*Images from wide-field endoscopy videos after topical application of fluorescence-labeled peptides. The top and bottom panels represent frames from white light and fluorescence, respectively. (A) The hyperplastic epithelium after QPI peptide application, (B) the lumen of a CPC;Apc bred mouse negative for Cre recombinase (control litter mate), (C) single adenoma after control peptide application, (D) single adenoma, and (E) multiple*

*adenomas in a CPC;Apc mouse after QPI peptide application. Used with permission [47].*

**120**

**Figure 3.**

after injection (**Figure 4**). In a pilot study in 15 patients at high risk of colorectal neoplasia, a total of 101 lesions were detected during first inspection with white light (WL), and an additional 22 were detected during second inspection with dual WL/fluorescence (FL). After immunohistochemical analysis, 36 hyperplastic lesions and 8 serrated polyps were identified. Most of these were visible in fluorescent mode (94 and 100%, respectively), and the majority (78 and 87%, respectively) showed increased fluorescence. From the data, GE-137 peptide showed some specificity to hyperplasia and serrated lesions in CRC mouse model and patients [48].

Overexpression of EGFR has been reported in as high as 97% of colonic adenocarcinomas, and it is a validated biomarker for CRC. In one study, the extracellular domain of EGFR (EGFR-ECD) was expressed and purified [49]. A library of M13 bacteriophage was used to select peptide candidates that bind specifically to EGFR-ECD. A peptide, QRHKPRE, that is specific for EGFR was developed and validated. Peptide binding to cells occurred within 2.46 minutes and had an affinity of 50 nM. A NIR fluorescence endoscope was used to perform *in vivo* imaging to validate peptide binding to spontaneous colonic adenomas in a *CPC;Apc* mouse model via topical administration (**Figure 5**). T/B ratios of polyps and flat lesions were 4.0 ± 1.7 and 2.7 ± 0.7, respectively. Subsequently, specific peptide binding to human colonic adenomas was assessed on immunohistochemistry and immunofluorescence. On human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.

In another study, a phage-derived peptide was tested for specific binding to sessile serrated adenomas (SSAs) in proximal colon which accounts for 35% in CRC. Joshi and team used phage display to identify a peptide that binds specifically to SSAs. Many SSA cells have the V600E mutation in BRAF. Therefore, peptide selection was performed with an M13 Ph.D.-7 phage display library using a biopanning strategy with subtractive hybridization with HT29 colorectal cancer cells containing the V600E mutation in BRAF [45]. Binding of fluorescently labeled peptide, KCCFPAQ, to colorectal cancer cells was evaluated with confocal fluorescence

#### **Figure 4.**

*WL and FL images of representative lesions. (a–c) The lesions that are visible in WL show increased fluorescence. (d) A lesion that is visible in WL has enhanced visibility in FL. (e, f) Flat lesions that were only visible in FL. Used with permission [48].*

#### **Figure 5.**

In vivo imaging of colon in CPC;Apc mouse. *(a) WL image of colon in CPC;Apc mouse shows the presence of polyp (arrow). (b) NIR fluorescence image after topical administration of QRH\*-Cy5.5 shows increased intensity from polyp (arrow) and several flat lesions (arrowheads). (c) Image with Cy5.5-control peptide (PEH\*-Cy5.5) shows minimal signal. (d) WL image shows no grossly visible lesions (polyps). (e) NIR fluorescence image with QRH\*-Cy5.5 shows the presence of flat lesions (arrowheads). (f) Image with Cy5.5 control peptide shows minimal signal. Used with permission [49].*

microscopy. The peptide showed an apparent dissociation constant of Kd = 72 nM and an apparent association time constant of K = 0.174/minute. Toxicity was also assessed in rats. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies. Subsequently, endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity.

#### *5.4.2 Confocal laser endomicroscopy*

Confocal laser endomicroscopy (CLE) is an endoscopic modality that based on tissue illumination using a low power laser and the subsequent detection of fluorescent light that is reflected back from the tissue through a pinhole. The term "confocal" refers to the alignment of both illumination and collection systems in the same plane. This alignment dramatically increases the spatial resolution of CLE and enables cellular imaging and evaluation of tissue architecture at the focal plane. CLE can be used to guide biopsies and has been demonstrated in a number of clinical studies to detect cancer in the digestive tract, bladder, cervix, ovary, oral cavity, and lungs. CLE requires the use of a fluorescent contrast agent to enhance visualization of cells. Contrast agents can be administered intravenously or topically. Intravenous fluorescein sodium and acriflavine are widely used contrast agents.

**123**

**Figure 6.**

*200 μm. Used with permission [50].*

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

derived from phage display are summarized below.

CLE can be performed shortly following injection with its fluorescence lasting approximately 30 minutes. However, due to the lack of specificity of conventional contrast agents, there is an increasing use of tissue-specific binding molecular probes in CLE. The studies that used CLE in combination with a targeting peptide

In another study, Qiu and team demonstrated vertical cross-sectional (XZ-plane) images of NIR fluorescence with a handheld dual-axis confocal endomicroscope that revealed a specific binding of a Cy5.5-labeled peptide (LTTHYKLGGGSK-Cy5.5) to premalignant colonic mucosa in mice [50]. This targeting peptide was selected using in vivo phage display technology in a *CPC;Apc* mouse model which developed adenomas spontaneously in the distal colon. NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance as shown in **Figure 6**. The peptide showed specific binding and distinguished premalignant colonic mucosa from

Gastric cancer vessels may have many differentiating characteristics compared to normal vessels. However, identification of gastric cancer vascular endothelial cells is difficult. Co-culture of gastric cancer cells and vascular endothelial cells was suggested to simulate gastric solid tumor mass. In a study, GEBP11, a nine amino

*Vertical cross-sectional image of colonic dysplasia. (a) Chemical structure of LTTHYKL peptide with GGGSK linker and Cy5.5 fluorophore. (b) NIR fluorescence image from CPC; Apc mouse colon ex vivo shows vertically oriented dysplastic crypts. (c) The border between normal colonic mucosa and dysplasia shows increased contrast from specific binding of the LTT\*-Cy5.5 peptide. (d) Corresponding histology (H&E), scale bar*

Hsiung et al. used an M13 phage library to identify peptides that would specifically bind dysplastic colonic mucosa from fresh human colonic biopsies [39]. A peptide (VRPMPLQ ) that bound to colonic dysplasia was identified, synthesized, and conjugated with fluorescein for in vivo testing in a pilot study in patients undergoing routine colonoscopy using a flexible-fibered confocal microscope. Fluorescence images and videos of bound topically administered peptide collected in vivo showed that the selected peptide bound more strongly to dysplastic colonocytes than to the adjacent normal mucosa in the same subject with 81% sensitivity

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

and 82% specificity.

normal mucosa.

#### *Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

*Bacteriophages - Perspectives and Future*

microscopy. The peptide showed an apparent dissociation constant of Kd = 72 nM and an apparent association time constant of K = 0.174/minute. Toxicity was also assessed in rats. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies. Subsequently, endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa

In vivo imaging of colon in CPC;Apc mouse. *(a) WL image of colon in CPC;Apc mouse shows the presence of polyp (arrow). (b) NIR fluorescence image after topical administration of QRH\*-Cy5.5 shows increased intensity from polyp (arrow) and several flat lesions (arrowheads). (c) Image with Cy5.5-control peptide (PEH\*-Cy5.5) shows minimal signal. (d) WL image shows no grossly visible lesions (polyps). (e) NIR fluorescence image with QRH\*-Cy5.5 shows the presence of flat lesions (arrowheads). (f) Image with Cy5.5-*

Confocal laser endomicroscopy (CLE) is an endoscopic modality that based on tissue illumination using a low power laser and the subsequent detection of fluorescent light that is reflected back from the tissue through a pinhole. The term "confocal" refers to the alignment of both illumination and collection systems in the same plane. This alignment dramatically increases the spatial resolution of CLE and enables cellular imaging and evaluation of tissue architecture at the focal plane. CLE can be used to guide biopsies and has been demonstrated in a number of clinical studies to detect cancer in the digestive tract, bladder, cervix, ovary, oral cavity, and lungs. CLE requires the use of a fluorescent contrast agent to enhance visualization of cells. Contrast agents can be administered intravenously or topically. Intravenous fluorescein sodium and acriflavine are widely used contrast agents.

with 89% sensitivity and 92% specificity.

*control peptide shows minimal signal. Used with permission [49].*

*5.4.2 Confocal laser endomicroscopy*

**122**

**Figure 5.**

CLE can be performed shortly following injection with its fluorescence lasting approximately 30 minutes. However, due to the lack of specificity of conventional contrast agents, there is an increasing use of tissue-specific binding molecular probes in CLE. The studies that used CLE in combination with a targeting peptide derived from phage display are summarized below.

Hsiung et al. used an M13 phage library to identify peptides that would specifically bind dysplastic colonic mucosa from fresh human colonic biopsies [39]. A peptide (VRPMPLQ ) that bound to colonic dysplasia was identified, synthesized, and conjugated with fluorescein for in vivo testing in a pilot study in patients undergoing routine colonoscopy using a flexible-fibered confocal microscope. Fluorescence images and videos of bound topically administered peptide collected in vivo showed that the selected peptide bound more strongly to dysplastic colonocytes than to the adjacent normal mucosa in the same subject with 81% sensitivity and 82% specificity.

In another study, Qiu and team demonstrated vertical cross-sectional (XZ-plane) images of NIR fluorescence with a handheld dual-axis confocal endomicroscope that revealed a specific binding of a Cy5.5-labeled peptide (LTTHYKLGGGSK-Cy5.5) to premalignant colonic mucosa in mice [50]. This targeting peptide was selected using in vivo phage display technology in a *CPC;Apc* mouse model which developed adenomas spontaneously in the distal colon. NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance as shown in **Figure 6**. The peptide showed specific binding and distinguished premalignant colonic mucosa from normal mucosa.

Gastric cancer vessels may have many differentiating characteristics compared to normal vessels. However, identification of gastric cancer vascular endothelial cells is difficult. Co-culture of gastric cancer cells and vascular endothelial cells was suggested to simulate gastric solid tumor mass. In a study, GEBP11, a nine amino

#### **Figure 6.**

*Vertical cross-sectional image of colonic dysplasia. (a) Chemical structure of LTTHYKL peptide with GGGSK linker and Cy5.5 fluorophore. (b) NIR fluorescence image from CPC; Apc mouse colon ex vivo shows vertically oriented dysplastic crypts. (c) The border between normal colonic mucosa and dysplasia shows increased contrast from specific binding of the LTT\*-Cy5.5 peptide. (d) Corresponding histology (H&E), scale bar 200 μm. Used with permission [50].*

acid vascular homing peptide, was screened and identified using Ph.D.C7C phage display peptide library kit panning against Co-HUVECS cells [51]. Liu et al. used FITC-GEBP11 to identify gastric cancer in mouse model [52]. A whole-body fluorescent imaging was first used to screen for the strongest specific fluorescent signal in xenograft models after tail vein injection of FITC-GEBP11. A specific signal was observed in both subcutaneous and orthotopic xenograft models in vivo, whereas the group injected with FITC-URP, a control peptide, showed no fluorescent signals. In addition, neoplastic and nonneoplastic gastric mucosae obtained from the patients were incubated with FITC-GEBP11 or FITC-URP for 30 minutes and were scanned with CLE. A specific signal of GEBP11 was observed in 26/28 neoplastic human specimens and in 8/28 samples of nonneoplastic specimens (p < 0.01).

Sturm and team developed a peptide (ASYNYDA) that binds specifically to high-grade esophageal dysplasia and adenocarcinoma using phage display technology [53]. After peptide specific binding validation in human esophageal cancer specimens, they applied peptide topically and performed confocal endomicroscopy in 25 patients. The targeting peptide showed 3.8-fold greater fluorescence intensity for esophageal neoplasia compared with Barrett's esophagus and squamous epithelium with high sensitivity and specificity. The peptide revealed no toxicity in animals or patients.

In a pilot study, Palma and team identified dysplasia lesions in ulcerative colitis (UC) patients using CLE and a fluorescently labeled peptide [54]. A phage-derived peptide (VRPMPLQ ) was synthesized and conjugated with fluorescein. Eleven suspected dysplasia specimens were collected from nine UC patients. Specimens were stained with the peptide and subsequently inspected by CLE. The CLE images were correlated to histological results from specialists. The peptide showed a different pattern on dysplastic mucosa compared to nondysplastic lesions. However, due to several restrictions of this study, further studies on larger UC patients are required for systematic validation.

ErbB2 expression in early breast cancer can predict tumor aggressiveness and clinical outcomes in patients. Up to 30% of all breast cancers express ErbB2, also known as HER2. Immunohistochemistry is commonly used to evaluate ErbB2 expression, but it has limitations due to tumor heterogeneity. Therefore, the use of a specific biomarker for ErbB2 is increasingly popular. One study used a NIR-labeled ErbB2 peptide and a handheld dual-axis CLE to detect in human xenograft breast tumors and human specimens [55]. As a result, they found significantly greater peptide binding to xenograft breast cancer in vivo and to human specimens of invasive ductal carcinoma that express ErbB2 ex vivo. From the data, a miniature dual-axis confocal fluorescence endomicroscope with ErbB2-specific peptide could be implemented to support future image-guided surgery.

#### **5.5 Multimodality imaging**

Radiation-induced pulmonary fibrosis (RIPF) is a serious side effect of radiation therapy, especially in lung and breast cancers. Computed tomography (CT) imaging is currently utilized to identify and monitor RIPF. However, anatomical change interference is a major limitation of CT. Therefore, RIPF detection and observing techniques need to be improved. Collagen accumulation is common in fibrosis. In one study, a collagen-targeting peptide was fabricated to maximize the visualization of fibrosis using fluorescence endomicroscope imaging [37]. The probe showed moderate binding ability to collagen in a fibrosis in vitro binding assay and on lung tissue specimens. The probe showed a similar binding pattern on lung specimens compared to antibody. But its sensitivity was not as good as the collagen-binding antibody.

**125**

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

In another study, Zhang and team evaluated the potential applicability of GEBP11 peptides in gastric cancer diagnosis and radiotherapy. They developed iodine 131-labeled GEBP11 peptides and derivatives [56]. The clinical potential of GEBP11 peptides was determined with multimodality imaging methods. Cerenkov and SPECT imaging showed significantly higher tumor uptake for 131I-2PEG- (GEBP11)3 trimer compared to monomer. Higher tumor accumulation and better T/B ratio of 131I-2PEG-(GEBP11)3 trimer were observed. Treating with 131I-2PEG- (GEBP11)3 trimer exhibited a significant tumor growth suppression compared to control and monomer groups. The tumor volume and vasculature decreased significantly after treatment with 131I-2PEG-(GEBP11)3 trimer, resulting in prolonged survival time. In addition, 131I-2PEG-(GEBP11)3 trimer showed no significant hepatic or renal toxicity. In conclusion, 131I-2PEG-(GEBP11)3 trimer could be a potential ligand used to identify gastric cancer and in antiangiogenic therapy.

The discovery and development of therapeutic drugs and diagnostic probes are a time-consuming, expensive, and complex process. The processes involve experts from a wide range of disciplines such as medicinal chemistry, biochemistry, molecular biology, medicine, and pharmacology. It has been estimated that from about 10,000 new chemical entities identified, only one will reach the market in an average time of 16 years. Phage display, and particularly peptide phage display, has played a major role in the development pipeline for bringing peptide therapeutics into the clinic. Phage-derived peptides play an important role in disease detection and therapy, including in clinical imaging. The potential of peptides in preclinical and clinical molecular imaging is tremendous. Molecular imaging offers invaluable opportunities to explore complex disease-related biological processes at the molecular level in vivo. The emergence of current molecular imaging technologies is dependent not only on the progress of imaging systems but, more importantly, also on molecular imaging probes. Peptide-based imaging has now become an established approach in nuclear imaging, and its application is expanding to other imaging modalities. Considering the emergence of novel library designs and innovative selection strategies, we are confident that phage-derived peptides will continue to be promising biomarkers for early cancer detection, in metabolic abnormalities,

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

**6. Future prospects**

and in personalized medicine.

*Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

In another study, Zhang and team evaluated the potential applicability of GEBP11 peptides in gastric cancer diagnosis and radiotherapy. They developed iodine 131-labeled GEBP11 peptides and derivatives [56]. The clinical potential of GEBP11 peptides was determined with multimodality imaging methods. Cerenkov and SPECT imaging showed significantly higher tumor uptake for 131I-2PEG- (GEBP11)3 trimer compared to monomer. Higher tumor accumulation and better T/B ratio of 131I-2PEG-(GEBP11)3 trimer were observed. Treating with 131I-2PEG- (GEBP11)3 trimer exhibited a significant tumor growth suppression compared to control and monomer groups. The tumor volume and vasculature decreased significantly after treatment with 131I-2PEG-(GEBP11)3 trimer, resulting in prolonged survival time. In addition, 131I-2PEG-(GEBP11)3 trimer showed no significant hepatic or renal toxicity. In conclusion, 131I-2PEG-(GEBP11)3 trimer could be a potential ligand used to identify gastric cancer and in antiangiogenic therapy.

#### **6. Future prospects**

*Bacteriophages - Perspectives and Future*

animals or patients.

for systematic validation.

**5.5 Multimodality imaging**

acid vascular homing peptide, was screened and identified using Ph.D.C7C phage display peptide library kit panning against Co-HUVECS cells [51]. Liu et al. used FITC-GEBP11 to identify gastric cancer in mouse model [52]. A whole-body fluorescent imaging was first used to screen for the strongest specific fluorescent signal in xenograft models after tail vein injection of FITC-GEBP11. A specific signal was observed in both subcutaneous and orthotopic xenograft models in vivo, whereas the group injected with FITC-URP, a control peptide, showed no fluorescent signals. In addition, neoplastic and nonneoplastic gastric mucosae obtained from the patients were incubated with FITC-GEBP11 or FITC-URP for 30 minutes and were scanned with CLE. A specific signal of GEBP11 was observed in 26/28 neoplastic human specimens and in 8/28 samples of nonneoplastic specimens (p < 0.01). Sturm and team developed a peptide (ASYNYDA) that binds specifically to high-grade esophageal dysplasia and adenocarcinoma using phage display technology [53]. After peptide specific binding validation in human esophageal cancer specimens, they applied peptide topically and performed confocal endomicroscopy in 25 patients. The targeting peptide showed 3.8-fold greater fluorescence intensity for esophageal neoplasia compared with Barrett's esophagus and squamous epithelium with high sensitivity and specificity. The peptide revealed no toxicity in

In a pilot study, Palma and team identified dysplasia lesions in ulcerative colitis (UC) patients using CLE and a fluorescently labeled peptide [54]. A phage-derived peptide (VRPMPLQ ) was synthesized and conjugated with fluorescein. Eleven suspected dysplasia specimens were collected from nine UC patients. Specimens were stained with the peptide and subsequently inspected by CLE. The CLE images were correlated to histological results from specialists. The peptide showed a different pattern on dysplastic mucosa compared to nondysplastic lesions. However, due to several restrictions of this study, further studies on larger UC patients are required

ErbB2 expression in early breast cancer can predict tumor aggressiveness and clinical outcomes in patients. Up to 30% of all breast cancers express ErbB2, also known as HER2. Immunohistochemistry is commonly used to evaluate ErbB2 expression, but it has limitations due to tumor heterogeneity. Therefore, the use of a specific biomarker for ErbB2 is increasingly popular. One study used a NIR-labeled ErbB2 peptide and a handheld dual-axis CLE to detect in human xenograft breast tumors and human specimens [55]. As a result, they found significantly greater peptide binding to xenograft breast cancer in vivo and to human specimens of invasive ductal carcinoma that express ErbB2 ex vivo. From the data, a miniature dual-axis confocal fluorescence endomicroscope with ErbB2-specific peptide could

Radiation-induced pulmonary fibrosis (RIPF) is a serious side effect of radiation therapy, especially in lung and breast cancers. Computed tomography (CT) imaging is currently utilized to identify and monitor RIPF. However, anatomical change interference is a major limitation of CT. Therefore, RIPF detection and observing techniques need to be improved. Collagen accumulation is common in fibrosis. In one study, a collagen-targeting peptide was fabricated to maximize the visualization of fibrosis using fluorescence endomicroscope imaging [37]. The probe showed moderate binding ability to collagen in a fibrosis in vitro binding assay and on lung tissue specimens. The probe showed a similar binding pattern on lung specimens compared to antibody. But its sensitivity was not as good as the collagen-binding

be implemented to support future image-guided surgery.

**124**

antibody.

The discovery and development of therapeutic drugs and diagnostic probes are a time-consuming, expensive, and complex process. The processes involve experts from a wide range of disciplines such as medicinal chemistry, biochemistry, molecular biology, medicine, and pharmacology. It has been estimated that from about 10,000 new chemical entities identified, only one will reach the market in an average time of 16 years. Phage display, and particularly peptide phage display, has played a major role in the development pipeline for bringing peptide therapeutics into the clinic. Phage-derived peptides play an important role in disease detection and therapy, including in clinical imaging. The potential of peptides in preclinical and clinical molecular imaging is tremendous. Molecular imaging offers invaluable opportunities to explore complex disease-related biological processes at the molecular level in vivo. The emergence of current molecular imaging technologies is dependent not only on the progress of imaging systems but, more importantly, also on molecular imaging probes. Peptide-based imaging has now become an established approach in nuclear imaging, and its application is expanding to other imaging modalities. Considering the emergence of novel library designs and innovative selection strategies, we are confident that phage-derived peptides will continue to be promising biomarkers for early cancer detection, in metabolic abnormalities, and in personalized medicine.

#### **Author details**

Supang Khondee1 and Wibool Piyawattanametha2,3\*

1 School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand

2 Faculty of Engineering, Department of Biomedical Engineering, King Mongkut's Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand

3 Institute for Quantitative Health Science and Engineering (IQ ), Michigan State University, Michigan, USA

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. 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.

**127**

*Targeting Peptides Derived from Phage Display for Clinical Imaging*

multimeric RGD peptides for targeting tumors. Journal of Medicinal Chemistry.

[11] Comeau AM et al. Exploring the prokaryotic virosphere. Research in Microbiology. 2008;**159**(5):306-313

[12] Ofir G, Sorek R. Contemporary

[13] Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska

B. Bacteriophages and phage-derived proteins, application approaches. Current Medicinal Chemistry.

[14] Murphy FA et al. Virus Taxonomy: Classification and Nomenclature of Viruses. Vol. 10. New York: Springer Science & Business Media; 2012

[15] Russel M, Lowman HB, Clackson T. Introduction to phage biology and phage display. In: Phage Display: A Practical Approach. New York: Oxford University

[16] Barbas CF, et al. Phage Display: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 2001.

[17] Pal G, Fellouse FA. Methods for the construction of phage-displayed libraries. In: Phage Display in Biotechnology and Drug Discovery. Florida: CRC Press; 2005. pp. 131-162

[18] Zeri AC et al. Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy. Proceedings of the National Academy of Sciences.

2003;**100**(11):6458-6463

[19] Bratkovic T. Progress in phage display: Evolution of the technique and

phage biology: From classic models to new insights. Cell. 2018;**172**(6):1260-1270

2015;**22**(14):1757-1773

Press; 2004. pp. 1-26

pp. 1-24

2006;**49**(7):2268-2275

*DOI: http://dx.doi.org/10.5772/intechopen.84281*

[1] Lee S, Xie J, Chen X. Peptidebased probes for targeted

molecular imaging. Biochemistry.

[3] Wu AM, Olafsen T. Antibodies for molecular imaging of cancer. The Cancer Journal. 2008;**14**(3):191-197

[4] Derda R et al. Diversity of phagedisplayed libraries of peptides during panning and amplification. Molecules.

[5] Smith GP. Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science. 1985;**228**(4705):1315-1317

[6] Zhang ZF et al. Screening and selection of peptides specific for esophageal cancer cells from a phage display peptide library. Journal of Cardiothoracic Surgery. 2014;**9**(1):76

[7] Witt H et al. Identification of a rhabdomyosarcoma targeting peptide by phage display with sequence similarities to the tumour lymphatic-homing peptide LyP-1. International Journal of Cancer.

[8] Hui X et al. Specific targeting of the vasculature of gastric cancer by a new tumor-homing peptide CGNSNPKSC. Journal of Controlled

[9] Wang W et al. Near-infrared optical imaging of integrin αvβ3 in human tumor xenografts. Molecular Imaging.

[10] Ye Y et al. Design, synthesis, and evaluation of near infrared fluorescent

2009;**124**(9):2026-2032

Release. 2008;**131**(2):86-93

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*Targeting Peptides Derived from Phage Display for Clinical Imaging DOI: http://dx.doi.org/10.5772/intechopen.84281*

#### **References**

*Bacteriophages - Perspectives and Future*

**126**

**Author details**

Supang Khondee1

University, Michigan, USA

provided the original work is properly cited.

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. 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,

and Wibool Piyawattanametha2,3\*

Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand

1 School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand

2 Faculty of Engineering, Department of Biomedical Engineering, King Mongkut's

3 Institute for Quantitative Health Science and Engineering (IQ ), Michigan State

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[3] Wu AM, Olafsen T. Antibodies for molecular imaging of cancer. The Cancer Journal. 2008;**14**(3):191-197

[4] Derda R et al. Diversity of phagedisplayed libraries of peptides during panning and amplification. Molecules. 2011;**16**(2):1776-1803

[5] Smith GP. Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science. 1985;**228**(4705):1315-1317

[6] Zhang ZF et al. Screening and selection of peptides specific for esophageal cancer cells from a phage display peptide library. Journal of Cardiothoracic Surgery. 2014;**9**(1):76

[7] Witt H et al. Identification of a rhabdomyosarcoma targeting peptide by phage display with sequence similarities to the tumour lymphatic-homing peptide LyP-1. International Journal of Cancer. 2009;**124**(9):2026-2032

[8] Hui X et al. Specific targeting of the vasculature of gastric cancer by a new tumor-homing peptide CGNSNPKSC. Journal of Controlled Release. 2008;**131**(2):86-93

[9] Wang W et al. Near-infrared optical imaging of integrin αvβ3 in human tumor xenografts. Molecular Imaging. 2004;**3**(4):15353500200404148

[10] Ye Y et al. Design, synthesis, and evaluation of near infrared fluorescent multimeric RGD peptides for targeting tumors. Journal of Medicinal Chemistry. 2006;**49**(7):2268-2275

[11] Comeau AM et al. Exploring the prokaryotic virosphere. Research in Microbiology. 2008;**159**(5):306-313

[12] Ofir G, Sorek R. Contemporary phage biology: From classic models to new insights. Cell. 2018;**172**(6):1260-1270

[13] Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B. Bacteriophages and phage-derived proteins, application approaches. Current Medicinal Chemistry. 2015;**22**(14):1757-1773

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[41] Li ZJ et al. A novel peptide specifically targeting the vasculature of orthotopic colorectal cancer for imaging detection and drug delivery. Journal of Controlled Release. 2010;**148**(3):292-302

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[49] Zhou J et al. EGFR overexpressed in colonic neoplasia can be detected on wide-field endoscopic imaging. Clinical and Translational Gastroenterology. 2015;**6**(7):e101

[50] Qiu Z et al. Targeted vertical crosssectional imaging with handheld nearinfrared dual axes confocal fluorescence endomicroscope. Biomedical Optics Express. 2013;**4**(2):322-330

[51] Liang S et al. Screening and identification of vascular-endothelialcell-specific binding peptide in gastric cancer. Journal of Molecular Medicine. 2006;**84**(9):764-773

[52] Liu L et al. In vivo molecular imaging of gastric cancer in humanmurine xenograft models with confocal laser endomicroscopy using a tumor vascular homing peptide. Cancer Letters. 2015;**356**(2):891-898

[53] Sturm MB et al. Targeted imaging of esophageal neoplasia with a fluorescently labeled peptide: First-in-human results. Science Translational Medicine. 2013;**5**(184):184ra61-184ra61

[54] De Palma GD et al. Detection of colonic dysplasia in patients with ulcerative colitis using a targeted fluorescent peptide and confocal laser endomicroscopy: A pilot study. PLoS One. 2017;**12**(6):e018050e9

[55] Gao Z et al. In vivo near-infrared imaging of ErbB2 expressing

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**130**

## *Edited by Renos Savva*

Bacteriophages are viruses that utilise bacterial cells as factories for their own propagation and as safe havens for their genomic material. They are capable of equipping bacteria with properties that bestow environmental advantages. They are also capable of specifically and efficiently killing bacteria.Bacteriophages are resilient in a wide diversity of environments, presumed to be as ancient as life itself, and are estimated to be the most numerous biological entities on the planet. Their overarching capacity to survive via molecular adaptation is supported by an arsenal of encoded enzymatic tools, which also enabled biotechnology.This volume includes contributions that describe bacteriophages as nanomachines, genetic engineers, and also as medicines and technologies of the future, including relevant production and process issues.

Published in London, UK © 2020 IntechOpen © iunewind / iStock

Bacteriophages - Perspectives and Future

Bacteriophages

Perspectives and Future

*Edited by Renos Savva*


**6. Site effects in landslide zones**

In order to compare the results from recordings in a natural cavity with those from records performed in a cavity having a simpler geometry, ambient noise was also recorded inside, over, and in the neighborhood (≈30 m) of two artificial tunnels. Both tunnels are located close to the Catania urban area, dug at about 4 m from the topographic surface and having a length of about 100 m, but different height. One of them (height of about 4 m) is dug in massive lavas and the other (height of about 7 m) is excavated in altered lavas. HVNRs show that in the smaller tunnel (Fig. 17a), dug in massive lavas, spectral peaks are significantly less pronounced than those observed in the tunnel, dug in altered lavas, having a greater height (Fig. 17b). In this tunnel, H/V spectral peaks, in the frequency range 4.0–7.0 Hz, obtained from measure‐ ments performed over and inside, attain values of about 8 and 4 units, respectively, therefore confirming the observation, aforementioned for the Petralia grotto, that inside the cavity spectral peaks are less pronounced than those observed when measurement is performed over the cavity. It is noteworthy that the spectral ratios from measurements in both tunnels show peaks that however are more pronounced than those observed from H/V performed in and over the study grotto. This is possibly related to the height that in both tunnels is significantly greater than in the studied cavity, considering also that the lava characteristics (altered lavas) of the Petralia grotto area are comparable to those of the lava where the greater artificial tunnel

124 Engineering Seismology, Geotechnical and Structural Earthquake Engineering

**Figure 17.** Spectral ratios HVNR from measurements performed in two underground tunnels having different height.

The results so far described are quite complex but nevertheless some interesting considerations

**•** The size of the vertical section and geometry of the cavities seem to play an important role. According to experimental data, it appears evident that only cavities having height greater

**•** Results of spectral ratios at Petralia grotto (having height of about 2.5 m) show spectral ratio peaks less pronounced than those observed in grottoes and tunnels having height of about 4 and 7 m. Such behavior, in our opinion, need to be tested and interpreted with the support of numerical modeling, to investigate also about possible amplifications/deamplification of

the vertical component of motion as highlighted by processing earthquake data.

than about 4 meters show significant H/V spectral peaks.

is dug.

can be inferred:

Landslide phenomena, besides exposing the affected areas to a considerable natural risk, imply the occurrence of significant variations in the local seismic response. To investigate such features, Fekruna Bay, in the area of Xemxija (Fig. 18), was selected. Xemxija is a seaside village and marina on the northeastern part of Malta and it is a very important site for touristic attractions, as well as cultural and historical heritage. The study area spans a couple of square kilometres. More than half of it is intensely built, while the remaining area consists of meadows and agricultural land. The area is characterized by a geology and topography that varies over small spatial scales. Its geomorphologic features are the result of the combined effect of the lithology, tectonics and coastal nature that shaped the region, and such features contribute towards the degree of geological instability of the whole area and particularly to the cliff sections.

The outcropping local geology in the Fekruna bay (Pedley *et al*., 2002) is characterised by the Upper Coralline Limestone (UCL) and the Blue Clay (BC) formations. Underneath the BC, a carbonatic formation, the Globigerina Limestone formation (GL) consisting mainly of loosely aggregated planktonic foraminifers, and a Lower Coralline Limestone formation (LCL), which consists of massive biogenic limestone beds, are present. This geolithologic sequence gives rise, in the north coast of Malta, to lateral spreading phenomena which take place within the brittle and heavily jointed and faulted UCL formation overlying the BC which consists of softer and unconsolidated material (Mantovani *et al.,* 2012). The UCL formation is characterized by a prominent plateau scarp face, whereas BC produces slopes extending from the base of the UCL scarp face to sea level. It is well known that lateral spreading usually takes place in the

We recorded ambient noise at 27 sites using a 3-component seismometer (Tromino, www.tro‐ mino.eu) and directional effects were investigated by rotating the NS and EW components of motion by steps of 10 degrees starting from 0° (north) to 180° (south). Moreover, a direct estimate of the polarization angle by using the covariance matrix method (Jurkevics, 1988) and the time-frequency (TF) polarization method (Burjánek *et al*., 2010; 2012) was achieved.

Speedy Techniques to Evaluate Seismic Site Effects in Particular Geomorphologic Conditions: Faults, Cavities,

Landslides and Topographic Irregularities http://dx.doi.org/10.5772/55439 127

A dense microtremor measurement survey was carried out focusing attention on the NE part of the bay, in which there is major evidence of slope instability and in which a high level of cliff fracturing is evident. Recording sites were located in order to sample the area as uniformly as possible. Moreover, several recording sites where chosen on a linear deployment for investigating the role of the fractures in the HVNR behaviour. The measurements were performed in three different zones: indeed, we carried out one set of recordings in a stable platform and far away from the cliff edge, a set of measurements in the fractured area along the cliff, and another set on the landslide body (see Fig. 18 for measurement points location). Examples of the spectral ratios obtained in the three different zones are shown in Figure 19a. The results allowed us to identify a region, away from the cliff edge, where the HVNR peaks are around a stable frequency of about 1.5 Hz. These fundamental peaks may be generally associated with the interface separating the BC layer from the underlying carbonates of the Globigerina Limestone formation (GL). Moreover, the presence of the BC layer gives rise to a velocity inversion since it has a lower shear wave velocity with respect to the overlying UCL formation. This causes the HVNR values to drop below 1 unit over a wide frequency range (Di Giacomo et al., 2005). The origin of the resonance peak as linked to the BC/GL interface was confirmed by the results obtained through a 1D modelling, performed by computing the synthetic HVNR curves (Fig. 19b). To compute the synthetic spectral ratios we considered that ambient vibrations wavefield can be represented by the superimposition of random multi modal plane waves moving in all the directions at the surface of a flat 1-D layered visco-elastic solid, as in Herrmann (2002) formulation, extending the modal summation up to the fifth mode. We also applied initial constraints on the thicknesses and elastic parameters of the layers using borehole logs data and we took shear waves velocity values from a separate preliminary study carried out in the same area using the ReMi, MASW and Refraction methods (Panzera *et al.,* 2011a). Similar behaviour to the above is observed in the spectral ratio obtained at sites close to the cliff edge and all around the identified fractures, but with slightly different features at the high-frequency interval. We observe indeed a clear and predominant peak at around 1.5 Hz, which is associated with the interface between BC and GL, and several smaller peaks at higher frequency (> 9.0 Hz). The fact that these peaks are not visible in the unfractured region leads us to postulate that they may be associated with the presence of fractures and of blocks almost detached from the cliff and therefore free to oscillate. Finally, the sites on the rock-fall area show a different HVNR behaviour with respect to the measurements taken on the plateau. In this area it is possible to identify HVNRs showing bimodal dominant peaks at low frequen‐ cy, in the range 1.0-3.0 Hz, as well as pronounced peaks at about 3.0 Hz and at frequency higher than 9.0 Hz. The bimodal peaks at low frequency (1.0-3.0 Hz) can, in our opinion, be associated

**6.1. Results and discussion**

**Figure 18.** Geo-lithologic map of the north-eastern part of the Xemxija bay (modified from MEPA Various Authors, 2004). The insets show the location of the study area and a sketch of lateral spreading effects along the Xemxija coast.

lateral extension of cohesive rock masses lying over a deforming mass of softer material where the controlling basal shear surface is often not well defined.

A preliminary study of the area, focusing our attention on the risk of landsliding and rockfalls, was carried out using the ambient noise HVNR technique in order to characterize the rock masses behavior in the presence of fractures linked to the landslide body. This type of measurements can be done quickly and with a high spatial density, providing a fast tool for setting the dynamic behavior of the rock outcropping. Generally, this spectral ratio exhibits a peak, corresponding to the fundamental frequency of the site whereas, it is less reliable as regards its amplitude. Nevertheless, the HVNR curve contains valuable information about the underlying structure, especially as concerns the relationship between VS of the sediments and their thickness (Ibs-Vonseth and Wholenberg 1999; Scherbaum *et al*. 2003). Recordings of ambient noise and the use of the HVNR technique has recently had widespread use in studying landslides (*e.g.* Del Gaudio *et al*., 2008; Burjánek *et al*., 2010; Del Gaudio and Wasowski, 2011; Burjánek *et al*., 2012).

We recorded ambient noise at 27 sites using a 3-component seismometer (Tromino, www.tro‐ mino.eu) and directional effects were investigated by rotating the NS and EW components of motion by steps of 10 degrees starting from 0° (north) to 180° (south). Moreover, a direct estimate of the polarization angle by using the covariance matrix method (Jurkevics, 1988) and the time-frequency (TF) polarization method (Burjánek *et al*., 2010; 2012) was achieved.

#### **6.1. Results and discussion**

lateral extension of cohesive rock masses lying over a deforming mass of softer material where

**Figure 18.** Geo-lithologic map of the north-eastern part of the Xemxija bay (modified from MEPA Various Authors, 2004). The insets show the location of the study area and a sketch of lateral spreading effects along the Xemxija coast.

A preliminary study of the area, focusing our attention on the risk of landsliding and rockfalls, was carried out using the ambient noise HVNR technique in order to characterize the rock masses behavior in the presence of fractures linked to the landslide body. This type of measurements can be done quickly and with a high spatial density, providing a fast tool for setting the dynamic behavior of the rock outcropping. Generally, this spectral ratio exhibits a peak, corresponding to the fundamental frequency of the site whereas, it is less reliable as regards its amplitude. Nevertheless, the HVNR curve contains valuable information about the underlying structure, especially as concerns the relationship between VS of the sediments and their thickness (Ibs-Vonseth and Wholenberg 1999; Scherbaum *et al*. 2003). Recordings of ambient noise and the use of the HVNR technique has recently had widespread use in studying landslides (*e.g.* Del Gaudio *et al*., 2008; Burjánek *et al*., 2010; Del Gaudio and Wasowski, 2011;

the controlling basal shear surface is often not well defined.

126 Engineering Seismology, Geotechnical and Structural Earthquake Engineering

Burjánek *et al*., 2012).

A dense microtremor measurement survey was carried out focusing attention on the NE part of the bay, in which there is major evidence of slope instability and in which a high level of cliff fracturing is evident. Recording sites were located in order to sample the area as uniformly as possible. Moreover, several recording sites where chosen on a linear deployment for investigating the role of the fractures in the HVNR behaviour. The measurements were performed in three different zones: indeed, we carried out one set of recordings in a stable platform and far away from the cliff edge, a set of measurements in the fractured area along the cliff, and another set on the landslide body (see Fig. 18 for measurement points location). Examples of the spectral ratios obtained in the three different zones are shown in Figure 19a. The results allowed us to identify a region, away from the cliff edge, where the HVNR peaks are around a stable frequency of about 1.5 Hz. These fundamental peaks may be generally associated with the interface separating the BC layer from the underlying carbonates of the Globigerina Limestone formation (GL). Moreover, the presence of the BC layer gives rise to a velocity inversion since it has a lower shear wave velocity with respect to the overlying UCL formation. This causes the HVNR values to drop below 1 unit over a wide frequency range (Di Giacomo et al., 2005). The origin of the resonance peak as linked to the BC/GL interface was confirmed by the results obtained through a 1D modelling, performed by computing the synthetic HVNR curves (Fig. 19b). To compute the synthetic spectral ratios we considered that ambient vibrations wavefield can be represented by the superimposition of random multi modal plane waves moving in all the directions at the surface of a flat 1-D layered visco-elastic solid, as in Herrmann (2002) formulation, extending the modal summation up to the fifth mode. We also applied initial constraints on the thicknesses and elastic parameters of the layers using borehole logs data and we took shear waves velocity values from a separate preliminary study carried out in the same area using the ReMi, MASW and Refraction methods (Panzera *et al.,* 2011a). Similar behaviour to the above is observed in the spectral ratio obtained at sites close to the cliff edge and all around the identified fractures, but with slightly different features at the high-frequency interval. We observe indeed a clear and predominant peak at around 1.5 Hz, which is associated with the interface between BC and GL, and several smaller peaks at higher frequency (> 9.0 Hz). The fact that these peaks are not visible in the unfractured region leads us to postulate that they may be associated with the presence of fractures and of blocks almost detached from the cliff and therefore free to oscillate. Finally, the sites on the rock-fall area show a different HVNR behaviour with respect to the measurements taken on the plateau. In this area it is possible to identify HVNRs showing bimodal dominant peaks at low frequen‐ cy, in the range 1.0-3.0 Hz, as well as pronounced peaks at about 3.0 Hz and at frequency higher than 9.0 Hz. The bimodal peaks at low frequency (1.0-3.0 Hz) can, in our opinion, be associated

with the contact between the rockfall and detritus unit and the BC formation, as well as to the interface between BC and the underlying GL formation.

to the vibration of smaller blocks can be observed at higher frequencies (9.0-40.0 Hz). Further‐ more, we obtained a direct estimate of the polarization angle through the full use of the threecomponent vector of the noise wave-field. General behavior of the noise wave-field, in this frequency range is shown through rose diagrams, whereas, in order to distinguish between properties of low and high frequency components of the signal, strike versus frequency polar plots were obtained. The examples shown in Figure 19d and e show that the maxima of the horizontal polarization occur in the north-east to east-north- east direction, although in some cases the high frequency directionality is more complex. As observed by Burjánek et al. (2010), high-frequency ground motion can indeed reflect the vibration of smaller blocks that imply both different resonant frequencies and directions. The polarization observed for the sites located away from the unstable areas (see e.g. #25 in Figure 19) show a trend with more dispersed and variable directions. The boundaries of the landslide area therefore appear well defined by the polarization pattern and as postulated by Kolesnikov et al. (2003) the landslide activity is characterized by strong horizontal polarization in a broad frequency band. In our study, a tendency seems evident for the entire landslide body to generally vibrate with a northeast azimuth and it may be assumed that, during a strong earthquake the ground motion would be amplified in this direction. Studies of Burjánek *et a*l. (2010; 2012) point out that the ambient noise polarization take place at about 90 degree angle to the observed fractures which are perpendicular to the sliding direction. In the present study the polarization angle is parallel to the opening cracks, which appears in contrast to the above mentioned results. A possible explanation of our findings is that there exists a prevailing north-easterly sliding direction of the landslide body which is strongly affecting the polarization direction especially in the 1-10

Speedy Techniques to Evaluate Seismic Site Effects in Particular Geomorphologic Conditions: Faults, Cavities,

Landslides and Topographic Irregularities http://dx.doi.org/10.5772/55439 129

In Figure 20 we summarize the above results into a tentative draft profile, located as shown in Fig. 18, which illustrates the main geological features and hypothesizes the shape of the landslide body. The bottom panel shows a 2-D diagram obtained by combining all the ambient noise measurements along the profile. Moving along the profile from measurement point #5 to #17, it is interesting to observe the increasing amplitude of the HVNR at frequencies greater than 6.0-7.0 Hz. It can also be noticed that, especially between 50 and 60 m along the profile (sites #1, #2 and #3) the influence of the fracture zone is evident (see dashed area in Fig. 20). This is associated with the vibrational mode of the almost detached blocks. Along the cross section, at distances ranging from 60 to 100 m, it is possible to note both the presence of the bimodal peak associated with the two interfaces detritus/BC and BC/GL, as well as the high frequency peaks most probably associated with the vibration of large blocks that have been

detached from the cliff-face and are now partially or totally included in the BC.

As final considerations, it is noteworthy to observe that this study highlights the importance of evaluating the local seismic response in presence of slope instabilities related to landslide hazard. The instability processes that could be potentially triggered are linked to both slow mass movements, which might normally occur in tens or hundreds of years, and to sudden rockfall in the case of ground shaking due to moderate-to-strong earthquake activity. It is

Hz frequency range.

worth noting that:

**Figure 19.** a) Example of HVNR results obtained at some recording sites located in the not fractured zone (#25), in the cliff area (#13), and in the landslide zone (#14); (b) results obtained through a 1D modelling, performed in sites locat‐ ed on the non-fractured zone, by computing the synthetic HVNR curve; (c) rotated HVNR, (d) rose diagrams and (e) polar plots obtained at recording sites #25, #13 and #14.

Inspection of directional effects (see examples in Fig. 19c) show that they are clearly evident in the cliff fracture zones,at an angle of about 40°-60° N in all the considered frequency range, although some variability in azimuth is observed at high frequency (>9 Hz) at site #13. On moving away from the cliff edge, the rotated HVNR show a slight change of the directional resonance angle and an amplitude decrease of the rotated spectral ratios at high frequency. Such a behavior could be linked to the increase of rock stiffness and a reduction of the blocks' freedom to oscillate. Finally, it is evident that the directionality pattern observed in the rotated HVSRs performed on the landslide body is quite complex. The general trend has a prevailing direction of about 40°-60° N at low frequency (1.0-9.0 Hz), similarly to what is observed in the fractured zone, whereas different resonant frequencies and directions that could be ascribed to the vibration of smaller blocks can be observed at higher frequencies (9.0-40.0 Hz). Further‐ more, we obtained a direct estimate of the polarization angle through the full use of the threecomponent vector of the noise wave-field. General behavior of the noise wave-field, in this frequency range is shown through rose diagrams, whereas, in order to distinguish between properties of low and high frequency components of the signal, strike versus frequency polar plots were obtained. The examples shown in Figure 19d and e show that the maxima of the horizontal polarization occur in the north-east to east-north- east direction, although in some cases the high frequency directionality is more complex. As observed by Burjánek et al. (2010), high-frequency ground motion can indeed reflect the vibration of smaller blocks that imply both different resonant frequencies and directions. The polarization observed for the sites located away from the unstable areas (see e.g. #25 in Figure 19) show a trend with more dispersed and variable directions. The boundaries of the landslide area therefore appear well defined by the polarization pattern and as postulated by Kolesnikov et al. (2003) the landslide activity is characterized by strong horizontal polarization in a broad frequency band. In our study, a tendency seems evident for the entire landslide body to generally vibrate with a northeast azimuth and it may be assumed that, during a strong earthquake the ground motion would be amplified in this direction. Studies of Burjánek *et a*l. (2010; 2012) point out that the ambient noise polarization take place at about 90 degree angle to the observed fractures which are perpendicular to the sliding direction. In the present study the polarization angle is parallel to the opening cracks, which appears in contrast to the above mentioned results. A possible explanation of our findings is that there exists a prevailing north-easterly sliding direction of the landslide body which is strongly affecting the polarization direction especially in the 1-10 Hz frequency range.

with the contact between the rockfall and detritus unit and the BC formation, as well as to the

**Figure 19.** a) Example of HVNR results obtained at some recording sites located in the not fractured zone (#25), in the cliff area (#13), and in the landslide zone (#14); (b) results obtained through a 1D modelling, performed in sites locat‐ ed on the non-fractured zone, by computing the synthetic HVNR curve; (c) rotated HVNR, (d) rose diagrams and (e)

Inspection of directional effects (see examples in Fig. 19c) show that they are clearly evident in the cliff fracture zones,at an angle of about 40°-60° N in all the considered frequency range, although some variability in azimuth is observed at high frequency (>9 Hz) at site #13. On moving away from the cliff edge, the rotated HVNR show a slight change of the directional resonance angle and an amplitude decrease of the rotated spectral ratios at high frequency. Such a behavior could be linked to the increase of rock stiffness and a reduction of the blocks' freedom to oscillate. Finally, it is evident that the directionality pattern observed in the rotated HVSRs performed on the landslide body is quite complex. The general trend has a prevailing direction of about 40°-60° N at low frequency (1.0-9.0 Hz), similarly to what is observed in the fractured zone, whereas different resonant frequencies and directions that could be ascribed

interface between BC and the underlying GL formation.

128 Engineering Seismology, Geotechnical and Structural Earthquake Engineering

polar plots obtained at recording sites #25, #13 and #14.

In Figure 20 we summarize the above results into a tentative draft profile, located as shown in Fig. 18, which illustrates the main geological features and hypothesizes the shape of the landslide body. The bottom panel shows a 2-D diagram obtained by combining all the ambient noise measurements along the profile. Moving along the profile from measurement point #5 to #17, it is interesting to observe the increasing amplitude of the HVNR at frequencies greater than 6.0-7.0 Hz. It can also be noticed that, especially between 50 and 60 m along the profile (sites #1, #2 and #3) the influence of the fracture zone is evident (see dashed area in Fig. 20). This is associated with the vibrational mode of the almost detached blocks. Along the cross section, at distances ranging from 60 to 100 m, it is possible to note both the presence of the bimodal peak associated with the two interfaces detritus/BC and BC/GL, as well as the high frequency peaks most probably associated with the vibration of large blocks that have been detached from the cliff-face and are now partially or totally included in the BC.

As final considerations, it is noteworthy to observe that this study highlights the importance of evaluating the local seismic response in presence of slope instabilities related to landslide hazard. The instability processes that could be potentially triggered are linked to both slow mass movements, which might normally occur in tens or hundreds of years, and to sudden rockfall in the case of ground shaking due to moderate-to-strong earthquake activity. It is worth noting that:

**7. Local site effects linked to the topography**

major axis of the ridge, are often observed (Spudich *et al.*, 1996).

formed by sand and gravel, laying over a marly clays basement.

2002; Pagliaroli *et al.*, 2007).

The evaluation of the local seismic response when affected by the presence of topographic irregularities is particularly important, from the engineering point of view, mostly since a number of historical villages in Italy are erected on the top of natural reliefs. The influence of the topography on ground motion is linked to the sharpness of the ridge crest (Géli *et al*., 1988; Bard and Riepl-Thomas, 1999). Amplification effects are generally linked to the focalization of seismic waves at topmost part of a hill, due to the existence of diffraction, reflection, and conversion of the incident waves (Bard, 1982). They appear also frequency-dependent so that resonance phenomena occur when the wavelength of the incident wave is comparable to the horizontal dimension of the hill. In addition, significant directional effects, transverse to the

Speedy Techniques to Evaluate Seismic Site Effects in Particular Geomorphologic Conditions: Faults, Cavities,

Landslides and Topographic Irregularities http://dx.doi.org/10.5772/55439 131

Several analytical and numerical methods have been developed to study incoming seismic waves when crossing a hill shaped morphology (*e.g.*, LeBrun *et al.*, 1999; Paolucci, 2002). Although experimental studies using earthquake instrumental records are relatively few, the use of earthquake data has shown to be a successful tool for the evaluation of topographic effects, as well as artificial explosions and ambient noise records, processed with the HVNR technique (*e.g.*, Borcherdt, 1970; LeBrun *et al.*, 1999; Poppeliers and Pavlis,

The present study was performed in two reliefs having different morphologic and geologic features aiming to discriminate the topographic from the stratigraphic effects, using experi‐ mental techniques based on earthquake and ambient noise recordings in order to test at the same time the reliability of ambient noise recordings, processed through HVNR techniques, to estimate topographic effects. The first relief investigated is the area of Ortigia (downtown Siracusa, Sicily). It is a hill shaped peninsula, mostly formed by a carbonate sequence, elongated in the N-S direction, reaching a length of about 1,500 m, with a maximum height of 30 m a.s.l., having a transverse section width of about 700 m (Fig. 21). The second test area is the university campus (S. Sofia hill), a ridge located in the northern part of Catania (Fig. 22). The S. Sofia hill has a gentle topography with a flat surface at the top, it is elongated for about 700 m in NW-SE direction with a maximum height of 40 m. Its longitudinal section (B-B' in Fig. 22) is asymmetric, consequently the northwestern side is quite gentle with respect to the southeastern part. On the other hand, the transverse section is more regular and symmetric (A-A' in Fig. 22), with a base having width of about 500 m. This area has a more complex geology. The most frequently cropping out lithotype is basaltic lava that in pre-historical and historical times flowed onto the valleys originally existing in the sedimentary formations,

In both areas several ambient noise measurements were performed, processing data with spectral ratio techniques and evaluating the directional effects as well. Moreover, in the S. Sofia area three permanent stations located respectively on the top of the hill, along the slope and at a reference site, about three kilometers away from the study area (see Fig. 22a, b). The area was monitored for about two years and a set of 44 local and regional seismic events, having a good signal-to-noise ratio was selected and processed using HVSR and SSR techniques.

**Figure 20.** a) Cross section along the A-B profile in Fig. 27; b) 2-D diagram obtained combining all the ambient noise measurements along the profile as a function of distance (x axis) and frequency (y axis); c) HVSR results at the record‐ ing sites located across the profile.


*3.4. Mechanical Testing*

The mechanical testing served to compare the effects of three main variables on the biocomposite material's properties: fungal species (*Pleurotus ostreatus* and *Coprinus comatus*), substrate particle sizes (with or without micro-particles), and post-growth treatment (dried, baked, compacted then dried, and compacted then baked). The acronyms given to each sample are shown in Table 1.