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Šošić-Jurjević Et Al 2015
Šošić-Jurjević Et Al 2015
Experimental Gerontology
journal homepage: www.elsevier.com/locate/expgero
Institute for Biological Research Sinia Stankovi, University of Belgrade, Despot Stefan Blvd. 142, 11000 Belgrade, Serbia
Institut fr Experimentelle Endokrinologie, Charit - Universittsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany
a r t i c l e
i n f o
Article history:
Received 7 April 2015
Received in revised form 9 September 2015
Accepted 10 September 2015
Available online 15 September 2015
Keywords:
Testosterone
Estradiol
Pituitary-thyroid axis
Middle age
Rat
a b s t r a c t
We previously reported that orchidectomy (Orx) of middle-aged rats (1516-month-old; MA) slightly affected
pituitary-thyroid axis, but decreased liver deiodinase (Dio) type 1 and pituitary Dio2 enzyme activities. At
present, we examined the effects of subsequent testosterone-propionate treatment (5 mg/kg; Orx + T), and
compared the effects of testosterone with the effects of estradiol-dipropionate (0.06 mg/kg; Orx + E) treatment.
Hormones were subcutaneously administered, daily, for three weeks, while Orx and sham-operated (SO)
controls received only the vehicle. The applied dose of T did not alter serum TSH, T4 and T3 concentrations in
Orx- MA, though it increased TSH when administrated to Orx young adults (2.5-month-old; Orx-YA). However,
pituitaries of OrxMA + T rats had higher relative intensity of immunouorescence (RIF) for TSH; in their thyroids we found increased volume and height of follicular epithelium, decreased volume of the colloid and higher
RIF for T4-bound to thyroglobulin (Tg-T4). Liver Dio1 activity was increased. E-treatment did not affect serum
hormone levels, pituitary RIF for TSH, or liver Dio1 activity in Orx-MA rats. Thyroids had decreased relative
volume and height of follicular epithelium, increased relative volume of the colloid, decreased volume of
sodium-iodide symporter-immunopositive epithelium and lower RIF for Tg-T4. Detected changes were
statistically signicant. In conclusion, androgenization enhanced pituitary TSH RIF, thyroid activation and
liver Dio1 enzyme activity in Orx-MA, without elevating serum TSH as in Orx-YA rats. Estrogenization induced
pituitary enlargement with no effect on pituitary TSH RIF, serum TSH or liver Dio1 activity. E also induced
alterations in thyroid histology that indicate mild suppression of its functioning, and contributed to thyroid
blood vessel enlargement in Orx-MA rats.
2015 Elsevier Inc. All rights reserved.
1. Introduction
Serum testosterone (T) levels in men decline progressively with age.
This is associated with numerous symptoms and poor health condition,
including type-2 diabetes, higher incidence of cardiovascular disease,
and increased mortality (Samaras et al., 2013).
http://dx.doi.org/10.1016/j.exger.2015.09.010
0531-5565/ 2015 Elsevier Inc. All rights reserved.
86
For IFC staining of pituitary TSH and thyroid Tg-T4, Alexa Fluor 488
donkey anti-rabbit IgG (Invitrogen Life technologies, CA, USA; 1:200)
was applied as secondary antiserum. The sections were mounted with
Mowiol 488 (Sigma-Aldrich, St. Louis, MO, USA). The IFC was detected
using a confocal laser scanning microscope Leica TCS SP5 II Basic (Leica
Microsystems CMS GmbH; Germany).
All incubation steps were performed at room temperature in a dark
humid chamber. For washes and antibody dilutions, 0.1 mol/l phosphate saline buffer (PBS; pH 7.4) was applied. Representative sections,
which were processed in the same way as described above using PBS
instead of the primary antibodies in the incubation, were used for the
control purposes.
2.4. Quantitative image analyses
A quantitative image analysis was performed on captured images
of IFC-stained sections taken from anterior, central, and posterior
part of pituitary or thyroid glands (23 slides per each tissue level/
organ/animal, n = 6). Relative intensity of uorescence (RIF) was
determined from measurements of one hundred TSH-IFC stained
pituitary thyrotroph cells or fty thyroid Tg-T4 IFC-stained follicles per
each tissue level/organ/animal. Measurements were performed using
the Quantify option in LAS AF Lite software (Leica Microsystems CMS
GmbH; Germany), as previously reported (Miler et al., 2014). In brief,
IFC-stained regions of interest (ROI) were encircled with drawing tool.
Two other immune negative spots in proximity to ROI were also rounded for background subtraction. The mean value of the two repeated
measurements for each pituitary thyrotroph cell or thyroid follicle
was calculated and then, after subtraction of background uorescence,
used for RIF determination.
2.5. Stereological and morphometrical analyses
Stereological analyses of thyroid sections were carried out by pointcounting method (Weibel, 1979) as previously described (Miler et al.,
2014). Briey, four to ve 5 m-thick sections from the anterior, central
and posterior parts of each animal's thyroid were analyzed. Sections
stained with hematoxylin-eosin (HE), Novelli, or NIS (IHC) were used
for morphometric study. The measurements were carried out using a
newCAST stereological software package (VIS Visiopharm Integrator
System, version 3.2.7.0; Visiopharm; Denmark), at objective magnication of 20 (for HE or Novelli stained sections) or 40 (for NIS IHC
stained sections).
The counting area was dened using a mask tool; test grid with uniformly spaced test points and lines was provided by the new-CAST software. Test points hitting the colloid, epithelium and interstitium were
determined. Relative volume densities (VV) were calculated as the
ratio of the number of points hitting each tissue component divided
by the number of points hitting the reference space, i.e. analyzed thyroid
section: VV (%) = Pp/Pt 100 (Pp, counted points hitting the tissue
component, Pt, total of points of the test system hitting reference
space). Relative volume densities (VV) were calculated for each tissue
component, representing VV of the colloid, epithelium and interstitium.
The relative volume density of blood vessels in interstitial tissue
fraction was determined on Novelli-stained sections as the ratio of the
number of points hitting blood vessels and capillary network divided
by the number of points hitting the interfollicular space.
NIS positivity was interpreted as membrane or cytoplasmic staining. The relative volume density of NIS-immunopositive epithelium
(membrane + cytoplasmic) was estimated as the ratio of the number of points hitting NIS-immunopositive membranes and/or NISimmunopositive cytoplasm divided by the number of points hitting
the reference space (epithelium + colloid + interstitium).
As to morphometry, the follicular epithelium cell height (; m) was
calculated according to (Bogataj et al., 1977) from the formula =
3Pe Lt/2Pt (Ie + Ii + (Ie + Ii)1/2 (Pe counted points on epithelium,
87
Lt total length of the test line, Pt total of points of the test system, Ie
and Ii external and internal intersections of the structures with the
test line, respectively). The activation index (AI) is the VV epithelium/
VV colloid ratio and indicates TSH-mediated activation of the thyroid
follicles (Kalinik et al., 1988).
2.6. Ultrastructural analyses
For electron microscopy, thyroid tissue samples (810 samples/
thyroid lobe/animal, n = 6) were immersed in 4% glutaraldehyde
24 h and post-xed in 1% osmium tetroxide for 1 h. Specimens were
then dehydrated through a graded series of ethanol (30100%) and
embedded in Araldite (Agar scientic, Cambridge, UK). Ultrathin sections were cut with a Diatome ultra 45 diamond knife (Diatome,
Switzerland). Five grids with ultrathin sections from 4 randomly chosen
tissue samples/thyroid lobe/animal were stained with uranyl acetate
and lead citrate (Chemapol, Prague, Czech Republic) and examined
under a MORGAGNI 268 (FEI Company, USA) transmission electron
microscope.
2.7. Hormone analyses
Concentration of TSH in sera was measured with radioimmunoassay
(RIA), using rat TSH kit (Immunodiagnostics systems GmbH, Germany),
and RIA T4 and triiodothyronine (T3) kits developed by INEP (Zemun,
Serbia). According to the manufacturer, the calibration range of TSH
RIA was up to 30 ng/ml and the lower limit of detection was
1.03 ng/ml. The calibration range of T4 RIA was up to 500 nmol/l, with
the lower limit of detection of 6 nmol/l. For T3 it was up to 10 nmol/l,
with the lower limit of detection of 0.3 nmol/l. All samples were measured in duplicate within a single assay, with intra-assay coefcient of
variation of 6.1% for TSH, 4.7% for T4 and 5.3% for T3.
2.8. 5- Iodothyronine deiodinase activity measurements
Liver protein samples were prepared (oi-Jurjevi et al., 2012)
and Dio1 enzyme activities measured precisely as previously described (Renko et al., 2012). In brief, reaction mixture contained
40 g of liver microsomal proteins, 10 l of water or 10 mmol 6-npropyl-2-thio-uracil (PTU) and 50 l of freshly prepared substrate
mix (10 mol rT3 (Sigma-Aldrich, MO, USA), 0.2 mol KPO4 (pH 6.8),
2 mmol ethylenediaminetetraacetic acid, and 80 mmol dithiothreitol).
Enzyme reaction lasted for two hours at 37 C. After centrifugation (4
C, 15.000 g, 15 min), supernatant was used for quantication of released iodide. Dowex W50-X2 resin columns served for separation of
intact rT3 and the deiodinated breakdown products from the released
iodide. The iodide content was determined by the Sandell-Kolthoff reaction, using cerium solution [22 mmol (NH4)4Ce(SO4)4 and 0.44 mol
H2SO4] and arsenite solution (25 mmol NaAsO2,0.8 mol NaCl, and
0.5 mol H2SO4). The changes in absorption (OD at 415 nm) were determined at reaction starting point and after 20 min. All protein samples
were assayed in triplicate. The tubes that contained PTU, Dio1 enzyme
inhibitor, were used for background subtraction. Calculation of the enzyme activities was performed as previously described (Renko et al.,
2012).
2.9. Statistical analyses
Statistical analyses of all obtained results were performed using
GraphPad Prism v.6 for Windows (San Diego, CA, USA). To test the normality of distribution, the sets of results obtained for all experimental
groups were rstly subjected to KolmogorovSmirnov normality test,
while the equality of variance was tested by Bartlett's test. After conrmation of Gaussian distribution and homogeneity of variance, results on
concentration of hormones in sera and body weight gain were subjected
to two-way analysis of variance (ANOVA) and then to Tukey's multiple
88
Fig. 1. Concentration of TSH (A), total T4 (B) and T3 (C) in sera of control sham-operated (SO), orchidectomized (Orx), testosterone (Orx + T) and estradiol (Orx + E) treated
orchidectomized young adult (YA) and middle-aged (MA) rats. The values are means SD (n = 6); statistics: two way ANOVA, Tukey's multiple comparisons post hoc test, p b 0.05 versus
Orx or SO rats of the corresponding age.
comparison test for post hoc comparisons. For all other results, we used
one-way ANOVA for comparative evaluations, followed by Tukey's multiple comparison tests (versus Orx or SO control groups). Statistical signicance was set at p b 0.05. The data are presented as the means SD.
3. Results
3.1. Concentration of hormones in sera
Age- and treatment- related changes in serum concentrations of
TSH, total T4 and T3, which served as markers of pituitary-thyroid axis
functioning, are summarized in Fig. 1.
In case of concentration of TSH in sera, the effect of treatment was
signicant (p = 0. 02, two way ANOVA). The interaction of treatment
and age almost reached the statistical signicance (p = 0.07, two way
ANOVA). In case of concentration of T4 and T3 in sera, the effects of animals' age were signicant (for T4 p = 0.0005; for T3 p = 0.0009, two
way ANOVA), while the effect of treatment, or the interaction between
and age and the treatment were not signicant.
In Orx-YA animals TSH was 35% (p = 0.03, Tukey's multiple comparisons post hoc test) lower in comparison with the value obtained for SOYA controls. Subsequent T-treatment reversed this change (p = 0.03,
Tukey's multiple comparisons post hoc test) in comparison to value obtained for Orx-YA group (Fig. 1A). Concentration of TH (T4 and T3)
remained unaltered when compared Orx-YA + T with the control
Orx- or SO-YA values.
However, in MA animals, orchidectomy or subsequent T- treatment
did not change hormone levels (Fig. 1A-C).
E-treatment at both examined age did not affect serum hormone
levels in comparison to Orx controls of the corresponding age. Serum
TSH level in Orx-YA + E group remained at the level of Orx-YA controls
(36% lower, p = 0.02, Tukey's multiple comparisons post hoc test, in
comparison to value obtained for SO-YA rats; Fig. 1).
Table 1
Effects of testosterone (T) and estradiol (E) treatments on body weight, body weight gain, pituitary and thyroid weight in orchidectomized middle-aged (Orx-MA) rats.
Groups
Precastration
body mass (g)
Pretreatment
body mass (g)
Final body
mass (g)
Body
weight (mg)
Absolute pituitary
weight (mg)
SO-MA
Orx-MA
698 85
692 40
684 58
639 64
701 71
623 76
17.1 1.9
17.4 2.7
Orx-MA + T
707 54
664 60
697 67
Orx-MA + E
662 66
641 86
623 79
16 16
-(60 28)
(p b 0.0001)
37 10
(p = 0.03)
-(10 16)
Absolute thyroid
weight (mg)
2.4 0.7
2.9 0.6
33.0 2.6
34.0 1.7
5.5 0.5
5.7 0.5
16.5 2.8
2.5 0.3
32.0 2.5
4.4 0.8
46.0 8.9
(p b 0.0001)
7.7 2.1
(p b 0.0001)
33.0 1.3
5.0 0.5
The values are means SD (n = 6); statistics: for weight gain two way ANOVA and Tukey's multiple comparisons post hoc test were applied (for Orx-MA p b 0.05 versus precastration
body mass, for Orx + T and Orx + E groups p b 0.05 versus pretreatment body mass); for organ weights one way ANOVA and Tukey's multiple comparisons post hoc test were applied, p b
0.05, versus Orx-MA and SO-MA rats.
89
Fig. 2. Prolactin (Prl; brown)- and thyrotropin (TSH; red)-immunopositive cells in the pars distalis of the pituitary gland from the control sham-operated (A) orchidectomized (B), testosterone (C) and estradiol (D, E) treated orchidectomized middle-aged rat. Light microscopy, double immunohistochemical staining of Prl and TSH.
basal pole. In the apical region a limited number of small dense exocytotic granules were visible. Dilated cisternae of rough endoplasmic reticulum (RER) were noted in Orx-MA but not in SO-MA group. A great
number of large dense highly osmiophilic bodies throughout the cytoplasm and colloid droplets of smaller size were noted in the thyrocytes
of both control MA groups.
In Orx-MA + T group, thyroid tissue was composed of follicles lined
by taller follicular epithelium (columnar epithelium) and smaller portion of luminal colloid in comparison to both Orx- and SO-MA controls
(Fig. 4C). The following parameters increased in comparison to the control Orx-MA values: The relative VV of the interstitium (by 9%, p =
0.008, one way ANOVA, Tukey's multiple comparisons post hoc test;
Fig. 6), and AI (by 11%, p = 0.02, and 30%, p = 0.002, respectively,
one way ANOVA, Tukey's multiple comparisons post hoc test; Fig. 6).
The relative VV of the colloid decreased (by 23%, p = 0.009; one way
90
Fig. 3. TSH-immunouorescence in the thyrotroph cells of pars distalis of the pituitary gland from the of control sham-operated (A; SO-MA), orchidectomized (B; Orx-MA), testosterone
(C; Orx-MA + T) and estradiol (D; Orx-MA + E) treated orchidectomized middle-aged rat; Confocal microscopy, immunouorescence for TSH. The values for relative intensity of
TSH immunouorescence (RIF; E) are means SD (n = 6); statistics: one way ANOVA, Tukey's multiple comparisons post hoc test, p b 0.05 versus Orx-MA rats.
ANOVA, Tukey's multiple comparisons post hoc test; Fig. 6). The same
parameters were changed in the same manner in comparison to the
values obtained for SO-MA controls (Fig. 6).
When examining blood vessels on Novelli-stained thyroid sections
(Fig. 5), we detected enlargement of interfollicular larger set of capillaries, as well as of smaller ones more closely adherent to the follicles
in the thyroids of Orx-MA + T group (Fig. 5C); Vv of blood vessels within
the interstitial tissue was 29% higher (p = 0.0008; one way ANOVA,
Tukey's multiple comparisons post hoc test; Fig. 6) in comparison to
the value obtained for Orx-MA animals. VV of blood vessels was similarly increased in comparison with the value obtained for SO-MA controls
(p = 0.0004; one way ANOVA, Tukey's multiple comparisons post hoc
test).
Increased height of thyrocytes in comparison to both Orx- and SOMA controls was easily distinguishable at TEM micrographs. Numerous
mitochondria between the dilated cisternae of RER were present in the
apical part of the cytoplasm, as well as small dense vesicles and quite big
colloid droplets, indicating intense reabsorption of colloid (Fig. 7C).
Histological analysis of thyroid tissue in Orx-MA + E rats revealed
distended follicles, composed of attened follicular epithelium and signicant amount of colloid in the lumen (Fig. 4D). The following parameters decreased in comparison with the values obtained for the control
Orx-MA group (Fig. 6): relative VV of the epithelium (by 9%, p = 0.03;
one way ANOVA, Tukey's multiple comparisons post hoc test) and
(by 13%, p = 0.003; one way ANOVA, Tukey's multiple comparisons
post hoc test). VV of the colloid increased (by 15%, p = 0.002, one way
ANOVA, Tukey's multiple comparisons post hoc test) in comparison
with the Orx-MA. The same morphometric parameters were similarly
changed in comparison with the values obtained for SO controls, except
VV of the colloid, which were not signicantly different when compared
with the values obtained for SO-MA controls (Fig. 6). In thyroid tissue of
Orx + E group we also detected enlargement of interfollicular capillars,
91
Fig. 4. Thyroid gland of control sham-operated (A), orchidectomized (B), testosterone (C)- and estradiol (D) - treated orchidectomized middle-aged rat. Light microscopy, hematoxylin-eosin.
Fig. 5. Thyroid gland of control sham-operated (A), orchidectomized (B), testosterone (C)- and estradiol (D) - treated orchidectomized middle-aged rat. Light microscopy, Novelli
histochemical staining. Black arrows indicate large blood vessels, and white arrows small vessels.
92
Fig. 6. The relative volume density (VV; %) of follicular epithelium (A), interstitium (B) and colloid (C), height of thyroid follicular cells (;m; D), activation index (AI; E), and VV of blood
vessels within the interstitium (%; F) of the thyroid tissue in sham-operated (SO-MA), orchidectomized (Orx-MA), testosterone (Orx-MA + T) and estradiol (Orx-MA + E) treated
orchidectomized middle-aged rats. The values are means SD (n = 6); statistics: one way ANOVA, Tukey's multiple comparisons post hoc test, p b 0.05 versus Orx-MA or SO-MA rats.
93
Fig. 7. Thyroid follicular cells from control sham-operated (A), orchidectomized (B), testosterone (C)- and estradiol (D)- treated orchidectomized middle-aged rat. N, nucleus; RER, rough
endoplasmic reticulum; DB, dense bodies; CD, colloid droplets; C, colloid; AM, apical membrane; M, mitochondria; V, small dense vesicles; CC, thyroid C cell; Cy, cystic membrane-bound
dilatiation lled with lower electron-density material. Transmission electron microscopy.
Fig. 8. Immunohistochemical staining of sodium-iodide symporter (NIS) in the thyroid gland of control sham-operated (A), orchidectomized (B), testosterone (C)- and estradiol
(D)- treated orchidectomized middle-aged rat. Black arrows point to basolateral NIS immunostaining. Light microscopy.
94
Fig. 9. Immunohistochemical staining of vascular endothelial growth factor (VEGF) in the thyroid gland of control sham-operated (A), orchidectomized (B), testosterone (C)- and estradiol
(D)- treated orchidectomized middle-aged rat. Black arrows indicated occasional VEGF-immunopositive cells in blood vessels. Light microscopy.
4. Discussion
The results obtained in this study indicate direct and independent
stimulatory effect of T on: (i) pituitary TSH production, (ii) thyroid tissue activation, and (iii) increased Dio1 activity and metabolism of TH
in the liver of Orx-MA rats. The TSH levels in sera of Orx-MA + T
group did not increase as we detected in Orx-YA + T rats, in line with
our hypothesis on age-related decline in pituitary TSH cells responsiveness to T. Estrogenization caused pituitary enlargement, hypertrophy
and hyperplasia of lactotrophs, with no apparent effect on pituitary
TSH-immunostaining. Moreover, E had mild suppressive effect on
thyroid tissue, but it did not affect serum TSH and TH hormone levels,
or liver Dio1 enzyme activity in comparison with Orx-MA rats.
We previously reported that the doses of T and E applied in this experiment had bone-protective effect in our Orx-MA model (Filipovi
et al., 2013). Besides the benecial effect on bone tissue, the applied
doses of sex steroids, which induced approximately 10-fold increase
of serum T or E levels, respectively (Filipovi et al., 2013), were aimed
to challenge thyroid homeostasis of MA rats. The same dose of testosterone propionate was previously reported to increase serum TSH in young
male rats (Ross, 1990). Another estradiol ester, estradiol benzoate, at
dose of 0.25 mg/kg b.w. decreased the pituitary pool of TSH when administered to young euthyroid males, and increased serum TSH when
administered to hypothyroid rats (Boado et al., 1985).
-treatment induced mild weight gain and restored the body weight
to pre-castration level. The obtained result is in line with Woodward
(1993), who conrmed that anabolic effect of testosterone persists in
adrenalectomized or hypophysectomized animals. E-treatment did
not alter body weight, but induced substantial increase in pituitary
weight in comparison to the control value. This increase is probably
due to E-induced hypertrophy and hyperplasia of lactotrophs, as well
as hypertrophy of blood vessels, detected under our experimental
95
Fig. 10. Immunohistochemical staining of thyroglobulin (Tg) in the thyroid gland of sham-operated (A), orchidectomized (B), testosterone (C)- and estradiol (D)- treated orchidectomized
middle-aged rat. Light microscopy.
96
Fig. 11. Immunouorescence for thyroxine bound to thyroglobulin (Tg-T4) in the thyroid gland of control orchidectomized (A), testosterone (B)- and estradiol (C)- treated
orchidectomized middle-aged rat and the relative intensity of TSH (RIF; D). Confocal microscopy. The values for relative intensity of TSH immunouorescence (RIF; E) are means
SD (n = 6); statistics: one way ANOVA, Tukey's multiple comparisons post hoc test, p b 0.05 versus Orx-MA or SO-MA rats.
comparison to Orx-MA group. Our data are in line with hypothesis that
E-mediated inhibition of iodine transport may be one of the reasons
why women have a higher incidence of goiter than men (Furlanetto
et al., 1999).
However, results on colloid volume, basolateral NIS- and colloid TgT4-immunopositivity in the thyroids of Orx-MA + E group were more
comparable to SO-MA group. Taken together, our histological data,
besides differential effect of T and E, indicate that the thyroid gland of
MA males has a great capacity to adapt to manipulations with sex steroids, being more sensitive to Orx and subsequent androgenization,
then to estrogenization. Banu et al. (2002) found that sex steroids
stimulated thyrocyte proliferation in immature and young adult
rats in a gender specic manner: T stimulated thyrocyte proliferation
in males and immature females, while E had stimulatory effect in females but inhibited proliferation in male (immature and young
adult) rats. More recent research on human thyroid cancer tissue
showed that AR is posttrancriptionaly regulated by miR-124a, also
97
obtained changes did not signicantly affect pituitary TSH IFC expression or concentration of TSH in sera. In the thyroid, we did not nd histopathological changes. Structural and ultrastructural analyses, together
with IHC and IFC studies, indicate mild suppression of thyroid functioning, without the expected accompanying effect on concentration of TH
in sera or liver Dio1 enzyme activity. The obtained data are original
and indicate differential effect of E in comparison to T.
Declaration of Interest
The authors declare that there is no conict of interest.
Fig. 12. Enzyme activity of type 1 iodothyronine deiodinase (Dio1) in the liver of control
sham-operated (SO), orchidectomized (Orx), testosterone (Orx + T)- and estradiol
(Orx + E)- treated Orx middle-aged (MA) rats; enzyme activities were determined
using the nonradioactive iodide-release assay. The values are means SD (n = 5); statistics: one way ANOVA, Tukey's multiple comparisons post hoc test, p b 0.05 versus Orx-MA
or SO-MA rats.
Acknowledgments
This research was supported by grants from the Ministry of Education and Science of the Republic of Serbia (No. 173009) and the
Deutsche Forschungsgemeinschaft (DFG-GK 1208, TP3, RE3038/1-1),
Charit Universittsmedizin Berlin. B. . Jurjevi was supported by
European Society for Endocrinology (ESE) Short-Term Fellowship. The
authors would like to thank Mr. Kristijan Jurjevic for his assistance
with English proofreading.
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