrate the ability to either overcome or prevent drug resistance. The role of the SUMOylation pathway in Multiple Myeloma is only beginning to be understood and appears to be a novel therapeutic target that can be used not only to treat MM but also to overcome drug resistance. Future studies will address the

[1] Hideshima T, Bradner JE, Chauhan D, Anderson KC. Intracellular protein degradation and its therapeutic implications. Clin Cancer Res 2005;11:8530-8533. [2] Hershko, A. and Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 1998; 67,

[3] Kirkin V, Dikic I. Ubiquitin networks in cancer. Curr Opin Genet Dev. 2011, 21:21-28. [4] Kerscher O, Felberbaum R, Hochstrasser M. Modification of proteins by ubiquitin and

[5] Hideshima T, Bradner JE, Wong J, et al. Small-molecule inhibition of proteasome and

[6] Mitsiades CS, Davies FE, Laubach JP, et al. Future directions of next-generation novel

[7] Kopito, R R. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol.

[8] Pang, T. The Role of Ubiquitin in Autophagy-Dependent Protein Aggregate Processing

aggresome function induces synergistic antitumor activity in multiple myeloma.

therapies, combination approaches, and the development of personalized medicine

ubiquitin-like proteins. Annu Rev Cell Dev Biol. 2006;22:159–180.

Proc Natl Acad Sci U S A 2005;102(24):8567-8572.

in myeloma. J Clin Oncol 2011 29:1916–1923.

Genes and Cancer 2010; 779- 786.

inhibitors and further advance the treatment of MM.

role of this emerging pathway in myelomagenesis.

**6. Conclusions**

**7. References**

425-479.

2000; 10:524-30.

**7**

Keita Kirito

*Japan*

*University of Yamanashi*

**Therapeutic Approaches for Targeting Hypoxia-**

Hypoxia-inducible factor (HIF) is a transcription factor that is a master regulator of cellular responses to hypoxia and regulates many genes that are required for adaptation to hypoxia. 1HIF is composed of two subunits, HIFα and HIFβ. HIFα is composed of three family members, HIF-1α, HIF-2α and HIF-3α. 2Among these family members, HIF-1α and HIF-2α play crucial roles in hypoxic responses. HIF activity is regulated via two mechanisms in response to hypoxia. First, HIFα expression is dependent on oxygen levels. 1 Under normoxic conditions, HIFα is hydroxylated at specific proline residues via prolylhydroxylase domain proteins (PHDs). The enzymatic activities of PHDs are dependent on oxygen levels. Hydroxylation of the proline residues of HIFs increases the interaction of HIFα with the von Hippel-Lindau (VHL) proteins, which recruit an E3 ubiquitin-protein ligase. In turn, HIFα is ubiquitinated and subsequently degraded by the proteasome. In hypoxic conditions, PHD activity is suppressed and HIFα degradation is reduced. In addition to protein levels, oxygen levels control the transcriptional activities of HIFs. 2The factors that inhibit HIF (FIHs) play central roles in the process. At normoxic conditions, FIHs catalyze hydroxylation of asparagine residues of HIFα, which represses the interaction of HIF with the transcriptional coactivator CBP/P300. Using these mechanisms, HIF is

Under physiological conditions, HIFs are active during hypoxia. However, HIFs can be activated in many types of cancer cells, even under normoxic conditions, by oncogene products, impaired activities of tumor suppressor genes,3 or the accumulation of metabolic glucose intermediates. 4 HIFs are known to be activated in multiple myeloma cells. The hypoxic microenvironment of multiple myeloma bone marrow and oxygen-independent mechanisms contribute to abnormal activation of HIFs in myeloma cells. Asosingh and colleagues reported HIF-1α expression in 5T2 MM cells in the mouse myeloma model. 5These researchers showed that the hypoxic microenvironment of the bone marrow contributed to HIF-1 activation, especially in the initial stages of the disease. 5 Hypoxiainduced HIF-1α expression in myeloma cells in multiple myeloma bone marrow was

activated during hypoxia under normal physiological conditions.

**2. Abnormal activation of HIFs in multiple myeloma**

**1. Introduction**

**1.1 Regulation of HIF function**

**Inducible Factor in Multiple Myeloma**

[30] Catley L, Weisberg E, Kiziltepe T, et al.(2006) Aggresome induction by proteasome inhibitor bortezomib and alpha-tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH589 are synergistic in myeloma cells. Blood 2006, 108:3441– 3449.


**2. The role of skull pulse expanding in mechanism of circulatory supply of brain function**

The system of cerebral metabolic supply is dependent on the interaction of a number of elements some of them are determined by the skull properties. One of the most significant in this direction is clarifying the role of CSF movement to this system inside cranium and significance at this process of the bio-mechanical properties of the skull. Critical position in the role of the skull in support of circulatory-metabolic supply of brain functioning determined by the ability of the skull to accept an additional volume of blood during the phase of systolic increase in central arterial pressure.

Indeed, arterial blood pressure consists of two components. One is the steady state of flow through the brain, determined by the basal tone of the brain blood vessels. The second occurs with each heart- beat, which initiated by arterial pressure increases and so drives the pulse volume into the cranium. It is this component that is influenced by the level of cranial compliance, which in turn depends on the volume flexibility of the skull.

This increase in arterial pressure is short – about 0.1s. This means that the cranium needs to accommodate the increase in systolic blood volume very quickly in order to use it to drive cerebral circulation. This possibility, predicted by mathematical simulation of the cerebrovascular system [12], should be follow from biomechanical properties of the skull as united bio-mechanical system.

### 66 Injury and Skeletal Biomechanics

That is important statements, because from the beginning of XIX Century up to the second part of XX Century the most of investigators belong to conception, named "Monroe-Kelly" doctrine, which on the base of majority of anatomical investigations, declare, that skull is fully rigid cavity. However, when this statement has been included to the model, it didn't function. However, when some possibility for pulse change of intracranial volume has been included to the model, it starts to work. It was the first indicator, which shows the importance of pulse change of internal skull cavity. Some years later the presence of pulse dependence on "Pressure-Volume" relation for the skull have been shown by invasive technique [13].

The Role of Skull Mechanics in Mechanism of Cerebral Circulation 67

**3-5ml of stroke volume**

*and CSF - mobility*

*Depends on expanding of skull*

**6-7ml of stroke volume**

**(Pulsatile blood flow)**

*Depends on Vascular Resistance*

**(Steady state blood flow)**

**Figure 1.** Distribution of steady state and fluctuating of stroke volume at normal physiological

CSF pressure, which is a real source of forces for brain expanding.

This means, that through an average sized human brain (1200g) – about 600ml flows every minute and 110-130 ml of blood is about 20% of total brain blood supply. In case of skull is completely rigid, this additional stroke volume will by out from cerebral circulation, because additional blood couldn't inflow to the skull due to contra-pressure, determined by unchangeable volume of cranial cavity. Therefore, brain blood flow may be diminished up to 15-20%. Direct evidence of the presence of pulse skull expanding have been received by coupling of Rheoencephalogram (REG) – method, based on recording of electrical impedance between electrodes, placed in fronto-mastoid position to human head and transcranial dopplerogram (TCD) of basement of the Middle Cerebral artery [17). REG reflect changes of intracranial blood/CSF volume, because electrical resistance of these media are significantly less to compare with brain tissue and pulse blood volume fluctuations inside skull if they are have taken place, will change common electrical resistance between electrodes [14]. TCD reflect pulse changes of linear velocity inside intracranial large arteries [18], which by Poiseuille low are proportional to volume fluctuations of these arteries, surrounded by CSF. Volume fluctuations of large brain arteries could be compensated by skull expanding due to transmitting of arterial to surrounding

Computed aid analysis of simultaneously recorded REG and TCD pulse give the possibility to establish "Pressure-Volume" dependence for the skull for systolic phase of pulse cycle [17], which show, that this dependence is nearly to linear and in normalized coordinates equal to line with angle to horizontal coordinate 30-40o(Fig.2a) Linearity at the most cases of this dependence indicate, that pulse changes of intracranial pressure and volume are linear, which is permits to conclude, that SCF replacements inside skull during systolic part of cardiac cycle practically absent. Therefore, the fact of the presence of systolic skull pulse expansion has taken place and it is one of element of mechanism, which is responsible for circulatory-metabolic support of brain functioning and is possible quantitative to express as

conditions.

**100**

**cm/s**

**(∆P)**

**TCDG**

**80**

**60**

**40**

**20**

**0**

Follow this data, the skull could be accepted as a nearly rigid container with limited capabilities to accommodate internal volume changes in response to increases in arterial pressure. However, because investigations of Marmarou have been provided when comparative slow changes of intracranial pressure as respond to infusion to the skull artificial CSF, when volume compensation is provided also by CSF outflow to some spaces, connected with the skull by comparatively narrow gapes spinal cord, volumes, determined by arachnoid membranes of cranial nerves.

Therefore, it was important to evaluate skull itself volume reserves, in other words dynamic skull volume capabilities. Really, it is important to have the possibility to accept by the skull an extra volume of blood following the systolic increase in arterial pressure, which is in addition to the steady state level of the brain blood flow. The most rapid component of change of arterial blood pressure is evoked in systolic phase duration about 0.1s. During this time any replacements of CSF inside skull couldn't be provided [14].

There are skull expansions due to, perhaps, articular comparative mobility of skull bone in sutures of invisible value; investigations have shown, that these changes of articular bone position are less then 0.5 geometric degree [15], and this, as it follows from calculation, could increase internal volume of skull up to 3-6 ml.

Calculation of additional – systolic blood volume, which should accept cranial cavity may be provided, taking into account, than brain "portion" of stroke volume is about 10 ml. That is means, that steady stroke volume is 5 -7 ml and pulse component is 5-3 ml (Fig.1). Namely this the last volume of blood should accepted by skull during systolic phase of cardiac cycle. Comparatively to total volume of cranial cavity this volume is very small. Averaged size human brain is about 1200 ml. This value should be closely to internal volume of the skull. This means, that expand of internal volume of skull about 0.3% of initial volume to accept systolic portion of blood, could give the additional blood, which is necessary for normal functioning of circulatory –metabolic brain supply. Taking into account, that heart rate is usually 60-70 beats per minute, this additional blood volume will be about pulse evaluation due to of skull expanding could be about 110-130 ml per minute. It is known, that normal brain blood made using some relatively simple calculations. As well it was established in the middle of the last Century, brain takes 50-65ml of blood volume flows through 100g of brain mass in 1 minute [16].

66 Injury and Skeletal Biomechanics

technique [13].

by arachnoid membranes of cranial nerves.

That is important statements, because from the beginning of XIX Century up to the second part of XX Century the most of investigators belong to conception, named "Monroe-Kelly" doctrine, which on the base of majority of anatomical investigations, declare, that skull is fully rigid cavity. However, when this statement has been included to the model, it didn't function. However, when some possibility for pulse change of intracranial volume has been included to the model, it starts to work. It was the first indicator, which shows the importance of pulse change of internal skull cavity. Some years later the presence of pulse dependence on "Pressure-Volume" relation for the skull have been shown by invasive

Follow this data, the skull could be accepted as a nearly rigid container with limited capabilities to accommodate internal volume changes in response to increases in arterial pressure. However, because investigations of Marmarou have been provided when comparative slow changes of intracranial pressure as respond to infusion to the skull artificial CSF, when volume compensation is provided also by CSF outflow to some spaces, connected with the skull by comparatively narrow gapes spinal cord, volumes, determined

Therefore, it was important to evaluate skull itself volume reserves, in other words dynamic skull volume capabilities. Really, it is important to have the possibility to accept by the skull an extra volume of blood following the systolic increase in arterial pressure, which is in addition to the steady state level of the brain blood flow. The most rapid component of change of arterial blood pressure is evoked in systolic phase duration about 0.1s. During this

There are skull expansions due to, perhaps, articular comparative mobility of skull bone in sutures of invisible value; investigations have shown, that these changes of articular bone position are less then 0.5 geometric degree [15], and this, as it follows from calculation,

Calculation of additional – systolic blood volume, which should accept cranial cavity may be provided, taking into account, than brain "portion" of stroke volume is about 10 ml. That is means, that steady stroke volume is 5 -7 ml and pulse component is 5-3 ml (Fig.1). Namely this the last volume of blood should accepted by skull during systolic phase of cardiac cycle. Comparatively to total volume of cranial cavity this volume is very small. Averaged size human brain is about 1200 ml. This value should be closely to internal volume of the skull. This means, that expand of internal volume of skull about 0.3% of initial volume to accept systolic portion of blood, could give the additional blood, which is necessary for normal functioning of circulatory –metabolic brain supply. Taking into account, that heart rate is usually 60-70 beats per minute, this additional blood volume will be about pulse evaluation due to of skull expanding could be about 110-130 ml per minute. It is known, that normal brain blood made using some relatively simple calculations. As well it was established in the middle of the last Century, brain takes 50-65ml of blood volume flows through 100g of

time any replacements of CSF inside skull couldn't be provided [14].

could increase internal volume of skull up to 3-6 ml.

brain mass in 1 minute [16].

**Figure 1.** Distribution of steady state and fluctuating of stroke volume at normal physiological conditions.

This means, that through an average sized human brain (1200g) – about 600ml flows every minute and 110-130 ml of blood is about 20% of total brain blood supply. In case of skull is completely rigid, this additional stroke volume will by out from cerebral circulation, because additional blood couldn't inflow to the skull due to contra-pressure, determined by unchangeable volume of cranial cavity. Therefore, brain blood flow may be diminished up to 15-20%. Direct evidence of the presence of pulse skull expanding have been received by coupling of Rheoencephalogram (REG) – method, based on recording of electrical impedance between electrodes, placed in fronto-mastoid position to human head and transcranial dopplerogram (TCD) of basement of the Middle Cerebral artery [17). REG reflect changes of intracranial blood/CSF volume, because electrical resistance of these media are significantly less to compare with brain tissue and pulse blood volume fluctuations inside skull if they are have taken place, will change common electrical resistance between electrodes [14]. TCD reflect pulse changes of linear velocity inside intracranial large arteries [18], which by Poiseuille low are proportional to volume fluctuations of these arteries, surrounded by CSF. Volume fluctuations of large brain arteries could be compensated by skull expanding due to transmitting of arterial to surrounding CSF pressure, which is a real source of forces for brain expanding.

Computed aid analysis of simultaneously recorded REG and TCD pulse give the possibility to establish "Pressure-Volume" dependence for the skull for systolic phase of pulse cycle [17], which show, that this dependence is nearly to linear and in normalized coordinates equal to line with angle to horizontal coordinate 30-40o(Fig.2a) Linearity at the most cases of this dependence indicate, that pulse changes of intracranial pressure and volume are linear, which is permits to conclude, that SCF replacements inside skull during systolic part of cardiac cycle practically absent. Therefore, the fact of the presence of systolic skull pulse expansion has taken place and it is one of element of mechanism, which is responsible for circulatory-metabolic support of brain functioning and is possible quantitative to express as value of Tang. of angle of "Pressure-Volume" dependence in normalized coordinates. This dependence corresponds also to meaning "Cranial Compliance", applied to systolic phase of cardiac cycle, or in other words, "Dynamic Cranial Compliance" (DCC). Direct evidence of the role of small increasing of intracranial volume to DCC have been received by observation on neurosurgical patients just before and after trepanation, provided for the next neurosurgery [19]. These investigation have shown, that small 6-10cm2 "window" in skull bone with saved brain cover membrane, when real intracranial volume increase to a few (3-6 ml) due to deformation of brain cover membrane, could change significantly DCC (Fig.3). The significance of this mechanism was confirmed during many years experience in skull trepanation, which is used for increase of intracranial volume up to the present time.

The Role of Skull Mechanics in Mechanism of Cerebral Circulation 69

brain mass, which have been shown by MRI investigations [22]. Investigations, provided with aging persons, after 65-70 by comparing results of blood flow measurements, and DCC measurements and determination level of dementia level by psychophysiological computerized method "Prognosis", show (Fig. 4), that level of decrease of cognitive brain function and DCC are proportional but not closely correlate with values of level of brain blood that even small [17]. It is possible to conclude, that biomechanical properties of the skull determine its expanding due to pulse increase of intracranial pressure permits to accept additional volume of blood during systolic increase of central arterial pressure, with play sometimes significant role in supporting circulatory-metabolic supply of brain

**Figure 4.** Dependence between value of DCC , Cerebral blood flow and CSF – mobility in aging human

During diastolic part of cardiac cycle biomechanical properties of the skull also play role in supporting of cerebral blood circulation due to energy, collected during systolic increase of central arterial pressure. In this phase the role of CSF replacements is increase. In the beginning, they, in coupling with skull biomechanics, provide the distribution of pulse blood volume inside skull and, then, support pulse outflow of venous blood from the skull. Thus, evaluation of DCC for the skull during each cardiac cycle is comprised of the initial interval as a rapid and nearly linear increase of arterial pulse pressure which lasts from 0.05 - 0.15s and perfectly reflects "Pressure-Volume." dependence, or DCC, which is determined by the equivalent elasticity of the cranium due to the biomechanical of the skull structure

groups which are different by level of cognitive disfunction.

functioning.

elements

**Figure 2.** Averaged changes of DCC for different age groups, showing the decline in CCe in the middleage group.

**Figure 3.** Averaged DCC values before and after trepanation. Changes in the biomechanical elasticity of the skull allow the cranium to accept additional blood with each pulse stroke.

All above data permits to conclude, that without property of rapid expanding of the skull when DCC =0, brain blood flow will decrease with the same indices of arterial and venous central pressure to significant value – up to 15-20%., which is significant for brain functioning. The known data shows, that even some less significant decrease of DCC may in some cases reflect to brain activity. This suggestion is confirmed by observations, which shows, that DCC gradually decrease up to age 40-50 and, then increase again-Fig. 2b,c [20]. That abnormal decrease of angle of normalized curve widely vary for different persons and its pronounced values are correlate with some neurological symptoms (headache, decreased working capabilities). It is important to mention, that an increase of DCC after 55-60 Years (Fig.2c) is not connected with the skull properties. It is determining by aging decrease of brain mass, which have been shown by MRI investigations [22]. Investigations, provided with aging persons, after 65-70 by comparing results of blood flow measurements, and DCC measurements and determination level of dementia level by psychophysiological computerized method "Prognosis", show (Fig. 4), that level of decrease of cognitive brain function and DCC are proportional but not closely correlate with values of level of brain blood that even small [17]. It is possible to conclude, that biomechanical properties of the skull determine its expanding due to pulse increase of intracranial pressure permits to accept additional volume of blood during systolic increase of central arterial pressure, with play sometimes significant role in supporting circulatory-metabolic supply of brain functioning.

68 Injury and Skeletal Biomechanics

∆**V 1.0**

**0.5**

**0**

age group.

value of Tang. of angle of "Pressure-Volume" dependence in normalized coordinates. This dependence corresponds also to meaning "Cranial Compliance", applied to systolic phase of cardiac cycle, or in other words, "Dynamic Cranial Compliance" (DCC). Direct evidence of the role of small increasing of intracranial volume to DCC have been received by observation on neurosurgical patients just before and after trepanation, provided for the next neurosurgery [19]. These investigation have shown, that small 6-10cm2 "window" in skull bone with saved brain cover membrane, when real intracranial volume increase to a few (3-6 ml) due to deformation of brain cover membrane, could change significantly DCC (Fig.3). The significance of this mechanism was confirmed during many years experience in skull trepanation, which is used for increase of intracranial volume up to the present time.

**Figure 2.** Averaged changes of DCC for different age groups, showing the decline in CCe in the middle-

**0.5**

**0.5 1.0 0.5 1.0**

**CCe=1,26±023 CCe=0,69±0,16 CCe=0,78±0,18**

**1.0**

**0 0**

∆**V** ∆**V**

**0.5**

∆**P** ∆**P**

**1.0**

**Age: 18-25 40-50 65-75**

**0.5**

**1.0**

∆**P**

**Figure 3.** Averaged DCC values before and after trepanation. Changes in the biomechanical elasticity of

All above data permits to conclude, that without property of rapid expanding of the skull when DCC =0, brain blood flow will decrease with the same indices of arterial and venous central pressure to significant value – up to 15-20%., which is significant for brain functioning. The known data shows, that even some less significant decrease of DCC may in some cases reflect to brain activity. This suggestion is confirmed by observations, which shows, that DCC gradually decrease up to age 40-50 and, then increase again-Fig. 2b,c [20]. That abnormal decrease of angle of normalized curve widely vary for different persons and its pronounced values are correlate with some neurological symptoms (headache, decreased working capabilities). It is important to mention, that an increase of DCC after 55-60 Years (Fig.2c) is not connected with the skull properties. It is determining by aging decrease of

the skull allow the cranium to accept additional blood with each pulse stroke.

**Figure 4.** Dependence between value of DCC , Cerebral blood flow and CSF – mobility in aging human groups which are different by level of cognitive disfunction.

During diastolic part of cardiac cycle biomechanical properties of the skull also play role in supporting of cerebral blood circulation due to energy, collected during systolic increase of central arterial pressure. In this phase the role of CSF replacements is increase. In the beginning, they, in coupling with skull biomechanics, provide the distribution of pulse blood volume inside skull and, then, support pulse outflow of venous blood from the skull.

Thus, evaluation of DCC for the skull during each cardiac cycle is comprised of the initial interval as a rapid and nearly linear increase of arterial pulse pressure which lasts from 0.05 - 0.15s and perfectly reflects "Pressure-Volume." dependence, or DCC, which is determined by the equivalent elasticity of the cranium due to the biomechanical of the skull structure elements

It is important to emphasize that changes of the steady component of brain blood flow, determined by the perfusion pressure, are independent of the pulsatile component. The total brain blood supply is determined by the superposition of the steady state perfusion pressure (average level of arterial pressure) and the components of blood flow, which are in turn determined by the biomechanical properties of the skull and the mobility of cerebrospinal fluid within the skull.

The Role of Skull Mechanics in Mechanism of Cerebral Circulation 71

on the base of modern technology, have been appeared at the end of XX Century. Firstly, direct observations demonstrated, that skull represents a complicated mechanical moveable system. This suggestion is based on investigations, represented device with needles inserted through cover skull tissues in human head and fixed in skull bone. Invisible movements of upper end of the needle were registered by means of small mirror fixed there, which are

**Figure 5.** Principle of transforming individual regions of image series into the amplitude-time plot: (a) input of the image series into a computer and specification of the analyzed region; (b) collocation of images and creation of the intermediate image; and (c) transformation of the intermediate image into

deflected laser beam, focused to the mirror [28].

the amplitude-time plot.


**About the Editor**

#### **Michael S. Zhdanov**

Michael S. Zhdanov holds degrees in geophysics, physics, and mathematics from the Moscow State University. He is Distinguished Professor at The University of Utah, Director of the Consortium for Electromagnetic Modeling and Inversion (CEMI) sponsored by major international energy resources, petroleum and mining corporations and research organizations. His contributions are reflected in more than 300 articles, 16 books published in English, Russian, and Chinese, and dozens patents. He is an Honorary Gauss Professor of the Gottingen Academy of Sciences, Full ¨ Member of the Russian Academy of Natural Sciences, Fellow of the Electromagnetics Academy USA, and Honorary Professor of the China National Centre for Geological Exploration Technology. Professor Zhdanov received one of the highest awards of the international Society of Exploration Geophysicists, Honorary Membership Award, in recognition of his distinguished contributions to exploration geophysics.


**3. Results and Discussion**

Treatments were designed to investigate tar reduction (removal) efficiencies of wood shavings filter (dry filter) with and without syngas cooling and bubbling systems. Accordingly, the tar contents were measured at the inlet and outlet of the cleaning systems for the three treatments: (i) wood shavings filter; (ii) wood shavings filter after a heat exchanger; and (iii) wood shavings filter after an oil bubbler. Pressure drop, an important indicator in determining the tar absorbed by filter medium, was measured across the wood shavings filter over time. Similarly, because the temperature of syngas plays a vital role in condensation and deposition of tar, temperatures were recorded at the inlet and outlet of the cleaning systems. The gasifier was purposefully operated at low temperatures, which resulted in high syngas tar content to test the capabilities of the cleaning systems. The removal of tar can involve condensation or absorption process. In this experiment, the removal of tar by wood shavings involves condensation. Whereas, the removal of tar by oil is an absorption-based process because the solvent (oil) dissolves the tar products.


**5.2 Multi stakeholder model**

The primary responsibility of Zimplats is to meet the shareholders' demands and at the same time, the company takes into cognisance the interests of its stakeholders which can include the following:


**3. Proposed method**

*Artificial Intelligence - Latest Advances, New Paradigms and Novel Applications*

each map but keeps the important information.

fully connected layer like neural network.

ƒ(x) = max(0,x).

**Step-2: Classification**

**2.2 Association rule**

b.Activation layer: this layer is used to increase non-linearity of the network without affecting receptive fields of convolution layer. The output is

c.Pooling layer: this layer reduces the number of parameters when the images are too large. This operation means that the layer reduces the dimensionality of

This process executes image classification. There are two main layers as follows:

a.Fully connected layer (FC layer): to connect every neuron in one layer to every neuron in another layer. We flattened our matrix into vector and feed it into a

b.Softmax layer: A special kind of activation layer, usually at the end of FC layer

At this stage, the pretrained models such as *AlexNet, VGGNet, GoogLeNet*, and *ResNet* will be applied in order to integrate the CNNs. The procedure is to merge the multiple CNNs with fixed number of CNNs and trained them after merging.

Association rule is utilized in data mining phase as it is a machine learning method which finds the relationship between the items based on the frequency of item sets [18, 19]. To do this, a mathematical algorithm is needed to arrange the

There are three measures in association rule task as shown in **Figure 2**:

c.Lift: It indicates how the *antecedent* and the *consequent* are related to one another. If the Lift value is 1, it means that the *antecedent* and the *consequent* are independent. If the Lift value is less than 1, it means that the *antecedent* has negative effect on occurrence on the *consequent*. In addition, if the Lift value is more than 1, it means that the *antecedent* and the *consequent* are dependent.

a.Support: It indicates how often the items appear in the data-set.

b.Confidence: It indicates how often a rule is found to be true.

outputs. It produces a discrete probability distribution vector.

frequent item sets, for extracting association rule of food sets.

**76**

**Figure 2.**

*Basic terminologies of association rule measure.*

As the uncertainty of convolutional architecture due to the fix number of CNNs and the interpretability after merging of CNNs [20]. Simpler and more highly scalable method to integrate the multiple networks is necessary and therefore there are three features in consideration - liability score for the system, no further training after integration, and high scalability. Deep neural network (DNN) may be an alternative choice to solve this problem [21]. It provides the neural basis for efficient visual object recognition in humans. Its architecture comprises more than three layers. Each of layers contains a combination of convolution, max-pooling and normalization stages, whereas these layers are fully connected. It inherently fuses the process of feature extraction with classification into learning using Fuzzy Support Vector Machine (FSVM) and enables the decision making [22, 23]. Many efforts were made to speed up both the training time as well as inference time of DNNs. Since the flexibility and the performance scalability to deal with various types of networks are crucial requirement of the DNN accelerator design [24], we therefore proposed a scalable architecture for integrating different deep neural networks with a reliability score to increase the probability to return correct class as the result of food recognition. The reliability score allowed the integrated DNN to select a suitable recognition result obtained from the different DNNs that were independently constructed. In this study, we evaluated the feasibility of our proposed method.

The frequent set of foods extracted from food images was applied to a data mining algorithm such as the *Apriori* algorithm for the food recommendation process.

**Figure 3** shows the schematic diagram of food recommender. This system would recommend a set of foods suitable for users based on association rule, which were extracted by food set mining from food images.

The process of food recognition and extraction of association rule is given in **Figure 4**. Firstly, the food set images from database were collected. Secondly, the reliability score was calculated by Eq. (1). Then the food recognition and

**Figure 3.** *Schematic diagram of food recommender.*

**Figure 4.**

*Process of recognition of food set images and extraction of association rule.*

segmentation from the images were operated using DNNs. Finally, the extraction of association rule was obtained as the frequent food sets.

$$\text{Reliability score} = \mathbf{A} \times \mathbf{C} \times \mathbf{I} \times \mathbf{D} \tag{1}$$

where A, C, I, and D represented the accuracy of the learning process, number of classes, number of leaning images, and difference between learning data and test data, respectively.

We combined several DNNs and calculated new probability with reliability scores as shown in **Figure 5**. Each DNN has different reliability score depending on following factors:

**79**

necessary.

**Figure 6.**

predicted result was provided.

*Workflow of proposed food recommender.*

the phase of data mining.

DNNs in this experiment.

False Negative respectively.

**4. Experimental and evaluation**

*A Food Recommender Based on Frequent Sets of Food Mining Using Image Recognition*

If the learning data and the test data are similar, the accuracy will be high but it may be not reliable. As we test with the unseen data, the DNN may predict wrong answer. Therefore, the difference between the learning data and the test data is

We calculated the reliability score and the weight output (W) of fully connected layer as the new probability using Eq. (2). The label of the new probability as the

Accordingly, the food set could be recognized from food images using different DNNs with the reliability scores as shown in **Figure 6**. Based on the recognition results, transactions of food were formed and applied to conventional association in

We implemented our prototype of merging the results from different DNNs using MATLAB. The performance of recognition accuracy and the relationship between the recognition accuracy and the number of classes were evaluated. We used the dataset of food images for creating the DNNs and evaluated them as shown in **Table 1**. Three DNNs with different number of recognition classes for each dataset were created as given in **Table 2**. In addition, we made each DNN from *Scratch* by applying the transfer learning using *GoogLeNet*. Accordingly, we totally had 18

Recognition accuracy was calculated using the following equation:

New probability W Reliability score = ´ (2)

Accuracy TP TN / TP TN FP FN = + + ++ ( ) ( ) (3)

where TP, TN, FP, and FN were True Positive, True Negative, False Positive, and

*DOI: http://dx.doi.org/10.5772/intechopen.97186*

```
Figure 5.
The feature of proposed method.
```
*A Food Recommender Based on Frequent Sets of Food Mining Using Image Recognition DOI: http://dx.doi.org/10.5772/intechopen.97186*

#### **Figure 6.** *Workflow of proposed food recommender.*

*Artificial Intelligence - Latest Advances, New Paradigms and Novel Applications*

segmentation from the images were operated using DNNs. Finally, the extraction of

where A, C, I, and D represented the accuracy of the learning process, number of classes, number of leaning images, and difference between learning data and test

We combined several DNNs and calculated new probability with reliability scores as shown in **Figure 5**. Each DNN has different reliability score depending on

Reliability score A C I D = ´ ´´ (1)

association rule was obtained as the frequent food sets.

*Process of recognition of food set images and extraction of association rule.*

data, respectively.

**Figure 4.**

following factors:

• number of classes

• accuracy of the learning process

• difference between learning data and test data

• number of learning images

**78**

**Figure 5.**

*The feature of proposed method.*

If the learning data and the test data are similar, the accuracy will be high but it may be not reliable. As we test with the unseen data, the DNN may predict wrong answer. Therefore, the difference between the learning data and the test data is necessary.

We calculated the reliability score and the weight output (W) of fully connected layer as the new probability using Eq. (2). The label of the new probability as the predicted result was provided.

$$\text{New probability} = \mathbf{W} \times \text{Reliability score} \tag{2}$$

Accordingly, the food set could be recognized from food images using different DNNs with the reliability scores as shown in **Figure 6**. Based on the recognition results, transactions of food were formed and applied to conventional association in the phase of data mining.


**4.4. Raman-active modes calculation in C60 peapods**

low- and high-frequency component associated to the vibrations along the circumferential direction (G−) and the G direction of the nanotube axis (G+), respectively. In both metallic and semiconducting nanotubes, the former component is found to be dependent on the diameter of a nanotube, while the latter does not exhibit this dependence [40]. In the Raman spectra of isolated semiconducting and metallic SWCNT, the D-band and G'-band features are both

Raman spectroscopy was employed to further characterize the peapod samples and to obtain structural information on the inserted fullerene inside tubes. Many groups have performed the Raman experiments of filling SWCNTs with C60 or C70 molecules [12, 42–46] since the first synthesized by Smith et al. [4]. **Figure 5** presents two examples of peapod experimental

Usually, experimental Raman spectra have been obtained on an ensemble of peapods organized into bundles. Nevertheless, the analysis of these experimental results for bundles of C60 peapods [8, 44] leads to the well-established conclusion: the Peapod Radial Breathing-Like Mode (PRBLM) of the peapod having a tube diameter close to 1.37 nm was split into two components, downshifted and upshifted, respectively, with respect to the position of the RBM in empty SWCNT. The upshift was possibly assigned to the stress feeling by the tubes from the inside C60. For diameters between 1.45 and 1.76 nm, a single PRBLM measured

**Figure 5.** RBM Raman spectra taken for heat-treated C70 (top) and C60 (bottom) peapods (from reference [46]).

observed.

**4.3. Raman experiments on C60 and C70 peapods**

80 Fullerenes and Relative Materials - Properties and Applications

Raman spectra, one for C60@SWCNT and the other for C70@SWCNT.

The Raman cross section was calculated assuming that scattering can be described within the framework of the bond polarizability model [52]. Recently, we calculated the Raman spectra of infinite homogeneous bundles (crystal) of C60 peapods [49]. In order to reach a 100% factor filling, 20 C60 molecules are located in a length of 19.8 nm [17.7 nm] of a (10,10) [(13,13)] nanotube and the number of carbon atoms of the tube (10,10) [(13,13)] is close of 3240 [3744] atoms. For the C60@(10,10) (C60@(13,13)) peapod, a 100% factor filling corresponds to a concentration of 37% (32%) (concentration is the ratio between carbons in C60 and carbons in the tube. In **Figure 6b** are displayed the calculated polarized ZZ Raman spectra of individual C60 peapods and crystal of C60 peapods, respectively, for linear and zigzag configuration of the C60 molecules inside the tube: (bottom) linear chain of C60 confined into a (10,10) SWCNT (tube diameter close to 1.36 nm), (top) zigzag chain of C60 confined into a (13,13) SWCNT (tube diameter close to 1.76 nm).

for the individual C60@(13,13) peapod at 133 and 125 cm−1, respectively, shifts to 140 and 127 cm−1 in the infinite bundle of C60@(13,13) peapod. A new peapod bundle breathing-like mode (PBBLM) is also calculated in the Raman spectra of an infinite bundle of C60 peapods, which arises from the BBLM in SWCNT bundle [50]. Concerning the main Raman-active modes of C60 (e.g., modes located around 270 cm−1 (Hg) and 495 cm−1 (Ag)), independently of the configuration of C60 molecules inside the tube, the van der Waals interactions between

Structural and Vibrational Properties of C60 and C70 Fullerenes Encapsulating Carbon Nanotubes

http://dx.doi.org/10.5772/intechopen.71246

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**Figure 7** shows the PRBLM and PBBLM frequency dependencies as a function of the tube diameter. These dependencies are first compared with those of the RBLM and BBLM in unfilled bundles of tubes. The behavior of the PRBLM in linear peapods is qualitatively the same as those of RBLM in SWCNT bundle. However, the PRBLM frequency versus diameter relation deviates from the scaling law stated for the RBLM frequency in SWCNT bundle, especially for small diameters. For zigzag peapod, the PRBM(1) dependence as a function of the diameter is close to that of the RBLM in bundle of SWCNT. The PRBLM(1) downshifts when the tube diameter increases. Concerning the PBBLM frequency, it decreases with increasing the tube diameter and close to the frequency of BBLM, except at small diameters where a more significant increase of the PBBLM frequency is calculated. Our calculations based on DFT have clarified that the shift in the RBM frequencies of nanotubes containing fullerenes strongly depends on the diameters of the nanotubes. DFT calculations have also clarified that the shift in the RBM frequencies of nanotubes containing fullerenes strongly depends on the

More recently, Raman calculations are extended to a larger range of diameters in which C60

**Figure 7.** Diameter dependence of the frequencies of PRBLM (for d < Dc = 1.45 nm) and PRBLM(1) (for d > Dc) (stars), and PRBLM(2) (cross). Diameter dependence of the frequency of RBLM (open circles) and BBLM (open diamonds) in bundles of SWCNTs. The critical diameter Dc between linear (tube diameters lower than Dc) and zigzag (tube diameters greater than Dc) peapods is identified by the vertical-dashed line. (b) Schematic representation of homogeneous filling

mode of the different tubes within a bundle. The filling factor is related to the ratio *h* ⁄ *H* (see text).

molecules can adopt a double helix and a layer of two molecules [22].

peapods have no significant effect on these modes.

diameters of the nanotubes [51, 53–57].

*4.4.1. Diameter dependence of the Raman spectrum of C60 peapods*

In the intermediate and high-frequency regions, the calculated Raman spectra of individual peapods organized into infinite bundles do not show significant differences. It can be emphasized that a splitting of the Ag(1) and Ag(2) totally symmetric C60 modes was observed experimentally [45]. This splitting was considered as signature of the mobility of some encapsulated C60 molecules inside nanotubes [45]. In our calculations, the C60 molecules are kept at fixed positions in the considered peapods, and no splitting of the totally symmetric C60 modes is found.

Next, we show in **Figure 6b** the low-frequency range of Raman spectra of C60@(10,10) and C60@(13,13) peapods where the main changes are identified. We found that the PRBLM of individual C60@(10,10) linear peapod on one hand and the PRBLM(1) and PRBLM(2) of individual C60@(13,13) zigzag peapod on the other hand are slightly upshifted in bundles of C60@ (10,10) peapods and C60@(13,13) peapods, respectively. We calculated a shift of the RBLM from 170 cm−1 in the individual C60@(10,10) peapod to 186 cm−1 in the infinite bundle of C60@ (10,10) peapods. In the case of C60@(13,13), the PRBLM(1) and PRBLM(2) doublet calculated

**Figure 6.** The ZZ calculated Raman spectra of individual (curve (a)) and homogeneous bundles (curve (b)) of C60@(13,13) (top) and C60@(10,10) (bottom) (filling factor 100%). The stars give the positions of the RBM and RBLM in individual SWCNT and crystal of SWCNT, respectively. Cross indicates the frequency of Bundle Breathing Like-Mode (BBLM) in crystal of SWCNT. (left) Breathing modes range, (middle) region of the radial C60 modes, (right) range of tangential modes: Ag(2) mode of C60 and G-modes of the nanotubes.

for the individual C60@(13,13) peapod at 133 and 125 cm−1, respectively, shifts to 140 and 127 cm−1 in the infinite bundle of C60@(13,13) peapod. A new peapod bundle breathing-like mode (PBBLM) is also calculated in the Raman spectra of an infinite bundle of C60 peapods, which arises from the BBLM in SWCNT bundle [50]. Concerning the main Raman-active modes of C60 (e.g., modes located around 270 cm−1 (Hg) and 495 cm−1 (Ag)), independently of the configuration of C60 molecules inside the tube, the van der Waals interactions between peapods have no significant effect on these modes.


**1. Introduction**

Urban areas are city areas characterized by a dense human population of mixed age, sex, family and household structure, ethnic, cultural, religious diversity, educational and income levels,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and high built-up area with technological and economic advancement. Rural areas, on the other hand, are open broad areas of land located far from towns and cities, which are composed of extensive bushes between large crop fields and livestock herds and sparse housing and population density. Between the urban and rural settings lies peri-urban zone whose population, livestock, crops and land use features are influenced by the proximate interface. Part of peri-urban area adjacent to urban area has features resembling urban features, while its other side assumes the rural characteristics. There is no distinct line separating the peri-urban from urban and rural settings, but a slow zone of change [1]. The gradual transition from peri-urban to urban setup is moving constantly away from city center toward the rural direction due to persistent urbanization pressure, especially in developing countries.

Urban and peri-urban livestock farming is expanding in developing countries primarily due to high demand for protein of animal origin to feed the rapidly growing urban populations, but also to generate income of livestock keeping households [2]. It is also a diversification tactic to spread livelihood risks in adverse situations [3]. Some urban and peri-urban dwellers continue to keep livestock to maintain their rural cultural values [4]. The expansion of urban and peri-urban livestock farming, which is reflected as an increase in number of both the livestock and households involved in keeping livestock, and rapid urban human population growth has increased the chance of contact between humans, animals and manure. Urban areas of Morogoro in Tanzania, for example, had a cattle population of 2618 in 1996 [5], which almost doubly increased to 4170 in 2006 [6]. By 2008, the cattle population in Morogoro urban was 19,099, and among them, 4425 were dairy cattle [7]. This cattle population hiked up to 49,625 in 2012 [8]. Rapid urban population growth is primarily caused by influx of people from rural areas either as migrants or as commuters [1, 9]. For instance, Tanzania's annual population growth rate between 1988 and 2002 was 3% with the urban population size increasing from 18% in 1988 to 23% in 2002 [10]. Moreover, population size and growth in rural and urban areas of Morogoro region from 2002 to 2012 show that the rural population grew by 23.7% from 1,279,513 in 2002 to 1,582,434 compared to 34.2% growth in urban population from 473,849 in 2002 to 636,058 in 2012 [8]. In this region, the general population density changed from 24 persons per square kilometer in 2002 to 31 persons per square kilometer in 2012 [11]. As a result of increased human and animal density, the chance of contact between humans and livestock has increased. The growth in animal population and concomitant increase in manure production, in shrinking space separating humans, livestock and manure, require appropriate livestock and manure management practices taking into account that livestock harbors zoonotic pathogens [12].

Four decades ago, before the expansion of urban and peri-urban livestock farming, free open communal cattle grazing system required minimal effort to manage manure [3, 13]. Cattle freely grazed during daytime and were confined during the night for security. Most manure was left scattered everywhere except for a small amount which was applied on crop fields [3, 14]. To date, the manure management practices have changed to adapt to densely populated areas where the space separating humans from animals and their wastes has decreased. A question arises: does this change consider prevention of animal and human from pathogen exposure as well as environmental contamination? This chapter describes assessment of manure management practices and risks of contact and transmission of cattle manure-related zoonotic pathogens between cattle, humans and the environment in urban and peri-urban areas of Morogoro region of Tanzania. This report forms a basis for developing strategies to improve urban and peri-urban livestock farming practices in order to safeguard human, animal and ecosystem health in settings similar to study area.


*Article*

### **Ancient Environmental Preference and the Site Selection Pattern Based on the Edge E** ff**ect and Network Structure in An Ecosystem**

### **Jianfeng Zhu 1,2, Lijun Yu 1,\*, Yueping Nie 1, Fang Liu 1,2, Yu Sun 3, Yuanzhi Zhang 2,4 and Wenping Song 5**

Received: 22 November 2019; Accepted: 27 December 2019; Published: 31 December 2019

**Abstract:** Archaeological sites are facing serious threats from environmental changes in the background of urban sprawl. More e fforts are needed to enhance the cognition of human–environment interactions for better conservation. Under the traditional geomantic view, the environmental preference involved was presented to guide ancient life. In this study, we analyzed the edge e ffect and network structure of two periods in an ecological transition zone where the ancient sites were located. From the cases of Gouzhang and Yinxian, the separability of edge intensity indicated the different site selection patterns because of the discrepancy of patch fragmentation and ecological structure. Additionally, the different trends of the edge effect were thought to be related to the complexity of the ecological network. Besides that, the ancient cities located in or around the high-centrality terrain in the network of closed space could have provided the convenience of accessing living materials from early ecosystems. In practice, the comprehensive methods based on geomantic and ecological analysis proved effective when used to explore possible areas of the undiscovered archaeological sites. What is more important is that traditional environmental perceptions could be integrated into a scientific system of the ecological landscape and contribute more to archaeological research and the study of ancient culture.

**Keywords:** site selection; archaeological sites; edge e ffect; ecological network; geomantic environment