Matrilin-2 is a member of von Willebrand factor A
containing extracellular matrix proteins in which the cDNA-derived
sequence shows similar domain organization to cartilage matrix
protein/matrilin-1, but information on the protein structure is
limited. Here we studied the oligomerization potential of a
synthetic peptide
NH2-ENLILFQNVANEEVRKLTQRLEEMTQRMEALENRLKYR-COOH corresponding to the C-terminal sequence of mouse matrilin-2. The
central portion of this sequence shows a periodicity of hydrophobic residues occupying positions a and d of a
heptad pattern (abcdefg)n, which is characteristic
for
-helical coiled-coil proteins. Circular dichroism spectroscopy
revealed a high
-helical content, and the shape of the spectra is
indicative for a coiled-coil conformation. Chemical cross-linking and
size exclusion chromatography suggest a homotrimeric configuration.
Thermal denaturation in benign buffer shows a single cooperative
transition with
H0 =
375 kJ/mol. Melting
temperatures Tm varied from 38 to 51 °C within a
concentration range of 10 to 85 µM, which is about
35 °C lower than determined for a peptide corresponding to the
C-terminal domain of matrilin-1. The data suggest that despite the low
sequence identity within this region, matrilin-2 will form a homotrimer
as matrilin-1 does.
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INTRODUCTION |
Matrilins form a subfamily of extracellular matrix proteins
containing von Willebrand factor A-like domains. Its prototype member,
matrilin-1 (also known as cartilage matrix protein), is specifically
localized in some types of the hyaline cartilage (1) where it can
interact with aggrecan and collagen type II fibrils (2-4), but it can
also form a filamentous network by itself (5). Its primary structure
contains two N-terminal sequence segments with similarities to the von
Willebrand factor A domain separated by an epidermal growth factor-like
domain and a short unique C-terminal domain (6-9) that was recently
found to be responsible for the oligomerization into homotrimers by
assembling into a three-stranded
-helical coiled coil (4, 10, 11). Meanwhile, cDNA-derived sequences of two different gene products have been established that show a similar domain structure and are
consequently named matrilin-2 and matrilin-3 (12-14). Within the
matrilin-2 sequence, the two von Willebrand factor domains are
separated by 10 epidermal growth factor-like domains, whereas matrilin-3 lacks the second von Willebrand factor domain and contains four epidermal growth factor-like tandem repeats. In contrast to
matrilin-1, matrilin-2 is absent from epiphyseal and other cartilages,
but is found in high abundance in the limbs, calvaria, uterus, and
heart and in lower amounts in skeletal muscle, brain, and skin (12).
Expression studies suggest a similar tissue distribution for matrilin-3
as found for matrilin-1. It mainly co-localizes with collagen type II,
especially in the periphery of cartilage in sternum, femur, and trachea
(13, 14).
Currently the oligomerization state of the new matrilins is unknown.
Preliminary data from SDS-polyacrylamide gel electrophoresis run under
nonreducing conditions revealed only that matrilin-2 is not a monomeric
protein (12). Although the overall sequence similarity between the
different matrilins suggests a common evolutionary origin, it is lowest
for the C-terminal domain. Only 11 and 13 of the 38 C-terminal residues
of matrilin-2 and -3, respectively, are identical with those of
matrilin-1 of the same species, and only two residues are identical in
all known matrilin sequences in corresponding positions. To elucidate
the oligomerization potential of matrilin-2, we synthesized a peptide
corresponding to the 38 C-terminal residues of mouse matrilin-2 and
analyzed it by circular dichroism
(CD)1
spectroscopy, chemical cross-linking, and gel
filtration chromatography. Our data indicate that this sequence domain
can assemble into a homotrimeric
-helical coiled coil similar to
matrilin-1 although with a lower thermal stability.
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EXPERIMENTAL PROCEDURES |
Peptide Synthesis and Purification--
Peptide synthesis was
performed by solid-phase chemistry on a Milligen/Biosearch model 9050 synthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. The five arginine residues were protected by
pentamethylchroman-sulfonyl. Cleavage from the resin and deprotection
were carried out applying a two-step procedure as described (11).
Purification was performed by reversed phase HPLC using a YMC C18
column (20 × 250 mm) eluted with a linear binary gradient of
acetonitrile/water from 25 to 50% containing 0.1% trifluoroacetic
acid (7 ml/min) where the peptide eluted at around 45%
acetonitrile/water. The purified peptide gave a single absorption peak
at 220 nm when analyzed on an analytical C18 column. Peptide identity
was confirmed by laser desorption mass spectrometry performed by the
Protein and Carbohydrate Structure Facility of the University of
Michigan (Ann Arbor, MI). Concentrations were determined
spectrophotometrically assuming A1
cm1% = 3.32 at 276 nm as calculated from the
amino acid composition (15).
Circular Dichroism Spectroscopy and Thermodynamic
Analysis--
CD spectra were recorded on an Aviv model 62DS
spectrophotometer equipped with a five-cell holder and a
Hewlett-Packard Peltier temperature controller. Spectra are normalized
to mean residue ellipticities ([
]MRW) with
Mr = 124 as derived from the sequence. Thermal
transition curves were recorded at 222 nm from 0 to 85 °C in
0.2 °C intervals with a 0.2-min equilibration time. They were normalized to the fraction of folded peptide F with F
= (
u (T))/(
n
(T)
u(T)) where
n and
u represent ellipticities of the fully folded and unfolded species, respectively, corrected for their temperature dependence by
linear extrapolation of the low and high temperature baselines, and
is the observed ellipticity.
Transition curves were interpreted assuming a two-state mechanism in
which three unfolded chains u combine to a native
-helical coiled-coil trimer n (16). With the total
peptide concentration c0 = cu
+ 3cn and the degree of conversion to the
coiled-coil F = 3cn/c0,
the equilibrium constant K follows as K
=cn/cu3 = F/3c02 (1
F)3. With
G0
=
RT ln K =
H0
T
S0, it follows that at the
transition midpoint (F = 0.5 and T = Tm) (Equation 1),
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(Eq. 1)
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where
G0,
H0,
S0,
R, and T are the standard free energy, enthalpy,
entropy, gas constant, and absolute temperature, respectively. Thus,
H0 was calculated from the slope of
1/Tm versus ln (0.75 c02) for
Tm determined at different peptide concentrations. From single transition curves, Tm and
H0 were evaluated by a nonlinear
least-squares Marquardt-Levenberg fitting algorithm (Equation 2) of
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(Eq. 2)
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with
H0 and Tm as
variables.

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Fig. 1.
Coiled-coil probability of the C-terminal
domain of mouse matrilin-2. The sequence of the 38 C-terminal
residues was analyzed for its coiled-coil-forming potential using the
programs Coils (solid line) (21), version 2.2, applying the
MTIDK matrix and using a 2.5 weight on residues in heptad positions
a and d, PairCoil (dashed line) (22),
and MultiCoil (20), which differentiates between the possibilities of
forming a two-stranded (dotted line) or three-stranded
(dashed-dotted line) conformation. In each case, a window
size of 28 residues was used, and all algorithms agreed in the
assignment of uninterrupted heptad positions as indicated above the
sequence corresponding to peptide MTR2-C38. The first peptide residue
corresponds to position 919 of the full-length sequence (12). Putative
intrachain (spacing i i + 3; i i + 4, brackets) and interchain (heptad positions
g-2d', boxed, thick
bracket) ionic interactions are indicated. Hydrophobic residues in
heptad positions a and d are printed in
reverse font. For comparison, the 36 and 38 C-terminal
residues of mouse matrilin-1/cartilage matrix protein (MTR1, Ref. 9)
and matrilin-3 (MTR3, Ref. 14), respectively, are included.
Asterisks denote residues identical with MTR2, highlighting
the low sequence identity within this region.
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Fig. 2.
CD spectra of peptide MTR2-C38. Spectra
were recorded at 0 °C in the absence (solid line
) and presence (dashed line) of
50% trifluoroethanol (peptide concentration: 20 µM in
150 mM NaF, 20 mM
KH2PO4/NaOH, pH 7.2). The inset
shows the ellipticity ratios
[ ]222/[ ]208 determined at different
peptide concentrations in the same buffer without
trifluoroethanol.
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Crosslinking, Gel Electrophoresis, and Gel
Filtration--
Chemical cross-linking was performed at different
ionic strengths adjusted by NaCl using
bis(sulfosuccinimidyl)suberate BS3 and disuccinimidyl
glutarate (Pierce), which are homobifunctional N-hydroxysuccimide ester analogs with a spacer arm length of
1.14 and 0.77 nm, respectively. The peptide
(cfinal = 100 µM, in 20 mM KH2PO4/NaOH, pH 7.2, plus NaCl)
was incubated at various cross-linker concentrations for 1 h at
25 °C. The reaction was stopped by adding a 10-fold excess of
Tris-HCl contained in the sample buffer, and the aggregation state was
analyzed by Tris/Tricine SDS gel electrophoresis (17) using 16%
acrylamide gels containing 5 M urea and a 3% stacking gel.
Gels were stained with Coomassie Brilliant Blue G250 in 5%
formaldehyde added to keep the peptide in the gel (18).
Gel filtration was performed using a Superdex 75 prep grade column
(1 × 12 cm; Amersham Pharmacia Biotech) equilibrated in 0.25 M NaCl, 20 mM
KH2PO4/NaOH, pH 7.2, at 4 °C. The column was eluted at 12 ml/h and calibrated using standard proteins of known viscosity radius, and results were analyzed as described (11, 19).
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RESULTS |
Peptide Design and Coiled-coil Prediction--
Although the
overall domain structure of matrilin-2 is similar to that of
matrilin-1, the low sequence similarity within its C-terminal domain
(Fig. 1) makes it uncertain whether the
oligomerization state is the same. The common 3-4-3-4 spacing of
hydrophobic residues allows to predict that in matrilin-2 this sequence
region will also form an
-helical coiled coil. When analyzed by
different algorithms, the probability for coiled-coil formation is
similarly high for both protein domains. The MultiCoil program (20),
aimed to differentiate between dimeric and trimeric coiled coils,
assigns the highest probabilities for a two-stranded conformation.
To determine the secondary structure, stability, and oligomerization
state of matrilin-2, we synthesized a peptide, designated MTR2-C38,
corresponding to the 38 C-terminal residues of the mouse sequence (Fig.
1). Within the mature protein, the first peptide residue is adjacent to
Cys-Lys-Cys-918, the cysteines of which might form interchain disulfide
bridges as it was shown for the corresponding residues of chicken
matrilin-1 (10, 23). The sequence differs from that of human matrilin-2
by three structurally related residues (L923M, V927L, K954R). All four
coiled-coil predictions agree in their assignment of heptad positions
(abcdefg)nto each residue where positions
a and d are occupied by hydrophobic amino acids
except for Gln-25 (Fig. 1). Within an
-helical coiled coil, these
residues come into close contact as knobs filling holes in the center
and stabilize the oligomer by hydrophobic interactions (for details,
see e.g. Ref. 24). Further stabilization can arise from
intrahelical ionic interactions between oppositely charged side chains
of the type i
i + 3 and i
i + 4 (Fig. 1) (25). The single putative interchain ionic
pair Arg-945/Asp-950 might be crucial for oligomerization specificity
and chain orientation as it was found for a corresponding peptide
resembling the matrilin-1 C-terminal domain (26).
Secondary Structure of Peptide MTR2-C38--
When analyzed by far
ultraviolet CD spectroscopy, MTR2-C38 shows a spectrum characteristic
for a high
-helical content with extrema around 192, 208, and 222 nm
(Fig. 2), although the amplitudes are
slightly lower than observed for a corresponding peptide resembling the
matrilin-1 C-terminal domain (11). In 50% trifluoroethanol, which
disrupts the tertiary and quaternary structure and stabilizes single
-helices (27), the amplitudes increase, specifically at 208 nm. The
relatively small increase indicates that the peptide is almost fully
-helical in benign buffer. As the n-
* transition (222 nm) is mainly indicative for the
-helical content, whereas the
-
* transition (208 nm) polarizes parallel to the helix axis, the
ellipticity ratio [
]222/[
]208
reflects whether the
-helix is monomeric or forms a coiled coil (28,
29). For MTR2-C38, this ratio significantly depends on the
concentration and is greater than 0.98 at peptide concentrations above
20 µM, which is compatible with the assumption that
MTR2-C38 forms an
-helical coiled coil (Fig. 2, inset).
The lower ratio of 0.87 observed at 5 µM might indicate
some dissociation that is also reflected in the lower ellipticities
observed (data not shown).
Oligomerization State of Peptide MTR2-C38--
The specific
homotrimeric association of matrilin-1 has been shown by electron
microscopy of the reduced and unreduced native protein (4),
site-directed mutagenesis on recombinantly overexpressed mini-gene
matrilin-1 proteins (10), and analysis of peptides by analytical
ultracenrifugation, size exclusion chromatography, and chemical
cross-linking (11, 26). To test whether despite the low sequence
identity (Fig. 1), matrilin-2 can also assemble into a homotrimer, we
performed cross-linking studies on MTR2-C38 using BS3 and
disuccinimidyl glutarate, which differ in their spacer arm length. Fig.
3A shows the reaction products
analyzed by SDS gel electrophoresis. At all cross-linker
concentrations, major bands corresponding to a monomeric, dimeric, and
trimeric state appeared, but small amounts of higher oligomerization
states were detected at increased cross-linker concentrations. The
migration pattern is essentially uneffected from the ionic strength in
the 100-750 mM NaCl range (not shown).

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Fig. 3.
Oligomerization state of peptide
MTR2-C38. A, MTR2-C38 (100 µM) was
incubated with various concentrations (top row, in
mM) of the cross-linkers disuccinimidyl glutarate (DSG)
(left) and BS3 (right) for 1 h and
analyzed by SDS Tris-Tricine gel electrophoresis. The position of
molecular weight marker proteins are shown on the left, and
the assignment of monomer (M), dimer (D) and
trimer (T) positions are on the right. B, upon
gel filtration on a Superdex 75 column, the MTR2-C38 elution position
corresponds to a viscosity radius of 2.0 nm ( ) when injected at high
concentration (c = 85 µM), whereas at low
concentration, it elutes corresponding to a radius of 1.5 nm ( ,
c = 8.5 µM). The calibration standards
(Ref. 19) are bovine serum albumin (1), ovalbumin (2), trypsin
inhibitor (3), carbonic anhydrase (4), myoglobin (5), and cytochrome
c (6). The reduced retention volume
Kav = (Ve
V0)/(Vt V0) was determined assuming that the elution
positions Ve of laminin and acetone indicate the
void volume V0 and total volume
Vt of the column, respectively.
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To test whether the different gel bands observed after cross-linking
reflect a heterogeneous population of oligomers in solution, the
noncross-linked peptide was analyzed by size exclusion chromatography. A relatively short column was used to diminish dilution effects. When
injected at a concentration of 85 or 8.5 µM, the peptide eluted as a single sharp symmetric peak; in the former case, it eluted
between the positions of carbonic anhydrase and myoglobin, and in the
latter case, between those of cytochrome c and acetone (Fig.
3B). These results indicate that at the lower concentration, the peptide dissociates into monomeric chains of random-coil
conformation, whereas at the higher concentration, they elute as an
oligomer of extended structure with a hydrodynamic radius greater than that of a protein of globular shape as myoglobin
(Mr = 17,200). When a similar peptide
corresponding to the 36 C-terminal residues of matrilin-1 (11) was
chromatographed under the same conditions, it eluted in the position of
carbonic anhydrase.
The combined data from cross-linking and gel chromatography are most
consistent with the assumption that MTR2-C38 forms a rod-shaped
homotrimer in solution. The heterogeneity observed upon cross-linking
is probably due to the reaction mechanisms of BS3 and
disuccinimidyl glutarate. Their principal targets are primary amines,
such as the amino group at the N terminus of the peptide chains and the
-amino group of lysine residues (30). The human matrilin-1 domain
has four lysines. According to their optimized positioning into
heptads, one is located near the center of the coiled coil (heptad
positions g). Within MTR2-C38, the two lysines are probably
more exposed to the surface of the coiled coil in heptad positions
b and c (Fig. 1). Thus the probability for an intermolecular cross-link between them is substantially lower, and
besides one cross-link between the N-terminal amino groups covalently
stabilizing a dimer, at least one further cross link is necessary to
observe a trimer on SDS gels. The population of peptides running in the
dimer position (Fig. 3A) might include intermolecular cross
links between both the N termini and one or two of the lysines, which
excludes their native third chain partner from participating in a
covalent structure and lets this chain run in the monomeric position. A
similar heterogeneity of bands on SDS gels upon cross-linking with
BS3, where the majority of the material runs in the monomer
position, has also been observed for a rat mannose-binding protein
fragment, the crystal structure of which shows a three-stranded
-helical coiled coil containing four lysine residues (31).
Thermal Stability of Peptide MTR2-C38--
To measure the thermal
stability and evaluate the nature of the folding/unfolding transition
of MTR2-C38, the CD signal at 222 nm was monitored at peptide
concentrations ranging from 5 to 85 µM upon raising the
temperature. The melting curves showed a sigmoidal shape indicating a
cooperative unfolding from a coiled-coil to random-coil conformation.
The first derivatives d
/dt exhibit a single minimum that
is compatible with the assumption of a single transition (data not
shown). Transition curves were converted to the fraction of folded
protein (Fig. 4A). Assuming a
first order transition reflecting the unfolding of three peptide chains in a coiled-coil conformation into single random-coil chains, the data
were analyzed for the melting temperature and transition enthalpies by
nonlinear least-squares fitting to Equation 2. As expected for the
unfolding of noncovalently associated peptide chains, the melting
temperature varied with concentration ranging from 38.2 °C
determined at 5 µM to 50.9 °C at 85 µM
(Fig. 4B). These temperatures are about 35 °C lower than
those of a corresponding peptide of matrilin-1 at similar
concentrations (11). From the slope of 1/Tm
versus ln (0.75 c02), the
enthalpy change results to
375 kJ/mol, which is consistent with
H0 values resulting from fitting the single
transition curves but significantly lower in its amount than the
508
kJ/mol reported for the matrilin-1 peptide (11). Assuming that the
entire peptide chain participates in the coiled-coil conformation, a
mean value of
3.3 kJ/mol/residue results that is within the range of
1.8 to
4.6 kJ/mol/residue measured calorimetrically for a large
number of coiled-coil proteins (32).

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Fig. 4.
Thermal denaturation of peptide
MTR2-C38. A, the thermal transition was recorded by
following the CD signal change at 222 nm and normalized to the fraction
of folded peptide (left ordinate, straight line;
(c) = 42 µM). The dotted line
represents the same curve plotted as ln K(T)
(right ordinate) derived from the fraction folded, assuming
a two-state transition from a three-stranded -helical coiled coil to
random-coil monomers. H0 and
Tm values were derived from such curves by
least-squares fitting as described under "Experimental Procedures"
(Equation 2). B, from the slope of the straight line of the
reciprocal transition temperature versus ln (0.75 c2) results
H0 = 375 kJ/mol (Equation 1). The
inset shows the same values plotted as
Tm(c).
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DISCUSSION |
The
-helical coiled coil is a ubiquitous structural domain
found in many intra- and extracellular proteins within which it specifically serves as a region for polypeptide chain recognition and
oligomerization (for recent reviews, see Refs. 24 and 33). Despite its
relatively simple heptad sequence motif where hydrophobic residues are
predominently located in a 3-4-3-4 spacing, it is difficult to predict
the correct oligomerization state. Thus the multidimensional scoring
approach for identifying and distinguishing between dimeric and
trimeric coiled coils implemented in the MultiCoil program (20) favors
a dimer assembly for matrilin-2 (Fig. 1) as well as for matrilin-1
(data not shown). For matrilin-1, different approaches have
conclusively shown that it forms a homotrimeric
-helical coiled coil
(4, 10, 11), and the data presented here make this the most likely
assembly state also for matrilin-2. Some important rules for predicting
the association state of coiled coils were derived from the systematic
exchange of hydrophobic residues in heptad positions a and
d within the yeast transcription factor GCN4. The geometry
of the C
-C
bond of the core residues is parallel to the C
-C
vector of the opposing hole in heptad position a and
perpendicular in position d for the dimeric variant, whereas
the trimeric variant shows acute geometry of these residues, and in the
tetrameric one, a is perpendicular, and d is
parallel (34). Although the periodicity of hydrophobic residues is less regular within the matrilin C-terminal domains, the predominance of
leucine residues assigned to positions a and d
according to these rules is consistent with a trimeric state. It has
been shown, however, that a single residue, Arg-487 of the human
matrilin-1 sequence, which is probably involved in an interchain ionic
interaction with Glu-492, is crucial for trimer formation; when
replaced by Gln within a peptide resembling the C-terminal domain,
tetramer formation was observed at physiological pH and ionic strength (26). Interestingly, the positively charged character of Arg-487 is
conserved within all matrilin sequences. The corresponding residues of
matrilin-2 studied here are Arg-945/Glu-950 (Fig. 1, thick
bracket) and are identical both in the mouse and human sequence
(12). The matrilin-3 sequences contain a lysine instead of arginine in
this position (13, 14).
When compared with the peptide corresponding to the C terminus of
matrilin-1, MTR2-C38 is considerably less stable with
Tm values about 35 °C lower, and the enthalpy
change differs by ~130 kJ/mol (11). This is most probably due to the
weakening in the stabilizing interactions between hydrophobic core
residues. Matrilin-2 residues Ala-928, Met-942, and Met-946, which are
assigned to heptad positions a and d (Fig. 1),
are considerably less hydrophobic than the corresponding amino acids
(Val-470, Val-484, Leu-488) of matrilin-1. Based on model peptides, it
was found that depending on its position, an Ala-Ala interaction can
decrease the stability by 
Gu ~ 13 kJ/mol
when compared with a Leu-Leu interaction (29). Although methionine
residues are relatively rare in coiled-coil domains, their preference
to occupy heptad positions a or d is less
pronounced than observed for other hydrophobic amino acids (21, 35),
which might indicate that their contribution to stability is weak.
The most conserved residue pair within the currently known C-terminal
sequences of matrilins is Phe-Gln-925 of matrilin-2 (exceptions:
Phe-Glu in mouse matrilin-1; Leu-Gln in chicken matrilin-3). Interestingly, Gln is specifically found near the N terminus of many
trimeric in contrast to dimeric coiled coils (36). Indeed, Gln residues
in the heptad position a are thought to specify a trimer
assembly (37). Heteronuclear NMR assignments for the chicken matrilin-1
C-terminal domain containing the two cysteine residues preceding the
coiled-coil region indicate that they form symmetric disulfide bonds in
which the first cysteine of one chain is linked to the second cysteine
of a neighboring chain, contributing a stabilizing energy of

Gu ~ 4 kJ/mol. They are incompatible with a
coiled-coil conformation, but this starts at the following residue and
lasts up to near the C terminus (23). In the reduced form, however, the
N termini of this domain are less ordered (23). These results suggest
for MTR2-C38 that the ~10 N-terminal residues are not well ordered
and thus account for the less than fully
-helical structure as
determined by CD spectroscopy (Fig. 2).
We are grateful to Mr. Jay E. Gambee
(Shriners Hospital for Children, Portland, Oregon) for peptide
synthesis and Dr. Jean Baum (Rutgers University, Piscataway, NJ) for
access to the spectrophotometer. We especially thank Dr. Barbara
Brodsky (University of Medicine and Dentistry of New Jersey, Robert
Wood Johnson Medical School, Piscataway, NJ) for continuous discussion,
constructive criticism, and generous support without which this study
would not have been possible.