**2.3 Hot injection method**

The stability of inorganic perovskites is significantly higher than that of hybrid perovskite crystals. By changing the organic cation with an inorganic one (e.g., Cs),

#### **Figure 2.**

*(a) Schematic illustration of Set up for LARP; (b) starting materials and shape of perovskite nano-crystals; (c) image of typical solution containing CH3NH3PbBr3 nano-structures (reproduced with permission of Ref. [26]).*

#### *Perovskite and Piezoelectric Materials*

chemical and thermal stability of Perovskite material can be increased greatly (**Figure 3**) [31, 32]. It is possible to obtain various crystal phases by changing the temperature resulting a shifting in the optical responses. The hot injection method is a widespread method for synthesizing inorganic PNSs. In a typical synthesis procedure, a solution of PbX2 (X = I/Br/Cl), in octadecene (ODE) along with oleic acid and oleyl amine is prepared. During stirring, Cs-oleate is injected into that solution quickly under dry condition at 140–200°C [32]. To quench the reaction (after 5–10 sec), the reactor is cooled with an ice bath. With this method, it is easy to obtain uniform nanocubes with the size of 4–15 nm edge length representing high PLQY, up to 50–90% and a very narrow emission band (12–42 nm) in the visible region (410–700 nm). As it was mentioned before, the optical properties of the NPSs are related to shape, size,

#### **Figure 3***.*

*Schematic illustration of Hot-injection method (left-hand), image of Cs2SnI6 samples under UV-light (righthand) and possible crystal shapes obtained by hot-injection method (reproduced with permission of Ref. [33]).*

#### **Figure 4.**

*Schematic illustration of perovskite nano-crystal preparation by the template-assisted method (reproduced with permission of Ref. [36]).*

**53**

**Figure 5.**

*Perovskite Nanoparticles*

perovskites.

*DOI: http://dx.doi.org/10.5772/intechopen.94588*

**2.4 Template-assisted method**

applications such as lasers [36, 37].

**2.5 Emulsion method**

capping agent.

and surface chemistry of them due to the significant changes in the band structures. Therefore, by the variation of the surfactants, ligands, reaction temperature and time, the perovskite precursor composition can be adjust [33] to realize the formation of nanowires, nanoplatelets, spherical dots, and nanorods (**Figure 3**) [33–35]. A critical point for the hot injection is the reaction temperature effecting the size of the NPSs. To overcome this disadvantage, new methods have been developed for hybrid

In this synthesis approach, NP formation is induced by a specific substrate such as mesoporous silica and aluminum oxide film as a template [36–38]. NPSs is obtained on the films which exhibits intensive light. This technique is suitable for formation of mono disperse NPSs with various narrow emissions (full wind of half maximum (FWHM) is less than 40 nm) bands from green (FWHM: 22 nm) to near infrared (FWHM: 36 nm) by template optimization [37–39]. In a recent study, perovskite nanocrystals have been prepared in a nano-porous structure. By this kind of strategies, it is possible to confine PNSs (<10 nm) without any capping agents (**Figure 4**). In other words, the emission wavelength of perovskite nanoparticles can be arranged precisely for sophisticated photonic

In order to control the crystallization of perovskite and obtain uniform NPs, emulsion synthesis method was modified [40]. By using this method, it is possible to tune the size of PNSs under 10 nm with an PLQY up to 92% [41]. In this method, an emulsion is prepared with two immiscible solvents. After that, a demulsifier is added into this for initiating solvent mixing and start crystallization (**Figure 5**). For this procedure, DMF and n-hexane are very good candidates as immiscible solvents while tert-butanol or acetone is used as demulsifier solvent [41]. This method is suitable to obtain PNSs in solid state which can be used in another solvent matrix for an application later. Furthermore, long alkyl ammonium halides are used as

*(a) Schematic illustration of preparation of nano-crystals by emulsion method; (b) ternary phase diagram (DMF/OA/n-hexane); (c) image of a typical CH3NH3PbBr3 emulsion, colloidal NP solution and solid-state*

*powder of CH3NH3PbBr3 NPs (reproduced with permission of Ref. [41]).*

### *Perovskite Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.94588*

*Perovskite and Piezoelectric Materials*

chemical and thermal stability of Perovskite material can be increased greatly (**Figure 3**) [31, 32]. It is possible to obtain various crystal phases by changing the temperature resulting a shifting in the optical responses. The hot injection method is a widespread method for synthesizing inorganic PNSs. In a typical synthesis procedure, a solution of PbX2 (X = I/Br/Cl), in octadecene (ODE) along with oleic acid and oleyl amine is prepared. During stirring, Cs-oleate is injected into that solution quickly under dry condition at 140–200°C [32]. To quench the reaction (after 5–10 sec), the reactor is cooled with an ice bath. With this method, it is easy to obtain uniform nanocubes with the size of 4–15 nm edge length representing high PLQY, up to 50–90% and a very narrow emission band (12–42 nm) in the visible region (410–700 nm). As it was mentioned before, the optical properties of the NPSs are related to shape, size,

*Schematic illustration of Hot-injection method (left-hand), image of Cs2SnI6 samples under UV-light (righthand) and possible crystal shapes obtained by hot-injection method (reproduced with permission of Ref. [33]).*

*Schematic illustration of perovskite nano-crystal preparation by the template-assisted method (reproduced*

**52**

**Figure 4.**

*with permission of Ref. [36]).*

**Figure 3***.*

and surface chemistry of them due to the significant changes in the band structures. Therefore, by the variation of the surfactants, ligands, reaction temperature and time, the perovskite precursor composition can be adjust [33] to realize the formation of nanowires, nanoplatelets, spherical dots, and nanorods (**Figure 3**) [33–35]. A critical point for the hot injection is the reaction temperature effecting the size of the NPSs. To overcome this disadvantage, new methods have been developed for hybrid perovskites.


*2.5. Uncertainty Analysis*

For comparison with and uncertainty analysis of other possible variable values than those considered in the base case scenario (Table 2), we also evaluate alternative scenarios of uncertain variable values. Table 3 compares the base and the alternative scenarios and lists their variable value differences for the total regional catchment; Supplementary Table S1 does the same for the other four study catchments.

With regard to *P* data, the alternative scenarios outlined in Table 3 (named in the first and explained in the second column, in direct comparison with the base case) consider possible corrections for *P* undercatch bias [37], or for both undercatch and orographic biases [38]. Moreover, the *ET*/*P* ratio used for estimating average *ET* in the recent period 1990–2009 ranges from 0.55 to 0.72 among the local catchments with available data across Greece (Table 1). The base case scenario considers the mean value of *ET*/*P*, whereas two alternative *ET*/*P* scenarios consider corresponding minimum and maximum values (Table 3); the *ET*/*P* values are multiplied with *P* in each data grid cell to obtain grid-cell values of *ET* over each catchment.

Furthermore, considerable uncertainty about irrigation variables may stem from the estimation of water use (withdrawal) per irrigated area in the 1990–2009 period (*Iww2*). For example, for the total regional catchment (Table 3), *Iww2* may be as large as 747 mm/year if the recent irrigated area in its calculation is estimated from the national reported irrigated area [30] (*Aai2* = 13,963 km2) instead of 591 mm/year in the base case scenario that estimates *Iww2* from GMIAv5.0 [32] (*Aai2* = 11,046 km2). The resulting uncertainty range of *Iww2* is then ±78 mm/year.

With this uncertainty range given for *Iww2*, a corresponding uncertainty range of ±78 mm/year may also be expected for *Iww1*, yielding an even wider uncertainty range for the temporal change in *Iww* by considering different possible combinations of *Iww2* and *Iww1* values in the base and alternative scenarios (as outlined in Table 3). Finally, also the value of the irrigation fraction *αirr* = *Aai1*/*Aai2* differs if the recent total irrigated area (*Aai2*) is estimated from the GMIAv5.0 data [32] (11,046 km2 for the regional catchment; base case scenario), or from the national reported data [30] (13,963 km2 for the regional catchment; alternative scenario).


**6. Conclusion**

The MoM-based waveguide port approach was developed to model waveguide port excitation problems on arbitrary conducting and composite geometries. The developed approach was validated for modeling radiation and coupling problems for coaxial ports by comparing the simulated results with those obtained by other approaches and measurements. The approach has been applied to practical EMC problems for microwave antennas fed by coaxial connectors. A good agreement between the simulated and measured results has been demonstrated. The efficiency of the developed approach for solving various complex problems with waveguide excitation has been verified.


**Section 2**

**Animal Models of Liver Regeneration**

120 Liver Regeneration

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**7**

*Japan*

**Analbuminemic Rat Model**

Katsuhiro Ogawa and Mitsuhiro Inagaki

**for Hepatocyte Transplantation**

*Departments of Pathology & Surgery, Asahikawa Medical University,*

Orthotropic whole and split liver transplantation are successful and well-established treatments for liver disease. However, the short supply of donor organs is a major obstacle to the widespread use of this therapy (Merion, 2010). Recent research has focused on cell transplantation as a therapy for liver disease. Although only a small number of hepatocytes can be transplanted into the liver by transfusion into the portal circulation, the transplanted cells can regenerate the normal hepatic tissue when liver growth is impaired as a result of continuous hepatic damage (Roy-Chowdhury & Roy-Chowdhury, 2011; Gilgenkranz, 2010). A number of animal models have demonstrated that much of the liver can be replaced via repopulation by a small number of transplanted hepatocytes, which restore normal liver functions and improve recipient survival. Hepatocyte transplantation is therefore expected to be used as a therapy for human liver disease. To optimize the therapeutic application of hepatocyte transplantation, however, number of problems such as the methods to monitor and evaluate the functionality of the transplanted cells and the suppression of host

The cell transplantation model, which uses Fischer344 (F344) rats as donors and F344 congenic analbuminemic (F344-alb) rats as recipients, provides an excellent system for the investigation of cell transplantation, because no immunosuppressants are required for cell transplantation. In addition, the transplantability can be accurately evaluated with immunohistochemistry to stain for albumin, using PCR to detect albumin mRNA and genomic DNA, and by measuring serum albumin protein. In this review, we describe the hepatocyte transplantation system that uses F344 and F344-alb rats to study the intrahepatic

Analbuminemic rats (Nagase analbuminemic rats, NARs) were first established from Sprague Dawley (SD) rats by Nagase et al. (1979). NARs show extraordinarily low serum albumin, hyperlipidemia and hormonal changes. However, their growth and reproduction rates do not differ from those of normal rats. The amount of total serum protein in NARs is similar to that seen in normal rats due to the increase in proteins other than albumin.

immunological responses against the transplanted cells require mitigation.

**1. Introduction**

hepatocyte transplantation.

**2. Nagase analbuminemic rats (NARs)**


Fruit Tree Physiology

(Rosaceae) using chloroplast *ndhF* and nuclear ribosomal ITS sequences. Journal of Systematics and Evolution.

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2008;**46**:322-332

Prunus

**42**

**45**

**Chapter 3**

**Abstract**

and anaerobic conditions.

**1. Introduction**

Response of Growth Inhibitor

Paclobutrazol (PBZ; IUPAC name: (2RS, 3RS)-1-(4-chlorophenyl)-4, 4-dimethyl-2-(1H-1, 2, 4-triazol-1-yl) pentan-3-ol) is a triazol derivative and an antagonist of gibberellins. It has been shown to inhibit shoot growth in various perennial fruit trees. Paclobutrazol application reduced the number of shoots, transforming trees into a more desirable, spur-type growth habit as the vegetative sink was reduced. This compound induces an early and intense flowering, diminishing vegetative growth and reducing the extension of buds, allowing for ripening and the initiation of apical buds inflorescence. Besides, it also increases fruit set, the years following application as a carryover effect. An increase in return bloom is a common response to paclobutrazol treatment and has been reported for various fruit crops. Paclobutrazol is widely used to advance harvest maturity in various fruit crops and it improves fruit quality in terms of accelerated colour development, delayed and synchronized fruit maturation and control of preharvest fruit drop. It is known to improve fruit physical and fruit chemical characteristics. Fruit calcium is increased for 2–3 years due to carry over effect. It helps in the maintenance of better fruit quality during storage and influences nutrient uptake in various fruit crops including stone fruits. It has been characterized as an environmentally stable compound in soil and water environments with a long half-life under both aerobic

**Keywords:** fruit set, paclobutrazol, flowering, shoot growth, fruit quality

One of the most important elements in fruit orchard management is growth control. Excessive vigour reduces light penetration, yield, fruit quality and an increase in cost of pruning and pest control [1]. On the other hand, many cultivars may set very large number of fruits with unacceptably smaller size and often serious reduction in return bloom and fruit set may occur in the following year leading

to biennial bearing. About 20–80% of the fruit which initially sets drop-off from the tree during various stages of development. Thus, various plant growth regulators are used for the control of vegetative growth, flowering of young trees, thinning of flowers and fruits, delaying fruit abscission, regulation of fruit ripening and improvement of fruit production and quality in bearing trees [2]. Various chemicals are used in horticulturally advanced countries to reduce the amount of pruning. Among the various growth control chemicals tested, paclobutrazol (PB) is one of the most successful and widely used in fruit trees that can retard tree

Paclobutrazol in Fruit Crops

*Naira Ashraf and Moieza Ashraf*


**3.1.5 Summary of the LUND experiment**

The results of the Lund experiments reveal that a single fraction radiotherapy session of 5 or 15 Gy combined with immunization by i.p. injection of irradiated syngeneic tumour cells induces a significant anti-tumour response to intra cranial implanted glioblastoma tumours

**RT 5 Gy + Imu**

**RT 15 Gy + Imu**

**0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32**

**Time after inoculation / days**

**Mann-Whitney 2-tailed test versus Control**

**Tumour weight g**

**Controls No Treatment**

and a combination of radiation therapy and immunization (upper panel).

**Num. Rats**

Fig. 4. Survival plot of intra-cerebral implanted N32 tumours: Controls (Lower panel); Immunization with syngeneic N32 tumours cells (2nd panel); radiation therapy (3rd panel),

> **Median Survival time days**

**Control** 12 19 3 0.19 0.16 **IMU IFN** 13 19 6 NS 0.25 0.23 **RT 5 Gy** 6 19.5 2 NS 0.18 0.10 **RT 15 Gy** 13 23 2 P<0.001\*\*\* 0.16 0.13 **RT 5 Gy +IMU IFN** 7 19 2 NS 0.14 0.09 **RT 15 Gy + IMU IFN 13 27 3 P<0.0001\*\*\* 0.30 0.28**  Table 4. Number of rats, mean survival time, and the significance of Mann-Whitney 2-tailed test versus control is shown in columns 2-4. In the last column is given the tumour weight at time of death of intra cerebral N32 tumours treated with syngeneic IFN transfected tumour

cells (IMU IFN), radiation therapy (RT) and their combination (RT + IMU IFN).

The results of the Lund experiments reveal that a single fraction radiotherapy session of 5 or 15 Gy combined with immunization by i.p. injection of irradiated syngeneic tumour cells induces a significant anti-tumour response to intra cranial implanted glioblastoma tumours

**Immunized IFN**

**RT 15 Gy**

**RT 5 Gy**

**3.1.5 Summary of the LUND experiment**

*Number of living rats with N32 tumors*

**Type of treatment**

in Fischer-344 rats. In the rats, which were inoculated with N32 tumour cells, the combination of 15 Gy single fraction radiation therapy with immunization of IFN- secreting syngeneic cells resulted in increased survival time by about 40% (p<0.001). But none of these rats survived longer than 30 days. In the group inoculated with N29 tumour cells and treated with 5 Gy RT combined with immunization the survival time was significantly increased by 87% (p=0.003), and 75% of the animals survived for more than 170 days. The difference in response of N29 and N32 cell lines indicate that there is difference in immune response in different clones of glioma.

#### **3.2 The Hungarian experience of single fraction RT and Immunization with (GM-CSF, IL-4, IL-12) in a mouse glioma (Gl261) brain tumour model**

In Hungary a study was performed in a mouse glioma (Gl261) brain tumour model with single fraction radiotherapy combined with administration of cytokine-producing cancer cell vaccines (Lumniczky, et al. 2002). Their brain tumour bearing mice were treated with various cytokine producing vaccines made by in vitro transduction of Gl261 tumour cells with different genes such as: IL-4, IL-6, IL-7, GM-CSF, TNF. Immunotherapy alone with vaccines producing either IL-4 or GM-CSF resulted in complete remission in 20–40% of the mice. By combining immunotherapy using (GM-CSF, IL-4, IL-12) producing vaccines with local tumour radiotherapy (single fraction 6 Gy X-ray radiations) about 80–100% of the glioma-bearing mice were cured. The high efficiency of the combined treatment was maintained even under suboptimal conditions when neither of the individual modalities alone cured any of the mice (Lumniczky, et al. 2002). Their results are in good agreement the survival rate of 75% (p<0.05) achieved in the Lund study of N29 tumours in rats treated with IFN- secreting vaccine combined with 5 Gy single fraction RT (B. R. R. Persson, et al. 2010).

#### **3.3 The U.S. experience of radiation therapy combined with vaccination of mice with Glioma or mammary carcinoma**

#### **3.3.1 Combining radiation therapy with blockade of the CTLA-4 pathway**

The cytotoxic T lymphocyte-associated protein CTLA-4 is involved in the immune regulatory mechanisms that control the early stage of the T cell response. It has previously been demonstrated that blockade of the CTLA-4 protein enhance anti-tumour responses both in experimental systems and in clinical trials (Chambers, et al. 2001; Egen, et al. 2002).

In a mouse model of the poorly immunogenic metastatic mouse mammary carcinoma 4T1, however, neither anti-tumour response nor survival-time was affected by using an anti-CTLA-4 monoclonal antibody for blocking the CTLA-4 protein. But anti-CTLA-4 monoclonal antibody administration combined with one 12 Gy fraction of radiation therapy, inhibited the growth of the primary irradiated tumour. Also the survival-time of the mice was significantly increased by this combined treatment (Demaria, et al. 2004; Demaria, et al. 2003; Demaria, et al. 2005b).

Another investigation of the effects of systemic CTLA-4 blockade with monoclonal antibody (9H10) to CTLA-4 employed in a mice model with well-established glioma, showed that CTLA-4 blockade confers long-term survival in 80% of treated mice (Fecci, et al. 2007). Thus the combination of local RT with CTLA-4 blockade might be applied as radio-immunemodulating therapeutic strategy also against glioma.

Radiation Immune Modulation Therapy of Glioma 375

rejecting challenge tumours. Antitumor immunity was associated with an increased number of tumour-infiltrating lymphocytes (TILs) in brain tumours and increased tumour-specific production of IFN. Since anti-CD137 therapy is already used in clinical trials it was suggested to be studied further in combination with local hypo-fractionated (2x4 Gy)

The expression profiles of CD4(+) and CD8(+) T cells and Treg from patients with newly diagnosed *glioblastoma multiforme* are quite different when compared with normal healthy volunteers (Learn, et al. 2006). But how various absorbed dose or various fractionation pattern or methods of radiation delivery can affect T-cell populations and alternative regulatory molecules in glioma patients is still under debate (Chiba, et al. 2010; Teitz-

**4.1 Effects of concomitant temozolomide and radiation therapies on WT1-specific**

Like many other solid tumours, glioma have been found to express a protein characteristic for Wilms' tumour 1 (WT1) (Hashiba, et al. 2007). A peptide based immunotherapy targeting the WT1 gene has successfully been used in patients with recurrent glioma. The clinical response indicates that CD8(+) cytotoxic T lymphocytes (CTLs) are the main effectors of this WT1 vaccination (Oka, et al. 2004). A phase II clinical trial of the WT1 vaccination for patients with recurrent malignant glioma resulted in a partial response rate of 9.5% but none complete response. The median length of period with progression-free

In planning for a clinical trial of WT1 vaccination involving patients with newly diagnosed malignant glioma, it is also aimed to combine concurrent radiation /TMZ therapy with WT1 immunotherapy. The critical question is, however, if the depletion of lymphocytes caused by the current standard radiation/TMZ treatment is a drawback for a combination with WT1 immunotherapy. Therefore a clinical study was performed in order to determine how the concomitant radiation/TMZ therapy affects the WT1-specific T-cells and other T-cells in terms of their frequencies and total numbers. This study concluded that, even after the decrease of the absolute numbers of lymphocytes, the fraction of WT1 specific T-cells was stable. They concluded that it may the possible to apply WT1 immunotherapy after the end

In another clinical study of 8 patients with primary glioma it was found that concomitant radiation/TMZ therapy integrated with autologous dendritic cell-based immunotherapy was feasible and well tolerated. The median progression-free survival (PFS) was 75% and at 6 months and 50% at 18 months. The median time of survival for all patients is 24 months. One patient was still free from progression or recurrence at 34 months (Ardon, et al. 2010).

A single fraction of high dose radiation therapy has been demonstrated to dramatically increase the priming of T-cell in draining lymphoid tissues, which increased the action of the CD8(+) T cells and lead to reduction and eradication of the primary tumour or distant

**4.2 Treatment recurrent malignant glioma with hypo-fractionated radiotherapy**

radiation therapy for clinical translation (Newcomb, et al. 2010).

Tennenbaum, et al. 2008; Verastegui, et al. 2003).

survival was 20 weeks (Izumoto, et al. 2008).

**combined with immune therapy**

of 6 weeks of radiation/TMZ therapy (Chiba, et al. 2010).

**T-cells in malignant glioma**

**4. Clinical studies of combining radiation and immune therapy**