**1. Introduction**

The ability of microorganisms to degrade a variety of environmental pollutants and to use them as growth substrates is a powerful tool to clean up hazardous contaminants that are causing irreversible damage to the biosphere. Many hydrocarbons such as most alkanes can be e fficiently degraded by several microorganisms [1] and many aromatic compounds can serve as growth substrates for a large variety of bacteria [2]. On the other hand, cycloalkanes seem to be more persistent to microbial attack [3]. Tetralin (1,2,3,4-tetrahydronaphthalene) is a bicyclic molecule composed of an aromatic and an alicyclic ring. It is naturally found in di fferent crude oil reservoirs and also produced for industrial purposes from naphthalene by catalytic hydrogenation or from anthracene by cracking. It is widely used as a degreasing agent and solvent for fats, resins and waxes, as a substitute for turpentine in paints, lacquers, and shoe polishes, and in the petrochemical industry in connection with coal liquefaction [3]. Like other cyclic hydrocarbons, tetralin is toxic due to its lipophilic character, which facilitates its accumulation within the biological membranes causing changes in their structure and function, and to the formation of hydroperoxides inside the cells [4]. Its hydrophobic character, which reduces tetralin bioavailability, along with the fact that concentrations greater than 100 μM are toxic for microbial cultures might be the reasons why it has been di fficult to isolate pure cultures of microorganisms able to grow on tetralin as the only carbon and energy source [3]. Tetralin utilization by microorganisms has long been reported by mixed cultures or by pure cultures through co-oxidation of tetralin in the presence of mixed substrates [5]. Reports in the literature describing poor growth on tetralin with pure cultures are from *Pseudomonas stutzeri* AS39 [6] and *Corynebacterium* sp. strain C125 [7]. By identifying accumulated intermediates, it was suggested that they might attack the molecule of tetralin in di fferent ways. In one, the alicyclic ring is initially hydroxylated and then oxidized [6] whereas in the other the aromatic ring is dioxygenated and subsequently cleaved in the extradiol position [7]. The degradation pathway has not been further studied in any of these strains. The main source of knowledge about tetralin degradation has been provided *by Sphingopyxis granuli* strain TFA (formerly *Sphingomonas macrogolitabida* strain TFA) where the catabolic pathway has been completely characterized at biochemical, genetic and regulatory levels. A similar degradation pathway has been described in *Rhodococcus* sp. strain TFB based on the induction in tetralin-grown cells of homologous enzymes to the TFA ones. These two di fferent bacteria were isolated from the river Rhine sediments after selection for growth on tetralin. TFA is a Gram-negative α-proteobacterium, whose genome is available and shows characteristics of oligotrophic bacteria, great plasticity and presence of other degradative genes that might confer new capabilities to this bacterium [8]. Moreover, TFA is the first of its genus, generally described as aerobic, in which the ability to grow in the absence of oxygen using nitrate as a terminal electron acceptor has been demonstrated [8]. This capacity might allow TFA to invade other niches. TFB is a Gram-positive Actinobacterium from the *Nocardiaceae* family refractory to genetic manipulation, although a proteomic approach has allowed the identification of all the tetralin gene clusters. In this review, we summarize the genetic organization, the biochemistry of the tetralin biodegradation pathway and its connection with the central metabolism via production of pyruvate and acetil-CoA in both strains. We also highlight the contributions that the study of the tetralin degradation genes regulation in each strain has made to understand the regulation of gene expression of other degradation pathways, and to the knowledge of bacterial mechanisms of gene regulation in general.

#### **2. Identification and Organization of Tetralin Degradation Gene Clusters**

As mentioned above, the tetralin biodegradation pathway has been elucidated only in two bacteria isolated from the same environmental niche, *Sphingophyxis granuli* strain TFA and *Rhodococcus* sp. strain TFB. In this section, we review the di fferent methodology employed to identify *thn* genes and the organization of the *thn* gene clusters in both strains.

#### *2.1. Organization of the Gene Clusters in TFA*

A Tn5 or miniTn5*Km* transposon mutant isolation approach was employed to identify *thn* genes in TFA. A gene bank of TFA was constructed [9] and used to complement transposon insertion mutants. Cosmids containing genes that complemented the growth deficiency on tetralin of transposon mutants were obtained and sequenced ([10] and references therein). As shown in Figure 1, the regulatory and structural *thn* genes consist of 21 genes comprised in a 14.3-kb DNA fragment and organized in four closely linked operons (B, C, H and M). The divergent operons C and B have two internal promoters *PR* and *PH.*

Operon C contains five genes: *thnC*, encoding an extradiol dioxygenase, *thnA3* and *thnA4*, coding for the ferredoxin and the ferredoxin reductase components of the complex catalysing the initial dioxygenase reaction, and the regulatory genes *thnRY*. ThnR belongs to the LysR-type transcriptional regulators (LTTR) family. In most cases, the gene encoding the LTTR is divergently transcribed to the regulated operon [11]. Thus, *thn* genes have an unusual organisation in TFA being that the regulatory genes are part of a structural operon while also being transcribed from the internal and constitutive promoter *PR*. In turn, *thnY* codes for a ferredoxin reductase-like protein, which has a transcriptional regulatory function. This is unusual for an electron transport protein, thus representing another particularity of the tetralin biodegradation genes of TFA (see below).

**Figure 1.** Organization of the chromosomal *thn* genes in TFA. The arrows show the directions of gene transcription. Bent arrows represent the identified promoters. Dashed lines represent internal promoters.

Operon B comprises 12 genes: *thnB*, which encodes a *cis*-dihydrodiol dehydrogenase, *thnD*, coding for the key hydroxylase that renders a linear compound, *thnE,* encoding a hydratase, *thnF,* encoding an aldolase, *thnA1* and *thnA2*, encoding the α- and β-subunits of the initial dioxygenase, respectively, and *thnG*, coding for a non-acylating aldehyde dehydrogenase. Five additional genes (*thnHIJKL*) are comprised within operon B although also transcribed from the internal promoter *P H*. These genes encode proteins similar to those involved in β-oxidation pathways.

Finally, operon M comprises *thnM*, encoding a TonB-dependent protein of unknown function, *thnN*, encoding a putative glutaryl-CoA dehydrogenase and *thnO* and *thnP* which encode proteins similar to the α- and β-subunits of a flavoprotein, respectively [10].

A few years ago, the complete genome sequence of TFA was obtained and analysed [8]. The presence of large plasmids in strains of the sphingomonas group able to degrade di fferent contaminants is very common. In fact, a number of "degradative megaplasmids" have been described in this group of bacteria that contribute to their catabolic flexibility. Some examples are pNL2, pCAR3, pSWIT02, pCHQ1, pISP0, and pISP1, which code for genes involved in the degradation of aromatic hydrocarbons, carbazole, dibenzo-p-dioxin and γ-hexachlorocyclohexane, respectively [12]. In contrast, no plasmids were detected in TFA by experimental approaches, which was later confirmed when the genome was sequenced. The *thn* genes were found within a chromosomal region identified as a putative genomic island and relatively close to a *tra*/*trb* gene cluster. Although more data are needed to be conclusive, those regions could be considered as integrative and conjugative elements (ICEs) [13] which might have played an important role in the final structure of the TFA genome. The analysis performed in 2016 to define the core genome of the *Sphingopyxis* genus showed that all tetralin degradation proteins, except ThnA4, ThnY and ThnM, are part of this core genome [8].

#### *2.2. Organization of the Gene Clusters in TFB*

TFB is a bacterium refractory to genetic manipulation and *thn* gene identification had to be carried out by reverse genetics using a proteomic approach [14]. This method has also been used to identify biodegradation genes in other bacteria of environmental interest [15]. Soluble proteins present in extracts of TFB cells grown on glucose or on tetralin were compared using 2D-DIGE (two-dimensional di fference gel electrophoresis). Di fferentially expressed proteins present in tetralin cell extracts were identified by MALDI-MS(MS) (maldi-assisted laser desorption/ionization tandem mass spectrometry) or ESI-IT MS/MS (electrospray ionization-ion trap tandem mass spectrometry). Eight proteins with probable roles in the conversion of tetralin into a linear compound were identified, ThnD being one of them. Degenerated primers based on the ThnD-obtained peptide sequences were designed to amplify a DNA fragment of the putative *thnD* gene, which was used to screen a TFB cosmid library. A DNA fragment comprising 18 putative *thn* structural genes and two more coding for the putative two-component regulatory system *thnST* were sequenced. *thnS* encodes for a membrane sensor protein and ThnT is similar to members of the LuxR family of regulators. As shown in Figure 2, TFB *thn* structural genes are organized into two operons, *thnA1A2A3UA4B* and *thnA1'A2'CGQILWVDEF*, transcribed from *PA1* and *PA1'* promoters, whereas the regulatory operon is divergently transcribed to the structural ones from the *Ps* promoter.

**Figure 2.** Organization of the chromosomal *thn* genes in TFB. The arrows show the directions of gene transcription. Bent arrows represent the identified promoters.

All genes encoding enzymes are similar to those found in TFA except *thnU*, coding for a putative sterol transfer protein, *thnQ*, encoding a pimeloyl-CoA synthase, *thnW* coding for a 3-hydroxyacyl-CoA dehydrogenase and *thnV* coding for an acyl-CoA dehydrogenase (see Table 1). TFB *thn* genes are highly similar to *bph* and *etb* genes in *R. jostii* sp RHA1, which are located on pRHL1 and pRHL2 plasmids. Similarly, using pulse field electrophoresis analysis, two large plasmids, pTFB1 and pTFB2, with estimated sizes of 1100 and 280 kb, respectively, have been identified in TFB, being the *thn* genes located on pTFB1 [14].

**Table 1.** Homology between TFA and TFB proteins.

TFA proteins were used as query in BlastP analysis with default parameters. \*Identity of ThnLTFB and ThnWTFB to ThnLTFA N and C-terminal part, respectively.

#### **3. Tetralin Degradation Pathway and Enzymes Involved**

In this section, the main features of the tetralin degradation pathway in TFA and TFB are reviewed. Although the degradation pathway is essentially the same in both bacteria (Scheme 1), di fferences have been found in some of the enzymes. The proteins involved in the degradation pathway to convert tetralin into pimelic acid were quite similar in both bacteria except for ThnA4 (Table 1). This similarity ranges from 65% of identity for ThnC proteins to 36% for ThnG enzymes. In all cases, coverage is higher than 90%. The main di fference between TFA and TFB pathways was found in those proteins involved in pimelic acid conversion to central metabolites via a β-oxidation pathway [14,16]. It should be noted that the tetralin degradation pathway has been characterised in detail only in strain TFA. The existence of similar enzymes led to the conclusion that a similar pathway should operate in TFB.

Aerobic biodegradation of tetralin starts by a common mechanism quite well described for degradation of aromatic compounds, which is the insertion of oxygen into the aromatic ring of tetralin by a non-heme iron-containing dioxygenase [17]. These multicomponent ring-hydroxylating enzymes require Fe2+ and an electron supply in addition to molecular oxygen. These enzymes establish an electron-transport chain that allows the transfer of electrons from NAD(P)H, via flavin and [2Fe-2S] redox centers, to the terminal oxygenase containing the catalytic site. Bacterial oxygenases has been classified into classes I, II, and III according to the number of components and the nature of the redox centers of the electron transport chain [18]. Class I consists of two components, the reductase and the oxygenase. The reductase contains a chloroplast type [2Fe-2S] cluster and a flavin, FMN in class IA and FAD in class IB. Class II consists of three-component enzymes but in this case, the flavin and [2Fe-2S] redox centers of the reductase are on a separate flavoprotein and ferredoxin, respectively. Class III consists of three-component enzymes in which the reductase harbours both FAD and a chloroplast type [2Fe-2S] cluster and transfer electrons to an intermediary Rieske type ferredoxin rather than directly to the oxygenase component [18]. The tetralin dioxygenase system belongs to class III, composed of the dioxygenase complex itself, formed by ThnA1 and ThnA2, the α and β subunits of the initial tetralin dioxygenase, respectively, and two electron transfer components, the ferredoxin ThnA3 and the ferredoxin reductase ThnA4. Both ThnA1TFA and ThnA1TFB amino acid sequences contain two Cys and two His residues, (CXHX15–17CX2H), that co-ordinate the Rieske-type [2Fe-2S] cluster and the other catalytic residues that coordinate the mononuclear iron, identified in other crystallized dioxygenases, such as the one from *Pseudomonas* sp. NCIB 9816–4 [19]. ThnA3 is homologous to the Rieske-type ferredoxin components that are electron transfer intermediates between the NAD(P)H-dependent ferredoxin reductases and the dioxygenase components. ThnA4TFA TFA is homologous to the class III NAD(P)H-ferredoxin reductases of degradative pathways. The modular structure of these ferredoxin reductases is composed of a N-terminal domain that contains a conserved Cys-X4-Cys-X2-Cys-X29/30-Cys motif that binds a plant-type [2Fe-2S] cluster, and a central and C-terminal domains with the conserved motifs for flavin and NAD(P)H binding, respectively [20]. However, ThnA4TFB is 81.5% identical to EtbAd from *Rhodococcus jostii* RHA1, a protein with Pyr\_redox\_2 domains which are present in pyridine nucleotide-disulphide oxdoreductases, a family that includes both class I and class II oxidoreductases.

Both ThnA4TFA and ThnA3TFA have been purified, their redox centres spectroscopically confirmed, the specific sequence in the electron transfer reaction from ThnA4red to ThnA3ox has been analysed using stopped-flow spectroscopy, and their midpoint reduction potentials have been determined. These results agree with the electron transfer from NADPH to FAD and then to [2Fe-2S]) in ThnA4, and subsequently to [2Fe-2S] cluster in ThnA3 [21]. Interestingly, this transfer of electrons is important not only for the initial oxidation of tetralin but also for the correct regulation of the *thn* genes expression (see Section 4), since a new electron transfer route from ThnA4 and ThnA3 to the regulatory protein ThnY may control the activity of the regulators of *thn* genes in response to particular substrates. The absence of ThnY in TFB and the di fferent regulatory system for *thn* gene expression in this bacterium might explain the lack of ThnA4 conservation between TFA and TFB. Experimental evidence that ThnA1A2A3A4TFA are directly involved in the initial dioxygenation of tetralin is provided by accumulation of 2HT (2-hydroxy-5,6,7,8-tetrahydronaphthalene), which could be determined by HPLC, in the supernatant of a TFA mutant lacking all *thn* genes supplied with tetralin and overexpressing ThnA1A2A3 [17]. Although ThnA4 is not essential for 2HT production, maximal production of 2HT is achieved only in its presence. According with this, mutants in ThnA1, ThnA2, or ThnA3 are unable to grow on tetralin. ThnA4 is partially dispensable and its function is apparently substituted by other ferredoxin reductases present in TFA, as it has been described for other ring-hydroxylating dioxygenases [18].

The next reaction of the catabolic pathway is catalysed by the *thnB* gene product. ThnB is a *cis*-dihydrodiol dehydrogenase highly homologous to those involved in phenanthrene degradation. A ThnBTFA mutant is unable to grow on tetralin. However, when this mutant is grown in minimal medium containing 8 mM β-hydroxybutyrate (βHB) and tetralin as carbon sources, a condition that allows growth of Thn-mutants while expressing the *thn* genes, a mixture of 1HT (1-hydroxy-5,6,7,8-217 tetrahydronaphthalene) and 2HT (2-hydroxy-5,6,7,8-tetrahydronaphthalene) were detected by high-performance liquid chromatography (HPLC) in the supernatant of the growth culture after acidification. Formation of 1HT and 2HT indicated that the metabolite accumulated in ThnB mutant corresponds to *cis*-dihydrodiol 1,2-dihydroxy-1,2,5,6,7,8-hexahydronaphthalene (1,2-DHT) since dihydrodiols are unstable under acidic conditions and this results in the formation of rearomatized monohydroxylated derivatives [17].

ThnC is the extradiol dioxygenase required for tetralin utilization. Extradiol dioxygenases are key enzymes in the aerobic degradation of many aromatics compounds. They function by cleaving the meta-position of the C–C bond of catechol derivatives, leading to the aromatic ring opening with the concomitant incorporation of two oxygen atoms [22]. ThnCTFA has been purified, and biochemically characterized. The estimated molecular mass of ThnC indicated that it is a decamer [23], which is di fferent from the octameric structures reported for other extradiol dioxygenases closely related to ThnC, such as BphC [24], NahC [25], and EtbC [26]. TFA *thnC* mutants are unable to grow on tetralin and accumulate 1,2-DHT in the supernatant when growing on 8 mM βHB and tetralin [23]. The product of the reaction catalysed by ThnC is 4-(2-oxocyclohexyl)-2-hydroxy-buta-2,4-dienoic acid (OCHBDA), a yellow ring product resulting from the cleavage proximal to the alicyclic ring of 1,2-DHT that exhibits the typical behaviour of a hydroxymuconic semialdehyde. In addition to 1,2-DHT, the range of substrates of ThnC is relatively wide. It showed activity towards 1,2-dihydroxynaphthalene (1,2-DHN), the intermediate in the naphthalene biodegradation pathway, and to a lesser extent, towards 2,3-dihydroxybiphenyl (2,3-DHB), the intermediate in the biphenyl biodegradation pathways, and the methylated catechols, 3-methylcatechol or 4-methylcatechol [23,27]. This broad substrate specificity of ThnC is also shown by other 1,2-dihydroxynaphthalene dioxygenases [25]. ThnC and these enzymes cluster together in group 2 of the I.3.E subfamily of two-domain extradiol dioxygenases, which preferentially cleave bicyclic substrates [23,28]. As shown by a metagenomic survey, dioxygenases of the I.3.E subfamily are the ones with the broadest substrate specificity [29]. Mutational analysis of ThnCTFA showed that it is possible to obtain altered ThnC proteins with modified positions surrounding the substrate-binding site (Q198H, G206M, N213H, A282R and A282G) that improved activity and/or specificity for 1,2-DHT, thus indicating that ThnC activity is not maximized for this substrate.

Hydrolases of *meta*-cleavage pathways are a class of β-ketolases which catalyse the hydrolysis of linear C-C bonds of vinylogous 1,5-diketones formed by the dioxygenative *meta*-ring cleavage of arenes. Most of them belong to the α/β-hydrolase-fold superfamily of enzymes. These enzymes are structurally, functionally and mechanistically related and generally utilize a nucleophilic residue (a serine in most cases)-acidic residue-histidine catalytic triad to hydrolyse the C-C bond adjacent to a carbonyl [30]. The serine hydrolase ThnD from TFA has been purified and biochemically characterized [31]. ThnDTFA is an octameric enzyme with an optimum reaction temperature at 65 ◦C. Moreover, it shows high stability at room temperature and heat treatment at 70 ◦C for 2 h resulted in 78% of the initial activity. ThnDTFB has also been heterologously expressed in *Escherichia coli* and purified, and its optimal temperature is 70 ◦C [14]. This stability at high temperature is a biotechnological interesting property given the use of this class of enzymes in organic synthesis [32]. ThnDTFA and ThnDTFB catalyse the hydrolysis of OCHBDA rendering the dienol 2-hydroxydec-2,4-diene-1,10-dioicacid as the reaction product. In fact, a TFA insertion mutant defective in ThnD accumulates OCHBDA [31]. It has been suggested that ThnD could be involved in biphenyl meta-cleavage pathways since it is highly similar to BphD enzymes and, in addition to OCHBDA, ThnD also e fficiently hydrolyses 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), the ring fission product of the biphenyl meta-cleavage pathway. In fact, the *Km* for OCHBDA and for 6-phenyl-HODA are 26.3 mM and 34.8 mM, respectively [31]. ThnDTFB hydrolase is the most similar to BphD hydrolases and even more active towards 6-phenyl-HODA than towards OCHBDA, which showed just 40% of the activity obtained with 6-phenyl-HODA [14]. ThnDTFA does not hydrolyse 6-methyl-HODA, aldehydes HODA or 5-methyl-HODA, ring fission products of 3-methylcatechol, catechol and 4-methylcatechol, equivalent intermediates of *meta*-cleavage pathways of mono-aromatic compounds. These compounds have in common the small substituents in C-6. Interestingly, since the bond of the vinylogous β-diketone connecting C-5 and C-6 in OCHBDA is within the alicyclic ring of tetralin derivative, hydrolysis of this bond by ThnD renders a single linear dicarboxylate product instead of the two products usually generated by these hydrolases (Scheme 1). Concomitantly, this hydrolysis breaks open the recalcitrant alicyclic ring, thus rendering a more easily catabolised product. This confers unique properties to the tetralin degradation pathway, since ThnD makes unnecessary the recruitment of enzymatic activities involved in degradation of alicyclic rings for tetralin complete mineralization.

Both ThnE and ThnF showed similarities with the corresponding hydratases and aldolases involved in the catabolic degradation of homo-protocatechuate, and their substrates and products of the reactions they catalyse have been identified [33]. GC-MS analysis of the reaction products using OCHBDA as a substrate and crude extracts of an *E. coli* strain overproducing ThnD and ThnE, identified a C10 dicarboxylic compound with two hydroxyl groups, the enol tautomer 2,4-dihydroxydec-2-ene-1,10-dioic acid (DHDDA) as the major product resulting from the hydration reaction [33]. Although it has been suggested that both the dienol and the keto tautomers of the ThnD product could be substrates of ThnE, in TFA the enol form is the most probable since when purified hydrolase ThnDTFA was used to transform OCHBDA, only the dienol tautomer reaction product was detected [31].

ThnF is a class II aldolase encoded by the *thnF* gene, whose start codon in TFA is just six nucleotides downstream of the stop codon of *thnE* gene [33]. A similar situation is found in TFB where *thnF* start codon is within the *thnE* coding sequence [14]. This indicates that translational coupling is highly probable for these genes. Aldolases are an evolutionarily diverse class of enzymes that are typically divided into two groups based on their catalytic mechanism. The Class I aldolases utilize a Schi ff base mechanism to stabilize a carbanion intermediate, whereas the Class II enzymes utilize divalent metal ions to stabilize an enolate intermediate. Within each mechanistic class of aldolases, further diversity exists with respect to the sequence, structure, and substrate specificities. Pyruvate-specific enzymes are within the Class II containing an octahedral coordinated divalent metal cofactor, which is usually Mg2+, Mn2+ or Co2+ [34]. Transformation of OCHBDA by *E. coli* extracts of enriched in ThnD, ThnE, and ThnF enzymes from TFA resulted in accumulation of two compounds as a result of the cleavage reaction, pimelic semialdehyde, identified by GC-MS, and pyruvate, which was spectrophotometrically estimated [33].

For complete oxidation, pimelic semialdehyde needs to be further metabolized into compounds that can enter the Krebs cycle. A first oxidation of pimelic semialdehyde leading to the synthesis of pimelic acid is enzymatically carried out by ThnG, which is similar to the non-acylating NAD-dependent aldehyde dehydrogenases involved in the biodegradation pathways of aromatic compounds. Aldehyde dehydrogenases (ALDHs) catalyse the oxidation of aldehydes to their corresponding carboxylic acids and all of them require either NAD or NADP as a cofactor [35]. The catalytic activity of ThnG has been assayed in crude extract of TFA and in a ThnGTFA *E. coli* overproducing strain [10]. ThnG shows ALDH activity depending on pimelic semialdehyde and NAD as an oxidant but not with NADP, and its activity does not show coenzyme A or dithiothreitol dependence, supporting the non-acylating ALDH activity of ThnG. Indeed, the primary structure of ThnGTFA contains the four invariant residues

(Gly228, Gly281, Glu381, and Phe383) found in this type of dehydrogenases, which are critical for binding of the nicotinamide ring of NAD and catalytic activity. These residues are also present in ThnGTFB in similar locations. The participation of ThnG in the catabolism of tetralin is clear since ThnG activity and *thnG* gene expression as part of the B operon is induced in TFA cells growing on tetralin. However, as it happens in *thnA4* mutants, TFA *thnG* mutants are able to grow on tetralin. Therefore, it seems that this role can be assumed by other aldehyde dehydrogenases present in TFA that could also use pimelic semialdehyde as a substrate [10]. Since ThnG is not an acylating enzyme, the formation of pimeloyl-CoA to enter a β-oxidation pathway needs an intermediate step consisting in the activation of the pimelic acid molecule.

Based on the homology of the *thnHIJKL* gene product with enzymes that participate in β-oxidation pathways, a complete β-oxidation pathway for pimelic acid has been described in TFA. The steps involved are: initially, ThnH, a protein similar to enzymes that belong to the "CaiB/BaiE" family of CoA transferases that catalyse the reversible transfer of CoA groups from CoA thioesters to free acids, would activate pimelic acid yielding pimeloyl-CoA. In TFB, the same step seems to be catalysed by ThnQ, a protein similar to pimeloyl-CoA synthases that catalyses the binding of a CoA-SH molecule to pimelic acid. In TFA, the resulting pimeloyl-CoA molecule would be the substrate of ThnJK, which encodes proteins similar to the large and small subunits of a pimeloyl-CoA dehydrogenase, respectively. This enzyme might oxidise pimeloyl-CoA to pimeloyl-2,3-dehydroacyl-CoA acid. In TFB, ThnV could be the acyl-CoA dehydrogenase responsible of catalysing this step. In TFA, a bifunctional enzyme (ThnL) carrying the enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase domains located at its N and C termini, respectively, could convert the pimeloyl-2,3-dehydroacyl-CoA acid into pimeloyl-3-ketoacyl-CoA in two steps. In TFB, ThnL would render pimeloyl-2,3-hydroxyacyl-CoA, which would be oxidised to the ketoacyl-CoA by ThnW. Finally, in both bacteria, ThnI, encoding an acetyl-CoA acetyl transferase, would cleave pimeloyl-3-ketoacyl-CoA into the central metabolites acetyl-CoA and glutaryl-CoA. In TFA, ThnN, encoding a putative glutaryl-CoA dehydrogenase, could catalyse the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, which could be further converted into acetyl-CoA. Moreover, two extra proteins, ThnO and ThnP encode proteins similar respectively to the α- and β-subunits of flavoproteins, which serve as specific electron acceptors for primary dehydrogenases of the β-oxidation pathway and transfer electrons to the respiratory chain. Proteins similar to ThnN, ThnO and ThnP have not been found associated to *thn* genes in TFB. Mutations in *thnH*, *thnI*, *thnJ* or*thnK*, did not a ffect the growth of TFA on tetralin, or on sebacic or pimelic acids [10]. Thus, redundant β-oxidation pathways should be present in TFA cells. Actually, there are 39 proteins annotated in TFA genome within the GO terms GO:0006635 (fatty acid beta-oxidation) and GO:009062 (fatty acid catabolic process) [8].

Taking all the steps together, degradation of one molecule of tetralin by this pathway renders one molecule of pyruvate, one of acetyl-CoA and one of glutaryl-CoA (Scheme 1). These molecules can be used either as energy or carbon source. The use of the same degradation strategy by two non-phylogenetically related bacteria and di fferent gene arrangements could mean that it is the most efficient pathway to obtain carbon and energy source from tetralin.

Interestingly, *thn* genes are also induced by naphthalene in both TFA and TFB [36,37]. However, and despite the ThnC activity towards 1,2 DHN, TFA is unable to grow on naphthalene while TFB can use it as the only carbon and energy source [37]. In fact, Thn proteins are the most prominent in extracts of naphthalene-grown TFB cells. Because of that, it has been proposed that ThnA1A2A3A4, ThnB and ThnC proteins also participate in the naphthalene degradation pathway in TFB and that the TFB naphthalene degradation ability actually results from the combination of di fferent catabolic pathways [37].

**Scheme 1.** Tetralin catabolic pathway.  Proteins in brown correspond to those identified in TFB. Red lines show the cleavage of the rings.

#### **4. Regulation of the** *thn* **Gene Expression**

In this section we review the regulatory elements and their function controlling the expression of the tetralin degradation genes in TFA and TFB. In both strains, tetralin degradation genes are subjected to a specific induction by tetralin but also to an over-imposed global control, which dictates the use of other preferential carbon sources when present. The regulatory mechanisms behind these responses are summarized.

#### *4.1. Specific Regulation: Elements Involved in Tetralin Induction*

#### 4.1.1. Tetralin Induction in TFA

When TFA cells are growing on tetralin as the sole source of carbon and energy, the expression of the *thn* genes from *PC* and *PB* promoters is induced more than 100-fold compared to when growing on the preferential carbon source βHB [38]. In many degradative routes, induction of the pathway requires an intermediate in the catabolism of the substrate [39–41]. However, induction of *thn* genes is exerted directly in response to tetralin, the substrate of the catabolic pathway, since some mutants unable to transform it are still able to activate *thn* gene transcription [36]. Three regulatory proteins achieve this specific response to tetralin. ThnR, a LysR-type regulator involved in the recognition of potential effector molecules, ThnY, the ferredoxin like reductase that has evolved to function as a ThnR co-activator, both essential for *thn* gene expression, and ThnA3, the ferredoxin that delivers electrons to the tetralin dioxygenase and that prevents promiscuous induction by molecules that are not substrates of the catabolic pathway [38]. Details of each regulatory element are described below.

ThnR has exclusive properties. Firstly, unlike other LysR-type regulators, it does not repress its own synthesis, but activates its own expression in a positive circuit of regulation, by being part of the same transcriptional unit as the catabolic genes *thnCA3A4* located upstream (Figure 1) [38]. It has been proposed that these positive auto-regulation circuits may allow a faster switch on/off of the regulatory systems in response to the inducer molecules, for example, in environments where the amount of substrate is limited [42]. This might be the case for tetralin considering its tendency to accumulate into biological membranes [4]. Secondly, ThnR needs the regulatory protein ThnY for transcription activation [17]. In this way, ThnR is one of the few examples of LysR-type activator that requires an additional protein to activate transcription [43]. Additionally, ThnY allows the system to integrate other signals, such us the presence of potential inducers that, being similar to tetralin, are not substrates of the pathway and thus preventing a wasteful production of useless catabolic enzymes (see ThnA3 modulation below).

The molecular mechanism responsible for *thn* gene induction in the presence of tetralin has been characterized. According to it, basal levels of ThnR and ThnY are provided by the weak constitutive promoter *PR*, which in turn are responsible for the activation of the four promoters, *PB*, *PC*, *PH* and *PM*, when tetralin is present [10]. Footprinting analyses have shown that ThnR binds to contiguous high affinity and low affinity sites in each promoter region [16]. A meticulous analysis of ThnR binding to the *thnB*–*thnC* intergenic region, (*PB*–*PC* promoters), led to a detailed molecular characterization of the activation mechanism mediated by ThnR and the importance that both primary and secondary binding sites have on transcription activation from each promoter. These results showed that mutations in the primary binding sites of each promoter can affect not only the transcriptional activation of its respective promoter but transcription from the other one, and vice versa. This transcriptional coordination is mediated by a complex structure of ThnR molecules that interact among them when bound to their respective sites in both promoters via a DNA loop, which is detected as a higher order structure in electrophoretic mobility shift assays (EMSA) [10]. For this DNA loop to be formed, primary binding sites of each promoter have to be aligned in the same face of the helix, since insertions of 4 and 6 bp between them prevented the formation of the complex structure and cooperative transcription activation. Furthermore, the DNA loop functions by compensating non-productive ThnR-DNA interactions at an activating site [16,44].

Similarly to ThnR, *PR* also provides basal levels of ThnY, which are increased from the inducible *PC* promoter in a ThnR-dependent manner when tetralin is present [45]. As mentioned above, ThnY is essential for expression of each of the four *thn* promoters controlled by ThnR. ThnY is homologous to the Class III of ferredoxin reductases of degradation of aromatic compounds [18], such as benzoate 1,2-dioxygenase reductase [46], naphthalene dioxygenase reductase [47] and carbazole dioxygenase reductase [48]. These ferredoxin reductases function by accepting electrons from NAD(P)H and transferring them through the FAD and [2Fe-2S] cluster, to an intermediary Rieske-type ferredoxin, which finally transfer the electrons to the dioxygenase. Purified ThnY contains a non-covalently attached FAD and one plant-type [2Fe-2S] cluster characteristic of this class of ferredoxin reductases [45]. Regarding these features, ThnY is similar to the ferredoxin reductase ThnA4 of the tetralin dioxygenase [21]. However, despite having these redox centers, ThnY does not behave like a NAD(P)H-dependent ferredoxin reductase, since neither can be reduced directly by NAD(P)H nor does it have the enzymatic activities of these enzymes, such as diaphorase and/or cytochrome *c* reductase activity. Actually, ThnY lacks a proper NAD(P)H binding in its C-terminal domain [45], such as the conserved glycine signature (GGXGXXP) proposed to be involved in the binding of the adenosine-5-phosphate groups of NADH or 2´-phospho-AMP of NADPH [20]. Knowing why ThnY is necessary for ThnR-mediated transcription activation has been one of the most interesting findings on *thn* genes regulation. Footprint and DNA binding assays revealed that when oxidised, ThnY does not alter the footprint pattern generated by ThnR but it does increase the affinity of ThnR for the *PC* promoter region and stabilize the structure of the protein-DNA complex, as a new defined complex is detected in its presence in EMSA assays. It has been proposed that ThnY co-activates transcription by interacting with ThnR [45]. This regulatory function of ThnY is of remarkable interest since it is the first example of a ferredoxin like reductase of the dioxygenases systems that works as an additional sensor regulating gene expression [45,49].

The role of ThnY as a new redox sensor linking cellular metabolism to gene expression has also come from the experiments carried out with the ferredoxin ThnA3 [16,21,50]. As mentioned above in Section 3, ThnA3 is a Rieske-type ferredoxin that functions by transferring electrons to the tetralin dioxygenase, but in addition to this catalytic function, ThnA3 has a regulatory role as a discriminator. There are several lines of evidences that show that ThnA3 reports to ThnY whether a molecule is a good

or a bad substrate of the tetralin dioxygenase through its redox state and that this communication is established by modifying the redox state of ThnY. Firstly, ThnA3 has a discriminatory role since in *thnA3* mutants, when only ThnR and ThnY are present, the *thn* promoters are activated promiscuously, producing high levels of wasteful expression with e ffector molecules that are not substrates of the first step of the tetralin catabolic pathway, such as *cis*-decalin, cyclohexane, *trans*-decalin, or benzene [16]. This phenomenon of gratuitous induction has also been described in other degradative pathways [51]. Secondly, reduced ThnA3 has a negative e ffect on *thn* expression since mutants strains lacking either the α (ThnA1) or β (ThnA2) subunits of the dioxygenase, a condition resulting in an accumulation of the reduced form of ThnA3, failed to induce properly the expression of *thn* genes even in the presence of the substrate tetralin [16]. Under this physiological condition, reduced ThnA3 is fully available to interact with ThnY. Thirdly, ThnA3 is the electron partner of ThnY. ThnY variants, ThnY-C40S and ThnY-N201G,S206P, a ffected in the iron sulfur cluster and in the degenerated NAD(P)H binding domain, respectively, render ThnA3 useless for signal transduction as the discrimination capacity is completely lost and the expression of *thn* genes occurs at high levels even in the presence of molecules other than tetralin. Thus, the discriminatory function of ThnA3 is exerted through the redox protein ThnY [50]. By using stopped-flow spectrophotometry and determination of midpoint reduction potentials of each redox cofactor within ThnA4, ThnA3 and ThnY, it has been possible to show that ThnY can actually be reduced by ThnA3, according to the following ordered steps during the electron transfer reactions [21]:

#### NADH → ThnA4FAD → ThnA4[2Fe-2S] → ThnA3[2Fe-2S] → ThnYFAD → ThnY[2Fe-2S].

Purified ThnA4 behaves as a typical ferredoxin reductase of dioxygenase systems, able to be reduced preferentially by NADH, able to reduce the ferredoxin ThnA3, and with midpoint potentials for *E*ThnA4FADox/hq and *E*ThnA4SFeox/red of −200 mV to −150 mV ranges respectively [21], similar to other reported NAD(P)H ferredoxin reductases of the dioxygenase systems [52]. Similarly, ThnA3 behaves like the one electron carrier Rieske-type [2Fe-2S] ferredoxin although with an *E*ThnA3SFeox/red (−112 ± 5 mV) [21] less negative than reported ferredoxin [53]. *E*ThnYFADox/hq and *E*ThnYSFeox/redwith with values of −131 ± 8 mV and −136 ± 8 mV, respectively, are less negative too than those of other ferredoxin reductases counterparts and slightly more negative than that of ThnA3. However, ThnY can only be reduced by ThnA3red but not by NAD(P)H. The redox potentials also highlight that in order to reduce ThnY, the reduced form of ThnA3 has to be predominant, thus allowing the equilibrium of the redox reaction to change toward ThnY [21]. Figure 3 shows the regulatory model for *thn* genes expression which implies two electron transport routes: (i) NAD(P)H-ThnA4–ThnA3–ThnA1/ThnA2 that occurs in the presence of tetralin, in which tetralin dioxygenase acts as an electron sink through the dioxygenation reaction. Reduction of ThnY by ThnA3 is minimal thus allowing ThnR and ThnYox to activate the *thn* promoters and (ii) the regulatory NAD(P)H–ThnA4–ThnA3–ThnY that occurs in the presence of non-e fficient substrates of the dioxygenase, thus leaving an abundant ThnA3red form fully available to interact with ThnY and to impair *thn* expression. This represents an unprecedented regulatory system in which components of an electron transfer system of a catabolic pathway prevent its gratuitous induction by molecules that cannot be e ffectively metabolized.

**Figure 3.** Model for specific regulation and modulation of *thn* genes transcription in TFA. (**a**) In the presence of the tetralin, electrons are preferentially delivered to the dioxygenase and ThnYox and ThnR can activate transcription from the inducible *thn* promoters. (**b**) In the presence of compounds that are non-substrates of the dioxygenase, ThnA3 is accumulated in the reduced state and electrons are redirected towards ThnY instead of the dioxygenase impairing *thn* genes transcription.

#### 4.1.2. Tetralin Induction in TFB

Semiquantitative RT-qPCR analysis of *thn* genes expression in TFB showed the induction of structural and regulatory genes in tetralin-grown cells in three operons (Figure 2). In glucose- or glucose plus tetralin-grown cells, regulatory genes were expressed at low level while transcription of structural genes was undetected in glucose-grown cells and partially repressed in glucose plus tetralin-grown cells [14]. Primer extension experiments allowed to establish the transcription start points for *thnS* and *thnA1* (which is the same as *thnA1'*) and the putative sequences of the divergent promoters [54]. An 18 bp sequence, similar to those present in biphenyl-inducible promoters, was found in *thnA1* and *thnA1'* promoters. Point mutations in this sequence negatively a ffected tetralin induction of a *thnA1::gfp* translational fusion indicating that those nucleotides could be recognised by a transcriptional activator. Moreover, introduction of extra copies of *thnST* into TFB resulted in higher expression of *thnA1::gfp* in all conditions. Taken together, those results suggested that the membrane sensor ThnS recognises a signal in the presence of tetralin, and that, in response, the regulator ThnT binds the promoters to induce *thn* gene expression in TFB [54].

#### *4.2. Carbon Catabolite Repression*

One of the major drawbacks of microbial degradation of pollutant compounds is the repression of the catabolic genes in the environment due to the presence of preferential carbon and energy sources. However, from an energetic point of view, this phenomenon can be regarded as a control system, which prevents expression of biodegradation capabilities when they are dispensable, thus improving the bacterial adaptation to its nutritional and energetic needs. This regulatory phenomenon is known as carbon catabolite repression (CCR) and can be exerted in di fferent ways. Regulation of gene expression at both transcriptional and translational levels has been described in di fferent biodegradation pathways [55]. CCR of *thn* genes has been demonstrated in TFA and TFB [14,38]. However, it is exerted by di fferent carbon sources, which are metabolized via distinct central pathways, and through di fferent mechanisms.

#### 4.2.1. Carbon Catabolite Repression in TFA

In TFA, the presence of βHB or fatty acids, such as sebacic acid, in the medium containing tetralin prevents the induction of *thn* genes [38]. Regarding the regulatory mechanisms, despite all the efforts invested in its hunt, like transposon mutagenesis or a ffinity binding assays, no transcriptional regulators responsible of CCR have been found in TFA. However, a clear connection between CCR and the energetic status of the cell has been demonstrated in this bacterium (Figure 4). Transposon mutants a ffecting in *phaC* gene, coding for a poly(3-hydroxybutyrate) synthase, are unable to synthetize the carbon storage PHB (polyhydroxybutyrate) granule and show a partial release of *thn* genes CCR [56]. Conversely to what happens in the wild-type, *phaC* mutants prematurely induce *thn* genes in the presence of βHB, although maximal induction is never reached. Homo- and heterologous complementation restores the wild type phenotype indicating that the amount of PHB granule is an internal signal of the energetic status of the cell that prevents *thn* genes expression in CCR conditions. However, CCR in TFA does not only rely on PHB synthesis.

Recently, a new element involved in CCR of TFA *thn* genes has been identified. A trans-encoded small non-coding RNA (sRNA), 61-nt long, named SuhB, and highly conserved in *Sphingopyxis* genus, was annotated in TFA genome and experimentally identified by dRNA-seq and Northern blot experiments [8,57]. SuhB deletion mutants exhibit *thn* gene de-repression in CCR conditions [57]. sRNAs are 50–250 bp long transcripts acting as bacterial riboregulators of mRNAs encoded elsewhere in the genome. The presence of hundreds of these molecules has been demonstrated in a range of bacteria post-transcriptionally controlling the expression of genes involved in a wide range of important processes, including cell division, responses to abiotic stimuli, quorum sensing and virulence [58,59]. sRNAs a ffect translation and/or stability of their mRNA targets and most of them are Hfq-dependent. Hfq is a RNA-binding protein that facilitates the binding of the sRNA to its target and/or protects it from degradation [60,61]. In TFA growing under high growth rate conditions, SuhB binds the 5' UTR of *thnR* mRNA, thus negatively a ffecting its translation in an Hfq-dependent manner. As result, *thn* genes are silenced even if tetralin is present in the medium. The lack of SuhB or Hfq provokes a premature induction of *thn* genes in CCR conditions. However, only partial induction, even lower than that reached in *phaC* mutants, is achieved [56,57]. It has been demonstrated that *suhB* expression is regulated by the growth rate, being elevated at high growth rates, which would imply a high-energy status of the cells. In a *phaC* mutant, the absence of PHB granule could be detected as a low-energy status, *suhB* will not be transcribed and *thn* genes would be induced in the presence of tetralin.

The high conservation of SuhB in other *Sphingopyxis* strains that do not bear *thn* genes suggests that this sRNA must be controlling other targets in these bacteria and that its involvement in the regulation of the tetralin degradation genes should have appeared more recently. This is another interesting example of how existing bacterial posttranscriptional regulatory mechanisms can be incorporated into the regulatory system of probably horizontally acquired metabolic pathways.

**Figure 4.** Elements involved in carbon catabolite repression in TFA. High growth rate conditions like growth on a preferential carbon source like βHB and the presence of PHB granules have a positive effect on *suhB* transcription. This sRNA, together with Hfq, a ffects negatively ThnR translation in those conditions impeding induction of *thn* promoters (*PC*, *PB*, *PH* and *PM*) in the presence of tetralin. The presence of PHB granules themselves have a negative e ffect on *thn* induction. Doted and solid lines represent unknown and known mechanism, respectively.

#### 4.2.2. Carbon Catabolite Repression in TFB

CCR in TFB seems to be exerted in a more canonical way (Figure 5). In the presence of glucose, a FNR/CRP-like protein binds to the *thnS-thnA1* intergenic region [54]. A palindromic nucleotide sequence (CTGTGT-N6-TCACAG) similar to the *E. coli* CRP binding site was found overlapping the -10 and +1 sequences of *thnA1.* Thus, the binding of the FNR/CRP-like protein would prevent e ffective transcription of *thn* genes. In fact, point mutants in this sequence prevents binding of the FNR/CRP-like protein and provokes a partial relief of CCR of a *thnA1::gfp* translational fusion in TFB [54]. Partial de-repression could be due to the presence of a similar sequence at the *thnS* transcription start point to which the FNR/CRP could also bind, thus preventing *thnST* transcription. In fact, *thnS* expression, measured by RT-qPCR, is 5-fold higher in tetralin-grown cells than in glucose or glucose plus tetralin grown cells. The CCR regulatory protein of strain TFB would then act as a transcriptional repressor of *thn* genes in CCR conditions.

**Figure 5.** Elements involved in carbon catabolite repression in TFB. In the absence of glucose and presence of tetralin, phosphorylated ThnS transfer the P group to ThnT which binds the ThnT box and induces *thnA1* transcription. In the presence of glucose, a CRP-like protein binds CRP-like binding sites present at *thnS* and *thnA1* promoters provoking a reduction in *thnS* transcription and impeding *thnA1* transcriptional activation by phosphorylated ThnT.

## **5. Concluding Remarks**

The organic solvent tetralin is composed of an aromatic and an alicyclic ring, none of which is easily biodegraded in nature. The biodegradation pathways described to degrade alicyclic and aromatic rings are completely different and use different enzymatic activities. However, a similar strategy for tetralin biodegradation has evolved in unrelated bacteria by which a single enzymatic team, typically involved in biodegradation of one aromatic ring, is able to break open both rings of the tetralin in two subsequent steps, thus rendering products easily catabolised by many bacteria. Apparently, the *Rhodococcus* strain also uses part of this enzymatic team to degrade naphthalene, thus representing an interesting way of reutilising bacterial enzymatic activities for different purposes. Although the biodegradation strategy and the enzymatic activities are the same, elements regulating the expression of the *thn* genes are completely different in both strains, thus supporting the view that catabolic pathways and their regulatory systems evolve independently. Particularly worth highlighting is the exquisite and sophisticated *thn* gene regulation observed in strain TFA, in which three different regulatory circuits act together at transcriptional and posttranscriptional levels to optimise the gene expression levels to the environmental conditions. One of the circuits involves transcription activation of different promoters in a non-canonical way for a LysR-type regulator, which implies cooperative transcription from divergent promoters. The other circuit affecting transcription constitutes an unprecedented regulatory system aimed to prevent gratuitous induction by related molecules that are not substrate of the catabolic pathway. Finally, the third system uses a less abundant type of regulators, an sRNA, to control *thn* genes' expression. Sophistication of these regulatory systems suggests that this biodegradation pathway has not been assembled recently as a response to human activity but bacteria might have been exposed to tetralin a long time ago, possibly as a component in the oil reservoirs.

The work presented here shows the usefulness of using a combination of different methodological approaches such as mutagenesis, mutant isolation, gene cloning, functional complementation, biochemical characterization and purification of enzymes, high throughput proteome analysis and chemical identification of metabolites to discover and characterize new mechanisms of microbial degradation. The knowledge gained substantially contributes to improving our understanding of important processes such as new metabolic capabilities and their regulatory mechanisms that control integrated physiological responses in bacteria other than the main models.

**Author Contributions:** Conceptualization, F.R.-R.; Original draft preparation, B.F. and F.R.-R.; Writing—review B.F., E.S. and F.R.-R.

**Funding:** This research was funded by Ministerio de Ciencia, Innovación y Universidades, grant numbers BIO2011-24003 and BIO2014-57545-R, and The APC (Article Processing Charge) was waived by MDPI.

**Acknowledgments:** We wish to thank Guadalupe Martín and Nuria Pérez for their technical support and all members of the laboratory for their insights and suggestions.

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


There are and have been many equipment for Voice Range Profles, e.g., Wevosys and Xion. We controlled the frequency and intensity of our phonetograph systematically with equipment from Brüel & Kjaer. The apparatus gave a tone with a sound that was constructed to give a tone resembling a tone from a piano and was made so that it could only measure a response variation of Hz in that semitone region—not in tones nearby. We developed software to draw lines between the measured standard tones of C-E-G-A-C and included the lowest and highest single-end tone. Average Voice Range Profles and semitone ranges could then be made. Standard deviations could be made for the lowest and highest tone, and the software in the equipment calculated the mean and standard deviations hereof. The mean yearly Voice Range Profles and ranges can be used for comparison with other parameters (Figs. 4.24 and 4.23).

The Voice Range Profles usable in singing were given on the abscissa in some of the fgures (Figs. 4.20 and 4.21). These measures are much more variable than the physiological ones, being dependent on many phenomena (talent, training, category, country). Singing category analyses and calculations were made separately, and no signifcant differences were found before nor after the pubertal pediatric/hormonal changes (Figs. 4.43 and 4.44). Also, here average Voice Range Profles and ranges were given as well as standard deviations for the lowest and highest tones.

The Voice Range Profles were standardized in the material of the girls and boys in the presented stratifed study. This means that a detailed statistical comparison with other pubertal parameters in the same normal test persons could be made. The total semitone range, the lowest tone, and the average Voice Range Profle area were used. The total semitone range and the lowest tone were analyzed based on the traditional chromatic tone scale of 12 semitones. The lowest tone could also be given in Hz. As for the calculation of the area—the engineer of the phonetograph wanted to use the diatonic scale of 7 semitones (C-D-E-F-G-H). Conversion to chromatic scale can be made. In this study, this was not considered needed since the main purpose of the measure was to use the results to describe how voice changes are related to other parameters of puberty.

Voice Range Profles in the eighth school year, where the boys mostly get pubertal voice changes, were made of three boys whom all had child's voice ranges at the beginning of the school year. Boy number three had a totally changed Voice Range Profle in December, and in boys one and two, a total change happened at the frst and last measure, respectively. In this period, no statistically specifed relationship to serum testosterone could be found (Figs. 4.24 and 4.25). The point of the old versus the new register change as presented in the fgures should be noted.

Before the use of Voice Range Profles as a method for simultaneous registration of the tonal and dynamic range of voice, the development of the voice was mostly described by the F0 and total tonal range. Already at an early stage in the history of phoniatrics, investigations of the tonal range for normal school children were carried out [7]. A summary of the results of research into children's voices was proposed at the Conference of Logopedics and Phoniatrics in 1936 and subsequently performed by Weiss [8]. This summary covers a period of 4.000 years and shows that people concerned themselves almost exclusively with boys' and eunuchs' voices. The average age for the change of voice was 14.5 years; the fundamental frequency in continuous speech (F0) for boys dropped by about an octave and for girls by about 1/3 octave. Frank and Sparber and Wendler et al. arrived at comparable results [9, 10]. Blatt discussed the topic of voice training during puberty [11].

Komiyama et al. performed an analysis of Voice Range Profles during puberty [12]. They did not, however, make any comparisons with other pubertal phenomena and fxed the lower measurement limit for intensity at 60 dB(A). In our investigations, the intensity of the voice during soft singing was signifcantly lower than 60 dB(A), and thus the measurements are not comparable.

Meuser and Nieschlag showed that the type and category of voice for adult men (tenor, baritone, bass) are related to the serum testosterone level [13]. Large and Iwata found differences between the formants, which depended on the voice type of adults [14]. We also believe that a distinction between the types of voice must be made if an exact artistic appraisal of the development of the voice during the time of puberty is to be achieved. We did not fnd hormonal related voice categories in childhood in this study. This aspect could in the future possibly also be considered in investigations of the pathology of the voice. Pedersen et al. made a followup on voice disorders [15].

Klingholz et al. carried out Voice Range Profles on members of the Tölzer boys choir; in addition, Konzelmann et al. investigated the Voice Range Profles of choirboys [16, 17]. A summary of the literature can be found in the thesis of Bühring [18]. Behrendt followed the development of the falsetto register of the boys of the Thomanerchor school until adulthood but did not relate the phenomena to other parameters [19]. Hacki used the shouting voice measurements in Voice Range Profles and electroglottography for studying dysfunctions [20, 21]. As referred to in chapter 2, tone ranges were measured in a big German population study during childhood and adolescence in 2021 (Figs. 11 and 12) [22]. It was concluded that two octaves (24 semitones) were the average during childhood and adolescence.

Details of the development of Voice Range Profles have now been presented in a stratifed randomized study and statistically compared with the fundamental frequency in running speech (mean F0), as dependent on pediatric and hormonal development in puberty, in one (the same) population.

#### **5.3 The Speaking Voice**

The development of voice in childhood and adolescence is depending on several parameters in a connection, where the mean fundamental frequency (F0) is one out of at least the given ones: the lowest measurable semitone, the semitone range in continuous speech, the total semitone range, and the Voice Range Profle. Results have been presented of the voice parameters combined with the pediatric and hormonal parameters that are usable in further studies on voice development also in pathology (e.g., Figs. 4.26, 4.27, 4.30, 4.31, 4.32, 4.33 and 4.34). A detailed overview of the relations between mean F0, tonal semitone range during speech, total semitone range, and height is given in boys in Fig. 4.28a. In Fig. 4.28b, the relationship between mean F0 and serum testosterone is given. All these details can vary differently in pathology.

As presented in Figs. 4.24 and 4.25, the register shift is abrupt in boys during artistic singing. It is related to serum testosterone. As shown in Fig. 4.29, it changes from 627 Hz when serum testosterone is under 1 nmol/L to 321 Hz when over 10 nmol/L. The register shifts in girls were not in focus but can also be seen, as illustrated in Fig. 4.19d. The defnition of an artistic singing Voice Range Profle is made by the pupils themselves, in dialog with the teacher and examiner if they seldomly were in doubt.

Summaries of the scientifc work which relates to the fundamental frequency of the speaking voice in children have been made by Baken and Schultz–Coulon et al. [23, 24]. Among others, Fairbanks et al., Michel et al., Hollien and Malcik, Hollien and Shipp, Hollien, Hollien et al., Fitch and Holbrook, McGlone and McGlone, and Coleman et al. have studied the development of the fundamental frequency of the speaking voice in children, without however also investigating the tonal range of the speaking voice [25–33].

Vuorenkoski et al. have compared the average fundamental frequency of the speaking voice with hormonal levels in children with endocrinological diseases [34]. Bastian and Unger investigated the fundamental frequency of the speaking voice in the different stages of puberty [35]. Harries et al. used laryngographic measurements on boys and found a good correlation between the sudden drop in frequency seen between Tanner stages 3 and 4 [36]. Lundy et al. used the singing power ratio as an objective means of quantifying the singers' formant; the values were not signifcantly different between the sung and spoken samples in young singing students [37].

In the literature, there are not many studies in which the process of bodily maturation in connection with hormonal development has been related to the important secondary sexual characteristic that the voice constitutes. Barlow and Howard used the closed quotient with electrolaryngographic measurements on 127 children with measurable effects on training [38, 39]. Amir et al. and Amir and Biron-Shental showed that it is a good idea to make supplemental sex hormone evaluations in different medical vocal conditions [40, 41]. They also showed that oral contraceptives might stabilize the voice. Cheyne et al. suggest normative values for electroglottography [42].

It is possible that calculations based on new mathematical models can reveal unknown aspects of hormonal regulation of the voice [43–46]. This would also be interesting for the quantitative differentiation between physiological and pathological voice development [47–49]. For voice research, the employment of technologies and the interpretation of the measurement results from a biological point of view are of the greatest importance.

#### **5.4 Puberty Stages and Hormonal Status Analysis**

Tables have been made with the traditional division in prepubertal, pubertal, and postpubertal results with the dependent voice parameters as a supplement. Statistical differences in girls were found between the groups for E1 (estrone), E1 sulfate, DHEAS, and androstenedione. This corresponded to a signifcant difference between the groups for the semitone range in continuous speech, the lowest tone, and the Voice Range Profle area (Fig. 4.30). In boys, the yearly changes were given in the androgens and voice parameters (Fig. 4.31).

The question of hormone-related variations in categories of singing was answered in Figs. 4.32 and 4.33. There was not a signifcant hormonal or pediatric difference of categories (soprano versus altos, tenors versus basses) at this stage of life.

Laryngologists are asked for the prediction of pubertal voice changes in girls and boys—for many reasons, with one being related to the aspect of child soloists. The mean F0 was used to fnd predictive results in both sexes.

Predictive calculations of the mean F0 are described in Figs. 4.34 and 4.35. In girls, the expansion of the semitone range in continuous speech (from 3.7 semitones prepubertal to 4.2 pubertal and 5.2 postpubertal) had an overall predictive value together with E1SO4 (estrone sulfate) of *P* < 0.05. A division in premenarche and post-menarche changed the picture. Pre-menarche E1SO4 was still signifcant, but also height and pubic hair stage were signifcant. After menarche, semitone range in continuous speech was a predictive factor, together with age and time after menarche (Fig. 4.34). The results were based on logarithmic calculations. Interestingly, there was a linear correlation of SHBG with menarche, *r* = 0.93. SHBG is predicting mean F0 change in boys: a boy in Tanner stages 2–4, with a mean F0 of 210 Hz and SHBG of 91 nmol, is in puberty based on logarithmic calculations (Fig. 4.35).

Puberty is defned as the period during which the ability to reproduce is attained. In practice work, it is related to the development of secondary sexual characteristics. The normal development of humans during puberty is a very complex process. Howard et al. have produced a survey article that is partly based on an investigation by Tanner and Whitehouse [2, 50]. The development of the voice is described as "the breaking of the voice" at the age of about 14.5 years and the defnite attainment of an adult voice about a year later. The body size of Danish children was reported by Andersen and later by Roed et al. and Hertel et al., and it matches our measurements [51–53].

In the book edited by Brook, it is highlighted that knowledge of the development of the heart and lungs is limited, and the development of these organ systems until now has only been related to body size and to the development of secondary sexual characteristics [54]. Similar remarks apply to the pediatric literature on voice development. Hägg and Taranger characterize the voice as childish, pubertal, or adult. Karlberg and Taranger describe the breaking of the voice in relation to the stage of puberty at an age of 14.5 years. Heinemann's work is concerned with abnormal processes in the development of the voice during puberty [55–57]. Kahane analyzed the development of the thyroid cartilage in relation to body size [58]. Potassium metabolism increases in close relationship to the level of sexual hormones and depends more on the stage of puberty than on age [59]. Hirano et al. measured the growth of the vocal cords during the time of puberty [60, 61].

Normal endocrinological development is controlled by the gonadotropin-releasing hormone from the hypothalamus. Through the infuence of this decapeptide, LH and FSH are released from the frontal lobes of the hypophysis. They regulate the growth of the testes and the ovaries. Sex hormones are produced by these organs. Our methods of measurement have been described by Lykkesfeldt et al.; they were carried out at the Danish Statens Seum Institut and are comparable to Binder [62, 63]. The measurements are also comparable to those of other authors [64]. A review of SHBG has been given [65].

With the method by means of which one can perform hormonal analysis on saliva, possibilities are open for investigating the close relationship between hormonal changes and voice [66]. New insights into the relationships between cerebral regulation and development of the voice in physiological and pathological cases will also make it possible in the future to explore the phenomenon of the change of voice from a neurophysiological point of view [67–70]. One further perspective of this is that we may expect to discover a new understanding of the psychology of music [71– 74].

Niedzielska et al. compared the change of voice with pathological activation of the gonads in male puberty [75]. Abitbol et al. found that the harmonics are hormonally dependent in female puberty [76]. Breteque and Sanchez analyzed the deepening of the speaking voice in boys and showed the individual nature of the related change of the singing voice [77]. Charpy underlines the concept that voice breaking does exist in adolescent females [78]. Chernobelsky shows that electroglottograms are highly effective in training vocal registers in deaf children also [79]. Chan documented electroglottography improvements in the voices of training kindergarten teachers [80].

Wiskirska-Wonica et al. studied the delay of voice break in adolescent boys [81]. Van Lierde et al. found no statistically signifcant difference for females, using the dysphonia severity index (DSI) between resonance parameters in the menstrual cycle in 24 healthy young professional voice users [82]. There are other changes of sounds during childhood: Harmonics-to-noise ratio was examined in 9–18-year-olds with no signifcant changes noticed in females. A transition in harmonics-to-noise ratio was seen in males at the age of 14–15 years [83]. Wide intersubject variation was found in a study of female adolescents in an exploratory study using LTAS and inverse fltering [84]. These measures are related to the development of the lips and jaw. The authors used 3D motion analysis of children with typical speech development compared with children with sound and speech disorders [85].

The fundamental frequency (F0) of voice is naturally an interesting biological parameter not only in childhood puberty, which is the limit of this study, but also during menopause. Truuverk and Pedersen investigated the Voice Range Profle of the speaking voice and its relationship to androgen and estrogen in amateur female choir singers in the World Festival Choir [86]. A connection was found between high estradiol and a larger area in the Voice Range Profle for the speaking voice. Russell et al. analyzed the tonal range of the speaking voice in adult women and obtained similar results [87].

#### **5.5 Further Results from the Statistical Analysis**

Further statistical calculations were used to fnd out how tight the connections were between voice development and pediatric/hormonal development.

In Fig. 4.36, in girls, all voice parameters, mean F0, total semitone range, semitone range in continuous speech, and lowest tone were shown, related to the pediatric/hormonal parameters. Mean F0 in continuous speech, lowest semitone, time after menarche, height, and E2 (estradiol) were not related to the Voice Range Profle area. In boys, the total semitone range, mean F0 in continuous speech (F0), semitone range in continuous speech, and lowest frequency were all signifcantly related to age and the Voice Range Profle area (Fig. 4.37).

Figure 4.38 illustrates the development of the F0 in continuous speech (in Hz as abscissa) and the relations to Tanner pubic hair stage, testis volumes, serum testosterone, and sex hormonebinding globulin (SHBG).

Figure 4.39 shows in girls the connection between voice parameters and pediatric/hormonal measurements: F0 is related to the lowest tone and E1 (estrone). Nearly all voice parameters are related to age. The semitone range during speech (the F0 tone range) is related to the total semitone range, the Voice Range Profle area, and some of the pubertal as well as hormonal parameters including E1. The lowest semitone is among others also related to E1.

Illustrations were also given with the Voice Range Profle area as abscissa in semitone time dB(A) on a diatonic scale for girls in Fig. 4.40 and for boys in Fig. 4.41. The biological connections between the voice parameters and the pediatric/hormonal measures are illustrated here. In a study of four Thomaner school boys, the prepubertal sopranos and pubertal hormonal values were as in Copenhagen. The prepubertal sopranos had Voice Range Profles of the same confguration as in Copenhagen although with less variance in the ranges (Fig. 4.42). In Figs. 4.43 and 4.44, the average voice categories in girls and boys in Copenhagen are presented with ranges.

Based on an overview in Fig. 4.45, a yearly difference between mean fundamental frequency (F0) in continuous speech in boys and girls is found after 13 years of age. This is also the case for the lowest biological tone. The semitone range in continuous speech and the average Voice Range Profles are illustrated.

Overall, the results are usable to help youngsters understand their own voices and discuss their voice-related possibilities. This normal material is basic in a future where voice as a biomarker in pathology in many genetic syndromes deviates from normal [88].

Another use of this book will be in restoring voices in treated child neoplasms. Peripheral precocious puberty is one of the frst clinical manifestations of chorion-gonadotropin-secreting intracranial tumors [89]. Deepening on the voice, genetic hyperandrogenism was found in a 13-year-old girl [90]. An 11-year-old girl presented to her oncologist with a recent voice change and increased leg hair growth due to a Sertoli-Leydig cell tumor with androgen excess [91]. Ovarian hilus cell hyperplasia in a girl with Turner's syndrome and progressive virilization including voice was treated with gonadectomy [92]. Wendler glottoplasty of voice feminization was carried out in a young female patient with irreversible voice changes due to a treated adrenocortical adenoma [93]. Deep learning methods probably can help in future voice diagnostics and treatment [1, 94].

#### **References**

and 45, X/46, X, +Mar karyotype. Kurume Med J. 1994;41(3):155–9. https://doi.org/10.2739/kurumemedj.41.155.

research in paediatrics 2022;95(suppl 2):1–616. doi: https://doi. org/10.1159/000525606.

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **6 Answers to the Questions Posed**

#### **Core Messages**

Measurements that include high-speed flms, Voice Range Profles, and conversational speech fundamental frequency provide important information which—when interpreted in conjunction with pediatric and hormonal parameters—can expand our understanding of the way in which the development of the voice proceeds.

Considering the information which we have accumulated from our investigations, we can answer the questions which we posed at the start of this work in the following way:

• How do high-speed videos change during childhood, from the prepubertal over the pubertal to the postpubertal period?

High-speed videos have some characteristics in childhood around the pubertal period, where irregularity of the margins of the vocal folds can suggest two child and two adult registers. The vocal folds are hardly ever shiny and often thickened, with a rear glottal insuffciency.

• How do the tonal range, dynamic range, and Voice Range Profle of the voice develop for girls and boys?

The Voice Range Profle in tones times dB(A) changes gradually for both sexes during childhood. In puberty, they temporarily decrease in the age range around 13.5 and 14.5 years; this phenomenon is more pronounced for boys than for girls. Voice Range Profles of the various categories (soprano, alto, tenor bass) are not signifcantly related to pediatric and hormonal development in childhood. The yearly development of average Voice Range Profles is presented; they are signifcantly related to estrogens and androgens.

• How does the fundamental frequency of the speaking voice (F0) develop for boys and girls?

The speaking voice (F0) changes in both sexes during childhood, especially in puberty. For boys, the change is dependent on the serum testosterone level (and predicted by the fall of the sex hormone-binding globulin (SHBG)), and for girls on the estrone level, predicted by the rise hereof and the enhanced semitone range during speech.

In the male group, the focus is on the mean fundamental frequency of the speaking voice (mean F0) dropping one octave during a period of around 8 months, while in girls, the focus is mainly on the semitonal range of the speaking voice which expands to fve semitones in Hz one octave higher up than boys, which means a double activity of the vocal folds compared to boys. For both sexes, the age parameter was around 13.5 and 14.5 years.

• How does the relationship between the voice parameters and the pediatric stages change in boys and girls?

The voice changes take place during stages 2–4 of puberty, during the period when the testosterone level rises in boys related to a fall on the mean F0 of one octave. For girls, the drop in the fundamental frequency of the speaking voice (mean F0) of one-third to one-fourth octave follows the increasing levels of estrone and estrone sulfate and the expanded semitonal range of the speaking voice. The timing of puberty and the way in which it proceeds are different for boys and girls.

• How do the androgen and estrogen hormones relate to childhood development stages of voice change in girls and boys?

The voice changes during puberty depend on the testosterone level and the estrone level, respectively, for boys and girls, independently of age. Nevertheless, the changes of voice are hormone dependent in various ways and take for both sexes around 13.5 and 14.5 years. The falling level of SHBG signifcantly precedes (predicts) the drop in the fundamental frequency of the speaking voice (mean F0) in boys in pediatric stages 2–4. In girls, the increasing semitone range and estrone values precede (predict) the drop in mean F0.

Our results can be the basis for further research, e.g., for the voice also as a biomarker in pathology. Until now, it has not been possible to set up results for girls corresponding to boys. Apart from reasons of tradition, limited knowledge of girls' voices and the way in which they change during puberty have played a role. To achieve optimal understanding of the development of vocal expression for girls, one should take into consideration that the speaking voice should lie in the biologically determined frequency range that is correct for each individual; this is especially important in pathology [1, 2].

#### **References**

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

### **Index**

#### **A**

Acoustical measurements, 4, 14, 22 Acoustical modifcations, 25 Adolescence, 5, 23, 26, 29, 96, 123, 124 Adolescents, 5, 13, 14, 20, 24, 26, 27, 30, 128 Adrenal glands, 52 Adrenarche, 28, 30, 50, 52, 58, 81, 92, 118 Adult, 4, 5, 9, 12–14, 17, 21, 27, 29, 30, 57, 58, 78, 79, 81, 95, 101, 110, 118, 123, 126–128, 140 Alto, 79–81, 95, 96, 105, 126, 140 Amplitude, 15, 22, 25 Amplitude modulation, 15 Androgens, 1, 29, 30, 52, 94, 99, 103, 126, 128, 130, 139–141 Androstenedione, 47, 52, 94, 103, 125 Areola, 23 Artistic singing, 91, 124 Artistic singing rage, 79 Axillary hair, 23

#### **B**

Benign vocal fold lesions, 23

Biological, 31, 84, 91, 92, 117, 125, 128, 129 Biological factors, 21 Bodily maturation, 125 Boys, 1, 13, 19, 20, 23–30, 47, 48, 50, 52, 57–61, 63–71, 75–79, 81, 83–87, 89–96, 98–103, 105–107, 109–111, 117, 118, 121–126, 128, 129, 139–141 Breathiness, 24 Brook's Clinical Endocrinology, 23 Build-up, 52

#### **C**

Change of registers, 16, 78, 79, 91 Child's voice, 79, 81, 95, 122 Childhood, 1, 3, 5, 14, 20, 23, 24, 29, 50, 58, 66, 78–81, 83, 96, 99, 110, 111, 117, 118, 123, 124, 128, 139–141 Children, 4, 5, 7, 13–14, 20–23, 25, 26, 29, 30, 53, 58, 59, 78, 80, 86, 106, 110, 118, 122, 124–126, 128 Choirs, 5, 23, 59, 68, 96, 108, 110, 123, 128

© The Editor(s) (if applicable) and The Author(s) 2024 M. Pedersen, *Normal Development of Voice*, https://doi.org/10.1007/978-3-031-42391-8

Chromatic scale, 9, 51, 57, 81, 83, 87, 89, 111, 122 Classifcation, 4, 5, 15 Classroom voice, 21 Closed quotients, 5, 13, 22, 125 Computed tomography, 27 Computerized Speech Lab, 12 Connected speech, 21 Conversational voice, 21, 23, 24, 47, 51, 86–87 Correlation coeffcient, 53, 54, 97, 100, 101, 103

#### **D**

Decibels, 3, 9 Deep learning, 5, 118, 130 Dehydroepiandrosterone-sulphate (DHEAS), 52, 94, 102, 125 Diatonic scale, 9, 51, 78, 82, 83, 89, 104, 105, 111, 129 DiVAS, 9, 21 Documentation, 8, 9, 13, 20, 59 Down syndrome, 14 Duty cycle, 17 Dytran instruments, 22

#### **E**

Early puberty, 25 Electroglottography, 3–5, 14–22, 51, 86–92, 110, 123, 125, 128 Electroglottography curves, 15, 18, 19 Endocam 5562, 49 Endocrinological development, 127 Endocrinologists, 25 Estradiol, 47, 52, 128, 129 Estrogen, 1, 29, 30, 52, 128, 139–141 Estrone, 47, 52, 92, 94, 97, 103, 118, 125, 129, 141

Estrone sulphate, 47, 52, 57, 126, 141 Examples, 19, 20, 47, 49, 50, 57–59, 62–64, 66, 93, 99, 117

#### **F**

Falsetto, 110, 123 Female, 26–28, 30, 97, 103, 127, 128, 130 First harmonic, 21 Formant, 14, 20, 26, 27, 30, 110, 123, 125 Formant analysis, 20 Frequency, 3, 5, 17, 19, 22, 25, 51, 59, 80, 82–84, 93, 108, 121, 125, 129, 141 Fundamental frequency (F0), 3, 13–17, 19, 20, 23–25, 27, 29, 30, 47, 51, 53, 60–77, 81, 84, 86–94, 96–103, 106, 111, 117, 119, 122–124, 126, 128, 129, 139–141

#### **G**

Genetic, 14, 28, 92, 117, 130 Genetic infuences, 25 Genetic voice disorders, 14, 96, 99, 117 German children, 14, 21, 23 Girls, 1, 13, 20, 23–29, 47, 48, 50, 52, 57–68, 72–74, 78–80, 82, 83, 87, 88, 90–95, 97–104, 106, 108, 110, 111, 118, 120–122, 124–126, 129, 139–141 Glissando, 12 Gonadarche, 23 Gonadotropin-releasing hormone expressing (GnRH) neurons, 28

#### **H**

Half-octave, 13 Half steps, 12 Harmonics, 14, 127, 128 Highest frequency, 13 High register, 16 High-speed kymography, 6–7 High-Speed Videos (HSV), 1, 3–7, 17, 22, 25, 26, 47, 49, 50, 57–79, 117–120, 139, 140 Hormonal analysis, 23, 52, 92–98, 127 changes, 13, 20, 48, 121, 127 development, 3, 14, 20, 28, 30, 78, 86, 99, 123, 125, 128, 140 measurements, 129 stages, 3, 4, 28 status analysis, 52, 125

#### **I**

Insuffciency, 59, 62, 66, 118, 140 Irregular surfaces, 59

#### **J**

Jitter, 19

#### **K**

Kay Elemetrics, 12 Kay Pentax, 22 Kymography, 5–7, 59–77, 119

#### **L**

Levator muscle, 26 LingWAVES, 9 Literature, 3–5, 9, 12, 14, 20, 50, 58, 59, 92, 110, 120, 123, 125, 127 Logarithmic criteria, 53

Longitudinal study, 25, 26, 86 Loudness, 12, 61–68, 70, 71, 73–77, 119 Lower register, 16 Lowest tone, 20, 57, 78–82, 84, 86, 91, 92, 96, 101, 103, 111, 118, 120–122, 125, 129

#### **M**

Maculae fava, 27, 30 Magnetic resonance imaging, 27 Manhattan School of Music, 20 MATLAB, 20 Maximum frequency, 12 Maximum phonation frequency, 13 Medizinische Hochschule in Hannover, 19 Menarche, 23, 24, 58, 94, 97, 98, 126, 129 Menarcheal stages, 26 Menstruation, 23, 30 Middle of the vocal folds, 7, 59–64, 66–68, 70–77, 119 Minimum frequency, 12 Minimum intensity, 9, 12 Muscle tension dysphonia, 12 Music students, 16

#### **N**

Neovius, 12 Neuromotor disturbances, 14 Noninvasive methods, 21 Normal onset of puberty, 23

#### **O**

One-way multivariate analysis, 47, 53, 98 Optical coherence tomography (OCT), 5–6, 27 Otorhinolaryngologists, 25

#### **P**

Pedagogical training, 13 Pediatrics development, 3, 14, 20, 30, 59, 78, 84, 99, 106, 123, 128, 140 stages, 1, 3, 4, 21, 23, 50, 139–141 Pediatric voice disorders, 23, 58 Phonation, 5, 14, 15, 22, 26 Phonetograph, 9, 51, 82, 120–122 Phoniatrics, 12, 15, 20, 23, 30, 106, 122 Photocell, 17 Piezotronics, 22 Pitch break, 24 Pixels, 4, 6, 47, 49, 67, 120 Postpubertal, 21, 47, 50, 54, 57–59, 62–64, 66–71, 80, 84, 87, 92, 94–96, 104, 118, 125, 126, 140 Postpubertal boy, 6 Post-pubertal girls, 94 PRAAT, 25 Prediction, 25, 53, 93, 97, 98, 126 Predictive calculations, 47, 126 Prepubertal, 1, 21, 26, 27, 29, 30, 47, 50, 52, 57–59, 64–66, 68, 72–77, 80, 84, 87, 92, 94–96, 104, 106, 118, 125, 126, 129, 139, 140 Prepuberty, 28 Pubarche, 23 Pubertal, 1, 4, 20, 21, 25, 26, 29, 47, 50, 57–61, 63–67, 79, 83, 84, 86, 87, 90, 92–95, 98, 106, 107, 110, 111, 117–122, 125–127, 129, 139, 140 Pubertal boys, 4 Pubertal development, 28–30, 111, 117

Pubertal girl, 26, 29, 59–60, 62–65, 94 Pubertal stages, 59–65, 92 Puberty, 4–6, 19, 20, 23–26, 28–30, 48, 50, 52, 57–59, 61, 67, 68, 78, 79, 81, 84, 87, 92–99, 102, 105, 108, 110, 119, 120, 122, 123, 125–128, 130, 140, 141 Puberty suppression, 29 Pubic hair, 52, 91, 96–99, 102, 103, 105, 126, 129

#### **R**

Register, 3, 4, 16, 20, 22, 57, 78, 79, 91, 106, 110, 118, 120, 122–124 Register analysis, 3, 14–20, 86–92 Register change, 20, 122

#### **S**

Saliva, 29, 127 School, 1, 13, 47, 48, 51, 67, 84–86, 103, 106–110, 121–123 School class, 28, 47, 84, 85 School room, 51, 121 Semitones, 9, 18, 25, 47, 51, 57, 58, 81, 82, 84, 86–89, 91–93, 96, 97, 101, 103, 106, 111, 117, 118, 121–126, 129, 140 Serum testosterone, 29, 47, 52, 84, 90–92, 96, 99, 100, 102, 103, 105, 110, 122–124, 129, 140 Sex hormone binding globulin (SHBG), 52, 57, 84, 94, 96–98, 100, 102, 103, 105, 126, 127, 129, 140, 141

Sex steroids, 29 Sexes, 23, 57, 91–93, 99, 110, 120, 126, 140, 141 Shimmer, 19, 27, 30 Shouting voice, 21, 110, 123 Singers, 22, 51, 125, 128 Singing boys, 25 Singing tone range, 23 Sonar artists, 20 Soprano, 79, 80, 95, 96, 105–107, 126, 129, 140 Sound intensity meter, 8 Sound pressure level (SPL), 13, 50, 51 Speaking voice, 17–22, 51, 53, 84, 93, 94, 96–98, 100, 124, 128, 139–141 Speech studio, 22, 52 Spermarche, 24 Standard text, 21, 25, 47, 51, 86 Standard tones, 121 Standardization proposal, 8, 47, 50 Statens Serum Institute, 47, 52, 53 Statistical analysis, 15, 53, 99–111, 128 Stroboscopy, 4, 5, 15, 16, 19 Surface changes, 50 Surveys, 9, 27, 28, 120, 126 Sustained vowel, 14

#### **T**

Tanner stage, 21, 23, 24, 28, 29, 47, 48, 59, 98, 125, 126 Testes volumes, 24 Testosterone, 4, 20, 27, 29, 30, 52, 57, 58, 84, 96, 98, 141 The North Wind and the Sun, 18, 51 Thelarche, 23 Therapeutic intervention, 21 Thickness, 13, 50, 59, 64, 71, 118 Thomaner choir, 23, 68, 106, 107, 110, 123

Time-varying F0, 22 Tonal analysis, 20 Tone range, 9, 14, 23, 25, 27, 51, 78, 83, 91, 93, 96, 120, 123 Total tone range, 79, 90, 96, 103 Trained pubertal voices, 20 Training, 13, 22, 78, 82, 110, 111, 117, 121, 122, 125, 128 Transgender girls, 29 Trans-men, 21, 29 Treatment, 3, 4, 15, 21, 29, 31, 78, 117, 130 Two maxima, 57, 59, 65–67

#### **U**

Union of European Phoniatricians, 7, 47, 50 Upper register, 78, 118

#### **V**

Videokymography, 58 Videostroboscopy (VS), 4, 15, 17, 19, 21, 119 Vocal cords, 3, 15, 127 Vocal fold amplitude, 13 Vocal fold mucosa, 27 Vocal folds, 3–6, 13, 15, 17, 20, 22, 25–27, 30, 50, 52, 57–77, 79, 117, 118, 120, 140 Vocal instability, 25 Vocal intensity, 67 Vocal mutation, 25 Vocal performance, 12 Vocalgrama, 12 Vocally trained boys, 16 Vocal-tract length, 26 Voice categories, 48, 54, 94–96, 99, 103–106, 108–111, 123, 129 Voice Extent Measure (VEM), 9

Voice onset, 26 Voice range profle (VRP), 1, 3, 7–10, 12–14, 24, 29, 47, 50, 51, 57, 59, 78–88, 90–91, 93, 95, 96, 99, 101, 102, 106–110, 117–125, 128, 129, 139, 140 Voice range profle area, 83, 84, 89, 93, 94, 96, 99–101, 103–106, 111, 125, 129

#### **W**

Wevosys, 9, 121 Whistle register, 23

#### **X**

XION, 9, 21

#### **Y**

Young women, 21 Younger children, 26, 118


2.3. Fondaparin

Fondaparin (Arixtra®) is a synthetic pentasaccharide that closely resembles the pentasaccharide naturally occurring in the UFH and LMWH. It is an antithrombin-mediated factor Xa inhibitor that is devoid of any anti-factor IIa (thrombin) activity [15]. It is used for treating patients with acute coronary syndrome and heparin-induced thrombocytopenia. It is indicated also for certain patients with thrombophlebitis in a fixed dose of 2.5 mg daily s.c. Laboratory monitoring is not needed; however, if necessary fondaparin levels should only be determined using assays that use known fondaparin concentrations to generate their calibration curve. The use of fondaparin in patients with creatinine clearance below 30 mL/min is contraindicated [2]. side effect of vitamin K antagonist therapy is skin necrosis that develops at therapy initiation

An Overview of the Anticoagulant Drugs Used in Routine Clinical Practice

http://dx.doi.org/10.5772/intechopen.76206

5

Dabigatran etexilate (Pradaxa®) is a low-molecular weight prodrug that exhibits no pharmacological activity. After oral administration, dabigatran etexilate is converted to its active form, dabigatran, a potent, competitive and reversible direct thrombin inhibitor [18]. The binding of dabigatran to thrombin is specific and selective and includes both free and thrombus-bound thrombin. Maximal blood concentration of dabigatran is reached after 1–3 h after the intake [18]. About 35% of the drug is bound to plasma proteins. Eighty percent of dabigatran is excreted through the kidneys [18]. Dabigatran half-life is 14–17 h [18]. It is given in fixed doses of either 150 or 110 mg twice daily in patients with atrial fibrillation and 150 mg twice daily in patients with VTE [19, 20]. Prophylactic doses after total hip or knee replacement are 220 or

The anticoagulant effect of dabigatran is predictive and, therefore, requires no regular laboratory monitoring. During dabigatran therapy, APTT and thrombin time (TT) are prolonged, but these two tests can only offer a rough approximation of dabigatran blood concentration. In certain situations when dabigatran concentration needs to be assessed, a specific test must be

Rivaroxaban (Xarelto®) directly inhibits factor Xa. It selectively binds both free and prothrombin complex bound factor Xa and in this way inhibits thrombin and clot formation. Peak blood concentration is achieved after 1–3 h after drug ingestion. As much as 95% of the drug is bound to plasma protein. One-third of the drug is excreted through kidneys, the other two-thirds are metabolized in the liver. The drug half-life is 8–13 h [1, 24]. Therapeutic doses are 20 and 15 mg once daily for patients with atrial fibrillation [25]. Patients with VTE are treated with 15 mg twice daily for the first 3 weeks, followed by 20 mg once daily [26, 27]. The drug must always be ingested with food. The prophylactic dose for patients with total hip or knee replacement is

No laboratory monitoring of therapy is needed due to the predictive effect of the drug. Rivaroxaban prolongs PT; however, when an assessment of the drug blood level is needed, an

Apixaban (Eliquis®) directly and reversibly inhibits factor Xa. Maximal blood concentration of the drug is achieved 3–4 h after ingestion. As much as 87% of the drug is bound to blood protein. Twenty-seven percent of the drug is excreted through kidneys and the remainder through the liver. The drug half-life is 12 h [31]. Patients with atrial fibrillation are treated with

and is a consequence of acute thrombosis of subcutaneous venules and capillaries [1].

150 mg once daily with only half the dose given as the first dose after surgery [21].

used, such as modified thrombin time or a chromogenic assay [22, 23].

anti-Xa test calibrated to rivaroxaban should be used [30].

3.2. Direct oral anticoagulants

3.2.1. Dabigatran

3.2.2. Rivaroxaban

10 mg once daily [28, 29].

3.2.3. Apixaban


Cell 1—Border Cell

In this scenario the temperature difference showed the same affects on the current distribution, but considering the current of cell one in opposite direction. The temperature difference led to a lower current of cell one and an earlier starting of the crossing point, even compared to the reference measurement. The temperature gap in the thermal-coupled scenario affect more balanced cell currents compared to the reference. This led also to a lower SoC gap, which in turn reduced the current peak of cell two to *I*2 = 0.61 · *I*Batt. The isolation further decreases the current of cell one *I*1, which caused a higher discharge and charge rate of cell two compared to cell one.


**6. Results**

During its development and improvement, the UAM was used to perform a number of numerical experiments aimed at testing and comparison of calculation results with measurements and other models. The UAM successfully showed its ability to reproduce the general behavior of ionospheric and thermospheric parameters such as at low and high geomagnetic and solar activity conditions. A good agreement of the numerical simulations' results was achieved in comparison with observations by incoherent scatter radars located at various latitudes and longitudes (ISRs) [31], digital ionosonde CADI in the Voeykovo Main Geophysical Observatory [35], several chains of the ionospheric tomographical receivers [36–38], satellites, including CHAMP and GPS [3, 4, 26, 30, 39–42], as well as with empirical models such as various types of IRI, MSIS, HWM [42–47], etc. and other theoretical models [42].

**Numerical modeling of the upper atmosphere behavior during substorms.** These relatively short disturbances of the geomagnetic field have duration from about 0.5 to 3 h. They are generated at the magnetopause and in the magnetosphere tail via the magnetic field lines reconnection processes and connected with the polar upper atmosphere in the auroral zones via FACs including the current wedges. The substorm auroral currents are reflected by the auroral magnetic activity indexes *AL*, *AU* and *AE*. The UAM takes into account these indexes. This allows the modeling of the upper atmospheric behavior during substorms via the UAM simulations. The results were presented in [4, 48–53] including the cusp and auroral oval behavior, energetic magnetospheric electron precipitations, electric fields, current wedge and internal atmospheric gravity waves generation. The main role of the thermospheric heating due to the soft electron precipitation was shown for the thermosphere substorm effects.

**Numerical modeling of the upper atmosphere behavior during geomagnetic storms.** These geomagnetic disturbances are global and have duration of about several days. Usually they include several substorms on the background of the geomagnetic field depression created by the ring current development. The geomagnetic activity indexes Dst and Kp characterize the geomagnetic storms. These indexes are included in the UAM inputs as well. This allows to model the upper atmospheric behavior during magnetic storms via the UAM simulations. The calculation results were presented in [25, 26, 30, 36–38, 54–59] including the main ionospheric trough dynamics due to the ionosphere-magnetosphere convection and non-coincidence of the geographical and geomagnetic axes of the Earth. The physical mechanisms of the negative and positive F2-layer ionospheric storms (electron concentration decreases and increases, correspondingly) formation were described. The main role of the thermospheric composition (atomic to molecular neutral gas concentration ratio) and winds disturbances in the magnetic storm ionospheric effects was demonstrated in these UAM calculations.

In addition to the UAM testing and comparison with the observations for the different levels of solar and geomagnetic activities, the model has been widely used to study **the ionosphere response on the local sources in the lower atmosphere**, such as disturbances associated with the processes of the earthquakes' preparation processes [26, 60–63]. Numerical UAM calculations showed that the electric fields of 5–10 mV/m effects on the F2-layer plasma by the electromagnetic drift in the crossed geomagnetic field and the electric field of the seismic origin. The vertical electric current, flowing through the lower boundary of the ionosphere with the density of ~20 nA/m2 , is required for the generation of the seismogenic electric field of ~10 mV/m [60–64]. The important role of the aerosols over the tectonic faults was underlined in this process due to the very low recombination rate for the charged aerosols.

Thus, the UAM was tested and used in many helio- and geophysical situations. Nevertheless, the amount of the UAM simulations remains to be insufficient despite the IT progress. This is related with the specifics of the geophysics as science at all. The near-Earth space environment varies due to the solar, seismic and human activities. This does not allow performing the repeated fixed experiments as in usual physical laboratories to obtain correctly the standard statistical error estimates. Moreover, the observations themselves are very limited. None of them has 3D spatial and time resolutions satisfying to the requirements of the modern technical means of the Space weather practical usage. This was well known long ago [65] and such models as UAM are aimed solving this important problem.


**4.2 Security provided by the patient standard**

In this criterion, the experts argued that some improvements must increase safety, such as lateral supports on the back and seat. Another evaluator expressed concern about how safe an obese patient might be. The average grade for this criterion was very good.