*Article* **Conventional Industrial Robotics Applied to the Process of Tomato Grafting Using the Splicing Technique**

**José-Luis Pardo-Alonso 1, Ángel Carreño-Ortega 1,\*, Carolina-Clara Martínez-Gaitán 2, Iacopo Golasi 3 and Marta Gómez Galán 1**

Received: 10 November 2019; Accepted: 9 December 2019; Published: 12 December 2019

**Abstract:** Horticultural grafting is routinely performed manually, demanding a high degree of concentration and requiring operators to withstand extreme humidity and temperature conditions. This article presents the results derived from adapting the splicing technique for tomato grafting, characterized by the coordinated work of two conventional anthropomorphic industrial robots with the support of low-cost passive auxiliary units for the transportation, handling, and conditioning of the seedlings. This work provides a new approach to improve the efficiency of tomato grafting. Six test rates were analyzed, which allowed the system to be evaluated across 900 grafted units, with gradual increases in the speed of robots work, operating from 80 grafts/hour to over 300 grafts/hour. The results obtained show that a higher number of grafts per hour than the number manually performed by skilled workers could be reached easily, with success rates of approximately 90% for working speeds around 210–240 grafts/hour.

**Keywords:** tomato grafting; splice grafting technique; agricultural robot; automated grafting; agricultural machinery


**3.3 Finding 3: cave porosity zone height decreases with a decrease in extraction rate**

Four different scenarios were simulated with increasing velocities from 0.0001 to 0.0004 m/s by keeping the same rock mass properties (RM2). For the block cave model, the same velocities (extraction rates) are assigned at all the grid points, whereas in the panel cave model, different velocities are applied at grid points due to the inherent nature of the caving process. An increase in velocity value means an increase in material extraction rates. The simulated porosity values for different zones under different extraction rates for both block and panel cave models are shown in **Figures 8** and **9**.

In the real-world scenario, in a propagating cave, the cave height increases with increased production. It was evident from **Figures 8** and **9** that when the ore

**Figure 6.** *Porosity value profile for RM1 (left) and RM2 (right) for block cave mine model.*

**Figure 7.** *Porosity value profile for RM1 (left) and RM2 (right) for panel cave mine model.*

*Block Cave Mine Ventilation: Research Findings DOI: http://dx.doi.org/10.5772/intechopen.104856*

### **Figure 8.**

*Porosity value profile for RM2 under four different extraction rates (0.0001–0.0004 m/s) for block cave model.*

### **Figure 9.**

*Porosity value profile for RM2 under four different extraction rates (0.0001–0.0004 m/s) for panel cave model.*

extraction rate was increased, the cave zones height was increased. The mobilized zone porosity tends to be higher as it is the actively flowing region in the cave. In both block and panel cave models, the mobilized zone porosity value ranges from 0.35 to 0.40. The height of this zone is relatively high compared to the other zones due to its proximity to the drawpoints where the ore is extracted from the cave.

In the block cave model, when different velocities, from 0.0001 to 0.0004 m/s, were applied at the drawpoints, the porosity values in the mobilized zone were observed to be from 0.30 to 0.35. This could result from stagnant flow in the cave that subjected the material to re-compaction. If the extraction rate of material is not equal to the rate of cave propagation, the material will stagnate, re-compacted, and result in lower porosity.

### **3.4 Finding 4: cave porosity is affected by the fragmentation size**

Material extraction is simulated in a block cave model by opening drawpoints to extract a targeted mass of 100,000 metric tons. As soon as the drawpoint is open, due to gravity, the spheres (broken rock) will start flowing toward the drawpoints. Measurement locations are strategically placed to measure the porosities as the material flows through the cave. Porosity measurement histories are recorded at all the measuring locations (spheres) while extracting the material from the cave through the drawpoint. From the simulation results, it was found that the porosity change in the isolated extraction zone (IEZ) ranges from 0.39 to 0.56. In comparison, in the isolated draw zone (IDZ) insignificant porosity change (0.38–0.42) was observed. The random spike(s) seen on the graph shows the change in the porosity while the material is drawn from the cave.

To study the effect of particle size distribution on the porosity change, two fragmentation distributions with different mean particle sizes and standard deviations are applied for the block cave mine. **Table 1** illustrates the particle distributions considered for the simulations.

Scenario #1 is simulated by consecutively opening the six drawbells. When the target discharge mass is reached the assigned value in the system, the next drawpoint will open immediately as shown in **Table 2**.

Scenario #2 is conducted by opening the six drawbells randomly. The discharged mass is applied to control the opening of the drawbells for the random opening. A summary of discharge mass criteria and the drawbells are provided in **Table 2**.

The numerical model of porosity assessment using a discontinuum approach successfully modeled change in porosity associated with the fragmented rock mass flow in a mature cave. It was found that during material extraction, the porosity changes relatively higher in IEZ than in IDZ. These changes for IEZ range from 0.39 to 0.56 for a block cave and 0.38–0.48 for a panel cave.

The sensitivity analysis on particle size distribution concluded that fragmentation size affects cave porosity. In the case of Fragmentation #1, the change in

**Table 1.** *Gaussian particle size distributions for fragmentation #1 and #2.* *Block Cave Mine Ventilation: Research Findings DOI: http://dx.doi.org/10.5772/intechopen.104856*

**Table 2.**

*Discharge mass criteria for opening the drawpoints for two scenarios.*

porosity ranges from 0.40 to 0.48 in IEZ and from 0.38 to 0.56 for Fragmentation #2. Similarly, the draw control strategy also affects cave porosity. In the case of Scenario #1, the change in porosity ranges from 0.38 to 0.52 in IEZ and from 0.38 to 0.48 for Scenario #2 in IEZ.


incision by using "junction tunnels", which preserve the mucosa. The mucoperichondrium and mucoperiosteum are elevated bilaterally at the junction of the septum with the nasal wall. The upper lateral cartilage is released by preserving the mucosal integrity through submucosal dissection. Entrapment of mucosa and formation of nasal cysts should be prevented by maintaining intact the mucosal integrity under osteotomy or cartilage incision sites. (Johnson and Anderson, 1977; Ress, 1980) Creation of dead space is declined by trying to avoid undermining osteotomy sites.

Irrigation at the end of the procedure can provide additional clearing of remnants from the surgical field. Careful removal of all mucosal fragments from the osteotomy sites at the time of primary surgery is mandatory and can by preventative.

Complete surgical extirpation of cysts may lead to disfigurement of the nasal framework.

Direct reconstruction of the defect sets the goal of surgical treatment of this rare condition. Cartilaginous grafts (e.g., rib or concha grafts) are recommended for reconstruction of the nasal frame in such cases.

#### **5. Intraoperative findings**

On exploration the cystic mass could appear as a distinct capsule with no direct connection between it and the nasal mucosa, as it has been reported in the majority of the published cases. (Harley and Erdman, 1990; Romo T 3rd et al., 1999) On the other hand the cyst could be found either tightly attached to the surrounding tissues, or adherent to the overlying skin, or in connection with the underlying cartilaginous and bony nasal structures, or even attached to previously used autogenous grafts. (Kotzur and Gubisch, 1997; Tan Ergin and Akkuzu , 2000; Pausch et al., 2010)

The underlying nasal bone or cartilage in the area of the lesion can be depressed with an impression deformity, or eroded or even resorbed. [21,30,31] (Harley and Erdman, 1990; Tan Ergin and Akkuzu , 2000; Pausch et al., 2010)

During the operation the lesion can be removed removed either intact or perforated.If the latter happens, clear fluid or partly fatty fluid or mucous fluid or even thick white-yellowish liquid usually is yielded from the cystic wall. (Flaherty et al., 1996; Kotzur and Gubisch, 1997; Romo T 3rd et al., 1999)

Dead space or deformity of bone or cartilage could be created,in the operation field, after complete cyst removal. which has to be managed through careful suturing in layers and to be repaired, usually by using autogenous graft.

#### **6. Histopathology**

Macroscopically a mucous cyst appears as an oval shaped,soft smooth walled cyst, located subcutaneously, easily separated from its surrounding structures and filled with mucinous material. Microscopically the cyst wall is lined by ciliated columnar respiratory type epithelium with dispersed goblet cells. (Ntomouchtsis et al., 2010)

Due to the fact that diagnosis is based primarily on microscopic findings, particularly the lining of the cysts, similar cutaneous cystic formations can enter in the differential diagnosis. Cutaneous ciliated cysts show numerous papillary projections lined by a simple cuboidal or columnar ciliated epithelium, while mucin-secreting cells are absent. These cysts can be found on the lower extremities (females) or on the back (males) and measure several centimeters in diameter. (Elder et al., 2010)

incision by using "junction tunnels", which preserve the mucosa. The mucoperichondrium and mucoperiosteum are elevated bilaterally at the junction of the septum with the nasal wall. The upper lateral cartilage is released by preserving the mucosal integrity through submucosal dissection. Entrapment of mucosa and formation of nasal cysts should be prevented by maintaining intact the mucosal integrity under osteotomy or cartilage incision sites. (Johnson and Anderson, 1977; Ress, 1980) Creation of dead space is declined by trying

Irrigation at the end of the procedure can provide additional clearing of remnants from the surgical field. Careful removal of all mucosal fragments from the osteotomy sites at the time

On exploration the cystic mass could appear as a distinct capsule with no direct connection between it and the nasal mucosa, as it has been reported in the majority of the published cases. (Harley and Erdman, 1990; Romo T 3rd et al., 1999) On the other hand the cyst could be found either tightly attached to the surrounding tissues, or adherent to the overlying skin, or in connection with the underlying cartilaginous and bony nasal structures, or even attached to previously used autogenous grafts. (Kotzur and Gubisch, 1997; Tan Ergin and

The underlying nasal bone or cartilage in the area of the lesion can be depressed with an impression deformity, or eroded or even resorbed. [21,30,31] (Harley and Erdman, 1990; Tan

During the operation the lesion can be removed removed either intact or perforated.If the latter happens, clear fluid or partly fatty fluid or mucous fluid or even thick white-yellowish liquid usually is yielded from the cystic wall. (Flaherty et al., 1996; Kotzur and Gubisch,

Dead space or deformity of bone or cartilage could be created,in the operation field, after complete cyst removal. which has to be managed through careful suturing in layers and to

Macroscopically a mucous cyst appears as an oval shaped,soft smooth walled cyst, located subcutaneously, easily separated from its surrounding structures and filled with mucinous material. Microscopically the cyst wall is lined by ciliated columnar respiratory type

Due to the fact that diagnosis is based primarily on microscopic findings, particularly the lining of the cysts, similar cutaneous cystic formations can enter in the differential diagnosis. Cutaneous ciliated cysts show numerous papillary projections lined by a simple cuboidal or columnar ciliated epithelium, while mucin-secreting cells are absent. These cysts can be found on the lower extremities (females) or on the back (males) and measure several

Complete surgical extirpation of cysts may lead to disfigurement of the nasal framework. Direct reconstruction of the defect sets the goal of surgical treatment of this rare condition. Cartilaginous grafts (e.g., rib or concha grafts) are recommended for reconstruction of the

to avoid undermining osteotomy sites.

nasal frame in such cases.

**5. Intraoperative findings**

Akkuzu , 2000; Pausch et al., 2010)

1997; Romo T 3rd et al., 1999)

**6. Histopathology**

Ergin and Akkuzu , 2000; Pausch et al., 2010)

be repaired, usually by using autogenous graft.

centimeters in diameter. (Elder et al., 2010)

epithelium with dispersed goblet cells. (Ntomouchtsis et al., 2010)

of primary surgery is mandatory and can by preventative.

Fig. 2. Cyst filled with mucinous material (H-E ×20)

Fig. 3. The cyst wall is lined by ciliated columnar respiratory type epithelium with dispersed goblet cells (H-E ×200)

Mucous Cysts as a Complication of Rhinoplasty 143

The aetiology of this lesion remains unknown and controversial. Different theories of creation exist. The first hypothesis assumes the herniation of nasal mucosa in the direct postoperative period, for example after a patient blew his or her nose. (McGregor et al., 1958) The herniation or subsequent growth of nasal mucosa can take place through the infracture sites or incisions, although no reported cases mentioned any connection of a cyst

The most reasonable explanation is the proliferation of ectopic or displaced mucous membranes, followed by improper clearing of mucous epithelial remnants and bony or cartilage parts. This seeding of mucous tissue and remnants attached to bone or cartilage either in situ or as a part of an autogenous graft seems to be the main reason for which the mucous grafts grow and proliferate in their ectopic position (Shulman and Westreich, 1983;

Another aspect that has to be emphasized is that in some of the described cases in the literature, the location of appearance does not coincide with the osteotomy lines or even with the intervention field of a rhinoplasty. This could be explained by poor surgical technique during a close rhinoplasty, which can lead to extreme surgical trauma, violation of the intranasal mucosal lining and subsequent encystation of nasal mucosal epithelium, displaced at the time of surgery. (Zijlker and Vuyk, 1993; Dini et al., 2001; Ntomouchtsis et al., 2010) This explains the different locations described in the literature, and the absence of connections with the internal nasal lining that is observed and described during surgical

On the other hand, the reappearance of a cyst can also be attributed to a faulty surgical technique. (Ntomouchtsis et al., 2010) It is also hypothesized that cysts may develop by occlusion of sebaceous glands because of scar tissue formation. (Rettinger and Zenkel, 1997) In cleft lip nose rhinoplasty, however, cystic masses of the nose might have other origins, so congenital malformations or remnants of the nasolacrimal duct have also been described. Mucous cysts of the nose are not specific complications of cleft nose surgery, although they have been observed in this specific patients group. (Aikawa et al., 2008; Pausch et al., 2010)

In order to prevent iatrogenic cyst formation it is important to use an appropriate surgical technique. The manoeuvres must be executed carefully with respect to the soft and hard tissues of the operation field. The need for atraumatic and careful tissue dissection has been emphasized by virtually all authors who have described postrhinoplasty mucous cysts. By preserving the mucosal integrity and using sharp instruments one can hope to avoid the involuntary dispersion of tissues into other layers while dissecting. The mucosal lining must be kept intact during the rhinoplasty, or in any case of disruption a meticulously restoration is mandatory. Mucosal lining can be preserved when subperichondrial and subperiostal tunnels are being established over the septum and under the nasal dorsum before any surgical alterations will be made to the structures. (Zijlker and Vuyk, 1993) Completing of all osteotomies is also important for maintaining mucosal integrity during intranasal osteotomy and to decrease the chance of cyst formation. The osteotomy sites must be placed properly, and performed with adequate water injection and hydrodissection. The mucosa around the lateral osteotomy site should be elevated, to prevent entrapment. For security

to the normal mucous layer of the nose. (Mouly, 1970; Harley and Erdman, 1990)

Harley and Erdman, 1990; Kotzur and Gubisch, 1997; Dini et al., 2001)

**7. Theories of creation**

eradication.

**8. Prevention**

Bronchogenic cysts are lined by a mucosa consisting of ciliated pseudostratified columnar epithelium, while goblet cells may be interspersed. The wall frequently contains smooth muscle and mucous glands. Thyroglossal duct cysts differ from bronchogenic cysts in that they do not contain smooth muscle and they frequently contain thyroid follicles. Bronchogenic cysts are most commonly seen on the suprasternal notch and rarely on the anterior aspect of the neck or on the chin. Thyroglossal duct cysts are clinically indistinguishable from bronchogenic cysts, except that they are usually located on the anterior aspect of the neck. (Elder et al., 2010)

Apocrine hidrocystoma is a multi-loculated cystic lesion with occasional papillary projections. The inner surface of the cyst and the papillary projections are lined by a row of columnar secretory cells of variable height showing decapitation secretion indicative of apocrine secretion. Peripheral to the layer of secretory cells are elongated myoepithelial cells, their long axes running parallel to the cyst wall. Apocrine hidrocystoma occurs usually as a solitary, translucent cystic nodule on the face and occasionally on the ears, scalp, chest, shoulders, or vulva. Multiple apocrine hidrocystomas are rarely encountered. Frequently, the lesion has a blue hue, while the diameter ranges from 1 to 20 mm. (Elder et al., 2010)

Eccrine hidrocystoma shows a single cystic cavity, while the cyst wall usually shows two layers of small, cuboidal epithelial cells. In some areas, only a single layer of flattened epithelial cells can be seen, their flattened nuclei extending parallel to the cyst wall. Small papillary projections extending into the cavity of the cyst are observed rarely. Eccrine secretory tubules and ducts are often located below the cyst and in close approximation to it, and, on serial sections, one may find an eccrine duct leading into the cyst from below. However, no connection can be found between the cyst and the epidermis. In this condition, usually one lesion -- but occasionally several, and rarely numerous lesions -- are present on the face. As in apocrine hidrocystoma, the lesion consists of a small, translucent, cystic nodule 1 to 3 mm in diameter that often has a bluish hue. (Elder et al., 2010)

Cutaneous metaplastic synovial cysts show villous-like projections and a lining that resembles hyperplastic synovium (not epithelium). This type of cysts is rare and presents months or years after a surgery as solitary tender subcutaneous nodule. The cause of formation of cutaneous metaplastic synovial cysts remains unclear, but previous trauma usually precedes their onset. Taking in consideration this entity is significant, since a recent publication reports a case of cutaneous metaplastic synovial cyst on a patient's face after receiving hyaluronic acid filler injections. (Inchingolo et al., 2010)

 Foreign body inclusion cysts, even though called cysts inappropriately, microscopically they represent granulomatous reaction to bone or cartilage fragments following rhinoplasty. [34] (Chang and Jin, 2008)

Follicular cysts (epidermal, trichilemmal, dermoid, etc) can be often found on the face, regardless of any previous intervention, but can easily be excluded from the diagnosis since they are lined by stratified squamous epithelium resembling epidermis and filled with keratinous material. Even though we should be aware of this type of cysts since there have been reports of post rhinoplasty epidermal cysts. (Grocutt and Fatah, 1989; Tastan et al., 2010)

 All of the aforementioned cysts show similar clinical or pathological findings with the cutaneous mucous cysts, but a careful histological examination can spot the differences and exclude them from the diagnosis.

In conclusion, histological examination of all cysts that present on the face after an intervention is of paramount importance, not only to exclude malignancy, but also to narrow down the list of differential diagnoses.

#### **7. Theories of creation**

142 Rhinoplasty

Bronchogenic cysts are lined by a mucosa consisting of ciliated pseudostratified columnar epithelium, while goblet cells may be interspersed. The wall frequently contains smooth muscle and mucous glands. Thyroglossal duct cysts differ from bronchogenic cysts in that they do not contain smooth muscle and they frequently contain thyroid follicles. Bronchogenic cysts are most commonly seen on the suprasternal notch and rarely on the anterior aspect of the neck or on the chin. Thyroglossal duct cysts are clinically indistinguishable from bronchogenic cysts, except that they are usually located on the

Apocrine hidrocystoma is a multi-loculated cystic lesion with occasional papillary projections. The inner surface of the cyst and the papillary projections are lined by a row of columnar secretory cells of variable height showing decapitation secretion indicative of apocrine secretion. Peripheral to the layer of secretory cells are elongated myoepithelial cells, their long axes running parallel to the cyst wall. Apocrine hidrocystoma occurs usually as a solitary, translucent cystic nodule on the face and occasionally on the ears, scalp, chest, shoulders, or vulva. Multiple apocrine hidrocystomas are rarely encountered. Frequently, the lesion has a blue hue, while the diameter ranges from 1 to 20 mm. (Elder et al., 2010) Eccrine hidrocystoma shows a single cystic cavity, while the cyst wall usually shows two layers of small, cuboidal epithelial cells. In some areas, only a single layer of flattened epithelial cells can be seen, their flattened nuclei extending parallel to the cyst wall. Small papillary projections extending into the cavity of the cyst are observed rarely. Eccrine secretory tubules and ducts are often located below the cyst and in close approximation to it, and, on serial sections, one may find an eccrine duct leading into the cyst from below. However, no connection can be found between the cyst and the epidermis. In this condition, usually one lesion -- but occasionally several, and rarely numerous lesions -- are present on the face. As in apocrine hidrocystoma, the lesion consists of a small, translucent, cystic

nodule 1 to 3 mm in diameter that often has a bluish hue. (Elder et al., 2010)

receiving hyaluronic acid filler injections. (Inchingolo et al., 2010)

[34] (Chang and Jin, 2008)

exclude them from the diagnosis.

narrow down the list of differential diagnoses.

Cutaneous metaplastic synovial cysts show villous-like projections and a lining that resembles hyperplastic synovium (not epithelium). This type of cysts is rare and presents months or years after a surgery as solitary tender subcutaneous nodule. The cause of formation of cutaneous metaplastic synovial cysts remains unclear, but previous trauma usually precedes their onset. Taking in consideration this entity is significant, since a recent publication reports a case of cutaneous metaplastic synovial cyst on a patient's face after

 Foreign body inclusion cysts, even though called cysts inappropriately, microscopically they represent granulomatous reaction to bone or cartilage fragments following rhinoplasty.

Follicular cysts (epidermal, trichilemmal, dermoid, etc) can be often found on the face, regardless of any previous intervention, but can easily be excluded from the diagnosis since they are lined by stratified squamous epithelium resembling epidermis and filled with keratinous material. Even though we should be aware of this type of cysts since there have been reports of post rhinoplasty epidermal cysts. (Grocutt and Fatah, 1989; Tastan et al., 2010) All of the aforementioned cysts show similar clinical or pathological findings with the cutaneous mucous cysts, but a careful histological examination can spot the differences and

In conclusion, histological examination of all cysts that present on the face after an intervention is of paramount importance, not only to exclude malignancy, but also to

anterior aspect of the neck. (Elder et al., 2010)

The aetiology of this lesion remains unknown and controversial. Different theories of creation exist. The first hypothesis assumes the herniation of nasal mucosa in the direct postoperative period, for example after a patient blew his or her nose. (McGregor et al., 1958) The herniation or subsequent growth of nasal mucosa can take place through the infracture sites or incisions, although no reported cases mentioned any connection of a cyst to the normal mucous layer of the nose. (Mouly, 1970; Harley and Erdman, 1990)

The most reasonable explanation is the proliferation of ectopic or displaced mucous membranes, followed by improper clearing of mucous epithelial remnants and bony or cartilage parts. This seeding of mucous tissue and remnants attached to bone or cartilage either in situ or as a part of an autogenous graft seems to be the main reason for which the mucous grafts grow and proliferate in their ectopic position (Shulman and Westreich, 1983; Harley and Erdman, 1990; Kotzur and Gubisch, 1997; Dini et al., 2001)

Another aspect that has to be emphasized is that in some of the described cases in the literature, the location of appearance does not coincide with the osteotomy lines or even with the intervention field of a rhinoplasty. This could be explained by poor surgical technique during a close rhinoplasty, which can lead to extreme surgical trauma, violation of the intranasal mucosal lining and subsequent encystation of nasal mucosal epithelium, displaced at the time of surgery. (Zijlker and Vuyk, 1993; Dini et al., 2001; Ntomouchtsis et al., 2010) This explains the different locations described in the literature, and the absence of connections with the internal nasal lining that is observed and described during surgical eradication.

On the other hand, the reappearance of a cyst can also be attributed to a faulty surgical technique. (Ntomouchtsis et al., 2010) It is also hypothesized that cysts may develop by occlusion of sebaceous glands because of scar tissue formation. (Rettinger and Zenkel, 1997) In cleft lip nose rhinoplasty, however, cystic masses of the nose might have other origins, so congenital malformations or remnants of the nasolacrimal duct have also been described. Mucous cysts of the nose are not specific complications of cleft nose surgery, although they have been observed in this specific patients group. (Aikawa et al., 2008; Pausch et al., 2010)

#### **8. Prevention**

In order to prevent iatrogenic cyst formation it is important to use an appropriate surgical technique. The manoeuvres must be executed carefully with respect to the soft and hard tissues of the operation field. The need for atraumatic and careful tissue dissection has been emphasized by virtually all authors who have described postrhinoplasty mucous cysts.

By preserving the mucosal integrity and using sharp instruments one can hope to avoid the involuntary dispersion of tissues into other layers while dissecting. The mucosal lining must be kept intact during the rhinoplasty, or in any case of disruption a meticulously restoration is mandatory. Mucosal lining can be preserved when subperichondrial and subperiostal tunnels are being established over the septum and under the nasal dorsum before any surgical alterations will be made to the structures. (Zijlker and Vuyk, 1993) Completing of all osteotomies is also important for maintaining mucosal integrity during intranasal osteotomy and to decrease the chance of cyst formation. The osteotomy sites must be placed properly, and performed with adequate water injection and hydrodissection. The mucosa around the lateral osteotomy site should be elevated, to prevent entrapment. For security

Mucous Cysts as a Complication of Rhinoplasty 145

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the osteotomy sites should be thoroughly inspected at the completion of the procedure. (Liu and Kridel, 2003).

On the other hand many surgeons prefer not to elevate tunnels, as this additional stripping and dissection of periosteum decreases the structural support of the nasal framework after osteotomies are complete. They report better stability and more predictable outcomes with external perforated osteotomy, by using a small osteotome (2-4 mm) that simultaneously minimize disruption of periosteal surfaces, stabilize medial movement, reduce lateral wall collapse and decreases dead space. (Goldfarb et al., 1993; Rohrich et al., 2001; Rohrich et al., 2003) It as experimentally been shown that this technique decreases soft-tissue disruption or displacement compared to continuous osteotomies. (Byrne et al., 2003) It must also be emphasized that none of the reported postrhinoplasty cysts are connected to this surgical technique.

Meticulous removal of all bony, cartilaginous and mucous remnants is essential. It is essential that if osteocartilagenous grafts are used harvested from the nasal frame they must be prepared by removing the respiratory epithelium prior to implantation. (Raine et al., 2003) Irrigation at the end of the procedure can be an additional measure to clear the surgical field of remnants. (Gryskiewicz, 2001) The created dead space must be closed in layers. Intranasal incisions should be closed properly. Rhinoplasty is a very demanding procedure. In complicated cases, especially in revision surgery, the risk of complications is higher when performed by inexperienced surgeons. The best prevention of mucous cysts occurring after rhinoplasty is not only meticulous elimination of all bony, cartilage, and epithelial tissues and mucous parts, but also, and even more important, emphasis on a most atraumatic and careful operation. (Kotzur and Gubisch, 1997) The key to a good outcome is localizing the lesion and selecting the most appropriate procedure.

#### **9. Conclusion**

Relative to the high number of rhinoplasty procedures performed each year worldwide, the number of the 31 presented published cases is on the contrary very low. (Senechal et al., 1981; Lawsonet al., 1983; Toriumi and Johnson, 1990; Struijs and Bauwens, 2010 ) The only data reported regarding the frequency of postrhinoplasty mucous cysts are from two case reports, where there are count 1/6000 and 3/5000, a percentage of 0.02 and 0.06 respectively. (Kotzur and Gubisch, 1997; Dini et al., 2001) Otherwise there is a large number of unreported cases or the postrhinoplasty cyst is a really rare condition. (Karapantzos et al., 1999)

It is possible that intraoperative tissue dispersion would occur in large number of cases, but does not lead automatically to the development of cysts. Although the physiopathology is uncertain, it seems that entrapped fragments of epithelial tissue are proliferated, if specific local conditions are created. It remains unclear what factors influence the final formation in these rare cases.

#### **10. References**

Aikawa T, Iida S, Fukuda Y, Nakano Y, Ota Y, Takao K, Kogo M. Nasolabial cyst in a patient with cleft lip and palate. Int J Oral Maxillofac Surg. 2008;37:874–876.

Anastassov GE, Lee H. Respiratory mucocele formation after augmentation genioplasty with nasal osteocartilagenous graft. J Oral Maxillofac Surg 1999;57:1263–5.

the osteotomy sites should be thoroughly inspected at the completion of the procedure. (Liu

On the other hand many surgeons prefer not to elevate tunnels, as this additional stripping and dissection of periosteum decreases the structural support of the nasal framework after osteotomies are complete. They report better stability and more predictable outcomes with external perforated osteotomy, by using a small osteotome (2-4 mm) that simultaneously minimize disruption of periosteal surfaces, stabilize medial movement, reduce lateral wall collapse and decreases dead space. (Goldfarb et al., 1993; Rohrich et al., 2001; Rohrich et al., 2003) It as experimentally been shown that this technique decreases soft-tissue disruption or displacement compared to continuous osteotomies. (Byrne et al., 2003) It must also be emphasized that none of the reported postrhinoplasty cysts are connected to this surgical

Meticulous removal of all bony, cartilaginous and mucous remnants is essential. It is essential that if osteocartilagenous grafts are used harvested from the nasal frame they must be prepared by removing the respiratory epithelium prior to implantation. (Raine et al., 2003) Irrigation at the end of the procedure can be an additional measure to clear the surgical field of remnants. (Gryskiewicz, 2001) The created dead space must be closed in layers. Intranasal incisions should be closed properly. Rhinoplasty is a very demanding procedure. In complicated cases, especially in revision surgery, the risk of complications is higher when performed by inexperienced surgeons. The best prevention of mucous cysts occurring after rhinoplasty is not only meticulous elimination of all bony, cartilage, and epithelial tissues and mucous parts, but also, and even more important, emphasis on a most atraumatic and careful operation. (Kotzur and Gubisch, 1997) The key to a good outcome is

Relative to the high number of rhinoplasty procedures performed each year worldwide, the number of the 31 presented published cases is on the contrary very low. (Senechal et al., 1981; Lawsonet al., 1983; Toriumi and Johnson, 1990; Struijs and Bauwens, 2010 ) The only data reported regarding the frequency of postrhinoplasty mucous cysts are from two case reports, where there are count 1/6000 and 3/5000, a percentage of 0.02 and 0.06 respectively. (Kotzur and Gubisch, 1997; Dini et al., 2001) Otherwise there is a large number of unreported cases or the postrhinoplasty cyst is a really rare condition. (Karapantzos et

It is possible that intraoperative tissue dispersion would occur in large number of cases, but does not lead automatically to the development of cysts. Although the physiopathology is uncertain, it seems that entrapped fragments of epithelial tissue are proliferated, if specific local conditions are created. It remains unclear what factors influence the final formation in

Aikawa T, Iida S, Fukuda Y, Nakano Y, Ota Y, Takao K, Kogo M. Nasolabial cyst in a patient with cleft lip and palate. Int J Oral Maxillofac Surg. 2008;37:874–876. Anastassov GE, Lee H. Respiratory mucocele formation after augmentation genioplasty with nasal osteocartilagenous graft. J Oral Maxillofac Surg 1999;57:1263–5.

localizing the lesion and selecting the most appropriate procedure.

and Kridel, 2003).

technique.

**9. Conclusion**

al., 1999)

these rare cases.

**10. References**

Liu ES, Kridel RW. (2003). Postrhinoplasty nasal cysts and the use of petroleumbased

McGregor, M. W., O'Connor, G. B., and Saffier, S. Complications of rhinoplasty: I. Skin and

Mouly R. Le kyste mucoide, complication inhabtuelle de la rhinoplastie. Ann Chir Plast

Ntomouchtsis A, Kechagias N, Xirou P, Triaridis A, Xinou K, Vahtsevanos K. Recurrent

Pausch NC, Bertolini J, Hemprich A, Hierl T. Inclusion mucous cysts of the nose: a late

Raine C, Williamson SLH, McLean NR. Mucous cyst of the alar base: a rare complication

Rees T, ed. The osteocartilaginous vault. In: Aesthetic Plastic Surgery. Philadelphia, Pa: WB.

Rohrich RJ, Krueger JK, Adams WP, Hollier LH. Achieving consistency in the lateral nasal

Rohrich RJ, Janis JE, Adams WP, Krueger JK. An update on the lateral nasal osteotomy in

Romo T 3rd, Rizk SS, Suh GD (1999) Mucous cyst formation after rhinoplasty. Arch Facial

Senechal G, Senechal B, Mamelle G (1981) Une complication rare e tardive des rhinoplasties.

Shulman Y, Westreich M. Postrhinoplasty mucous cyst of the nose. Plast Reconstr Surg

Struijs B, Bauwens LJ.Post-rhinoplasty mucous cyst formation of the nasal dorsum. B-ENT.

Tardy ME. Rhinoplasty, In: Tardy ME, Kastenbauer ER. Head and Neck Surgery. Thieme,

Tastan E, Kavuzlu A, Demirci M, Sungu N. A unique case of a postrhinoplasty epidermoid

Toriumi DM, Johnson CM (1990) Revision rhinoplasty: case study. In: Toriumi DM, Johnson CM (eds) Open structure rhinoplasty. WB Saunders, Philadelphia, PA, pp 464–469 Zerris VA, Annino D, Heilman CB. Nasofrontal dermoid sinus cyst: report of two cases.

Tan Ergin N., Akkuzu B. Mucous cyst of the nasal dorsum. Rhinology 2000;38,206-7

Zijlker TD, Vuyk HD. Nasal dorsal cyst after rhinoplasty. Rhinology. 1993; 31:89 –91.

osteotomy during rginoplasty: An external perforated technique. *Plast. Reconstr.*

rhinoplasty: An anatomic endoscopic comparison of the external versus the internal

Rettinger G, Zenkel M. Skin and soft tissue complications. Facial Plast Surg. 1997;13:51-9 Riedel F, Bersch C, Hörmann K (2007) Dorsal nasal mass formation—postrhinoplasty cyst.

glabellar mucous cyst: a rare postrhinoplasty complication.Oral Maxillofac Surg.

complication after septorhinoplasty in two cleft lip patients. Cleft Palate Craniofac

ointments and nasal packing. Plast Reconstr Surg, 112, pp. 282–287.

subcutaneous tissues. J. Int. Coll. Surg. 30: 179, 1958.

following rhinoplasty. Br J Plast Surg 2003;56(2):176–7.

approach. *Plast. Reconstr. Surg.* 111: 2461, 2003.

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2010;6(4):295-8.

1995,pp239-302

### *Edited by Michael J. Brenner*

Rhinoplasty is one of the defining procedures of plastic and reconstructive surgery. Its roots stem from early efforts in nasal reconstruction to the emergence of modern rhinoplasty. This book describes the latest clinical and research perspectives in rhinoplasty and balances structural correction with aesthetic refinement. With treatises on rhinoplasty from a diverse set of thought leaders from around the world, the collective experience of this books' authors cover cosmetic and reconstructive approaches with a wealth of proven and innovative approaches ranging from minor refinement to major reconstruction. This diversity reflects the inherent complexity of the art and science of rhinoplasty. Discussion of structural approaches is balanced by consideration of judicious resection and refinement. The overarching goal is to instill an understanding of the subtleties of nasal structure and how the natural complexities of nasal anatomy can be adapted to maximize both function and natural appearance.

Photo by fabrizio siboldi / iStock

Rhinoplasty

Rhinoplasty

*Edited by Michael J. Brenner*


*3.3. Heterogeneity: Considering Di*ff*erent EV Types*

After the basic model analysis, we examined the effects of the policy incentives and infrastructures by separating PHEVs from BEVs. After the separation, the fast charging infrastructures have more apparent impacts for BEVs than those for PHEVs, as illustrated in Table 9 with macroeconomic factors controlled. The parameter means that a 1% increase in density of fast chargers can cause a 0.246% increase for BEV sales, while the effect for PHEV sales is not significant enough. The reason may be that PHEV drivers suffer much less from range anxiety.

The variable subsidy is significant for BEVs but not significant for PHEVs, which may due to the low subsidy PHEV get for its relatively low battery density. The average subsidies for BEVs (USD 6000) are twice the average for PHEVs (USD 3200). The models separating PHEV from BEV also further indicates the robustness for analysis [32].

\*\* *p* < 0.01, \* *p* < 0.05.


**1. Introduction**

Generally, a wing or tail of an aircraft will be damped by damping when the speed of it is low; when the speed of flight exceeds a certain value, small disturbance will cause vibration divergence and the structure to collapse in a matter of seconds or even tens of milliseconds. This phenomenon is called flutter. Flutter is the most important subject in aeroelasticity. It is a kind of self-excited vibration which can maintain the constant amplitude oscillation under the interaction of elastic force, inertia force and aerodynamic force when the lifting surface moves at a certain

speed in the air flow. At this time, the damping (g) equals 0. Due to the existence of system damping (g < 0), the vibration of the aircraft soon attenuates or even disappears completely with a small flight speed after being disturbed, but when the flight speed increases to a certain value, the amplitude caused by the disturbance just keeps the same. This speed is called the critical flutter speed, the vibration frequency at this time is called the critical flutter frequency and g = 0. In order to prevent flutter, the critical flutter velocity must be larger than the maximum flight velocity under all flight conditions and there must be some margin. **Figure 1** taken from a Chinese book written by Liu, C., F. and Qiu, J. [1] is an example of the wing flutter calculation: when the speed of flight, VCR, is 850 m/s, the wing is in constant amplitude vibration, and if the speed of flight is less than or greater than it, the wing vibration will attenuate or disperse.

Classical flutter calculations in frequency domain are performed using either the K method proposed by Bisplinghof, R. L., and Ashley, H. [2] or the P-K method proposed by Hassig, H. [3] and Lawrence, J. A., and Jackson, P. [4]. The K method is generally very fast and quite simple, but it has a downfall in that sometimes the frequency and damping values "loop" around themselves and generate multi-value frequency and damping as a function of velocity. The K method solution is only valid when g = 0 and the structural motion is neutrally stable and matches the aerodynamic motion with the neutral stability. The P-K method is acknowledged to provide more accurate modal damping values after the K method. Gradually, the P-K method has become one of the most widely used methods in aeroelastic engineering. After that, the p method, proposed by Abel, I. [5], improves the damping and frequency trends by taking into account the effect of nonzero damping by means of generalized aerodynamic forces, which are approximately valid for the damping-frequency area under consideration. At the end of the 1990s, the *μ* method studied by Lind, R., and Brenner, M. [6], was used to fitting procedures to transform the aerodynamics to the state space. At the beginning of the new century, a g method proposed by P. C. Chen [7] is used in the analytic property of unsteady aerodynamics and a damping perturbation approach. These two methods of P-K method and g method are different in the

**Figure 1.** *The wing flutter calculation, VCR = 850 m/s.*

### *Verification and Validation of Supersonic Flutter of Rudder Model for Experiment DOI: http://dx.doi.org/10.5772/intechopen.98384*

equation form, but they share the same stability criterion, i.e., an eigen root of aeroelastic equation is solved and the root with positive real part indicates flutter. Additionally, the g method uses a reduced-frequency sweep technique to search for the roots of the flutter solution and a predictor corrector scheme to ensure the robustness of the sweep technique. The g method includes a first-order damping term in the flutter equation which is rigorously derived from the Laplace-domain aerodynamics. And then, an improved g method proposed by Ju Qiu and Qin Sun [8], increases a second-order damping term in the flutter equation. It is also valid in the entire reduced frequency domain and up to the second order of damping. Recently, the H method proposed by Michaël H. L. [9], automatically extends the aerodynamic data obtained for purely oscillatory motions to damping and diverging oscillatory motions by means of a direct harmonic interpolation method, thereby improving the prediction of damping and frequencies. This procedure may assist the aeroelastician in making improved estimates of aerodynamic damping at g < 0 conditions to support flight flutter testing and probably offers potential for flight control system design or analysis. Brian P. Danowsky and etc. [10] showed that three different flutter suppression controllers were designed using a flight-test-validated aeroelastic aircraft model. The flight tests demonstrated that the flutter boundary could be successfully expanded using active control. Eli [11] indicated that AFS (Active flutter suppression) technology had the potential to lead to significant weight savings and performance gains.

In the new and challenging field of energy harvesting through fluid–structure instabilities, such as analysis in time domain, the coupled-mode flutter mechanism has been recently scrutinized. A lot of complicated aeroelastic characteristics are predicted by a structural-aerodynamic fully-coupled formulation, such as rotorcraft written by Bernardini G., Serafini J., Molica Colella M., and Gennaretti M. [12], a transport wing's wingtip introduced by Peng Cui and Jinglong Han [13], a transonic wing analyzed by Xiang Zhao, Yongfeng Zhu and Sijun Zhang [14]. Francisco Palacios, Michael R. Colonno, and etc. [15] showed an integrated platform for multi-physics simulation and design, e.g. flutter predictions by a loosely-coupled method, remained open source and serves as a starting point for new capabilities that will hopefully be contributed by users in both academic and industrial environments. Most recently, Leclercq T., Peake N., and de Langre E. [16] pointed out that flutter did not prevent drag reduction by reconfiguration. Eirikur Jonsson [17] put flutter and post-flutter constraints into the process of the aircraft design optimization. Sergey Shitov and Vasily Vedeneev [18] investigated the flutter boundaries of rectangular panels simply supported at all edges, and used potential flow theory to calculate the unsteady pressure.

Naturally, for every simulation and every test, it is desired to produce a model that could be accurate, studied by Samuel C. Mclntosh Jr., Robert E. Reed Jr. T. and William P. Rodden [19], so as to permit meaningfully calculated and experimental comparisons and to provide data for evaluating new theoretical or numerical techniques for treating aeroelastic problems. The studies of all cases suggest that at increased airspeeds the aircraft may develop unstable oscillations leading to a catastrophic failure, described by Jieun Song, Seung Jin Song and Taehyoun Kim [20], Jie Zeng and Sunil L. Kukreja [21], Thomas Andrianne and Grigorios Dimitriadis [22]. At all times, on one hand, there exist analytical models and tools that allow estimating the onset of the dynamic instability, but they are often subject to modeling uncertainties and limitations that can produce inaccurate, unreliable results. On the other hand, the test data are a direct reflection of the actual aircraft, to some degree, and hence can be used with confidence. Also, it is a common and required practice in aerospace industry to conduct flight flutter tests and validate the simulation effect of the analysis model before aircraft enter into service.

Since the beginning of the century, American Society of Mechanical Engineers guide to Verification and Validation (V&V) defined the goal of V&V process as to develop standards for assessing the correctness and credibility of modeling and simulation in computational science. According to the paper, for Validation, and Predictive Capability in Computational Engineering and Physics, written by William L. Oberkampf, Timothy G. Trucano, Charles Hirsch [23], a proposed question was how confidence in modeling and simulation should be critically assessed. Verification and validation (V&V) of computational simulations are the primary methods for building and quantifying this confidence. Briefly, verification is the assessment of the accuracy of the solution to a computational model. Validation is the assessment of the accuracy of a computational simulation by comparison with experimental data. In verification, the relationship of the simulation to the real world is not an issue. In validation, the relationship between computation and the real world, i.e., experimental data, is the issue. Furthermore, Verification is defined as following: the process of determining that a model implementation accurately represents the developer's conceptual description of the model and the solution to the model, while Validation means that the process of determining the degree to which a model is an accurate representation of the real world from the perspective of the intended uses of the model. Supanee Arthasartsri and He Ren [24], in A380 Aircraft Reliability Program also pointed out that the validation is a process determining whether the mathematical model describes sufficiently well the reality with respect to the decision to be made, which includes requirement validation and product validation, whereas, the purpose of Validation is to ensure that the requirements for a product are sufficiently correct and complete to achieve safety and to satisfy the needs of the customer within program constraints (e.g. cost, schedule). The Product Validation is to check if product meets the implicit needs of the customer. It is also to ensure the final product meets the requirement, for example, airworthiness certification of commercial aircraft. Additionally, V & V method was described in Guide for Verification and Validation in Computational Solid Mechanics, written by Schwer L. E. and etc. [25].

In **Figure 2**, it is evident to see the test has not been involved in the first stage of the V&V process, Verification and only is employed in the second step, Validation.

When V & V technique is widely used to aerospace engineering, civil engineering, auto engineering, etc., it obtains fruitful achievements. For instance, the example of V&V method implementation on GP7200 by Engine Alliance, which the results of reliability before entry into service exceeded the expectation requirements from FAA. With the entry into service of Airbus A380, the application of validation and verification method in safety and reliability program has been proven successful. The other example is Validation and Verification of the Lunar Atmosphere Dust Environment Explorer (LADEE) mission models and software. This program identified and prioritized a set of V&V technologies that significantly reduced the development cost and compressed the development schedule of emerging safety critical flight control systems. It is predicted that it could also be applied to other aerospace system design and to other industry that require the high level of safety and V&V Method would be used to provide the confidence in the results from simulation.

The present research uses V & V technologies to handle the flutter problem of the rudder. There is, however, a significant difference, that is to say, the experimental data were added to modify the simulated models, besides codes and software verification.

In the Verification process, the stiffness of the rudder axis could be adjusted during the flight or the experiment. The accuracy of the flutter-prediction results of these methods heavily depends on how good the model or mode estimations are.

*Verification and Validation of Supersonic Flutter of Rudder Model for Experiment DOI: http://dx.doi.org/10.5772/intechopen.98384*

**Figure 2.**

*Verification and Validation's activities and products.*

Therefore, the modes of the rudder directly influencing flutter speeds become very critical. In order to obtain a precise model to simulate two cases of supersonic flutter, the appropriate axis structures were provided by optimization process. Unlike traditionally aeroelastic systems of maximum performance and minimum weight, studied by Melike Nikbay, and Muhammet N. Kuru, [26], the objective was a minimal 1-order or 2-order error which was a ratio of test eigenvectors and calculated ones, constraints were the first and second natural frequencies, and design variables were stiffness of the different rudder axles. Finally, modes of the first bending and second twisting of the rudder were found out in Case one, and

these of the first torsion and second bending were extracted in Case two. In addition, it focused on finding a right analysis model by optimization method, according to experimental data of mode shapes studied by Zimmerman, N. H., and Weissenburer, J. T. [27]. Test investigations were first presented, followed by verifying the structural model and mode by optimization. Although two flutter cases both had the same flutter mechanism, classical bending-torsion flutter, they had their own critical flutter points, respectively.

The present work also aims at a deeper understanding of the phenomena by characterizing the tight interaction between the unsteady flow patterns in the flow-field and the response of the structure. After numerical aeroelastic simulations were finished, the test data were employed to validate flutter prediction to confirm the observed critical velocity and coupled-mode flutter in the validation stage of V& V technologies.


**2. Materials and Methods**

The systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [23], and the study was registered with PROSPERO (CRD42021281160).


**7. Radiotherapy treatment and implants**

*Oral and Maxillofacial Surgery*

**Figure 4.**

Three-dimensional printing has been used to produce anatomical models, surgical guides and templates, implants, and molds. The main advantages include: the possibility of preoperative planning, the precision of the process used and the time saved in the operating room. However, other studies report inconsistency in

*Photograph of surgical field after placement of zygomatic and Pterygomaxillary implants.*

The maxillofacial reconstruction with osseointegrated implants simulated in a biomodel, allows to determine the lengths and final positions of the implants, prepare the surgeon for the surgical procedure and minimize the possibilities of

Oral and facial deformity can cause functional and psychological deterioration in oncological patients. The aims of prosthodontic reconstruction are the rehabilitation of the shape, function and esthetics of the lost anatomical structures by means of artificial substitutes. The main facial subunits that require reconstruction due to malignant pathological processes involve the ear, forehead, eyes and brow, nose,

Currently, there are multiple workflows where they combine conventional prosthetic preparation with the use of facial scanners and custom 3D impressions

precision and additional costs in treatment [32].

**6. Prosthetic and facial reconstruction**

check, lips and chin [33].

errors favoring the results of the treatment (**Figures 3** and **4**)**.**

*Photograph showing implant-supported obturator placed and final photograph.*

**168**

**Figure 5.**

In early clinical stages, surgery is the first decision. However, adjuvant radiotherapy is sometimes indicated in cases of close excision margins (<5 mm), involved (<1 mm), and in suspicion or confirmation of lymph node metastasis with or without extracapsular extension [36]. In general, radiation therapy includes conventional radiation therapy or intensity modulated radiation therapy (IMRT). The latter is more convenient by precisely targeting radiation to a specific area and reducing the dose to nearby anatomical structures such as malar bone, grafts, implants, salivary glands, eyes, and spinal cord [37].

Schiegnitz's study suggests that radiation negatively affects implant survival, but there is no statistically significant difference in survival when implants are inserted 12 months before or after radiation therapy [38]. Other authors have shown favorable results. With implantation at least 6 weeks before radiotherapy since there is a surgical area with less hypoxia, hypovascularity and hypocellularity [27, 39] (**Figure 6**).

**Figure 6.** *Histogram showing trajectories, dose and volume used for adjuvant radiotherapy.*


**3. Bias in the perceived orientation of locomotor surfaces: Hills and ramps**

While there are clear and consistent visual and spatial biases in the perception of surfaces within reach of the hands, visual biases measured in the perception of locomotor surfaces have tended to be seem even more exaggerated. These exaggerations were initially documented by Kammann (1967) who proposed that they might reflect the heightened energetics of the gravitational vertical, and by Ross (1974), but were most extensively documented by Proffitt et al. (1995). Although many of the theoretical conclusions of Proffitt et al. have been called into question by more recent work, their basic documentation of the judged orientation of 8 distinct hills on their campus remains a useful basic source regarding the overestimation of hills. In particular, Proffitt et al. reported that paths with physical slants of 4°, 5° or 6° were typically perceived as being about 20° in orientation when viewed with gaze forward, as shown in Figure 3.

Fig. 3. Verbal estimates of the (visual) slants of eight hills from Proffitt et al. (1995), ± 1 SEM. Hills were viewed from the base with gaze forward.

Because the range of orientations in most locomotor settings is no more than ± 10° from horizontal, and physical/biomechanical limitations restrict bipedal locomotion to surfaces of less than about 35°, Proffitt et al. (1995) proposed that the exaggerated perception of hill orientation was an adaptive strategy to represent the range of human behavioural potential

These observations are particularly relevant to the present discussion because they emphasize that the form of the perceptual bias may not be a "vertical tendency." Rather, the linear gain of about 1.5 found in various measurements of perceived pitch between 0° and 45° from horizontal suggests that deviations from horizontal are being exaggerated and therefore that it is the horizontal that is special. Durgin and Li (2011a) have hypothesized that the expanded scaling (near horizontal) of the perceived pitch of gaze direction serves to expand the most highly utilized portion of the angular range for purpose of maintaining greater cognitive precision. Across a variety of contexts, they proposed that a gain of about 1.5 may provide efficient scaling for retaining greater precision in this lower half of the

range when sending neural pitch signals upstream to cognitive and motor areas.

with gaze forward, as shown in Figure 3.

Hills were viewed from the base with gaze forward.

**3. Bias in the perceived orientation of locomotor surfaces: Hills and ramps**  While there are clear and consistent visual and spatial biases in the perception of surfaces within reach of the hands, visual biases measured in the perception of locomotor surfaces have tended to be seem even more exaggerated. These exaggerations were initially documented by Kammann (1967) who proposed that they might reflect the heightened energetics of the gravitational vertical, and by Ross (1974), but were most extensively documented by Proffitt et al. (1995). Although many of the theoretical conclusions of Proffitt et al. have been called into question by more recent work, their basic documentation of the judged orientation of 8 distinct hills on their campus remains a useful basic source regarding the overestimation of hills. In particular, Proffitt et al. reported that paths with physical slants of 4°, 5° or 6° were typically perceived as being about 20° in orientation when viewed

Fig. 3. Verbal estimates of the (visual) slants of eight hills from Proffitt et al. (1995), ± 1 SEM.

Because the range of orientations in most locomotor settings is no more than ± 10° from horizontal, and physical/biomechanical limitations restrict bipedal locomotion to surfaces of less than about 35°, Proffitt et al. (1995) proposed that the exaggerated perception of hill orientation was an adaptive strategy to represent the range of human behavioural potential and energetic costs and thus to inform decisions about route selection during locomotion in the wild. A difficulty with this view is that perceived hill orientation decreases as one approaches a hill (Li & Durgin, 2010), and nearer portions of hills appear shallower than farther portions (Bridgeman & Hoover, 2008). In fact, as we will discuss below, there is continuity between orientation biases we have measured for small, near surfaces and those measured by Proffitt et al. for hills (Li & Durgin, 2010).

Proffitt further proposed that physical actions, such as stepping, were controlled by an *unbiased* perceptual representation (vision for action) that was contrasted with the exaggerated representation available for long-range cognitive planning. This view has since been challenged by studies of the haptic perception of locomotor inclines.


**5. Conclusions**

The improvement of diesel engines, as the main source of modern energy, is associated with a further increase in fuel efficiency, liter capacity, and reliability, with a significant reduction in emissions of toxic substances and carbon dioxide into the environment.

The proposed systematic approach to a comprehensive assessment of fuel efficiency and emissions toxicity allowed proposing a dimensionless criterion that takes into account the operating conditions of the engine. Using this criterion, an assessment of the efficiency of using alternative fuels is given and the prospect of such an assessment is shown when using hybrid fuels that include green hydrogen.

To identify the mechanisms of the formation of internal and external losses and substantiate the ways to achieve optimal heat use, the use of the anergy-energetic method of analysis is justified.

In order to increase the reliability of the most thermally stressed parts of highly accelerated engines, taking into account the operating conditions, the approach has been proposed, taking into account the complexity of factors affecting the physical and parametric reliability.

The directions for improving diesel engines, considered in the chapter, are only a part of a set of tasks, the solution of which seems to be extremely relevant from the point of view of preventing an energy crisis and, at the same time, are only a part of the possible ones for practical implementation.


**4. Epidemiology and world impact of HCV**

HCV infection is one of the main causes of chronic liver disease worldwide, and according to recent estimates, until now more than 185 million people around the world have been infected. In addition, annually there are three million of new infected people, and among them 350,000 die every year due to HCV-related disorders [18–21]. The prevalence of HCV varies greatly, depending on the geographical area and the population considered: in Western Europe, it ranges from 0.4% to 3%; in Eastern Europe and the Middle East, it is higher but not precisely known [22]. The majority of the infected people reside in Asian countries (Taiwan, Mongolia, and Pakistan), sub-Saharan Africa (Cameroon, Burundi, and Gabon), and the Eastern Medi‐ terranean (Egypt), which holds the highest frequency, with more than 20% [18]. HCV is a major global public health issue due to its high prevalence, long-term unpredictable disease pro‐ gression, and low diagnosis and treatment response rates. Despite the fact that HCV infection rates are decreasing, the clinical and economic impact of chronic HCV infection is expected to considerably grow in the next decade since a large population of individuals that acquired the virus in the 1960s developed disease-associated health issues through to the 1980s [23]. The dual therapy, based on the administration of peg-IFN and RBV, is successful only in 40–50% of patients infected with the GT-1, while untreated individuals or who failed treatment are at risk of developing severe liver injuries such as cirrhosis, liver transplantation, and hepatocel‐ lular carcinoma (HCC) [24]. In Europe, there are 30,000 people on the transplant waiting list but only 12,000 procedures per year, and the average cost of liver transplant in the United States varies between \$139,000 and \$400,000 [25]. Although HCV can be successfully treated by now using antiviral therapy based on the administration of new direct acting antivirals (DAAs), the economic burden of the disease, including complex regimens and the cost of treatment, remains high since health care costs continue to rise [26]. For this reason, many HCV-diagnosed patients around the world are left untreated or undertreated. A 2010 study performed on U.S. employments found that the cost of sick days and lower productivity per HCV-infected workers was US\$8,352 per year [25]. A U.S. survey by the American Gastroen‐ terological Association (AGA) indicated that the cost for 30,000 outpatient visits for HCV infection amounted to US\$24 million in the 1998 [27]. The median cost for treating one patient with dual therapy (peg-IFN and RBV) ranges from €7,517 to €21,229, depending on the virus genotype, plus the costs of the new DAAs are about US\$70,100 per quality-adjusted life years (QALY) for mild fibrosis and US\$36,300 per QALY for advanced fibrosis [28].


**Acknowledgements**

The authors are grateful to the co-authors of the previous articles for carrying out our joint research and to the Ministry of Education and Science of Ukraine for financial support.


**Acknowledgements**

Part of this work was performed in the Micro/Nanomachining Research Education Center (MNC) of Tohoku University. This work was supported by Cabinet Office, Government of Japan, Cross-ministerial Strategic Innovation Promotion Program (SIP), (funding agency: The New Energy and Industrial Technology Development Organization, NEDO) and also supported in part by JSPS KAKENHI for Young Scientists (Grant number: 20K15147).


*4.3. Variation Law of Safety Factor*

The safety factor can be used to reflect the possibility of slope instability to a certain extent. As the subgrade and foundation are influenced by rainfall, the pore-water pressure in the traditional subgrade decreased, which reduced the overall safety factor of the subgrade. The safety factor was reduced from 1.35 to 1.15 in one year as shown in Figure 10, and the decrease in the safety factor with time was nonlinear. The safety factor of the subgrade protected by the capillary barrier was almost unchanged. This was mainly due to the better blocking effect of the capillary barrier. In one year, the safety factor of the subgrade using a capillary barrier increased by 15% compared to the traditional subgrade.

**Figure 10.** Comparison of the safety factor in the different subgrades.

## **5. Conclusions**

In this study, we first proposed effective measures to block the infiltrating rainwater in the subgrade through a capillary barrier. Secondly, using the numerical simulation method, we conducted studies on the mechanism and stability of the capillary barrier used in the subgrade to control distress against wetting. The subgrade with a capillary barrier could effectively use the different permeability coefficients of the different types of soil to achieve an anti-seepage effect, which minimized the deformation of the subgrade and increased stability. Using the numerical simulation method, this paper analyzed the pore-water pressure distribution, settlement, and safety factor of the conventional subgrade and the subgrade protected by the capillary barrier. Through a comparison of the numerical results of the two subgrades, the following conclusions were drawn:


Maria Teresa Gonzalez Garza

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62989

#### **Abstract**

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[82] Vaccaro A, Tauffenberger A, Aggad D, Rouleau G, Drapeau P, Parker JA. Mutant TDP-43 and FUS cause age-dependent paralysis and neurodegeneration in C. elegans.

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with prion-like domains. Mol. Biosyst. 2015;11:2210–8.

2009;73:1176–9.

120 Update on Amyotrophic Lateral Sclerosis

PLoS One. 2012;7:e31321.

miRNAs are sequences of 20–22 nucleotides that participate in the development, growth, and cell differentiation by the regulation of the mRNAs.Their possible participation in the development of degenerative diseases has been extensively investigated. Results show quantitative changes in miRNA transcription, to the pathogenesis of various neurodegenerative diseases. In this chapter, the dysregula‐ tion of microRNAs reported in the samples taken from amyotrophic lateral sclerosis (ALS) animal model or ALS patients is analyzed. Moreover,their probable participa‐ tion in the pathogenesis of the disease is also analyzed.

**Keywords:** ALS, microRNA, miR-206, miR-338-3, miR-9a

## **1. Introduction**

miRNAs are small RNA molecules that do not encode proteins. They are sequences of 20–22 nucleotides that regulate gene expression. miRNAs were identified in 1993 by Lee et al. [1]. Since then numerous studies have been carried out in order to have a better understanding of their functioning as regulators and, in turn, regulating transcription. These molecules are transcribed as a longer RNA between 500 and 3000 nucleotides [2, 3]. Then, a serial enzymat‐ ic activity inside the nucleus allows the reduction in the number of nucleotides. These primiRNAs are then exposed to an enzymatic process by using Drosha-associated Pasha (also known as DGCR8), resulting in a precursor of 65–75 nucleotides in a loop called pre-miR‐ NA, which is transported to the cytoplasm. Finally, the Dicer enzyme breaks for mature singlestranded fragments comprise 19–25 nucleotides. The last enzymatic activity into cytoplasm provides an miRNA of 20–22 nucleotides with a complementary sequence of seven base pairs that enable an miRNA to bind to the target mRNA [3–6].

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

The consequence of that complementary interaction could provide the inhibition of translation or the reduction in the stability of the mRNA resulting in the decrease of the target protein expression [7]. The importance of these small molecules in gene expression has led to numer‐ ous studies, which aimed at better understanding of its functioning as regulators and, in turn, the regulation of transcription. Another characteristic is its possibility to target several mRNAs given a major potential to regulate gene expression or a specific mRNA that can be regulated by multiple miRNAs [8–10]. Although most authors observed a downregulation by miRNAs, there are examples of upregulation by miRNAs as well. The ability of a single miRNA to act both in repression and activation depends on the number of nucleotides that can mate or phase of the cell cycle in which it is generated [11–16].

In order to homogenize the nomenclature of miRNAs for the pre-miRNA and the pri-miRNA, they are referred to as uncapitalized "mir," for the mature form of the miRNA as miR and MIR for the gene that encodes them [17].

Considering the complexity of the capacity and functions of these small molecules, they have been linked to several diseases such as cancer [18, 19], autoimmune diseases [20, 21], cardio‐ vascular diseases [22–26], and, of particular interest to our group, are those related to neuro‐ logical diseases.

General reviews on the participation of miRNAs in neurodegenerative diseases have been emerging, and the research on specific neurological diseases confirms a dysregulation biogenesis of these small molecules [27–31]. Impaired concentration of blood miRNAs has been reported in patients with stroke, Alzheimer disease, Huntington disease, Parkinson Disease, and Amyotrophic Lateral Sclerosis (ALS) [30–46]. In this chapter, ALS disease will be analyzed in more detail.

The dysregulation of miRNAs in the samples from ALS patients has been reported by several authors. Until now, it is not clear that which one is the most important dysregulation. This chapter aims at analyzing those reports that shed light on the enrolled miRNAs and their participation in ALS pathology.

## **2. miR-b2403 and miR-b1336 downregulation**

During neuron degeneration in ALS diseases, many alterations on neurofilament have been observed such as the aggregate formation of the perikaryon and proximal axons. In addition, mRNA of low molecular weight neurofilament (NEFL) decreases [47–49]. A number of reports agree that an alteration of NEFL synthesis could contribute to the possible cause of neuron death in ALS patients. Recent reports on the expression analysis of miRNAs, performed on neurons recovered from sporadic ALS patients' spinal cord, confirm the downregulation of miR-b2403 and miR-b1336. These miRNAs modulate NEFL mRNA stability [50]. Consequent‐ ly, it could be the cause of the decreased mRNA, as previously reported in patients with spinal cord ALS.


*Ulrich Götz* is professor at Zurich University of the Arts (ZHdK), where he has headed the ZHdK subject area of Game Design since 2004. He was trained as an architect at Berlin University of the Arts and the Escola Tècnica Superior D'Arquitectura in Barcelona. He lectures and publishes on the comparative analysis of spatial design in architecture and game spaces. He has built up extensive experience in research and development of serious and applied games over years of cooperation with numerous partners from medical, therapeutical, educational and economic contexts. His university teaching focuses on the analysis and design of game mechanics, game concepts, motivation design, and spatial design in virtual environments.

*Günter Hack* holds a doctorate in communication sciences. He has worked as a lecturer at the University of Erfurt, Germany, and at the University of St. Gallen, Switzerland, and as a journalist for various publications including the Frankfurter Allgemeine Zeitung, Zeit Online and Spiegel Online. He currently works as a project manager for the Austrian public broadcasting service ORF, Vienna. He is the author of two novels: *ZRH* (2009) and *QUIZ* (2018).

*Margarete Jahrmann* is an artist, researcher and activist game designer, as well as founder of Area7lab and Ludic Society, a magazine series on experimental game art and ludic interventions. In 2020 she was awarded the media arts price of the City of Vienna, in 2019 a fellowship at the Center of Advanced Studies LMU Munich. She was artist fellow at the Leibniz-Zentrum für Literatur- und Kulturforschung Berlin in 2017. In 2010 she researched at the MIT gamebit Lab for her PhD, which she finished at the University of Plymouth with her work *Ludics for a Ludic Society. The art and Politics of Play*, introducing a specific Ludic method (ludic-society.net). She and the artist Max Moswitzer received the software arts award transmediale Berlin and the prix ars electronica with distinction in interactive arts for the game the anti-war-shooter *Nybble-Engine*. Jahrmann is professor of Game Design at Zurich University of the Arts (gamedesign. zhdk.ch/personen/team/margarete-jahrmann/) with a focus on persuasive games, political play and experimental game design, Univ.-Prof. Artistic Research PhD program University of Applied Arts Vienna (zentrumfokusforschung.uni-ak.ac. at/index.php/profile/team/). Since 2020 she leads the Austrian Science Fund FWF research project Neuromatic Game Art: Critical Play with Neurointerfaces. www.margaretejahrmann.net

*Hiloko Kato* studied German, art history and philosophy at the University of Zurich. In 2015 she received her doctorate on the topic "At the Edges of Texts". She collaborated in the project "Textualisation Cues" for the Swiss National Science Foundation (SNF). She works as a postdoc at the Chair of German Linguistics of the University of Zurich. In recent years, her research interests have shifted significantly towards game studies and human-nonhuman interaction, with pragmatic linguistics remaining the main methodological approach. In her current scientific projects, she takes an ethnomethodological and conversation analytic approach to playing and designing video games and to human-animal interaction both in real and virtual worlds.

*Mela Kocher* studied German literature, computer science and history, and wrote her dissertation on the topic of aesthetics and narratology in video games ("Follow the Pixel Rabbit!" 2007). Her post-doctoral research included studies at the University of San Diego, California, and the Mobile Life Institute in Stockholm, Sweden. There she followed up on her research on pervasive games and participatory transmedia storytelling. Since 2009 she has been with Zurich University of the Arts (ZHdK) as a senior researcher, teaching in the Master's program of the Game Design specialization and working on R&D projects, mainly in the area of serious and applied (urban) games. Since 2019 she has also been working with the e-learning team of the ZHdK. Besides that, she enjoys studying local cultural life and is a freelance writer for a small-town newspaper.

*David Krummenacher* is a game designer with a background in IT and film making. In 2018 he received a Master of Arts in Game Design from Zurich University of the Arts. His master's thesis "Storytelling in Pinball Machines" examined pinball machines in terms of their narrative elements and structures and applied the insights gained in a practical project called "Bosch – The Pinball Machine". He currently works as a senior game and experience designer at Zurichbased FitTech startup Sphery Ltd.

*Martin Lindner* is a consultant and author. He received his doctorate at Ludwig Maximilian University Munich and is a certified "Author of Interactive Learning Media" (Macromedia Academy, Munich). He has been working with digital learning and knowledge media for many years in the areas of research and development (microlearning, micromedia), e-learning and MOOCs (conception, implementation), new hybrid formats for learning and collaborative knowledge work (beyond the online/offline gap). Lindner is also an expert in the development and implementation of projects in e-learning, web learning and knowledge management in organizations, large corporations and medium-sized companies.

*Eugen Pfister* is project lead of the SNF-Ambizione research project "Horror-Game-Politics" at Bern University of Arts (hgp.hypotheses.org), in which he examines ideological transfer processes in digital horror games. He studied history and political sciences at the University of Vienna and at the Université Paris IV – Sorbonne. He wrote his dissertation in co-tutelle at the Johann Wolfgang Goethe University Frankfurt and at the Universita degli studi di Trento in the field of history of political communication. He researches and teaches the history of ideas in digital games, and is also a founding member of the "Arbeitskreis Geschichtswissenschaft und Digitale Spiele".

*Chris Polus* studied Business Informatics at FFHS and worked as IT consultant for major Swiss companies. In 2011 he switched to creating sound for games and movies and helped found and grow stillalive studios in Innsbruck, Austria, which now employs 37 people. Polus teaches sound design for game design students at Zurich University of the Arts (ZHdK). In his early days, he helped develop a multimedia software whose core is still used to run light and video walls at events like Eurovision Song Contest, and Cirque du Soleil shows in Las Vegas.

*Stefan Schmidlin* studied computer science and computational linguistics at EPFL (École polytechnique fédérale de Lausanne) and McGill University and holds an M.A. in Game Design from Zurich University of the Arts (ZHdK). He wrote his thesis on the topic of dominant games, investigating the manipulative and controlling properties of games. Currently, he works on games and interactive installations at the Insert Coin collective and as a researcher on serious and applied games at ZHdK. Recent projects he was involved in include Foodscape, a learning game about sustainability, BrainDriver, a racing game using brain signals as game input and Shock Fighter, an experimental game delivering electric shocks as a reward. As a board member of the IGDA Switzerland he promotes inclusivity and LGBTQ representation in the games industry.

*Beat Suter* is senior lecturer for Game Design at Zurich University of the Arts (ZHdK), Switzerland, specializing in concepts, storytelling, mechanics and the history of games – and he manages the GameLab (gamelab.zhdk.ch). He holds a doctorate in literature from the University of Zurich, with a focus on digital literature. For some years Suter has worked as lecturer for the Merz Academy Stuttgart, as project manager for a communications agency and as publisher of books and electronic literature (edition cyberfiction and netzliteratur.net). He also works as an independent scholar and curator, and as editor and author. Suter co-organizes the annual conference GameZ&RuleZ (gamezandrulez.ch). He is a founding member of the art group AND-OR that specializes in gameart and netart (www.and-or.ch). Recently, he co-authored a series of creative tools for game designers: two card sets, *IdeasForGames* and *StoriesForGames*, and a *Prototyping Box* (www.ideasforgames.org). www.cybersuter.ch

# Cultural Studies

Gabriele Klein

 Pina Bausch's Dance Theater Company, Artistic Practices and Reception

 May 2020, 440 p., pb., col. ill. 29,99 € (DE), 978-3-8376-5055-6 E-Book: PDF: 29,99 € (DE), ISBN 978-3-8394-5055-0

Elisa Ganivet Border Wall Aesthetics Artworks in Border Spaces

 2019, 250 p., hardcover, ill. 79,99 € (DE), 978-3-8376-4777-8 E-Book: PDF: 79,99 € (DE), ISBN 978-3-8394-4777-2

 Krista Lynes, Tyler Morgenstern, Ian Alan Paul (eds.) Moving Images Mediating Migration as Crisis

 May 2020, 320 p., pb., col. ill. 40,00 € (DE), 978-3-8376-4827-0 E-Book: available as free open access publication PDF: ISBN 978-3-8394-4827-4

All print, e-book and open access versions of the titles in our list are available in our online shop www.transcript-publishing.com

# Cultural Studies

Andreas Sudmann (ed.)

 The Democratization of Artificial Intelligence Net Politics in the Era of Learning Algorithms

 2019, 334 p., pb., col. ill. 49,99 € (DE), 978-3-8376-4719-8 E-Book: available as free open access publication PDF: ISBN 978-3-8394-4719-2

 Jocelyne Porcher, Jean Estebanez (eds.) Animal Labor A New Perspective on Human-Animal Relations

 2019, 182 p., hardcover 99,99 € (DE), 978-3-8376-4364-0 E-Book: PDF: 99,99 € (DE), ISBN 978-3-8394-4364-4

 Ramón Reichert, Mathias Fuchs, Pablo Abend, Annika Richterich, Karin Wenz (eds.) Digital Culture & Society (DCS) Vol. 4, Issue 1/2018 – Rethinking AI: Neural Networks, Biometrics and the New Artificial Intelligence

 2018, 244 p., pb., ill. 29,99 € (DE), 978-3-8376-4266-7 E-Book: PDF: 29,99 € (DE), ISBN 978-3-8394-4266-1

All print, e-book and open access versions of the titles in our list are available in our online shop www.transcript-publishing.com


*2.3. Extraction of Plant Materials*

#### 2.3.1. Preparation of Aqueous Ethanolic Extract

We extracted dried powdered plant material using aqueous ethanolic solvent (50%, for 6 h at 37 ◦C), then pooled the hydroalcoholic extract, and it dried under reduced pressure at 40 ◦C with a rotator evaporator. For hydroalcoholic extracts, the percentage yields of the crude extracts were 15.58% *w*/*w*.

#### 2.3.2. Standardization of Polyphenols in *P. maderaspatensis* by HPTLC

Standard solutions of ellagic acid, gallic acid, catechin, quercetin, rutin, and kaempferol were applied in triplicate on silica gel 60 F254 plates, using a CAMMAG Linomat-5 Automatic Sample Spotter. The plates were developed in a solvent system, toluene/ethyl acetate/formic acid/methanol (3:3:0.8:0.2 *v*/*v*) in a CAMAG glass twin trough chamber (20 × 10 cm) up to a distance of 8 cm, dried in the air, and scanned at 254 nm. The developed plates were dried in the air and scanned at 254 nm using CAMAG TLC scanner 3 and win CATS 4 software. The peak areas were recorded. Calibration curves of ellagic acid, gallic acid, catechin, quercetin, rutin, and kaempferol were obtained by plotting peak areas versus applied crude extracts, and the concentration of ellagic acid, gallic acid, catechin, quercetin rutin, and kaempferol was calculated, respectively.

#### 2.3.3. Estimation of Different Markers

Each sample solution (10 μL) was applied in triplicate on silica gel 60 F254 plates with CAMAG Linomat-5 Automatic Sample Spotter and the peak areas and absorption spectra were recorded. We calculated the number of bioactive compounds in *P. maderaspatensis* using the peak areas and the absorption spectra were recorded. The number of bioactive compounds in *P. maderaspatensis* was calculated using the respective standard calibration curves of ellagic acid, gallic acid, catechin, quercetin, rutin, and kaempferol.

#### 2.3.4. Fractionation of Potent Aqueous Ethanolic (50% *v*/*v*) Extracts of *P. maderaspatensis*

The aqueous ethanolic extract of *P. maderaspatensis* was dissolved in 10% distilled water and was successively fractionated thrice with hexane (3 × 600 mL), chloroform (3 × 600 mL), ethyl acetate (3 × 500 mL), and water-soluble fractions. The combined fractions of *P. maderaspatensis* were evaporated to dryness under reduced pressure at 40 ◦C in a rotary evaporator.

#### 2.3.5. Nitric Oxide Estimation of Various Fractions of *P. maderaspatensis*

Nitric oxide was measured as nitrite released from mouse macrophage cells, RAW 264.7. Cells were suspended in DMEM (Sigma) supplemented with 10% FCS, seeded in a

96-well culture dish at a density of 1 × 106 cells/well, and incubated for 48 h at 37 ◦C in an atmospheric of 5% CO2 and 95% humidity. After incubation, 100 μL of the medium was transferred from the surface of the cultures of each well and replenished with the same amount of fresh medium. Further incubation for 24 h was done with concentrations of 25, 50, and 100 μg mL−1 of alcoholic and aqueous-alcoholic (50% *v*/*v*) extracts/metabolites (100 and 200 μg mL−1) of *P. maderaspatensis* in the presence or absence of the indicated amount of LPS (10 μg mL−1). Nitric oxide production was estimated in terms of the amount of nitrite. After incubation, 100 μL from the surface of the cultures was transferred into a new plate and the equivalent amount of Griess reagent was added (1% sulfanilamide in 5% phosphoric acid, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride in 5% phosphoric acid). This plate was incubated for 10 min at room temperature and measured by an ELISA reader at 570nm. NO concentration was determined using a standard curve plotted using a known quantity of sodium nitrite. The outcome is obtainable in μM concentration obtained from the mean OD of triplicate wells of each group.

The percentage of NO inhibition/stimulation by the extracts was calculated as follows:

% inhibition = 100 × [([NO2 −] control − [NO2 −] sample)/[NO2 −] control

2.3.6. Isolation and Purification of Potent Fractions by Vacuum Liquid Chromatography

The air-dried whole plant of *P. maderaspatensis* was coarsely powdered, defatted with extraction procedure thrice. Using a rotary evaporator, the aqueous ethanolic extracts were pooled and dried at 40 ◦C under reduced pressure. The crude extracts of *P. maderaspatensis* were successively fractionated thrice with chloroform, ethyl acetate, and water. Ethyl acetate fraction was found to have significant immunomodulatory and hepatoprotective activity compared with other fractions. Hence, this fraction was subjected to vacuum liquid chromatography to separate various components present in the fraction. An amount of 16 gm of ethyl acetate fraction was mixed with a small amount of Silica gel G (Merck) to form a dry slurry, which was then loaded onto a sintered glass funnel filled with silica gel gas stationary phase. The column was eluted stepwise under vacuum with solvents of accelerating polarity, starting from pure toluene, and ethyl acetate mixture, to pure ethyl acetate. To elaborate, after initially eluting with 5% ethyl acetate in toluene, the ethyl acetate portion was increased by 5% increments up to 50% and then in 10% increments up to 100% ethyl acetate with a concomitant decrease in the toluene levels. After elution with ethyl acetate, elution with 5% methanol in ethyl acetate, the methanol portion was increased by 5% increments up to 100% methanol. The solvents were eluted until they ran clear of the funnel. The flow rate of solvent was monitored constantly (100 mL/min) throughout the experiment. Various fractions were collected individually and monitored by TLC to match homogeneity. Similar fractions having the same Rf values were combined and crystallized.

#### 2.3.7. Statistical Analysis

Statistical analysis was performed using Dunnett's multiple comparison tests and one-way analysis of variance (ANOVA) using Graphpad prism 5.0 (Graphpad Software, Inc., San Diego, CA, USA). The level of significance was set at *p* < 0.05.

#### 2.3.8. Maintenance of Cell Lines

HepG2 cell lines were grown in 25 cm2 tissue culture flasks containing minimum essential medium (MEM) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 ◦C in a CO2 incubator in an atmosphere of humidified 5% CO2 and 95% air. The cells were maintained by routine sub-culturing in 25 cm2 tissue culture flasks.

#### 2.3.9. Method for Passaging the Cells

All the reagents were brought to room temperature before use. Media was removed from the 80–90% confluent flasks by a 10 mL serological pipette. Cells in the T-75 flask were washed with 10 mL of PBS. Two milliliters of 0.1% trypsin EDTA was added to the flask. The flask was kept at 37 ◦C in the CO2 incubator for 2–3 min and was observed under a microscope for detachment. Six milliliters of growth medium were added to the flask for inhibition of trypsin action and re-suspended properly by pipetting. The cell suspension was collected in a 15 mL falcon tube and then centrifuged at 1200 rpm for 3 min. The supernatant was discarded and the pellet was resuspended in 3 mL of complete medium. Cells were counted, and then 0.2–0.4 million cells were kept in a T-25 flask for growing. The flasks were incubated in a CO2 incubator at 37 ◦C and the cells were periodically monitored for any morphological changes and contamination. After the formation of an 80–90% confluent monolayer, the cells were further utilized.