Julien Toulouse

Michel Caffarel

Peter Reinhardt

Philip E Hoggan

C J Umrigar

Quantum Monte Carlo calculations of electronic excitation energies: the case of the singlet n to pi* (CO) transition in acrolein

teaching and research institutions in France or abroad, or from public or private research centers.

I. INTRODUCTION

Quantum Monte Carlo (QMC) methods (see, e.g., Refs. 1-3) constitute an alternative to standard quantum chemistry approaches for accurate calculations of the electronic structure of atoms, molecules and solids. The two most commonly used variants, variational Monte Carlo (VMC) and diffusion Monte Carlo (DMC), use a flexible trial wave function, generally consisting for atoms and molecules of a Jastrow factor multiplied by a short expansion in configuration state functions (CSFs), each consisting of a linear combination of Slater determinants. Although VMC and DMC have mostly been used for computing ground-state energies, excitation energies have been calculated as well (see, e.g., .

The simplest QMC calculations of excited states have been performed without reoptimizing the determinantal part of the wave function in the presence of the Jastrow factor. It has recently become possible to optimize in VMC both the Jastrow and determinantal parameters for excited states, either in a state-specific or a stateaverage approach [6,7,9,10,12,14,15]. Although this leads to very reliable excitation energies, reoptimization of the orbitals in VMC can be too costly for large systems.

In this context, Bouabça et al. [13] studied how to obtain a reliable excitation energy in QMC for the singlet n → π * (CO) vertical transition in the acrolein molecule without reoptimization of the determinantal part of the wave function. The acrolein molecule is the simplest member of the unsaturated aldehyde family whose photochemistry is of great interest. They showed that a good DMC excitation energy can be obtained by using non- * julien.toulouse@upmc.fr reoptimized complete-active-space (CAS) wave functions if two conditions are fulfilled: a) The wave functions come from a state-average multiconfiguration self-consistentfield (MCSCF) calculation (using the same molecular orbitals for the two states is indeed expected to improve the compensation of errors due to the fixed-node approximation in the excitation energy); and b) a sufficiently large active space including all chemically relevant molecular orbitals for the excitation process is used. In comparison, all the small CAS wave functions and the large statespecific CAS wave functions (coming from two separate MCSCF calculations) were found to lead to quite unreliable DMC excitation energies, with a strong dependence on the size of the basis set. These results were obtained using standard all-electron QMC calculations with Gaussian basis sets, with orbitals appropriately modified near the nuclei to enforce the electron-nucleus cusp condition, in the same spirit as in Ref. 16.

In this work, we extend the study of Bouabça et al. by testing the use of a Slater basis set and the effect of reoptimization of the determinantal part of the wave function in VMC. The use of Slater basis functions is motivated by the observation that they are capable of correctly reproducing the electron-nucleus cusp condition as well as having the correct exponential decay at large distances. In contrast, Gaussian basis functions have no cusp at the nucleus and a too rapid decay at large distances. As regards the effect of reoptimization, conclusions about the validity of using non-reoptimized CAS wave functions are drawn. The paper is organized as follows. In Section II, we explain the methodology used. In Section III, we present and discuss our results. Finally, Section IV summarizes our conclusions.

II. METHODOLOGY

We are concerned with the vertical electronic transition in the acrolein (or propenal) molecule, CH 2 =CH-CHO (symmetry group C s ), from the spin-singlet ground state (symmetry A') to the first spin-singlet excited state (A"). This transition is identified as the excitation of an electron from the lone pair (n) of the oxygen to the antibonding π * orbital of the CO moiety. We use the s-trans experimental geometry of Ref. 17, obtained by microwave spectroscopy in the gas phase.

We use Jastrow-Slater wave functions parametrized as [18,19]

|Ψ(p) = Ĵ(α)e κ(κ) NCSF I=1 c I |C I , (1)
where Ĵ(α) is a Jastrow factor operator, e κ(κ) is the orbital rotation operator and |C I are CSFs. Each CSF is a symmetry-adapted linear combination of Slater determinants of single-particle orbitals which are expanded in Slater basis functions. The parameters p = (α, c, κ) that are optimized are the Jastrow parameters α, the CSF coefficients c and the orbital rotation parameters κ. The exponents of the basis functions are kept fixed in this work. We use a Jastrow factor consisting of the exponential of the sum of electron-nucleus, electronelectron, and electron-electron-nucleus terms, written as systematic polynomial and Padé expansions [20] (see also Refs. 21 and22).

For each state, we start by generating standard restricted Hartree-Fock (RHF), and state-average and state-specific MCSCF wave functions with a complete active space generated by distributing N valence electrons in M valence orbitals [CAS(N ,M )], using the quantum chemistry program GAMESS [23]. As in Ref. 13, we consider a minimal CAS(2,2) active space containing the two molecular orbitals n (A') and π * CO (A") involved in the excitation, and a larger CAS(6,5) active space containing the 5 molecular orbitals that are expected to be chemically relevant: π CO (A"), n (A'), π CC (A"), π * CO (A"), π * CC (A"). Note that, since the two states have different symmetries, the purpose behind using the state-average procedure is not the usual one of avoiding a variational collapse of the excited state onto the ground state, but rather to possibly improve the compensation of errors in the excitation energy by using the same molecular orbitals for the two states. We use the triple-zeta quality VB1 Slater basis of Ema et al. [24]. For C and O, this basis contains two 1s, three 2s, three 2p and one 3d sets of functions; for H, it contains three 1s and one 2p sets of functions. Each Slater function is actually approximated by a fit to 10 Gaussian functions [25][26][27] in GAMESS. Theses wave functions are then multiplied by the Jastrow factor, imposing the electron-electron cusp condition, and QMC calculations are performed with the program CHAMP [START_REF] Champ | a quantum Monte Carlo program written[END_REF] using the true Slater basis set rather than its Gaussian expansion. The wave function parameters are optimized with the linear energy minimization method in VMC [18,19,[START_REF] Umrigar | [END_REF], using an accelerated Metropolis algorithm [30,[START_REF] Umrigar | Quantum Monte Carlo Methods in Physics and Chemistry[END_REF]. Two levels of optimization are tested: optimization of only the Jastrow factor while keeping the CSF and orbital parameters at their RHF or MCSCF values, and simultaneous optimization of the Jastrow, CSF and orbital parameters. For all wave functions, even the state-average ones, we always optimize a separate Jastrow factor for each state, rather than a common Jastrow factor for the two states. Although the electron-nucleus cusp condition is not enforced during the optimization in our current implementation, the orbitals obtained from Slater basis functions usually nearly satisfy the cusp condition. Once the trial wave functions have been optimized, we perform DMC calculations within the short-time and fixed-node (FN) approximations (see, e.g., Refs. [START_REF] Grimm | [END_REF][33][34][35][36]. We use an efficient DMC algorithm with very small time-step errors [37]. For a given trial wave function, the evolution of the ground-and excitedstate total DMC energies and of the corresponding excitation energy when the imaginary time step τ is decreased from 0.01 to 0.001 hartree -1 is shown in Table I. While the time-step bias is clearly seen for the total energies, it largely cancels out for the excitation energy for all the time steps tested here and cannot be resolved within the statistical uncertainty. In the following, we always use an imaginary time step of τ = 0.001 hartree -1 . Note that the Jastrow factor does not change the nodes of the wave function, and therefore it has no direct effect on the fixed-node DMC total energy (aside from of course the time-step bias and the population-control bias). Improving the trial wave function by optimization of the Jastrow factor is nevertheless important for DMC calculations in order to reduce the fluctuations and to make the timestep error very small and the population-control bias negligible. Of course, when the Jastrow factor is optimized together with the CSF and/or orbital parameters, then it has an indirect effect through those parameters on the nodes of the wave function. E 0 calculated by different methods. Since the excited state is an spin-singlet open-shell state, it cannot be described by a restricted single-determinant wave function; however, we report single-determinant results for the ground state for comparison of total energies. We take our best estimates of the vertical excitation energy to be those obtained with the CAS(6,5) wave functions in DMC. They range from 3.86 to 3.89 eV, depending whether a state-average or state-specific approach is used and whether the determinantal part of the wave function is reoptimized in QMC. Previously reported calculations include (a) time-dependent density-functional theory (TDDFT): 3.66 eV [39] and 3.78 eV [38]; (b) complete-active-space second-order perturbation theory (CASPT2): 3.63 eV [39], 3.69 eV [40], and 3.77 eV [41];

III. RESULTS AND DISCUSSION

(c) multireference configuration interaction: 3.85 eV [42];

(d) different variants of coupled cluster: 3.83 eV [43], 3.93 eV [38], 3.75 eV [38]. The most recent experimental estimate is 3.69 eV, which corresponds to the maximum in the UV absorption band in gas phase and which is in agreement with previous experimental data [44][45][46][47]. Beside different treatment of electron correlation, the discrepancies between these values may be due to the high sensitivity of the excitation energy to the C=C and C=O bond lengths [38]. Moreover, the comparison with experiment relies on the approximation that the vertical excitation energy corresponds to the maximum of the broad UV absorption band. In view of all these data, a safe estimate range for the exact vertical excitation energy is from about 3.60 to 3.90 eV. Even without reoptimization of the CSF and orbital coefficients, our state-specific Jastrow-Slater CAS (6,5) wave functions give a DMC excitation energy, 3.88(2) eV, as accurate as the one obtained with the fully optimized wave functions, even though the total energies E 0 and E 1 are about 20 mhartree higher. Also, our nonreoptimized state-average Jastrow-Slater CAS (6,5) wave functions give an essentially identical DMC excitation energy of 3.89 [START_REF]Quantum Monte Carlo Methods in Physics and Chemistry[END_REF] eV. This agrees well with the DMC result of Bouabça et al. [13], 3.86 (7) eV, obtained with nonreoptimized state-average Jastrow-Slater CAS (6,5) wave functions with a Gaussian basis set.

Thus it appears possible to obtain an accurate excitation energy using non-reoptimized state-specific CAS (6,5) wave functions in DMC. This is different from what was observed in Ref. 13 where state-specific CAS (6,5) wave functions were found to give unreliable excitation energies. The difference is that we use here a Slater basis set rather than the Gaussian basis set employed in Ref. 13. Even though the Gaussian basis contains more basis functions than the VB1 Slater basis, it gives a higher DMC energy for both states and tends to favor one state over the other in state-specific calculations. This example shows the importance of using a well-balanced basis set in state-specific calculations, even in DMC.

We comment now on the results obtained with the CAS(2,2) wave functions. The state-specific MCSCF CAS(2,2) excitation energy, 3.31 eV, is a strong underestimate. The corresponding VMC and DMC state-specific calculations without reoptimization of the CSF and orbital coefficients, give slightly overestimated excitation energies, 3.94(2) and 4.06(2) eV, respectively. Whereas the state-average MCSCF CAS(2,2) calculation gives a much better excitation energy, 3.73 eV, compared to the state-specific MCSCF calculation, the non-reoptimized state-average CAS(2,2) wave functions do not seem to improve the excitation energies in VMC and DMC. In fact, they give a worse VMC excitation energy of 4.09(2) eV, and a DMC excitation energy of 4.01(2) eV which is not significantly better than with the non-reoptimized state-specific wave functions.

The excitation energies obtained from the CAS(6,5) wave functions depend very little on whether a) they are calculated in MCSCF, VMC or DMC, b) the stateaverage or the state-specific approach is employed, and c) the CSF and orbital coefficients are reoptimized or not in the presence of the Jastrow factor. In contrast, the excitation energies obtained from CAS(2,2) wave functions do depend on all of the above and, in particular the reoptimization of the CSF and orbital coefficients in the presence of the Jastrow factor significantly improves the VMC and DMC excitation energies, to 3.80(2) and 3.83(1) eV, respectively. The importance of reoptimizing in VMC the CAS(2,2) expansions but not the CAS(6,5) expansions suggests that the Jastrow factor includes important correlation effects that are present in CAS (6,5) but not in CAS [START_REF]Quantum Monte Carlo Methods in Physics and Chemistry[END_REF][START_REF]Quantum Monte Carlo Methods in Physics and Chemistry[END_REF].

Finally, we note that without reoptimization of the determinantal part of the wave functions, the groundstate VMC and DMC energies can actually increase when going from a single-determinant wave function to a CAS [START_REF]Quantum Monte Carlo Methods in Physics and Chemistry[END_REF][START_REF]Quantum Monte Carlo Methods in Physics and Chemistry[END_REF] or CAS (6,5) wave function. This behavior has been observed in other systems as well, e.g. in C 2 TABLE II. Ground-state energy E0, first excited-state energy E1, and vertical excitation energy E1 -E0 for the singlet n → π * transition in the acrolein molecule at the experimental geometry calculated by different methods using the VB1 Slater basis set. The QMC calculations are done with Jastrow-Slater wave functions using a single determinant (JSD), or a state-average (SA) or state-specific (SS) complete-active-space multideterminant expansion (JCAS). The lists of parameters optimized by energy minimization in VMC are indicated within square brackets: Jastrow (J), CSF coefficients (c), and orbitals (o). For comparison, the DMC results of Ref. 13 obtained with state-average CAS (6,5) and Si 2 [START_REF] Umrigar | [END_REF]. Of course, if the CSF and orbital coefficients are reoptimized in VMC, then the VMC total energies must decrease monotonically upon increasing the number of CSFs. In practice, it is found that the DMC also decrease monotonically although there is in principle no guarantee that optimization in VMC necessarily improves the nodes of the wave function.

IV. CONCLUSION

In this work, we have extended the study of Bouabça et al. [13] on how to obtain a reliable excitation energy in QMC for the singlet n → π * (CO) vertical transition in the acrolein molecule. We have tested the use of a Slater basis set and the effect of reoptimization of the determinantal part of the wave function in VMC and of the corresponding changes in the nodal structure in fixed-node DMC. Putting together the conclusions of the study of Bouabça et al. and the present one, we can summarize the findings on acrolein as follows: a) It is possible to obtain an accurate DMC excitation energy with non-reoptimized CAS wave functions, provided that a sufficiently large chemically relevant active space is used. In the case of a too small active space, reoptimization of the CSF and orbital coefficients in the presence of the Jastrow factor appears to be necessary in order to get a good DMC excitation energy. b) When using Gaussian basis sets of low or intermediate quality, reliable DMC excitation energies could be obtained only by using state-average wave functions (i.e., with the same molecular orbitals for the two states). In contrast, when using a good quality Slater basis set such as the VB1 basis, state-specific wave functions were found to also give reliable DMC excitation energies. Thus, this provides some support for using Slater, rather than Gaussian, basis sets in all-electron QMC calculations. Note that other authors also advocate the use of Slater basis sets in all-electron QMC calculations (see, e.g., Refs. [48][49][50].

It remains to check whether these conclusions are generally true for other systems. It would be indeed desirable for calculations on large molecular systems if ac-curate DMC excitation energies could be obtained with state-specific or state-average CAS expansions without the need of an expensive reoptimization of the determinantal part of the wave functions in QMC.

FIG. 1 .

 1 FIG. 1. Schematic representation of the singlet n → π * excitation in the CO moiety of the acrolein molecule.

  Table II reports the ground-state energy E 0 , the first excited-state energy E 1 , and the excitation energy E 1 -

TABLE I .

 I Ground-state energy E0, first excited-state energy E1, and vertical excitation energy E1 -E0 for the singlet n → π * transition in the acrolein molecule at the experimental geometry calculated in DMC with different time steps τ using the VB1 Slater basis set and a state-specific Jastrow-Slater CAS(6,5) wave function with Jastrow, CSF and orbital parameters optimized by energy minimization in VMC.

	τ (hartree -1 ) E0 (hartree) E1 (hartree) E1 -E0 (eV)
	0.01	-191.8734(4) -191.7312(4)	3.87(2)
	0.005	-191.8753(4) -191.7319(4)	3.90(2)
	0.0025	-191.8762(4) -191.7330(4)	3.90(2)
	0.001	-191.8769(3) -191.7350(3)	3.86(1)

  wave functions and a Gaussian basis set are also shown. Maximum in the UV absorption band in gas phase, Ref.38.

		E0 (hartree) E1 (hartree) E1 -E0 (eV)
	RHF	-190.83430261
	MCSCF CAS(2,2) SA	-190.82258836 -190.68568203	3.73
	MCSCF CAS(2,2) SS	-190.83891553 -190.71709289	3.31
	MCSCF CAS(6,5) SA	-190.88736483 -190.74691372	3.82
	MCSCF CAS(6,5) SS	-190.89520291 -190.75181511	3.90
	VMC JSD [J]	-191.7107(5)
	VMC JSD [J+o]	-191.7636(5)
	VMC JCAS(2,2) SA [J]	-191.7121(5) -191.5619(5)	4.09(2)
	VMC JCAS(2,2) SS [J]	-191.7099(5) -191.5652(5)	3.94(2)
	VMC JCAS(2,2) SS [J+c+o]	-191.7643(5) -191.6247(5)	3.80(2)
	VMC JCAS(6,5) SA [J]	-191.7182(5) -191.5747(5)	3.90(2)
	VMC JCAS(6,5) SS [J]	-191.7221(5) -191.5776(5)	3.93(2)
	VMC JCAS(6,5) SS [J+c+o]	-191.7795(5) -191.6342(5)	3.95(2)
	DMC JSD [J]	-191.8613(4)
	DMC JSD [J+o]	-191.8698(3)
	DMC JCAS(2,2) SA [J]	-191.8608(5) -191.7133(5)	4.01(2)
	DMC JCAS(2,2) SS [J]	-191.8606(4) -191.7113(4)	4.06(2)
	DMC JCAS(2,2) SS [J+c+o]	-191.8700(3) -191.7293(3)	3.83(1)
	DMC JCAS(6,5) SA [J]	-191.8568(5) -191.7138(5)	3.89(2)
	DMC JCAS(6,5) SS [J]	-191.8585(4) -191.7160(4)	3.88(2)
	DMC JCAS(6,5) SS [J+c+o]	-191.8769(3) -191.7350(3)	3.86(1)
	DMC JCAS(6,5) SA [J] a	-191.8504(20) -191.7086(23)	3.86(7)
	Experimental estimate b		3.69
	a QMC calculations with a Gaussian basis, Ref. 13.

b

ACKNOWLEDGMENTS

Most QMC calculations have been done on the IBM Blue Gene of Forschungszentrum Jülich (Germany) within the DEISA project STOP-Qalm. CJU acknowledges support from NSF grant number CHE-1004603.


M.D Nicoletta Fortunati
email: nfortunati@molinette.piemonte.it

M G Catalano

G Boccuzzi

R Frairia

SEX HORMONE BINDING GLOBULIN (SHBG), ESTRADIOL AND BREAST CANCER

SUMMARY

The human serum Sex Hormone-Binding Globulin (SHBG) plays an important role in breast cancer pathophysiology and risk definition, since it regulates the bioavailable fraction of circulating estradiol. We here summarize data reported over the years concerning the involvement of SHBG and SHBG polymorphisms in the definition of breast-cancer risk. We also report what is known about the direct action of SHBG in breast cancer cells, illustrating its interaction with these cells and the subsequent initiation of a specific intracellular pathway leading to cross-talk with the estradiol-activated pathway and, finally, to the inhibition of several effects of estradiol in breast cancer cells. In conclusion, as a result of its unique property of regulating the estrogen free fraction and cross-talking with the estradiol pathways, by inhibiting estradiol-induced breast-cancer cell growth and proliferation, SHBG is associated with a reduced risk of developing the neoplasm after estrogen exposure.

A c c e p t e d M a n u s c r i p t INTRODUCTION Estradiol, as other sex steroid hormones, circulates in human blood mainly bound to Sex Hormone-Binding Globulin (SHBG), a 373-amino-acid glycoprotein produced by the liver [START_REF] Hammond | Potential functions of plasma steroid-binding proteins[END_REF][START_REF] Rosner | The functions of corticosteroid-binding globulin and sex hormone-binding globulin: recent advances[END_REF]). The bioavailable fraction of estrogens and their access to target cells are directly regulated by SHBG [START_REF] Siiteri | The serum transport of steroid hormones[END_REF][START_REF] Toniolo | Steroid-protein interactions[END_REF]. Sex steroids are known to be involved in several human diseases, including hormone-dependent tumours, e.g. breast cancer [START_REF] Chen | Exogenous and endogenous hormones and breast cancer[END_REF]) and prostate cancer [START_REF] Bosland | The role of steroid hormones in prostate carcinogenesis[END_REF], obesity, cardiovascular disease and metabolic syndrome [START_REF] Hennekens | Risk factors for coronary heart disease in women[END_REF][START_REF] Jones | Testosterone and atherosclerosis in aging men: purported association and clinical implications[END_REF][START_REF] Bogers | Association of overweight with increased risk of coronary heart disease partly independent of blood pressure and cholesterol levels: a metaanalysis of 21cohort studies including more than 300 000 persons[END_REF][START_REF] Mattsson | Estrogens and glucocorticoid hormones in adipose tissue metabolism[END_REF][START_REF] Blouin | Androgens and body fat distribution[END_REF][START_REF] Renehan | Obesity and cancer: pathophysiological and biological mechanisms[END_REF]. Consequently, factors regulating bioavailable sex steroids are of key importance in the pathophysiologic background of these diseases, and SHBG has thus been the object of several studies about this issue.

We have long focused our interest on SHBG involvement in breast cancer; here we summarize what we and others have reported over the years.

SHBG AND BREAST CANCER RISK

A number of studies have concerned sex hormone and SHBG levels in women in relation to breast cancer risk. In a very early report, Moore and co-workers [START_REF] Moore | Serum concentrations of total and non-protein-bound oestradiol in patients with breast cancer and in normal controls[END_REF] observed that, while SHBG binding capacity was in the normal range in premenopausal breast-cancer patients, in postmenopausal breast-cancer patients it was significantly reduced. The reduction of SHBG levels in postmenopausal breast-cancer patients was later confirmed by other studies (Toniolo et al. 1995[START_REF] Zeleniuch-Jacquotte | Postmenopausal levels of oestrogen, androgen, and SHBG and breast cancer: long-term results of a prospective study[END_REF]) suggesting a role for SHBG. Furthermore, in these patients the free fraction of estradiol is increased, in relation to an absolute or relative reduction of SHBG [START_REF] Lipworth | Serum steroid hormone levels, sex hormone-binding globulin, and body mass index in the etiology of postmenopausal breast cancer[END_REF]. In addition, in a large study analyzing the individual data from nine prospective studies on 663 women who developed breast cancer and 1765 women who did not, the risk for breast cancer increased in a statistically significant manner with increasing concentrations of all sex A c c e p t e d M a n u s c r i p t hormones examined, while SHBG was associated with a decrease in breast cancer risk (P trend = .041) [START_REF] Key | Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies[END_REF]. More recently, in a case-controlled study within the European Prospective Investigation into Cancer and Nutrition (EPIC), (Kaaks et al. 2005b), SHBG levels in postmenopausal women who developed breast cancer were confirmed to be significantly lower compared with controls, while no significant difference was observed in premenopausal women (Kaaks et al. 2005a). SHBG, as one of the factors able to modulate estrogen balance, has thus to be taken into account in evaluating a woman's risk of developing breast cancer later in life. Some early studies also reported an interesting correlation between serum SHBG and estrogendependent breast cancer. Murayama and co-workers [START_REF] Murayama | Sex hormonebinding globulin and estrogen receptor in breast cancer: technique and preliminary clinical results[END_REF] first observed a correlation between SHBG and estrogen receptor (ER) in postmenopausal breast cancer patients, but not in ER-positive premenopausal breast cancer patients. This finding was later confirmed by the same authors, who observed the simultaneous presence of high plasma SHBG concentrations and estrogen receptors in hormone-dependent breast cancer [START_REF] Murayama | Sex hormone binding globulin as a reliable indicator of hormone dependence in human breast cancer[END_REF]) and by Plymate and co-workers [START_REF] Plymate | Relationship between sex hormone binding globulin and estrogen receptors in breast cancer[END_REF]) who reported higher SHBG levels in ER-positive than in ERnegative breast-cancer patients.

A c c e p t e d M a n u s c r i p t

SHBG IN BREAST CANCER CELLS: ROLE AND MECHANISM OF ACTION

SHBG IS DETECTED IN BREAST CANCER CELLS

The existence/presence of intracellular forms of SHBG has been evaluated in different tissues and cell types. As far as breast cancer is concerned, early immunohistochemical studies, using rabbit polyclonal antisera, showed the presence of SHBG antigen in breast cancer cells and tissue sections [START_REF] Bordin | Immunocytochemical localization of the sex steroid-binding protein of plasma in tissues of the adult monkey Macaca Nemestrina[END_REF][START_REF] Tardivel-Lacombe | Immunohistochemical detection of the sex steroid-binding plasma protein in human mammari carcinoma cells[END_REF][START_REF] Sinnecker | Immunohistochemical localization of sex hormone-binding globulin in normal and neoplastic breast tissue[END_REF][START_REF] Meyer | Intracellular sex hormone binding globulin (SHBG) in normal and neoplastic breast tissue -an additional marker for hormone dependency?[END_REF][START_REF] Germain | Use of confocal microscopy to localize the SHBG interaction with human breast cancer cell lines -a comparison with serum albumin interaction[END_REF]. The source of this immunoreactive SHBG has never been completely clarified; two possible explanations (uptake from the circulation or synthesis in loco) have been suggested but neither has been definitively proved.

Another group of studies focused on the demonstration of SHBG transcripts in tissues other than the liver, where SHBG is normally produced. SHBG mRNA has also been demonstrated in the placenta [START_REF] Larrea | Evidence that human placenta is a site of sex hormone-binding globulin gene expression[END_REF], in the endometrium and endometrial cancer [START_REF] Misao | Sex hormone-binding globulin mRNA levels in human uterine endometrium[END_REF][START_REF] Misao | Expression of sex hormone-binding globulin exon VII splicing variant messenger RNA in human uterine endometrial cancers[END_REF]). Furthermore, using RT-PCR Moore and co-workers [START_REF] Moore | Sex hormone binding globulin mRNA in human breast cancer: detection in cell lines and tumor samples[END_REF] detected SHBG mRNA in ZR-75-1, MCF-7 and MDA-MB-231 breast cancer cells, and in 11 (out of 30) breast tissue samples. Two different PCR products (300 and 500 bp) were reported and the DNA sequence of the 300 bp PCR product was consistent with alternate splicing of the SHBG mRNA. Although data concerning the cell lines are convincing, no evidence for mRNA translation was presented.

More recently, in tissue sections of human prostate, Hryb and co-workers [START_REF] Hryb | Sex hormone-binding globulin in the human prostate is locally synthesized and may act as an autocrine/paracrine effector[END_REF] demonstrated the presence of both SHBG (immunohistochemistry) and SHBG mRNA (in situ hybridization), suggesting that SHBG, at least in the prostate, is locally regulated and produced.

The same laboratory reported similar findings also in breast cancer (Khan et al. 2008).

Even though considerable effort has been made to understand the significance of the intracellular presence/production of SHBG, no consistent, conclusive and convincing evidence has been provided and this issue is still an open one.

A c c e p t e d M a n u s c r i p t 6 In contrast, two very interesting recent papers provide strong evidence in favour of SHBG uptake from the extracellular space. It has been reported that megalin, an endocytic receptor in the reproductive tissues, promotes cellular uptake of biologically-active androgens and estrogens bound to SHBG [START_REF] Hammes | Role of endocytosis in cellular uptake of sex steroids[END_REF]. Ng and co-workers also found that, in the endometrium, SHBG binds the carboxyl-terminal domains of fibulin-1D and fibulin-2 in a steroid-dependent manner, estradiol being the most effective ligand. In addition, SHBG co-immunoprecipitates with these fibulins in uterine extract; it is thus suggested that these matrix-associated proteins may therefore sequester plasma SHBG and control sex-steroid access to target cells [START_REF] Ng | Evidence that fibulin family members contribute to the steroid-dependent extravascular sequestration of sex hormone-binding globulin[END_REF]. The possibility that SHBG can interact with matrix-associated proteins is of considerable interest, since it could explain both previous data concerning the intracellular presence of SHBG and the findings reported in the next section about SHBG interaction with sex steroid target cells.

SHBG INTERACTS WITH BREAST CANCER CELL MEMBRANES

Starting from the early 1980s, numerous studies reported the existence of specific and high-affinity binding sites for SHBG on cell membranes from different human tissues. The binding site for SHBG was observed in plasma membranes from decidual endometrium [START_REF] Strel'chyonok | A recognition system for sex-hormonebinding protein-estradiol complex in human decidual endometrium plasma membranes[END_REF], normal and neoplastic endometrium (Fortunati et al. 1991;Fortunati et al. 1992), prostate [START_REF] Hryb | Testosterone-estradiol-binding globulin binds to human prostatic cell membranes[END_REF], human placenta [START_REF] Avvakumov | Interaction of blood sex steroid-binding globulin with cell membranes of human decidual tissue[END_REF], and epididymis [START_REF] Gueant | Evidence that androgen-binding protein endocytosis in vitro is receptor mediated in principal cells of the rat epididymis[END_REF]. Binding sites for SHBG have also been observed in the medial preoptic area and the medial basal hypothalamus in rats [START_REF] Caldwell | Evidence of sex hormone binding globulin binding sites in the medial preoptic area and hypothalamus[END_REF]. SHBG has also been reported to interact with membranes of estrogen-dependent MCF-7 breast cancer cells [START_REF] Porto | Binding of an extracellular steroid-binding globulin to membranes and soluble receptors from human breast cancer cells (MCF-7 cells)[END_REF]Fortunati et al.1993).

In all tissues and cell types, the binding characteristics of SHBG to cell membranes are consistent with the presence of a receptor structure, since all reports have described highly specific binding that is time-and temperature-dependent. The SHBG receptor has never actually been purified or cloned, but recent evidence concerning the interaction of SHBG with matrix-associated proteins A c c e p t e d M a n u s c r i p t 7 [START_REF] Ng | Evidence that fibulin family members contribute to the steroid-dependent extravascular sequestration of sex hormone-binding globulin[END_REF]) has opened up a new scenario, that deserves further study. In line with this, we suggest that SHBG interacts with cell membranes through a receptor able to connect extracellular SHBG to intracellular pathways, as will be described in detail.

The modalities of SHBG-membrane interaction have been exhaustively studied and described. As elegantly described by Hryb and co-workers [START_REF] Hryb | Solubilization and partial characterization of the sex hormone-binding globulin receptor from human prostate[END_REF][START_REF] Hryb | The control of the interaction of sex hormonebinding globulin with its receptor by steroid hormones[END_REF]), only steroid-free SHBG binds to cell membranes, whereas, if sex steroids bind first to SHBG, they prevent the SHBG-cell interaction; the extent of the inhibition is directly proportional to the magnitude of the association constant for the steroid-SHBG interaction. Once bound to the membrane site, SHBG can bind steroids with equal affinity as it does in solution. The SHBG domain interacting with cell membranes has also been identified; it has been localized to a ten-amino-acid stretch (TWDPEGVIFY) at the amino-terminal end of the SHBG molecule [START_REF] Khan | Delineation and synthesis of the membrane receptor-binding domain of sex hormone-binding globulin[END_REF]). This region is referred to as the laminin-G domain of human SHBG and is the most highly conserved portion of the molecule, both across species and in related proteins such as protein S, laminin A, merosin, and Drosophila crumbs protein [START_REF] Gershagen | A cDNA coding for human sex hormone binding globulin. Homology to vitamin K-dependent protein S[END_REF][START_REF] Joseph | Sex hormone-binding globulin, androgen-binding protein, and vitamin K-dependent protein S are homologous to laminin A, merosin, and Drosophila crumbs protein[END_REF][START_REF] Grishkovskaya | Crystal structure of human sex hormone-binding globulin: steroid transport by a laminin G-like domain[END_REF]. Moreover, the structural integrity of the O-glycosylation site in Thr 7 of this SHBG domain is critical for significant biological effects to be achieved in breast cancer cells: it has been observed that mutant SHBG lacking Thr 7 O-glycosylation at its amino terminus fails to produce any biological effect, whereas the activity of SHBG mutants lacking the two N-glycosylation sites located at the carboxy-terminus of the protein does not differ from that of the wild type protein (Raineri et al.2002).

It may thus be concluded that SHBG interacts with cell membranes, binding to matrix-associated proteins. SHBG laminin-G domain, located at the amino-terminus portion of the protein, is the membrane-binding domain, and its carbohydrate chain at Thr 7 contributes to its structural stability, allowing correct interaction with cell membranes and induction of biological effects, at least in breast cancer cells.

SHBG CROSS-TALKS WITH ESTRADIOL IN BREAST CANCER CELLS

A number of reports indicate that the interaction of SHBG with breast cancer cell membranes is closely related to cell sensitivity to estrogens. The binding sites for SHBG have been described in MCF-7 breast cancer cells, which are positive for the estrogen receptor (ER ), [START_REF] Porto | Binding of an extracellular steroid-binding globulin to membranes and soluble receptors from human breast cancer cells (MCF-7 cells)[END_REF]Fortunati et al. 1993), while no binding site was detectable on MDA-MB 231 breast cancer cells, which are ERα-negative and estrogen-insensitive [START_REF] Fissore | The receptormediated action of sex steroid binding protein (SBP, SHBG): accumulation of cAMP in MCF-7 cells under SBP and estradiol treatment[END_REF]). In addition, in tissue samples from breast cancer patients, SHBG binding was detectable in 75% of ERα-positive samples and in 37% of ERα-negative samples. Samples binding SHBG and expressing ERα were also characterized by a significantly lower proliferation rate than samples unable to bind SHBG [START_REF] Catalano | Sex steroid binding protein receptor (SBP-R) is related to a reduced proliferation rate in human breast cancer[END_REF].

The binding of SHBG to MCF-7 cell membranes generates a cascade of signals; the first event to be described was increased intracellular cAMP. The accumulation of cAMP induced by SHBG binding has been described both in prostate cancer cells (Nahkla et al. 1990;[START_REF] Rosner | Sex hormone-binding globulin. Binding to cell membranes and generation of a second messenger[END_REF]) and in breast cancer cells [START_REF] Fissore | The receptormediated action of sex steroid binding protein (SBP, SHBG): accumulation of cAMP in MCF-7 cells under SBP and estradiol treatment[END_REF][START_REF] Becchis | The additionally glycosylated variant of human sex hormone-binding globulin (SHBG) is linked to estrogen-dependence of breast cancer[END_REF]. SHBG caused a significant increase of cAMP in both cell types, this effect being induced by DHT in the former and by estradiol in the latter cells. The elevation in intracellular cAMP suggests that the SHBG binding site is related to a G-protein membrane receptor [START_REF] Nakhla | Sex hormone-binding globulin receptor signal transduction proceeds via a G protein[END_REF]. The biological effects of SHBG appear to be mediated by cAMP and its target PKA (Protein Kinase A): they are lost in MCF-7 cells if PKA is blocked by the specific inhibitor, PKI (6 -22), (Fortunati et al. 1996;[START_REF] Fazzari | The control of progesterone receptor expression in MCF-7 breast cancer cells: effects of estradiol and sex hormone-binding globulin (SHBG)[END_REF].

The immediate target of PKA after SHBG activation in breast cancer cells has not yet been identified, but it has been suggested that increased PKA levels may inhibit the MAP kinase pathway. It has also been reported that SHBG counteracts estradiol-induced ERK activation [START_REF] Catalano | Sex hormone-binding globulin antagonizes the anti-apoptotic effect of estradiol in breast cancer cells[END_REF], which is one of the MAP kinases involved in controlling cell proliferation.

ERK activity is suppressed by both cAMP and PKA (Filardo et al.2002) and inhibition of ERK causes inhibition of the anti-apoptotic effects caused by estradiol [START_REF] Catalano | Sex hormone-binding globulin antagonizes the anti-apoptotic effect of estradiol in breast cancer cells[END_REF]) and reduces ERα transcription efficiency [START_REF] Kato | Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase[END_REF].

A c c e p t e d M a n u s c r i p t 9 It has also been reported that membrane-associated ER-α is responsible for rapid E 2 -induced cAMP accumulation and subsequent activation of the downstream PKA pathway, and that cAMP-activated PKA activity is associated with inhibition of breast cancer cell proliferation [START_REF] Zivadinovic | Membrane estrogen receptor-α levels in MCF-7 breast cancer cells predict cAMP and proliferation responses[END_REF]. Besides the classical pathway mediated by nuclear ER, estradiol it thought to bind to a putative membrane ER [START_REF] Marquez | Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells[END_REF] and to trigger specific intracellular signal transduction pathways [START_REF] Razandi | Plasma membrane estrogen receptors signal to antiapoptosis in breast cancer[END_REF] leading to activation of MAP kinase, that transmits and amplifies signals involved in both cell proliferation and apoptosis [START_REF] Pearson | Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions[END_REF][START_REF] Santen | The role of mitogen-activated protein (MAP) kinase in breast cancer[END_REF][START_REF] Freeman | The role of the mitogen-activated protein kinase cellular signaling pathway in tumor cell survival and apoptosis[END_REF]. Furthermore, MAP kinase can directly catalyze the phosphorylation of serine 118 of nuclear ERα, thus increasing ER transcriptional efficiency [START_REF] Kato | Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase[END_REF].

Estradiol and SHBG membrane-initiated pathways can thus cross-talk at different levels (at the membrane or at the ERK site) with the ultimate result of inhibiting cell proliferation and inducing apoptosis [START_REF] Catalano | Sex hormone-binding globulin antagonizes the anti-apoptotic effect of estradiol in breast cancer cells[END_REF][START_REF] Fortunati | Sex hormone-binding globulin (SHBG) and estradiol cross-talk in breast cancer cells[END_REF].

The mechanism of action of SHBG in breast cancer cell is summarized in Figure 1.

SHBG INHIBITS ESTRADIOL EFFECTS IN BREAST CANCER CELLS

SHBG-estradiol cross-talk is thus likely to interfere with the biological effects of estradiol in breast cancer cells, and several lines of evidence reported over the years substantiate this suggestion.

Firstly, SHBG has been widely demonstrated to inhibit estrogen-dependent cell growth. Estradiol is known to induce and maintain the proliferation of breast cancer cells [START_REF] Dickson | Hormones and breast cancer in vitro[END_REF]). Through its nuclear receptor ERα, it regulates the transcription and expression of many genes involved in cell proliferation, inducing positive regulators of cell growth and inhibiting negative regulators (Frasor et al. 2003;[START_REF] Thiantanawat | Signaling pathways of apoptosis activated by aromatase inhbitors and antiestrogens[END_REF]). In addition, by interacting with its membrane receptor [START_REF] Marquez | Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells[END_REF], it activates ERK and inhibits Jun kinase, causing the induction of bcl-2 and thus the inhibition of apoptosis in MCF-7 breast cancer cells [START_REF] Razandi | Plasma membrane estrogen receptors signal to antiapoptosis in breast cancer[END_REF].

The ability of SHBG to inhibit estradiol-induced cell proliferation was first reported in 1993,

A c c e p t e d M a n u s c r i p t 10 together with one of the first descriptions of the SHBG binding site on MCF-7 cells (Fortunati et al. 1993). This observation was subsequently further confirmed in our laboratory and this effect of SHBG in MCF-7 cells was described in detail. The antiproliferative effect of SHBG is detectable when the correct sequence of binding is followed; that is, SHBG first binds to its membrane on MCF-7 cells, and then estradiol binds at the steroid-binding site of SHBG; the inhibition of estradiol-induced proliferation of MCF-7 cells is mediated by increasing intracellular cAMP and activating its target, PKA (Fortunati et al. 1996). SHBG is also reported to completely reverse the anti-apoptotic effect of estradiol [START_REF] Catalano | Sex hormone-binding globulin antagonizes the anti-apoptotic effect of estradiol in breast cancer cells[END_REF]) and the facilitating effect of SHBG on apoptosis is one of the mechanisms involved in the inhibition of MCF-7 cell proliferation.

Moreover, the structural integrity of the O-glycosylation site in Thr 7 at the amino-terminal end of SHBG is required to block estradiol antiapoptotic effect and to induce cell proliferation [START_REF] Raineri | O-Glycosylation of human sex hormone-binding globulin is essential for inhibition of estradiolinduced MCF-7 breast cancer cell proliferation[END_REF].

In view of estradiol's central role in gene expression, the ability of SHBG to modulate estradiolcontrolled genes in breast cancer cells was also evaluated [START_REF] Catalano | Sex hormone-binding globulin selectively modulates estradiol-regulated genes in MCF-7 cells[END_REF]). SHBG is effective on a small number of genes, all involved in cell growth, apoptosis control and cell estrogendependence. In detail, SHBG inhibits estradiol up-regulation of bcl-2, c-myc, EGF-R, PR, and its down-regulation of ERα. The effect on bcl-2 expression may be one of the mechanisms elicited by SHBG to restore apoptosis in breast cancer cells under the action of estrogen. The proto-oncogene bcl-2 inhibits apoptosis [START_REF] Thompson | Apoptosis in the pathogenesis and treatment of disease[END_REF][START_REF] Craig | The bcl-2 gene family[END_REF] and is over-expressed in breast cancer [START_REF] Nahta | Bcl-2 antisense oligonucleotides: a potential novel strategy for the treatment of breast cancer[END_REF]. The ability of SHBG to down-regulate bcl-2 can counteract its effects, normally amplified by estradiol, in breast cancer cells. As reported for bcl-2, SHBG also inhibits estradiol induction of c-myc expression. In breast cancer cells the proto-oncogene c-myc, which is up-regulated by estradiol [START_REF] Shang | Molecular determinants for the tissue specificity of SERMs[END_REF], acts on multiple targets that are key cell-cycle regulators; in particular, it increases the expression of cyclin E and CDK4 (Santoni-Rugiu 2000; [START_REF] Hermeking | Identification of CDK4 as a target of c-MYC[END_REF]. Also in this case SHBG can abrogate the magnifying effect of estradiol on positive regulators of cell cycle and growth. It is also reported that SHBG inhibits estradiol A c c e p t e d M a n u s c r i p t 11 induction of EGF-R, a member of the epidermal growth factor family of trans-membrane receptors [START_REF] Rjr | The ErbB/HER receptor protein-tyrosine kinases and cancer[END_REF]. Over-expression of EGF-R in breast cancer, which might be induced by estradiol [START_REF] Berthois | Regulation of epidermal growth factor-receptor by estrogen and antiestrogen in the human breast cancer cell line MCF-7[END_REF], is a negative prognostic factor [START_REF] Spyratos | Epidermal growth factor receptors and prognosis in primary breast cancer[END_REF]) and, in addition, is involved in a bidirectional cross-talk with estrogen receptors; activation of the EGF-R-derived signalling pathway could amplify the effect of estradiol in breast cancer [START_REF] Levin | Bidirectional signalling between the estrogen receptor and the epidermal growth factor receptor[END_REF]. Therefore, by abrogating estradiol induction of EGF-R expression, SHBG breaks the loop between the two pathways, reducing breast cancer cell growth. Furthermore, SHBG is also effective on both ER and PR expression. First, it prevents estradiolinduced down-regulation of ERα, an effect that could also be related to apoptosis induction and cell growth inhibition, since both phenomena peak when ERα expression is increased and bcl-2 reduced [START_REF] Truchet | Interconnections between E2-dependent regulation of cell cycle progression and apoptosis in MCF-7 tumors growing on nude mice[END_REF][START_REF] Detre | Time-related effects of estrogen withdrawal on proliferation-and cell death-related events in MCF-7 xenografts[END_REF]. With regard to PR modulation, in MCF-7 cells, estradiol strongly upregulates PR levels, measured either by gene expression or by functioning protein [START_REF] Berkenstam | Hormonal regulation of estrogen receptor messenger ribonucleic acid in T47Dco and MCF-7 breast cancer cells[END_REF][START_REF] Jensen | Differential regulation of specific genes in MCF-7 and the ICI 182 780-resistant cell line MCF-7/182R-6[END_REF]Frasor et al. 2003). The estradiol-induction of PR gene expression and protein levels is inhibited by SHBG [START_REF] Fazzari | The control of progesterone receptor expression in MCF-7 breast cancer cells: effects of estradiol and sex hormone-binding globulin (SHBG)[END_REF][START_REF] Catalano | Sex hormone-binding globulin selectively modulates estradiol-regulated genes in MCF-7 cells[END_REF].

Finally, the effect of SHBG on gene expression is highly selective, depending on its interaction with cells, and restricted to genes associated to cell growth and estrogen-sensitivity.

SHBG POLYMORPHISMS IN BREAST CANCER

Some nucleotide variations in the human SHBG gene have been described, in both coding and regulatory sequences. Two different mutations (P156L and 326) leading to an apparent absence of SHBG in one individual with symptoms of androgen excess have been reported [START_REF] Hogeveen | Variations in the human sex hormone binding globulin (SHBG) gene associated with hyperandrogenism and ovarian dysfunction[END_REF], but these mutations appear to be extremely rare.

On the contrary, a single-nucleotide polymorphism, Asp327Asn (D327N), which introduces an additional consensus site for N-glycosylation, is present in the SHBG gene exon 8 [START_REF] Power | Molecular analyses of a human sex hormone-binding globulin variant: evidence for an additional carbohydrate chain[END_REF]) and is found worldwide [START_REF] Van Baelen | Genetic variation of human sex hormonebinding globulin: evidence for a worldwide bi-allelic gene[END_REF]). This substitution leads to an increase in the A c c e p t e d M a n u s c r i p t 12 half-life of human SHBG [START_REF] Cousin | Human variant sex hormone-binding globulin (SHBG) with an additional carbohydrate chain has a reduced clearance rate in rabbit[END_REF]) and may be associated with higher SHBG levels in variant allele carriers.

A further polymorphism in the SHBG gene 5'-flanking region has been reported [START_REF] Hogeveen | Human sex hormone-binding globulin promoter activity is influenced by a (TAAAA)n repeat element within an alu sequence[END_REF]. It is a pentanucleotide repeat polymorphism [PNRP (TAAAA) n ] within the human SHBG promoter that has a marked effect on its transcriptional activity in vitro in HepG2 cells. The number of TAAAA repeat elements ranged from 6 to 10 in the original report by Hogeveen and co-workers, but later studies found 11 repeats [START_REF] Xita | Association of the (TAAAA)n repeat polymorphism in the sex hormone-binding globulin (SHBG) gene with polycystic ovary syndrome and relation to SHBG serum levels[END_REF][START_REF] Cousin | Influence of SHBG gene pentanucleotide TAAAA repeat and D327N polymorphism on serum sex hormone-binding globulin concentration in hirsute women[END_REF]). This polymorphism has been studied in depth in women affected with Polycystic Ovary Syndrome (PCOS) who presented a significantly greater frequency of longer (TAAAA)n alleles (more than eight repeats) than normal women who had shorter alleles (less than eight repeats); carriers of the longer allele genotypes had lower SHBG levels than those with shorter alleles [START_REF] Xita | Association of the (TAAAA)n repeat polymorphism in the sex hormone-binding globulin (SHBG) gene with polycystic ovary syndrome and relation to SHBG serum levels[END_REF], Xita et al. 2008). Moreover, in these patients a strong disequilibrium linkage between the D327N v allele and the eight-TAAAA repeat was observed [START_REF] Cousin | Influence of SHBG gene pentanucleotide TAAAA repeat and D327N polymorphism on serum sex hormone-binding globulin concentration in hirsute women[END_REF]). Longer (TAAAA)n repeats in the SHBG gene promoter are also reported to be associated with more severe CAD in women undergoing coronary angiography [START_REF] Alevizaki | The importance of the (TAAAA)n alleles at the SHBG gene promoter for the severity of coronary artery disease in postmenopausal women[END_REF].

As far as the general population is concerned, Haiman and co-workers [START_REF] Haiman | Common genetic variation in the sex steroid hormone-binding globulin (SHBG) gene and circulating shbg levels among postmenopausal women: the Multiethnic Cohort[END_REF] evaluated the association between the (TAAAA)n repeat polymorphism, Asp327Asn polymorphism, and SHBG levels in a population of African-American, Native Hawaiian, Japanese, Latina, and Caucasian healthy postmenopausal women from the Multiethnic Cohort Study (n = 372). They found suggestive evidence of linkage disequilibrium between the Asn327 allele and the eight-repeat allele in all populations except the African-Americans. The individual genotypic classes contributed modestly to the overall prediction of SHBG levels, but carriers of the six-repeat allele were found to have significantly lower SHBG levels than non-carriers.

The Asp327Asn polymorphism has also been widely studied in relationship to breast cancer risk, and results from different studies are to some extent divergent, but nevertheless intriguing. In a study on both familial and sporadic breast cancer in Polish and Nordic populations (Försti et al. A c c e p t e d M a n u s c r i p t 13 2002) the 327Asn allele carriers correlated to an overall reduced breast cancer risk, but statistical significance was not attained. Dunning and co-workers [START_REF] Dunning | Polymorphisms associated with circulating sex hormone levels in postmenopausal women[END_REF] reported no significant association between the Asn variant and breast cancer risk, although they observed increased serum SHBG levels and a reduced estradiol to SHBG ratio in the same subjects. In a large population-based case-control study (1,106 cases, 1,180 controls) in postmenopausal women, Cui et al. [START_REF] Cui | Association of breast cancer risk with a common functional polymorphism (Asp327Asn) in the sex hormone-binding globulin gene[END_REF]) observed a significant association of the Asp327Asn polymorphism with reduced breast cancer risk. Furthermore, a significantly higher frequency of the polymorphism was observed in postmenopausal patients with ER-positive breast cancer than in ER-negative [START_REF] Becchis | The additionally glycosylated variant of human sex hormone-binding globulin (SHBG) is linked to estrogen-dependence of breast cancer[END_REF]) and more recently Costantino and co-workers [START_REF] Costantino | Molecular mechanisms of the D327N SHBG protective role on breast cancer development after estrogen exposure[END_REF]) observed a significantly higher frequency of the polymorphism in postmenopausal women taking Hormone Replacement Therapy (HRT) who did not develop breast cancer than in their counterparts who did, suggesting a protective role of D327N SHBG in estrogen-dependent breast cancer. The protective role of D327N SHBG in estrogen-dependent breast cancer was further supported by data obtained in vitro [START_REF] Costantino | Molecular mechanisms of the D327N SHBG protective role on breast cancer development after estrogen exposure[END_REF]). In MCF-7 cells, D327N SHBG is more effective than wild type protein in inhibiting estradiol-induced cell proliferation and anti-apoptosis, and this is due to the fact that D327N SHBG binds to MCF-7 cells to a greater extent than does wild type protein. Lastly, D327N causes greater induction of the second messenger cAMP and stronger inhibition of the estradiol-induced Erk ½ phosphorylation, and these data provide evidence for the mechanism of D327N SHBG protective action in breast cancer.

In a recent very large study [START_REF] Thompson | Identification of common variants in the SHBG gene affecting sex hormone-binding globulin levels and breast cancer risk in postmenopausal women[END_REF]) ten single nucleotide polymorphisms (SNPs) within or close to the SHBG gene were found to be significantly associated with SHBG levels, as was the (TAAAA) n polymorphism. At least 3 SNPs showed associations with SHBG levels that were highly significant, though relatively small in magnitude. In particular, rs6257 (here referred as to D356N, but normally referred as to D327N) is a potential breast cancer susceptibility variant. In addition, there was no evidence of any association between the (TAAAA) n polymorphism and breast cancer risk.

A c c e p t e d M a n u s c r i p t 14 A study is currently in progress in our laboratory on the (TAAAA) n repeat distribution and role in breast cancer patients [START_REF] Piccioni | The (TAAAA)n repeat of Sex Hormone-Binding Globulin gene promoter in breast cancer patients: distribution and implication[END_REF]). We have found that the distribution of the (TAAAA) n alleles differs significantly between breast cancer patients (79.3% with ≤ 8 repeats; 20.7% > 8 repeats) and healthy controls (59.8 % with ≤ 8 repeats; 40.2 % > 8 repeats). There is also a strong disequilibrium linkage between D327N polymorphism and the 8 TAAAA repeat in breast cancer patients, confirming other studies.

Overall, it can be concluded that relationships between the genetic determinants of SHBG and breast cancer are complex, but are worthy of considerable research effort since genetic SHBG variations in breast cancer may account for different risk profiles.

CONCLUSIONS AND FUTURE PERSPECTIVES

The body of information about the involvement of SHBG in breast cancer has been growing since the very first reports in the early 1980s. At the same time, our knowledge about the role and the mechanisms of action of the protein in breast cancer cells has been increasing and, although it is still far from exhaustive, we can now delineate quite a detailed scenario. SHBG plays a protective role in the exposure of breast cells to estrogens. Thanks to its unique property of regulating estrogen free fraction and to cross-talk with estradiol pathways in breast cancer cells, SHBG smoothes estrogen effects and has a pivotal effect in reducing breast cancer cell growth and proliferation (Figure 2). Structural alterations to the protein, such as the introduction of an additional carbohydrate chain in D327N SHBG, can modify its functional properties, in some cases increasing its protective function. Finally, SHBG and its polymorphism may correspond to different profiles of breast cancer risk that are not still fully understood.

We believe that this field deserves further study, especially with the aim of clarifying the putative interaction of SHBG with matrix associated proteins in breast cancer, and the role of SHBG in the definition of estrogen-dependence of breast cancer.

  A c c e p t e d M a n u s c r i p t 8

  Fissore F, Fazzari A, Berta L, Giudici M, Frairia R. 1991. Sex steroid-binding protein interacts with a specific receptor on human premenopausal endometrium membrane: modulating effect of estradiol. Steroids 56: 341-346. Fortunati N, Fissore F, Fazzari A, Berta L, Benedusi-Pagliano E, Frairia R.1993. Biological relevance of the interaction between sex steroid binding protein and its specific receptor of MCF-7 cells: effect on the estradiol-induced cell proliferation. J Steroid Biochem Mol Biol 45: 435-444. Fortunati N, Fissore F, Fazzari A, Becchis M, Comba A, Catalano MG, Berta L, Frairia R. 1996. Sex steroid binding protein exerts a negative control on estradiol action in MCF-7 cells (human breast cancer) through cyclic adenosine 3',5'-monophosphate and protein kinase A. Endocrinology 137: 686-692. Fortunati N, Fissore F, Fazzari A, Piovano F, Catalano MG, Becchis M, Berta L, Frairia R. 1999. Estradiol induction of cAMP in breast cancer cells is mediated by foetal calf serum (FCS) and sex hormone-binding globulin (SHBG). J Steroid Biochem Mol Biol 70: 73-80. Fortunati N, Frairia R, Fissore F, Berta L, Fazzari A, Gaidano G. 1992. The receptor for human sex steroid binding protein (SBP) is expressed on membranes of neoplastic endometrium. J Steroid Biochem Mol Biol 42: 185-191. Frasor J, Danes JM, Komm B, Chang KCN, Lyttle R, Katzenellenbogen BS. 2003. Profiling of estrogen up-and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 144: 4562-4574.

Figure 1 -

 1 Figure 1 -Mechanism of action of SHBG in breast cancer cells and cross-talk with estradiol

Figure 2 -

 2 Figure 2 -SHBG, estradiol and breast cancer.

Figure

  Figure

ACKNOWLEDGEMENTS

Thanks to: Laura Berta, Annamaria Fazzari, Federica Fissore, Marzia Becchis, Elisabetta Benedusi-Pagliano, Alessandra Comba, Patrizia Ferrera, Mariangela Raineri, Silvia Bertino,Lucia Costantino, Mariateresa Pugliese. Special thanks to our maestro, Gianpiero Gaidano.


Chris Kimble

Building Effective Virtual Teams: How to Overcome the Problems of Trust and Identity in Virtual Teams

This article explores some of the challenges faced when managing virtual teams, in particular the role played by trust and identity in virtual teams. It outlines why teams and virtual teams have become a valuable part of the modern organization and presents ten short case studies that illustrate the range of activities in which virtual teams can be found. Following this, the article examines some of the common problems encountered in virtual team working. It discusses two broad classes of solutions. The first are solutions that are essentially technical in nature (i.e., where changes to or improvements in technology would help to solve or ameliorate the problem); the second are more organizationally based (i.e., where the root of the problem is in people and how they are managed). The article concludes that both the technical and the organizational solutions need to be considered in parallel if an attempt to build an effective virtual team is to be successful.

Introduction

Workgroups of various sorts are the fundamental building blocks of the organization. In the traditional organization, these groups would be functional departments, like sales, engineering, or manufacturing. However, in recent years, many companies have begun to move toward a style of working that is explicitly cross-functional and built upon flatter organizational structures. Instead of the traditional functional areas and hierarchies, these companies are moving toward team-based structures, where groups of people take responsibility for a particular organizational deliverable. In some cases, these may be whole products or services; in other cases, they may be sub products or some other element of the organization's value chain.

In an era of increasing globalization, international trade, and fast communications networks, these team-based structures may consist of members that are located in different buildings, different cities, or even different continents. Teams that operate in this way are sometimes called "virtual teams." This article will discuss some of the challenges that organizations face when managing virtual teams and, in particular, will examine the role played by trust and identity. It will explore how organizations respond to these challenges under two broad headings: those that are principally "technical" problems in the sense that technology can help to solve or reduce the effect the problem has and those that are "organizational," where the solution to the problem lies mainly with people and how they are managed, before going on to discuss how these challenges should be dealt with in practice.

Teams

Teams are organizational units that share a common goal and whose members have a feeling of mutual responsibility for the results that the team produces. Thus, to some extent, teams are self-managing, as to work in this way requires a degree of mutual commitment if the team is to achieve its goal. Because teams are not fixed, they can be more easily formed and disbanded than traditional hierarchical structures; thus, teams and team working offer a flexible, dynamic, and efficient way of organizing working life.

Teams also allow members to share information that would previously have never crossed the walls of the traditional functional silos based on departments. C. Kimble. Building effective virtual teams: How to overcome the problems of trust and identity in virtual teams. Global Business and Organizational Excellence, 30(2), 2011, pp. 6-15. DOI: 10.1002/joe.20364

In essence, teams provide vehicles for learning from others. To learn, people need time to reflect and a safe environment. By working together over a period of time, team members are able to develop a sense of trust and shared identity that increases their ability to share and learn from each other. If a team can develop a sense of trust and mutual respect, then people feel able to share their thinking, the reasons behind their conclusions, and even the doubts that they have about their conclusions. Together they can build on each other's ideas, create new ideas, and develop new insights.

Team-working strategies have been used in a variety of industries, from mobile phones to banking and financial institutions. Some of the earliest examples of this can be found in manufacturing, where teams of multi-skilled operators worked together to build a complete subassembly [START_REF] Gyllenhammar | People at work[END_REF]. However, the same phenomenon can be found in product development, where people from different areas of the company work together to design new products, and in the service sector, where people with different backgrounds work together to provide a range of different services for a client.

Virtual Teams

Although examples of the move toward team-based organizational structures can be found dating back to the 1970s, the nature of modern teamwork has changed significantly. Thanks in part to the power of information technology and fast and reliable communications networks, organizations have become more geographically distributed, and firms have increasingly begun to work in partnerships that span different industries. Where once teams were about relationships inside an organization, now they frequently include those that were previously considered to be outside, such as customers, suppliers, and other organizations with whom they collaborate. For example, [START_REF] Kimble | Innovation and knowledge sharing across professional boundaries: Political interplay between boundary objects and brokers[END_REF] describe how two firms who were potentially competitors were able to cooperate with each other and their clients to build a customer relationship management system that neither would have been able to build on its own. The term virtual team can be applied to a number of different types of groups. Team membership may be relatively stable (e.g., in an established sales team) or change on a regular basis (e.g., in project teams). Members may be drawn from the same organization or from several different organizations (e.g., when projects involve consultants or external assessors). Team members may work in close proximity (e.g., in the same building) or geographically distantly (e.g., in different countries) and, similarly, team members may work at the same or at different times (e.g., depending on whether the team members are in the same time zone).

However, despite the widespread use of mobile telephones, computers, and the Internet, truly virtual teams, in the sense that they only ever "meet" together through technology, are relatively rare. A far more common scenario is one where team members continue to engage in face-to-face contact of one form or another throughout the life of the team. Most "virtual" teams operate in multiple modes: sometimes face to face, sometimes via electronic communication, sometimes interacting with each other directly, and sometimes working as individuals. Managing such teams means managing a whole spectrum of different communication strategies and techniques, as well as managing the human and social processes that nourish and support the esprit de corps that makes a team a team.

However, before we go on to look at the problems of managing virtual teams, we will first look at some case studies that illustrate the range of virtual teams that can be found in different sectors [START_REF] Kimble | Effective virtual teams through communities of practice[END_REF]. The information about these virtual teams was collected through face-to-face interviews, combined with other forms of correspondence such as e-mails, faxes, company reports, and phone calls. C. Kimble The first example (see case 1 in Exhibit 1) is a virtual team that involves a CASE (Computer-Aided Software Engineering) tool supplier and its main customer in the United Kingdom. The supplier develops a range of software applications for customers in aerospace and defense, telecommunications, electronics, systems software, and manufacturing. As part of the services it provides, it offers constant, high-quality technical support to its customers. In the past, this was provided by the supplier sending experts directly to customers' sites, but now a virtual team-based solution has enabled the company to find a more effective way of supporting its customers as well as provide them with greater responsiveness and flexibility.

The task of supporting these systems is a complex undertaking, potentially involving interactions at many different technical and managerial levels, and requiring high levels of interactivity among geographically dispersed individuals. In order to provide the level of support that is required, high specification workstations, which have the ability to support a wide range of applications simultaneously, need to be networked together because problem solving often requires simultaneous step-by-step operations on CASE tools at both ends of a link. A multimedia communications link, with audio-visual capability, supports direct interaction between users and experts. Taking this approach, the geographical flexibility of the experts and the responsiveness of the service has improved significantly, especially in the case of mission-critical situations such as a systems breakdown.

C. Kimble. Building effective virtual teams: How to overcome the problems of trust and identity in virtual teams. Global Business and Organizational Excellence, 30(2), 2011, pp. 6-15. DOI: 10.1002/joe.20364

The system now covers three sites: the head office in southeast England, a remote office in the north of England, and a large customer site in the West Midlands. The services offered by the company include tele-support for tool use (such as installation, problem identification, and decisions about tool-use and tool-method issues), tele-guidance for systems engineering methods (such as queries, method teaching, and modeling alternatives), and tele-reviews of analysis and design models (via synchronized model browsing).

Similar applications were identified in two other companies: one in a team of software developers in Northern Ireland who develop software remotely for a client in London (case 2 in Exhibit 1) and the other in a team of software engineers in Scotland (case 3 in Exhibit 1) who work from home on a variety of software-development projects. In all three cases, both the audio-visual and data communications facilities of the systems were essential if the teams were to work together effectively.

A different approach to virtual teams was identified in a large law firm in Germany with multiple offices (case 4 in Exhibit 1). The provision of the full range of professional legal services in remote locations, with only small branch offices and a limited number of clients, is expensive. The result is a lower level of service in rural areas. This often has a selfreinforcing effect, since poorer-quality services discourage firms that have more specialized legal needs from locating in these areas. However, this situation has been alleviated to some extent by the use of dedicated telecommunications networks that support the provision of legal services in branch offices through providing direct communication links to larger offices in more central locations.

In this case, the solution was developed between a main office and two branch offices in northern Germany. The intention was not only to provide enhanced services to remote locations but also to reverse the trend for experienced legal experts to migrate to the cities by offering them the possibility of providing their services from more remote rural locations. The idea behind the innovation was that a particular legal expert would not have to be based in the main office but could provide services from a branch office. This application required reasonable quality videophones that could support the sort of direct exchanges one might find in any legal consultancy. In addition, the system also needed to support the simultaneous viewing of images or documents, and the transmission of large volumes of case file data. Despite various difficulties, the system has significantly improved the geographical flexibility of the firm's legal experts and the quality and responsiveness of the firm's service to customers.

Similar virtual teams were identified in other sectors. In France, a business services company (case 5 in Exhibit 1) set up a system to support communication among its central office in Paris, three satellite offices in the suburbs, and several regular clients. The system enabled direct communication, and the simultaneous viewing and editing of word-processed documents, through an audio-visual data link with clients. This allowed complex editing and formatting issues on documents to be quickly resolved. Direct communications between the central and satellite offices of the company were also used for the allocation and scheduling of work. This system helped to improve some existing problems, such as errors and delays in correcting typed documents, which were major elements constraining the further expansion of the company.

In southern Italy, a system was developed to link together several academic and research institutions to provide a full range of research, training, and consultancy services for industry (case 6 in Exhibit 1). The system they use supports the transmission of large volumes of multimedia information (including audio, images, graphics, text, and data) in real time.

Another example can be found in a market research firm (case 7 in Exhibit 1). Here a new system was developed to support collaboration within a team of market researchers, C. Kimble. Building effective virtual teams: How to overcome the problems of trust and identity in virtual teams. Global Business and Organizational Excellence, 30(2), 2011, pp. 6-15. DOI: 10.1002/joe.20364 consultants, and a manager who all work from their own homes. The system supported communication between a central office and remote workers and offered facilities for information storage, the configuration of a central database, and the preparation of client reports.

In Scotland, a system was developed between a large central hospital and a small clinic on a remote island (case 8 in Exhibit 1). The system is used to transmit high-quality X-ray images, together with other audio, visual, and text support, in real time in order to facilitate remote diagnosis by medical experts in the central hospital. Similarly, in Greece, a new system was developed to provide a full-time medical consultancy involving a major teaching hospital in a large urban area and some small clinical units based in remote rural areas (case 9 in Exhibit 1). The final case study was of a homework-based telephone enquires service in Portugal (case 10 in Exhibit 1) where a data network was used to support the management and supervision of home-based workers and to enable communication between co-workers in order to provide support and guidance and to help avoid problems of social isolation.

The creation of virtual teams in these case studies brought a range of benefits to the organizations and individuals concerned, including increased geographical flexibility for team members; more efficient and effective use of expert time; improved responsiveness; increased productivity; more satisfying working environments; and reduced costs. However, the process of creating and operating these teams was not problem-free, and to achieve their full potential, a number of barriers needed to be overcome.

In all of the case studies, there were a number of relatively straightforward technical problems, ranging from unreliable systems and incompatible networks to slow computers and poor response times at certain times of the day. However, the issues of trust and identity posed a more serious impediment to the effectiveness of these virtual teams. The issue of trust was most apparent where team members needed to share work-in-progress electronically. For example, the software developers in cases 2 and 3 were reluctant to share half-finished programs with others. Similarly, the consultants and market researchers (cases 6 and 7) were often unwilling to share half-written reports with colleagues.

Even when team members are prepared to share information and knowledge with each other, the time and effort required to manage communication can be a serious problem. This is mainly associated with the issue of identity: knowing whom one is in communication with, and precisely how that communication should be managed. This problem is often seen in medical settings, such as cases 8 and 9, where a number of different professions need to communicate across professional boundaries. For example, Kindberg, Bryan-Kinns, and Makwana (1999) describe the technical and professional trade-offs that clinicians need to make when they are unable to ascertain exactly who their reports will be seen by and in what context they will be read.

In the sections that follow, we will discuss some of the challenges faced in managing virtual teams under two broad headings. The first concerns problems that are in some way related to the technology that is used. These are not technological problems per se, but the human and managerial problems that the perceived deficiencies in the technology cause. The second are essentially organizational problems, where the issue is more concerned with people, and how they are managed in virtual environments.

Technological Problems

Establishing trust in online transactions is a well-recognized problem in e-commerce; it is also a significant challenge for virtual teams. Online communication lacks the richness of face-to-face interaction. Relying solely on online communication tends to inhibit participation C. Kimble. Building effective virtual teams: How to overcome the problems of trust and identity in virtual teams. Global Business and Organizational Excellence, 30(2), 2011, pp. 6-15. DOI: 10.1002/joe.20364 and the creation of trust and the sense of mutual responsibility that characterizes teamwork. [START_REF] Jarvenpaa | Communication and trust in global virtual teams[END_REF] have highlighted the importance of the first "online impression," which can set the tone for much of the later discussion. [START_REF] Jarvenpaa | Toward contextualized theories of trust: The role of trust in global virtual teams[END_REF] argue that trust has both a direct and a mediating effect on team effectiveness. Given this problem, most virtual teams will still tend to favor holding occasional face-to-face meetings. For example, a study by [START_REF] Kimble | Dualities, distributed communities of practice and knowledge management[END_REF] that dealt with a virtual team spread across three continents found that although the team had access to some of the most sophisticated and up-to-date communications technology available, the "boost" provided by face-to-face meetings was needed to sustain it through extended periods of online communication.

Studies have identified two principal modes of interaction in virtual teams [START_REF] Sivunen | Team leaders' technology choice in virtual teams[END_REF]. The first is sometimes termed "hot" virtual working and is also known variously as "closely coupled," "tightly coupled," or "online" working. This is interaction in the sense that we would normally think of it -that is, synchronous, fluid, and requiring the active participation of the other members of the team. The second is "cold" virtual working, which is sometimes termed "loosely coupled" or "off-line" working. This form of interaction is work in the sense that it is part of some collective activity directed toward a shared goal or common purpose, but it is work that is performed individually. In general, it does not require the active presence of the other members of the group and can be performed alone and asynchronously.

During the life of a team, the interaction between its members moves repeatedly between these modes of communication. For example, [START_REF] Ribeiro | Quantum phenomena in communities of practice[END_REF] noted how people in the groups they studied would sometimes use the techniques of cold distributed working even when they shared the same physical office space. However, while their use of technology was "fluid and almost transparent" (Ribeiro et al., 2010, p. 27), their decision about when to use it was not. The members of the groups were happy to use technology to maintain day-today contact, yet when it became too difficult or involved delicate decisions, face-to-face meetings were still the preferred method of communication. When face-to-face meetings are feasible, this need not be a problem, but in situations where they are either too costly or would be inefficient, as in the example of the software support teams in case 1, other solutions need to be found.

The technological solution to the problem of not being able to meet somebody face to face relies on the creation of what is called social presence. The classic definition of social presence is the degree of awareness of other people in an interaction and the subsequent recognition of interpersonal relationships [START_REF] Short | The social psychology of telecommunications[END_REF]. Now social presence, or co-presence, is more commonly taken to mean the degree to which one can form a sense that one is interacting with another individual. Early attempts to deal with this problem relied on trying to provide "contextual information" about the other person, usually in the form of textual descriptions; later attempts relied on avatars (computer-generated animated images) to simulate co-presence. Now this problem is most often dealt with by some form of teleconferencing. In case 1, where establishing a close working relationship quickly was critical, great efforts were made to create a sense of co-presence. However, in other cases, such as case 5, this was of less importance, and document management software that supported the simultaneous editing of documents was simply augmented by standard tele-conferencing facilities.

Organizational Problems

As we have seen, without effective communication effective teamwork becomes difficult. While some aspects of this problem can be dealt with by technological means, others are more fundamentally rooted in the ways in which people work and are managed. Identity, for C. Kimble. Building effective virtual teams: How to overcome the problems of trust and identity in virtual teams. Global Business and Organizational Excellence, 30(2), 2011, pp. 6-15. DOI: 10.1002/joe.20364 example, plays a critical role in communication, where knowing the identity of those with whom you communicate is central to creating a shared understanding. Yet in virtual teams, the status and identity of a person can be ambiguous, as many of the basic cues that exist in the physical world may be absent; in the virtual world, one can have as many electronic personas as one has the time and energy to create. At the individual level, this can lead to the problems of trust outlined earlier, but at the level the team, it can lead to difficulties in establishing a sense of collective identity and can inhibit effective communication among team members.

Identity helps to establish shared meanings through providing a common perspective on, for example, where somebody's job fits within the wider organization. Without this common ground, it becomes difficult to share knowledge effectively. At a more fundamental level, unless shared meanings can be established, even "common sense" words and terms become open to different interpretations, as the same word may be used in different ways in different settings. Identity in this sense is defined by a group rather than by an individual. Within a group, a shared interest or a common domain of knowledge provides the "common sense" definitions for the words and terms used by the group. Sharing that group's identity implies not only a commitment, but also shared knowledge and shared competencies.

Teams are mainly focused on achieving a task rather than building a sense of identity. Consequently, we need to look elsewhere for a different type of group structure if we are to solve the problems of building and maintaining group identity.

As Exhibit 2 illustrates, Communities of Practice are a different cut on the organization's structure that emphasizes the learning that people do rather than the functional unit they report to or the project they are working on [START_REF] Wenger | Communities of practice: Learning as a social system[END_REF]. Because membership in a Community of Practice is based on a shared interest, it can cross organizational boundaries and span structures and hierarchies. Communities of Practice are "not just places where local activities are organized, but where the meaning of belonging to broader organizations is negotiated and experienced" (Wenger, 1996, p. 25). Thus, for example, people who work in cross-functional teams might form a Community of Practice to keep in touch with their peers and keep up to date with what is happening in their particular field of specialist expertise.

Richard McDermott coined the phrase "the double-knit organization" [START_REF] Mcdermott | Learning across teams: The role of communities of practice in team organizations[END_REF] to describe an organization that combines teams with Communities of Practice and by doing so overcomes some of the problems of maintaining a sense of identity online. He argues that cross-functional teams focus on outputs such as products, processes, or market segments, while Communities of Practice focus on working together to solve shared problems, to learn, and to build a body of knowledge. He sees Communities of Practice as a way to maintain a technical focus within a broader discipline, while cross-functional teams serve to unite those disciplines around a common product. Instead of sharing product or process-specific information via team leaders, Communities of Practice share knowledge and standardize practices across teams.

C. [START_REF] Pemberton-Billing | Distributed design teams as communities of practice[END_REF] used the concept of a Community of Practice to highlight some of the root causes of the problems they found in their study of distributed design teams, such as the problems that a hierarchical client/supplier relationship caused when attempting to create a common sense of purpose for the team. Similarly, research by [START_REF] Hildreth | Going the extra half-mile: International communities of practice and the role of shared artifacts (Unpublished doctoral dissertation)[END_REF] has indicated that the willingness to go "the extra half mile" in a Community of Practice can help overcome many of the problems associated with issues of trust and identity in virtual working and enable relationships in virtual teams to develop quicker, go further, and provide a sound basis for subsequent hot and cold electronic collaboration.

Conclusions

The goal of this article was to discuss some of the challenges that organizations face when managing virtual teams. One of the main conclusions must be that there are no easy answers. To ensure that virtual teams work effectively, you need to address both the people issues and the technology, rather than look for the answer in one or the other. Davis refers to this as "the Tao of leadership in virtual teams" [START_REF] Davis | The Tao of leadership in virtual teams[END_REF] and describes managing virtual teams as managing a paradox. He notes, "Numerous paradoxes exist in virtual teams. Attempts to focus on one side of the paradox yield only limited success" (Davis, 2004, p. 57). Some problems can be ameliorated by improving or updating technology, and others by finding new ways to organize workgroups. However, truly effective virtual teams will only be built upon understanding the limitations of virtual working on a human scale and finding ways, both technological and managerial, to overcome them.

While it is clear that, because virtual teams are composed of groups of people who must work together as a single cohesive entity, technology alone can never provide all of the solutions, the role of technology cannot be ignored. Thus, [START_REF] Breu | Making organisations virtual: The hidden cost of distributed teams[END_REF] describe how an attempt to create virtual teams in the public sector floundered because of an unreliable and inadequate technological infrastructure. Similar problems can be found in the private sector, where a visit to a client's site can mean that resources that are readily available in the headquarters are no longer accessible.

Although technology cannot be ignored, it is not always the case that particularly sophisticated technology is needed. For example, [START_REF] Henttonen | Managing distance in a global virtual team: The evolution of trust through technology-mediated relational communication[END_REF] A different solution to the problems of virtual working is to blend virtual teams and Communities of Practice in a "double-knit" organization. Even here, we find a paradox. Just as McDermott concedes that teams can become the new functional silos because a team's focus on fulfilling a task can lead to isolation and team myopia [START_REF] Mcdermott | Learning across teams: The role of communities of practice in team organizations[END_REF], so others have found that his solution to the problem, Communities of Practice, can have the same shortcomings. Hislop (2003), for example, found that certain types of knowledge, although valuable to others, never moved beyond the boundaries of the community. Similarly, while the self-managing characteristics of teams can be a bonus, the same characteristic in a Community of Practice can be a problem. [START_REF] Gongla | Where did that community go? Communities of practice that "disappear[END_REF], for example, found numerous examples of Communities of Practice that simply "disappeared" when the interests of the community and the interests of the organization diverged. While some of the theoretical concepts from Communities of Practice might help to inform and give insights into the way that virtual teams operate, Communities of Practice alone are not the whole solution.

The desire to communicate and work with others in groups is part of human nature, but the rapid development of communications technology, in all of its forms, has added a new dimension to this basic desire. Within a single generation, we have moved from fixed location, one-to-one communication by telex and telephone to a whole range of different possible modes of communication, all of which have been opened up by the sudden and rapid expansion of digital networks. Perhaps, to paraphrase Mark Twain, Cairncross's reports of "the death of distance" [START_REF] Cairncross | The death of distance: How the communications revolution will change our lives[END_REF] have been greatly exaggerated, but there is no doubt that, technically at least, working at a distance is no longer the challenge that it once was. The challenge to us as human beings is in how we adapt and respond to these new opportunities.

Main activity Intra or inter- organizational team Synchronous or asynchronous communication Geographical spread Number of geographical locations Case 1

	Exhibit 1. The Case Studies
		Software	Inter-	Synchronous	National /	3-5
		support	organizational		International
	Case 2	Software	Both	Both	National	2-5
		development
	Case 3	Software	Intra-	Both	Regional	3-8
		development	organizational
	Case 4	Legal	Intra-	Mostly	Regional	3
		services	organizational	synchronous
	Case 5	Secretarial	Intra & Inter-	Synchronous	Local	2-4
		services	organizational
	Case 6	Research and	Inter-	Both	Regional	2-5
		consultancy	organizational
	Case 7	Home based	Intra-	Both	Local	2-5
		market	organizational
		research
	Case 8	Hospital and	Inter-	Synchronous	Regional	2
		medical	organizational
		services
	Case 9	Hospital and	Inter-	Synchronous	Regional	2-3
		medical	organizational
		services
	Case	Enquiries for	Intra-	Both	Regional	2 +
	10	home based	organizational
		workers
	4
		. Building effective virtual teams: How to overcome the problems of trust and
	identity in virtual teams. Global Business and Organizational Excellence, 30(2), 2011, pp. 6-
	15. DOI: 10.1002/joe.20364

Virtual Teams in Practice: Some Illustrative Case Studies

  Kimble. Building effective virtual teams: How to overcome the problems of trust and identity in virtual teams. Global Business and Organizational Excellence, 30(2), 2011, pp. 6-15. DOI: 10.1002/joe.20364 Exhibit 2. Comparison of Communities of Practice and Teams (Adapted From[START_REF] Wenger | Communities of practice: The organizational frontier[END_REF] Other research has also indicated that Communities of Practice may be one way to make some inroads into the complexities and challenges of virtual working. Pemberton-Billing,

		What's the	Who belongs?	What holds it	How long does it
		purpose?		together?	last?
	Community of Practice	To develop members' capabilities and to build and exchange knowledge	Members select themselves	Passion, commitment, and identification with the group's expertise	As long as there is interest in maintaining the group
	Team	To accomplish a specified task	Employees assigned by senior management	The project's milestones and goals	As long as the project

  describe how, in a global virtual team where most of the members had never met each other, traditional means of communication such as telephone and e-mail were more popular than Web based collaborative tools and groupware. It seems strange that although the concept of C. Kimble. Building effective virtual teams: How to overcome the problems of trust and identity in virtual teams. Global Business and Organizational Excellence, 30(2), 2011, pp. 6-15. DOI: 10.1002/joe.20364 a virtual team only became reality thanks to advances in information technology, the technology itself often seems to play but a small part.