*Article* **Space- and Ground-Based Geophysical Data Tracking of Magma Migration in Shallow Feeding System of Mount Etna Volcano**

**Marco Laiolo 1,\*, Maurizio Ripepe 2, Corrado Cigolini 1, Diego Coppola 1, Massimo Della Schiava 2, Riccardo Genco 2, Lorenzo Innocenti 2, Giorgio Lacanna 2, Emanuele Marchetti 2, Francesco Massimetti 1,2 and Maria Cristina Silengo 2**

Received: 29 April 2019; Accepted: 16 May 2019; Published: 18 May 2019

**Abstract:** After a month-long increase in activity at the summit craters, on 24 December 2018, the Etna volcano experienced a short-lived lateral effusive event followed by a rapid resumption of low-level explosive and degassing activity at the summit vents. By combining space (Moderate Resolution Imaging Spectroradiometer; MODIS and SENTINEL-2 images) and ground-based geophysical data, we track, in near real-time, the thermal, seismic and infrasonic changes associated with Etna's activity during the September–December 2018 period. Satellite thermal data reveal that the fissural eruption was preceded by a persistent increase of summit activity, as reflected by overflow episodes in New SouthEast Crater (NSE) sector. This behavior is supported by infrasonic data, which recorded a constant increase both in the occurrence and in the energy of the strombolian activity at the same crater sectors mapped by satellite. The explosive activity trend is poorly constrained by the seismic tremor, which shows instead a sudden increase only since the 08:24 GMT on the 24 December 2018, almost concurrently with the end of the infrasonic detections occurred at 06:00 GMT. The arrays detected the resumption of infrasonic activity at 11:13 GMT of 24 December, when tremors almost reached the maximum amplitude. Infrasound indicates that the explosive activity was shifting from the summit crater along the flank of the Etna volcano, reflecting, with the seismic tremor, the intrusion of a gas-rich magma batch along a ~2.0 km long dyke, which reached the surface generating an intense explosive phase. The dyke propagation lasted for almost 3 h, during which magma migrated from the central conduit system to the lateral vent, at a mean speed of 0.15–0.20 m s−1. Based on MODIS and SENTINEL 2 images, we estimated that the summit outflows erupted a volume of lava of 1.4 Mm3 (±0.5 Mm3), and that the lateral effusive episode erupted a minimum volume of 0.85 Mm3 (±0.3 Mm3). The results presented here outline the support of satellite data on tracking the evolution of volcanic activity and the importance to integrate satellite with ground-based geophysical data in improving assessments of volcanic hazard during eruptive crises.

**Keywords:** MODIS data; SENTINEL-2 images; infrasonic activity; open-vent activity; fissural eruption; long- and short-term precursors


**6.2. Mitochondrial phosphate carrier**

The role of mitochondrial phosphate carrier (PiC, SLC25A3) is importing inorganic phosphate into the mitochondrial matrix. A part of ATP synthasome enables efficient energy production, since Pi is essential for F1 F0 -ATP synthase to catalyze formation of ATP from ADP. For PiC, two isoforms differing in alternative splicing of mutually exclusive exon are documented in tissue-specific expression pattern. PiC-A is expressed in heart and skeletal muscle, while PiC-B is expressed in liver, kidney, and other tissues [128]. The PiC gene is located on chromosome 12q23.1. In 2007, the presence of a homozygous mutation in PiC-A was found in two siblings of nonconsanguineous Turkish parents. Given the role of PiC in energy production, the clinical manifestation of PiC deficiency is associated with multisystemic disorder characterized by muscle hypotonia, lactic acidosis, severe hypertrophic cardiomyopathy, and shortened lifespan [129, 130]. Besides that, PiC has been suggested to impact mtPTP opening [131].

#### **6.3. Mitochondrial pyruvate carrier**

increased glycolysis, maintaining mitochondrial membrane potential, and finally prevention of apoptosis [115]. The effect of hexokinase has been described in Section 4.1. Similarly, the ATP synthasome, a complex of ANT, F0F1-ATPase, and phosphate carrier (PiC), facilitates a mechanism for adenine nucleotide and pyrophosphate release. Under pathological conditions, the imported ATP may also be hydrolyzed by F0F1-ATPase to maintain mitochondrial membrane potential [115]. This type of hydrolysis has also been reported in order to prevent neuro-

Roussel et al. [119] found isoforms ANT1 and 2 to mediate uncoupling by fatty acids and to lower mitochondrial membrane potential in heart and skeletal muscle in rats. ANT is inhibited by competitive displacement of the nucleotide by long-chain acyl-carnitines (LCAC) [120]. LCAC may accumulate under pathological conditions with excess lipid supply, obesity, and mitochondrial β-oxidation defects [121]. ANT inhibition is thought to contribute to

The ANT function in subcellular compartment energy supply is important and therefore it is not surprising that the altered structure and subsequently properties of this solute carrier protein associated with DNA mutations are also associated with serious clinical conditions. Mutations linked to mitochondrial disorders with autosomal recessive inheritance cause mitochondrial myopathy and cardiomyopathy presented in childhood or early adulthood. It is characterized by lactic acidosis, fatigue, proximal muscle weakness, and exercise intolerance [123, 124]. Several different autosomal-dominant mutations in ANT1 gene have been associated with an adulthood-onset disorder, autosomal-dominant progressive external ophthalmoplegia, characterized by ptosis, restriction of eye movement, and accumulation of clonally expanded mtDNA deletions in postmitotic tissues [125, 126]. Thompson et al. [127] have recently described recurrent *de novo*–dominant mutation with severe early-onset of mitochondrial disease. Mutations in the gene encoding ANT1 are associated with the presence of mtDNA deletions. The most likely mechanism of how the defective carrier affects the appearance of mtDNA mutations is the unsufficient adenine nucleotide availability for dATP

The role of mitochondrial phosphate carrier (PiC, SLC25A3) is importing inorganic phosphate into the mitochondrial matrix. A part of ATP synthasome enables efficient energy

ADP. For PiC, two isoforms differing in alternative splicing of mutually exclusive exon are documented in tissue-specific expression pattern. PiC-A is expressed in heart and skeletal muscle, while PiC-B is expressed in liver, kidney, and other tissues [128]. The PiC gene is located on chromosome 12q23.1. In 2007, the presence of a homozygous mutation in PiC-A was found in two siblings of nonconsanguineous Turkish parents. Given the role of PiC in energy production, the clinical manifestation of PiC deficiency is associated with multisystemic disorder characterized by muscle hypotonia, lactic acidosis, severe hypertrophic cardiomyopathy, and shortened lifespan [129, 130]. Besides that, PiC has been suggested to

F0

degeneration [117] and in activated macrophages [118].

262 Mitochondrial Diseases

mitochondrial defects in metabolic syndrome [122].

synthesis and consequent imbalance in dNTP pools [127].

**6.2. Mitochondrial phosphate carrier**

production, since Pi is essential for F1

impact mtPTP opening [131].

Important one is another of the mentioned carriers. Pyruvate is the end product of glycolysis in the cytosol. In mitochondria, pyruvate entering the tricarboxylic acid cycle supports the ATP generation but also serves as a link to anabolic pathways for lipid, amino acid biosynthesis, and gluconeogenesis. The main sources of pyruvate in the cytoplasm are reactions catalyzed by pyruvate kinase. Two more sources are lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), which are important to mention in terms of linking metabolic pathways between tissues. Reversible transamination of pyruvate and glutamate to alanine and α-ketoglutarate catalyzed by ALT converts the pyruvate from muscles into a transport form, alanine, which is reused in the liver for gluconeogenesis. LDH reversibly catalyzes reduction of pyruvate to lactate concurrently with oxidation of NADH to NAD+ . Enzyme gains importance especially in muscle tissue in conditions requiring excessive energy production and in cancer cells. During increased need for ATP for muscle activity, the energy requirements to support continued muscle activity exceed mitochondrial capacity for ATP production. As glycolysis requires NAD+ , ATP production is limited when NAD+ depletes and NADH accumulates. In this case, LDH ensures ATP production in glycolysis by regenerating NAD+ . With a steady supply of NAD<sup>+</sup> , and until acidosis becomes limiting, glycolysis can produce ATP to support work rates exceeding those that could be supported by oxidative phosphorylation alone [132]. The lactate is transported to the circulatory system from where it is taken up by the liver and converted back into pyruvate.

The cross-connection of pyruvate with catabolic and anabolic pathway in mitochondria depends on its passage through mitochondrial membranes. Pyruvates cross MOM through VDAC; however, transport through MIM requires specific carrier. Although the existence of carrier was known earlier, the existence of genes on chromosome 6q27 encoding of mitochondrial pyruvate carrier (MPC) formed by hetero-oligomeric complex of two proteins, MPC1 and MPC2, has been revealed recently. Both proteins are needed for sufficient activity [133, 134]. Proteins do not contain any sequence homology to other mitochondrial carriers. Instead, they have been proposed to belong to the PQ-loop/MtN3/MPC superfamily [135]. Members of PQ-loop family are located in a variety of organelles performing diverse functions. They combine common characteristic features that are seven transmembrane domains and two conserved glutamine motifs. Subunits MPC1 and 2 contain three of seven transmembrane domains and are only half size of other PQ-loop family members [136]. Pyruvate uptake has been proposed to be coupled with the electrochemical gradient, occurring with the symport of one proton, or exchange with one hydroxide ion [137].

MPC activity increases in response to glucagon and decreases in response to insulin. Adrenaline and cortisol also have been found to increase pyruvate carboxylation by increasing mitochondrial pyruvate import [138]. The MPC2 transcript levels have been found to increase up to 1.5-fold under fasting conditions. In a physiological response to fasting (e.g., excessive exercise or prolonged food deprivation), hepatic mitochondrial ketone export and pyruvate import through MPC allows to enhance hepatic gluconeogenesis and maintains membrane potential [132]. Conversely, unregulated rate of gluconeogenesis contributes to chronic hyperglycemia in diabetes. Except for some substances (e.g., α-cyano-4-hydroxy cinnamate, UK-5099, and several thiazolidinediones) acting as specific MPC inhibitors, inhibitory effects of α-ketoacids and phenylpyruvate were found [139, 140]. The accumulation of phenylpyruvate in phenylketonuria prevents pyruvate transfer to mitochondria. Malate was shown to significantly increase mitochondrial pyruvate uptake while not affecting affinity. A genetic background of diminished pyruvate utilization due to pyruvate transport deficiency accompanied by lactic acidosis resulting also from the described metabolic possibilities of pyruvate are the mutations in MPC1 and/or MPC2 genomic loci [133, 141].

to periods of increased levels of glutamate. High levels of glutamate cause complex I inhibition through receptor-stimulated Ca2+ overload, which is an attribute of acute neuropathologies [151]. MPC1 and MPC2 are highly expressed in brown adipose tissue compared with other tissues [152]. Brown adipocytes use predominantly fatty acids as an energy source for uncoupled respiration and thermogenesis, which requires replenishment of oxaloacetate through pyruvate carboxylation to enter citric acid cycle. MPC is supposed to be important in shifting between

Nuclear Encoded Mitochondrial Proteins in Metabolite Transport and Oxidation Pathway…

http://dx.doi.org/10.5772/intechopen.72937

265

Alteration in pyruvate metabolism plays a conspicuous role in heart disease. Heart muscle predominantly metabolizes fatty acids, ketone bodies, lactate, and glucose depending on their availability and neurohormonal signaling. Up to 95% of the heart's ATP generation comes from mitochondrial oxidation, and typically approximately 60–90% of this mitochondrial ATP production comes from fatty acids, whereas 10–40% is from pyruvate oxidation. The myocardium is a significant consumer of lactate even at the maximum load, because of specific expression of LDH-B isoform preferring reaction catalysis toward pyruvate [153]. It was found that acute stress (such as ischemia) and chronic stress (hypertrophy and heart failure) change substrate availability and metabolism [143]. Reduction in pyruvate oxidation leads to increased anaerobic glycolysis and lactate formation. However, age-related decrease in MPC activity was observed [154]. Shift in substrate utilization in order to maintain citric acid cycle can lead to serious states of energy deficiency called "starved heart." The effect was observed in cancer treatment with doxorubicin reducing carnitine transport followed by reduced fatty acid oxidation [155].

The tricarboxylate carrier (SLC25A1) catalyzes an electroneutral exchange of the dibasic form of a tricarboxylic acid (citrate, isocitrate, and cis-aconitate) with proton for another tricarbox-

of the citrate carrier (CiC) results from formation of a link between carbohydrate catabolism and lipogenesis. The CiC overlaps with oxoglutarate carrier ((OGC) SLC25A11) by transporting the 2-oxoglutarate in exchange for malate and malonate [157]. CiC facilitates transport of citrate across MIM, followed by passive diffusion through VDAC in MOM into the cytosol. In the cytosol, fatty acids and cholesterol are synthesized from citrate. Citrate also acts as inhibitor of phosphofructokinase 1, thus affecting the rate of glycolysis, positive allosteric modulator of acetyl-CoA carboxylase in fatty acid synthesis pathway, and serves as a substrate for the formation of malate, the conversion of which into pyruvate facilitates NADPH production necessary for lipogenesis [158]. High CiC mRNA levels in liver, kidney, and pancreas; lower levels in heart, skeletal muscle, and placenta; and no detectable mRNA in brain and lung were detected [128]. High liver and kidney CiC mRNA levels are supposed to be due to gluconeogenesis and lipogenesis. Moreover, CiC plays a role in gluconeogenesis from lactate where phosphoenolpyruvate carboxykinase is located in mitochondria. Similarly as in other animals, mitochondrial phosphoenolpyruvate isoforms are present mainly in the liver, kidney, and adipose tissue [159]. The relatively high CiC mRNA level in pancreas could be explained with the role of CiC in regulation of insulin secretion. On the other hand, the low CiC mRNA level in skeletal muscle correlates to the very low activity of gluconeogenesis and fatty acid synthesis [160].

, dicarboxylate (malate and succinate), or phosphoenolpyruvate [156]. An importance

formation and oxidation of fatty acids in fat cell metabolism.

**7.1. Mitochondrial di- and tricarboxylic acid transport**

*7.1.1. Tricarboxylate carrier*

ylate-H+

A 13C metabolic flux analysis of cells after transcriptional or pharmacological inhibition of MPC, published by Vacanti et al. [142], revealed that inhibition of MPC activity leads to shift from glucose to amino acid and fatty acid oxidation. Citric acid cycle and fatty acid synthesis were maintained due to malic enzyme flux, glutaminolysis, fatty acid, and branched chain amino acid oxidation. Alternatively, pyruvate interconversion into gluconeogenic substrates (e.g., alanine) that can enter mitochondria independently of the MPC could compensate for loss of the MPC [143].


**Marcin Kami ´nski**

Marcin Kaminski has been a Full Professor in the Department of Structural Mechanics, Ł ´ od´ z´ University of Technology, since 2015. He has been the Head of the Discipline Civil Engineering, Geodesy & Transportation in the Faculty of Civil Engineering, Architecture & Environmental Engineering, since 2019. His main research includes the stochastic finite element method, numerical modeling of random composites including homogenization theory, reliability assessment and optimization of civil engineering structures, stochastic ageing processes, and, recently, an application of probabilistic entropy in engineering computations.

He spent a postdoctoral study at Rice University in Houston, USA, and was a visiting professor at Leibniz-Institute of Polymer Research in Dresden, Germany, and also at Politecnico di Milano, Italy. He authored more than 200 research papers in various journals including the *International Journal for Numerical Methods in Engineering, Composite Structures, International Journal of Solids & Structures, and also Computers & Structures*. He published two monographs: "Computational Mechanics of Composite Materials", Springer, 2005; and "The Stochastic Perturbation Method for Computational Mechanics", Wiley, 2013. He serves as the Associate Editor of Mechanics Research Communications and a member of the editorial board in *Acta Mechanica, Computers & Structures, Journal of Composites Science* and *SCI*.

He has been the recipient of fellowships from the Foundation for Polish Science, J. Argyris Award from ECCOMAS and Elsevier, J.T. Oden Scholarship at The University of Texas at Austin, and also some Polish national or academic awards including Bronze Cross of Merit (1998), Silver Cross for the Longstanding Service (2017), Medal of Commission of Education (2022), and also very recently The Research Prize of Ministry of Education & Science in 2023. He has recently been recognized (since 2021) as the World's Top 2% Scientists by Stanford University, CA, USA.


**3. Results**

Fifty-five consecutive HHT patients with a pulmonary shunt (grade > 1) on TTCE were asked to participate. Two patients declined and therefore 53 patients were included in this study representing 105 PAVMs. Forty-five PAVMs had a feeding artery diameter of ≥2 mm (42.9%). Out of these, 11 PAVMs had a feeding artery diameter of ≥3 mm. The 45 PAVMs with a FAD ≥ 2 mm were present in 24 patients (45.3%). Patients' demographics are shown in Table 1.

**Table 1.** Baseline patient characteristics.

#### *3.1. Per-Patient Analysis*

With the threshold set at a diameter of the feeding artery of ≥2 mm, R1 correctly diagnosed PAVMs in 22 patients based on CE-MRA. The absence of PAVMs was correctly diagnosed in 28 patients; there were two false negative cases and one false positive case (sensitivity 92% (73–99%) and specificity 97% (82–100%)). Reader 2 also correctly diagnosed PAVMs in 22 patients. The absence of PAVMs was correctly diagnosed in 18. Similar to R1, R2 had two false negative cases, but 11 false positive cases (sensitivity 92% (73–99%) and specificity 62% (42–79%)). Both readers correctly diagnosed PAVMs in all patients when the threshold of ≥ 3 mm was applied. Results are presented in Tables 2 and 3.

**Table 2.** Contingency table of Reader 1 and 2 per-patient analysis.

#### *3.2. Per-PAVM Analysis*

One hundred and five PAVMs were identified in a total of 954 segments (53 patients × 18 segments per patient) on CT, of which 45 PAVMs had a feeding artery diameter of ≥2 mm. R1 correctly diagnosed 43 of 45 PAVMs and had seven false positives (sensitivity 96% (85–99%) and specificity 99% (98–100%)) on CE-MRA. R2 identified 42 of 45 PAVMs and had 33 false positives (sensitivity 93% (82–99%) and specificity 96% (95–97%)). As in the per-patient analysis, all PAVMs were identified by both readers when the threshold was set at ≥3 mm. Results are presented in Tables 3 and 4.

Data are presented as number, (%) and mean ± SD; HHT = hereditary hemorrhagic telangiectasia; PAVM = pulmonary arteriovenous malformation; \* diagnosis only based on Curacao criteria.

**Table 3.** Sensitivity, Specificity, NPV and PPV of CE-MRA.

Data are in percentages, data in parenthesis are 95% confidence intervals. PAVM = pulmonary arteriovenous malformation, CE-MRA = contrast enhanced magnetic resonance angiography, NPV = negative predictive value, PPV = positive predictive value.

**Table 4.** Contingency table of Reader 1 and 2 per-PAVM analysis (18-segment model).

## **4. Discussion**

In this study, CE-MRA showed a high sensitivity and negative predictive value for the diagnosis of PAVMs with a FAD ≥ 2 mm in patients with PAVMs and a TTCE confirmed significant pulmonary shunt. PAVMs with a FAD ≥ 2 mm were diagnosed by chest CT in 45% of patients with at least a moderate pulmonary shunt on TTCE. PAVMs with a FAD ≥ 2 mm were found in 43% using CE-MRA, with a sensitivity of 92% and a NPV between 90 and 93%.

Earlier, we have shown that, in the presence of a moderate or severe shunt on TTCE, the chest CT is negative for PAVMs in 55% and 8%, respectively [13]. Chest CT has an important role in the detection and follow-up of PAVMs in HHT. Its high spatial resolution enables imaging of feeding arteries, sacs and draining veins and thereby is able to accurately diagnose and assess treatment effect of PAVMs. However, the use of chest CT comes at the expense of ionizing radiation, which is an important disadvantage in this cohort of HHT patients, who are generally young and are likely to require several follow-up CT scans during their lifetime. In addition, metal artifacts hamper evaluation of PAVMs with CT after coil embolization. MRA is not limited by radiation burden and also seems to perform well with respect to the evaluation of PAVM persistence.

In the last two decades, a handful of studies have been published reporting on the feasibility of MRI in the management of PAVMs. In 2008, Schneider et al. reported a study including 203 HHT patients evaluating CE-MRA as a screening procedure for the detection of PAVMs [16]. They showed that CE-MRA is more sensitive in detecting PAVMs compared to pulmonary angiography (PA), which was the reference standard. Overall, there were 40 patients identified in whom 119 PAVMs were found with screening and follow-up using CE-MRA. Interestingly, with PA only 92 PAVMs were found (77%). As pointed out by the authors, there were important limitations in this study. First, patients with a negative CE-MRA were not compared to the reference standard. Second, there was no comparison to chest CT as the most important diagnostic tool in current algorithms. A third limitation was that the lower threshold for detection was defined as a sac diameter of at least 5 mm instead of feeding artery diameter ≥ 2 mm which is advocated in the guidelines [3].

Although not the subject in our study, MRA could also have a role in the follow-up of embolized PAVMs. Several studies have demonstrated the feasibility of (CE)-MRA for detecting PAVM persistence [17–20]. Kawai et al. used time-resolved CE-MRA to assess PAVMs and correlated with the recanalization rate on chest CT and PA [18]. The sensitivity (96%) and specificity (96%) were excellent, with good interobserver agreement. Important limitations of this study were the low number of included PAVMs (*n* = 28) and that 3 PAVMs could not be imaged by CE-MRA because of field of view limitation. For the detection of PAVM perfusion after embolotherapy, Hamamoto et al. investigated the feasibility of time-spatial labeling inversion pulse MRA (time-SLIP MRA or arterial spin labeling), a cinematic MRI method in which the inversion pulse facilitates the determination of blood flow without a contrast medium [20]. In 11 patients, 38 PAVMs were imaged with the time-SLIP MRA technique that was compared to PA as reference standard. The sensitivity and specificity of time-SLIP MRA for detecting perfusion after embolization were both 100%, with an excellent inter-observer agreement. Although very promising, time-SLIP MRA is also limited by the relatively small field of view. In this small study, four PAVMs could not be imaged because of this.

MRA is a very promising imaging method for PAVMs but there are limitations. Our study and previous studies have shown that (CE)-MRA is a feasible method to detect PAVMs. It performs well compared to chest CT in the detection of PAVMs with a FAD ≥ 2 mm. However, the number of false positives needs to be improved. The experience of the operator seems to play an important role in this, as there is a large di fference between both operators in the number of false positives. With respect to the detection of perfusion of embolized PAVMs, MRA seems to perform superior compared to CT because it is less influenced by the presence of micro coils [17,18]. This is attributable to the fact that micro coils are of platinum material which has few paramagnetic e ffects. MRA could therefore be feasible for the postembolization follow-up as well. As mentioned above, with CE-MRA and time-SLIP MRA, it is possible that the field of view is not large enough to cover the whole lung. The time-SLIP MRA technique often requires targeted imaging and thus knowledge of the location of the PAVM of interest. This means that either extra contrast administration during the MRA investigation or a chest CT scan prior to the MRA investigation will be necessary. Considering these limitations, the relative high costs and limited availability of MR imaging, it may be too early to use (CE)-MRA for routine evaluation of PAVMs.

Our study has several limitations. MRA acquisitions were performed on a 1.5 T system. With a 3 T system, higher signal-to-noise ratios can be achieved. This could result in superior image quality based on a higher spatial and temporal resolution. In addition, it enables a broader coverage of the lung reducing the risk of false negative readings. Unfortunately, a 3 T system was not available during the study. Furthermore, there was a di fference in experience in reading CE-MRA investigations between both readers. This study showed that image interpretation is reader-dependent, mainly resulting in false positives by the less experienced reader. Reader 1 has both a large experience in chest radiology and PAVM embolotherapy. He represents the level of experience that can be expected in an expert center. Reader 2, although being trained in an expert center, has less experience and could be regarded as representing nonexpert centers. HHT guidelines recommend that PAVM screening, diagnosis and treatment should be performed in expert centers. Furthermore, both readers were aware of the fact that the subjects had a positive TTCE and thus a high chance of having PAVMs with a FAD ≥ 2 mm. This could have induced a review bias lowering the threshold for diagnosing PAVMs. Finally, a limitation was the relatively small number of patients in this study. However, it is largest cohort of consecutive patients investigated so far representing a reasonably number of PAVMs with a FAD ≥ 2 mm.


**5.3.1** *β***-blockers**

Contact allergy to topical *β*-blockers is a well-recognized side-effect of glaucoma treatment [**38-41**]. Sensitization may be singly to agents such as timolol, befunolol, levobunolol, or, more rarely, to multiple *β*-blockers in a single patient.

A closed patch test, usually used in clinical practice for the diagnosis of allergic contact dermatitis, is often sufficient to show *β*-blockers contact allergy. However, there may be difficulties in obtaining positive patch tests to *β*-blockers, as showed in earlier reports [**42- 43**]. Poor penetration through intact skin on the back, where patch testing is normally applied, may be a factor.

#### **5.3.2 Antiviral drugs**

Topical antiviral drugs are frequently used, but although repeated applications can lead to contact reactions **[44-45]**, adverse cutaneous reactions are not commonly observed. Allergic contact dermatitis caused by acyclovir is rare, with only 20 studies reported **[46-48].** Because of the doubtful reactions with antiviral, especially acyclovir, and in view of the suggestive clinical history, we recommend the scratch–patch test followed by repeated open application test (ROAT).


**7. References**

[1] Huang K (1987) Statistical Mechanics. John Wiley & Sons, New York; Munster A (1969) Statistical Thermodynamics (volume I). Springer-Verlag, Berlin; Ditlevsen PD (2004) Turbulence and Climate Dynamics. Frydendal, Copenhagen.

© 2012 Opeyemi, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Opeyemi, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Stochastic Modelling of Structural Elements**

One of the primary goals of structural engineers is to assure proper levels of safety for the structures they design. This seemingly simple task is complicated by uncertainties associated with the materials with which the structure was designed and the loads they must resist, as well as our inaccuracies in analysis and design. Structural reliability and probabilistic analysis/design are tools which can be employed to quantify these uncertainties and inaccuracies, and produce designs and design procedures meeting acceptable levels of safety. Recent researches in the area of structural reliability and probabilistic analysis have centred on the development of probability-based design procedures. These include load modelling, ultimate and service load performance and evaluation of current levels of safety/reliability in design (Farid Uddim, 2000; Afolayan,

Deterministic methods are very subjective and are generally not based on a systematic assessment of reliability, especially when we consider their use in the entire structure. These methods can produce structures with some "over designed" components and perhaps some "under designed" components. The additional expense incurred in constructing the over designed components probably does not contribute to the overall reliability of the structure, so this is not a very cost-effective way to produce reliable structures. In other words, it would be better to redirect some of the resources used to build the over designed components toward strengthening the under designed ones. Therefore, there is increasing interest in adopting reliability-based design methods in civil engineering. These methods are intended to quantify reliability, and thus may be used to develop balance designs that are both more reliable and less expensive. Also, according to Coduto (2001), the methods can be used to better evaluate the various sources of failure and use this information to develop design and construction methods that are both more reliable and more robust - one that is insensitive to variations in materials, construction

1999; Afolayan, 2003; Afolayan and Opeyemi, 2008; Opeyemi, 2009).

David Opeyemi

**1. Introduction**

http://dx.doi.org/10.5772/45951

techniques, and environment.

Additional information is available at the end of the chapter


**4. Discussion**

A photosynthetic lifestyle allows organisms to use light as an energy source for growth and proliferation. However, this benefit comes at a price, that is, the risk of 1O2 generation by energy transfer from (bacterio-) chlorophyll to molecular oxygen (3O2) within photosynthetic complexes. It is suspected that purple bacteria from the genus *Rhodobacter* address this problem by avoiding strong pigmentation under high light and/or high oxygen conditions, a response that is mainly regulated by light- and oxygen-sensing proteins [2,3]. Despite regulation of pigmentation in response to oxygen tension and light, *Rhodobacter* species cannot completely avoid 1O2 stress in their natural aquatic environments. They have therefore evolved strategies to counteract 1O2 and to deal with the resulting damages [1,65]. *R. sphaeroides* is a well-studied model organism with regard to the photooxidative stress response [70], and was compared here to *R. capsulatus* using transcriptomics and proteomics. Intriguingly, the two *Rhodobacter* species elicit very similar responses in the light of functional categories. Many proteins with an increased abundance upon 1O2 exposure are involved in oxidation–reduction processes and DNA damage repair (Figure 8). Furthermore, several methionine sulfoxide reductases (MsrA and MsrB orthologs) and a variety of proteases were increased, which was also confirmed on the transcript level (Figure 7). Since proteins are the main targets of 1O2 [15], we conclude that the repair of proteins and the removal of damaged proteins is essential to survive this particular stress, and that efficient protein maintenance likely represents a key feature of the response to 1O2 in many organisms. In *R. sphaeroides*, the 1O2 stress response is mainly controlled by the alternative sigma factors RpoE and RpoHII [10,18–21]; we have reason to believe that this also holds true for *R. capsulatus*. Firstly, both *rpoE* and *rpoHII* have elevated transcript levels upon 1O2 exposure (Table 1), and secondly, an *rpoHII* deletion strain is more sensitive to 1O2 than the *R. capsulatus* wild type (Figure 6). Hence, the basic regulatory principles of the photooxidative stress response might be very similar in both *Rhodobacter* species.

Despite the aforementioned similarities, there are remarkable di fferences between *R. sphaeroides* and *R. capsulatus*. Even though both species fall into the same clade within a phylogenetic tree based on a core genome of 580 orthologous proteins [71], synteny analysis revealed that the genetic organization of orthologous genes on the chromosome is considerably di fferent, with the exception of, e.g., the photosynthetic gene cluster (Figure S8). This pronounced genomic rearrangement is not observed between di fferent *R. sphaeroides* strains (e.g., strains 2.4.1, KD131, and WS8N), but between di fferent genera within the *Rhodobacteraceae* (e.g., between *Rhodobacter* and *Roseobacter*). Hence, our analyses suggest that *R. capsulatus* and *R. sphaeroides* are, from a genomic point of view, more distantly related than expected from their common lifestyle and the arrangement of the photosynthetic gene cluster [71]. Another remarkable finding concerns the genetic context of the *rpoE-chrR* locus in *R. capsulatus*, which is in close proximity to the photosynthetic gene cluster (Figure 4). This genetic arrangement was not found in other *Rhodobacteraceae* species, and can therefore be considered as unique. It is worth noting that RpoE from *R. capsulatus* shares higher identity with the extra-cytoplasmic function sigma factor SigK from *Mycobacterium tuberculosis* (34%), compared to only 24% identity with *R. sphaeroides* RpoE. The same is true for *R. capsulatus* ChrR, which shares 25% identity with *M. tuberculosis* SigK antisigma factor RskA, but only 13% with *R. sphaeroides* ChrR. Phylogenetic trees further support the special position of the *R. capsulatus* RpoE and ChrR proteins within the *Rhodobacteraceae*family (data not shown). These findings raise the question of whether the *rpoE-chrR* locus in *R. capsulatus* and *R. sphaeroides*

originated from a common ancestor and was then intensely remodeled in *R. capsulatus*, or whether *R. capsulatus* has received a *sigK-rskA*-like locus from another bacterial lineage (e.g., Gram-positives like *M. tuberculosis*) via horizontal gene transfer.

*R. capsulatus* not only displays a unique colocalization of the photosynthetic gene cluster near the *rpoE-chrR* locus; it also differs to *R. sphaeroides* on the physiological level with regard to its pigmentation. a higher pigmentation of *R. capsulatus* under high oxygen conditions obviously allows much faster adaptation to occur to phototrophic conditions compared to *R. sphaeroides* (Figure 1B). Despite this higher pigmentation, *R. capsulatus* has no disadvantage when exposed to sudden photooxidative stress (Figure 1C). Low expression of BChl *a* and carotenoid biosynthesis genes under aerobic conditions is reflected by low BChl *a* and carotenoid levels in both *R. capsulatus* and *R. sphaeroides* (Figure 2A). Upon photooxidative stress, Bchl *a* and carotenoid levels decreased in *R. sphaeroides*. By contrast, in *R. capsulatus,* only BChl *a* levels declined, while carotenoid levels remained fairly constant (Figure 2B), resulting in an increasing Crt:BChl *a* ratio in the course of 1O2 exposure (Figure 2D). These differences in Crt:BChl *a* ratio may be the reason for lower 1O2 levels in *R. capsulatus* under photooxodative stress considering the 1O2 quenching ability of carotenoids (Figure 3A). Increased carotenoid biosynthesis under conditions of photooxidative stress is a strategy that is used by some microorganisms, including the deltaproteobacterium *Myxococcus xanthus* and the yeast *Pha*ffi*a rhodozyma*, in order to avoid extensive cellular damages by direct quenching [72,73]. Accumulation of carotenoids upon 1O2 exposure was, however, not observed in *R. sphaeroides* [27], and omics data even revealed declining transcript and protein levels of genes from the photosynthetic gene cluster, including genes for carotenoid biosynthesis [28]. Interestingly, RNA-seq revealed that three genes within the photosynthetic gene cluster had increased transcript levels in *R. capsulatus*, which applies to *crtI* (log2 fold change of ~2.3), *crtB* (log2 fold change of ~2.0), and *tspO* (log2 fold change of ~1.3). Induction of *crtI* was validated by qRT-PCR (Figure 5). The three genes form an operon between the carotenoid biosynthesis genes *crtA* and *crtC*. TspO is an outer membrane protein, which controls efflux of porphyrin intermediates, and thereby negatively modulates expression of photosynthesis genes, likely through AppA [74]. The increased expression of *tspO* might enhance porphyrin efflux under photooxidative stress conditions, limiting both expression of photosynthesis genes and accumulation of potential porphyrin-derived photosensitizers, which may support adaptation to this particular stress. CrtB is a phytoene synthase and CrtI is a phytoene desaturase, which catalyze the reaction of precursors to phytoene (CrtB) and from phytoene to zeta-carotene and neurosporene (CrtI). The carotenoids spheroidene (SE) and spheroidenone (SO) are then synthesized in subsequent steps from neurosporene. Even though a switch from SE to SO (catalyzed by CrtA) might be an important adaption to photooxidative stress, we could not detect a strong increase for *crtA* mRNA in the RNA-seq data. It is conceivable that enhanced CrtB and CrtI levels are needed to provide sufficient amounts of neurosporene as a precursor for SE and SO biosynthesis to maintain carotenoid levels in *R. capsulatus* (Figure 2B). By contrast, in *R. sphaeroides crtI* and *crtB* transcript levels do not increase upon photooxidative stress (Figure 5) [28], which even coincides with a decrease in carotenoid levels (Figure 2B). Obviously, both *Rhodobacter* species use different strategies to adapt to high light regimes. Since *R. capsulatus* initially grows better than *R. sphaeroides* both after a shift to phototrophic conditions (Figure 1B) and upon photooxidative stress (Figure 1C), elevated carotenoid levels are expected to be advantageous to photosynthetic bacteria in rapidly changing environments.

Besides the protective function of carotenoids, *cbiX* might also play a more crucial role in defending against photooxidative stress in *R. capsulatus* than in *R. sphaeroides*. Our omics data show that *cbiX* is strongly increased on both transcript and protein level in *R. capsulatus* (log2 fold change of ~3). The RSP\_1566 protein of *R. sphaeroides* shows 47% identity to CbiX, but neither protein nor transcript levels change significantly in response to 1O2 [28], and *cbiX* transcripts were not even detected by qRT-PCR (Figure 5). The *cbiX* gene is annotated as a cobaltochelatase which incorporates cobalt into sirohydrochlorin to form cosirohydrochlorin, an early precursor of vitamin B12 [75]. B12 has special functions in the formation of the photosynthetic apparatus in *Rhodobacter*, i.e., it is required for the

conversion of protoporphyrin IX to Mg-protoporphyrin monomethyl ester [76]. Furthermore, it is needed by the antirepressor AerR to efficiently bind to repressor CrtJ, thereby inducing *bch* gene expression in *R. capsulatus* [77,78]. Theoretically, increased CbiX levels favor B12 biosynthesis and, consequently, derepression of *bch* genes via AerR. However, increased *bch* expression would then increase photooxidative stress, but this was not observed. a structural homology search for *R. capsulatus* CbiX using Phyre2 suggests that it might rather act as a ferrochelatase involved in the biosynthesis of siroheme. This assumption is supported by a study by Bali and colleagues [69], showing that CbiX functionally replaces characteristic siroheme biosynthesis enzymes, which are missing in alphaproteobacteria, including *R. sphaeroides*. As siroheme is a cofactor of nitrite and sulfite reductases, an increased siroheme production might also explain the high accumulation of the mRNA for sulfite reductase CysI (log2 fold change of ~3.2). Increased sulfur assimilation might help to counteract the 1O2-caused depletion of glutathione and abundant damages on sulfur-containing amino acids. More importantly, however, the insertion of Fe2+ into sirohydrochlorin reduces the concentration of free iron, which would otherwise be available for both the Fenton reaction and the formation of the photosensitizer protoporphyrin IX. Hence, additional stress is prevented by changing the flux through tetrapyrrole pathways [79–81]. As a conclusion, the induction of both the *crtIB-tspO* operon and *cbiX* might represent a successful strategy to respond to photooxidative stress, which was specifically invented in *R. capsulatus* to support adaptation. The question of whether other phototrophs have evolved similar strategies will be an exciting subject for future studies.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-2607/8/2/283/s1, Figure S1: Analysis of *Rhodobacter* transposon mutants (*R.c.* Tn5 and *R.s.* Tn5) lacking important carotenoids, Figure S2: Growth of *R. capsulatus* and *R. sphaeroides* with and without methylene blue in the dark or under high light conditions, Figure S3: Pigmentation of *R. capsulatus* and *R. sphaeroides* under microaerobic conditions, Figure S4: SOSG fluorescence in cell-free reactions, Figure S5: Correlation analysis of RNA-seq replicates, Figure S6: PCA of RNA-seq and LC-MS/MS replicate samples, Figure S7: Correlation analysis of LC-MS/MS replicates, Figure S8: Synteny plot comparing *Rhodobacter* genomes, Table S1: Strains used in this study, Table S2: Transcriptome data for *Rhodobacter* upon photooxidative stress, Table S3: Primers used for qRT-PCR in this study, Table S4: LC-MS/MS data for *R. capsulatus* upon photooxidative stress.

**Author Contributions:** Conceptualization, G.K.; methodology, M.K.L., A.M.N., A.K., M.B., B.A.B. and G.K.; software, M.B.; validation, M.K.L.; formal analysis, M.K.L., A.K., M.B. and B.A.B.; investigation, M.K.L., A.M.N., M.V., A.K.; data curation, M.K.L., A.K. and M.B.; writing—original draft preparation, M.K.L., B.A.B. and G.K.; writing—review and editing, M.K.L., B.A.B. and G.K.; visualization, M.K.L. and B.A.B.; supervision, B.A.B. and G.K.; project administration, G.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We thank Petra Dersch (University of Münster, Germany) for generous support with RNA-seq and corrections on the manuscript, and Andrew Lang (Memorial University of Newfoundland, Canada) for providing strains.

**Conflicts of Interest:** The authors declare no conflict of interest.


**Chapter 3**

*Intellectual Property Rights - Patent*

[cited: 02 June 2019]

[cited: 02 June 2019]

2006;**34**(17):3268-3283

business employment. SSRN Electronic Journal. 2016. pp.1-68. Available from: http://www.ssrn.com/abstract=2887104 Carpenter MP, Narin F, Woolf P. World

patent information [Internet]. WIPO jointly with the Commission of European Communities, Vol. 3; 1979. pp. 160-163. Available from: https://econpapers.repec.org/article/ eeeworpat/v\_3a3\_3ay\_3a1981\_3ai\_3a4\_3 ap\_3a160-163.htm [cited: 02 June 2019]

[24] Wagner RP, Parchomovsky G. Patent portfolios. SSRN Electronic Journal. 2004. Available from: http:// www.ssrn.com/abstract=582201 [cited:

[25] Aristodemou L, Tietze F. Citations as a measure of technological impact: A review of forward citation-based measures. World Patent Information.

technological changes using engineering heuristics in the telecommunications switching industry. Research Policy.

[26] Martinelli A. An emerging paradigm or just another trajectory?

Understanding the nature of

2012;**41**(2):414-429

02 June 2019]

2018;**53**:39-44

[17] Turner JL. Patent thickets, trolls and unproductive entrepreneurship. SSRN Electronic Journal. 2011. Available from: http://www.ssrn.com/abstract=1916798

[18] UNCTAD. Second generation biofuel markets: Satet of play, trade and developing country perspectives. In: United Nations Conf Trade Dev [Internet]. 2016. p. 69. Available from: http://unctad. org/en/PublicationsLibrary/ ditcted2015d8\_en.pdf

[19] Hamelinck CN, Faaij APC. Outlook for advanced biofuels. Energy Policy.

[20] Ferreira L. Em decisão histórica, EUA e China comprometem-se a cortar gases estufa em pacto global: 11/12/2011- UOL Notícias. UOL Ciência e Saúde, em Durban [Internet]. 2011. Available from: https://noticias.uol.com.br/ciencia/ ultimas-noticias/redacao/2011/12/11/ em-decisao-historica-eua-e-chinacomprometem-se-a-cortar-gases-estufaem-pacto-global.htm [cited: 02 June

[21] Tomei J, Helliwell R. Food versus fuel? Going beyond biofuels. Land use policy [Internet]. 1 Nov 2016;**56**: 320-326. Available from: https://www. sciencedirect.com/science/article/abs/ pii/S0264837715003579 [cited: 02 June

[22] Van Eck NJ, Waltman L. VOS viewer: Visualizing scientific landscapes [Internet]. 2016. Available from: http:// www.vosviewer.com/ [cited: 12 April

[23] World Intellectual Property Organization. MP, Commission of the European Communities. F, Woolf P,

**32**

2019]

2019]

2017]


**Abstract**

This chapter focuses on some new definitions and methodologies of electric drives that are facing new challenges raised by alternative fuel vehicles. It starts with the objectives, fundamentals, and current research issues of alternative fuel vehicles based electric drives, before moving on to new definitions of unified modeling of the entire electric drive system and design of the proposed DC active power filter aimed at energy storage system chaotic current elimination. Next, novel motor control strategies taking into account alternative fuel vehicle operations are presented for improvement of sensorless drive and flux weakening control performance. Finally, conclusions of this chapter are drawn.

**Keywords:** Alternative fuel vehicles, unified modeling, DC active power filter, slid‐ ing mode observer, flux weakening control


**13. Effects on the histaminergic system**

Histamine acts in conjunction with serotonin to regulate the circadian rhythm [31]. Histaminergic neurons originate in the tuberomammillary nucleus of the hypothalamus and make extensive projection throughout the central nervous system, including the spinal cord (**Figure 8**) [37]. Significant increase in histamine

#### **Figure 8.**

*The ascending histaminergic pathway originates in the hypothalamus and extends to the medial portion of the temporal cortex and prefrontal cortex, while the descending pathway modulates the activity of the spinal cord. AN: accumbens nucleus, GP: globus pallidus, S: striatum, and T: thalamus.*

levels have been documented in patients with hepatic encephalopathy [31]. Histamine is produced from the amino acid histidine [37]; hyperammonemia increases both the concentration of histidine and the activity of its membrane transporter into the histaminergic neurons, stimulating the synthesis of histamine [31].