**6. Interpretation of Cronbach's alpha**

The interpretation of reliability is the correlation of the test with itself. When the estimate of reliability increases, the portion of a test score related to the error will decrease. Wise interpretation of alpha needs an understanding of the interrelatedness of items and whether the items measure a single latent trait or construct. Exam or test with different content materials such as integrated courses, for example, in the musculoskeletal system course, although is dominated by anatomy it contains other subjects of basic medical and clinical sciences that have different contains. Therefore, interpretation of such a course exam needs deep looks beyond the alpha figure. It was reported that KR20 of 0.7 is acceptable to short test (less than 50 items) and KR20 of 0.8 for an extended test (more than 50 item-test) [25]. Moreover, it was documented that a multidimensional exam does not have a lower (**Table 1**) alpha value than a unidimensional one [30].

A low alpha value can be due to a smaller number of items, reduced interrelatedness between items, or heterogeneous constructs [22]. A high value of alpha can suggest exam reliability, and some items are non-functional as they are testing the same content but in a different guise or repeated ones [16, 22]. Also, a high value indicates items with high interrelatedness, indicating a limited coverage of the content materials [22].


4.5.1. Scanning Electron Microscopy (SEM)

SEM is an analytical technique used for the characterization of surface morphology of API and excipients microscopically, in particular, differences in crystal habits and the shape of the samples [128,129]. SEM does not convey any information in respect of the chemical structure and/or thermal behaviour of compounds, and is used with other thermal and spectroscopic methods such as DSC, TGA, and FTIR to identify incompatibilities between API and excipients [129].


*3.1. E*ff*ect of the FYM*

Application of the FYM to the maize three years before wheat was sown (see the crop rotation description in Section 2.2.) significantly affected average wheat yields in all experimental sites (Table 4).

**Table 4.** Comparison of average grain yields (t ha−1) as affected by locality, year, and fertilizer treatment.

Mean values with the standard error of the mean SE, followed by the same letter (A vertically, a horizontally), are not significantly different (α < 0.05).

The average grain yield increase between the control and FYM treatments ranged from 0.48 t ha−1 in Lukavec to 0.78 t ha−1 in Caslav. However, the highest percentage increase was recorded in Lukavec (20%), followed by Caslav (16%) and Ivanovice (13%). The differences in average grain yieldswere not achieved by the only application of the FYM, as the effect of the year also significantly affected the compared grain yields. While the overall effect of the fertilizer treatment and weather conditions on average yield was equal in Ivanovice (49% for fertilizer treatment; 51% for weather conditions) and Caslav (47% for fertilizer treatment; 52% for weather conditions), the higher effect of the fertilizer treatment was recorded in Lukavec (62% for fertilizer treatment; 35% for the weather conditions), showing a lower fluctuation of grain yields between the years.

#### *3.2. E*ff*ect of the Mineral N Applied without P and K Mineral Fertilizers*

In comparison with the control, application of mineral N fertilizers applied without mineral P and K fertilizers significantly increased the average grain yields in all experimental stations (Table 5).

**Table 5.** Comparison of average grain yields (t ha−1) as affected by locality, year, and fertilizer treatment.

Mean values with SE, followed by the same letter (A vertically, a horizontally), are not significantly different (α < 0.05).

The average grain yield increase was 2.79 t ha−1 (+63%) for FYM+N1 treatment and 3.37 t ha−1 (+76%) for FYM+N2 treatment in Ivanovice. The effect of the fertilizer treatment on overall grain yields was dominant (86%) when compared with the effect of the season (9%). The difference between FYM+N1 and FYM+N2 treatments was significant. According to the quadratic model, the maximum grain yield was reached at FYM+70 kg N ha−1 (Figure 2a).

**Figure 2.** The average wheat grain yield as affected by fertilizer treatment ((**a**)—control, FYM+N1, FYM+N2; (**b**)—control, FYM+N1PK, FYM+N2PK, FYM+N3PK), and locality over the period 2015–2018.


*4.9. Real-Time PCR*

Real-time PCR was performed using the QuantStudio 6 Flex Real-Time PCR System (ABI, Thermo Fisher, Shanghai, China) and Roche LightCycler ® 480 (Roche, Switzerland), and the following PCR program: Denaturation at 95 ◦C for 10 min; amplification for 45 cycles at 95 ◦C for 15 s; annealing and extension at 60 ◦C for 1 min. 2× SYBR Green qPCR Master Mix (#B21203, Bimake, Shanghai, China) were used for the detection of RT-qPCR. Primer sequences are shown in Table 1. Specific amplifications for certain PCR reactions were assessed using a melting curve. One negative control reaction, in which the cDNA template was replaced by water, was performed to avoid potential contamination. The sample from each well was repeated three times, and the comparative Ct (2−ΔΔCt) value method was used for relative quantification. GAPDH (NM\_002046.6) was used as the reference gene.

**Table 1.** Primer used for SYBR Green I qRT-PCR validation.

#### *4.10. Apoptosis and Mitochondrial Membrane Potential Analysis*

The analysis was performed by Servicebio Co., Ltd. (Wuhan, China). Briefly, the cells with transfections were collected through trypsin digestion. Then, the cells were incubated by annexin V-FITC and propidium iodide (PI). Then, the apoptosis rates were detected through flow cytometry (Ex = 488 nm, FL1 (Em = 525 ± 20 nm) and FL2 (Em = 585 ± 21 nm)). Flow Jo software was used to analysis the rates of cells in di fferent conditions. For mitochondrial membrane potential (MMP) analysis, JC-1 probe was used. Briefly, the cells were collected and counted. The cells were then incubated with 10 μg/mL JC-1 probe at 37 ◦C for 20 min. Cells were detected through flow cytometry (Ex = 488 nm, FL1 (Em = 525 ± 20 nm) and FL2 (Em = 585 ± 20 nm)). Flow Jo software was used for the analysis.

#### *4.11. Bioinformatics and Data Analysis*

The survival predication was performed using the GEPIA database (http://gepia.cancerpku.cn/). The prognosis analysis and gene expression analysis were performed according to the construction of the creator of this database [59].

#### *4.12. Dual-Luciferase Reporter Assay*

The promoter region of *PLIN5* was amplified by PCR with total DNA of HepG2 cells. The primers were used as following, F: 5-GAAAACTGGATCGGATGAATTGG-3 and R: 5-CACCCCCGCCGGTCCCGC-3. Then, the promoter region was cloned into the pGL3-basic vector. Then, the reconstructed vector was co-transfected with the ATFs expression or pcDNA3.1 (control) vectors and TK vector into HepG2 cells seeded into the 12-well plate. Moreover, pGL3-basic vector was used as the negative control and pGL3-CMV vector was used as the positive vector. Luciferase enzymatic activity was measured by a microplate reader from a multi-wavelength measurement system (PE Enspire, PerkinElmer, Germany) using a dual-luciferase reporter assay system (#RG027, Beyotime Biotechnology, Nanjing, China). The relative light unit (RLU) was normalized by a control group. The result was showed by the relative RLU. All transfections were performed in triplicate, and the data are expressed as the means ± SD.


**Author details**

Mislav Radić1 and Josipa Radić2

1 Division of Rheumatology and Clinical Immunology, University Hospital Centre Split, University of Split School of Medicine, Split, Croatia

2 Division of Nephrology, University Hospital Centre Split, University of Split School of Medicine, Split, Croatia

### **References**

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of the syndrome, and might be defined as rheumatoid vasculopathy. Several disease-related mechanisms may be involved in the development of premature vascular damage in RA. Increased synthesis of proinflammatory mediators, autoantibodies against endothelial cell components, perturbations in T-cell subsets, genetic polymorphisms, hyperhomocysteinemia, oxidative stress and abnormal vascular repair are associated with atherosclerosis in RA.

Vascular involvement in SLE may be of inflammatory or thrombotic origin. Both mechanisms involve the immune system. The activation and consequent endothelial lesions play a very important role in the disease pathogenesis. The common hypothesis for vasculopathy in SLE concerns the endothelial deposition of circulating immune complexes. There are many various autoantibodies in SLE as circulating immune complexes which directly or indirectly affect endothelial cells, causing chronic vessel wall damage. Furthermore, vasculitis in SLE is proatherogenic condition and is characterized by leucocytes activation and production of

Although SSc is considered a fibrosing disease, vascular involvement plays a major role in pathogenesis and organ dysfunction. SSc vascular disease involves vasculopathy with luminal occlusion, thrombosis and vasospasm. The vascular pathology in SSc is not necessarily an inflammatory process and would be better be characterised as a vasculopathy in the absence of vasculitis. In the current pathogenic model of SSc, a vascular injury of unknown cause leads to endothelial apoptosis and initiates the process of SSc vasculopathy. Histopathology of SSc vasculopathy reflects the underlying pathogenesis, with myofibroblast proliferation and matrix deposit in the subendothelial layer leading to obliterative thickening of vessel walls. Inflammatory infiltrates are absent, and the internal elastic lamina remains intact. In contrast to vasculopathy, concurrent vasculitis in SSc shows histopathological evidence of inflamma‐

tion with presence of mononuclear infiltrates and destruction of the vascular wall.

1 Division of Rheumatology and Clinical Immunology, University Hospital Centre Split,

2 Division of Nephrology, University Hospital Centre Split, University of Split School of

[1] Medsger, T. A. Jr. Natural history of systemic sclerosis and the assessment of disease activity, severity, functional status, and psychologic well-being. Rheumatic diseases

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176 Updates in the Diagnosis and Treatment of Vasculitis

and Josipa Radić2

University of Split School of Medicine, Split, Croatia

clinics of North America. (2003). , 29(2), 255-73.

**Author details**

Medicine, Split, Croatia

Mislav Radić1

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**Chapter 8**

**Treatment of ANCA-Associated Vasculitis in Adults**

Vasculitis is the general term used to describe diseases associated with inflammation of the blood vessels. This inflammation results in end-organ ischemia and damage with lifethreatening consequences. Treatment is tailored to the type of vasculitis the patient has, prognostic features and disease severity. Two main treatment phases are recognized: induction of remission, and maintenance of remission. In this chapter we will focus on the treatment of ANCA-associated vasculitis (AAV), namely: Granulomatosis with polyangiitis (GPA), formerly Wegener's Granulomatosis; Eosinophilic granulomatosis with polyangiitis (EGPA), formerly Churg-Strauss Syndrome, and Microscopic Polyangiitis (MPA). Patients with Polyarteritis nodosa (PAN) were included in the initial therapeutic trials of these diseases,

We introduce first the historical use of glucocorticoids, which are uniformly incorporated in the treatment protocols of therapeutic trials. Cyclophosphamide is recommended for the induction of remission in AAV, and in particular for generalized and severe disease. CY‐ CLOPS, a trial of oral versus intravenous cyclophosphamide, demonstrated that intravenous dosing was as effective in inducing remission with a reduced cumulative dose, and with fewer episodes of leucopenia, but in long-term follow-up relapse was more common in the intrave‐ nous treatment group. NORAM compared the use of methotrexate compared to oral cyclo‐ phosphamide for induction of remission in patients with limited GPA, and concluded that methotrexate was nearly as effective as cyclosphosphamide in achieving remission, but in long-term follow-up more corticosteroids and further immunosuppressive agents were required. More recently, rituximab use for the induction of remission was studied in the RAVE and RITUXVAS clinical trials. Rituximab was proven to be effective as cyclophosphamide, but without a reduction in the rate of infection as had been expected. Plasma exchange in combi‐ nation with oral cyclophosphamide for patients with severe renal involvement significantly decreases the risk of end-stage renal disease compared to intravenous steroids and oral

> © 2013 Fifi-Mah and Barnabe; licensee InTech. This is an open access article 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.

> © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and reproduction in any medium, provided the original work is properly cited.

therefore some of the studies results have been applied to that population as well.

cyclophosphamide, but without a significant difference in patient survival.

Aurore Fifi-Mah and Cheryl Barnabe

http://dx.doi.org/10.5772/54290

**1. Introduction**

Additional information is available at the end of the chapter


**3. Conclusions**

From the evidence presented, it can be concluded that technology needs to apply a principle of responsibility with the aim of benefiting society and not just the scientific field. To support ethics, stakeholders need to be made aware of informed consent and professional codes of conduct, which are unknown in most technological fields. Professionals should be made aware that their work should be guided by ethical and moral standards and carried out without having negative effects on users.

Finally, it is recommended that for future studies the search for articles not be limited to a single language, as international sources are an important support when searching for other publications, and there are several areas of research in this field, such as ethical frameworks for emerging technologies, cybersecurity, ethical regulation, artificial intelligence. As a limitation, we mention the scarcity of information found on the web at a local level that would allow the analysis of intervention groups from the same cohort.