*Article* **Translational Frameshifting in the** *chlD* **Gene Gives a Clue to the Coevolution of the Chlorophyll and Cobalamin Biosyntheses**

**Stepan Kuznetsov 1 , Alexander Milenkin 1 and Ivan Antonov 2,3,\***

**Abstract:** Today, hundreds of prokaryotic species are able to synthesize chlorophyll and cobalamin (vitamin B12). An important step in the biosynthesis of these coenzymes is the insertion of a metal ion into a porphyrin ring. Namely, Mg-chelatase ChlIDH and aerobic Co-chelatase CobNST are utilized in the chlorophyll and vitamin B12 pathways, respectively. The corresponding subunits of these enzymes have common evolutionary origin. Recently, we have identified a highly conserved frameshifting signal in the *chlD* gene. This unusual regulatory mechanism allowed production of both the small and the medium chelatase subunits from the same gene. Moreover, the *chlD* gene appeared early in the evolution and could be at the starting point in the development of the chlorophyll and B12 pathways. Here, we studied the possible coevolution of these two pathways through the analysis of the chelatase genes. To do that, we developed a specialized Web database with comprehensive information about more than 1200 prokaryotic genomes. Further analysis allowed us to split the coevolution of the chlorophyll and B12 pathway into eight distinct stages.

**Keywords:** chlorophyll; cobalamin; vitamin B12; frameshifting; evolution; chelatase; chlIDH; cobNST

#### **1. Introduction**

The early evolution of photosythesis has been an intriguing but complex research subject for many years [1,2]. Clearly, the appearance of photosythesis required the ability of the ancient prokaryotes to synthesize chlorophyll (CHL). Chlorophyll is a relatively complex organic compound that includes Mg2+ ion in its tetrapyrrole ring [3]. Modern organisms utilize a number of different enzymes to produce chlorophyll from a metal-free tetrapyrrole intermediate Uroporphyrinogen III (Uro) [4]. On the other hand, other species utilize several alternative pathways that can turn Uro into cobalamin (vitamin B12), heme, or coenzyme F430 (Figure 1). These coenzymes contain different metal ions in their tetrapyrrole rings [5]. Namely, cobalamine (vitamin B12), haem, and coenzyme F430 include Co2+, Fe2+ and Ni2+, respectively. The enzymes that insert those ions into the corresponding organic rings are called chelatases.

The biosynthesis of Mg2+-containing chlorophylls and bacteriochlorophylls requires the magnesium chelatase that performs the insertion of the magnesium ion (Mg2+) into protoporphyrin IX [6]. This enzyme consists of small (I), medium (D), and large (H) subunits [7,8]. The corresponding genes are called *chlI*, *chlD*, and *chlH* in the genomes of chlorophyll-producing organisms and *bchI*, *bchD*, and *bchH* in bacteriochlorophyll producers. To reduce the number of different gene names in this work, we will use the *chlI*, *chlD*, and *chlH* terminology for both groups of genes.

**Citation:** Kuznetsov, S.; Milenkin, A.; Antonov, I. Translational Frameshifting in the *chlD* Gene Gives a Clue to the Coevolution of the Chlorophyll and Cobalamin Biosyntheses. *Microorganisms* **2022**, *10*, 1200. https://doi.org/10.3390/ microorganisms10061200

Academic Editors: Robert Blankenship and Matthew Sattley

Received: 9 April 2022 Accepted: 2 June 2022 Published: 11 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Overview of the existing biochemical pathways that produce (Bacterio)chlorophyll, Heme, Cobalamin, and Coenzyme F430 from Uroporphyrinogen III (Uro). The magnesium-chelatase ChlIDH inserts Mg2+ ion in the Protoporphyrin IX (Proto). There are two alternative pathways to synthesize Cobalamin. The CobNST chelatase inserts Co2+ in the aerobic path. It has been suggested that ChlIDH and CobNST have common evolutionary origin.

Interestingly, the cobalamin biosynthesis pathway has two alternative routes—the anaerobic and aerobic ones (Figure 1) [9]. Strict anaerobes only posses the anaerobic B12 pathway. On the other hand, cobalamin-producing facultative anaerobes or aerobic organisms usually possess both routes and switch between them depending on the current living conditions [10]. From the evolutionary point of view, it is logical to assume that the aerobic route could only evolve when free oxygen was available in the Earth's atmosphere, e.g., only after the CHL pathway and oxygenic photosynthesis have already existed. Moreover, the interplay between the CHL pathway and the aerobic B12 route is not limited to the oxygen itself. It has been well established that the cobalt chelatase CobNST from the aerobic route resembles the magnesium chelatase ChlIDH as it also consists of the small, medium and large subunits encoded by *cobS*, *cobT*, and *cobN* genes, respectively [11]. Additionally, the *chlI* and *chlD* from the chlIDH chelatase are likely to be homologous to the *cobS* and *cobT* genes of the cobNST chelatase [12]. The similarity between the large subunit genes (i.e., *chlH* and *cobN*) has also been reported [13].

Later studies have revealed additional existing strategies to encode the cobNST enzyme. First, Rodionov et al. [14] have suggested that in some species, the products of the *chlI* and *chlD* can replace the *cobS* and *cobT* genes and function as the small and medium subunits of the aerobic Co-chelatase. Moreover, a functional programmed ribosomal frameshifting (PRF) found in some *chlD* genes may allow the production of both the small and the medium subunits of the cobNST chelatase from the same gene [15,16]—see Supplementary Figure S1. Altogether, this indicates a close connection between the CHL pathway and the aerobic B12 route suggesting their possible coevolution [17].

Here, we attempted to organize all the different strategies to encode cobNST and chlIDH enzymes and suggest a logical model of the CHL and aerobic B12 pathways coevolution. Our strategy was to pinpoint possible prokaryotic genomes that may still be using the ancient strategies to encode Mg- and/or Co-chelatases. Such "living fossil" species can be used to reconstruct the early stages of the CHL and B12 pathway evolution.

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

The Chelatase DB was developed using the Django framework implemented in Python. To populate the database the tBLASTn [18] tool was used to identify the genomes containing *chlD* gene(s). The translations of the three *chlD* genes with the validated frameshifting signals were used as queries [16]. To allow the identification of the other frameshifted *chlD* genes, two separate tBASTn searches (E-value threshold = 10−6 ) were performed using the N-terminal (i.e., before the frameshift) or the C-terminal (after the frameshift) parts of each query protein. The genomic regions with adjacent hits (in the correct order) with up to one frameshift were classified as *chlD* genes.

In order to identify the small and the large chelatase subunit genes as well as the other genes from the chlorophyll and cobalamin biosynthesis pathways, tBLASTn search was performed for a set of reference query proteins. Given the similarities between different chelatase subunits, we imposed additional constraints on their lengths. Namely, we required that the small chelatase subunits were between 250 aa and 500 aa, medium were between 500 aa and 800 aa, and large were between 1000 aa and 2000 aa (these values corresponded to the lengths of the annotated chelatase subunits). The identified genes were automatically annotated (i.e., assigned a gene name such as *chlD*, *bchD*, or *cobT*) by the best reciprocal hit approach.

We used the identified representative sets of the genes from the CHL and B12 pathways to predict whether a given prokaryotic genome possessed the corresponding biochemical pathway. Additionally, the automatically assigned genotype of the species mentioned in the text was double-checked with the information from the KEGG database (the "Porphyrin metabolism" section) [19]. The phylogenetic tree was constructed using RAxML version 8.2.12 [20]. Subsequent phylogenetic analyses and visualizations were performed using the "ape" R package [21]. The RNA secondary structures were predicted using the RNAfold web server [22] available at http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi (accessed on 1 April 2022).

All the data files and scripts used to generate figures and tables from the article are available at https://github.com/vanya-antonov/article-chelatase-db (accessed on 1 April 2022).

#### **3. Results**

Our recent analysis [16] as well as earlier studies [8,14,23] have suggested that there were several different strategies to encode aerobic cobalt chelatase cobNST. Interestingly, in a number of species, the products of the Mg-chelatase genes (*chlD* and/or *chlI*) functioned as corresponding subunits of the cobNST complex (Supplementary Figure S1). To explore the diversity of the strategies utilized by different prokaryotes to encode chelatases, we developed a specialized Web database called Chelatase DB (http://ivanya.com/chelatase/ (accessed on 1 April 2022)). For each prokaryotic genome, the Chelatase DB provided information about the number of the small, medium, and large subunit genes, the existence of the chlorophyll and/or B12 biosynthesis pathway and reference to the KEGG database for detailed analysis. Additionally, the database contained a collection of the frameshifting signals predicted in some *chlD* genes.

To identify the possible starting point in the evolution of the CHL pathway, the *chlD* gene encoding medium subunit of the Mg-chelatase was analyzed. We chose this gene because it was likely to appear early in the evolution, since it may have been present in the genome of the last universal common ancestor (LUCA) [24]. Thus, it was possible that the modern genes encoding medium chelatase subunits were descendants of the putative *chlD* gene from LUCA. Our previous analysis [16] of the frameshifted *chlD* (fs-*chlD*) genes has demonstrated that such genes were present in all major prokaryotic phyla (including archaea, proteobacteria, and actinobacteria)—Supplementary Figure S2. Indeed, on the phylogenetic tree of 286 medium subunit genes all the fs-*chlD* genes were located near the root of the tree (Supplementary Figure S3). Thus, the modern *chlD*, *bchD*, and *cobT* genes may have originated from the ancient fs-*chlD* gene.

Our next goal was to use the functionality of the Chelatase DB to reveal a possible evolutionary trajectory of the CHL and B12 pathways with respect to the corresponding chelatase genes. Namely, we inspected different chlIDH/cobNST genotypes and matched them with the corresponding predicted phenotypes (i.e., the ability to synthesize CHL and/or B12). This allowed us to define eight stages in the evolution of CHL and B12 pathways as well as *chlD* gene—see Table 1. Since fs-*chlD* gene had an ancient origin, it was logical to assume that it appeared in the evolution before the complete CHL and B12 pathways existed. Therefore, we search the Chelatase DB for the genomes with fs-*chlD* genes that did not have the genes from the CHL and B12 pathways. Indeed, we identified several such species including *Spirochaeta thermophyla* and *Brevefilum fermentans* (Table 1, stage 1). All of these bacteria that corresponded to the conditions of the prechlorophyll era were anaerobic [25]. Moreover, according to the global prokaryotic phylogentic tree, both Spirochaetes and Chloroflexi were among the most ancient prokaryotic phyla that are present today [26]. The presence of the frameshifting signals in the fs-*chlD* genes from *S. thermophyla* and *B. fermentans* suggested that they produced two proteins from the same mRNA (Supplementary Figure S4). In the *chlD* genes from various taxa −1 frameshifts have been observed more frequently than +1 frameshifts [16]. The recoding signals in these genes usually consisted of AT-rich "slippery sites" together with a stimulatory RNA secondary structures located downstream. More info about the frameshifting mechanism can be found elsewhere [27]. Due to the stochastic nature of the programmed frameshifting, ribosomes reading the same mRNA can produce two different proteins that allows cotranslational assembly of protein complexes. Consequently, it was likely that the two proteins produced from fs-*chlD* mRNA interacted with each other—similarly to the CobS and CobT subunits [28]. However, the function of this protein complex was unclear since the genomes of the corresponding bacteria completely lacked the CHL and B12 pathways as well as the large chelatase subunit gene (either *chlH* or *cobN*). Together, these observations indicated that the *S. thermophyla* and *B. fermentans* could be living prokaryotic fossils and their genotypes can be considered as the starting point in the evolution of the CHL and B12 pathways.

It has previously been reported that the *chlI* gene encoding the small chelatase subunit was homologous to the N-terminal part of the *chlD* gene [29]. Therefore, the next putative evolutionary event was duplication of the N-terminal part of the fs-*chlD* gene and formation of a separate *chlI* gene. In this case, the frameshifting signal in the fs-*chlD* was likely to disappear since there was no need to produce both proteins from a single gene. Accordingly, we found another prokaryotic group that had *chlD* and *chlI* genes but still lacked the CHL and B12 pathways (Table 1, stage 2). Interestingly, the ability of the cotranslational interaction between the products of these proto-*chlI* and proto-*chlD* genes was possible since they were located next to each other on the genome and can be present in the same polycistronic mRNA. Although the function of the proteins produced from these two genes was still unclear, the corresponding genotypes can be considered as the next stage in the early evolution of CHL biosynthesis. Moreover, we identified several Chloroflexi species (from the Anaerolineaceae group) with a potential intermediate genotype between Stage 1 and 2. Namely, *Anaerolinea thermophila UNI-1*, *Ornatilinea apprima*, *Longilinea arvoryzae*, and *Levilinea saccharolytica* possessed the fs-chlD genes with characteristic frameshifting signals but also had a separate *chlI* gene. Interestingly, these species were also strict anaerobes and thermophiles suggesting their ancient origin [30]. Thus, there were a number of living prokaryotes that provided insights into the early evolution of the modern Mg-chelatase enzyme.

A good representative case of the stage 3 was photosynthetic proteobacteria *Polynucleobacter duraquae* that had the *chlH* gene encoding the large subunit of the Mg-chelatase (Table 1, stage 3). Similar to the genomes from stage 2, all three chelatase subunit genes were located at the same genomic locus, possibly allowing cotraslational assembly of the Mg-chelatase from a polycystronic mRNA. According to the KEGG database, *P. duraquae* possessed all the major genes required for CHL biosynthesis. It was likely that this bacteria was only capable of anoxygenic photosynthesis [31] supporting its relatively early position in our proposed evolutionary system.

*Microorganisms* **2022**, *10*, 1200

2

2

2

2

2

2

1

(Archaea)

(Archaea)

**8**

**8**

*Thiocystis violascens* (Proteobacteria)

*Pseudomonas aeruginosa* (Proteobacteria) *Methanocaldococcus fervens*

*Pseudomonas aeruginosa* (Proteobacteria) *Methanocaldococcus fervens*

Mg- (Co-?)chelatase (small and medium subunits)

chlH

**4**

*Halorhodospira halophila* (Proteobacteria)

Small and medium subunits of Cochelatases

Small and medium subunits of Cochelatases

Yes Yes?

Yes

cobN

cobN

Yes

Yes

**Table 1.** The possible stages of the evolution of the genes encoding Mg-/Co-chelatases as well as CHL and B12 pathways. **Function of B12**

**B12**

At stage 4, we placed anoxygenic proteobacteria [32] that in addition to chlorophyll biosynthesis also had the B12 pathway (Table 1, stage 4). These bacteria possessed anaerobic B12 pathway as well as many genes from the aerobic route. However, it was unclear if they were able to synthesize cobalamin using oxygen since these species were anaerobic. Additionally, their genome did not encode the large subunit of the aerobic Co-chelatase (the *cobN* gene). Thus, if the B12 biosynthesis was possible through the aerobic route, then the Mg-chelatase from these species might be able to function as a Co-chelatase as well.

Modern Cyanobacteria (such as *Synechococcus elongatus*) were at the next stage in our model because they were able to perform oxygenic photosynthesis, encoded large subunits for both Mg- and Co-chelatases, and could produce cobalamin via anaerobic and aerobic routes (Table 1, stage 5). Interestingly, Cyanobacteria did not have the *cobS* and *cobT* genes that encode the small and the medium subunits of the cobNST chelatase. It has been suggested that the corresponding subunits from the Mg-chelatase (*chlI* and *chlD*) could substitute them [14].

A number of oxygenic phototrophic Proteobacteria had the most advanced genotype with respect to the Mg- and Co-chelatases (Table 1, stage 6). In comparison to Cyanobacteria, their genomes contained *cobS* and *cobT* genes that were likely duplicated from the *chlI* and *chlD* and subsequently specialized to function as subunits of the cobNST chelatase. Since many of these bacteria (e.g., *Rhodospirillum rubrum*) were able to live under both aerobic and aerobic conditions, their genomes encoded both routes of B12 biosynthesis [33].

Starting from stage 1 to stage 6, we observed gradual development of the gene sets encoding chlIDH and cobNST chelatases. On the other hand, a number of modern heterotrophic Actinobacteria (such as *Mycobacterium tuberculosis*) did not have the CHL pathway but encoded both pathways of B12 biosynthesis (Table 1, stage 7). However, they utilized the *chlI* and *chlD* genes, rather than *cobN* and *cobS*, to encode the small and the medium subunits of their aerobic Co-chelatase. This suggested that their chelatase genotypes originated from the Cyanobacteria where *chlI* and *chlD* genes performed similar function. Thus, we hypothesized that Actinobacteria from stage 7 adopted some genes from the Cyanobacterial CHL pathway (stage 5) and lost the rest of the CHL biosynthesis genes. Finally, stage 8 represented further simplification of the chelatase genotype (Table 1, stage 8) where a frameshifting signal in the fs-*chlD* gene allowed the production of both the small and the medium subunits of the Co-chelatase for the aerobic route of B12 biosythesis [16].

#### **4. Discussion**

In this paper, we presented a model for the possible coevolution of the CHL and B12 (aerobic route) pathways (Figure 2). To develop this model, we assumed that among the thousands of the sequenced prokaryotic genomes, there were examples of so-called "living fossils", i.e., the direct descendants of the species from the early days of cellular life on Earth. Our analysis was based on the *chlD* gene due to its likely early appearance in the evolution [24]. We hypothesized that the frameshifted version of this gene was the starting point in the early evolution of CHL pathway. Namely, we identified this gene in several bacteria that could be considered as living fossils (stage 1). We also identified several other bacteria where a part of *chlD* gene was probably duplicated producing the proto-*chlI* gene (stage 2). Importantly, the stage 1 and 2 phlyla (Spirochaetes, Chloroflexi, Thermodesulfobacteria, and Nitrospirae) that appeared in the evolution before the Proteobacteria from the stage 3 and 4 [26] supporting our suggested evolutionary order. Interestingly, species from stage 1 and 2 did not have B12 or CHL pathways as well as either large subunit genes. On the other hand, the fs-*chlD*, *chlI* and *chlD* genes from these species can produce two proteins that may interact with each other and form a separate complex [28]. To our knowledge, the function of the *chlD* + *chlI* complex (without the a subunit protein) has not been studied and its function is unknown. Thus, further studies of these genomes may reveal the new function for the corresponding proteins.

**Figure 2.** A putative chain of events that could happen between different stages during the evolution of the CHL and aerobic B12 pathways.

The appearance of the CHL and the aerobic B12 pathways as well as oxygenic photosynthesis was observed in Cyanobacteria at stage 5 of our system. Clearly, the aerobic B12 pathway could only appear after the oxygenic photosynthesis; however, we were not able to find any oxygenic phototroph without the aerobic B12 pathway. Although we believe that such putative bacteria existed at some evolutionary point, they probably adopted the more efficient aerobic B12 pathway after its appearance and therefore could not be found today. Proteobacteria at stage 6 represented the most advanced chelatase genotypes where all the subunits of the chlIDH and cobNST enzymes were encoded by specialized genes. On the other hand, a possible reduction of the Cyanobacterial genomes could gave rise to the modern Actinobacteria that utilized *chlI* and *chlD* genes to encode small and medium cobNST subunits. Indeed, according to the global phylogenetic tree, Actinobacteria had a common ancestor with Cyanobacteria which appeared in the evolution first [26]. Finally, we observed that some prokaryotes utilized the ancient strategy to encode both the small

and medium subunits of the aerobic Co-chelatase using frameshifting signal in the fs-*chlD* gene (stage 8). The *chlD* genes with frameshifting signals in these genomes likely originated via horizontal gene transfer rather than the signals evolving once again in the conventional *chlD*/*bchD* genes. This is supported by the fact the fs-*chlD* genes do not group with the other genes without frameshifts (Supplementary Figure S3).

Another interesting question was related to the anaerobic cobalamin biosynthesis pathway—e.g., whether it appeared before or after the CHL biosynthesis pathway. Interestingly, the Chelatase DB included several archaeal species (such as *Methanobacterium paludis* and *Thermoplasma acidophilum*) that contained the *chlD* gene (without a frameshift) as well as the anaerobic B12 pathway. Again, the function of the *chlD* gene in these organisms was unclear, but the presence of the anaerobic B12 route may suggest that this pathway existed before the development of the CHL pathway.

To conclude, our analysis suggested a logical model for the coevolution of CHL and B12 pathways. We pinpointed specific prokaryotic species that can be considered as living fossils and their further study may shed a new light on the early evolution of these pathways. To our knowledge, the function of the *chlI* and *chlD* proteins without the large subunit (*chlH* or *cobN*) remained unexplored, and here, we suggested some model organisms where this could be studied.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/microorganisms10061200/s1, Figure S1. The main strategies utilized by different prokaryotic species to encode Mgand Co-chelatases; Figure S2. The number of the identified *chlD* genes in the genomes from different prokaryotic phyla; Figure S3. The reference phylogenetic tree of the annotated medium chelatase subunit proteins and as well as the long products of the fs-*chlD* genes; Figure S4. Frameshifting signlas identified in the fs-*chlD* genes from *Spirochaeta thermophila* DSM 6578 (A) and *Brevefilum fermentans* (B); Figure S5. A putative chain of events that could happen between different stages during the evolution of the CHL and aerobic B12 pathways.

**Author Contributions:** S.K., A.M. and I.A. formulated and evaluated the ideas and designed the website and the database structure. S.K. and A.M. conducted the research and implemented the Web database. I.A. supervised the research and and wrote the original draft. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by the Russian Science Foundation grant number 20-74-00128 to IA.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The website of the database with all the information about 1207 prokaryotic genomes with the *chlD*, *bchD*, and *cobT* genes together with the corresponding information about the predicted frameshifts and frameshifting signals are available at http://ivanya.com/ chelatase/ (accessed on 1 April 2022).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Abbreviations**

The following abbreviations are used in this manuscript:


**2.6 Eyes**

As shown in **Figure 2**, proptosis, stare, lid lag, globe lag, dry eyes, photosensitivity, corneal ulceration, loss of vision (uncommon) are features of Graves associated orbitopathy (GAO) [13].


**5. Recommendation**

The World Bank recommends that cost recovery of waste management services should be ensured and the revenue collection of waste management fees from waste producers should be increased [31]. Also, a transparent procurement process for private sector participants should be established and performance-based contracts should be developed. The government should include the people in the decision making process to foster acceptance of new policies and initiatives. Finally, the act of keeping the environment safe and clean by proper disposal of wastes can be enhanced if regulatory measures are enforced on the people. In lieu of this, public health o fficers (PHOs) need to enforce compliance with these regulations by the residents. This will go a long way in achieving sustainable waste management because people will not want to be victims of law for fear of the penalty attached to it [28,39].

**Author Contributions:** Conceptualization of the research work came from D.O.O. and the methodology adopted was carried out by the authors: D.O.O. and C.O.N. All the authors played a contributing role in the use of the software, and the validation of the work was carefully checked by D.O.O. The formal analysis and investigation was done by D.O.O. Resources and data curation was carried out by D.O.O., and C.O.N. While the writing of original draft preparation was done by D.O.O. and C.O.N. Writing-review editing was done by D.O.O. Visualization, supervision and project administration was done by D.O.O.

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

**Acknowledgments:** The authors are grateful to the management of Covenant University for providing an enabling environment in doing the report of this research work. We also appreciate the various authors of resource materials that were used which provided relevant information that led to the success of this work.

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


**2.1. Study area: Mustafa Kemal ATATURK'S Mausoleum, ANITKABIR**

The Anitkabir mausoleum, was completed in 1953 in Ankara, Turkey (Figure 1). It hosts state ceremonies during national festivals, and represents the Turkish people and Ghazi Mustafa Kemal ATATÜRK, the founder of the Republic of Turkey (Figure 1b). Anitkabir was con‐ structed in three phases. The first part is an entrance road, called the Lion Road, which is 262m long and has a total of 24 lion statues along each side, representing power and peace (Figure 1c). The second part is a ceremonial square, and the third is the Mausoleum (Figure 1c).

The monument groups (three women, three men) and twenty-four lion statues of Anitkabir are mainly composed of white travertine from Pinarbasi, Kayseri, Turkey. The white-coloured travertine has a banded and spongy texture under the microscope [23]. It is mainly composed of calcite, aragonite with a small amount of salt, recrystallized calcite, gypsum and plant relicts. Table 1 shows the modal mineralogical composition and physical properties of this travertine. Micro-fractures were observed under a polarizing microscope, especially at the rim of the vesicular of the rocks (Figure 2).

**Table 1.** Mineralogical composition and physical properties of white-coloured travertine in Anitkabir.

**Figure 1. (a)** Geographical location of the study area, **(b)** Ghazi Mustafa Kemal ATATURK, the founder of the Republic of Turkey, **(c)** the ANITKABIR monument, Ankara-Turkey, **(d)** three large male statues at the beginning of the Lion Road, which is the entrance to Anitkabir, on the left side, **(e)** female statues facing the male group, **(f)** 24 lion statues, representing power and peace, sit on each side of the Lion Road.

**Figure 2. (a)** A lion statue, **(b)** view close to surface of lion, and **(c)** microphotograph of white-coloured travertine.

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**Figure 2. (a)** A lion statue, **(b)** view close to surface of lion, and **(c)** microphotograph of white-coloured travertine.

**Figure 1. (a)** Geographical location of the study area, **(b)** Ghazi Mustafa Kemal ATATURK, the founder of the Republic of Turkey, **(c)** the ANITKABIR monument, Ankara-Turkey, **(d)** three large male statues at the beginning of the Lion Road, which is the entrance to Anitkabir, on the left side, **(e)** female statues facing the male group, **(f)** 24 lion statues,

110 Imaging and Radioanalytical Techniques in Interdisciplinary Research - Fundamentals and Cutting Edge Applications

representing power and peace, sit on each side of the Lion Road.

**Figure 3.** Data measurements on the female statues.

#### **2.2. GPR survey description**

The human statues were divided into several subparts such as skirt or under waist, between waist and neck, arms, trousers, etc. for GPR survey. Some parts were divided into additional subparts (Figures 3 and 4) according to their figures, in order to enable true 3D imaging and protect the profile line position, because topography correction was not possible on the statues. In addition, three profiles spaced 10 cm apart were arranged along the backs of lion statues. The data acquisition scheme is shown in Figure 5, in which GPR data profiles gathered on the body of the first female statue were split into two parts, called skirt and upper part between waist and neck.

Profiles were spaced at 10 cm on each subpart, and were lined with a paper band sticker. First, data survey tests were carried out to determine the recording time-window according to the approximate thicknesses of the statues. A RAMAC CU II GPR system equipped with a 1.6- GHz bistatic shielded antenna was employed on all the statue groups. Transmitter–receiver antenna offset was 0.05m. Trace spacing was 0.0044m and time-sampling interval per trace on each profile was 0.0327 ns.

**Figure 4.** Data acquisitions on the male statues.

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**Figure 4.** Data acquisitions on the male statues.

**Figure 3.** Data measurements on the female statues.

The human statues were divided into several subparts such as skirt or under waist, between waist and neck, arms, trousers, etc. for GPR survey. Some parts were divided into additional subparts (Figures 3 and 4) according to their figures, in order to enable true 3D imaging and protect the profile line position, because topography correction was not possible on the statues. In addition, three profiles spaced 10 cm apart were arranged along the backs of lion statues. The data acquisition scheme is shown in Figure 5, in which GPR data profiles gathered on the body of the first female statue were split into two parts, called skirt and upper part between

112 Imaging and Radioanalytical Techniques in Interdisciplinary Research - Fundamentals and Cutting Edge Applications

Profiles were spaced at 10 cm on each subpart, and were lined with a paper band sticker. First, data survey tests were carried out to determine the recording time-window according to the approximate thicknesses of the statues. A RAMAC CU II GPR system equipped with a 1.6- GHz bistatic shielded antenna was employed on all the statue groups. Transmitter–receiver antenna offset was 0.05m. Trace spacing was 0.0044m and time-sampling interval per trace on

**2.2. GPR survey description**

waist and neck.

each profile was 0.0327 ns.

#### **2.3. GPR data processing**

Data processing was performed on 2D GPR profile data sets for each part of the statues with the REFLEXW program (ver. 5.5) developed by Sandmeier Scientific Software [30]. Start-time correction, then de-wow and background removals were applied to all the profile data in order to protect the true time scale, remove very low frequency effect and average amplitude knowledge respectively. The amplitude decay compensation was applied to all traces of the whole data set by using the same small-scale linear gain function. A second-order band-pass Butterworth filter was used for the whole data set to eliminate low-frequency artifacts and high-frequency noise. The resulting synthetic hyperbolas were matched with diffraction patterns throughout the profiles to determine average velocity of the electromagnetic (EM) wave. The best matching hyperbola provided the velocity of 0.12 m/ns. Finally, Kirchhoff migration was applied to the radargrams using average velocity, in order to carry diffracted electromagnetic (EM) waves true locations.

The quality of GPR images is strongly dependent on appropriate correction of the attenuation effects, usually supplied by time-varying gain. However, historically, the use of amplitude gain in basic processing of GPR data has been highly subjective and also very much displaying methodology [31]. There are various methods available for amplitude gain for GPR data. Traditional time-varying gain is carried out using linear, exponential functions, etc. functions, including ground wave amplitudes. However, this operation is not linear. The time-gained GPR data cannot recover the original information. Selection of the gain function depends mostly on the user and the quality of the GPR data. Both exaggerated linear and exponential time-gain change not only the amplitude range for each time step but also amplitude shape. Exaggerated time gain to image 2D GPR data can result in erroneous interpretation, especially when using such data to construct a 3D volume.

Time-gained signal is

$$s(\mathbf{x}, t) = r(\mathbf{x}, t)h(t) \tag{1}$$

$$h(t) = a, 1 < a \le \left| \frac{r\_{\text{max}}}{r\_{\text{min}}} \right| \tag{2}$$

**Figure 5.** GPR data acquisition scheme of the first female statue.

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Where, *h* (*t*) , is a time gain function, |*r*max | and |*r*min | are maximum- and minimum amplitude of the reflected/diffracted wave *r*(*x*, *t*), respectively.*h* (*t*), has a constant decimal value *a* between 1 and 2, and also is a linear function between 1 and *a* for a 2D data profile [32].

Our approximation concerns 2D or 3D image simplification in determining buried archaeo‐ logical remains and discontinuities such as fractures. Therefore, we assigned a new amplitude– colour range for 2D radargrams of the GPR profiles and opaque range in order to identify anomalies and display the data set in a transparent 3D data volume. Figure 6a indicates some processed radargrams of the profiles of the skirt of the first female statue, shown in Figure 5. **2.3. GPR data processing**

electromagnetic (EM) waves true locations.

when using such data to construct a 3D volume.

Time-gained signal is

Data processing was performed on 2D GPR profile data sets for each part of the statues with the REFLEXW program (ver. 5.5) developed by Sandmeier Scientific Software [30]. Start-time correction, then de-wow and background removals were applied to all the profile data in order to protect the true time scale, remove very low frequency effect and average amplitude knowledge respectively. The amplitude decay compensation was applied to all traces of the whole data set by using the same small-scale linear gain function. A second-order band-pass Butterworth filter was used for the whole data set to eliminate low-frequency artifacts and high-frequency noise. The resulting synthetic hyperbolas were matched with diffraction patterns throughout the profiles to determine average velocity of the electromagnetic (EM) wave. The best matching hyperbola provided the velocity of 0.12 m/ns. Finally, Kirchhoff migration was applied to the radargrams using average velocity, in order to carry diffracted

114 Imaging and Radioanalytical Techniques in Interdisciplinary Research - Fundamentals and Cutting Edge Applications

The quality of GPR images is strongly dependent on appropriate correction of the attenuation effects, usually supplied by time-varying gain. However, historically, the use of amplitude gain in basic processing of GPR data has been highly subjective and also very much displaying methodology [31]. There are various methods available for amplitude gain for GPR data. Traditional time-varying gain is carried out using linear, exponential functions, etc. functions, including ground wave amplitudes. However, this operation is not linear. The time-gained GPR data cannot recover the original information. Selection of the gain function depends mostly on the user and the quality of the GPR data. Both exaggerated linear and exponential time-gain change not only the amplitude range for each time step but also amplitude shape. Exaggerated time gain to image 2D GPR data can result in erroneous interpretation, especially

( ) max

,1 *r ht a a*

min

*r*

Where, *h* (*t*) , is a time gain function, |*r*max | and |*r*min | are maximum- and minimum amplitude of the reflected/diffracted wave *r*(*x*, *t*), respectively.*h* (*t*), has a constant decimal value *a* between 1 and 2, and also is a linear function between 1 and *a* for a 2D data profile [32].

Our approximation concerns 2D or 3D image simplification in determining buried archaeo‐ logical remains and discontinuities such as fractures. Therefore, we assigned a new amplitude– colour range for 2D radargrams of the GPR profiles and opaque range in order to identify anomalies and display the data set in a transparent 3D data volume. Figure 6a indicates some processed radargrams of the profiles of the skirt of the first female statue, shown in Figure 5.

*s xt r xt ht* ( , , ) = ( ) ( ) (1)

= <£ (2)

**Figure 5.** GPR data acquisition scheme of the first female statue.

A linear colour scale of the radargrams (Figure 6b) indicates the amplitude range, which begins with maximum negative polarity and ends with maximum positive polarity. The blue colour range represents maximum negative amplitude, while the purple colour range represents the positive amplitudes according to their values. It is known that the maximum positive and maximum negative amplitude ranges in the amplitude–colour scale represent the fractures and cavities filled with air or buried archaeological remains in soil. Therefore, it is necessary to check the maximum amplitude ranges on the time slices to identify these target objects. We applied a new approximation to eliminate the weak reflections that are characteristic of cemented rock [7, 23], and to activate the fractures and cavities represented by strong reflec‐ tions/diffractions on the radargrams (Figure 6c). This involved assigning a new colour scale for the amplitude range of the processed profile data set by means of a new amplitude–colour

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**Figure 7. (a)** A linear functioned amplitude–colour scale and **(b)** re-functioned colour scale of the same amplitude range.

**Figure 8. (a)** A linear functioned opacity for the linear functioned amplitude–colour scale and **(b)** A re-arranged opac‐ ity function using the same amplitude–colour scale to activate only maximum amplitude range and remove all others.

function rather than linear amplitude–colour function (Figure 6d, Figure 7).

**Figure 6.** a) Processed 2D radargrams of the GPR profiles 1 to 5, acquired from the skirt of the first female statue, using (b) selected linear amplitude–colour function, (c) the same radargrams using (d) re-arranged amplitude–colour scale of (b) to reveal small cavities and fractures on the radargrams.

A linear colour scale of the radargrams (Figure 6b) indicates the amplitude range, which begins with maximum negative polarity and ends with maximum positive polarity. The blue colour range represents maximum negative amplitude, while the purple colour range represents the positive amplitudes according to their values. It is known that the maximum positive and maximum negative amplitude ranges in the amplitude–colour scale represent the fractures and cavities filled with air or buried archaeological remains in soil. Therefore, it is necessary to check the maximum amplitude ranges on the time slices to identify these target objects. We applied a new approximation to eliminate the weak reflections that are characteristic of cemented rock [7, 23], and to activate the fractures and cavities represented by strong reflec‐ tions/diffractions on the radargrams (Figure 6c). This involved assigning a new colour scale for the amplitude range of the processed profile data set by means of a new amplitude–colour function rather than linear amplitude–colour function (Figure 6d, Figure 7).

**Figure 7. (a)** A linear functioned amplitude–colour scale and **(b)** re-functioned colour scale of the same amplitude range.

**Figure 8. (a)** A linear functioned opacity for the linear functioned amplitude–colour scale and **(b)** A re-arranged opac‐ ity function using the same amplitude–colour scale to activate only maximum amplitude range and remove all others.

**Figure 6.** a) Processed 2D radargrams of the GPR profiles 1 to 5, acquired from the skirt of the first female statue, using (b) selected linear amplitude–colour function, (c) the same radargrams using (d) re-arranged amplitude–colour

116 Imaging and Radioanalytical Techniques in Interdisciplinary Research - Fundamentals and Cutting Edge Applications

scale of (b) to reveal small cavities and fractures on the radargrams.

limitation allowed simplification and made only fractures and native cavities visible on the radargrams, as seen in Figure 6c. The horizontal x-axis of Figures 6a and 6c indicate the distance along the measuring profile. The vertical axis shows depth range, which represents the thickness of the statue skirt from front surface to back surface, transformed by using average

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Generally, interactive visualization is carried out by constructing 3D data volumes of parallelaligned 2D GPR data sets to show the target objects. The 3D data volume can be displayed as slices, including profiles, times (or depths) and common traces of the profiles; or separated sub-blocks are rendered as solid iso-volumes with linear opacity, determined by the amplitude of the anomalies. The buried fractures or cavities can be defined on the interactive slices, particularly on depth slices with location, and shapes according to depth. Therefore, it was necessary to check the most meaningful depth slices and profiles to define the structures of the subsurface if the area is small and complex. However, the obtained results could be further

Our aim was to obtain a good 3D data volume display, which was a critical part of interpreting the GPR data set. The 3D image is able to present a view of subsurface features such as a fracture or cavity, in addition to objects such as industrial and/or archaeological remains, etc. This imaging could be achieved by a transparent 3D half bird's-eye view revealing only buried objects. Therefore, firstly, transparency could be achieved by constructing an opacity function instead of linear opacity determined by the amplitude scale (Figure 8). The horizontal axis of the opacity function was the amplitude scale starting with maximum negative amplitude and ending with maximum positive amplitude; the vertical axis represented opacity coefficients of the amplitude range [11, 23]. Thus, any amplitude range could be highlighted or minimized by the appointed opacity coefficient. The REFLEXW program allows the opacity coefficient to be chosen between one (maximum opacity) and zero (transparent) (Figure 8b) [26]. A trans‐

parent view could be obtained only by eliminating the unwanted amplitude range.

location and shapes according to depth (thickness).

Therefore, the amplitude range was important. Because it was known that the maximum amplitudes represented discontinuities, the weak amplitude range was eliminated by giving these a zero opacity value, and transparent 3D imaging was obtained. The transparency was achieved by allocating an opaque interval to the amplitude scale, similar to the re-arranged amplitude–colour approximation for interested profile range or time range for the solid 3D GPR data volume. This visualization type was applied to both the statues and archaeological remains in this chapter. Figure 9a indicates traditional, solid depth-slices, while Figure 9b indicates transparent depth slices at 15 cm, 38 cm and 51 cm of the data set for the skirt of the first female statue. These slices were used to control micro-fractures and cavities according to the skirt thickness. The horizontal x-axis and y-axis of slices indicate the profile sequence and the distance along the measuring profile respectively. Locations of the micro-cavities could be seen on the slices. It is necessary to carefully check interactive slices in order to determine

velocity of the EM wave.

improved.

**2.4. Transparent 3D half bird's-eye view of GPR data set**

**Figure 9. (a)** Traditional solid depth slices at 15 cm, 38 cm and 51 cm, **(b)** Transparent depth slices for the skirt of the first female statue (same three depths).

The horizontal axis of the amplitude–colour function (Figure 7) is the amplitude scale of the GPR data, whereas the vertical axis represents colour categories from 0 to 255. The colour limitation allowed simplification and made only fractures and native cavities visible on the radargrams, as seen in Figure 6c. The horizontal x-axis of Figures 6a and 6c indicate the distance along the measuring profile. The vertical axis shows depth range, which represents the thickness of the statue skirt from front surface to back surface, transformed by using average velocity of the EM wave.


**5. Conclusions**

Canonical correlation analysis is an optimized multivariate technique for evaluating the existence or non-existence of relationships between complex groups of variables. In this study it proves to be a powerful tool to study the relationship between the selected set of carcass traits and the proximate, lipid and mineral components, particularly when it is expected that certain degree of interaction exists among these three groups of chemical variables. This work demonstrates an important relationship between backfat thickness, marbling and the content of total lipids and fatty acids in beef LDL muscle from cattle fed on pastures under tropical conditions. Instead, carcass traits are poorly associated with beef mineral content. These findings allow for the possibility to develop selection criteria based on BFT and/or marbling to sort carcasses with differing nutritional values. For the experience gained in beef carcass grading in Venezuela, the evaluation of marbling levels requires more intense training and supervision of graders than the BFT measurement. Moreover, marbling is seldom used to grade beef carcasses in the developing, tropical countries. Therefore, it is more practical to use BFT in future regression analyses to explain the variation in lipid composition of beef *longissimus* muscle.

Further analyses are needed to determine the potential influence of sex condition on the magnitude of the associations among carcass traits and beef fatty acid composition.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/foods10061364/s1, Figure S1: Graphical description of sample preparation and chemical analyses; Figure S2: Dendrogram for samples from the hierarchical cluster analysis identifying clusters. The samples were numbered randomly from 1 to 109; Figure S3: Projection of variables from the subgroup proximate composition by clusters; Figure S4: Projection of variables from the subgroup mineral content by clusters; Figure S5: Projection of variables from the subgroup lipid profile by clusters; Figure S6: Projection of variables from the subgroup carcass traits by clusters.

**Author Contributions:** N.H.-L. designed the investigation and field data that supported this research. L.A.d.M. was responsible for the selection of chemical procedures and training of personnel who conducted the chemical analyses and collected lab data that support this research. S.U.-B. conducted the chromatographic analysis of the samples. N.J.-T., E.M.-V. and L.A.d.M. performed statistical analyses, and tabulated results. L.A.d.M., N.J.-T., M.G.-M. and N.H.-L. interpreted, designed, and revised the structure of the manuscript. All authors searched and reviewed the literature, discussed the contents of the manuscript, and approved submission. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Consejo de Desarrollo Científico y Humanístico de la Universidad del Zulia (CONDES-LUZ), AND Facultad de Agronomía, Universidad del Zulia, Venezuela.

**Institutional Review Board Statement:** Data collection was carried on in compliance with the criteria for animal care and welfare described in the Bioethics and Biosafety guide of the Fondo Nacional de Ciencia, Innovación y Tecnología (FONACIT) of Venezuela.

**Data Availability Statement:** Data are not available in public datasets, please contact the authors.

**Acknowledgments:** The authors are grateful to Matadero Industrial Centro Occidental (MINCO) for their valuable support during the live and carcass data collection, and donation of beef samples. We thank Martin OConnors for providing further training in carcass evaluation according to the USDA standards. The authors also gratefully acknowledge the cooperation of Daniel Huerta-Sánchez (Department of Economics and Finance, Florida Gulf Coast University) in the review of technical English to improve the statistical discussion, and Javier Aracena in the editing of the supplementary figures.

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


Preface

Despite revolutionary advances in medical science, some diseases are still difficult to manage and treat, particularly rare diseases. Thus, much current medical research focuses on identifying molecular mechanisms and cell signaling pathways of disease to develop new treatments. The complexities of pathogenicity and damage of diseases need to be fully understood to design more accurate treatment and control methods.

The immune system is the first line of defense against disease in the human body. Thus, upgrading immune responses is necessary to cure many diseases. In the case of diseases in which infectious agents are involved in pathogenesis, increasing the quality of immune responses sometimes leads to increased resistance to infectious agents and promotes escape routes from the immune system. As such, it is necessary to identify the exact pathways of the immune system to increase the precision of practical responses to diseases such as sarcoidosis.

Sarcoidosis is a rare immune-mediated disease that affects multiple organs in the body. Written by experts in the field, this book presents an overview of the disease from an immunologic perspective. It discusses the cell signaling pathways and molecular mechanisms involved in sarcoidosis as well as pathophysiology, diagnosis, management, and treatment of the disease. The book begins with an introduction to sarcoidosis, including definitions and a description of the disease's immunopathology. Subsequent chapters discuss the disease in more detail as well as highlight recent advances in the field.

The material covered contributes to a better understanding of the immunopathology and molecular pathways of sarcoidosis. This book is a useful resource for researchers and medical professionals both in the lab and in the field.

### **Dr. Seyyed Shamsadin Athari** Faculty of Medicine, Department of Immunology, Zanjan University of Medical Sciences, Zanjan, Iran

### **Dr. Entezar Mehrabi Nasab**

Department of Cardiology, School of Medicine, Tehran Heart Center, Tehran University of Medical Sciences, Tehran, Iran

Department of Cardiology, School of Medicine, Valiasr Hospital, Zanjan University of Medical Sciences, Zanjan, Iran

## **Chapter 1**

## Introductory Chapter: Sarcoidosis – New Perspectives

*Seyyed Shamsadin Athari and Entezar Mehrabi Nasab*

## **1. Introduction**

Sarcoidosis is polygenic, multifactorial, and inflammatory multisystem disease, which preferentially affects the lung and thoracic lymph nodes with the noncaseating epithelioid granulomas formation and is characterized by the presence of noncaseating granulomas. The incidence and prevalence assessment of sarcoidosis is difficult because many patients can be asymptomatic and the disease can also remit spontaneously. In addition to the lungs, the skin, spleen, liver, lymph nodes, heart, and nervous system have all been shown to be affected by sarcoidosis. In 1889, Ernest Besnier described the cutaneous hallmarks of chronic sarcoidosis as lupus pernio. Later, Caesar Boeck used the term sarkoid (sarcoid) for the first time when he observed that the lesions were similar to sarcoma, but benign [1, 2].

### **2. Sarcoidosis immunopathology**

Pathogenesis of sarcoidosis involves both the innate and adaptive immune systems. In patients with sarcoidosis, the activated macrophages and T cells release chemokines and cytokines involved in the formation of the granuloma [such as the tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), interleukin-18 (IL-18), and IL-12]. The clinical appearance of sarcoidosis varies depending on the specific organ involved. It may present with a wide range of clinical functions from asymptomatic to fatal. The sarcoidosis' etiology is still unknown, but many studies have shown that an unknown antigen processed by active macrophages stimulates an immune response that is regulated by T lymphocytes and macrophages. Several studies suggest that not only unknown antigens are responsible for sarcoidosis, but also genetic susceptibility, environmental factors, and in some instances, autoimmunity [2, 3].

Worldwide, the prevalence and incidence of sarcoidosis are not well known. Sarcoidosis affects individuals of all ages irrespective of ethnicity or race, with maximum incidence among people aged 20–39 years, and also, quite more prevalent in nonsmokers, women, and in rural communities. The most common comorbidities encountered in sarcoidosis patients are obesity, coronary heart disease, asthma, hyperlipidemia, diabetes, thyroid disease, osteoporosis, hypertension, chronic renal disease, and chronic obstructive pulmonary disease (COPD). Also, sarcoidosis was often reported in patients with certain autoimmune diseases including Sjogren's syndrome ankylosing spondylitis, autoimmune thyroid disease, and systemic sclerosis. Many studies have hypothesized the role of genetic susceptibility, putative antigens, environmental factors, and autoimmunity in the development of sarcoidosis, but no single cause has been identified to date [2–4]. Although no diseasespecific auto-antibodies (Igs) have been observed, it has been presented that the

### *Sarcoidosis - New Perspectives*

major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) possess an autoantigen that is recognized by the T cells in sarcoidosis patients. Autoimmunity presents a novel spectrum for immunopathogenesis of sarcoidosis and may help elucidate sarcoid etiology. It was mentioned that sarcoidosis has overlapped with some autoimmune diseases, including autoimmune thyroid disease, rheumatoid arthritis, Sjogren's syndrome, and ankylosing spondylitis; thus, study of the immunopathogenesis of sarcoidosis patients and its correlation with other autoimmune diseases could open new avenues for investigating the underlying causes of this disease [4, 5].

**In** the most cases in sarcoidosis patients, the growth of these granulomas establishes the primary abnormality. Sarcoid granulomas are ordered, structured masses comprising epithelioid cells, macrophages and their derivatives, giant cells, and T cells. It is presumed that in sarcoidosis, infiltrating T-reg cells fail to reduce the exaggerated inflammatory response, thereby contributing to persistence and integrity of granuloma. Also, T-reg cells release transforming growth factor-β (TGF-β), which may contribute to fibrosis and granuloma. Th17 cells are linked to the sarcoidosis pathogenesis and are recruited to the disease site and involved in the construction of the granuloma. The balance between Th17 and T-reg cells is thought to be disrupted in sarcoidosis and also, is an important factor in sarcoidosis prognosis [2, 4, 6].

Although a wide range of biomarkers has been proposed for screening of sarcoidosis, none is recommended per se in the clinical practice because of the insufficient sensitivity and specificity. Proposed sarcoidosis biomarkers include many cytokines, chemokines, and mediators that are derived from macrophage or and lymphocyte, such as IL-2R, chitotriosidase, neopterin, angiotensin-converting enzyme, and lysozyme, but there is no single reliable biomarker with proven unequivocal prognostic value [1, 3, 4, 6].

Despite extensive research over the past several decades, the sarcoidosis etiology remained unknown. Numerous potential etiological agents have been identified. In recent years, there was an extraordinary increase of data; however, they have very heterogenous characteristics that are used to personalized medicine purpose posing many challenges. The enormous amount of data, applied to the study of sarcoidosis, needs to be dynamically organized and recovered. The integration of the recent deep data will allow to fill the gap between genotype and phenotype, avoiding falsenegative and false-positive results. Early diagnosis is important to treat this disease; therefore, there is a fundamental need to develop robust diagnostic tools for the diagnosis and prognosis of sarcoidosis. Recently, new strategies of the sarcoidosis' diagnoses, including FDG-PET scans, HRCT technologies, EBUS, and TBNA, have enhanced the prognosis. More focus should be on the development of noninvasive biomarkers. Corticosteroids play a main role in the treatment of sarcoidosis, but if used for a long time, they can cause many side effects. Second-line and targeted therapies can be promising alternatives for treating sarcoidosis in the near future [5–7]. In this field, precision medicine is the new hope, and it is necessary to be monitored closely for progress toward targeted interventions. For better disease management, multifaceted approaches remain the best practice to ensure competent and effective patient care.

Therefore, in this book, study of new aspect of sarcoidosis was tried, to introduce new way to design advanced methods for prevention, diagnosis, treatment, and control of sarcoidosis.

*Introductory Chapter: Sarcoidosis – New Perspectives DOI: http://dx.doi.org/10.5772/intechopen.105554*

## **Author details**

Seyyed Shamsadin Athari1 \* and Entezar Mehrabi Nasab2,3

1 Faculty of Medicine, Department of Immunology, Zanjan University of Medical Sciences, Zanjan, Iran

2 Tehran Heart Center, Department of Cardiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

3 Department of Cardiology, School of Medicine, Valiasr Hospital, Zanjan University of Medical Sciences, Zanjan, Iran

\*Address all correspondence to<EMAIL_ADDRESS><EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. 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.

## **References**

[1] Sweiss NJ, Salloum R, Gandhi S, Alegre M-L, Sawaqed R, Badaracco M, et al. Significant cd4, cd8, and cd19 lymphopenia in peripheral blood of sarcoidosis patients correlates with severe disease manifestations. PLoS ONE. 2010;**5**(2):e9088

[2] Jain R, Yadav D, Puranik N, Guleria R, Jin J. Sarcoidosis: Causes, diagnosis, clinical features, and treatments. Journal of Clinical Medicine. 2020;**9**:1081

[3] Landi C, Carleo A, Cillis G, Rottoli P. Sarcoidosis: Proteomics and new perspectives for improving personalized medicine. Expert Review of Proteomics. 2018;**15**(10):829-835

[4] Albert M, Adrianto I, Datta I, Iannuzzi MC, Trudeau S, Li J, et al. Association of HLA-DRB1 with sarcoidosis susceptibility and progression in African Americans. American Journal of Respiratory Cell and Molecular Biology. 2015;**53**(2): 206-216

[5] Chen ES, Moller DR. Etiologies of Sarcoidosis. Clinical Reviews in Allergy & Immunology. 2015;**49**(1):6-18

[6] Kiszałkiewicz J, Piotrowski WJ, Brzeziańska-Lasota E. Selected molecular events in the pathogenesis of sarcoidosis – recent advances. Pneumonologia i alergologia polska. 2015;**83**(6):462-475

[7] Wang V, Jiang F, Kallepalli A, Basen T, Yusin J, Krishnaswamy G. Sarcoidosis. Annals of Allergy, Asthma and Immunology. 2018;**121**(6):662-667

## **Chapter 2**


**3. The diffractive screen helping to fulfill the observation field in multiple projections**

The surface which, like a plane mirror or a hologram, should generate a light ray distribution which is equivalent to the originally generated by a three-dimensional object must have the capability to emit in a large angular field with directional intensity and color values corresponding to the scene which is being reconstructed. Each ray is the component of a luminous point to be constructed outside the surface, generally, in front of it. The threedimensional image which floats in front of a screen is the one that most impresses the public, even more when the observer can pass his hand through.

Fig. 1. Left: Ordinary diffusion at a screen. Right: Rays in an ideal three-dimensional display.

The capability to make such an element within a so-called pixel for a TV or computer display does not yet exist. A theoretical way to achieve it was patented (4) based on the

plane may correspond to the Fourier transformation of the transmission or reflection surface properties. It is not completely correct to suppose that, because the Fourier transformation can only be applied in a paraxial approximation, and the angles we need to achieve do not always have this limitation. But, assuming the hypothesis, we could obtain the desired surface profile by using the inverse Fourier Transformation of a light distribution that we

How can one construct such a microstructure on the surface? Which machining technique

We understand that if we make the hologram of a surface of any profile the surface of the resulting hologram is effectively a diffuser with the diffracted light intensity profile of the original surface. It is a fact that diffractive elements made by holography begin to be employed for designing illuminating systems, working with monochromatic or nearly monochromatic light (2). Although the efficiency of an easily replicated hologram is less than 25%, the concentration of the light strictly at a desired region may compensate and even overcome the problem in many cases, and holographic diffusers are on sale by many companies, one of the applications being the internal illumination of computer displays (3).

**3. The diffractive screen helping to fulfill the observation field in multiple**

Fig. 1. Left: Ordinary diffusion at a screen. Right: Rays in an ideal three-dimensional

The capability to make such an element within a so-called pixel for a TV or computer display does not yet exist. A theoretical way to achieve it was patented (4) based on the

The surface which, like a plane mirror or a hologram, should generate a light ray distribution which is equivalent to the originally generated by a three-dimensional object must have the capability to emit in a large angular field with directional intensity and color values corresponding to the scene which is being reconstructed. Each ray is the component of a luminous point to be constructed outside the surface, generally, in front of it. The threedimensional image which floats in front of a screen is the one that most impresses the

define as the desired one on the lighted plane.

**projections**

display.

would be useful? Could we replicate it in a rapid and cheap way?

public, even more when the observer can pass his hand through.

location of a 2D micro-display at the focus of a small lens, each micro-pixel element being a ray generator. The number of pixels of a modern display should correspond to the number of volumetric pixels, also called "voxels". Each voxel is the origin of many rays and each ray comes from a sub-pixel of the ideal complete screen, so that we may think these as "ray pixels". An image in a volumetric space with the present pixel display capability for each voxel involves the need for much more pixelation at the emitting surface. The required resolution can be considered as the square of the present capability of 2D screens.

The addressing of those micro-pixels could be facilitated by coding techniques because in each micro-display one pixel's position would always have a corresponding pixel position in its neighbor micro-display, both beginning to a unique three-dimensional display point so that its color and intensity would be very close. Usually, colour does not change with the observation perspective, and the intensity angular change is a relationship which corresponds to a diffusion surface law that could be predetermined and does not need to be received as object information, as it happens in 3D computer drawing rendering.

A fixed relationship interconnects then each family of micro-pixels, each one belonging to one specific micro-pixel display element, and its implementation could be automatic to reduce the need for transmitted information. As such a system is not yet possible, the Holografika company (5) produces one in which a certain number of projectors is located behind a diffracting screen whose function is to produce a diffuse lobe so that as the observer moves, the transition of the light coming from one projector to the light coming from the neighbor projector does not have dark regions. The space region for the observer is then continued and no dark regions are present. The number of projectors must be about 18 and until now only images made by the computer have been shown and animated through simple movements. We can understand that the name "holographic display" given by the company to the system can only be accepted within the popular meaning described above; no light interference is present during the process, neither in practice nor in concept. It is the only commercial system claiming to have the appearance of a continuous parallax. It also claims that the light is directed so that the observer's eye focalizes effectively at the point where the image is represented, eliminating the difference between convergence and accommodation, one important element of visual discomfort.

To support such an assertion, it is necessary to prove that more than one ray exiting from different positions on the screen, those rays converging at the image, are seen by one eye. The commercial system has only a horizontal parallax, so that a certain degree of vertical astigmatism should be present in direct proportion to the eye's aperture. A similar system employing LEDs instead of projectors was proposed (6)

### **4. Non-diffractive screens for stereo imaging**

Dennis Gabor asserted (1) that he and Semioj Ivanov simultaneously studied the possibilities of achieving a screen which could eliminate the need of special goggles. The original idea was developed almost fifty years before by Gabriel Lippmann (1), who employed thin cylindrical transparent elements assembled side by side. Gabor's description is very complete, but it was Ivanov who succeeded in installing the first (and still the only) 3D auto-stereoscopic cinema (7) in 1945.

Based on two cinema projectors placed side by side, the 3 m x 4 m screen was made of a set of thin vertical cylindrical lenses whose surfaces were as shown in the upper part of Figure

After having performed some tests he thought that, if the depth of the scene could be limited, the observer could see the scene normally when one eye is receiving any view or even when the eyes are at positions when inverted views were received. That is, the

monocular or pseudoscopic image could be seen as an orthoscopic one.

Fig. 3. Elements and vision zones at Ivanov's autoestereopic system (from Ref.1).

certainly possible to perceive the depth in such situations.

**5. Diffractive screens for stereo imaging**

In my test of similar situations employing diffractive screens, I could mention a few people who could tolerate those cases. When there is lateral movement of the camera or some relative movement between objects, some 3D perception can occur to be explained by a sort of Pulfrich effect. For people who are very keen on 3D observation, as Gabor would be, it is

Once holography was discovered and its first practical applications performed, the possibility of employing a hologram not for creating images but for generating vision zones of projected images appeared. The roughness properties that Gabor wanted for the screen to

2, where the light that is focused at a short distance of the back surface is reflected with different angular orientation due to the different transversal position of each projector and in a multiple way because each lenticular element receives the reflected light with a different angle. Figure 3 shows the conical distribution of the elements necessary to converge the whole scene at the observer's positions. It is to be noted that much accuracy is needed to keep the distance corresponding to the right and left eyes positions so that there are limitations to the positions where observers can be, something common to every stereo system, if mainly auto-stereoscopic.

Fig. 2. Surface structure of Ivanov's screen (from Ref.1).

It is reported (1, 7) that Ivanov's theater could receive 180-200 spectators and that some movies were made for it, but it was discontinued because of the fact that the spectator needs to keep his or her head in an almost fixed position.

After Ivanov died Gabor tried to improve the technique by experiencing with mirrored elements with only vertically diffusing properties, eliminating the need for the conical assembly of elements, but he finally concluded that the production of such a screen would be too expensive and abandoned the idea.

2, where the light that is focused at a short distance of the back surface is reflected with different angular orientation due to the different transversal position of each projector and in a multiple way because each lenticular element receives the reflected light with a different angle. Figure 3 shows the conical distribution of the elements necessary to converge the whole scene at the observer's positions. It is to be noted that much accuracy is needed to keep the distance corresponding to the right and left eyes positions so that there are limitations to the positions where observers can be, something common to every stereo

system, if mainly auto-stereoscopic.

Fig. 2. Surface structure of Ivanov's screen (from Ref.1).

to keep his or her head in an almost fixed position.

be too expensive and abandoned the idea.

It is reported (1, 7) that Ivanov's theater could receive 180-200 spectators and that some movies were made for it, but it was discontinued because of the fact that the spectator needs

After Ivanov died Gabor tried to improve the technique by experiencing with mirrored elements with only vertically diffusing properties, eliminating the need for the conical assembly of elements, but he finally concluded that the production of such a screen would After having performed some tests he thought that, if the depth of the scene could be limited, the observer could see the scene normally when one eye is receiving any view or even when the eyes are at positions when inverted views were received. That is, the monocular or pseudoscopic image could be seen as an orthoscopic one.

Fig. 3. Elements and vision zones at Ivanov's autoestereopic system (from Ref.1).

In my test of similar situations employing diffractive screens, I could mention a few people who could tolerate those cases. When there is lateral movement of the camera or some relative movement between objects, some 3D perception can occur to be explained by a sort of Pulfrich effect. For people who are very keen on 3D observation, as Gabor would be, it is certainly possible to perceive the depth in such situations.


*3.2. RSTT against Di*ff*erent Counterbody Materials*

The additives ZDDP are highly optimized to work efficiently on metallic (mostly steel) surfaces as anti-wear agents [12,17–19]. Since in the previous experiments (shown in Section 3.1) balls made of 100Cr6 steel were used as counterbodies, the question remains open, whether the beneficial tribological effect originates from the binding of the additive molecules to the oxidized titanium alloy surface or to the surface of the steel ball. Hence, in a second set of experiments the material of the counterbody was systematically varied in order to use also non-metallic, i.e., oxide- (Al2O3) or nitride-based (Si3N4) ceramics, which feature different chemical compositions and were available in a similar surface quality as the 100Cr6 steel balls.

Figure 5 gathers the results by showing the graphs of the CoF and OM and SEM micrographs of the corresponding wear tracks on polished and fs-laser-processed Ti6Al4V surface after the RSTT in VPX oil + 0.5% RC 3180 against balls made of 100Cr6 steel [left column, (a)], Al2O3 ceramic [middle column, (b)] and Si3N4 ceramic [right column, (c)].

**Figure 5.** Tribological performance of fs-laser-processed Ti6Al4V surfaces after RSTT (normal force 1.0 N, stroke 1 mm, frequency 1 Hz, cycles 1000) in RC 3180-additivated VPX oil (VPX oil + 0.5% RC 3180) against 10 mm balls made of different materials. RSTT against 100Cr6 steel (metallic) counterbody (**a**), Al2O3 (oxide ceramic) counterbody (**b**) and Si3N4 (non-oxide ceramic) counterbody (**c**). The top row provides the coefficient of friction (CoF) as function of the number of sliding cycles. The middle row shows optical micrographs of the corresponding wear tracks after the tribological tests and the bottom row depicts detailed scanning electron microscope (SEM) micrographs taken at the centre of the wear tracks. The capitalized labels indicate tests performed on polished surfaces (**A**) or fs-laser-processed (LSFL-covered) surfaces (**B**). Common scale bars are provided at the left.

For a direct comparison, Figure 5a displays again the results obtained for the 100Cr6 steel balls previously shown in Figure 2 (left column). The CoF's measured in the fs-laser-processed regions (B, blue curves) with both ceramic counterbodies (Al2O3, Si3N4) are as small as those obtained for the metallic steel balls, see Table 3. In all cases, the LSFL endured the RSTT, see the high-resolution SEM images for the associated wear tracks (B) in Figure 5, bottom row. The CoF's recorded in the polished reference surfaces (A, red curves) always start between ~0.4–0.6. However, after less than 200 cycles the CoF's are remarkably lower for the ceramic counterbodies when compared to the metallic steel ball. Nevertheless, the averaged CoF always shows a clear reduction between the polished surfaces and the fs-laser-processed regions, although the reduction ratio is smaller for both ceramic counterbodies. The reduced CoF's on the polished surfaces when using ceramic balls compared to the steel balls are also reflected in the size of the corresponding wear tracks (compare the tracks A in the OM, middle row, Figure 5).

**Table 3.** Mean values and standard deviations of the coefficient of friction (CoF) corresponding to the entire test (RSTT in Ti6Al4V, normal force 1.0 N, stroke 1 mm, frequency 1 Hz, cycles 1000) and resulting wear volumes for different lubricants and tested surfaces [reference (polished) and fs-laser-processed (LSFL-covered)] using different counterbody materials.

The quantitative values of the averaged CoF's along with the estimated wear volumes provided in Table 3 prove that the difference in the counterbody material (chemical composition as well as hardness) and the tribological contact characteristics estimated from an elastic (Hertzian) deformation model [20] (contact pressure, contact radius, sample-ball deformation, see Table 1) are not the dominating effect in our RSTT featuring the beneficial tribological effect. Hence, it is likely that the laser-induced oxidation and the interplay with surface topography are the crucial aspects here. Inferring that a minimum oxygen-containing layer thickness and an enlarged surface area are required together with the additives ZDDP [14] to form a sufficiently thick anti-wear layer during RSTT, which prevents a direct contact of the two sliding tribological bodies. These findings are supported by Figure 6, which visualizes the values gathered in Table 3.

#### **4. Conclusions**

Low spatial frequency LIPSS (LSFL) were uniformly processed by Ti:sapphire fs-laser pulses on Ti6Al4V titanium alloy samples. The tribological performance of the surfaces [reference (polished) vs. fs-laser-processed (LSFL-covered)] was determined in linear reciprocating sliding tribological tests against 100Cr6, Al2O3 and Si3N4 balls as counterbodies in two different lubricants [VPX oil and VPX oil + 0.5% of an anti-wear (ZDDP) additive, RC 3180]. Subsequently, the wear tracks were characterized by OM, SEM, STEM, and confocal profilometry. For the specific testing conditions here, a reduction by a factor of 4–5 for the coefficient of friction and by a factor >2500 for the wear volume was observed in RC 3180-additivated VPX oil, while the tribological tests against 100Cr6 balls showed no beneficial influence of LSFL-covered surfaces with VPX oil lubrication. These new results proved the similar behaviour between VPX oil and paraffin oil (free of any additives) and between RC

3180-additivated VPX oil and commercial VP1 oil containing ZDDP as anti-wear agent. This clearly evidences our previous speculation that only the admixture of the specific ZDDP molecules account for the reproduction of the beneficial tribological behaviour on the laser-processed Ti6Al4V surfaces. Additionally, no significant influence of the counterbody material (metal vs. ceramics) was observed, thereby implying that the positive effect is mainly caused by the presence of the 2-ethylhexyl zinc dithiophosphate molecules in the lubricant along with a nanostructured and oxidized layer on the laser-processed surfaces. The interplay between the sample topography (featuring an enlarged surface area and a confinement of the lubricant) and the local chemistry in the tribological contact area—via formation of a protective surface layer on the LSFL—prevents a direct contact of the two sliding bodies, finally resulting in reduced friction and wear.

**Author Contributions:** Conceptualization, D.S. and J.B.; Methodology, J.B. and D.S.; Validation, all authors; Formal analysis, J.J.A. and N.S.; Investigation, all authors; Resources, J.B., N.S., and D.S.; Data curation, J.J.A. and N.S.; Writing—original draft preparation, J.J.A. and J.B.; Writing—review and editing, all authors; Visualization, J.J.A., N.S., and J.B.; Supervision, D.S., J.B., A.Z., I.L., and A.A.; Project administration, D.S., J.B., A.Z., and I.L.; Funding acquisition, J.J.A., A.Z., J.B. and D.S.

**Funding:** This work was supported by the ERASMUS+ program of the European Union (KA103). This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 814494 (project "i-TRIBOMAT") and grant agreement No. 665337 (project "LiNaBioFluid"). The Surface Technologies research group (Mondragon University, Faculty of Engineering) gratefully acknowledges the financial support given by the Red Guipuzcoana de Ciencia Tecnología e Innovación 2018 program through the project ASEFI (Orden Foral Número 218/2018).

**Acknowledgments:** The authors would like to thank C. Neumann (BAM 6.3) for help with profilometric sample characterizations, S. Benemann (BAM 6.1) for the SEM characterizations, R. Koter (BAM 6.4) for the laser surface processing of a sample copy, and S. Binkowski (BAM 6.3) for polishing the titanium samples. The STEM analysis was performed by I. Dörfel (BAM 5.1). The femtosecond laser processing was performed within the frame of German Science Foundation (Deutsche Forschungsgemeinschaft, DFG) project under Grant KR 3638/1-2.

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


**Abstract**

This chapter will cover the fundamentals of nuclear thermal propulsion systems, covering basic principles of operation and why nuclear is a superior option to chemical rockets for interplanetary travel. It will begin with a historical overview from early efforts in the early 1950s up to current interests, with respect to fuel types, core materials, and ongoing testing efforts. An overview will be provided of reactor types and design elements for reactor concepts or testing systems for nuclear thermal propulsion, followed by a discussion of nuclear thermal design concepts. A section on system design and modeling will be presented to discuss modeling and simulation of driving phenomena: neutronics, materials performance, heat transfer, and structural mechanics, solved in a tightly coupled multiphysics system. Finally, it will show the results of a coupled physics model for a conceptual design with simulation of rapid startup transients needed to maximize hydrogen efficiency.

**Keywords:** neutronics, high temperature, multiphysics, griffin, MOOSE, nuclear thermal propulsion, interplanetary, NASA