Monja Knoll
email: monja.knoll@port.ac.uk

Lisa Scharrer
email: lisa.scharrer@port.ac.uk

Alan Costall
email: alan.costall@port.ac.uk

Running head: Analyses of infant-and foreigner-directed speech Are actresses better simulators than female students? The effects of simulation on prosodic modifications of infant-and foreigner-directed speech 1

Keywords: IDS, simulated speech, actresses, hyperarticulation

Previous research has used simulated interactions to investigate emotional and linguistic speech phenomena. Here, we evaluate the use of these simulated interactions by comparing speech addressed to imaginary speech partners produced by psychology students and actresses, to an existing study of natural speech addressed to genuine interaction partners. Simulated infant-(IDS), foreigner-(FDS) and adult-directed speech (ADS) was obtained from ten female students and ten female actresses. These samples were acoustically analysed and rated for positive vocal affect. Our results for affect for actresses and student speakers are consistent with previous findings using natural interactions, with IDS rated higher in positive affect than ADS/FDS, and ADS rated higher than FDS. In contrast to natural speech, acoustic analyses of IDS produced by student speakers revealed a smaller vowel space than ADS/FDS, with no significant difference between those adult conditions. In contrast to natural speech (IDS > ADS/FDS), the mean F 0 of IDS was significantly higher than ADS, but not than FDS. Acoustic analyses of actress speech were more similar to natural speech, with IDS vowel space significantly larger than ADS vowel space, and with the mean FDS vowel space positioned between these two conditions. IDS mean F 0 of the actress speakers was significantly higher than both ADS and FDS. These results indicate that training plays an important role in eliciting natural-like speech in simulated interactions, and that participants without training are less successful in reproducing such speech. Speech obtained from simulated interactions should therefore be used with caution, and level of experience and training of the speakers should be taken into account.

Introduction

An underlying dilemma in research is to remain proximate to the actual phenomenon under investigation, while at the same time achieving effective experimental control.

In linguistic and emotional speech studies, researchers have attempted to resolve this dilemma by using simulated speech to obtain standardised speech samples for comparison across different experimental conditions. In such studies, speakers are provided with specific instructions to address an imaginary audience or perform a particular scenario (e.g., [START_REF] Biersack | Fine-tuning speech registers: A comparison of the prosodic features of child-directed and foreigner-directed speech[END_REF][START_REF] Papousek | Tone and intonation in Mandarin babytalk to presyllabic infants: Comparison with registers of adult conversation and foreign language instruction[END_REF][START_REF] Picheny | Speaking clearly for the hard of hearing I: intelligibility differences between clear and conversational speech[END_REF][START_REF] Smiljani | Production and perception of clear speech in Croatian and English[END_REF][START_REF] Trainor | Is infant-directed speech prosody a result of the vocal expression of emotion?[END_REF]. However, the effects of such simulated interactions, for instance on prosodic modifications, are not well understood and merit further examination.

One area in which such methodologies have recently been used is research into prosody of speech directed to infants (IDS) and relevant comparison groups. These speech recipient groups will form the focus of the present investigation. IDS is acoustically and phonetically modified compared to adult-directed speech (ADS) (e.g. [START_REF] Kitamura | Pitch and communicative intent in mother's speech: Adjustments for age and sex in the first year[END_REF][START_REF] Kuhl | Early language acquisition: Cracking the speech code[END_REF][START_REF] Kuhl | Crosslanguage analysis of phonetic units in language addressed to infants[END_REF][START_REF] Stern | The prosody of maternal speech: Infant age and context related changes[END_REF], and is characterised by exaggerated pitch contours, increased mean pitch, hyperarticulated vowels and high emotional affect (e.g. [START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF][START_REF] Fernald | Expanded intonation contours in mothers' speech to newborns[END_REF][START_REF] Stern | The prosody of maternal speech: Infant age and context related changes[END_REF][START_REF] Trainor | Is infant-directed speech prosody a result of the vocal expression of emotion?[END_REF]. Acoustic modifications in IDS may have linguistic, emotional-affective and attentional functions (e.g. [START_REF] Fernald | Acoustic determinants of infant preference for motherese speech[END_REF][START_REF] Fernald | Expanded intonation contours in mothers' speech to newborns[END_REF]Kitamura andBurnham, 1998, 2003;[START_REF] Kuhl | Crosslanguage analysis of phonetic units in language addressed to infants[END_REF]Papousek et al., 1991). A growing body of research has attempted to separate these functions by comparing IDS with speech addressed to other linguistic (e.g. foreigners (FDS): [START_REF] Papousek | Tone and intonation in Mandarin babytalk to presyllabic infants: Comparison with registers of adult conversation and foreign language instruction[END_REF][START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] or emotional groups (e.g. pets or partners: [START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF][START_REF] Trainor | Is infant-directed speech prosody a result of the vocal expression of emotion?[END_REF], and a variety of different methodologies has been used in these investigations. For instance, IDS has been compared to ADS using student speakers and imagined interaction partners (simulated speech; e.g. [START_REF] Biersack | Fine-tuning speech registers: A comparison of the prosodic features of child-directed and foreigner-directed speech[END_REF][START_REF] Papousek | Tone and intonation in Mandarin babytalk to presyllabic infants: Comparison with registers of adult conversation and foreign language instruction[END_REF] or using mothers in natural interactions with real interaction partners [START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF][START_REF] Englund | Infant directed speech in natural interaction -Norwegian vowel quantity and quality[END_REF][START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF].

Simulated speech, which usually takes place in a laboratory context, offers a greater potential than natural speech to control factors such as speaker variability, room acoustics, speech content and consistency of recordings (e.g. [START_REF] Englund | Infant directed speech in natural interaction -Norwegian vowel quantity and quality[END_REF]. Using speakers' simulated speech to relevant groups is also more time efficient and convenient than using genuine interaction partners. In the case of IDS and FDS, it might be particularly difficult to find an infant or foreign confederate that is available over a prolonged period of time. However, resulting interactions of simulated speech might be contrived and unnatural. For instance, a person's ability to simulate speech addressed to certain listener groups might depend both on their previous exposure to these groups, and their 'acting' ability. The advantage of natural interactions is that they are genuine rather than contrived.

In view of these considerations, there is clearly a need to evaluate natural and simulated comparative IDS-based research to determine whether the two differ and in what way. For instance, two recent studies [START_REF] Biersack | Fine-tuning speech registers: A comparison of the prosodic features of child-directed and foreigner-directed speech[END_REF][START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] used these different methodologies to investigate differences between IDS (or childdirected speech), FDS and speech addressed to a British adult control group (ADS). [START_REF] Biersack | Fine-tuning speech registers: A comparison of the prosodic features of child-directed and foreigner-directed speech[END_REF] used simulated speech of student speakers, whereas [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] used natural interactions with mothers as speakers. With regards to fundamental frequency (F 0 ), both studies reported similar findings in that IDS achieved higher F 0 measures than the adult conditions. However, because [START_REF] Biersack | Fine-tuning speech registers: A comparison of the prosodic features of child-directed and foreigner-directed speech[END_REF] study reported findings on speech rate and F 0 range modifications, and Uther et al.'s (2007) study concentrated on examining hyperarticulation of vowels (vowel space expansion due to shifts in Formant 1 (F1) and Formant 2 (F2), see [START_REF] Kuhl | Crosslanguage analysis of phonetic units in language addressed to infants[END_REF]) and affective measures, the two studies are not directly comparable.

Two studies that attempted to compare natural and simulated interactions were carried out by [START_REF] Fernald | Expanded intonation contours in mothers' speech to newborns[END_REF] and [START_REF] Schaeffler | Comparing vocal parameters in spontaneous and posed child-directed speech[END_REF]. [START_REF] Fernald | Expanded intonation contours in mothers' speech to newborns[END_REF] found that F 0 contours in natural IDS were more exaggerated than F 0 contours in simulated IDS (both of which consisted of 'free speech' produced by mothers), but that simulated IDS presented more exaggerated F 0 contours than ADS.

Both interaction types used the same mothers, who had previous experience with their infants, and should therefore be better at producing simulated IDS than for instance a student speaker without such experience. Nonetheless, these authors were unable to replicate the occurrence of increased F 0 in simulated IDS. In contrast, [START_REF] Schaeffler | Comparing vocal parameters in spontaneous and posed child-directed speech[END_REF] found that child-directed speech with a real interlocutor (produced by mothers) differed only in terms of shimmer and harmonics-to-noise ratio from simulated childdirected speech (without interlocutor, produced by non-mothers), but not in terms of frequencies (F 0 , F1, F2). However, compared to [START_REF] Fernald | Expanded intonation contours in mothers' speech to newborns[END_REF] 'free speech' study, [START_REF] Schaeffler | Comparing vocal parameters in spontaneous and posed child-directed speech[END_REF] used target sentences and booklets for both their interactions, which are not entirely spontaneous.

Although both studies represent important steps in comparing simulated speech and speech directed to a real interlocutor, it remains unclear to what extent simulated interactions are comparable to genuine natural speech interactions. An understanding of the similarities and differences of these two approaches is crucial in determining whether findings of simulated studies are close approximations of natural interactions.

If simulated and natural speech are comparable, then other comparative speech types (e.g. hearing impaired speech) could be investigated using simulated interactions, particularly considering the investment in time and effort of natural interactions. We aimed to investigate this possibility by comparing results from an earlier study of natural IDS, FDS and ADS produced by mothers [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF]; see section 1.1 for summary) with recordings of simulated IDS, FDS and ADS by female student speakers. Student speakers were chosen because they constitute the most easily available speaker group in psychological research, and have been previously used in simulated speech research (e.g. [START_REF] Biersack | Fine-tuning speech registers: A comparison of the prosodic features of child-directed and foreigner-directed speech[END_REF][START_REF] Papousek | Tone and intonation in Mandarin babytalk to presyllabic infants: Comparison with registers of adult conversation and foreign language instruction[END_REF].

However, students may vary in their ability to simulate speech without a speech partner, or may even be unable to convincingly perform such a task. As a speaker group, they might perceive the situation as contrived because they are required to carry out a task that might be unfamiliar and uncomfortable particularly if they are not used to role-play. By comparison, a group that may be better suited for such a task are actresses that have undergone professional training, and should thus be more used to role-play and simulated interactions (e.g. use of bluescreen or telephone conversations without interaction partners). Recordings of actors' speech have already been extensively and successfully used in the area of vocal emotion (e.g. [START_REF] Banse | Acoustic profiles in vocal emotion expression[END_REF][START_REF] Kramer | Elimination of verbal cues in judgments of emotion from voice[END_REF][START_REF] Scherer | Vocal communication of emotion: A review of research paradigms[END_REF][START_REF] Scherer | Emotion inferences from vocal expression correlate across languages and cultures[END_REF][START_REF] Scherer | Multimodal expression of emotion: Affect programs or componential appraisal patterns?[END_REF][START_REF] Wallbott | Cues and channels in emotion recognition[END_REF]. Although a study by [START_REF] Viscovich | Acoustical analysis of posed prosodic expressions: Effects of emotion and sex[END_REF] found that results of affective prosody produced by actors may be generalisable to people without professional training, actors are generally considered to be better at portraying different basic emotions than lay people [START_REF] Scherer | Vocal communication of emotion: A review of research paradigms[END_REF]. It is thus reasonable to expect that actresses, due to their professional training, may be better at imagining talking to infants and foreigners that are not present, than psychology students without such training. We intended to assess this possibility by comparing actresses' simulated speech samples with those of the students and the natural data set of [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF].

1.1 Summary of [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] To provide the reader with a better understanding of the comparative natural data set [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF], we will present a summary of this study here. The [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] study attempted to clarify the functional role of IDS by investigating the independence of specific acoustic components (mean F 0 , vowel hyperarticulation and duration, affect) across IDS, ADS and FDS. Speakers consisted of 10 southern English mothers (mean age 30.7) in interactions with their infants, a foreign (Chinese) and southern English confederate (both adult females). All interactions were recorded in the mothers' houses, and mothers were left alone with their infant or the confederate during the 5 to 10 minute interactions. Mothers were provided with a toy sheep, shark and shoe to elicit specific target words, but the interactions were otherwise natural and spontaneous. These target words were chosen based on previous research [START_REF] Andruski | The acoustic structure of vowels in mothers' speech to infants and adults[END_REF][START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF], to elicit the three corner vowels /a/, /i/, /u/ for the detection of vowel hyperarticulation and to control for differing F1/F2 values due to co-articulation (the sound 'sh' preceded each corner vowel). [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] found IDS and FDS to be characterised by vowel hyperarticulation compared to ADS. However, ADS and FDS exhibited significantly lower mean F 0 and vowel duration than IDS, with no significant differences between the adult conditions. IDS also obtained higher ratings of emotional affect compared to the adult conditions, while FDS contained less positive vocal affect than ADS. The authors concluded that, consistent with Kuhl (e.g. [START_REF] Kuhl | Crosslanguage analysis of phonetic units in language addressed to infants[END_REF][START_REF] Kuhl | Early language acquisition: Cracking the speech code[END_REF], vowel hyperarticulation is a didactic device of IDS, independent of F 0 values and affect, and suggested that vowel hyperarticulation may serve a similar purpose in FDS.

Aims of the present study

The aim of the present study was to investigate similarities and differences in prosodic If simulated speech is a close approximation of natural speech, and based on the findings of [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF], we hypothesise that vowel space (hyperarticulation) of both speaker groups in IDS and FDS will be greater relative to their vowel space in ADS, with no difference between IDS and FDS. Secondly, we hypothesise that the speakers' mean F 0 values and vowel duration will be greater in IDS relative to ADS and FDS, with no difference between the adult conditions. Thirdly, affective ratings of positive vocal affect of IDS will be higher relative to ADS and FDS, with ADS achieving higher ratings than FDS. However, if role-play experience and the ability to act are important for producing simulated speech samples that are viable for psychological research, as indicated by [START_REF] Scherer | Vocal communication of emotion: A review of research paradigms[END_REF], actresses would be expected to perform more similarly to the mothers' natural speech than student speakers. In that case, we hypothesise that actress speakers will produce the same pattern of prosodic modification results for IDS, FDS and ADS as described above, with student speakers differing in at least one of these measures.

Method

Participants

Speakers

The speakers of the student interactions consisted of 10 females with a mean age of 22.9 years (sd 8.84) recruited from the student population of the University of Portsmouth. None of these speakers had previous acting experience. The speakers of the actress interactions consisted of 10 professional actresses with a mean age of 28.4 years (sd 13.06) recruited from theatres and acting schools in London. All actresses had undergone professional acting training (including voice coaching) and had been acting for more than a year (mean 5.6 years) with most of their acting work carried out in the theatre. Each of the speakers was asked to indicate their previous exposure to each of the speech groups (Likert-scale; 1 (not much exposure) to 5 (a lot of exposure)), the type of exposure, the difficulty of the task and their frustration with it, and which speech recipient group they perceived as the easiest and the most difficult.

Furthermore, we collected information about how they perceived their voice to have changed, and their prediction of how other people would rate their voice on positive affect in each of the speech groups. All speakers were British citizens with comparable southern English accents, who had been living in the south-east of England for most of their lives (> 16 years).

Listeners (raters)

For the ratings of vocal affect, we recruited different participants for both student and actress imaginary interactions, as both sets of interactions were not carried out simultaneously. We recruited 40 students in total (20 for the student interactions; 20 for the actress interactions) via the participant pool of the Department of Psychology, University of Portsmouth. Participants consisted of 8 males and 12 females with a mean age of 27.25 years (sd 10.37) for the ratings of the student interactions. For the ratings of the actress interactions, participants consisted of 6 males and 14 females with a mean age of 26.25 years (sd 10.12). None of the participants was hearing impaired, and all were British citizens. These participants were required to rate lowpass filtered speech samples of the interactions for vocal affect. Inter-rater reliability was found to be high (student interactions: reliability coefficient = .81; actress interactions: reliability coefficient = .88).

Acoustic analyses and filtering

A total of 346 words were analysed for the student interactions, and 417 words were analysed for the actress interactions (compared to 496 words in [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF].

Acoustic analyses were carried out using Praat 4.5.16 [START_REF] Boersma | Praat: doing phonetics by computer (Version 4.5.16[END_REF] and centred on the corner vowels /a/, /i/ and /u/ of the target words shark, sheep and shoe. Vowel sounds were firstly extracted using Soundforge 6.0, and the resulting sound samples were then analysed for mean F 0 , vowel duration, and F1 and F2 values (taken at the midpoint of the vowel). Mean F1/F2 values were then used as x/y coordinates to plot bivariate vowel triangles for /a/, /i/ and /u/ for each speaker's production of IDS, ADS and FDS, from which vowel triangle area was calculated to detect 'hyperarticulation' of the vowels. Plotting and calculation of the vowel space was carried out in Autocad 2000 (a software application for 2D and 3D drafting, design and modelling). The procedures used were identical to those of [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] and follow those of previous studies [START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF][START_REF] Kuhl | Crosslanguage analysis of phonetic units in language addressed to infants[END_REF].

For the affective analysis, approximately 30 seconds of each speech sample were low-pass filtered (Hann window) with a cut off point of 1000 Hz, and smoothing at 100 Hz. We used this level of low-pass filtering to keep our study comparable to [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] who also employed a 1000 Hz filter. Otherwise, the speech samples received no further modification. The 30-second speech samples were selected from the same part of the recording for each of the speaker's interactions (location on tape at approximately the 0.20 to 0.50 second mark). The first 20 seconds of the interactions were disregarded as we assumed that speakers might speak more fluently and natural later on the recording. Ideally, low-pass filtering should remove the intelligibility of speech without impairing its affective features, and the procedure has recently been used in IDS research (e.g. [START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF][START_REF] Kitamura | Pitch and communicative intent in mother's speech: Adjustments for age and sex in the first year[END_REF].

Procedure

Speech samples

The recordings were carried out in the laboratory with digital recording equipment.

All speakers were required to produce simulated speech directed to an imagined infant, a British and a foreign adult in separate interactions. For the infant interaction, the speakers were instructed to imagine that they were talking to a close family member (ideally to their own child). However, we provided no example or idea of what IDS should sound like. For the British and foreign adult interactions, speakers were instructed to imagine talking to a female stranger in her early twenties. In the foreign interactions they were instructed to imagine that the person was a female Chinese exchange student who had been living in the UK for less than two months, and that they might encounter some communication problems. It is important to note here that we did not explicitly instruct our participants to produce clear speech in either of these interactions. To keep these interactions consistent with [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] and to elicit the same target words, we supplied the speakers with three toys (a shark, a sheep and a shoe). The speakers were encouraged to use these toys for the interactions (e.g. to play with them with the imaginary infant, or to explain which toy they would buy for an infant in the adult interactions). Apart from the toy stimuli, the speakers were encouraged to construct and invent their own scenarios, which ideally should have resulted in free speech. Similar to the mothers in [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF], the speakers were left alone during the interaction. The order of the interactions for each speaker was counterbalanced, and each interaction lasted for approximately three minutes. Speakers were given 10 minutes between each interaction to prepare for the next interaction.

Affective ratings

Raters listened to 30 low-pass filtered speech samples (10 each in IDS, FDS and ADS for both imaginary student and actress interactions). Using a five point Likert-scale ( 1(not at all) to 5 (extremely)), participants were instructed to rate subjectively how much positive vocal affect they could detect in each 30-second speech sample. The presentation order of the speech samples was randomised (using Microsoft Office Excel 2003). This order was presented to the first half of the participants, and then reversed for the remainder of the participants. Each participant was supplied with two test trials of the filtered speech to familiarise themselves with the filtered sound.

Results

The data for the acoustic and affective analyses were subjected to 2 x 3 mixed measures ANOVAs with the speakers (actresses and students) as between-subjects factor (conditions) and the three speech recipient groups (IDS, ADS and FDS) as the within-subjects factor. Differences between groups were further explored with planned contrasts. Where any deviations from the assumptions of ANOVA were found, the ANOVA analyses were complemented by non-parametric tests. It should be noted here that ADS represented a baseline condition in the original study, with FDS as a linguistic comparison group to IDS.

Previous exposure to groups

We investigated whether actress and student speakers differed in self-reports of how much exposure they had to infants and foreigners. The means indicated that actresses rated themselves as having had more exposure to infants (mean 3.1, sd 1.2) and foreigners (mean 3.0, sd 1.2) than student speakers (IDS mean 2.9, sd 1.4; FDS mean 2.7, sd 1.5), and that overall both speaker groups rated themselves to have had more exposure to infants than to foreigners. However, these differences were not statistically significant, although it should be noted that these are self-reports and a reliable quantification of previous exposure would be difficult to achieve. In an attempt to further define whether the speakers differed in the type of exposure they reported, we divided their qualitative responses into four categories for IDS (casual (e.g. on the street, not much contact), friends (friends of the participants who had an infant), family (e.g. niece, nephew), own child (included older children)) and FDS (casual (e.g. on the street; not much contact), work (worked regularly with foreigners), friends (friends were foreigners), family (e.g. stepfather)). Distribution of the speakers in each of these categories can be found in Table 1. Fisher's exact test indicated that actresses and students did not report significantly different exposure to these categories. As these categories were not necessarily progressive, we collapsed them further into 'casual' (indicating little contact) and 'regular' (indicating regular contact with IDS and FDS, and encompassed the remaining categories for these speech recipient groups). However, we found no statistical difference between these categories for the actresses and the students.

Table 1 about here

There was a difference between student speakers and actresses in their perception of the difficulty, frustration and discomfort of the task. Student speakers (Median = 2.5, 2.5, 3.0 respectively) rated the task as more frustrating (U = 14.00, p < .002, r =-.648), difficult (U = 24.00, p =.025, r = .459) and uncomfortable (U = 8.00, p < .001, r = -.741) than the actresses (Median = 1.0, 2.0, 1.0 respectively). Eight of the ten student speakers reported that they found IDS the easiest condition (two reported ADS), and all of the student speakers reported FDS as the most difficult condition. Similarly eight of the ten actress speakers reported IDS as the easiest condition (two reported ADS), and the same number of actresses reported FDS as the most difficult condition (two reported ADS).

Acoustic analyses

F1/F2 vowel hyperarticulation

Mean formant values for the three corner vowels and the resulting vowel triangles for each speech recipient group for students and actresses can be found in Figure 1. We found a significant main effect of condition for vowel space (F (1, 18) = 14.136, p = .001, = .440), with the actress speakers producing greater vowel spaces across the three speech recipient groups than the student speakers (see Fig. 1 and2). We did not find a significant main effect of speech recipient groups, but a significant interaction between the conditions and the three speech recipient groups (F (2, 36) = 7.852, p = .001, = .304), which is due to an interaction between IDS and ADS (F (1, 18) = 19.889, p = .001, = .525).

Figure 1 about here

We explored these interaction effects further with planned contrasts for each of the groups. In the student imaginary interactions, IDS vowel space was significantly smaller than both ADS (F (1, 9) = 6.196, p = .034, = .408) and FDS (F (1, 9) = 8.982, p = .015, = .499; Fig. 2), with no significant difference between the adult conditions. In contrast, IDS vowel space in the actress interactions was significantly larger than ADS (F (1, 9) = 13.935, p = .005, = .608), but no significant differences between FDS and IDS, and between FDS and ADS were found (Fig. 2).

Figure 2 about here

The above results were confirmed with a Friedman two-way analysis of ranks on the effect of speech recipient groups for students ( 2(2) = 7.800, p =.018) and for actresses ( 2(2) = 12.200, p =.001). Wilcoxon tests showed that IDS vowel space was significantly lower than FDS (z = -2.176, p = .021), and ADS (z = -2.495, p = .028), with no difference between ADS and FDS for the student speakers. ADS vowel space was significantly lower than IDS (z = -2.889, p = .001) and FDS (z = -2.565, p = .006), with no difference between IDS and FDS for the actress speakers.

Vowel duration

Our analyses for vowel duration revealed a significant main effect for condition (F (1, 18) = 8.574, p = .009, = .323), with students achieving significantly higher vowel duration (mean 0.114 seconds, sd 0.024) than actresses (mean 0.091, sd 0.023).

However, we found no significant main effect for speech recipient groups, or interaction effect between speech recipient groups and conditions. This is in contrast to [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF], where IDS achieved significantly higher vowel duration than either ADS or FDS with no difference between those two conditions.

Fundamental frequency (F 0 )

We found no significant main effect of conditions, but a significant main effect of speech recipient group (F (2, 36) = 20.898, p < .001, = .537). IDS achieved significantly higher F 0 values than FDS (F (1, 18) = 25.684, p < .001, = .588) and ADS (F (1, 18) = 40.123, p < .001, = .690), with no difference between the adult conditions (Fig. 3). However, these main effects must be interpreted in light of the significant interaction between the conditions and speech recipient groups (F (2, 36) = 3.910, p = .029, = .178). As can be seen in Figure 3, there is a difference in the two conditions due to an interaction between IDS and FDS (F (1, 18) = 6.251, p = .022, = .258).

Figure 3 about here These effects are further demonstrated by comparing the differences between the three speech recipient groups for each of the conditions. In the student interactions, IDS achieved significantly higher mean F 0 than ADS (F (1, 9) = 15.424, p = .003, =

.632), but there was no significant difference between IDS and FDS, or between FDS and ADS (Fig. 3). In contrast to this, actress-produced IDS mean F 0 was significantly higher than both FDS (F (1, 9) = 22.678, p = .001, = .716) and ADS (F (1, 9) = 25.035, p = .001, = .736), with no difference between the adult conditions. These results were identical when confirmed with Friedman two-way analysis of ranks, Wilcoxon sign rank tests and the Chi-square approximations to the Friedman statistics for each group (F-ratio comparison between the two conditions).

Affective analyses

Raters

There was a significant difference for ratings of positive vocal affect between the speech recipient groups (F (2, 76) = 62.927, p < .001, = .623). IDS obtained significantly higher ratings of positive vocal affect than ADS (F (1, .38) = 61.012 p <

.001, = .616) or FDS (F (1, 38) = 101.411, p < .001, = .727). ADS also achieved significantly higher ratings than FDS (F (1, 38) = 6.958, p = .012, = .155). We did not find a significant main effect for condition, or a significant interaction between speech recipient groups and conditions. As such the two speaker groups did not differ in ratings of positive vocal affect (Fig. 4).

Figure 4 about here

Speakers' self ratings

We also asked the speakers how they thought other people would rate their speech samples on positive vocal affect for each of the three speech types. We included this question to investigate their awareness of the effect their simulated speech might have on naïve raters, and to compare the results with the perceptual ratings of the low-pass filtered speech. Interestingly, there was no difference between actresses and student speakers, in that both groups were conscious that their speech would be rated differently depending on who they had imagined talking to (F (2, 36) = 16.251, p < .001, = .474; see Fig. 5). Both groups assumed that their speech to the infants would be rated more positively than FDS (F (1, 18) = 33.800, p < .001, = .653) and ADS (F (1, 18) = 12.898, p = .002, = .417). Interestingly, both actress and student speakers assumed that their speech to foreigners would be rated less positively than ADS (F (1, 18) = 6.050, p = .043, = .209). These results were identical when confirmed with Friedman two-way analysis of ranks, Wilcoxon sign rank tests and the Chi-square approximations to the Friedman statistics for each group (F-ratio comparison between the two conditions). These findings of the self-ratings are matched by the actual results of the naïve raters, and show that both speaker groups had some understanding of how their speech would be perceived.

Figure 5 about here

Discussion

The aim of our study was to investigate the validity of simulated infant-and foreignerdirected speech of student and actress speakers by comparing it to natural speech produced by mothers. Our results suggest that, while some of the acoustic and affective aspects of natural speech samples can be reproduced in simulated speech conditions, others cannot. We also found a distinct difference between student speakers and actresses in their ability to simulate the relevant speech types.

F1/F2 vowel hyperarticulation and duration

Hyperarticulation of vowels has been found to play an important part in the development of infants' vowel categories and therefore speech acquisition (e.g. [START_REF] Andruski | The acoustic structure of vowels in mothers' speech to infants and adults[END_REF][START_REF] Kuhl | Crosslanguage analysis of phonetic units in language addressed to infants[END_REF]. Not only do mothers across diverse languages use these acoustic features in interactions with their infants (e.g. [START_REF] Andruski | Point vowels in Japanese mothers' speech to infants and adults[END_REF][START_REF] Kuhl | Crosslanguage analysis of phonetic units in language addressed to infants[END_REF], but it has also been found that mothers' F1/F2 vowel expansion is related to their infants' skills in speech discrimination [START_REF] Liu | An association between mothers' speech clarity and infants' speech discrimination skills[END_REF]. Uther et al.'s (2007) finding of the occurrence of hyperarticulation in IDS and FDS provided further support for the linguistic role of this acoustic feature in both speech groups. However, we were unable to replicate the occurrence of hyperarticulation in either IDS or FDS with our student speakers. If we consider ADS as a normal baseline form of speech, our findings are particularly interesting as the students in our study in effect hypoarticulated in IDS, in that they produced a smaller vowel space than in ADS or FDS. This finding is difficult to explain, but may relate to the speakers' limited experience with infants, and lack of feedback from the 'imaginary' infant. It is notable that the speakers in the imaginary ADS and FDS conditions reported that their 'dialogue' was uttered in an explanatory fashion which focused on the target words (e.g. explained where the animal (shark) might be found, or the material from which it was made). In contrast, in IDS the speakers tended to bracket the target words with typical 'baby sounds', and made less of an attempt to teach the words to their imaginary infant compared to mothers in the original study. The speakers may have used clearer (hyperarticulated) speech in the two adult conditions as part of a more instructional 'teacher' manner of speaking.

The results for vowel hyperarticulation of the actresses followed the trend of the natural speech samples of [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF]. Actresses clearly expanded their vowel space when they imagined talking to an infant compared to when they were talking to a British adult, which is consistent with previous research (e.g. [START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF][START_REF] Kuhl | Crosslanguage analysis of phonetic units in language addressed to infants[END_REF]. Interestingly, nine out of the ten actresses also expanded their vowel space when they imagined talking to the foreigner than when they were talking to the British adult. Overall, the vowel space of actresses was more expanded than that of the students or of the mothers (see [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF]. This could be explained by the observation that actresses use exaggerated language for their work -particularly if they are carrying out a lot of theatre work -or that they overemphasise appropriate cues (e.g. [START_REF] Scherer | Vocal communication of emotion: A review of research paradigms[END_REF]. Interestingly, neither the actress nor the student speakers replicated the greater vowel duration of IDS compared to the adult conditions in the natural speech samples. This finding supports Uther et al.'s (2007) suggestion that vowel hyperarticulation and duration are independent of each other.

Nonetheless, our results show that actresses were capable of reproducing the expanded vowel space previously found in natural speech samples (e.g. [START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF][START_REF] Kuhl | Crosslanguage analysis of phonetic units in language addressed to infants[END_REF], Uther et al, 2007), and previously assumed to have been an unconscious process that might need the presence of an infant. Despite their slightly higher mean age, the actresses did not report more exposure to infants or foreigners than the students, although they reported less discomfort, frustration and difficulty with the task, which could be due to their acting experience and training. As such it can be assumed that their training/experience gave them an advantage over the students. This assumption would be consistent with [START_REF] Scherer | Vocal communication of emotion: A review of research paradigms[END_REF] claim that actors are better than lay people at producing posed emotions that are similar to natural vocal emotions.

Fundamental frequency (F 0 )

Our F 0 findings support this possibility, as our results for the actress interactions are concordant with those of the natural interactions [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF]. The results of F 0 for the student interactions are slightly different. Although we found increased mean F 0 in student-produced IDS compared to ADS, we found no difference between FDS and IDS, or FDS and ADS. Compared to previous research (e.g. [START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF][START_REF] Fernald | Expanded intonation contours in mothers' speech to newborns[END_REF]Grieser and Kuhl, 1988;[START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF][START_REF] Werker | A cross-language investigation of infant preference for infant-directed communication[END_REF] using mothers and infants, the mean difference between IDS and both adult conditions for the student speakers is lower than expected.

The student F 0 findings are surprising, as even previous studies using imaginary scenarios found significantly increased F 0 measures in IDS (or child-directed speech) compared to both adult conditions (e.g. [START_REF] Biersack | Fine-tuning speech registers: A comparison of the prosodic features of child-directed and foreigner-directed speech[END_REF][START_REF] Papousek | Tone and intonation in Mandarin babytalk to presyllabic infants: Comparison with registers of adult conversation and foreign language instruction[END_REF]. However, our findings are comparable to [START_REF] Fernald | Expanded intonation contours in mothers' speech to newborns[END_REF], who also reported a lack of increased F 0 in their simulated IDS compared to ADS. Similar to [START_REF] Fernald | Expanded intonation contours in mothers' speech to newborns[END_REF] and to keep our methods consistent with those of Uther et al. (2007), we instructed our speakers to produce their own imaginary 'free speech', whereas previous research generally used standardised sentences or utterances (e.g. Papousek andHwang, 1991, Schaeffler et al., 2006). This methodological difference could potentially be responsible for our dissimilar findings, as the speakers in our present sample might have been unable to produce the increased F 0 in IDS, because they were distracted by the additional task of inventing their own scenarios. We are currently in the process of investigating standardised sentence production in the same speaker population. However, because the increased F 0 in IDS has been associated

with high emotional affect (e.g. [START_REF] Fernald | Expanded intonation contours in mothers' speech to newborns[END_REF][START_REF] Kitamura | Pitch and communicative intent in mother's speech: Adjustments for age and sex in the first year[END_REF], it seems also possible that the lack of a real infant in the imaginary interactions impeded the full use of this affective-acoustic feature by student speakers. We suspect that the increase of F 0 may be one of the acoustic features that are difficult to replicate without previous experience, feedback from a real infant or without professional training (e.g. actresses). Future research should investigate this possibility by using real infants or toddlers as speech recipients in the students' simulated interactions.

Emotional affect

Interestingly with regards to emotional affect, the results for both actresses and student speakers were similar to those of the mothers. IDS was consistently rated higher for positive vocal affect than either adult condition. Although these findings follow the same trend as those of the natural speech samples [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] the difference in ratings between IDS and the adult conditions for the actresses and particularly for the students was smaller than in the natural speech samples. It may be that, although only slightly higher than ADS in the student interactions, the increased mean F 0 in IDS played an important role in the raters' decision to rate the IDS samples higher. This would also be consistent with the notion that voice pitch (F 0 ) serves as a vehicle for the heightened affect in IDS (e.g. [START_REF] Fernald | Acoustic determinants of infant preference for motherese speech[END_REF][START_REF] Kitamura | The infant's response to vocal affect in maternal speech[END_REF]. Another possibility is that the speakers in the imaginary IDS still used exaggerated F 0 contours, albeit to a lesser degree than the mothers in the natural speech interactions. We are currently in the process of investigating this possibility by using new methods we recently evaluated (Eigenshape analysis; [START_REF] Knoll | Good performers know their audience! Identification and characterisation of pitch contours in infant and foreigner-directed speech[END_REF] to compare the F 0 contours in our imaginary samples with those of the original mothers.

We observed the same trend for the difference of emotional affect between the two adult conditions as [START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF], where FDS was rated to contain less positive vocal affect than ADS for both student and actress interactions. One of the reasons for this finding could be an intentional reduction in speech rate by the speakers in FDS compared to ADS. It has been found that listeners tend to evaluate slower speech rate more negatively than faster speech rate (particularly younger speakers: 20-22 years old; [START_REF] Stewart | Attitudes toward younger and older adult speakers: Effects of varying speech rates[END_REF], and that they also rate speakers with a faster speech rate as more competent and social attractive than slower speakers [START_REF] Street | The influence of speech rate stereotypes and rate similarity or listeners' evaluations of speakers[END_REF]. It is possible that the speakers in the current study (with a similar mean age to the speakers in [START_REF] Stewart | Attitudes toward younger and older adult speakers: Effects of varying speech rates[END_REF] reduced their speech rate in the imaginary foreign interaction, and that the raters used this modification as a cue for their negative evaluation of FDS.

This possibility would be consistent with [START_REF] Biersack | Fine-tuning speech registers: A comparison of the prosodic features of child-directed and foreigner-directed speech[END_REF] earlier study based on simulated speech, in which speakers modified (reduced) their speech rate for an imaginary foreigner. It may be that a slower speech rate is the most accessible and obvious modification to enhance intelligibility for a foreign speech partner, and speakers may therefore use it in natural as well as in imaginary situations. It was not possible to test this alternative explanation through comparison, as speech rate was not investigated in Uther et al.'s (2007) study. However, we did ask our participants how they perceived their voices to have changed in each interaction, and the most common response for FDS was that they reduced their speech rate. We are currently in the process of investigating the speech rate in the three samples. Interestingly both student and actress speakers accurately predicted how people would rate their speech samples, suggesting that speakers may have an inherent understanding of how their speech is perceived.

Conclusions

Our results show that only some aspects of natural speech can be reproduced with simulated speech conditions, and that a successful reproduction of other acoustic aspects may depend on the experience and training of the speakers. It seems that affective modifications (quantified by raters' perception of emotional affect) can be elicited in simulated speech regardless of the speaker's background and experience.

Certain crucial acoustic modifications such as hyperarticulation are not encountered in student imaginary IDS, but can be partly replicated by actresses with the necessary training. Our results firstly add further support to previous findings [START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF][START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] that vowel hyperarticulation in IDS and FDS occurs independent of F 0 modifications and affect. Secondly our results suggest that the use of imagined partners in speech research is probably only valid as a first step before following up with investigations using real interactions, and should depend on the experiences and training of the speakers. Since hyperarticulation is thought to be an unconscious modification in IDS (e.g. [START_REF] Burnham | What's new pussycat? On talking to babies and animals[END_REF][START_REF] Uther | Do you speak E-n-g-l-i-s-h? A comparison of foreigner-and infant-directed speech[END_REF] we suspect that such unconscious modifications are those most likely to be lost (as in the case of the student speakers), or that they at least require some previous training (e.g. actresses) or explicit instructions in order to be elicited. As such, the two-way dynamic feedback between speaker and listener and professional experience/training are probably fundamental in the process of generating appropriate speech modifications. The importance of providing either genuine speech partners or using professional speakers for speech research should therefore not be underestimated if the results of such studies are required to be generalisable to real-world situations.

  modifications in IDS and FDS resulting from the use of different methodologies (simulated versus natural interactions) and speaker populations (students versus actresses). Specifically, we aimed to replicate the linguistic and affective results of the earlier natural IDS, FDS and ADS data set of Uther et al. (2007) with student and actress speakers' simulated speech. To facilitate comparison, the analyses of the present study are restricted to the same acoustic and affective measurements used by Uther et al. (2007).

Figure captions Figure 1 .

 captions1 Figure captions

Fig. 2 .

 2 Fig. 2. Interaction between conditions (actresses and students) and speech recipient

Fig. 3 .

 3 Fig. 3. Interaction between conditions (actresses and students) and speech recipient

Fig. 4 .

 4 Fig. 4. Representation of positive vocal affect for imaginary student and actress IDS,

Fig. 5 .

 5 Fig. 5. Comparison of self-ratings of positive vocal affect for student and actress

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

 12345 Figure1

Table captions Table 1 .

 captions1 Comparison of qualitative self-reports of exposure to IDS and FDS for student and actress speakers (values represent frequency counts).

Acknowledgements

We thank Dr Maria Uther (Brunel University, UK), Dr Stig Walsh (NHM London) and Dr Darren Van Laar (University of Portsmouth) for useful comments on the project. Our particular thanks go to David Bauckham and the 'The Bridge Theatre Training Company' for their invaluable help in recruiting the actresses used in this study. This research was supported by a grant from the Economic and Social Research Council (ESRC) to Monja Knoll.


Dr Rita Carini
email: carini@med.unipmn.it

Gianluca Baldanzi

Elisa Alchera

Chiara Imarisio

Miriam Gaggianesi

Caterina Dal Ponte

Paola Nitti

Cinzia Domenicotti

Wim J Van Blitterswijk

Emanuele Albano

Gianluca Baldanzi1

Elisa Alchera2

Chiara Imarisio2

Miriam Gaggianesi1

Caterina Dal Ponte2

Paola Nitti3

Cinzia Domenicotti3

Wim J Van Blitterswijk4

Emanuele Albano2

Andrea Graziani1

Rita Carini2

NEGATIVE REGULATION OF DIACYLGLYCEROL KINASE THETA MEDIATES ADENOSINE-DEPENDENT HEPATOCYTE PRECONDITIONING

Keywords: Hepatocytes, Adenosine, RhoA, Hypoxia, Cytoprotection A2aR Adenosine A2a receptors PKC Protein kinase C DGK Diacylglycerol kinase

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Adenosine production and release by stressed cells has been shown to play an important role in modulating tissue damage and repair [START_REF] Fredholm | Adenosine, an endogenous distress signal, modulates tissue damage and repair[END_REF] . Among these effects, adenosine is regarded as a key mediator in the development of cell tolerance to ischemia and reperfusion injury triggered by ischemic preconditioning [START_REF] Peralta | The protective role of adenosine in inducing nitric oxide synthesis in rat liver ischemia preconditioning is mediated by the activation of adenosine A 2 receptors[END_REF][START_REF] Carini | Recent insights on the mechanisms of liver preconditioning[END_REF][START_REF] Cohen | Adenosine: trigger and mediator of cardioprotection[END_REF] . Ischemic preconditioning (IP) has been proposed as a simple procedure to reduce ischemia/reperfusion injury following hepatic resection and orthotopic liver transplantation [START_REF] Banga | Ischaemic preconditioning in transplantation and major resection of the liver[END_REF] . However, the application of IP in hepatic surgery is limited by the high inter-individual variability of the responses [START_REF] Deoliveira | Ischemic preconditioning: promises from the laboratory to patients, sustained or disillusioned?[END_REF] , indicating the need for a better inside in the mechanisms involved.

The importance of adenosine in hepatic preconditioning is supported by the observation that targeted deletion of hepatic ecto-5'-nucleotidase blocks adenosine generation during hypoxia and abolishes liver protection by IP [START_REF] Hart | Extracellular adenosine production by ecto-5'-nucleotidase protects during murine hepatic ischemic preconditioning[END_REF] . The hepatocyte response to adenosine autocrine loop has been characterized to some extent showing that the stimulation of A2A adenosine receptors (A2aR) is coupled to the combined activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the novel protein kinases C (nPKC), namely, PKCδ and PKCε that on their turn lead to the stimulation of a network of protein kinases including p38 MAP kinase (p38 MAPK) [START_REF] Carini | Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes[END_REF][START_REF] Carini | Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning[END_REF] . The membrane recruitment and activation of PKCδ and PKCε is fully dependent on their direct interaction with diacylglycerol generated by A2aRinduced activation of phospholipase C-γ, through a mechanism requiring both Src and PI3K activities [START_REF] Carini | Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes[END_REF][START_REF] Carini | Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning[END_REF] . These finding are consistent with central role of nPKCs in the development of the protective action of ischemic preconditioning in several tissues [START_REF] Hausenloy | Reperfusion injury salvage kinase signalling: taking a RISK for cardioprotection[END_REF][START_REF] Bright | The role of protein kinase C in cerebral ischemic and reperfusion injury[END_REF][START_REF] Um | Role of protein kinase C in intestinal ischemic preconditioning[END_REF] .

An increasing body of evidence suggests that diacylglycerol signalling is also regulated by diacylglycerol kinases (DGKs), which metabolize diacylglycerol to phosphatidic acid [START_REF] Mérida | Diacylglycerol kinases: at the hub of cell signalling[END_REF] .

DGKs are a family of enzymes encoded by ten distinct genes, grouped in five structurally related classes [START_REF] Mérida | Diacylglycerol kinases: at the hub of cell signalling[END_REF] . Accordingly, the genetic ablation of some DGK isoforms results in the deregulated activation of specific diacylglycerol-mediated signalling pathways [START_REF] Mérida | Diacylglycerol kinases: at the hub of cell signalling[END_REF] . These observations suggest that the regulation of DGK activity may provide a strict control of the kinetic as well as of the extent of diacylglycerol signalling. However, direct evidence that extracellular ligands control intracellular diacylglycerol signalling by negative regulating specific DGK isoforms is still missing. Thus, we set to investigate whether negative regulation of a DGK activity might contribute to diacylglycerol-dependent activation of PKCδ/ε upon adenosine-induced hepatic preconditioning.

RESULTS

Hypoxic preconditioning and activation of adenosine receptors negatively regulate

DGKθ in primary hepatocytes

We first investigated whether preconditioning of rat hepatocytes affected the total diacylglycerol kinase activity. Figure 1A shows that hepatocyte preconditioning, obtained by the 10 min exposure to hypoxia followed by 10 min re-oxygenation, reduced total DGK activity by about 50%. A similar effect was also induced by hepatocytes treatment with either adenosine (10 μM) or CGS21680 (1 μM), a specific adenosine A2a receptor (A2aR) agonist (Fig. 1B andC). Concomitantly CGS21680 increased at least 2 folds the intracellular diacylglycerol concentration (Fig. 1D). In order to identify the DGK isoforms putatively regulated by A2aR, DGK expression was investigated in primary hepatocytes by RT-PCR. DGKζ, θ and δ were the most expressed isoforms, while DGKα was expressed at low levels and DGKβ, γ , ε, η and ι were not expressed (Fig 2). We obtained no positive signals with several primer pairs for rat DGKκ  (not shown). As the suppression of DGK-1, the C. Elegans orthologue of DGKθ, positively regulates diacylglycerol in response to the serotonin receptor stimulation [START_REF] Mcmullan | Rho is a presynaptic activator of neurotransmitter release at pre-existing synapses in C. elegans[END_REF][START_REF] Nurrish | Serotonin inhibition of synaptic transmission: Galpha(0) decreases the abundance of UNC-13 at release sites[END_REF] , we set to investigate the regulation of DGKθ in response to A2aR activation. The DGKθ enzymatic activity measured in immunoprecipitates obtained using two distinct anti-DGKθ antibodies raised against different epitopes was significantly reduced upon CGS21680 stimulation (Fig. 3A

RhoA-dependent inhibition of DGKθ mediates adenosine A2a receptor-induced protection of hepatocytes from hypoxia-induced cell death

Biochemical evidence both in neuroblastoma [START_REF] Pacini | Muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells elicits phosphoinositide and phosphatidylcholine hydrolysis: relationship to diacylglycerol and phosphatidic acid accumulation[END_REF] and in C. Elegans motoneurons [START_REF] Mcmullan | Rho is a presynaptic activator of neurotransmitter release at pre-existing synapses in C. elegans[END_REF] shows that the enzymatic activity of DGKθ and of its orthologue DGK-1 is strongly inhibited upon direct interaction with the small GTPase RhoA [START_REF] Houssa | Diacylglycerol kinase theta binds to and is negatively regulated by active RhoA[END_REF] . Thus we set to investigate the interaction between active RhoA and DGKθ in PC12 cells, , a rat cell line expressing A2aR [START_REF] Mori | Adenosine A2A receptor facilitates calcium-dependent protein secretion through the activation of protein kinase A and phosphatidylinositol-3 kinase in PC12 cells[END_REF][START_REF] Huang | Activation of protein kinase A and atypical protein kinase C by A(2A) adenosine receptors antagonizes apoptosis due to serum deprivation in PC12 cells[END_REF] transiently co-transfected with FLAG-tagged DGKθ and myc-tagged RhoA.

Consistently with previously published data [START_REF] Houssa | Diacylglycerol kinase theta binds to and is negatively regulated by active RhoA[END_REF] , FLAG-DGKθ co-immunoprecipited with constituitvely active myc-RhoA-V14, but not with inactive myc-RhoA-N14 mutants from unstimulated cells (Fig. 4). Moreover , FLAG-DGKθ specifically co-precipitated with myc-RhoA in anti-myc immunoprecipitates from CGS21680 stimulated cells, but not from unstimulated cells (Fig. 4). In the whole these data confirmed that indeed DGKθ is recruited in a complex with active RhoA. We then moved to investigate whether RhoA regulated DGKθ activity upon A2aR activation in primary rat hepatocytes. To this aim, we first verified whether RhoA was regulated by activation of A2aR. Indeed upon 15 minutes treatment, CGS21680 increased by 50% RhoA-GTP complex, as measured by pull down assay with immobilized rothekin-Rho Binding Domain (Fig. 5A-B). Such A2aR-induced activation of RhoA was insensitive to the addition of pertussis toxin (data not shown).

Next, in order to verify whether DGKθ inhibition downstream from A2aR was mediated by RhoA-GTP, primary hepatocytes were treated with the cell permeable analogue of Clostridrium botulinum-derived C3 transferase (1 μM), a highly specific RhoA inhibitor stabilizing RhoA in the inactive GDP-bound form. Inhibition of RhoA entirely reverted CGS21680-induced inhibition of DGKθ (Fig. 5C), demonstrating that RhoA-GTP mediated the inhibition of DGKθ triggered upon A2aR activation. Moreover, the inhibition of RhoA by C3 tranferase abolished the protection against hypoxic injury in hepatocyte treated with CGS21680 (Fig. 5D). In the whole these data demonstrated that the cytoprotective signalling downstream from A2aR requires the activation of RhoA-GTPase followed by the inhibition of DGKθ upon binding of RhoA to its catalytic site.

In order to provide further support to the claim that RhoA-mediated inhibition of DGKθ contributed to A2aR-dependent signalling in preconditioning, we down-regulated the expression of DGKθ in HTC cells by specific siRNAs. HTC cells are a rat hepatocyte-derived cell line that, similarly to primary hepatocytes, undergoes to a A2aR-mediated preconditioning [START_REF] Alchera | Adenosine-dependent activation of hypoxia-inducible factor-1 induces late preconditioning in liver cells[END_REF] ζ). Upon 60 min exposure to hypoxia, un-transfected cells and cells transfected with control siRNA underwent a significant loss of cell viability (Fig. 6B). Conversely, HTC cells transfected with either DGKθ specific siRNAs were fully protected from hypoxic damage, as were the cells pre-treated with CGS21680 (Fig. 6B). Thus, these data demonstrate that specific down-regulation of DGKθ isoform in HTC cells provides a sufficient signalling to induce tolerance to hypoxic damage.

Pharmacological inhibition of DGK is sufficient to activate signals leading to hepatocyte resistance to hypoxic damage.

The identification of the key role played by the negative regulation of DGKθ in mediating adenosine-induced preconditioning, let us to investigate whether direct modulation of diacylglycerol turnover through pharmacological inhibition of DGK, could induce hepatocytes resistance to hypoxic damage. The DGK inhibitor R59949 in known to preferentially inhibit DGKα, β, γ and δ isoforms [START_REF] Jiang | Selectivity of the diacylglycerol kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1piperidinyl)ethyl]-2,3-dihydro-2-thioxo4(1H) quinazolinone (R59949) among diacylglycerol kinase subtypes[END_REF] , whereas DGKζ, one of the major DGK isoform expressed in hepatocytes, is resistant to R59949 [START_REF] Jiang | Selectivity of the diacylglycerol kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1piperidinyl)ethyl]-2,3-dihydro-2-thioxo4(1H) quinazolinone (R59949) among diacylglycerol kinase subtypes[END_REF] and no information is available on the R59949 sensitivity of the others DGK isoforms. Inhere we show that R59949 at 10 μM, but not at lower concentrations, effectively inhibited hepatocyte DGKθ activity as assayed "in vitro" in anti-DGKθ immunoprecipitates (Fig. 7A). Accordingly, in the same cells, 10 μM R59949 induced a sustained increase of intracellular diacylglycerol concentration (Fig. 7B). Pre-treatment with R59949 also protected primary hepatocytes from hypoxia-induced cell death (Fig. 7C), while it did not affect viability of control cells.

Moreover in primary hepatocytes as well as in HTC cells R59949 increased PKCδ and PKCε activities in both membrane and cytosolic fractions (Fig. 8A). The extent of both diacylglycerol accumulation (Fig. 1D) and enzymatic activation of PKCδ and PKCε was highly similar to that induced by CGS21680-mediated activation of A2aR (Fig. 8A).

Moreover neither pharmacological inhibition of DGK nor A2aR stimulation promoted the enzymatic activities of PKCα, β1 and β2, which are regulated by both calcium and diacylglycerol (Fig. 8A). We previously showed that PKCδ/ε-dependent phosphorylation of p38 MAPK is required for A2aR-induced protection from hypoxic damage [START_REF] Hart | Extracellular adenosine production by ecto-5'-nucleotidase protects during murine hepatic ischemic preconditioning[END_REF] . Consistently, R59949 mediated inhibition of DGK stimulated the dual phosphorylation Thr 180 /Tyr 182 of p38 MAPK (Fig. 8B). The effect induced by DGK inhibition was comparable to that obtained by the activation of A2aR and was prevented by the PKC inhibitor chelerythrine (50 μM) (Fig. 8B). Moreover, the inhibition of either PKC or p38 MAPK kinase with, respectively chelerythrine or SB203580 (5 μM), abolished R59949-induced protective effects against hypoxic hepatocyte death (Fig. 7C). All together these findings indicate that the pharmacological inhibition of DGK provides sufficient diacylglycerol accumulation to trigger PKC-mediated signals leading to resistance to hypoxic damage.

DISCUSSION

It is increasingly clear that the development of ischemic preconditioning in many tissues, including the liver, requires the activation of a complex network of signals comprising cellsurface receptors, redox signals and diverse arrays of protein kinases such as reperfusion injury salvage kinases (RISK), that rapidly re-modulate several cell functions to better cope with energy loss, oxidative stress and ion unbalances associated with hypoxia and reperfusion [START_REF] Carini | Recent insights on the mechanisms of liver preconditioning[END_REF][START_REF] Cohen | Adenosine: trigger and mediator of cardioprotection[END_REF][START_REF] Hausenloy | Reperfusion injury salvage kinase signalling: taking a RISK for cardioprotection[END_REF][START_REF] Murphy | Mechanisms underlying acute protection from cardiac ischemiareperfusion injury[END_REF][START_REF] Hausenloy | Preconditioning and postconditioning: united at reperfusion[END_REF] . Among these protein kinases, PKCδ and PKCε play a key role by coupling adenosine receptors to down-stream stress activated protein kinases Erk1/2 and p38 MAP kinases ,3,4,26 . Moreover, translocation of PKCε to mitochondria has been implicated in preventing mitochondrial permeability transition upon myocardial reperfusion [START_REF] Jabůrek | Mitochondrial PKC epsilon and mitochondrial ATPsensitive K+ channel copurify and coreconstitute to form a functioning signaling module in proteoliposomes[END_REF] . PKCδ and PKCε membrane recruitment and activation is fully dependent on their direct interaction with diacylglycerol, generated by adenosine-induced activation of phospholipase C-γ and diacylglycerol analogues fully mimic the activation of the signals that onset ischemic preconditioning [START_REF] Carini | Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes[END_REF][START_REF] Carini | Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning[END_REF][START_REF] Galiñanes | Diacylglycerolinduced protection against injury during ischemia and reperfusion in the rat heart: comparative studies with ischemic preconditioning[END_REF] . However, the accumulation of cellular diacylglycerol is dependent also on the rate of its rapid metabolism to phosphatidic acid by DGKs [START_REF] Mérida | Diacylglycerol kinases: at the hub of cell signalling[END_REF] . Recent data indicate that the genetic deletion of specific DGK isoforms results in a sustained diacylglycerol signalling in different cell systems and tissues [START_REF] Mérida | Diacylglycerol kinases: at the hub of cell signalling[END_REF] . Moreover, the role of DGKs as terminator of diacylglycerol-mediated signals has been recently underscored by the finding that several DGK isoforms are recruited to the activated muscarinic receptors, where they rapidly metabolize diacylglycerol similarly to the action of phosphodiesterase toward cAMP [START_REF] Nelson | Targeting of diacylglycerol degradation to M1 muscarinic receptors by betaarrestins[END_REF] . However, so far, no direct biochemical evidence is available for a receptor-mediated negative regulation of any DGK isoform. In this study we show that in hepatocytes both hypoxic preconditioning and A2aR stimulation lead to a strong decrease of total DGK activity that contributes to diacylglycerol accumulation. In particular, we demonstrate that DGKθ enzymatic activity that is, along DGKζ the most abundant isoform in hepatocytes, is strongly inhibited upon A2aR activation. This provides the first demonstration of a negative regulation of a DGK isoform by extracellular ligands. However, we can not rule out that other DGK isoforms beside DGKθ may be inhibited upon hepatocyte A2aR activation. Nevertheless, the demonstration that specific genetic down-regulation of DGKθ expression is sufficient to protect HTC hepatoma cells from hypoxic damage, indicates that the negative regulation of the DGKθ isoform may be sufficient to trigger the onset of cytoprotective signals upon A2aR activation by hypoxic preconditioning.

The localization and function of DGKθ have been previously reported to be regulated by several G-protein coupled receptors, although the molecular mechanisms underlying such a regulation have not been fully investigated. For instance, in vascular smooth muscle cells purinergic receptors promote PKC-mediated recruitment of DGKθ to the plasma membrane, while norepinephrine stimulates DGKθ activity at lipid rafts [START_REF] Van Baal | Translocation of diacylglycerol kinase theta from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C[END_REF][START_REF] Clarke | Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonistspecific role of PI3-kinaseAm[END_REF][START_REF] Bregoli | Nuclear diacylglycerol kinase-theta is activated in response to alpha-thrombin[END_REF][START_REF] Tu-Sekine | Modulation of diacylglycerol kinase theta activity by alpha-thrombin and phospholipids[END_REF] . Inhere we demonstrate that upon ligand-stimulation of A2aR, RhoA is activated, binds and inhibits DGKθ . Such a Rho-A signalling is required for A2aR-induced inhibition of DGKθ as well as for the development of cytoprotection. These findings are consistent with the evidence that both DGKθ and its C. Elegans orthologue, DGK-1, are inhibited upon the direct binding of active RhoA to their catalytic domain [START_REF] Houssa | Diacylglycerol kinase theta binds to and is negatively regulated by active RhoA[END_REF][START_REF] Los | Structureactivity relationship of diacylglycerol kinase theta[END_REF] . Moreover, in motoneurons of C.

Elegans, the activation of the RhoA orthologue, Rho-1, leads to diacylglcyerol accumulation which then allows the recruitment and activation of PKC-1 and UNC-13 proteins [START_REF] Mcmullan | Rho deep in thought[END_REF] . Thus, our finding that in hepatocytes RhoA-mediated inhibition of DGKθ triggers cytoprotective signalling downstream from A2a receptors, unveils the general relevance of a novel Rho-mediated signalling pathway linking G-protein coupled receptors to the negative regulation of DGKθ activity. Such a novel pathway is conserved from C.

Elegans to different mammalian tissues and may enable G-protein coupled receptors to regulate the extent and the kinetics of diacylglycerol-mediated signalling, by fine tuning the enzymatic activity of DGKθ. This is also the first evidence for a direct activation of RhoA by A2aR, albeit in hepatic stellate cells A2aR has been recently reported to mediate RhoA inhibition through the cAMP-mediated activation of protein kinase A [START_REF] Sohail | Adenosine induces loss of actin stress fibers and inhibits contraction in hepatic stellate cells via Rho inhibition[END_REF] . It is known that Gprotein coupled receptors activate RhoAGTPases, by coupling the pertussis toxin insensitive Gα12/13 or Gαq/11 proteins to, respectively p115 or Trio RhoGEFs [START_REF] Sah | The role of Rho in G proteincoupled receptor signal transduction[END_REF] .

Moreover, previous evidence indicates that A2aR couples to Gα12/13 in endothelial cells [START_REF] Sexl | Stimulation of the mitogen-activated protein kinase via the A2A-adenosine receptor in primary human endothelial cells[END_REF] . In our hands, A2aR-induced activation of hepatocyte RhoA, is pertussis toxin insensitive, suggesting that it may be mediated through either Gα12/13 or Gαq/11 [START_REF] Hermans | Biochemical and pharmacological control of the multiplicity of coupling at Gprotein-coupled receptors[END_REF] and ruling out the involvement of inhibitory G (Gαi) proteins, which instead transduce A2aRinduced Src-mediated signalling to PI 3-kinase and PLC-γ [START_REF] Carini | Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning[END_REF] . These findings indicate that A2aRs by interacting with, respectively, Gαi or Gα12/13(q/11) GTPases promote both the PLC-γ-mediated diacylglycerol synthesis and, at the same time, inhibit diacylglycerol catabolism by DGK (Fig. 9).

Thus, the accumulation of diacylglycerol required for preconditioning-induced PKC δ/ε activation, results from the fine tuning of the enzymes responsible for both diacylglycerol generation and catabolism. Such an interpretation is consistent with a growing body of evidence suggesting that the temporal and spatial regulation of intracellular second messengers depends upon the simultaneous reciprocal regulation of both activating and de-activating enzymes, such as adenylate cyclase and phosphodiesterase for cAMP, and PI3-kinase and PTEN for PI(3,4,5)P3 [START_REF] Houslay | Regulation of hepatocyte adenylate cyclase by amylin and CGRP: a single receptor displaying apparent negative cooperatively towards CGRP and simple saturation kinetics for amylin, a requirement for phosphodiesterase inhibition to observe elevated hepatocyte cyclic AMP levels and the phosphorylation of Gi-2[END_REF][START_REF] Gericke | Regulation of the PTEN phosphatase[END_REF] . Moreover, these observations underscore the relevance of negative regulators for the transduction of the complex signals required for the onset of the cytoprotective machinery in ischemic preconditioning. In this latter respect, a decline in the activity of PTEN is evident in parallel with PI3K activation during brain and myocardium preconditioning [START_REF] Cai | PTEN activity is modulated during ischemia and reperfusion: involvement in the induction and decay of preconditioning[END_REF][START_REF] Omori | Enhanced phosphorylation of PTEN in rat brain after transient middle cerebral occlusion[END_REF] . By unveiling that the RhoA-mediated negative regulation of DGKθ is essential for A2aRdependent hepatocyte preconditioning, these results suggest that the pharmacological inhibition of DGK may likely trigger cytoprotection. Indeed, the treatment of primary hepatocytes with the DGK inhibitor R59949 is sufficient to induce adequate diacylglycerol accumulation to activate diacylglycerol-dependent nPKCs, fully protecting hepatocytes from hypoxic damage. Upon pharmacological inhibition of DGK, adequate diacylglycerol levels likely build from the unstimulated basal phospholipids turnover, even in absence of a receptor-mediated activation of PLC-γ. At the concentrations used in this study, R59949 inhibits several DGK isoforms including DGKθ, DGKδ and DGKα, but does not affect DGKζ, which is along with DGKθ the most abundant DGK isoform expressed in hepatocytes. Thus, the inhibition of DGKθ may be largely responsible for R59949-induced protection, although we can not rule out the involvement of other DGKs, such as DGKδ and DGKα.

In conclusion, the data presented characterize a novel signalling pathway in the mechanisms by which adenosine promotes hepatic preconditioning involving DGKθ downregulation. These findings indicate DGK modulation as a potential target of new pharmacological strategies aimed to induce tolerance to ischemia/reperfusion injury in organs such as the liver, the myocardium and the kidney that relay on nPCK activity for the development of the cytoprotective effects of preconditioning.

MATERIALS AND METHODS

Hepatocyte preparation and treatments. Primary hepatocytes were isolated by collagenase (Sigma, MI, Italy) liver perfusion of fed male Wistar rats (180-250 g weight) (Harlan Italy 8 .

The use and care of the animals were approved by the Italian Ministry of Health.

Hepatocytes were suspended in Krebs-Henseleit-HEPES (KHH) medium (final cell density of 10 6 /mL) and preconditioned by exposure to 10 min of hypoxia (95% N2 -5% CO2) followed by 10 min re-oxygenation (95% air -5% CO2) or by 15 min treatment with CGS21680 (Sigma, Milan, Italy) as previously described [START_REF] Carini | Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes[END_REF] . Hypoxic damage was evaluated by exposing hepatocytes to 90 min hypoxia immediately after preconditioning procedures.

Control cells were maintained under normoxic atmosphere (95% air -5% CO2). The inhibitors: SB203580, chelerythrine (Sigma, Milan, Italy) and the cell-permeable Clostridrium boltulinum-derived C3 transferase (Citoskeleton, Denver, CO, USA) were added respectively, 5, 15 and 120 min before preconditioning. R59949 (Sigma, Milan, Italy) was added 15 min before hypoxic exposure.

Cell cultures and treatments. HTC rat hepatoma cell line was obtained from the European RhoA/C-GTP Pull-Down Assay. RhoA/C-GTP pull-down assays were performed according to Ren et al. [START_REF] Ren | Determination of GTP loading on Rho[END_REF] . Briefly 10 6 hepatocytes were washed in ice-cold PBS and lysed with 1 ml GST-fish buffer (50 mM Tris-HCl, pH 7.2, 1% Triton X-100, 5% sodium deoxicolate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, supplemented with fresh 1 mM Na3VO4, protease inhibitors, and 1 mM dithiothreitol). 5% of each sample was directly denatured in Laemmli buffer for whole cell lysate proteins analysis. The remaining clarified lysates were incubated for 60 min with purified GST6 Rhotekin-RBD at 4°C, pre-coupled to glutathione-Sepharose beads (GE Healthcare, UK). After 4 washes with 1 mL of Tris buffer (1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, protease inhibitors) samples were resuspended in Laemmli buffer, heat-denatured, and separated by SDS-PAGE in a 12% polyacrylamide gel. RhoA/C was visualized by western blotting and quantified using Versadoc with Quantity One software (Biorad, Milan, Italy). RhoA/C activation was calculated as GTP-Rho ratio to Rho in total cell lysate.

RT-PCR determination of rat DGK isoforms RNA was extracted from 10 6 hepatocytes or HTC cells using charge switch total RNA cell kit (Invitrogen, San Giuliano, MI, Italy).

Reference RNA was extracted from 200 μg rat brain using Tri-reagent (Applied Biosystems, Austin, Texas, USA). RNA aliquots (200 ng) were retrotranscribed using the RT-Kit (Applied Biosystems, Austin, Texas, USA). One tens of the cDNA obtained was Distribution normality of all groups was preliminary verified by Kolmogorov and Smirnov test. Significance was taken at 5% level.

Titles and legends to figures

  & 3B), while DGKθ protein expression was not affected (Fig 3). DGKθ involvement was further confirmed by the use of a well characterized anti-DGKθ neutralizing antibody 18 that decreased by about 80% the recovered DGKθ activity in immunoprecipitates (Fig 3C). All together, these data indicate that the activation of A2aR provides a signal which negatively regulates DGKθ activity, likely contributing to the sustained accumulation of diacylglycerol.

  . Down-regulation of DGKθ expression was obtained by transient transfection of two different specific siRNAs or a control siRNA and verified by western blot 48 hours after transfection. As shown in Fig 6A, both anti-DGKθ siRNAs specifically downregulated DGKθ and not the other mayor hepatocytes DGK isoforms (DGK α, δ and

(

  60:47:11:4) using TLC plates previously coated with potassium oxalate 1.3%, EDTA 5mM:methanol 3:2 and desiccated. [γ32P]-phosphatidic acid formation was identified by comigration with non radioactive phosphatidic acid formation standards stained by exposure to iodine vapours. Radioactive signals were detected and quantified by GS-250 Molecular Imager and Phosphor Analyst Software (Bio-Rad, Milan, Italy). The specific DGKθ-activity was evaluated by the same method after DGKθ immunoprecipitation with protein A Sepharose and two anti-DGKθ-specific antibodies recognising different DGKθ epitopes (Santa Cruz, Biotecnologies, CA, United States) according to Houssa et al. 20 . DGKθ-western blotting was performed using 25 μg of hepatocyte proteins and a specific anti-DGKθ-antibody (BD Biosciences, CA, United States) . To verify the effects of neutralizing antibodies on Dgk activity control IgG (Santa Cruz Biotecnologies, Milan, Italy) and anti-Dgkθ inhibitory IgG (50 μg/ml; BD Biosciences, CA, United States) were added to the samples 30 min before the assay. The results were acquired and quantified with GS-250 Molecular Imager and Phosphor Analyst Software (Bio-Rad, Milan, Italy).

  amplified using Go-Taq Master Mix (Promega, Milan, Iraly). The primers used for the DNA amplification of specific DGK isoforms were: Dgk-η LEFT PRIMER TTCAGAAGGCTGATGCTGTG RIGHT PRIMER CACATTAAACTGGGCCACCT blue in 30% CH 3 OH and 100 mM NaCl for 30 minutes. The plates were de-stained for 5 minutes in dyefree solution and the bands were acquired with Versadoc system. The band density was calculated with Quantity One software (Biorad, Milan, Italy). The amount of diacylglycerol was calculated using a standard curve of dioleyl-diacylglycerol (Sigma, Milan, Italy).Assay of PKCs activities. PKC isoforms were immunoprecipitated with rabbit polyclonal α, β, β1, δ and ε anti-antibodies (Santa Cruz Biotechnology, Santa Cruz,CA United States) and protein A Sepharose. After 3 washes in a buffer containing 10 mmol/L Tris HCl, 150 mmol/L NaCl, 10mmol/L MgCl 2 , and 0.5 mmol/L dithiotreitol, PKC activity was assayed by using 10 g of histone H1 as substrate and a reaction mixture containing 0.1 mmol/L ATP, 2 μCi/sample[32P]ATP (GE Healthcare, Cologno Monzese, MI, Italy), 1 g of phophatidylserine,and 0.4 μg of diacylglycerol as previously reported[START_REF] Carini | Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes[END_REF] .Analysis of p38 MAPK phosphorylation state. The phosphorylation state of p38 MAPK was assessed by using dual (Thr 180 /Tyr 182 ) phosphorylated p38 MAPK and total p38 MAPK antibodies (PhosphoPlus p38 MAPK antibody kit, New England BioLabs, Beverly MA, USA).Determination of cell viabilityCell viability was estimated by microscope-counting the hepatocyte excluding Trypan blue and by the determination of nuclear fluorescence staining with propidium iodide 8 .Data analysis and statistical calculations. The data were expressed as means ± S.D or ± SEM. Statistical analysis was performed by Instat 3 statistical software (GraphPad Software Inc, San Diego CA, USA) using one-way ANOVA test with Bonferroni's correction for multiple comparisons when more than two groups were analyzed.

Figure 1 :

 1 Figure 1: Hypoxic preconditioning and adenosine A2a receptor (A2aR) stimulation decrease diacylglycerol kinase (DGK) activity and promote diacylglycerol accumulation in isolated hepatocytes. DGK activity was evaluated in the homogenates of primary rat hepatocytes undergoing preconditioning by exposure to transient hypoxia/reoxygenation (panel A), 15 min preincubation with adenosine (10 μM) (panel B) or A2aR stimulation with CGS21680 (1 μM) (panel C). A representative determination of phosphatidic acid (PA) determination is shown together with the quantification of 5 independent experiments. Statistical significance: * p<0.05; ** p<0.01. Panel D shows the quantification of the hepatocyte diacylglycerol (DAG) content after treatment with CGS21680 (1 μM) for 15 or 60 minutes. The data are mean ± SEM of 5 replicates. Statistical significance *p<0.01.

Figure 2 :

 2 Figure 2: Characterization of the different diacylglycerol kinase (DGK) isoforms expressed in parenchimal liver cells. RT-PCR was used to verify the expression of the different DGKs in the HTC cell line and primary rat hepatocytes. Rat brain was used as positive control. GAPDH was used as loading control. The values above each bands represent the densitometric analysis normalized by the value of the brain. .

Figure 3 :

 3 Figure 3: Modulation of DGKθ by the adenosineA2a receptor agonist CGS21680 Panel A & B: DGKθ was immunopurified with two different anti-DGKθ antibodies (Dgkθ-1 and Dgkθ-2) from extracts of hepatocytes treated with CGS21680 (1 μM) for 15 or 30 minutes. DGKθ enzymatic activity was measured as reported in the method section. A representative experiment of phosphatidic acid (PA) determination is shown together with the quantification of 3 independent experiments. WB: represents Western blot analysis

Figure 4 :

 4 Figure 4: The adenosineA2a receptor agonist CGS21680 promotes DGKθ association with RhoA/C GTPase . DGKθ/RhoA association was evaluated in PC12 cells transfected with FLAG-DGKθ and either wild type myc-RhoA (wt) or constitutive active myc-RhoA-V14 (active) or inactive myc-RhoA-N19 (inactive) mutants. Following trasfection cells were serum starved for 24 hours and treated with CGS21680 for 30 min. Cells were then lysed and transfection verified by western blotting and protein association was assessed following immunoprecipitation with anti-myc specific antibodies using western blotting with anti-FLAG specific antibodies.

Figure 5 :

 5 Figure 5: CGS21680 promotes the activation of RhoA/C GTPase which is required for the inhibition of DGKθ and the protection of hepatocytes against hypoxic damage. Panel A: Primary rat hepatocytes were stimulated with CGS21680 (1 μM) for 30 minutes and active RhoA/C was purified by pull-down with GST-Rhotekin-RBD and visualized by western blotting with RhoA/C antibodies (middle panel). The GST and GST-Rhotekin-RBD loading was verified by western blotting with GST antibodies (lower panel). The RhoA/C content in total cell lysate is also shown (upper panel). Panel B. Densitometric quantification of RhoA/C activity in four independent experiments. Statistical significance: * p<0.05. Panel C: Effect of the RhoA inhibitor Clostridrium botulinum-derived C3 transferase (C3) (1 μM), on DGKθ activity in primary rat hepatocytes. The cells were pretreated with C3 for 120 minutes and then stimulated with CGS21680 (1 μM) for 30 minutes. The data are mean ± SEM of 5 replicates. Statistical significance *p<0.01 vs

Figure 6 :

 6 Figure 6: siRNAs DGKθ down-regulation in HTC cells protects against cell killing by hypoxia as the adenosine A2a receptor stimulation. HTC cells were trasfected with control siRNA or two different DGKθ siRNAs (siRNA1 and siRNA2) and submitted to 60 min incubation under either normoxic or hypoxic conditions. When indicated HTC were pre-treated 15 min with A2aR agonist CGS21680 (1 μM). Results are mean of 3 different experiments. Statistical significance: *p <0.05 versus hypoxia. The capacity of the different SiRNA to specifically block DGKθ expression was assessed by western blot (WB) using antibodies against different DGK isoenzymes. Equal amounts of proteins was assessed by using actin as loading control. One experiment representative of 3.

Figure 7 :

 7 Figure 7: The inhibition of DGK promotes diacylglycerol accumulation and protection against hypoxic injury in primary rat hepatocytes Panel A: DGKθ activity was evaluated following immunopurification with specific antibodies from extracts of hepatocytes incubation with increasing concentration of the DGK inhibitor R59949. The values are mean ± SEM from a representative experiment performed in quadruplicate. Panel B: Quantification of diacylglycerol content of primary rat hepatocytes after treatment with R59949 (10 μM) for 15 or 60 min. The data are mean ± SEM of 4 replicates. Statistical significance: * p< 0.05 Panel C: Viability of primary rat hepatocytes incubated 90 min under normoxic or hypoxic conditions in presence or absence of R59949 (10 μM). The effects of inhibiting PKC with cheletrytrine (50 μM) or

Figure 8 :

 8 Figure 8: DGK inhibition induces the activation of PKC δ/ε and p38 MAPK in both primary hepatocytes and HTC cells. Panel A: The activity of different PKC isoforms was measured in the immunoprecipitated fractions obtained from primary hepatocytes or HTC cells incubated 30 min with R59940 (10 μM) or CGS21680 (1 μM). The results are expressed as percentage of the values in the controls and are means of 3 different experiments SD. Statistical significance: *p< 0.05 vs controls. Panel B: The activation of p38 MAPK was estimated by Western blotting using antibodies against dual (Thr 180 /Tyr 182 ) p38 MAPK following 45 min incubation with R59940 or CGS21680. In some experiments cells were pre-treated with the PKC inhibitor cheletrytrine (50 μM). The relative intensity of phosphorylated and non phosphorylated bands was measured by videodensitometry and the results were expressed as ratios after normalization at 1 of the controls. A representative experiment is shown together with the quantification of 5 independent experiments. Statistical significance: *p<0.005 vs control or cells treated with R59940+cheletrytrine or CGS21680+cheletrytrine.

Figure 9 :

 9 Figure 9: Proposed role of DGKθ inhibition and phospholypase C activation in the recruitment of protein PKC dependent survival pathways responsible for inducing the resistance to hypoxia of precondutioned hepatocytes. During hepatocytes preconditioning, adenosine A2 receptor (a2aR) stimulation induces RhoA/C activation and a Rho A/C-dependent inhibition of DGKθ activity. The consequent decrease of diacylglycerol (DAG) metabolism to phosphatidic acid (PA) allows a stable increase of DAG intracellular level that maintains the activation of the DAG dependent cytoprotective signals.

This work has been supported by the Regional Government of Piedmont (Carini, Fondi Ricerca Sanitaria Finalizzata, 2006, 2007, 2009; Graziani Fondi Ricerca Sanitaria Finalizzata, 2008) by the University "Amedeo Avogadro", by MIUR (Graziani PRIN 2007TE8NFY) and AIRC (Graziani).

A siRNA with no matching sequences in the rat genome (Ambion, Austin, Texas, USA) was used as negative control. HTC cells were transfected using Lipofectamine 2000 (Invitrogen San Giuliano, MI, Italy) according to manufacturer recommendations.

Transfection efficiency was analyzed after 72 hours using BLOCK-iT Fluorescent Oligo (Invitrogen, San Giuliano, MI, Italy) procedure according to the producer instruction The transfection efficiency of HTC cells with the different siRNAs was more than 75%.

Diacylglycerol quantification. Hepatocyte content of diacylglycerol was evaluated in the lipids extracted from 2x10 6 hepatocytes as described by Bligh and Dyer [START_REF] Bligh | A rapid method of total lipid extraction and purification Can[END_REF] . Briefly Hepatocyte content of diacylglycerol was evaluated by extracting 2x10 [START_REF] Deoliveira | Ischemic preconditioning: promises from the laboratory to patients, sustained or disillusioned?[END_REF] hepatocyte pellets with 0.750 ml CHCl 3 :CH 3 OH (1:2 v/v) and the phases separated by the addition of with 0.25 ml CHCl 3 and 0.25 ml H 2 O followed by centrifugation at 1000 g for 5 minutes. The lower phase was dried, re-dissolved in 50 μl CHCl3 and spotted on a silica 60 TLC plate (Sigma, Milan, Italy). Diacylglycerol was eluted with diethylether : heptane : acetic acid (75:25:1 v:v:v) mixture, the plates were dried and stained with 0,003% Coomassie brilliant