Biogenic monoamine levels in the central nervous system of the sea hare, Aplysia kurodai

Camp. Biochem.

Physiol.

Vol. 103C,

No. 3, pp. 51 l-519,

0306-4492/92$5.00+ 0.00 0 1992Pergamon Press Ltd

1992

Printed in Great Britain

BIOGENIC MONOAMINE LEVELS IN THE CENTRAL NERVOUS SYSTEM OF THE SEA HARE, APLYSIA

KURODAI

NAOKUNI TAKEDA Department

of Biotechnology, Research and Development Center, COSMO Research Institute, Satte, Saitama, 340-01, Japan (Tel.: 048-42-221 I, Fax: 0480-42-3790) (Received 13 April 1992; accepted for publication 22 May 1992)

Abstract-l. By using a three-dimensional-coulometric HPLC system, biogenic monoamines and their metabolites were quantified simultaneously in the central nervous system of the sea hare, Aplysiu kurodui. 2. Precursor amino acids, tyrosine-4 (TYR-4) and tryptophan (TRP), and dopamine (DA), 3, 4-dihydroxyphenylacetic acid (DOPAC) and 5hydroxytryptamine (5-HT) were detected in all the ganglia examined. 3. Levels of these compounds in the cerebral, pedal and parieto-visceral ganglia were higher than those of the other ganglia examined. 4. In some ganglia, epinephrine (E), 3-0-methyldopa (30MD), 3-methoxytyramine (3-MT), dihydroxyphenylethleneglycol (DOPEG), metanephrine (MN), vanillic acid (VA), octopamine (OCT), kynurenine (KYN) and S-hydroxyindoleacetic acid (5-HIAA) were also detected. 5. The main metabolic pathways of biogenic monoamines were shown to be TYR-4-DA-DOPAC and TRP-5-HT-5-HIAA. Furthermore, following five pathways were also suggested to be present; TYR-4-DA-E-MN-VA, TYR-4--TYW-OCT, TYR-4--30MD, DA-3-MT, E-DOPEG and TRP-KYN.

INTRODUCTION Aplysia has widely been used for the research of neuroscience. As to the neurochemistry, neuropeptides concerning egg-laying have mainly been studied (e.g. Geraerts et al., 1988; Takeda, 1989a). In addition, the presence of some neuropeptides such as FMRFamide and met-enkephaline (Takayanagi and Takeda, 1988) arginine-vasotocin/argininevasopressin (Mizuno and Takeda, 1988a) and oxytocin (Mizuno and Takeda, 1988b) has been examined immunohistochemically. Reports of biogenic monoamines in the central nervous system of Aplysiu have appeared (e.g. Klemm, 1985). Dopamine (DA) has been detected biochemically in the visceral ganglion in large quantities (Carpenter et al., 1971). Octopamine (OCT) has also been detected in a single neuron (Saavedra et al., 1987) and has shown to occur large amounts in the baccal ganglion (Farnham et al., 1978). As to indolalkylamines, 5-hydroxytryptamine (5HT) has been detected in the central nervous system (Carpenter et al., 1971; McCaman et al., 1973). Catecholamines and 5-HT containing neurons have been found by fluorescent histochemistry (Rathouz and Mark, 1988; Salimova et al., 1987; Salimova et al., 1987). In particular, 5-HT cells in the central nervous system have been detected immunohistochemically (Goldstein et al., 1984; Ono and McCaman, 1984; Kistker et al., 1985; Fujii and Takeda, 1988; Takayanagi and Takeda, 1988; Goldstein and Schwartz, 1989; Kawata et al., 1989; Longley and Longley, 1989). 5-HT containing neurons have also been detected by the glyoxylic acid method (Tritt et aI., 1983) 5, 7-dihydroxytryptamine method (Jahan-Parwar et al., 1987), and retrograde labeling

method (McPherson and Blankenship, 1991). Compared with the histochemistry, biochemical researches on biogenic monoamines in Aplysiu were few in number and have been performed by the conventional methods. By applying a three-dimensional high performance liquid chromatography (HPLC) system with coulometric electrochemical detection (ECD) developed to the field of clinical chemistry, many compounds in the central nervous system of invertebrates (Takeda, 1989; Shimizu and Takeda, 1991; Shimizu et at., 1991; Takeda, 1991; Takeda and Svendsen, 1991; Takeda et al., 1991; Takeda, 1992) and lower vertebrates (Takeda and Takaoka, 1991) can simultaneously be detected. In the present paper, we tried to clarify the levels of biogenic monoamines and their metabolites in the central nervous system of the sea hare, Aplysia kurodui by using the present HPLC system. MATERIALS AND

METHODS

Sample preparations and treatments

The central nervous system of the adult sea hare, Aply& kurodai examined consists of the following ganglia, buccal ganglion, cerebral ganglion, pedal ganglion, pleural ganglion parieto-visceral (abdominal) ganglion and bag cell cluster (Fig. 1). Each extirpated ganglion was put into a solution of 300 ul of 0.4 N nerchloric acid (PCA), 20 ul of 50 mM EDTA and 10 ~1 of 0.2 N sodium hydrogensulfite and homogenized with a Physcotron (Niti-On; Tokyo, Japan). The homogenates were centrifuged at 3000 rpm for 10min and the supematants collected were filtered. Then, 80 ~1 was injected on column. In the central nervous system of Aplysiu kurodui, ganglion area can clearly be distinguished from the connective tissue by its orange color. As the connective tissue in the central nervous system except the nerve cords was very thick, the weight of each ganglion was 511

N. TAKEDA

512

HPLC

Fig. 1. Schematic drawing of the central nervous system in the sea hare, Aplysia kurodai. (A) Buccal ganglion; (9) cerebral ganglion; (C) pleural ganglion; (D) pedal ganglion; (E) bag cell cluster; (F) parieto-visceral ganglion. (a) Nerve tract I; (b) nerve tract II.

greatly affected even by a slight difference of cutting positions. In the present paper, therefore, levels were expressed not as per mg but as per each ganglion to remove weighing errors and to get a more valid value. In the nerve cords, however, a part of long thread-like structure was cut and weighed, and levels were expressed as per mg.

Table

with ECD

The analytical apparatus used was a three-dimensional HPLC system (Coulochem Electrode Array System, ESA, Bedford, MA, U.S.A.). The system consists of a gradient HPLC system and 16 high sensitivity coulometric ECD coupled with a compatible computer. The concept and inherent advantage of multi-electrode HPLC system have been described elsewhere (Matson et al.. 1984). In this study. a reverse phase C,, column (4.60 x 150 mm. NBS column: Niko Bioscience, Tokyo, Japan) was used. Two mobile phases, A and 9. were employed in a solvent profile that contained both linear and step gradients. Solvent system A consisted of 0.1 M sodium phosphate and IO mg/ml of sodium dodecyl sulfate at a pH of 3.35, while mobile phase B consisted of methanol-water (I : 1, V/V) and 50 mg/l sodium dodecylsulfate at a pH 3.45. Together, this protocol permitted simultaneous separation of 26 compounds. The 16 serial electrodes were set in an incremental 60 mV array that ranged from 0 to 900 mV. The column and electrodes were maintained at a temperature of 34°C throughout the runs. Data from each electrode were collected on the computer and stored on the hard disk for later analyses. Typically, each compound would be detected at three electrodes, with an average ratio in peak height between the electrodes of 1: 6: I. However, the exact ratio was specific for each compound and could be used to establish the purity of the compound presented in unknown peaks in the sample that eluted from the column at the same time as a known standard. Unknown peaks found in the sample were matched with standards using both retention time and the oxidation electrode. As nearly all compounds would spread over at least two electrodes, ratio could be calculated and used to compare standards against unknown peaks giving a “ratio accuracy” measurement. A ratio accuracy of 100% mean that the unknown compound detected at a certain retention time spread over the array of electrodes in exactly the same way as the standard and was therefore pure. In biological samples there are always some other ions and trace metabolites which shift this ratio slightly and in this study a ratio accuracy of over 70% was taken as being sufficiently close to the pure standard. Final concentration data were calculated by comparing the dominant peak of the standard to the dominant peak ofthe unknown in the sample.

1. List of standards

used Oxidation

Potential

(mV) Abbreviation

Compound

First ~_____

Second

DOPEG VMA NE L-DOPA MHPG OCT E TYR-4 NMN DOPAC 30 MD DA 5.HTP MN

300 480

EPN

120

-

5-HIAA

I80

750

NACETS-HT KYN

I80 800

700 -

VA

480

-

HVA

450

-

TYRA

620

-

22: 5-hydroxytryptamine

5.HT

23: 3-methoxytyramine

3-MT

180 450

700 -

24: N-methyl-S-hydroxytryptamine

NMET

25: Melatonin

MEL

300 600

700 -

TRP

600

I: Dihydroxyphenylethleneglycol 2: Vanillylmandelic

acid

3: Norepinephrine 4: L-DOPA 5: 3-methoxy-4-hydroxyphenylethylene 6: Octopamine 7: Epinephrine 8: Tyrosine-4 9: Normetanephrine IO: 3,4-dihydroxyphenylacetic

acid

I I: 3-O-methyldopa 12: Dopamine 13: S-hydroxytryptophan 14: Metanephrine 15: Epinine 16: 5-hydroxyindoleacetic

acid

17: N-acetyl-5-hydroxytryptamine 18: Kynurenine 19: Vanillic

acid

20: Homovanillic 2

acid

I : Tyramine

26: Trvotoohan

glycol

180 300

600

180 I50

-

450

-

620

-

I80

-

650 480 150 450 I50 650 -

-

Biogenic

levels in 4plysia

monoamine

-------2_\

I I1,lI I I I I I

513

f,

I/ I I I I I I I I I I I I I[, I li,li-!h, I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

12

i

6

Time

( Min

:

I

18

)

Fig.2. A standard chromatogram including 26 compounds analysed (full current: 5pA; see text and Table 1). (1) DOPEG; (2) VMA; (3) NE; (4) L-DOPA; (5) MHPG; (6) OCT; (7) E; (8) TYR-4 (9) NMN; (IO) DOPAC; (11) 30MD; (12) DA; (13) 5-HTP; (14) MN; (15) EPIN; (16) 5-HIAA; (17) NACETSHT; (18) KYN; (19) VA; (20) HVA; (21) TYRA; (22) 5-HT; (23) 3MT; (24) N-MET; (25) MEL; (26) TRP.

Chemicals Chemicals for analyses were of analytical reagent grade. All compounds were purchased from Sigma (U.S.A.). These were as follows; tyrosine-4 (TYR-4), L-dopa (LDOPA), dopamine, 3-0-methyldopa (30MD), 3-methoxytyramine (3-MT), norepinephrine (NE), epinephrine (E), metanephrine (MN), normetanephrine (NMN), 3-methoxy4-hydroxyphenylethylene glycol (MHPG), 4-hydroxy-3methoxy-mandelic acid (VMA), vanillic acid (VA), 3, 4dihydroxyphenylacetic acid (DOPAC), dihydroxyphenyl-

ethyleneglycol (DOPEG), homovanillic acid (HVA), tyramine (TYRA), octopamine (OCT) and epinine (EPIN) in catecholamine system and tryptophan (TRP), kynurenine (KYN), 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamaine, 5-hydroxyindoleacetic acid (5-HIAA), N-acetyl5-HT (NACETS-HT) melatonine (MEL) and N-methy-5-hydroxytryptamine (N-MET) in indolalkylamine system. These compounds are shown in Table 1 with the specific oxidation potentials. A chromatogram including these standards, obtained in the manner outlined above, is presented in Fig. 2.

A

0 Time Fig. 3.A: A representative

( Min.)

chromatogram of buccal ganglion in the central Aplysia kurodai (full current, 5 PA).

nervous

system of the sea hare,

N. TAICEDA

514 RESULTS

Although 26 compounds were analyzed (Fig 2), surely detected compounds were few in number. From 7 to 9 compounds were detected in every nervous tissue. Representative chromatograms of each ganglion and nerve tract in the central nervous system are shown in Figs 3 and 4. Characteristically, many high peaks were localized in higher voltages of early retention times, although standard peaks were few in number in that area. In general, each chro-

matogram was similar in pattern. Among them, somewhat complicated pictures were found in the main ganglia, such as the cerebral ganglion, pedal ganglion and parieto-visceral ganglion. The levels of detected compounds are summarized in Table 2 and 3. High levels of precursor amino acids, TYR-4 and TRP, were obtained in all of the nervous tissues. DA, DOPAC and 5-HT were the main compounds found in all the ganglia and nerve tracts examined. As to catecholamines, levels of DA were high in the pedal ganglion, parieto-visceral

-_

Fig. 3.8: A

Time ( Min.) representative chromatogram of cerebral ganglion in the central nervous system of the sea hare, Aplysia kurodai (full current, 5 PA).

Time ( Min.) Fig. 3.C: A representative chromatogram of pleural ganglion in the central nervous system of the sea hare, Aplysia kuroabi (full current, 5 PA).

Biagenic monoamine fevels in Ap&Gu ganglion and cerebral ganglion lar, DA in the pedal ganglion

in order. In particuwas higher in levels

than that in the other ganglia. In the nerve tract I, DA contents were higher than those of the nerve tract II. Although the level was low, E was detected in the parieto-visceral, cerebral and pleural ganglia and bag cell clusters. DOPEG was also found in the nerve tract II, pedal and parieto-visceral ganglia. 30MD was found in the peda1 and parieto-visceral ganglia. In the nerve tract II and cerebral ganglion, 3MT was detected. TYRA was found in the buccal, cerebral,

Fig. 3.0: A re~re~utatjve ~hromato~m

51s

pedal and parieto-visceral ganglia. In the buccal ganglion and the bag cell cluster, MN was detected. VA was detected in the pedal ganglion. In the nerve tract I, VMA was detected. Only in the buccal ganglion, TYRA and OCT were found. As to indolalkylamines, levels of 5-HT were high in the pedal ganglion, parieto-visceral ganglion and cerebral ganghon and bag cell cluster in order. In the nerve tract I, 5-HT levels were higher than those of the nerve tract II. In the pedal and cerebral ganglia, S-MT and 5-HIAA were found. In the buccal ganglion and

of pedal ganglion in the centrai nervous system of the sea hare,

Aplysiu kurodoi ffull current, 5 pA).

._;._. .. .. ..-_...__

Time Fig. 3.E: A ~~m~u~~ve

i[\._--

.I._----

~--

( Min.)

chromatogram of ~~eto-~~rai ganglion in the ceutral nervous system of the sea hare, ~~~y~~ukurodai (full current, 5 fitA).

N. TAKEDA

516

Time

f Min.)

Fig. 3(F). A representative chromato~ram of bag cell cluster in the central nervous system of the sea hare, Aplysia kurodui (full current, 5 pA). bag cell cluster, 5-HTP was detected. NACETSHT was detected in the bag cell and nerve tract II. In the pleural ganglion and nerve tract I and 11. KYN was found. DlSCUSSlON

This is the first report to quantify a wide range of biogenic monoamines and related metabolites simul-

---__.1’,

!! -------

taneously in the central nervous system of Aplysiu. Different from conventional HPLC, the present HPLC system can separate many compounds not only by retention times but also voltages, as a threedimension. It makes it possible to represent peak purity by the ratio accuracy. Therefore, it can be decided whether a value is acceptable or not by considering the ratio accuracy. In each specified retention time, many unknown compounds were

Ii

Time ( Min. ) Fig. 4.A: A representative chromatogram of nerve tract I in the central nervous system of Aply& kurodai (full current, 5 FA). Nerve tract I is a part of the nerve tract which runs from the cerebral ganglion to the pedal ganglion, see Fig. la.

Biogenic

levels in Aplysiu

monoamine

Time

517

( Min.) Fig. 4(B)

Fig. 4.B: A representative chromatogram of nerve tract in the central nervous system of Apiysiu kurudui (full current, 5 FA). ‘Nerve tract II is part of the nerve tract which runs from the pleural ganglion to the parieto-visceral ganglion, see Fig. I b. Table 2. Biogenic

amine labels in the central

nervous

system of the sea hate. Ap&sio kuradai

(A)

Compounds (TYR-4)’ DA DOPAC 3-M-f DOPEG E MN TYRA $$,* S-HT 5-HTP SHIAA KYN

Buccal ganglion (n =9) ng/ganglion [Mean + S.D.) 230.2 + 112.0 19.9 & 6.5 1.1 +0.9 -

Ratio

I

2

/0.89:0.?4) ~0.94:0.68) (0.26:0.?9) -

0.7 0.6 3.4 139.9 8.0 0.2

I: 0. I i. 0.2 i: I.1 _t 24.9 i: 3.4 + 0.03 -

433.6 * 53.4 277.4 + 53,O IO.2 + 4.0 5.2 + 1.2 0.2 & 0.04 4.0 10.8 0.4 + 0.8 282.9 rt: 18.3 106.3 + 23.9 0.6 + 0.1

5-HTP NACETS-HT KYN *--Precursor

6.6 f 2.7

f * f * f +

(0.98:0.71) (0.98:0.12) (0.71:ERR)

-

0.2 * 0.1 -

-

(0.80:0.44) (0.87:0.19) (0.75: 0.791 (0.88: ERR) (0.87: ERR)

Pleural ganglion (right plus left; n = 16) ng/ganglion Ratio (Mean _+ S.D.) I 2

44.5 18.3 7.1 0.09 0.1 0.01

248.2 f 29.2 31.8 29.1 -

I .3 + 0.4

Parieto-visceral ganglion (n = tot ngjgangfion Ratio {Mean f SD.1 f 2

(0.74<.34) f0.00:0.77) 0.89 :0.42) (0.71~0.29) (0.90: ERR)

+ 132.4 k 11.2 + 2.3 * 5.7 1.9 i: 0.3 2.3 + 0.7 k.5 to.2 394.2 k 32.0 39.4 k 6.2 -

(0.94GRRJ

27.5 * 27.0

amino acid.

381.6 64.1 12.4 0.1 6.7 1.2

(0.97:o.OO) (0.00: 0.75) (0.92: ERR) (0.97:0.81) f0.88:0.8?) 10.7 f : 0.08)

Pedal ganglion (right pllrs left; n = 14) ngJgangtion Ratio (Mean +. SD.) 1 2 (TY R-4)* DA DOPAC DOPEG VA E MN 3UMD TYRA (TRP)* S-HT S-HIAA

Cerebral ganglion (n = IO) ngjganglion Ratio (Mean + SD.) 1 2

698.1 153.8 18.6 10.7

(0.83:0.50) (0.98: 0.52) (0.98: ERR) (0.74: ERR) (0.80: ERR) (0.85 : 0.4) (0.71 :O.OO) (0.88 : 0.52) (0.94: 0.69) f0.66:ERR)

106.7 * 2.2 3.6 _t 0.5 2.1 f 0.4

&x99%.74) (0.91 FO.82) (0.3&:0.69)

Bag cell clusters In = 16) ng#uster Ratio (Mean k SD.) 1 2 114.2 k 57.2 11.3k9.0 0.6 f 0.2 -

qI.90: 0.79) (0.97 !0.74) (0.96: ERR) -

0.1 kO.0 0.6 2 0.1

(0.92;RRf [0.83:0.81)

49.8 + 14.1 14.9 + 0.7

(0.98 : 0.89) (0.85 : 0.74)

0.1 kO.05 0.2 i 0.08

(0.95:0.50) (O.#J:O.ao) -

N. TAKEDA

518

Table 3. Biogenic amine levels in the nerve tracts of the sea hare, Aplysia kurodai Nerve tract I* (N = 10) Ratio Pglmg (Mean f SD.) 1 2

Compounds (TYR-4) # DA DOPAC DOPEG VMA 3-MT (TRP) # 5-HT NACETS-HT KYN

7906.80 + 8784.56 581.52 + 550.27 5.59 f 6.27

(0.80:0.62) (0.93 : 0.64) (0.14:0.66)

223.90 k 213.56

(0.83: 0.32)

7983.44 f 7285.82 5985.91 f 7917.17

(0.95:0.67) (0.75:0.X3)

163.04 i_ 175.89

(0.58:0.57)

Nerve tract IIt (N = 10) Ratio pg/mg (Mean + S.D.) I 2 5971.66 206.83 19.31 84.65

? k + f

1643.65 80.96 24.55 58.79

(0.96:0.73) (0.93:0.80) (0.94:0.23) (0.73:0.13)

2.95 3892.61 367.16 22.01 54. I4

f + + + f

0.73 754.08 100.84 11.21 14.05

(0.75:0.56) (0.97:0.79) (0.86:0.76) (0.85:0.02) (0.69:0.43)

*A part of the nerve tract which runs from the cerebral ganglion to the pedal ganglion (Fig. la). tA part of the nerve tract which runs from the pelural ganglion to the parieto-visceral ganglion (Fig. lb). # Piecursor amino acid.

usually coeluted with the target compounds, because biological samples are usually wide in matrix. Therefore, hitherto obtained levels of compounds determined simply by retention times have usually been considered to be overestimated (e.g. Klemm, 1985). Therefore, the levels obtained by the present HPLC system may be somewhat lower for their high accuracy than those measured by the conventional two-dimensional HPLC system. Based on the present results, the metabolic pathways of biogenic monoamines in the central nervous system of Ap/ysiu are shown in Fig. 5. As to catecholamines, TYR-4-DA-DOPAC was shown to be a main metabolic pathway. Furthermore, TYR-430MD, DA-E, DA-3-MT and TYR-4-TYRAOCT pathways were suggested to be present. The last pathway was found only in the buccal ganglion. As the buccal ganglion has been reported to develop after hatching (Kriegstein, 1977), this pathway was shown to be introduced later in the nervous system of

(4

TYR-4 9

30MD

Y

TYRA

*

9

OCT

DA Y 3MT

+

y

E Y

DOPAC

DOPEG

3 MN 9 VA

0)

TRP 9 KYN

+ 5HT

NACETSHT

5HIAA

Fig. 5. Metabolic pathways of biogenic monoamines in the central nervous system of the sea hare, Apiysiu kurodai. (A) Catecholamines; (B) indolalkylamines.

Aplysiu. In addition to this, E-DOPEG and EMN-VA pathways were shown to be present as fine routes. As to indolalkylamines, TRP-5-HT was shown to be a main metabolic pathway. Sometimes, a route from 5-HT to NACETS-HT was found to develop. Furthermore, the TRP-KYN pathway was also suggested to be present. The number and levels of detected compounds were large in the cerebral, pedal and parieto-visceral ganglia. It seems reasonable to see this from the view point of ontogeny: the cerebral-pedal ganglia system appeared first among the ganglia in the early development and the other ganglia developed after hatching (Kriegstein, 1977). With the progress of morphogenesis, the pedal and parieto-visceral ganglia appeared to control mainly the abdominal region. For a neural control, higher ranking cerebral ganglion appears to synthesize and transport large amounts of transmitters to control lower ranking ganglia such as the buccal, pleural, pedal and parietovisceral ganglia. As the pathway of neurotransmitters, the nerve tracts, including a bundle of axons, were shown to contain large amounts of compounds. Similar aspects seem also to be applied to lower ranking ganglia and nerve tracts in order. It seems reasonable to consider that the number and amounts of neurotransmitters are due to the numbers of target tissues to control. By using the present HPLC system, the levels of biogenic monoamines in the central nervous system of invertebrates have been examined in Hydra mugnipapillata (Takeda and Svendsen, 1991), Ciona intestinalis (Takeda, 1992), terrestrial isopods such as Lygia, Porcellionides and Armadillidium (Takeda, 1990), insects such as Bomby.u (Takeda et al., 1991), Periplaneta (Shimizu et al.. 1991) and Mamestra (Shimizu and Takeda, 1991), and several other invertebrate species including pulmonate gastropods such as Achatina, Euhadra and Limax (Takeda, 1989b). In particular, detected compounds in pulmonata were more in number and higher in levels than those of the opisthobranchia Aplyxiu. Although the data are fragmentary, the principle of the developmental aspects of the metabolic pathway of invertebrates may be drawn; precursor amino acids such as TYR and TRP, 5HT and DA were constantly present in the nervous system. With the progress of phylogeny, biosynthetic pathways tend to advance, these were the TYRTYRA-OCT system, the DA-DOPAC system and the DA-NE-E system. Compared with the compounds

Biogenic

monoamin le levels in Aplysia

detected in other species, metabolic pathways in molluscs have also a tendency to develop with the advances of phylogeny. Although it is impossible to identify the unknown peaks without the standard compounds, many unknown peaks which appeared in the early retention times have a possibility to contain new neuroactive substances in the central nervous system of Aplysia. Acknowledgements-The author wishes to express his thanks to the Misaki Marine Biological Station of Tokyo University for supplying Aplysiu. Thanks are also due to Mr Hideo Takaoka, Director of the present Department. COSMO Research Institute, for his encouragement throughout the course of the present work.

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