**6. Innate immunity abnormalities in GCA**

Immune responses are initiated by the recognition of foreign molecular structures, such as invading pathogens, by the antigen presenting cells (APCs) of the innate immune system. Tissue macrophages and dendritic cells (DCs) represent the main classes of professional APCs and are characterized by the membrane expression of germ-line receptors (pattern recognition receptors, PRRs). These receptors are able to recognize specific molecular patterns of exoge‐ nous and/or endogenous foreign proteins, known as pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs).

Upon recognition of a certain PAMP or DAMP, dendritic cells become differentiated and activated and produce cytokines, which are able to recruit neutrophils and macrophages, activate the adaptive immune system and trigger the complement cascade. The physiological goal of early innate immune response is to control and demarcate infection and prevent microbe spreading and further tissue damage.

it has been shown that DCs' depletion abrogates vasculitis, thus confirming the critical role of

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The activation of vascular DCs is mediated via the ligation of their TLRs. It has been shown that certain infectious agents are able to legate to specific Toll-like receptors, such as TLR-4 (LPS) or TLR-5 (flagellin). The ligation of a PAMP (or DAMP), such as LPS or flagellin, to the extracellular portion of the TLR provokes the activation of the intracellular TRAM motif and the consequent activation of an intracellular phosphorylation cascade (second message). The final result is the activation of the NfKB, which enters the nucleus and induces certain genes. This mechanism leads to the translation of pro-inflammatory molecules with autocrine or paracrine actions, expression of co-stimulatory receptors on cell surface and production of

Additional research, in regard to the role of TLRs in GCA pathogenesis, led to some very interesting results. It is well known that GCA shows an impressive, yet unexplained, predi‐ lection for specific sites of the vasculature, such as the 2nd to 5th aortic branches. [1] Histopa‐ thologic studies demonstrated that DCs express different type of TLRs in different arteries. [30]

The distribution of TRLs in the vessel wall is highly determined by the embryological origin of the tissue. The aortic arch and its branches derive from the ectoderma, whereas the de‐ scending aorta derives from mesodermal cells. The heterogeneity of the immune response in GCA is believed to be strongly influenced by the specific type of TLRs, whose expression varies in the different blood vessels. Indeed, in an experimental study, Pryshchep et al showed that the distribution of TLRs, in various sites of the vascular tree, determine the extent and profile of the inflammatory reactions. [30] DCs, with differential surface expression of TLRs, display a marked heterogeneity in their immune-regulatory functions, providing a possible clue toward the tissue tropism of GCA. Furthermore, the immunological identity of blood vessels, as defined by the expression of a vessel-specific profile of TLRs, has been considered to determine the nature of the inflammatory reaction in various types of vasculitides. [21]

In this context, it has been shown that TLR-4, abundantly expressed on adventitial DCs, recognize LPS from bacterial pathogens. Upon recognition, IFN-γ is produced in large amounts and leads the subsequent mononuclear infiltration in all layers of the arterial wall. This all-layer inflammation characterizes panarteritis, with granuloma formation, which is

On the contrary, when TLR-5 recognizes flagellin, the elicited inflammatory response is characterized by the sole infiltration of the adventitia (periarteritis). In this case, disruption of the elastic lamina and subsequent luminal occlusion is typically lacking. [31] Clinical obser‐ vations have suggested that periarteritis rarely accompanies systematic inflammatory proc‐ esses, such as aneurysm formation. Subtle alterations in inflammatory reactions guided by DCs with TLR-4 and/or TLR-5 overexpression may explain the differences in the clinical

typically found in biopsies of the temporal artery in GCA. [31]

phenotype of giant cell arteritis.

activated DCs in sustaining wall inflammation. [29]

antimicrobial substances. [26]

Recently, DCs were shown to initiate the immune response in GCA. [23] These cells lay dormant, in a ring-like structure around the adventitia-media border. It is suggested that, in normal arteries, DCs are sentinels that form a part of the first line immune defense of the vessel wall. [24]

The population of the immune cells in the adventitia of the large-vessel wall is mainly consisted of immature myeloid DCs, with a characteristically high threshold of activation. [25] In contrast to mature APCs that induce adaptive immunity, immature DCs do not express co-stimulatory molecules on their surface, such as CD80 and CD86. This condition is primarily responsible for maintaining an anergic state for T cells. In normal arteries, immature APCs are tolerogenic, thus supporting T-cell unresponsiveness. [21] They have been found to be positive for the S-100 protein and express the chemokine receptor CCR6. [26] As guardians of the immunoprivileged arterial wall, DCs are committed to protect the structural integrity of these vital and nonregenerative tissue structures.

However, in susceptible individuals, such as those bearing the HLA-DR4 allele or in older persons (immune-aging), an unknown instigator or a persistent stimulus activates DCs and initiates an innate immune response. In this context, certain antigens (derived from pathogens or locally formatted by tissue calcification) are considered to infiltrate the vessel wall adven‐ titia, through vasa vasorum, and activate immature DCs.

Physiologically, dendritic cells subsequently migrate to the local lymph nodes and clear the antigens, without triggering inflammation. [26] In GCA, however, for yet ill-defined reasons, the activated DCs remain *in situ* and mature in the vessel wall. [27] Existing evidence supports that the maturation of DCs is a very early step in the initiation of the vasculitic process and occurs long before the chronic phase of wall inflammation. In biopsy studies from patients with polymyalgia rheumatica (PMR), mature DCs, already expressing co-stimulatory mole‐ cules, were found in their temporal arteries, despite the absence of any clinically apparent sign of inflammation. [25] These observations are closely correlated with the fact that a great proportion of PMR patients will eventually develop giant cell arteritis.

The principal role of DCs in GCA pathogenesis lies, not only in initiating the inflammatory process, but, also, in perpetuating immune reactions. Dendritic cells, found in vasculitic lesions, are able to produce high amounts of IL-12 and IL-18 and up-regulate the release of IFN-γ from T cells. [22] In addition, dendritic cells, in inflamed arteries, can release the homing chemokines CCL19 and CCL21, which bind to the receptor CCR7. The expression of CCR7 results in the local arrest of activated DCs, which are no longer able to leave the tissue. Instead, they are trapped in the arterial wall and enforce an aberrant T-cell response. [28] Furthermore, it has been shown that DCs' depletion abrogates vasculitis, thus confirming the critical role of activated DCs in sustaining wall inflammation. [29]

activate the adaptive immune system and trigger the complement cascade. The physiological goal of early innate immune response is to control and demarcate infection and prevent

Recently, DCs were shown to initiate the immune response in GCA. [23] These cells lay dormant, in a ring-like structure around the adventitia-media border. It is suggested that, in normal arteries, DCs are sentinels that form a part of the first line immune defense of the vessel

The population of the immune cells in the adventitia of the large-vessel wall is mainly consisted of immature myeloid DCs, with a characteristically high threshold of activation. [25] In contrast to mature APCs that induce adaptive immunity, immature DCs do not express co-stimulatory molecules on their surface, such as CD80 and CD86. This condition is primarily responsible for maintaining an anergic state for T cells. In normal arteries, immature APCs are tolerogenic, thus supporting T-cell unresponsiveness. [21] They have been found to be positive for the S-100 protein and express the chemokine receptor CCR6. [26] As guardians of the immunoprivileged arterial wall, DCs are committed to protect the structural integrity of these vital and non-

However, in susceptible individuals, such as those bearing the HLA-DR4 allele or in older persons (immune-aging), an unknown instigator or a persistent stimulus activates DCs and initiates an innate immune response. In this context, certain antigens (derived from pathogens or locally formatted by tissue calcification) are considered to infiltrate the vessel wall adven‐

Physiologically, dendritic cells subsequently migrate to the local lymph nodes and clear the antigens, without triggering inflammation. [26] In GCA, however, for yet ill-defined reasons, the activated DCs remain *in situ* and mature in the vessel wall. [27] Existing evidence supports that the maturation of DCs is a very early step in the initiation of the vasculitic process and occurs long before the chronic phase of wall inflammation. In biopsy studies from patients with polymyalgia rheumatica (PMR), mature DCs, already expressing co-stimulatory mole‐ cules, were found in their temporal arteries, despite the absence of any clinically apparent sign of inflammation. [25] These observations are closely correlated with the fact that a great

The principal role of DCs in GCA pathogenesis lies, not only in initiating the inflammatory process, but, also, in perpetuating immune reactions. Dendritic cells, found in vasculitic lesions, are able to produce high amounts of IL-12 and IL-18 and up-regulate the release of IFN-γ from T cells. [22] In addition, dendritic cells, in inflamed arteries, can release the homing chemokines CCL19 and CCL21, which bind to the receptor CCR7. The expression of CCR7 results in the local arrest of activated DCs, which are no longer able to leave the tissue. Instead, they are trapped in the arterial wall and enforce an aberrant T-cell response. [28] Furthermore,

microbe spreading and further tissue damage.

96 Updates in the Diagnosis and Treatment of Vasculitis

wall. [24]

regenerative tissue structures.

titia, through vasa vasorum, and activate immature DCs.

proportion of PMR patients will eventually develop giant cell arteritis.

The activation of vascular DCs is mediated via the ligation of their TLRs. It has been shown that certain infectious agents are able to legate to specific Toll-like receptors, such as TLR-4 (LPS) or TLR-5 (flagellin). The ligation of a PAMP (or DAMP), such as LPS or flagellin, to the extracellular portion of the TLR provokes the activation of the intracellular TRAM motif and the consequent activation of an intracellular phosphorylation cascade (second message). The final result is the activation of the NfKB, which enters the nucleus and induces certain genes. This mechanism leads to the translation of pro-inflammatory molecules with autocrine or paracrine actions, expression of co-stimulatory receptors on cell surface and production of antimicrobial substances. [26]

Additional research, in regard to the role of TLRs in GCA pathogenesis, led to some very interesting results. It is well known that GCA shows an impressive, yet unexplained, predi‐ lection for specific sites of the vasculature, such as the 2nd to 5th aortic branches. [1] Histopa‐ thologic studies demonstrated that DCs express different type of TLRs in different arteries. [30]

The distribution of TRLs in the vessel wall is highly determined by the embryological origin of the tissue. The aortic arch and its branches derive from the ectoderma, whereas the de‐ scending aorta derives from mesodermal cells. The heterogeneity of the immune response in GCA is believed to be strongly influenced by the specific type of TLRs, whose expression varies in the different blood vessels. Indeed, in an experimental study, Pryshchep et al showed that the distribution of TLRs, in various sites of the vascular tree, determine the extent and profile of the inflammatory reactions. [30] DCs, with differential surface expression of TLRs, display a marked heterogeneity in their immune-regulatory functions, providing a possible clue toward the tissue tropism of GCA. Furthermore, the immunological identity of blood vessels, as defined by the expression of a vessel-specific profile of TLRs, has been considered to determine the nature of the inflammatory reaction in various types of vasculitides. [21]

In this context, it has been shown that TLR-4, abundantly expressed on adventitial DCs, recognize LPS from bacterial pathogens. Upon recognition, IFN-γ is produced in large amounts and leads the subsequent mononuclear infiltration in all layers of the arterial wall. This all-layer inflammation characterizes panarteritis, with granuloma formation, which is typically found in biopsies of the temporal artery in GCA. [31]

On the contrary, when TLR-5 recognizes flagellin, the elicited inflammatory response is characterized by the sole infiltration of the adventitia (periarteritis). In this case, disruption of the elastic lamina and subsequent luminal occlusion is typically lacking. [31] Clinical obser‐ vations have suggested that periarteritis rarely accompanies systematic inflammatory proc‐ esses, such as aneurysm formation. Subtle alterations in inflammatory reactions guided by DCs with TLR-4 and/or TLR-5 overexpression may explain the differences in the clinical phenotype of giant cell arteritis.

Nevertheless, independently of the mode of the initial stimulation, DCs become activated and subsequently produce cytokines with redundant and pleiotropic actions. In inflamed temporal arteries, DCs secrete pro-inflammatory cytokines, mainly IL-2, IL-6 and IFN-γ, which, in turn, mediate the recruitment of inflammatory cells, inhibition of cell migration, enhancement of T cell proliferation and stimulation of T and B cells. [32] The net result is further amplification of the immune response, through positive feedback loops.

**8. Th1 cells in GCA**

pro-inflammatory environment.

treated patients. [38]

destruction. [21]

**9. Th17 cells in GCA**

inflammatory bowel disease. [41]

Th1 cells represent the dominant cell population in the intramural lesions and the periphery of patients with untreated GCA. [37] These cells produce IFN-γ, as their signature cytokine, which, physiologically, has a critical role against viral and intracellular bacterial infections. Once called macrophage activating factor, IFN-γ target macrophages and provide a substantial

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IFN-γ committed T cells are considered to account for >20% of circulating CD4+ T cells, an almost 100% increase compared to age-matched healthy controls. [38] Corticosteroid therapy cannot affect the expansion of this subpopulation, indicating continuous signaling from the respective DCs. The underlying mechanism of this resistance involves the triggering of APCs that continue to release IL-12. Actually, both in the blood and the temporal arteries of GCA patients, IL-12 production continued unabated during the chronic phase of the disease in

At the tissue level, cytokine profiling in GCA temporal arteries has demonstrated robust expression of IFN-γ and an association with a defined disease phenotype. [32] In particular, high tissue IFN-γ levels are typical for patients with ischaemic complications, implicating its crucial participation in the process leading to luminal occlusion. Pathophysiological studies have correlated increased IFN-γ levels with the production of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF), which are molecules implicated in the intimal response that leads to lumen stenosis. [39, 40] VEGF may, in turn, promote IFN-γ production, thus leading to a vicious cycle of inflammation and structural stenosis. [39]

It is currently unknown which aspects of the granulomatous inflammation depend upon IFNγ. The ability of this cytokine to activate monocytes and macrophages certainly has a role in promoting the differentiation of lesional histiocytes. However, the profound differences in the clinical presentation of treated and untreated GCA patients suggest that IFN-γ is less relevant to the systemic manifestations of the disease and, instead, the major mediator of vessel wall

Th17 cells play an important role in antimicrobial immunity where they regulate the recruit‐ ment of neutrophils and facilitate protection against extracellular bacteria and fungi. Far beyond their role in host defense, Th17 cells have been implicated in the pathogenesis of several autoimmune and inflammatory disorders, such as rheumatoid arthritis, multiple sclerosis and

In untreated GCA patients, the frequency of Th17 cells is 10-fold elevated in the peripheral blood and they accumulate in the vascular infiltrates. [37] In healthy individuals, Th17 cells are infrequent and account for less than 0.3% of the circulating CD4+ T cells. In newly diag‐


Finally, the high selectivity values could be explained considering the interaction between the substrate triple or double bond (alkyne or alkene) with the complex species LUMO frontier orbital, and, additonally, for the heterogeneous system, with the support chemical sites. For both factors the stronger the interaction, the more favourable the hydrogenation process, but in each case a triple bond will give place to the strongest interaction. Thus, the selectivity to 1-heptene or *(Z)-*3-hexene will be kept in a very high value inasmuch as the alkyne concentration is high enough to consider the alkene hydrogenation negligible, after

On the other hand, regarding that [RhCl(NH2(CH2)12CH3)3] anchored on -Al2O3 or RX3 is the optimum catalyst, some practical-economical benefits can be mentioned: a) the easy and cheap way in which the catalyst is removed from the remaining solution after ending the hydrogenation reaction; b) the main product does not need further purification due to a possible contamination with a heavy metal compound as no complex leaching was detected; and lastly, c) there is no need for a costly temperature control due to the mild operational

greatly favoured because of the long hydrocarbon chain of the TDA ligand.

low oxidation state of the central element.

than in the bulk solution.

that the situation is reversed.

*3.5.2. Practical considerations*

conditions.

**Table 5.** 3-hexyne total conversion (XT), conversions to *(Z)-*3-hexene (X*(Z)*) and selectivities to *(Z)-*3 hexene (S*(Z)*), *(E)-*3-hexene (S*(E)*) and n-hexane (Sn) for the following catalysts: Lindlar, [RhCl(TDA)3] complex unsupported and anchored on RX3. 3-hexyne/Rh = 8.1 103.

*It can be remarked that for the 3-hexyne (non-terminal alkyne) partial hydrogenation, [RhCl(TDA)3] is a much better option than the Lindlar catalyst to obtain the desired product.*

### **3.5. The optimum catalytic system for both test reactions**

#### *3.5.1. Chemical considerations*

Based on the information obtained from the previous sections, it can be concluded that [RhCl(TDA)3], supported either on -Al2O3 or on RX3 in a "table" arrangement structure (Section 3.3), is the best option to carry out the 1-heptyne or 3-hexyne partial hydrogenation to obtain high conversion values. As it was said in Section 3.2, this complex has a d8 central element with a combination of ligands L'/L = 3 (L' = TDA, L = Cl), with a square planar geometry associated to a C2v local site symmetry. This type of coordination compound, at some point of the reaction mechanism, has to release one of the coordination sphere ligands; in this respect, the trans-effect series indicates that the labile ligand is TDA opposite to the Cl ligand. Some features related to the central atom, the TDA ligand, the complex coordination number, the site symmetry and the supported condition, could explain this optimum performance.

According to the Atomic Orbital Model (AOM) mentioned in Section 3.2, the (dz2)\* and (dx2 y2)\* are the HOMO/LUMO frontiers orbitals, respectively. The former, along the z axis with a high electron density, is ready to overlap with the hydrogen σ antibonding orbital, thus favouring H-H bond breaking and the latter, an empty orbital extended on the xy plane, is available to receive electron density from the substrate molecule triple bond, thus weakening its π bonds. These two factors, that are important in the hydrogenation catalytic cycle, will be particularly favoured in this case as the complex antibonding orbitals have considerably more metal than ligand character with a relatively high energy because of the low oxidation state of the central element.

On the other hand, the complex bonding molecular orbitals have a predominant character from the TASO/MOs of the ligands in the coordination sphere constituted by tridecylamine (an electron donating σ species) and chloride (an electron-withdrawing σ/π species) in a 3/1 ratio what means a net electronic enrichment on the Rh atom, a fact that will also contribute to the H-H bond rupture. Besides, the TDA ligand presents another remarkable feature related to the Dispersion forces which are relevant when the TDA/solvent interaction is analyzed. This is important as soon as the TDA molecule is released, due to the trans-effect, and it is simultaneously stabilized by the solvent via a solvatation process, a fact that is greatly favoured because of the long hydrocarbon chain of the TDA ligand.

Besides, the presence of a support, either -Al2O3 or RX3, can favour the catalytic process by activating some extra substrate molecules (1-heptyne or 3-hexyne) because of their interactions with the support surface via Acid/Base Lewis or Dipersion forces for -Al2O3 or RX3 respectively. In any case, this situation turns out to be an important factor as it makes the alkyne (1-heptyne or 3-hexyne) concentration around the supported complex higher than in the bulk solution.

Finally, the high selectivity values could be explained considering the interaction between the substrate triple or double bond (alkyne or alkene) with the complex species LUMO frontier orbital, and, additonally, for the heterogeneous system, with the support chemical sites. For both factors the stronger the interaction, the more favourable the hydrogenation process, but in each case a triple bond will give place to the strongest interaction. Thus, the selectivity to 1-heptene or *(Z)-*3-hexene will be kept in a very high value inasmuch as the alkyne concentration is high enough to consider the alkene hydrogenation negligible, after that the situation is reversed.

### *3.5.2. Practical considerations*

152 Hydrogenation

50

120

Reaction

Lindlar

[RhCl(TDA)3]

[RhCl(TDA)3]/RX3

Lindlar

[RhCl(TDA)3]

[RhCl(TDA)3]/RX3

*3.5.1. Chemical considerations*

optimum performance.

complex unsupported and anchored on RX3. 3-hexyne/Rh = 8.1 103.

*is a much better option than the Lindlar catalyst to obtain the desired product.*

**3.5. The optimum catalytic system for both test reactions**

time (min) Catalyst T (K) XT (%) X*(Z)* (%) S*(Z)* (%) S*(E)* (%) Sn (%)

**Table 5.** 3-hexyne total conversion (XT), conversions to *(Z)-*3-hexene (X*(Z)*) and selectivities to *(Z)-*3 hexene (S*(Z)*), *(E)-*3-hexene (S*(E)*) and n-hexane (Sn) for the following catalysts: Lindlar, [RhCl(TDA)3]

*It can be remarked that for the 3-hexyne (non-terminal alkyne) partial hydrogenation, [RhCl(TDA)3]*

Based on the information obtained from the previous sections, it can be concluded that [RhCl(TDA)3], supported either on -Al2O3 or on RX3 in a "table" arrangement structure (Section 3.3), is the best option to carry out the 1-heptyne or 3-hexyne partial hydrogenation to obtain high conversion values. As it was said in Section 3.2, this complex has a d8 central element with a combination of ligands L'/L = 3 (L' = TDA, L = Cl), with a square planar geometry associated to a C2v local site symmetry. This type of coordination compound, at some point of the reaction mechanism, has to release one of the coordination sphere ligands; in this respect, the trans-effect series indicates that the labile ligand is TDA opposite to the Cl ligand. Some features related to the central atom, the TDA ligand, the complex coordination number, the site symmetry and the supported condition, could explain this

275 48 44 92 3 5 290 53 49 93 3 4 303 57 53 93 4 3

275 76 61 79 13 8 290 81 64 80 13 7 303 82 69 84 13 3

275 87 85 98 2 0.3 290 89 87 98 2 0.3 303 92 90 98 1.5 0.5

275 60 46 77 6 17 290 66 45 68 9 23 303 73 44 61 11 28

275 94 55 58 21 21 290 98 58 59 20 21 303 98 62 64 20 16

275 99.9 82 82 9 9 290 99.9 81 81 8 11 303 99.9 82 82 8 10

> On the other hand, regarding that [RhCl(NH2(CH2)12CH3)3] anchored on -Al2O3 or RX3 is the optimum catalyst, some practical-economical benefits can be mentioned: a) the easy and cheap way in which the catalyst is removed from the remaining solution after ending the hydrogenation reaction; b) the main product does not need further purification due to a possible contamination with a heavy metal compound as no complex leaching was detected; and lastly, c) there is no need for a costly temperature control due to the mild operational conditions.

### **4. Conclusions**

Experimental results demonstrate that [PdCl2(NH2(CH2)12CH3)2] and [RhCl(NH2(CH2)12CH3)3], *d8* transition metal complexes, can be used as catalysts to partially hydrogenate 1-heptyne and 3-hexyne (terminal and non-terminal alkynes respectively) in homogeneous and heterogeneous systems (-Al2O3 and RX3 as supports) at mild operational conditions (P = 150 kPa and T up to 303K) with very good catalytic performances even better than the Lindlar catalyst. Analyses based on Elemental Composition, XPS, IR, Atomic Absorption Spectroscopy show that the active catalytic species is the complex itself in each case, with a minimum formula as written above.

Terminal and Non Terminal Alkynes Partial Hydrogenation

Catalyzed by Some *d8* Transition Metal Complexes in Homogeneous and Heterogeneous Systems 155

[3] Mastalir A, Király Z (2003) Pd nanoparticles in hydrotalcite: mild and highly selective

[4] Marín-Astorga N, Pecchi G, Fierro JLG, Reyes P (2003) Alkynes hydrogenation over Pd-

[5] Semagina N, Kiwi-Minsker L (2009) Palladium Nanohexagons and Nanospheres in

[6] Choudary BM, Kantam ML, Reddy NM, Rao KK, Haritha Y, Bhaskar V, Figueras F, Tuel A (1999) Hydrogenation of acetylenics by Pd-exchanged mesoporous materials. App.

[7] Li F, Yi X, Fang W (2009) Effect of Organic Nickel Precursor on the Reduction Performance and Hydrogenation Activity of Ni/Al2O3 Catalysts. Catal. Lett. 130:335-340. [8] Gruttadauria M, Noto R, Deganello G, Liotta LF (1999) Efficient semihydrogenation of the C-C triple bond using palladium on pumice as catalyst. Tetrahedron Lett. 40:2857-

[9] Lennon D, Marshall R, Webb G, Jackson SD (2000) The effects of hydrogen concentration on propyne hydrogenation over a carbon supported palladium catalyst studied under

[10] Lindlar H, Dubuis R, Jones FN, McKusick FC (1973) Palladium catalyst for partial

[11] Yu J, Spencer JB (1998) Discovery that quinoline and triphenylphosphine alter the

[12] Nijhuis TA, van Koten G, Kapteijn F, Moulijn JA (2003) Separation of kinetics and masstransport effects for a fast reaction: the selective hydrogenation of functionalized

[13] Huang W, Li A, Lobo RF, Chen JG (2009) Effects of Zeolite Structures, Exchanged Cations, and Bimetallic Formulations on the Selective Hydrogenation of Acetylene Over

[14] Badano JM, Quiroga M, Betti C, Vera C, Canavese S, Coloma-Pascual F (2010) Resistence To Sulfur And Oxygenated Compounds Of Supported Pd, Pt, Rh, Ru

[15] Nijhuis TA, van Koten G, Moulijn JA (2003) Optimized palladium catalyst systems for selective liquid-phase hydrogenation of functionalized alkynes. App. Catal. A: Gen. 238:

[16] Chen JG, Qi S-T, Humbert MP, Menning CA, Zhu Y-X (2010) Rational design of lowtemperature hydrogenation catalysts: Theoretical predictions and experimental

[17] Shiju RN, Guliants VV (2009) Recent developments in catalysis using nanostructured

[18] Volpe MA, Rodríguez P, Gigola CE (1999) Preparation of Pd/Pb/α-Al2O3 catalysts for selective hydrogenation using PbBu4: the role of metal-support boundary atoms and the

electronic properties of hyfrogenation catalysts. Chem. Comm. 1103-1104

catalysts for alkyne semihydrogenation. J. Catal. 220: 372-381.

Selective Alkyne Hydrogenation. Catal. Lett. 127 (3–4):334-338.

continuous flow conditions. Stud. Surf. Sci. Catal. 130: 245-250.

reduction of acetylenes. Org. Synth. Coll. 5: 880.

Zeolite-Supported Catalysts. Catal. Lett. 130: 380-385.

verification. Acta Phys. Chim. Sin. 26 (4): 869-876.

formation of a stable surface complex. Catal. Lett. 61: 27-32

materials. Appl. Catal. A: Gen. 356: 1-17.

alkynes. Catal. Today 79–80: 315-321.

Catalysts. Catal. Lett. 137: 35-44.

supported catalysts. Catal. Lett. 91 (1–2):115-121.

Catal. A; Gen. 181:139-144.

2858.

259-271.

Tetracoordinated electron-rich transition elements, as well as the presence and the relative quantity of a good electron donating ligand such as NH2(CH2)12CH3 with a long-chain hydrocarbon substituent and the heterogeneous condition, contribute to obtain a catalytic system with a high activity and selectivity performance. According to this and supported by experimental results, the optimum catalyst turns out to be [RhCl(NH2(CH2)12CH3)3] supported either on -Al2O3 or RX3. This behaviour can be understood in terms of Coordination Sphere parameters, Complex Dimensions, Local Site Symmetry, HOMO/LUMO frontier orbitals and some Support features

### **Author details**

Domingo Liprandi1,\*, Edgardo Cagnola1, Cecilia Lederhos2, Juan Badano2 and Mónica Quiroga1,2 *1Inorganic Chemistry, Departament of Chemistry, Faculty of Chemical Engineering, National University of Litoral (UNL), Santa Fe, Argentina, 2Institute of Catalysis and Petrochemistry Research, INCAPE (CONICET- UNL), Santa Fe, Argentina*

### **Acknowledgement**

UNL and CONICET financial supports are greatly acknowledged.

### **5. References**

<sup>\*</sup> Corresponding Author

[3] Mastalir A, Király Z (2003) Pd nanoparticles in hydrotalcite: mild and highly selective catalysts for alkyne semihydrogenation. J. Catal. 220: 372-381.

154 Hydrogenation

**4. Conclusions**

formula as written above.

**Author details**

**Acknowledgement**

**5. References**

Corresponding Author

 \*

*Argentina*

frontier orbitals and some Support features

*University of Litoral (UNL), Santa Fe, Argentina,*

Juan Badano2 and Mónica Quiroga1,2

App.Catal. A: Gen. 280:17-46.

Wiley-VCH, Darmstadt, p 375

Domingo Liprandi1,\*, Edgardo Cagnola1, Cecilia Lederhos2,

UNL and CONICET financial supports are greatly acknowledged.

Experimental results demonstrate that [PdCl2(NH2(CH2)12CH3)2] and [RhCl(NH2(CH2)12CH3)3], *d8* transition metal complexes, can be used as catalysts to partially hydrogenate 1-heptyne and 3-hexyne (terminal and non-terminal alkynes respectively) in homogeneous and heterogeneous systems (-Al2O3 and RX3 as supports) at mild operational conditions (P = 150 kPa and T up to 303K) with very good catalytic performances even better than the Lindlar catalyst. Analyses based on Elemental Composition, XPS, IR, Atomic Absorption Spectroscopy show that the active catalytic species is the complex itself in each case, with a minimum

Tetracoordinated electron-rich transition elements, as well as the presence and the relative quantity of a good electron donating ligand such as NH2(CH2)12CH3 with a long-chain hydrocarbon substituent and the heterogeneous condition, contribute to obtain a catalytic system with a high activity and selectivity performance. According to this and supported by experimental results, the optimum catalyst turns out to be [RhCl(NH2(CH2)12CH3)3] supported either on -Al2O3 or RX3. This behaviour can be understood in terms of Coordination Sphere parameters, Complex Dimensions, Local Site Symmetry, HOMO/LUMO

*1Inorganic Chemistry, Departament of Chemistry, Faculty of Chemical Engineering, National*

*2Institute of Catalysis and Petrochemistry Research, INCAPE (CONICET- UNL), Santa Fe,*

[1] Chen B, Dingerdissen U, Krauter JGE, Lansink Rotgerink HGJ, Móbus K, Ostgard DJ, Panste P, Riermeir TH, Seebald S, Tacke T, Trauthwein H (2005) New developments in hydrogenation catalysis particularly in synthesis of fine and intermediate chemicals.

[2] Elsevier CJ, Kluwer AM (2007) Homogeneous hydrogenation of alkynes and dienes. In: de Vries JG, Elsevier CJ (eds) Handbook of homogeneous hydrogenation. vol 1, ch 14.

[19] Zhang W, Li L, Du Y, Wang X, Yang P (2009) Gold/Platinum Bimetallic Core/Shell Nanoparticles Stabilized by a Fréchet-Type Dendrimer: Preparation and Catalytic Hydrogenations of Phenylaldehydes and Nitrobenzenes. Catal. Lett. 127 (3–4): 429-436.

Terminal and Non Terminal Alkynes Partial Hydrogenation

Catalyzed by Some *d8* Transition Metal Complexes in Homogeneous and Heterogeneous Systems 157

[35] Halpern J (1968) in: Homogeneous Catalysis. American Chemical Society: Chap. 1. [36] Kolthoff IM, Sandell EB, Meehan EJ, Bruckenstein S (1969) Quantitative Chemical

[38] Vogel AI (1951) A Text Book of Quantitative Inorganic Analysis, Longmans, Green and

[39] Livingstone S, in: Bailar JC Jr, Emeléus H., Nyholm R, Trotman-Dickenson AF (Eds.) (1973) The Chemistry of Ruthenium, Rhodium, Palladium, Osmium, Iridium and

[40] Mallat T, Petrov J, Szabó S, Sztatisz J (1985) Palladium–cobalt catalyst: phase structure and activity in liquid phase hydrogenations. Reac. Kinet. Catal. Lett. 29: 353-361.

[43] Nakamoto K (1986) Infrared and Raman Spectra of Inorganic and Coordination

[44] Silverstein RM, Clayton Basler G, Morril TC, Spectrometric Identification of Organic

[46] Rodríguez-Reinoso F, Linares-Solano A (1988) in: Chemistry and Physics of Carbon,

[47] Holland FA, Chapman FS (1976) Liquid Mixing and Processing in Stirred Tanks,

[49] Quiroga M, Liprandi D, Cagnola E, L'Argentière P (2007) 1-heptyne semihydrogenation catalyzed by palladium or rhodium complexes. Influence of: metal atom, ligands and

[50] Wagner CD, Riggs WM, Davis RD, Moulder JF (1978). In Handbook of X-ray Photoelectron Spectroscopy. Muilenberg. G.E., Ed. Perkin-Elmer: Eden Preirie, MN. [51] NIST X-ray Photoelectron Spectroscopy Database NIST Standard Reference Database 20, Version 3.5 (Web Version), National Institute of Standards and Technology, USA,

[52] Liprandi DA, Cagnola EA, Paredes JF, Badano JM, Quiroga M E (2012) A High (Z)/(E) Ratio Obtained During the 3-Hexyne Hydrogenation with a Catalyst Based on a Rh(I)

[53] L'Argentiere P, Quiroga M, Liprandi D, Cagnola E., Román Martínez MC, Díaz Auñón JA, Salinas Martínez de Lecea C. (2003) Activated carbon heterogenized [PdCl2(NH2(CH2)12CH3)2] for the selective hydrogenation of 1-heptyne. Catal. Lett. 87:

[54] Liprandi DA, Cagnola E A, Quiroga M E, L'Argentière PC (2009) Influence of the Reaction Temperature on the 3-Hexyne Semi-Hydrogenation Catalyzed by a

[55] Cotton FA, Wilkinson G (1988) Advanced Inorganic Chemistry, fifth ed, John Wiley and

Complex Anchored on a Carbonaceous Support. Catal. Lett. 142: 231–237.

Palladium(II) Complex. Catal. Lett. 128: 423–433

Sons, New York, pp 901-902.

Platinum, Comprehensive Inorganic Chemistry, Pergamon Press, Oxford.

[41] Borade R, Sayari A, Adnot A, Kaliaguine S (1990) J. Phys. Chem. 94: 5989.

[45] Pouchert CJ (1981) The Aldrich Library of Infrared Spectra Ed. (III): 1562 D.

[48] Le Page JF (1978) Catalyse de Contact, Editions Technip, Paris, Chap 2.

the homo/heterogeneous condition. Appl. Catal. A: Gen. 326:121–129.

[42] Scofield JH (1976) J. Electron. Spectrosc. Relat. Phenom. 8: 129.

Compounds, 4th Ed., Wiley, New York, parts I and III.

Compounds, 5th Ed. Wiley, New York, 1991, chapter III.

Vol. 21, Walker PL Jr. (Ed) ,Marcel Dekker, New York p1.

Reinhold, New York, Chap 5.

2007.

97 – 101.

Analysis, fourth ed., Interscience Publishers, New York. [37] Anderson SN, Basolo F (1963) Inorg. Synth. 7: 214-220.

Co, London.

hexyne. J. Catal. 279: 66-74.

667: 197-208.

Tod. 93-95: 445-450.

Chem. 197 (1-2): 37-50.

Gen. 274: 205-212.

152.

Technol .Biotechnol. 71: 285-290.

Ind. Eng. Chem. Res. 41: 4906-4910

alkynes. J. Molec. Catal. A: Chem. 178: 21-26.

Tetrahedron Lett. 29 (43): 5545-5548.

alkenes and 4-alkynes. Tetrahedron 58: 3911-3922.

[19] Zhang W, Li L, Du Y, Wang X, Yang P (2009) Gold/Platinum Bimetallic Core/Shell Nanoparticles Stabilized by a Fréchet-Type Dendrimer: Preparation and Catalytic Hydrogenations of Phenylaldehydes and Nitrobenzenes. Catal. Lett. 127 (3–4): 429-436. [20] Choi J, Yoon NM (1996) An excellent nickel boride catalyst for the cis-selective

[21] Crespo-Quesada M, Dykeman RR, Laurenczy G, Dyson PJ, Kiwi-Minsker L (2011) Supported nitrogen-modified Pd nanoparticles for the selective hydrogenation of 1-

[22] Costa M, Pelagatti P, Pelizzi C, Rogolino D (2002) Catalytic activity of palladium(II) complexes with tridentate nitrogen ligands in the hydrogenation of alkenes and

[23] de Wolf E, Spek AL, Kuipers BWM, Philipse AP, Meeldijk JD, Bomans PHH, Frederik P.M., Deelman B.J., van Koten G. (2002) "Fluorous derivatives of [Ru(COD)(dppe)]BX4 (X=F, Ph): synthesis, physical studies and application in catalytic hydrogenation of 1-

[24] Edvrard D, Groison K, Mugnier Y, Harvey PD (2004) The Pd4(dppm)4(H)22+ cluster: a precatalyst for the homogeneous hydrogenation of alkynes. Inorg. Chem. 43: 790-796. [25] Frediani P, Giannelli C, Salvini A, Ianelli S (2003) Ruthenium complexes with 1,1' biisoquinoline as ligands. Synthesis and hydrogenation activity. J. Organomet. Chem.

[26] Kerr JM, Suckling CJ (1988) Selective hydrogenation by novel palladium(II) complex.

[27] Park JW, Chung YM, Suh YW, Rhee HK (2004) Partial hydrogenation of 1,3 cyclooctadiene catalyzed by palladium-complex catalysts inmobilized on silica. Catal.

[28] Santra PK, Sagar P (2003) Dihydrogen reduction of nitroaromatics, alkenes, alkynes using Pd(II) complexes both in normal and high pressure conditions. J. Molec. Catal. A:

[29] L'Argentière PC, Cagnola EA, Cañón MG, Liprandi DA, Marconetti DV (1998), A nickel tetra-coordinated complex as catalyst in heterogeneous hydrogenation. J. Chem.

[30] Quiroga ME, Cagnola EA, Liprandi DA, L'Argentière PC (1999) Supported Wilkinson's complex used as a high active hydrogenation catalyst. J. Mol. Catal. A: Chem. 149: 147-

[31] Liprandi DA, Quiroga ME, Cagnola EA, L'Argentière PC (2002) A new more sulfurresistannt rhodium complex as an alternative to the traditional Wilkinson's catalyst.

[32] Cagnola EA, Quiroga ME, Liprandi DA, L'Argentière PC (2004) Immobilized Rh, Ru, Pd and Ni complexes as catalysts in the hydrogenation of cyclohexene. Appl. Catal.. A:

[33] Hamilton CA, Jackson SD, Kelly GJ, Spence R, Bruin D (2002) Competitive Reactions in

Alkyne Hydrogenation. App. Catal. A: Gen. 237: 201-209.

[34] Bailar J C, (ed) (1975) Comprehensive Inorganic Chemistry. Vol 3, p. 1234.

semihydrogenation of acetylenes. Tetrahedron Lett. 37 (7): 1057-1060.

[56] Purcell KF, Kotz JC (1977) Inorganic Chemistry, Holt-Saunders International Editions: Philadelphia, pp 543-549.

**Chapter 7**

© 2012 Maccarrone et al., licensee InTech. This is an open access chapter 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.

© 2012 Maccarrone et al., licensee InTech. This is a paper 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.

**Kinetic Study of the Partial Hydrogenation of**

M. Juliana Maccarrone, Gerardo C. Torres, Cecilia Lederhos, Carolina Betti,

Selective hydrogenation reactions are industrially used for the partial hydrogenation of unsaturated organic compounds in order to form more stable products or intermediate materials for different processes. The production of final organic products of high added value or intermediate compounds for the synthesis of fine chemicals is both of industrial and academic importance [1]. Alkenes are much appreciated products used in the food industry (flavours), the pharmaceutical industry (sedatives, anesthetises, vitamins) and in the perfumes industry (fragrances). They are also used for the production of biologically

The partial hydrogenation of acetylenic compounds using homogeneous or heterogeneous metallic catalysts provides a very viable and economically feasible way for the obtaining of these olefinic compounds. Selective catalysts and optimum operational conditions are necessary in order to avoid the complete hydrogenation of the unsaturated bond. Certain transition metals anchored on different solids have demonstrated to be very active and selective catalysts for this type of reaction. They also have the advantage that they can be operated under milder reaction conditions. It is well documented that palladium is a highly active catalyst for hydrogenation [3]. In this sense, Lindlar catalyst (Pd/CaCO3, 5 wt % of Pd modified with Pb(OAc)2) has been used since 1952 as an excellent commercial catalyst for this type of reactions [4]. The argued reasons for the differences in reactivity of Pd indicate that when the metal is electron deficient it becomes less active because alkynes are more

During decades a lot of research has been carried out modifying this type of catalysts in order to increase the activity and selectivity: different supports as alumina, coal, silica [5-7] have been tried while modified palladium [8], or nanoparticles of Pd have also been

Juan M. Badano, Mónica Quiroga and Juan Yori

active compounds [2], resins, polymers and lubricants, etc.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48699

**1. Introduction**

weakly adsorbed [5].

**1-Heptyne over Ni and Pd Supported on Alumina**

[57] Papp A, Molnár A, Mastalir A (2005) Catalytic investigation of Pd particles supported on MCM-41 for the selective hydrogenations of terminal and internal alkynes. Appl. Catal. A: Gen. 289: 256-266.


**5. BDNF and Brain Cancer: An Unexpected Role. An Oncogene or a Tumor Suppressor?**

The role of BDNF and its cognate receptor TrkB in cancer, including brain cancer, has been recognized for a long time [254]. In many types of cancers, BDNF and/or TrkB have been found expressed or in some cases over-expressed [255]. This is not surprising since growth factors, including neurotrophic factors, and their tyrosine kinase receptors have long been involved in tumors with different cell-dependent mechanisms, fostering proliferation, enhancing anti-apoptotic signaling, and making cells unresponsive to anti-proliferative stimuli [256]. The direct oncogenic activity of TrkB might also be due to the crosstalk with EGF receptors that together with its ligand is well-known to promote cell transformation. BDNF administration not only does phosphorylate TrkB but also EGFR [257]. In line with these observations, it has been recently shown that BDNF produced by glioblastoma (GBM) differentiated cells acts on GBM stem cells, fostering their growth through paracrine signaling [258].

However, recently another study showed that exposing mice to an enriched environment is able to decrease the growth of intracranial glioma, decreasing proliferation and invasion, and improving overall survival. Such an effect is achieved by means of both indirect and direct mechanisms. The former acts via natural killer cells of the innate immune system, whereas the latter utilizes BDNF stimulation of its truncated receptor TrkB.T1 on glioma cancer cells. BDNF binding the TrkB.T1 receptor signals to the Rho protein dissociation inhibitor (RhoGDI), the latter detaches from TrkB.T1 and binds to the small G protein RhoA, leading to its inhibition. The authors found that an enriched environment causes the synthesis of IL-15 and BDNF. When mice bearing the glioma and not housed in enriched environments were infused with BDNF, they reduced tumor size and macrophage infiltration. Thus, showing that at least in part, BDNF accounts for the oncolytic effect elicited by the enriched environment [259]. In a more recent study, the same group delved deeper into the mechanisms, finding that enriched environment changes glioma-associated myeloid cells. BDNF plays a central role by stimulating the production of IL-15 in microglia, which in turn stimulates the natural killer cells to produce IFN-γ. Natural killer cells were responsible for the switch to an oncolytic environment [260] (Figure 3).

Taken together, a scenario emerges where BDNF, acting on different cells is able to reorganize the brain microenvironment in such a way that it becomes resilient to neurodegeneration or oncolytic for tumors. In this regard, although supported by much more preliminary data, it seems that also other compounds might share these properties [261,262].


**4. Energy dispersive x-ray analysis**

With the help of the energy dispersive x-ray spectroscopy (EDX) was performed the elemental analysis of the polyester resin and waste fiber glass. The weight fraction ratio is composed on the total weight of the chemical substances analyzed. The predominance of silicon and aluminum can be observed in the **Figure 12**, after the elemental EDX analysis was done of the waste glass fiber [32]. Also, small amounts of carbon, oxygen, sodium, magnesium and calcium are detected. The obtained data are expressed in two ways, both atomic percent (At.%) and weight percent (Wt.%). The atomic and the weight percentages of the fiber glass elements are: C with 30.96At.%, 17.67Wt.%; O with 24.12At.%, 18.34Wt.%; Na with 0.34At.%, 0.37Wt.%; Mg with 0.38At.%, 0.43Wt.%; Al with 14.43At.%, 18.50Wt.%; Si with 21.12At.%, 28.19Wt% and

Ca with 8.66At.%, 16.50Wt.%. In **Table 2** are presented the elements on the surface of a waste

Recycling of Polymeric Composite Materials http://dx.doi.org/10.5772/intechopen.81281 117

**Element At.% Wt.%** Si 21.12 28.19 Al 14.43 18.50 Ca 08.66 16.50 O 24.12 18.34 C 30.96 17.67 Mg 00.38 00.43 Na 00.34 00.37

The EDX analysis of the matrix polymer is presented in **Figure 13**, [32]. The predominance of carbon, silicon and oxygen is obviously in this case study. Also, small amount of sodium, aluminum and calcium are detected. The atomic and the weight percentages of the polyester matrix

glass fiber.

**Table 2.** EDX analysis of the glass fiber.

**Figure 13.** EDX analysis of the matrix polymer.

**Figure 12.** EDX analysis of the chemical constituents from the glass fiber.

**Table 2.** EDX analysis of the glass fiber.

According to the **Figure 11**, it can be observe the achieved connection between polyester matrix, glass fibers and sand, because of the particles of sand and polyester resin that were well glued on the glass monofilaments. Thus, a composite material with low density and high mechanical properties has obtained. These types of materials allow one reuse of glass fiber waste. Using these types of materials at low temperatures increases their mechanical

With the help of the energy dispersive x-ray spectroscopy (EDX) was performed the elemental analysis of the polyester resin and waste fiber glass. The weight fraction ratio is composed on the total weight of the chemical substances analyzed. The predominance of silicon and aluminum can be observed in the **Figure 12**, after the elemental EDX analysis was done of the waste glass fiber [32]. Also, small amounts of carbon, oxygen, sodium, magnesium and calcium are detected. The obtained data are expressed in two ways, both atomic percent (At.%) and weight percent (Wt.%). The atomic and the weight percentages of the fiber glass elements are: C with 30.96At.%, 17.67Wt.%; O with 24.12At.%, 18.34Wt.%; Na with 0.34At.%, 0.37Wt.%; Mg with 0.38At.%, 0.43Wt.%; Al with 14.43At.%, 18.50Wt.%; Si with 21.12At.%, 28.19Wt% and

characteristics.

**4. Energy dispersive x-ray analysis**

116 Product Lifecycle Management - Terminology and Applications

**Figure 12.** EDX analysis of the chemical constituents from the glass fiber.

Ca with 8.66At.%, 16.50Wt.%. In **Table 2** are presented the elements on the surface of a waste glass fiber.

The EDX analysis of the matrix polymer is presented in **Figure 13**, [32]. The predominance of carbon, silicon and oxygen is obviously in this case study. Also, small amount of sodium, aluminum and calcium are detected. The atomic and the weight percentages of the polyester matrix

**Figure 13.** EDX analysis of the matrix polymer.

The new composite material contained grinded glass fiber waste, polyester matrix and sand all mixed together. After polymerization of the resin we obtain a composite material with superior mechanical properties. This material can be used in different applications, like: strengthening composite parts (ornamental garden stones, ornamental composites plates,

Recycling of Polymeric Composite Materials http://dx.doi.org/10.5772/intechopen.81281 119

This paper was supported by the project AMATUC HORIZONT 2020, contract nr. GA 691787, "Boosting the scientific excellence and innovation capacity in Additive Manufacturing of the

[1] Barbero EJ. Introduction to Composite Materials Design. USA: West Virginia University;

[2] Gay D. Matériaux Composites. 4th ed. Paris: Hezrmes; 1997. 640 p. ISBN2-86601-586-X [3] Berthelot JM. Matériaux Composites. Comportement mécanique et analyse des struc-

[4] Ch D. Structures composites. Calcul des plaques et des poutres multicouches. Paris:

[5] Jones RM. Mechanics of Composite Materials. 2nd ed. Philadelphia: Taylor and Francis;

[6] Kollar LP, Springer GS. Mechanics of Composite Structures. Cambridge: Cambridge

[7] Vasiliev VV, Morozov EV. Mechanics and Analysis of Composite Materials. London:

[8] Cobzaru P. Materiale compozite (Composite materials). Bucureşti: Didactică şi Pedagogică;

[9] Nemeş O, Lachaud F, Piquet R, Soporan VF, Tătaru O. The waste rubber used to improve the properties of composite materials. Studia UBB Chemia;**2008, 53**(3):25-30. ISSN:

tures. Paris: Editions TEC&DOC; 1999. 620 p. ISBN 2-7430-0349-9

garden furniture, additive materials and so on), polyester reinforced concrete.

TUC-N (AMaTUC)", 2016-2018, project director: Nicolae Balc.

Address all correspondence to<EMAIL_ADDRESS>
Technical University of Cluj-Napoca, Romania

1998. 336 p. ISBN 97e8-1-4200-7915-9

Hermes; 2000. 352 p. ISBN 2-7462-0114-3

University Press; 2003. 480 p. ISBN 978-0-511-05703-8

Elsevier Science; 2001. 41e2 p. ISBN 0-08-042702-2

1999. 519 p. ISBN 1-56032-712-X

2004. ISBN 973-30-1445-1

1224-7154. WOS:000263940900002

**Acknowledgements**

**Author details**

Emilia Sabău

**References**

**Table 3.** EDX analysis of the matrix.

elements are: C with 74.80At.%, 62.18Wt.%; O with 15.97At.%, 17.69Wt.%; Na with 0.29At.%, 0.47Wt.%; Al with 1.28At.%, 2.39Wt.%; Si with 4.78At.%, 9.30Wt.% and Ca with 2.87At.%, 7.98Wt.%.

In the table above, **Table 3**, are presented the elemental quantitative analyses that give us the polyester matrix elements on the surface.


4. Sliding mode control

In this section, the main work is to find a state feedback control law based on the sliding mode control technique for the class of multimotor systems.

Nonlinear systems are described as follows:

$$\frac{d}{dt}\mathbf{x} = A\mathbf{x} + B(\boldsymbol{\mu} + \boldsymbol{\mu}\_d(\mathbf{x}, t)) \tag{9}$$

where udð Þ x; t is the nonlinear term in system. Lemma 2 [10]: The sliding mode controller is described as follows

$$\mu = -\left[\mathbb{S}.A\mathbf{x} + \beta \text{sgn}(\sigma)\right] \tag{10}$$

based on the sliding surface:

$$\begin{aligned} \{\mathbf{x} : \sigma = \mathbf{S} \mathbf{x} = \mathbf{0}\}, \\ \mathbf{S} = \left(\mathbf{B}^T \mathbf{X}^{-1} \mathbf{B}\right)^{-1} \mathbf{B}^T \mathbf{X}^{-1} \mathbf{X} \end{aligned} \tag{11}$$

with X is satisfied, the LMI problem as follows:

$$\text{II}^T \{ AX + XA^T \} \text{II} < 0, X > 0,\\ \text{II} = \begin{bmatrix} 1 & 0 & 0 \end{bmatrix}^T \tag{12}$$

Remark 3. We obtain the sliding mode control for multimotor systems (2) based on Lemma 3 because it belongs to the class of systems (9).


Finally, the building code is a question of political commitment to the founding values of the EU and future geostrategic processes in the Western Balkans. It was probably not a coincidence that a building code was not practiced during the socialism regime in all eastern bloc countries, including the Socialist Federal Republic of Yugoslavia, Socialist Republic Bosnia-Herzegovina, and City of Sarajevo. The only question that remains unanswered is why, after the fall of socialismn and thirty years after the first multi-party parliamentary elections in Bosnia-Herzegovina since when we are officially in transition, the building code is still not a part of the spatial planning system? Albeit the building code is currently a utopia, the researcher profoundly believes that Sarajevo's spatial planning system will be significantly reformed and that the absence of a building code document will remain one ephemeral phase of the city's longevity.

## **6.2 Research Contributions**

The first scientific contribution of the present research is the selection of a research subject that might be useful in the SC spatial and land-use planning systems. However, the absence of a building code is a real problem in the SC, not an invented or hypothetical one. The absence of a building code, as a topic, has never beforehand been a reason for the systematic research of Sarajevo planning and coding system.

The second scientific contribution is the elaboration of the analysis of the coding and planning system in Sarajevo in a chronological and systematic order for the first time and for the first time in the English language.

Very few professionals from Sarajevo have written fragmentally and generally about problems and solutions for the Sarajevo spatial planning system and even less have written about the absence of building norms and standards, mostly in the previous century. Among the most significant ones are Architects Juraj Neidhardt, Muhamed Kadić, Ivan Taubman, Jahiel Finci, Aleksandar Levi, Mehmed Bublin, and certainly Midhat Aganović, former managing director of the City of Sarajevo Development Institute in the 1970s and the Institute for the City of Sarajevo Planning in the 1980s.

The third scientific contribution is a clearly defined methodology for the examination of a spatial and land-use implementation system sustainability in the absence of a building code, through a process evaluation of the building permit procedure in the SC.

The methodology framework of the combined comparative-historical method, with the relevant historical references and contemporary spatial planning systems and building permit procedures in Austria and Switzerland, empirical–analytical method with a representative sample of over 60,000 analyzed requests in the building permit procedure in the last decade in the SC, the institutional land-use implementation analysis, and the Delphi method conducted with local experts, represents an in-depth analysis that can provide a complete scientific gnosis on the justification of reintroducing the new building code in Sarajevo.

The fourth scientific contribution is the reopening of the constructive dialogue between European spatial planners and urban jurists about the spatial planning system in the European countries in transition, such as Bosnia-Herzegovina and its capital city Sarajevo, challenged by its past and present, albeit directed toward the EU future.

The aim is to develop a model for introducing a new building code to the SC spatial and land-use planning legislation, which is motivated by the will and professional dedication to find a new modus operandi for developing a sustainable city in the future.

## **6.3 Building Code Model Limitations and Future Research**

The limitations in the application of a proposed model are related to the absence of a national legislation to guide the implementation of public policies and an ineffective planning implementation system in BiH based on urban permits. In other words, an essential reform of the spatial planning system in BiH is needed as a precondition for introducing a building code document.

When speaking about the future sociopolitical and economic environment in BiH, in general, one has to mention its path to EU membership, particularly because the building code is primarily the European legislative heritage contributing to the founding value of the EU, which is a stronger rule of law.

A building code represents a challenge for the current transitional BiH spatial and land-use planning legislative systems. Albeit in the future, if applied, it may contribute to the strengthening of the legislative system and true integration of BiH to the continental European planning and coding systems. It would certainly be much easier to think of introducing the building code to the legislative framework of the SC in light of BiH becoming a member state when European acts and directives can become binding to BiH. The legislative gap between eastern and western Europe would be overcome, following the cohesion policy principles. Sarajevo and BiH would be a part of a wider European region.

The new model for the SC building code document may also be applied in the future spatial system in BiH and other BiH cities, considering their characteristics and specificities.

Future research relates to the several aspects of a spatial planning system as a whole and a building code in particular, such as integrative development, environmental protection, economy aspects of a building code, institutional land-use planning implementation evaluation, and regional planning; testing the hypothetical framework of this research in other southeastern European, Mediterranean, and global contexts; and creating informal instruments based on a building code, such as spatial concepts and technical guidelines.

## **Reference list**

Editorial

Commission of the European Communities. (1998). *Sustainable Urban Development in the European Union: A Framework for Action*. Brussels: CEC

aptation [Priručnik o tehničkim i obligacionim uvjetima za projektovanje i izvođenje radova na izgradnji, rekonstrukciji, sanaciji adaptaciji građevina visokogradnje]. Rabic, Sarajevo, BiH

Koolhaas, R. & Mau, B. (OMA). (1995). *S, M, L, XL.* Rotterdam: 010 Publishers

century: A territory of technical and political innovation]. *Les Annales de la recherche urbaine*, 1992. No.57-58, (p. 24-45)

*Grad, Ilidža i Vogošća) za period od 1986. do 2015. godine* [The City of Sarajevo Land-use Plan for the urban territory of Sarajevo (Stari Grad, Centar, Novo Sarajevo, Novi Grad, Ilidža i Vogošća) for the period 1986 – 2015]. Sarajevo Službene novine Kantona Sarajevo br. 4/90

In most European countries, spatial and land-use planning documentation (formal and informal) and building codes complement each other. The city of Sarajevo, however, has not had a building code over the last eight decades. The author, with many years of professional experience as a planner in Sarajevo, shows how this has affected the spatial planning system in all its segments, including through a weak building permission procedure. She compares Sarajevo's legal framework to the planning systems and building regulations of Slovenia, Vienna, Zurich, and Paris. Arguing that a building code is an inseparable part of land-use management and land-use planning implementation system as well as of sustainable, effective urban politics, the author proposes a model for a new building code and a comprehensive planning system for the Canton of Sarajevo.

With implications for spatial planning beyond Bosnia and Herzegovina, the book is highly relevant for planning policy and administration, but also for the scientific community: It addresses spatial and urban planners, jurists, architects, sociologists, and historians of architecture in Continental and South-East Europe.


**7. Discussion**

*Updates in Volcanology – Transdisciplinary Nature of Volcano Science*

Platy juvenile clasts related to penetration of magma into the host sediment are commonly extensively altered into laumontite and iron oxides (**Figure 11E**). The same assemblage typically replaces irregularly shaped juvenile clasts in peperites

*(A) juvenile clasts (white) altered to laumontite (Lmt), iron oxides (o) and chlorite (Chl). The composition of the mingling siliciclastic sediment (S) remained largely unaltered; (B) a juvenile clast altered to laumontite (Lmt), epidote (Ep) and actinolite (Ac); (C) a clast of fine-grained pyroclastic deposit incorporated in a lava flow has been altered to albite (Ab), quartz (Qtz), chlorite (Chl) and Fe-oxides (o). Some clasts underwent magma assimilation (ma); (D) the mingling fine-grained pyroclastic deposit (S) formed a large oval inclusion in a juvenile clast that has been partially altered into laumontite (Lmt). Smaller inclusions are altered to pumpellyite (Pmp) and quartz, some small spherical inclusions are unaltered (o) and the other replaced by chlorite (Chl). juvenile clasts (jc); (E) an inclusion of fine-grained pyroclastic deposit (s) in a juvenile clast altered to prehnite (Prh) and laumontite (Lmt); (F) inclusions of fine-grained pyroclastic deposit in a juvenile fragment (brownish) altered to pumpellyite (Pmp), epidote (Ep), quartz (Qtz) and albite (Ab). Volcanic*

The alteration of peperites related to coarse-grained host sediment is characterised by chlorite, interlayered chlorite-smectite with more than 90% of chlorite layers, quartz, and sometimes incipient epidote. Laumontite and other zeolites, and

formed during the emplacement of the Krmarica Sill (**Figure 11F**).

*glass in the juvenile clast is locally replaced by chlorite (Chl).*

pumpellyite, prehnite and epidote are uncommon (**Figure 12A**-**D**).

**316**

**Figure 13.**

The formation of alteration minerals in volcanic-hydrothermal systems is a complex process affected by temperature, pressure, composition of reacting fluids, porosity, permeability and initial composition of the host-rock, duration of hydrothermal activity and superimposed thermal (or hydrothermal) regimes [55, 56]. Nevertheless, there is a general relationship between temperature and the formation of alteration minerals, and some mineral assemblages can be used to interpret temperatures within a geothermal system (**Figure 14**). For heulandite and stilbite, laumontite, yugawaralite, pumpellyite and actinolite widely accepted temperature stability ranges are 100-120°C, 120-220°C, 172-234°C, 200-310°C, 220-310°C and 280-460°C [52, 55–65]. Incipient, fine-grained and poorly crystallised epidote has been encountered in hydrothermal systems of the Philippines [53, 55] and the Nisyros Island [63], respectively, in the temperature range of 180-220°C although epidote generally forms at temperatures higher than 240°C [64, 65]. The temperature stability range of 245-265°C has been reported for mixed-layer R0 and R1 chlorite-smectite from Nesjavellir geothermal field, Iceland [66].

### **Figure 14.**

*Authigenic calcium-aluminosilicate minerals that commonly serve as geothermometers in volcanichydrothermal systems, their chemical formulae and temperature stability ranges, compiled from [52, 55–65].*

The alteration of peperites from the Smrekovec Volcanic Complex indicates close relationship to the rock composition and texture, and therefore, the style of peperite formation. Porosity and permeability of peperites is, in general, lower than that of the host sediment. The types involving fine-grained host sediments have low permeability and low porosity, and in the stratovolcano-hosted hydrothermal system with convective-advective flow regime (**Figure 3**) they must have functioned as aquicludes unable to drain effectively the advective flow of hydrothermal fluids that largely controlled hydrothermal alteration of volcanic deposits [37]. In such hydrothermal system, peperites involving coarse-grained host sediments should be more extensively altered and contained authigenic minerals with higher temperature stability ranges than the types involving fine-grained host-sediments, but that is not the case. On contrary, in this type of peperites significant authigenic calcium-aluminosilicate minerals are often lacking and the most common alteration mineral is chlorite or interlayered chlorite-smectite.

In dispersed blocky peperite, matrix composed of siliciclastic host sediment is usually unaltered. The alteration of juvenile clasts often indicates only the reactions of devitrification of volcanic glass, and only juvenile clasts with perlitic cracks can be altered to laumontite and Fe-oxides (**Figure 9A**). The activity of hot fluids originating from heated pore waters can be assumed, but chemical gradients favourable for the formation of laumontite were attained only inside the juvenile clasts with perlitic cracks. Perlitic cracks apparently served as conduits for hot fluids that leached volcanic glass during the flow, and in this manner underwent the changes in chemical composition (e.g. [67]) that finally resulted in crystallisation of laumontite. Far more extensive alteration of blocky peperites involving fine-grained pyroclastic host sediment supports the forementioned explanation. The interaction of heated pore fluids and highly reactive host sediment apparently controlled geochemical evolution of so-formed hydrothermal solutions and the related extensive alteration of all constituents of peperite. Laumontite or mineral assemblages of laumontite, albite, quartz, pumpellyite, incipient epidote and chlorite, or laumontite, prehnite, quartz, chlorite and incipient epidote, or laumontite, analcime and interlayered chlorite-smectite indicate that temperature gradients were prerequisite but not sufficient for alteration to occur and that the main controlling factor were geochemical gradients.

Microglobular peperite has been interpreted as a frozen example of a fuelcoolant interaction (FCI) between magma and fluidised host sediment [5], and the

**319**

*Submarine Stratovolcano Peperite Syn-Formational Alteration - A Case Study of the Oligocene…*

temperatures of alteration reactions in direct contact with magma must have been higher than in blocky peperite. Microglobules of fine-grained pyroclastic sediment underwent alteration, and typically, smaller microglobules are commonly completely altered to authigenic mineral assemblages with higher temperature stability ranges (e.g. pumpellyite, epidote) than the larger ones that remained incompletely altered or altered to authigenic minerals with lower temperature stability ranges (e.g. laumontite) (**Figure 13D**). The relationship indicates that high-temperature

Despite of the complexity of alteration of volcanic deposits in lithofacies associations of the sections Krnes and Smrekovec G34 the assemblages of authigenic minerals in peperites are different than those in the adjacent underlying and overlying autoclastic, pyroclastic or resedimented volcaniclastic deposits irrespectively of their texture and grain-size (**Figures 4** and **5**). Particularly outstanding is the occurrence of pumpellyite, actinolite and epidote. In lavas pumpellyite and actinolite very rarely occur as the replacement of volcanic glass and other primary constituents although the temperatures in cooling lavas could be favourable for

If compared to the section Krnes, the alteration of volcanic deposits in the section Smrekovec G34 has been far more complex owing to the emplacement of the Kramarica Sill. Progressive reactions related to an elevated temperature regime are characterised by the occurrence of prehnite and indicate, at least in the lower half of the section, that the temperatures could have reached over 300°C. Yet, even in such elevated temperature regime the exclusive occurrence of pumpellyite and particularly actinolite in peperites indicates that higher temperatures and specific geochemical conditions related to the formation of peperites must have controlled the crystallisation of pumpellyite and actinolite. The occurrence of the same assemblage in peperites in the section Krnes is particularly important. Here, hydrothermal activity and temperature regime were mainly related to a deep igneous source and the associated convective-advective flow of hydrothermal fluids and elevated geothermal gradients in the area of stratovolcano, and they were not affected, at least significantly, by the emplacement and cooling of the Kramarica Sill. Maximum temperatures have been determined by the temperature stability of laumontite, namely, 234°C which is insufficient for the crystallisation of pumpellyite or actinolite. The alteration of juvenile clasts to laumontite in peperites indicates the presence of hydrothermal conditions although they cannot be conclusively ascribed to specific hydrothermal conditions and geochemical gradients related to

The alteration of peperites can be regarded as syn-formational hydrothermal, although it is local, specific and ephemeral lasting until thermal gradients persisted. Authigenic mineral assemblages developed in peperites from the Smrekovec Volcanic Complex are rare on a worldwide scale and have not been identified in such context yet. Geochemical evolution of heated pore fluids circulating in the vicinity of the source of heat controlled the formation of authigenic mineral assemblages and the presence of unstable, reactive volcanic material was crucial for

Peperites are commonly developed in submarine environments with contemporaneous volcanic activity and sedimentation. And although the occurrence and complex processes of formation have been studied and explained in many modern and ancient geological settings worldwide [4, 10–14], and particularly in the

*DOI: http://dx.doi.org/10.5772/intechopen.95480*

their formation.

the formation of peperites.

their formation and diversity.

**8. Conclusion**

conditions could not persist for a long period of time.

### *Submarine Stratovolcano Peperite Syn-Formational Alteration - A Case Study of the Oligocene… DOI: http://dx.doi.org/10.5772/intechopen.95480*

temperatures of alteration reactions in direct contact with magma must have been higher than in blocky peperite. Microglobules of fine-grained pyroclastic sediment underwent alteration, and typically, smaller microglobules are commonly completely altered to authigenic mineral assemblages with higher temperature stability ranges (e.g. pumpellyite, epidote) than the larger ones that remained incompletely altered or altered to authigenic minerals with lower temperature stability ranges (e.g. laumontite) (**Figure 13D**). The relationship indicates that high-temperature conditions could not persist for a long period of time.

Despite of the complexity of alteration of volcanic deposits in lithofacies associations of the sections Krnes and Smrekovec G34 the assemblages of authigenic minerals in peperites are different than those in the adjacent underlying and overlying autoclastic, pyroclastic or resedimented volcaniclastic deposits irrespectively of their texture and grain-size (**Figures 4** and **5**). Particularly outstanding is the occurrence of pumpellyite, actinolite and epidote. In lavas pumpellyite and actinolite very rarely occur as the replacement of volcanic glass and other primary constituents although the temperatures in cooling lavas could be favourable for their formation.

If compared to the section Krnes, the alteration of volcanic deposits in the section Smrekovec G34 has been far more complex owing to the emplacement of the Kramarica Sill. Progressive reactions related to an elevated temperature regime are characterised by the occurrence of prehnite and indicate, at least in the lower half of the section, that the temperatures could have reached over 300°C. Yet, even in such elevated temperature regime the exclusive occurrence of pumpellyite and particularly actinolite in peperites indicates that higher temperatures and specific geochemical conditions related to the formation of peperites must have controlled the crystallisation of pumpellyite and actinolite. The occurrence of the same assemblage in peperites in the section Krnes is particularly important. Here, hydrothermal activity and temperature regime were mainly related to a deep igneous source and the associated convective-advective flow of hydrothermal fluids and elevated geothermal gradients in the area of stratovolcano, and they were not affected, at least significantly, by the emplacement and cooling of the Kramarica Sill. Maximum temperatures have been determined by the temperature stability of laumontite, namely, 234°C which is insufficient for the crystallisation of pumpellyite or actinolite. The alteration of juvenile clasts to laumontite in peperites indicates the presence of hydrothermal conditions although they cannot be conclusively ascribed to specific hydrothermal conditions and geochemical gradients related to the formation of peperites.

The alteration of peperites can be regarded as syn-formational hydrothermal, although it is local, specific and ephemeral lasting until thermal gradients persisted. Authigenic mineral assemblages developed in peperites from the Smrekovec Volcanic Complex are rare on a worldwide scale and have not been identified in such context yet. Geochemical evolution of heated pore fluids circulating in the vicinity of the source of heat controlled the formation of authigenic mineral assemblages and the presence of unstable, reactive volcanic material was crucial for their formation and diversity.


*Article* **Particle-Size-Dependent Delivery of Antitumoral miRNA Using Targeted Mesoporous Silica Nanoparticles**

**Lisa Haddick 1, Wei Zhang 2, Sören Reinhard 2, Karin Möller 1, Hanna Engelke 1, Ernst Wagner 2 and Thomas Bein 1,\***

Received: 11 April 2020; Accepted: 27 May 2020; Published: 2 June 2020

**Abstract:** Multifunctional core-shell mesoporous silica nanoparticles (MSN) were tailored in size ranging from 60 to 160 nm as delivery agents for antitumoral microRNA (miRNA). The positively charged particle core with a pore diameter of about 5 nm and a stellate pore morphology allowed for an internal, protective adsorption of the fragile miRNA cargo. A negatively charged particle surface enabled the association of a deliberately designed block copolymer with the MSN shell by charge-matching, simultaneously acting as a capping as well as endosomal release agent. Furthermore, the copolymer was functionalized with the peptide ligand GE11 targeting the epidermal growth factor receptor, EGFR. These multifunctional nanoparticles showed an enhanced uptake into EGFR-overexpressing T24 bladder cancer cells through receptor-mediated cellular internalization. A luciferase gene knock-down of up to 65% and additional antitumoral effects such as a decreased cell migration as well as changes in cell cycle were observed. We demonstrate that nanoparticles with a diameter of 160 nm show the fastest cellular internalization after a very short incubation time of 45 min and produce the highest level of gene knock-down.

**Keywords:** mesoporous silica nanoparticles; core-shell; surface functionalization; cell targeting; size-dependent delivery; antitumoral microRNA (miRNA); confocal microscopy


*Article* **Canine Olfactory Detection of a Non-Systemic Phytobacterial Citrus Pathogen of International Quarantine Significance**

**Timothy Gottwald 1,\*, Gavin Poole 1,2, Earl Taylor 1, Weiqi Luo 1,2, Drew Posny 1,2, Scott Adkins 1, William Schneider 3 and Neil McRoberts 4**

Received: 19 August 2020; Accepted: 27 October 2020; Published: 9 November 2020

**Abstract:** For millennia humans have benefitted from application of the acute canine sense of smell to hunt, track and find targets of importance. In this report, canines were evaluated for their ability to detect the severe exotic phytobacterial arboreal pathogen *Xanthomonas citri* pv. *citri* (Xcc), which is the causal agent of Asiatic citrus canker (Acc). Since Xcc causes only local lesions, infections are non-systemic, limiting the use of serological and molecular diagnostic tools for field-level detection. This necessitates reliance on human visual surveys for Acc symptoms, which is highly inefficient at low disease incidence, and thus for early detection. In simulated orchards the overall combined performance metrics for a pair of canines were 0.9856, 0.9974, 0.9257 and 0.9970, for sensitivity, specificity, precision, and accuracy, respectively, with 1–2 s/tree detection time. Detection of trace Xcc infections on commercial packinghouse fruit resulted in 0.7313, 0.9947, 0.8750, and 0.9821 for the same performance metrics across a range of cartons with 0–10% Xcc-infected fruit despite the noisy, hot and potentially distracting environment. In orchards, the sensitivity of canines increased with lesion incidence, whereas the specificity and overall accuracy was >0.99 across all incidence levels; i.e., false positive rates were uniformly low. Canines also alerted to a range of 1–12-week-old infections with equal accuracy. When trained to either Xcc-infected trees or Xcc axenic cultures, canines inherently detected the homologous and heterologous targets, suggesting they can detect Xcc directly rather than only volatiles produced by the host following infection. Canines were able to detect the Xcc scent signature at very low concentrations (10,000× less than 1 bacterial cell per sample), which implies that the scent signature is composed of bacterial cell volatile organic compound constituents or exudates that occur at concentrations many fold that of the bacterial cells. The results imply that canines can be trained as viable early detectors of Xcc and deployed across citrus orchards, packinghouses, and nurseries.

**Keywords:** early detection; Asiatic citrus canker; latent class; information theory; field diagnostic; scent signature; direct assay; deployment

#### **1. Introduction**

Asiatic citrus canker (ACC) is a fruit, foliar, and twig lesion disease that has significant international, national, and local quarantine implications and has been the focus of multiple extensive eradication

programs. The disease is caused by the bacterium *Xanthomonas citri* pv. *citri* (Xcc). When pathogen infection is severe, leaf drop and up to 69% crop loss can occur due to fruit drop [1]. The bacteria disperse via meteorological events ranging from gentle rain to tropical storms and hurricanes. The more severe the meteorological event, the more effective the inoculum dispersal [2,3]. Hurricanes and tropical storms have been associated with long-range dissemination as well as local increase of the bacterium [2,4–6]. Xcc inoculum can also be transmitted mechanically by machinery and humans if the foliage is wet. Inoculum begins to exude from Xcc lesions within 1–5 min of becoming wet [4,6–8]. The maximum concentration of bacteria is exuded within the first 1–2 h period following the beginning of the rainfall event or wetting of the lesion, although inoculum is produced continuously at a lower concentration for the duration of the wetting period or storm [2–4]. Infection takes place when inoculum-laden water passes through stomata of foliage, fruit, or green twigs. Infection can occur through wounds as well, and it is highly exacerbated by the Asian citrus leafminer (*Phyllocnistis citrella* Stainton), whose feeding galleries create a labyrinth of wounds that expose susceptible leaf mesophyll tissues to splashed inoculum, greatly increasing the probability of infection by Xcc [9,10].

After infection occurs, bacteria propagate within the plant tissues, eventually forming small blister-like protrusions that become visible 5–7 days post-infection with close examination augmented by 10× magnification. Within 12–14 days, these protrusions erupt through the epidermis, forming 1–2 mm light brown erumpent lesions. As the lesions age, they darken to brown and develop water-soaked margins with a surrounding chlorotic halo [7]. The lesion center develops a raised spongy and corky appearance on adaxial and abaxial foliar surfaces. Older lesions can reach 1 cm in diameter and can coalesce to form mass infections.

Pathogen detection in the field is almost exclusively by human visual inspection. Confirmation of Xcc infection can be accomplished by serological or Polymerase Chain Reaction (PCR) assays, both of which require infected tissue. Therefore, such assays are of minimal use for a field survey of a pathogen that causes only local lesions, i.e., non-systemic infections. Visual inspection is tedious, labor intensive, and highly variable due to the elusiveness of Xcc lesions, especially when high in the canopy. Since mature citrus trees can have in excess of 100,000 leaves, finding initial infections visually in such a large canopy is challenging and uncertain. As a consequence, multiple infection cycles must occur before symptoms are of sufficient prevalence to permit visual detection. During a major epidemic in Florida (1996–2006), the detection time for Xcc by trained regulatory inspectors averaged 106 days post-infection [11]. In one regulatory exercise, 14 teams of two inspectors per team examined the same infected orchard in succession. No two teams found the same infected trees, each successive team found new infected trees previously undetected by prior teams, and no team found all known infections. [Riley, unpublished results]. Since it is highly improbable that all infected trees in an orchard will be found by visual inspection, the true incidence of infection cannot be determined, and thus, calculating the detection accuracy of visual inspection is not possible.

The unpromising results for human visual detection suggest the need for the deployment of a detection technology with much better performance. Genetic analyses indicate that modern-day canines (*Canis lupus familiaris*) were the first domesticated animal, arising from two separate wolf (*Canis lupus*) populations ≈ 15,000 years ago [12–17]. The mammalian (including canids) olfactory system is antediluvian, having evolved from early chemotactic receptors during the Precambrian over 600 million years ago. Acute olfaction evolved to enhance finding food, mates, detecting danger, avoiding predators, etc. [18]. An overarching advantage to the human use of canine olfaction is that all canids non-destructively interrogate their environment for a scent signature of interest. This scent signature is composed of a specific volatile organic compound (VOC) or complex of VOCs. In contrast, commonly used molecular or biochemical assays often require destructively sampling a small proportion of the host or environment and are specific and do not detect complex VOC composites.

Wolves employ acute vision and hearing when prey is in close proximity. However, when tracking prey, often over great distances, wolves resort to olfactory cues to locate widely dispersed and often low-density prey [19], as is true for domestic canines [20]. To find rare targets of human interest, we exploit the tracking expertise and acute sense of smell handed down to modern canines from wolves and their evolutionary predecessors. Nonetheless, scent detection is pervasive through the animal kingdom. Other vertebrates and invertebrate animals have been explored recently in the burgeoning science of scent detection research with diverse practical applications [21]. However, canines dominate as olfactory detector tools because of their unsurpassed domestication and easily deployed tracking skills.

Canines are used broadly to detect and locate a wide array of organic and inorganic odors, (e.g., explosives, drugs, accelerants, pollutants, toxins, pesticides), tracking humans and game animals, finding cadavers, and matching the scent of criminal perpetrators to crime scenes [22]. Canines are also proficient at medical human disease detection, especially cancer (malignant melanoma, non small-cell, small-cell lung, breast, prostrate, bladder, ovarian, and colorectal cancers) often with equivalent or superior sensitivity to current medical assays and can detect epileptic seizures prior to onset [22–25].

Relative to agricultural diseases, canines were recently trained extensively to detect *Candidatus* Liberibacter asiaticus (CLas), which is the causal pathogen of citrus Huanglongbing with greater than 99%, 96%, and 92% accuracy in field trials, commercial citrus orchards and citrus in residential properties, respectively [26]. In the same study, canines detected CLas in the Asian citrus psyllid, *Diaphorina citri,* that vectors the bacteria and in bacterial co-cultures devoid of plant or animal host cells. Additionally, canines have been trained to detect plum pox virus in commercial peach (*Prunus persica*) orchards [Gottwald unpublished] and the fungal pathogen *Ra*ff*aelea lauricola*, which is the cause of Laurel wilt disease of avocado (*Persea americana*) [27]. Thus, canines have been demonstrated to detect bacterial, viral, and fungal pathogens in plant hosts.

Canine detection of the plant pathogens as indicated above represents a novel extension of previous detection targets. For drugs, explosives, pollutants, etc., canines are trained to detect specific VOC scent signatures of the target compounds. For biologicals such as plants, plant parts, humans, and other animals, the canines are trained to recognize the volatilome, e.g., unique VOC complex, emitted by the target. Conversely, when trained to detect pathogenic organisms, the canines must detect one organism (bacterium, virus, or fungus) within another organism, i.e., the host plant or animal. The three plant pathogens discussed above, CLas, plum pox virus and *R. lauricola,* are all systemic within the vascular system of the host, and therefore, they can be completely or incompletely distributed within the plant as the infection progresses. In contrast, Xcc is non-systemic and causes only local lesions, which can range in incidence from a single lesion to thousands per tree and can be rare, highly aggregated, or diffused within the host.

In this study, we demonstrate proof of concept that canines can discriminate and detect Xcc and can be trained as viable detectors of the pathogen in agricultural environments. Here we document the detection of Xcc, an exotic bacterial pathogen by use of canine olfactory surveillance in orchards and packinghouses. Finally, we determine that canines can directly detect the target bacterium *in planta* and *in vitro*.

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

#### *2.1. Initial Sensitization Training of Canines for Xcc Scent Signature Recognition*

Eleven canines of various breeds were utilized for the various trials throughout the course of the study (Table 1).

**Table 1.** Canines trained to detect *Xanthomonas citri* pv.*citri* (Xcc) scent signature and utilized in this study.

At the onset of this study, Xcc was considered a quarantine pathogen in Florida [3,6,7,11], and neither the bacteria nor infected plant material could be transported to new locations for experimental purposes. Therefore, we used a Scent Transfer Unit (STU) (Model STU-100, Tolhurst Big "T" Enterprises, Lockport, New York, NY, USA, 14094) vacuum device commonly used in canine detection work to draw air at a constant rate (≈300 L/min) to collect volatile samples containing the "scent signature" of the target, in this case Xcc-infected plants, and deposit the scent onto a cotton "scent pad" [28]. Since the scent pad does not contain the pathogen (Xcc is only splash dispersed, generally by rain and not dispersed in dry air) nor any potentially infected plant material, it can be safely transported to non-endemic areas for testing. Scent pad samples were collected from non-infected and Xcc-infected Ruby Red grapefruit (*Citrus paradisii*) trees using the STU. A sterile 12.7 × 22.9 cm (5 × 9 inch) cotton pad was placed into the STU for 10 min by setting the STU within the canopy of a non-infected or Xcc-infected tree 5–10 cm from leaves, fruit and branches while air was pulled through the pad. Following each 10 min sampling period, scent pads were aseptically removed, placed in a volatile-proof plastic bag (K-pak), and heat sealed. The STU was disinfected with ethyl alcohol between each use. Multiple heat-sealed samples from infected and non-infected trees were segregated, placed in zip lock plastic bags, and stored at −20 ◦C until transported (Figure 1A–C).

Canine detection companies are for-profit ventures, and therefore, they are often reticent to undertake non-profitable and time-consuming basic research. Fortuitously, a canine detection company in California was willing to dedicate two canines to do an initial testing of canine detection of Xcc. In preliminary studies, it was not feasible to bring canines to the Xcc-infected sites due to quarantine constraints. Therefore, scent pad samples were collected in South Florida, sealed in a styrofoam shipping box with ice packs, and transported overnight to the California training facility.

One Belgian Malinois (Kimba) and one German Shepard (Tank), previously trained for criminology scent detection, were trained to the Xcc scent signature by imprinting, during which the canine was introduced to the target and other neutral scents. Relative to scent signature training, when the canine becomes interested in/reacts to the correct target, the experience is immediately encouraged with verbal and play rewards [18]. In our case, canines were imprinted to the scent signature presented on Xcc-infected scent pads to discriminate the scent from scent pads without the scent signature. To take advantage of the candid passion to search and track, a mixed population of mostly non-infected with a few Xcc-infected scent pad samples were arrayed in a series of rows of metal cans placed outdoors on metal stands (Figure 1D). Correctly alerting on the Xcc-infected pad placed in the bottom of a can resulted in the canine receiving verbal praise and a few moments of play with the handler and a ball

or "kong" (hard rubber ball on a short rope). Initial training used non-frozen scent pads. Canines subsequently detected both non-frozen and frozen scent pads from Xcc-infected trees. Thereafter, scent pads were stored frozen until use in training and assessment.

**Figure 1.** Initial training of canines for detection of the phytobacterial arboreal pathogen *Xanthomonas citri* pv. *citri* (Xcc), the causal agent of Asiatic citrus canker (Acc). (**A**) Xcc-infected red grapefruit fruit and (**B**) foliage. (**C**) Scent Transfer Unit (STU) used to draw in Xcc volatiles and deposit on cotton scent collection pad. (**D**) Canine "Kimba" training by interrogating a row of metal cans containing Xcc-positive and negative scent pads. (**E**,**F**) Detector canine NDD-1 and NDD-3 alerting on boxes containing Xcc-infected foliage at the USDA, APHIS, National Detector Dog Training Center.

Following initial scent training, a series of preliminary studies were conducted in which canines interrogated scent pads exposed to Xcc-infected red grapefruit for periods of 1, 5, 10, or 30 min versus pads exposed to non-infected trees for similar times, to determine if there was a lower threshold of exposure necessary to create scent pad training materials. Additionally, once the scent pads were removed from the heat-sealed plastic bags, the canines interrogated them every few days to determine the temporal viability of the scent pads as a training tool.

## *2.2. Training Canines for Detection of Xcc-Infected Plant Material*

During 2004–5, in collaboration with the U.S. Department of Agriculture, Animal and Plant Health Inspection Service (USDA, APHIS), National Detector Dog Training Center (NDDTC) in Orlando, Florida, we trained canines to detect Xcc-infected Duncan grapefruit seedlings. At the USDA, ARS laboratory in Fort Pierce, Florida, quarantine greenhouse, seedlings were infected by placing drops of Xcc inoculum, ≈106 to 108 cfu/mL prepared from pure cultures on young leaves and using a sterile needle to wound the leaf lamina, causing 5–10 wounds/leaf of five leaves/seedling. Seedlings were incubated in the greenhouse for ≈2–3 weeks until symptomatic and then transported under regulatory permit in sealed containers to the NDDTC in Orlando along with non-infected seedlings as training

materials where they were maintained until use. At the NDDTC, two beagles (NDD-1 and NDD-2), one German pointer (NDD-3), and one Labrador retriever (NDD-4) were trained to recognize the Xcc scent signature by a similar imprinting/reward method as described above. Scent detection proficiency was tested by placing excised branches or whole potted Xcc-infected or non-infected trees in closed cardboard boxes and arraying the boxes on the floor of the training facility (Figure 1E,F). In a second NDDTC study, the trained Labrador was transported to an Xcc-infected orchard in Indian River County, Florida. To avoid disease quarantine issues, leaves were collected from Xcc-infected and non-infected trees and placed into separate sealed plastic pipe containers with holes drilled in the sides to allow the volatiles but not infected plant material to escape for canine interrogation. The canine interrogated the containers, which were placed on the ground at the edge of the orchard. When the trial ended, the infected and non-infected plant materials were removed from the containers, discarded in the infected field, and the containers were disinfected. Due to quarantine issues, intermittent and uncoordinated testing by NDDTC personnel in absence of the authors, and uncoordinated replacement of canines assigned to the study, data were not consistently taken with the exception of an overall assessment of performance by NDDTC personnel.

In all subsequent studies, we collaborated with three commercially certified canine detector companies who specialize in trained detector canines for military, police and domestic clientele, for the detection of an array of targets including explosives, drugs, bed bugs, etc. This was important because professional canine handlers are themselves trained not to give any voluntary or involuntary cues to the canines while testing their performance. A single handler was used for all replications of most studies to avoid bias or variation due to handler. These companies trained nine canines (7 were used in this study) for the detection of Xcc via imprinting on scent pads from Xcc-infected trees or Xcc-infected grapefruit seedlings until canines gained proficiency at differentiating Xcc-infected from non-infected samples (Table 1).