PhD Emma Bestaven

PhD Etienne Guillaud

MD Aupy Jerome

MD, PhD Pierre Burbaud

PhD Jean-René Cazalets

MD, PhD Dominique Guehl
email: dominique.guehl@u-bordeaux.fr

EFFECT OF TRUNK MUSCLE STRENGTHENING ON GAIT PATTERN AND FALLS IN PARKINSON'S DISEASE

Keywords: Parkinson's disease, falls, gait, kinematic, rehabilitation

Background: The course of Parkinson's disease is characterized by gait disturbance and falls, which affect patients' quality of life and engender high healthcare costs. These factors are not greatly improved by levodopa therapy or deep brain stimulation of the subthalamic nuclei. Indeed, the symptoms may even worsen with these treatment. Physiotherapy may be the most appropriate treatment to reduce the incidence of falls in these cases; how ever,itsbenefitsaremodest. Objective: To assess the effectiveness of trunk muscle strengthening in 10 patients with Parkinson's disease being treated with deep brain stimulation of the subthalamic nuclei who are affected by gait disturbances and falls. Method: A standardized physiotherapy programme centred on trunk muscle strengthening was conducted. Its effectiveness was assessed using a clinical approach combined with video-based motion analysis. Results: After 4 weeks of trunk muscle strengthening, the gait item on the Unified Parkinson's Disease Rating Scale (UPDRS, part 3) together with several gait kinematic parameters (step length, walkingcycledurationvariability,gaitspeed)were significantlyimprovedandthenumberoffallsdecreased. Conclusion:Thesepreliminaryfindingssuggestthat physiotherapy centred on rachis mobility improves the quality of gait and reduces the number of falls in patients with Parkinson's disease who are being treated with deep brain stimulation of the subthalamic nuclei. This is a potentially useful supplement to the traditional physiotherapy approach, in addition to the pharmacological and surgical treatment of Parkinson's disease.

I n the course of Parkinson's disease (PD), axial symp- toms, such as gait and balance impairments with falls,

LAY ABSTRACT

Over time, patients with Parkinson's disease treated operatively with subthalamic nucleus deep brain stimulation develop drug-resistant gait disturbance and falls. Traditional physiotherapy programmes for reducing the incidence of falls in these patients have modest effects. Thus, a physiotherapy programme based on trunk exercises could be helpful, since optimal axial motility and gait and posture quality are strongly linked. A specific programme was used for 10 patients with Parkinson's disease treated operatively with subthalamic nucleus deep brain stimulation. The programme involved 2 physiotherapy sessions per day for 5 days a week over a period of 4 consecutive weeks. Quality of gait improved and the number of falls decreased significantly. In conclusion, specific standardized rehabilitation of axial motility is potentially a useful supplement to the traditional physiotherapy approach in order to reduce falls in patients with Parkinson's disease who have been treated operatively with subthalamic nucleus deep brain stimulation.

worsen. Up to 68% of patients with PD fall each year, with 50% falling repeatedly [START_REF] Pickering | A meta-analysis of six prospective studies of falling in Parkinson's disease[END_REF]. Falls may cause multiple fractures, which are costly for medical and health services, and may be life-changing for patients [START_REF] Englander | Economic dimensions of slip and fall injuries[END_REF].

Risk factors for falls in patients with PD are numerous, such as longer duration and higher severity of disease (for review see (3)). Levodopa is a potential risk factor for hip fractures, probably due to the occurrence of dyskinesia or orthostatic hypotension [START_REF] Kerr | Predictors of future falls in Parkinson disease[END_REF]. Levodopatherapy may also make patients more mobile and therefore more likely to fall, since they are still able to move around despite their postural instability. Unpredictable freezing of gait (FOG) at step initiation, half turn, or during ongoing gait, can also induce falls (4). On the other hand, the rate of falls tends to stabilize or decrease with disease progression, since patients are more disabled and less able to move [START_REF] Pickering | A meta-analysis of six prospective studies of falling in Parkinson's disease[END_REF].

Balance and gait disabilities are difficult to treat in patients with PD, since levodopatherapy is only partially effective [START_REF] Coelho | Late-stage Parkinson disease[END_REF][START_REF] Sethi | Levodopa unresponsive symptoms in Parkinson disease[END_REF]. Furthermore, deep brain stimulation (DBS) of the subthalamic nucleus (STN), which is an effective treatment for rigidity, akinesia and tremor, but less so for axial symptoms, can sometimes worsen FOG and postural instability [START_REF] Fasano | Axial disability and deep brain stimulation in patients with Parkinson disease[END_REF][START_REF] Grabli | Normal and pathological gait: what we learn from Parkinson's disease[END_REF][START_REF] Weiss | Nigral stimulation for resistant axial motor impairment in Parkinson's disease? A randomized controlled trial[END_REF][START_REF] Schlenstedt | Effect of high-frequency subthalamic neurostimulation on gait and freezing of gait in Parkinson's disease: a systematic review and meta-analysis[END_REF]. Physiotherapy therefore represents a complementary approach for improving gait and balance [START_REF] Bartolo | Four-week trunk-specific rehabilitation treatment improves lateral trunk flexion in Parkinson's disease[END_REF][START_REF] Cugusi | Effects of an adapted physical activity program on motor and non-motor functions and quality of life in patients with Parkinson's disease[END_REF][START_REF] Schenkman | Spinal flexibility and balance control among community-dwelling adults with and without Parkinson's disease[END_REF]. However, in some cases, it does not improve these symptoms in patients with PD, including those who have received STN stimulation. This preliminary study proposes an original standardized rehabilitation programme focused on trunk exercises in order to improve gait and reduce falls in previously operated patients with PD (STN-DBS) who continue to fall despite receiving conventional physiotherapy. In addition to a clinical approach, a three-dimensional (3D) kinematic system was used to objectively assess the influence of the rehabilitation programme on gait.

PATIENTS AND METHODS

Subjects

Ten patients with PD who had received STN-DBS were included in this study because they experienced gait disturbance and falls (minimum 1 fall/48 h) related to festination and/or FOG despite optimization of both pharmacological treatment and stimulation settings. Patients had to be receiving regular ambulatory rehabilitation (minimum of 2 sessions per week) from their practitioners, including gait rehabilitation and work on postural reflexes. Patients needing assistance for deambulation or with a disadvantaged family environment were excluded. All procedures performed in this study were approved by the local ethics committee from the University Hospital of Bordeaux. All patients provided written informed consent.

Rehabilitation protocol

The patients were hospitalized in the rehabilitation department of the University Hospital in Bordeaux for 4 weeks. During this period they were treated daily 5 days a week with 2 physiotherapy sessions per day. Each session lasted 90 min. The intensity of sessions was increased progressively over the 4 weeks until the end of the period of hospitalization. In the mornings, the physiotherapy session focused on trunk mobility only. Passive trunk stretching was followed by active trunk stretching and muscle strengthening exercises in extension, flexion and rotation. In the afternoons, patients were asked to perform the same trunk exercises as in the morning for 1 h, and to perform cardiovascular training for 30 min on a cycle ergometer (Motomed, Viva 2, RECK-Technik GmbH & Co. KG, Medizintechnik, Betzenweiler, Germany). The patients were encouraged to repeat the trunk exercises every day during both the physiotherapy session and in their hospital rooms, and to correct their position as soon as they could in the evenings and at the weekend. At discharge, care was provided by their usual practitioner; patients received the same regular ambulatory rehabilitation as prior to hospitalization and were encouraged to continue trunk exercises every day, together with daily walking for at least 30 min and cycling on a stationary bicycle for 30 min 3 times a week.

Clinical assessment

Patients were assessed in the morning in the drug-On (usual dose in the morning)/DBS-On condition only, 24 h before the beginning and 24 h after the end of the rehabilitation programme. The stimulation parameter settings and drug dosages were unchanged throughout the evaluation. The total score on the motor part of the Unified Parkinson's Disease Rating Scale (UPDRS, part 3), a composite axial score derived from the UPDRS 3 (items 18, 19, 20-face, 22-neck, 27-30), a gait score (item 30 of UPDRS 3) and a postural instability score (item 29 of UPDRS 3) were used to assess the clinical motor state of each patient. Activities-specific Balance (ABC) and Berg scales were used to assess confidence in balance and severity of imbalance [START_REF] Lusardi | Mobility and function in later life[END_REF][START_REF] Powell | The Activities-specific Balance Confidence (ABC) Scale[END_REF]. The ABC is a 16-item scale, and ratings consist of a percentage range from 0% to 100% for each item. A score < 67% corresponds to adults at risk of falling [START_REF] Lajoie | Predicting falls within the elderly community: comparison of postural sway, reaction time, the Berg balance scale and the Activities-specific Balance Confidence (ABC) scale for comparing fallers and nonfallers[END_REF]. The Berg is a 14item scale with a 5-point ordinal scale for each item, ranging from 0 to 4. "0" indicates the lowest level of function and "4" the highest level. The score out of 56 is interpreted as follows: 41-56 = low risk of falls, 21-40 = medium risk, 0-20 = high risk. Finally, a self-report questionnaire, in which the daily number of falls was noted systematically, was applied for 2 weeks before hospitalization and 3 weeks after discharge.

Quantitative assessment

Patients were assessed for gait and posture on the motion platform 24 h before the beginning and 24 h after the end of the rehabilitation protocol (INCIA CNRS UMR5287, Bordeaux University).

Three-dimensional kinematic gait analyses. Kinematic data were acquired with a motion analysis system (Elite, BTS Spa, Milan, Italy) composed of 8 infrared cameras placed throughout the room (acquisition frequency 100 Hz). Each patient had reflective markers attached to the skin bilaterally on anatomical landmarks: heads of the first and fifth metatarsals, lateral malleolus, lateral femoral condyle, greater trochanter, anterior superior iliac spine, and acromion. Additional markers were positioned on the lumbar vertebra L3, thoracic vertebra T10, cervical vertebra C7 and sternum. The motion analysis system calculated the position of the markers in 3D. With the volume required for the movement, the spatial error of the system was < 2 mm (mean 1.5 mm, SD 0.2). The following gait parameters Test procedure. Patients had to walk on the walkway at a comfortable speed. Gait events (heel contact and toeoff for each foot) were identified from kinematic data, and gait spatio-temporal parameters were calculated for each cycle and then averaged with a Matlab routine (Matlab 2013, Mathworks, US). Subjects performed 6 trials, which allowed the identification of at least 18 walking cycles for each participant.

Statistical analysis

Descriptive analyses of clinical and paraclinical data (means and SD) were performed using SPSS Statistic Software (IBM SPSS Statistics). Patients served as their own controls. Comparison of clinical/paraclinical data obtained before and after rehabilitation was performed with the non-parametric Wilcoxon signed-rank test, with a threshold of significance of 5%.

RESULTS

Epidemiological data and deep brain stimulation settings

Demographic data and DBS settings are shown in Table I. The population was relatively elderly (mean age 67.6, standard deviation (SD) 6.3 years) with a predominance of men (7 males (M)/3 females (F)) and a long mean (SD) duration of disease, of 18.8 (4) years.

Effects of rehabilitation on clinical parameters

Despite initial doubts and apprehension, no patient refused to perform or failed to complete the programme. No significant improvements were observed 4 weeks after rehabilitation regarding total UPDRS 3, postural stability and axial scores (Fig. 1; p = 0.20; p = 0.25 and p = 0.18, respectively). On the other hand, the gait item of the UPDRS 3 was significantly improved (mean 1.1 (SD 1) vs 0.7 (SD 0.7) before and after rehabilitation, respectively; p = 0.04).

Individual inspection revealed an improvement in the Berg scores for patient 8 and an improvement in the ABC scores for patients 3 and 7 after rehabilitation (Fig. 2A,B). However, statistical analysis of the population did not de- JRM-CC 2019, Vol. 2 monstrate any significant improvement in the Berg scores after rehabilitation (mean 48.7 (SD 2.8) and 49.6 (SD 2.8) before and after rehabilitation, respectively; p = 0.39; Fig. 2A). Similarly, the mean ABC scores were not significantly improved by rehabilitation (42.6 (SD 11.1) and 45.9 (SD 11.9) before and after rehabilitation, respectively; p > 0.40; Fig. 2B). On the other hand, the mean daily number of falls decreased significantly, from 3.5 (SD 3.2) to 1.3 (SD 1.4) after rehabilitation (p = 0.01; Fig. 2C).

Effect of rehabilitation on paraclinical parameters

Trunk posture. Two parameters related to trunk posture were analysed: the first (D1) was the horizontal distance between the C7 and L3 markers (Fig. 3A, sagittal deviation) and the second (D2) was the vertical distance between the right and left acromion markers (Fig. 3B, frontal deviation on a posterior view). There was a trend toward a decrease in D2 after rehabilitation (mean 19.8 (SD 17.3) mm and 11.1 (SD 6.8) mm before and after rehabilitation, respectively; p = 0.05). No significant change in D1 was observed after rehabilitation (mean 64.7 mm (SD 34.8) and 61.7 mm (SD 30.1) before and after rehabilitation, respectively; p = 0.89).

Kinematic data for gait. The mean time for the walking cycle, right and left feet support durations and double-stance time were decreased significantly after rehabilitation (Table II). Rehabilitation significantly increased walking speed, cadence and length of the left step (Table II). In addition, a coefficient of variation was calculated for the walking cycle duration and cadence (CV = SD/ mean × 100; ( 17)). Rehabilitation significantly reduced variability in the duration of the walking cycle and in gait cadence (Table II).

DISCUSSION

Rehabilitation targeting axial motility improved gait kinematic parameters and reduced the number of falls in patients with PD treated with STN-DBS. The gait item of the UPDRS 3 was significantly improved by trunk muscle strengthening, as confirmed by kinematic analysis. Walking cycle duration, and right-, left-and double-stance durations were significantly reduced by rehabilitation, together with an increase in left-step length, and an increase in gait speed and cadence. The latter was probably related to a significant decrease in variability in duration of walking cycles and a decrease in cadence variability after rehabilitation. The failure to increase right-step length was probably related to the higher rigidity and akinesia scores in the right lower limb than in the left lower limb in our population. Typically, parkinsonian gait is slow, with a shortened step length, reduced speed and an increase in variability of step length [START_REF] Delval | Kinematic angular parameters in PD: reliability of joint angle curves and comparison with healthy subjects[END_REF]. These gait parameter abnormalities seem to predict falling in patients with PD and elderly people [START_REF] Callisaya | Gait, gait variability and the risk of multiple incident falls in older people: a population-based study[END_REF]. Our patients experienced falls, although their Berg and ABC scores suggested a low risk of falling [START_REF] Lajoie | Predicting falls within the elderly community: comparison of postural sway, reaction time, the Berg balance scale and the Activities-specific Balance Confidence (ABC) scale for comparing fallers and nonfallers[END_REF]. This apparent discrepancy could be due to the fact that these scales were first designed to assess the risk of falling in elderly healthy patients. In addition, a ceiling effect on the Berg test was previously reported in patients with PD [START_REF] King | Comparing the Mini-BESTest with the Berg Balance Scale to Evaluate Balance Disorders in Parkinson's Disease[END_REF]. Furthermore, this test does not assess reactive postural control, which is important, as it is associated with a greater risk of falling. On the other hand, the number of self-reported falls decreased significantly after rehabilitation, suggesting an indirect effect of axial mobility on the risk of falling in these operated patients with PD. This could be due to postural changes related to trunk muscle strengthening and/or the improvement in gait quality, as mentioned above. The axial and postural stability items of the UPDRS 3 were not modified by axial rehabilitation. This apparent lack of effectiveness of axial rehabilitation on clinical scores could be due, in part, to the size of the sample and to the fact that these clinical items represent only crude measures of balance disability and are subject to variations in clinical performance and interpretation [START_REF] Maetzler | Quantitative wearable sensors for objective assessment of Parkinson's disease[END_REF]. Indeed, clinical assessment of axial signs, such as gait and balance, remains imprecise, subjective and semi-quantitative. On the other hand, patients tended to improve their posture in the frontal plane, as suggested by a clear trend (p = 0.05) to a decrease in frontal deviation (D2 distance). Trunk-specific rehabilitation treatment has been reported previously to improve lateral trunk flexion in patients with PD [START_REF] Bartolo | Four-week trunk-specific rehabilitation treatment improves lateral trunk flexion in Parkinson's disease[END_REF]. Postural abnormalities, notably mediolateral oscillations, are known to be associated with a higher risk of falling in patients with PD [START_REF] Rocchi | Effects of deep brain stimulation and levodopa on postural sway in Parkinson's disease[END_REF]. Thus, our data suggest that the tendency to postural improvement in the frontal plane observed after trunk muscle strengthening in our population could have contributed to a reduction in the number of falls.

Regarding the impact of gait quality on the number of falls, our patients especially seemed to fall due to gait akinesia with festination episodes and some freezing of gait. Consequently, improvement in gait parameters due to trunk muscle strengthening could explain the decrease in the number of falls. The mechanisms by which trunk muscle strengthening improves quality of gait remain poorly understood. An abnormal increase in activity of trunk muscles during walking was reported recently in patients with PD who fall [START_REF] Cole | Neuromuscular impairments are associated with impaired head and trunk stability during gait in Parkinson fallers[END_REF]. This increase in axial rigidity could lead to less independence of the pelvis, trunk and head. This could, in turn, induce disturbed balance during walking [START_REF] Cromwell | Head stabilization strategies in the sagittal plane during locomotor tasks[END_REF][START_REF] Cromwell | Horizontal plane head stabilization during locomotor tasks[END_REF]. Thus, we hypothesize that changes in axial motility induced by our physiotherapy programme may improve the coordination between the axial segments, and that this improvement could have a positive impact on gait quality. This hypothesis is in line with the notion that quality of gait and risk of falling depend closely on axial motility [START_REF] Grasso | Interactions between posture and locomotion: motor patterns in humans walking with bent posture versus erect posture[END_REF]. In their study, Stozek et al. reported gait improvement in patients with PD after a rehabilitation programme including various physical exercises, such as trunk rotations [START_REF] Stozek | The effect of the rehabilitation program on balance, gait, physical performance and trunk rotation in Parkinson's disease[END_REF].

In conclusion, trunk muscle strengthening should be an ongoing adjunct to PD medication and classical physical therapy in the context of gait abnormality with falls in patients with PD treated with STN-DBS.

Nevertheless, these preliminary data should be viewed with caution due to the small size of the patient sample. The therapy proposed in this study is probably more intensive than is generally available in routine care. However, no patient refused the therapy or failed to perform it completely. Clinical and paraclinical data were collected 24 h after the end of the rehabilitation programme, whereas the number of daily post-rehabilitation falls was assessed 3 weeks after discharge. Thus, even if this period is short, it should be taken into account before directly associating these 2 types of parameter. Our results were obtained in patients with advanced PD who were operated for the severity of the disease (STN stimulation). It would therefore be interesting to administer this programme to non-operated patients. Our data, obtained by movement platform analysis, do not exactly reflect the patient's environment, thus the parameters we measured are difficult to translate into routine daily practice. Finally, it remains to be investigated whether the reduction in falls and the improvement in gait found with our protocol have an impact on patients' quality of life, and how long the benefit lasts.

In conclusion, gait and balance impairments are frequent in the course of PD, and involve a high risk of falls. Dealing with these axial symptoms is a challenge that not only concerns improving the autonomy and quality of life of these patients, but also reducing the cost of medical and health services. These preliminary results are encouraging and suggest that specific rehabilitation focused on axial motility is a potentially useful supplement to the traditional approach (pharmacological/surgical) of treating the primary deficits in patients with PD treated with STN-DBS.

  www.medicaljournals.se/jrm-cc JRM-CC p. 3 of 6 Rachis rehabilitation in Parkinson's disease were assessed: walking speed, walking cycle duration, the variability coefficient of walking cycle duration, gait cadence, variability coefficient of cadence, double-stance duration, left-and right-stance duration, right-and left-step length, and right-and left-shifts of the trunk.

Fig. 1 .

 1 Fig. 1. Impact of rehabilitation on clinical data. (*p < 0.05); For the postural stability and gait items (C and E, respectively), some values were similar between the patients. This explains why some lines are superimposed and appear as missing lines.

Fig. 3 .

 3 Fig.3.Postural measures. (A) Measures in sagittal plane. D1: horizontal distance between C7 (7th cervical vertebra) and L3 (3 th lumbar vertebra) markers. (B) Measures in frontal plane. D2: vertical distance between right and left acromion markers. T10: 10 th thoracic vertebra.

Fig. 2 .

 2 Fig. 2. Impact of rehabilitation on gait scales and number of falls.

Table I .

 I Epidemiological data and deep brain stimulation settings

	Id	Age	Sex	PD duration, years LEDD, mg/d	DBS duration, years	NR	NL	VR	VL	PWR	PWL	Freq
	1	73	M	17	1,100	9	2	2	2.5	2.3	90	90	60
	2	73	M	24	400	8	2	2	2.8	2.8	90	90	130
	3	64	M	14	600	7	2	2	4.2	3.5	90	90	175
	4	62	M	16	1,355	11	2	1	3.4	3.4	60	60	60
	5	67	F	25	760	10	1	1	2.5	2.5	90	90	130
	6	54	F	21	650	11	1	1	4.6	3.2	60	60	130
	7	68	F	21	865	9	1	1	4.0	3.8	90	90	60
	8	70	M	19	1,050	9	1	1	2.1	2.3	90	60	140
	9	75	M	18	850	2	1	1	2.5	2.5	60	60	140
	10	70	M	13	1,228	3	1	1	3.2	3.0	60	60	130
	Mean	67.6		18.8	885.8	7.9	1.4	1.3	3.2	2.9	78.0	75.0	115.5
	SD	6.3		4.0	298.3	3.1	0.5	0.5	0.8	0.5	15.5	15.8	40.6

Id: identification number of patients; M: male; F: female; LEDD: levodopa equivalent daily dosage, mg/day

[START_REF] Tdbsfpsds | Deep brain stimulation of the subthalamic nucleus or the pars interna of the globus pallidus in Parkinson's disease[END_REF]

; NR: number of contacts used on right side; NL: number of contacts used on left side; VR: voltage (in volts) on right side; VL: voltage (in volts) on left side; PWR: Pulse width (in µs) on right side; PWL: pulse width (in µs) on left side; Freq: stimulation frequency; SD: standard deviation.

Table II .

 II Impact of rehabilitation on kinematic data of gait

		Before	After
		rehabilitation	rehabilitation
		Mean (SD)	Mean (SD)	p
	Walking speed, sec	807.3 (275.6) 915.6 (193.5) 0.03
	Walking cycle duration, sec	1.3 (0.2)	1.1 (0.1)	0.02
	Variability coefficient of walking
	cycle duration	6.7 (3.8)	3.8 (1.8)	0.03
	Cadence, steps/mn	96.7(11.2)	105.5 (11.1)	0.02
	Variability coefficient of cadence	6.4 (3.5)	3.7 (1.8)	0.03

Right stance duration, % 65.2 (8.4) 61.7 (4.8) 0.03 Left stance duration, % 65.3 (8.3) 60.1 (4.1) 0.007 Double stance duration, % 30.4 (16.7) 21.8 (8.8) 0.01 Length of right step, mm 496.0 (147.3) 511.4 (109.5) 0.07 Length of left step, mm 493.5 (125.9) 535.1 (102.7) 0.02 p: significance threshold values (Wilcoxon rank test).

ACKNOWLEDGEMENTS

The authors would like to thank the physiotherapists who cared for patients during the 4-week period of rehabilitation, and Ray Cooke for copyediting the manuscript.

The authors have no conflicts of interest to declare.


E Garcia

P Sciora

G Rimpault
email: gerald.rimpault@cea.fr

EVALUATION OF THE NEUTRONIC SUPERPHENIX START-UP COMMISSIONIG TESTS WITH TRIPOLI4

Keywords: Superphénix reactor, start-up commissioning tests, TRIPOLI-4

The Superphénix reactor was a Sodium-cooled Fast Reactor (SFR) developed under a European framework that intended to provide a general design of a commercial power plant. During the start-up of the reactor, six different batches containing dummy assemblies allowed to verify the main safety criteria at zero-power conditions tests at 180°C. The present paper presents a stochastic analysis of two start-up cores by comparing the experimental results with the performed calculations with TRIPOLI-4 and the JEFF 3.1.1 nuclear data evaluations. The main and back-up control rod evaluations, the critical mass, as well as the handling error experiment are examined. In addition, two particular irradiation tests are studied to determine the flux distribution of the core and finally, the assessment of a subcriticality analysis is performed to verify the validity of the former MSM factors. In general, the obtained results show good agreement with the experiment, having a general reactivity bias with the performed calculations, the flux distribution is also in great agreement with the tests with small discrepancies and the MSM evaluation permitted to observe better consistency with experimental data. The obtained results allowed to validate our model and will serve as reference calculation for further research.

INTRODUCTION

Since past decades, the generated amount of depleted uranium motivated the development of a technology that could maximize uranium utilization and hence, extend the fuel reserves for several centuries. The Sodium-cooled Fast Reactor (SFR) concept was the selected candidate to attend this need because of its neutronic properties and its breeding capabilities.

In France, the construction of an experimental facility called Rapsodie in 1959, followed by the demonstrator reactor Phénix, provided the basis knowledge of this technology. Subsequently, a large-scale reactor intended to consolidate the SFRs as a choice for electricity production by including the gained experience in the past reactors [START_REF] Rodriguez | Evolution of sodium technology R&D actions supporting French liquid-metal fast breeder reactors[END_REF]. This commercial size reactor was called Superphénix and it was developed under a European framework between France, Italy and Germany.

The construction of the plant began in 1975 and after ten years of construction, in 1985, the first phase of the commissioning tests was performed, in which sodium flow technical problems were successfully fixed. The second phase was devoted to the core loading and a set of tests to assess the safety criteria of the core. A particular core loading strategy enabled to minimize the time between the defined core patterns, in which a total of six batches accomplished the full loading of the Main control rod system Back-up control rod system Neutron guides Superphénix core, reaching the first criticality in a configuration known as Coeur de Première Divergence (C1D) on 6 September 1985 [START_REF] Gourdon | An Overview of Superphénix Commisioning Tests[END_REF]. Before the core reached full power, the safety commissioning tests in Superphénix were performed in a core named Coeur de Montée en Puissance (CMP). The main objective in the CMP core was to verify the safety criteria related to the control rod systems, as well as to evaluate the available reactivity reserves for the full power cycle.

Early analysis of Superphénix was based in diffusion approach with the former CARNAVAL-IV calculation scheme and implied a large number of corrections due to the existing calculation route, such as mesh, transport and heterogeneity corrections among others. Afterwards, in the 1990 decade, the Superphénix analysis led to the production of the adjusted library ERALIB1 [START_REF] Fort | Realisation and performance of the adjusted nuclear data library ERALIB1 for calculating fast reactor neutronics[END_REF]. This adjustment was done from JEF2.2 library with a simplified transport approach, and it consisted in cross-section corrections in nuclear data of a set of isotopes ( 235 U, 238 U, 239 Pu, 240 Pu, 241 Pu, 242 Pu, 56 Fe, 52 Cr, 58 Ni, 23 Na, and 16 O). Despite the obtained results with this library were close to measurements [START_REF] Rimpault | Développements Algorithmiques et Qualification du Système de Codes et Données Nucléaires ERANOS pour la Caractérisation des Réacteurs à Neutrons Rapides[END_REF], the ERALIB1 library misses all the nuclear data improvements that are coming from new evaluations since JEF2.2.

At the present day, the evaluation of the Superphénix core can be performed with the late developed neutronic platforms, which will bring a better comprehension of the core physics and the associated bias of the different calculation schemes. The present paper presents a stochastic analysis of the start-up cores by comparing the experimental results with the performed calculations. The TRIPOLI-4 code is used as the stochastic reference with the European nuclear data evaluations JEFF 3.1.1.

Chapter 2 presents the main characteristics of the Superphénix core as well as the general description of the batches under analysis and the performed tests of each of these core patterns. The chapter 3 provides a brief description of the model considerations in this paper. Obtained results are shown in chapter 4 and finally, in chapter 5 the concluding remarks are presented as well as the further research to be done.

THE START-UP CORES

Core Characteristics

The Superphénix core was composed by two concentric fissile regions with axial blankets at the top and the bottom of the fuel zone boundaries. Radially, fertile assemblies were placed surrounding the outer fuel core. Sintered UPuO 2 pellets were introduced into the fuel pin elements in the fissile assemblies and depleted uranium pellets were placed in the upper, lower and radial blankets. Figure 1 presents the core map of the Superphénix core in which the distribution of the fuel and blanket zones is shown. The main control rod system SCP (Système de Commande Principal) contained 21 assemblies divided in two curtains, one in the inner core and another in the interface of the inner and outer core. Each of the SCP control assemblies contained 31 absorber pins of B 4 C enriched at 90% in 10 B. The back-up control rod system SAC (Système d'Arrêt Complémentaire) was placed in the inner core and its objective was to assure the shutdown of the rector. Since this system was only used for shutdown and not for the reactor control, it has only two positions: inserted (core shutdown) and withdrawn position (core operation).

In order to assure the proper control of the reactor, three neutron guides were placed at 120 degrees of the outer core boundary. This neutron guides enabled the flux to be measurable enough in the bottom fission chambers of the core to provide precise reactivity measurements. However, a special assembly for online in-core measurements was used during these tests since the neutron flux in the reactor was very low and the nominal measurement system would not provide accurate measurements. It accounted a telescopic tube in the central channel of the reactor that could be inserted in the central assembly since the central fuel pin elements were not present in this element. It accounted three fission and activation foils to determine neutron flux distribution inside and around the core [START_REF] Gourdon | An Overview of Superphénix Commisioning Tests[END_REF] as it is discussed in next section.

The Selected Cores in the Checkerboard Loading Pattern

The loading of Superphénix core was performed by applying a strategy called the checkerboard pattern, which consisted in the gradual replacement of previously loaded dummy assemblies by fuel assemblies in predefined batches.

The whole core loading was performed in six different batches and it permitted to predict the first critical core, whose name was "Coeur de Premiere Divergence" (C1D). The last batch was also known as the "Coeur de Montée de Puissance" (CMP), in which an exhaustive experimental program was performed to assess the safety criteria of the core [5]

First divergence core

Once a predefined batch was loaded, reactivity measurements were done at zero-power conditions at 180°C. In each batch, the backup and main control rod systems were withdrawn to estimate the first critical core, which was in agreement with previously calculated results [START_REF] Flamenbaum | Superphénix Core-Loading Strategy Using the Checkerboard Pattern[END_REF]. The first divergence core (C1D) was composed of 325 fuel assemblies and 33 dummy assemblies placed in the inner core. Criticality was achieved with the SCP barely inserted and the withdrawn position of the SAC.

The Coeur de Montée en Puissance

The last batch was called Coeur de Montée en Puissance (CMP) in which a total of 18 dummy assemblies were placed next to the main control rod systems to limit the reactivity excess at this stage. In particular, the analyzed tests that are presented in this paper are:

Control rod evaluations

An exhaustive SCP analysis program allowed the estimation of reactivity reserve for the first power cycle and determined the main and back-up control rod worth. For the SAC evaluations, two assessments were performed:

 Being the reactor in critical state (SCP in critical position Zc) the SAC was inserted.  Being the reactor in subcritical state (SCP completely inserted) the SAC was inserted.

Handling error test

Another test to survey local criticality in the core was done. The handling error was performed to verify previous research concerning the amplification of this effect in large size cores. To assess this possibility the following procedure was done:  The SCP and SAC were inserted except for the control rod 13 (see Figure 3)  A fuel assembly was loaded instead of the dummy loaded beside control rod 13.

Transversal irradiation tests

During the start-up commissioning tests, a particular experiment was performed to determine the radial flux profile of the core. For this test, a series of special subassemblies containing measurement capsules instead of the central pins (replacement of 19 central fuel pins for the fuel assemblies and 7 fertile pins for the blanket assemblies [START_REF] Gourdon | An Overview of Superphénix Commisioning Tests[END_REF]) were radially loaded as shown in Figure 4. Fission and activation foils were installed inside these capsules, after the irradiation tests these detectors were analyzed to determine neutron flux distribution inside and around the core. The core and blanket rate distributions were measured by irradiating these assemblies at 3 MW for two hours [START_REF] Cabrillat | Power and Neutron Flux Distributions in the Core and Shielding[END_REF]. The experimental reactivity was determined by the Modified Source Multiplication (MSM) method, as stated in reference [START_REF] Gauthier | Measurement and Prediction of Control Rod Worth[END_REF], therefore, the available measurements of Superphénix are spatially corrected with the former CARNAVAL-4, which may be a source of bias. The MSM method was previously validated in the MASURCA facility, from which the experimental reactivity is related to the MSA (méthode de source approchée, approximated source method) and a spatial correction given by the MSM method. The sub criticality test consisted in measuring each of the control rods worth by independently extracting and inserting each of the control rods. As stated in section 2.1, a special assembly for online measurements contained three fission chambers with different axial position (see Figure 5), from which the reactivity was corrected with the MSM method. Currently the interest is to determine whether or not the reactivity worth improvement can be done with the TRIPOLI-4 code, based in previous references that show a better agreement with the MSM method when using a Monte Carlo method. Besides, the uncertainty impact by its inclusion in the experimental correction with no use of adjusted libraries (JEFF 3.1.1 in this case, ERALIB1 in former calculations).

PERFORMED ANALYSIS

The objective of the present paper is to evaluate the start-up commissioning tests Superphénix in a stochastic perspective. To accomplish this task, a developed model in the stochastic TRIPOLI-4 code [START_REF] Trama | Overview of TRIPOLI-4 version 7 continuous-energy monte-carlo transport code[END_REF] is validated to serve as further reference in this research. The TRIPOLI-4 code is a reactor physics probabilistic code based on the Monte Carlo method in which the neutronic behavior is reproduced by following the neutron histories in a defined geometry with assigned material characteristics. In our case, the specified geometry accounts a high heterogeneous description considering the geometrical and material implications due to the dilatation at 180°C.

Evaluated tests

Control rod evaluations

The first critical core presented a considerable amount of dummy assemblies, from which 18 were strategically placed next to the main control rod system and 33 were located in the inner core. In this core, criticality was obtained with main control rods barely inserted in the fuel core. For the C1D only the evaluation of the main control rod is presented. Secondly, the analysis concerning the CMP includes the evaluation of the main control rods and the back-up control system assessment as well as the handling error test.

For these cores, two fuel approaches are considered: the first considers the average fuel composition of the assemblies per fuel zone. The second assessment, called the per assembly composition approach, accounts each assembly composition by taking into account the heavy isotopes in each of these fuel assemblies. The composition of each assembly was measured in October 1984, and it was adjusted to accord the test's date by considering the 241 Pu decay to 241 Am. By comparing these approaches, the bias by considering average fuel in the core regions will be known as a modeling bias in fuel composition definition.

Transversal irradiation test

For the CMP, two different tests are presented, the first identified as Transversal Irradiation 1 (T1) accounts all of the control rods at the same axial position at the half height of the active core. The second irradiation test is recognized as Transversal Irradiation 2 (T2), with an axial displacement of the control rods curtains as seen in Figure 6. To analyze the radial flux distribution only five sub assembly positions of the internal core are selected, one for the outer core position and the last one in the fertile position.

Subcriticality analysis

The evaluation of each of the control rod worth in the SCP is done with the TRIPOLI-4 code accounting a new MSM spatial correction. Several references state the methodology of the MSM method [START_REF] Jean | Monte Carlo MSM correction factors for control ros worth estimates in subcritical and near-critical fast neutron reactors[END_REF], [START_REF] Blaise | Application of the Modified Source Multiplication (MSM) Technique to Subcritical Reactivity Worth Measurements in Thermal and Fast Reactor Systems[END_REF], [START_REF] Truchet | Application of the modified neutron source multiplication method to the prototype FBR Monju[END_REF], [START_REF] Naberejnev | Physics study of the TRADE: TRIGA Accelerator Driven Experiment[END_REF] and in this paper the objective is to observe the impact in the control rod worth for the subcritical analysis. The generation of MSM factors with a stochastic code requires the reference and the configuration reactivity calculations as well as the corresponding reaction rates.

RESULTS

In this section, results of the evaluations are presented in three subdivisions. First the control rod evaluations for SCP are presented for three positions (↑: withdrawn position, Zc: critical position and ↓: inserted position) and for the SAC, two evaluations are presented with the SCP in extracted and inserted position. Control rod worth is also discussed in these sections. Secondly, the flux distribution is given for two different tests, the T1 and T2, as it was described in the previous chapter and finally, the subcriticality analysis is presented with the corresponding evaluations of the MSM method.

Before commenting the results, a couple of remarks are needed in this section. The first concerns to the use of nuclear data evaluations and its impact on calculations. A previous analysis performed with ERANOS and the use of the COMAC V2 covariance matrix provided a nuclear data uncertainty propagation of almost 700 pcm, so even with the use of the Monte Carlo method this uncertainty is present for all the calculations. Therefore, one has not to be surprised for the presence of bias within this range for these evaluations.

The second remark involves the experimental uncertainty determination and its components. The considered sources to its evaluation are the inherent measurement uncertainty (counting rates uncertainty) which are unavoidable in all experimental tests. The reactivity reference uncertainty and the reactivity determination method (MSM method) are also accounted to the final uncertainty estimation, and of course the delayed neutron fraction (β eff ) uncertainty, being this very important for the final uncertainty determination. To evaluate the β eff , the IFP method already implemented in TRIPOLI-4 is used, however its uncertainty is not included for the present values, instead the former uncertainty evaluation with ERANOS code is taken. Further analysis includes the β eff uncertainty analysis with IFP method and its inclusion in the experimental uncertainty.

Control rod evaluations

The calculated reactivity for different SCP positions of the C1D core is presented in TABLE I. For the average composition approach a reactivity difference of about 560 pcm is registered for the extracted and the critical position of the SCP, while for the inserted position this difference is reduced about 140 pcm. When using the per assembly approach the difference between measurements and calculations are reduced about 100 pcm for the withdrawn and critical positions and for the inserted position this difference is reduced to 50 pcm. In consequence the control rod worth evaluation is different depending on the composition approach. However, one has to be aware that the inserted position of the SCP registers a large subcriticality state and hence a large uncertainty is related to this measurement. Control rod worth of the SCP in the C1D is shown in Table II. All the results are in good agreement with the measurements, remaining within the experimental uncertainty range. The fuel composition approaches have an impact in this evaluation, for instance, when using the average approach the total control rod worth has an error of about 1.6% however, when the per assembly composition approach is used, this error is reduced down to 0.5%, this means that a slight improvement of the control worth is observed with the per assembly approach. An overestimation of the control rods is seen for the Δρ (Zc to ↓) and Δρ (↑ to ↓) positions with TRIPOLI-4. Results for the SCP evaluation in the CMP are seen in Table III for three different positions, still one has to recall that the reactivity reserves of the CMP were not directly measured and they are related to the S curve of the main control rod system. When observing these evaluations a reactivity bias is also present, as in the C1D. Nevertheless, this bias seems to be reduced to about 100 pcm in general. For directly measured positions (Zc and ↓) a bias of 370 pcm is identified while for the reactivity reserves an increase of about 90 pcm is seen independently from the composition approach. The influence of the per assembly composition approach is not identical to the observed in the C1D, now only about 50 pcm reactivity decrease is seen compared to nearly 100 pcm in some positions of the C1D. Control rod worth of the SCP for the CMP is shown in Table IV. The evaluations are in good agreement with measurements within the experimental uncertainty range. For the evaluation involving the critical and the inserted position of the SCP a remarkable agreement is seen with diminutive discrepancy face to the measurements. The per assembly composition approach is very similar to the average approach evaluation with minimal differences, and contrariwise to the C1D evaluations, the analyzed composition approaches have no major influence on control rod worth for the CMP. An overestimation of the control worth is seen in all positions with TRIPOLI-4 with a maximum value of 106 pcm. The reactivity change from the C1D core to the CMP core can be evaluated at this point since the only modification is the loading of 33 fuel assemblies in the CMP core. In Table V two different conditions are evaluated for the reactivity reserves change from C1D to CMP, the extracted control rod position (SCP ↑) and the inserted control rod position (SCP ↓). Results remain within the experimental uncertainties, however one has to perceive the wide uncertainty experimental range.

Table III. Main control rod reactivity difference (C-E) in pcm in the CMP. Measurement in pcm with

For the extracted SCP position of the CMP presents a large experimental uncertainty due to the critical mass determination method, which contributes to the large uncertainty at this position. In the other hand, for the inserted position, the C1D has a large subcriticality state, which provides a large uncertainty range in this evaluation. However, the presented evaluations are within the experimental uncertainty range, with a major discrepancy of 107 pcm. For the withdrawn position of the SCP a better agreement is found with the per assembly approach with only 50 pcm difference while the average approach presents almost double reactivity difference with measurements. In the other hand, for the inserted SCP evaluation the average approach has a better agreement with 56 pcm difference, and this time the per assembly composition approach doubles the difference with measurements. In Table VII, as in the past evaluation, the reactivity bias is again present in a consistent manner. The per assembly composition approach shows the same trend that was pointed out before, a reactivity decrease is seen when compared to average approach. The control worth evaluation is in good agreement with the experimental data, remaining within the uncertainty range. This evaluation consists in calculating the reactivity difference between the withdrawn position of the all the control rods and the inserted position of all control subassemblies except for the 30 22 position, as described in Section 2.2.2. The evaluations are consistent and in good agreement with measurements, remaining within the experimental uncertainty range. A deviation of almost 100 pcm is seen between calculations and measurements. However, this difference is related to the withdrawn position of the SCP, as it was discussed in Table V. Finally, the composition approaches are consistent between them with practically no reactivity difference. As a final remark of the control rod evaluation, the CMP criticality reserves has to be revisited to check consistency of this evaluation given the discrepancy between measurements and calculations.

It is likely to correct this measurement since the S curve evaluation does not accounts spatial corrections, whose importance may be conducted in further research.

Transversal irradiation tests

Results for the Transversal irradiation 1 are shown in Figure 7 with the TRIPOLI-4 code. As stated before, in this test the control rods position was identical for all control rods and flux is normalized to the recorded flux in 30 29. The results presented in this section correspond to the flux at the midheight of the active core. The measurement uncertainties are also provided at each assembly position, however it does not includes another uncertainty sources such as the control rod axial position.

The TRIPOLI-4 evaluation presents very good agreement with the experimental data with less than 1% error, besides, the results are less dispersed than those compared to the former evaluation. The largest discrepancy is seen at the position 30 25, which is in the internal core at the half distance between the central assembly and the periphery of the internal core. The outer core position assembly (30 21) and the fertile position assembly (30 19) are well described with minimal error.

The contiguous assemblies to the control rods, 30 28 in the inner core and 30 21 in the outer core, permit to observe its flux distribution impact at the mid-height of the active core. The developed model in TRIPOLI-4 can describe with remarkable agreement the flux at this state, with less than 0.50%. Results concerning the Transversal Irradiation T2 are shown in Figure 8. The difference with the T1 irradiation test is the displacement of the control rods between them, as previously depicted in section 2.2.2. Results presented correspond to the recorded fission rate at the mid-height of the active core and as in the previous evaluation the presented values are normalized to the 30 29 flux position. Evaluations with TRIPOLI-4 are in great agreement with measurements with less than 1% error, however, C-E differences are more dispersed than in the previous evaluation.

- When comparing the T1 and T2 irradiation tests the flux distribution is clearly different, for instance, the flux in the position 30 28, which is next to the inner control rod curtain, is flatter in the T2 than in the T1 because this curtain is axially higher in the T2. In compensation, since the outer control curtain has a lower position in the T2 than in the T1 the flux in the positions 30 23 and 30 21 is clearly smaller for the T2. In both cases the TRIPOLI-4 model is capable of describe the flux with small difference when compared to the measurements. Finally, for all subassemblies positions the fission rate values remain within the experimental uncertainty range being this valid for both evaluations T1 and T2.

Subcriticality analysis

Results concerning the subcriticality test are compared to the developed model with TRIPOLI-4, and the results are compared with the former correction with CARNAVAL-IV and the present correction with TRIPOLI-4. Figure 9 presents the control rod worth difference from critical to inserted position for each of the 21 control subassemblies in the main control rod system. The inner curtain of the SCP is composed by six control rods (B1 to B6) from which an average bias of 40 pcm is observed with the CARNAVAL-IV corrections, however the values present important dispersion of about 25 pcm. For the outer curtain of the SCP, composed by 15 control subassemblies (B7 to B21) an average bias of about 42 pcm is observed, but a larger deviation of 58 pcm is seen.

Present evaluations show better agreement with minimal dispersions than the former evaluations. For the inner curtain a dispersion of only 3 pcm is seen, compared to the 25 pcm with former evaluations and an average of 41 pcm is seen for these rods, being consistent with past assessments. The outer control rod curtain presents larger dispersed values with a maximal 10 pcm value, however is well reduced compared to the former evaluations. For both evaluations, in general an average bias is identified of about 40 pcm, having good consistency with both evaluations. Figure 10 presents the control rod worth difference between measurements and calculation for the critical to extracted positions of the SCP. The former measurements corrected with CARNAVAL-IV present a considerable dispersion of about 71 pcm, being this 20 pcm larger than the seen in the previous evaluation. Conversely, to the previous evaluation the reactivity values are dispersed with the same behavior in the internal and external curtain. After applying the MSM correction with TRIPOLI-4 for these evaluations the reactivity dispersion is largely reduced to only 5 pcm difference, besides the C-E difference is also considerably reduced.

CONCLUSIONS

The evaluation of a selected list of the Superphénix start-up has been done with the Monte Carlo code TRIPOLI-4. Results provide a remarkable good agreement with the experimental data: control rod worth is well described for the main and back-up control rod systems (SCP and SAC) as well as the handling error, which is accurately reproduced. The radial flux profile was also analyzed for two different tests with good agreement against the experimental measurements and finally the evaluation of the MSM factors led to a better agreement and interpretation of the control rod worth in a subcriticality test.

The developed model with TRIPOLI-4 has now been validated against experimental data and its corresponding uncertainty, thus, it serves as calculation reference for future research concerning the Start-up of Superphénix. Moreover, further analysis has to be done to assess the impact of these evaluations when the use of another more recent nuclear data evaluation (JEFF 3.2 for instance).

Besides the nuclear data uncertainty propagation assessment is required to determine its impact on current evaluations, nevertheless the use of Monte Carlo codes to perform this task is often complex.

As with all Monte Carlo codes, the calculation time and the memory demands with TRIPOLI-4 is an important drawback, particularly when studying reaction rates. For this reason, the use of a deterministic code, such as ERANOS or the novel APOLLO3, is suggested to avoid these difficulties, besides, the use of its perturbation modules would enable performing detailed reactor physics analyses. A particular interest to perform such assessment with APOLLO3 appears for its considerable improvements for neutronic calculation and its novel treatment at different levels of the core [START_REF] Archier | New Reference APOLLO3© Calculation scheme for sodium cooled fast reactors: from sub-assembly to full-core calculations[END_REF].

In the other hand, the experimental uncertainty of these tests has to be revisited, to attribute a possible reduction of each of its sources and to determine whether or not a considerable uncertainty reduction is possible with the later neutronic tools.

Figure 1 .

 1 Figure 1. Superphénix core map

Figure 2 .

 2 Figure 2. Core layout of the C1D (left) and the CMP (right).

Figure 3 .

 3 Figure 3. Handling error test in the CMP

Figure 4 .Figure 5 .

 45 Figure 4. Core layout of the Irradiation tests T1 and T2 and experimental assembly measurement positions

Figure 6 .

 6 Figure 6. Control rod displacement for Irradiation tests T1 (left) and T2 (right)

Figure 7 .

 7 Figure 7. Fission rate of 239 Pu at mid-height of the active core for the irradiation test T1.

Figure 8 .

 8 Figure 8. Fission rate of 239 Pu at mid-height of the active core for the irradiation test T2.

Figure 9 .

 9 Figure 9. Control rod worth C-E difference between critical to inserted positions. In red the measurements corrected with former CARNAVAL-IV and in blue measurements corrected with TRIPOLI-4.

Figure 10 .

 10 Figure 10. Control rod worth C-E difference from critical to extracted positions. In red the measurements corrected with former CARNAVAL-IV and in blue measurements corrected with TRIPOLI-4.

Table I . Reactivity results in pcm (C-E ) of the C1D core for average and exact approaches.

 I

	Measurement in pcm with 1$ = 365 pcm and uncertainty at 1σ.
	Measurement	321 ± 22 pcm	0 pcm	-8221 ± 231 pcm
	Composition approach	SCP ↑ C-E	SCP Zc C-E	SCP ↓ C-E
	Average	562	566	424
	Per assembly	461	471	413

Table II . Control rod worth results (C-E and C/E) by position of the C1D core for average and exact approaches. Measurement in pcm with 1$ = 365 pcm and uncertainty at 1σ. Measurement 321 ± 22 pcm 8221 ± 231pcm 8542 ± 231pcm Composition approach Δρ (↑ to Zc) Δρ (Zc to ↓) Δρ (↑ to ↓)

 II

		C-E	C/E	C-E	C/E	C-E	C/E
	Average	-4	0.98	141	1.017	138	1.016
	Per assembly	-10	0.96	58	1.007	48	1.005

Table IV . Control rod worth results (C-E and C/E) by position of the CMP core for average and exact approaches. Measurement in pcm with 1$ = 370 pcm and uncertainty at 1σ.

 IV

	Measurement	3774 ± 264	4414 ± 151	8188 ± 294
	Composition	Δρ (↑ to Zc)	Δρ (Zc to ↓)	Δρ (↑ to ↓)
	approach	C-E	C/E	C-E	C/E	C-E	C/E
	Average	94	1.025	5	1.00	100	1.012
	Per assembly	90	1.025	15	1.00	106	1.013

Table V . Reactivity reserves difference between C1D and CMP cores in pcm. Measurement in pcm with 1$ = 370pcm and uncertainty at 1σ.

 V Evaluation of SAC is presented in Table VI and TableVIIfor the SCP in critical and inserted position respectively. A consistent reactivity bias is seen with the critical position of the SCP in TableIII, and the per assembly composition approach has the same tendency and is translated as a reactivity decrease of about 50 pcm. Despite the control rod worth is overestimated in both cases for almost 50 pcm this evaluation is within the experimental uncertainty range.

	Measurement	3453 ± 268 pcm	3807 ± 275 pcm
	Composition approach	C-E	SCP ↑	C/E	C-E	SCP ↓	C/E
	Average	-94	0.97	-56		0.98
	Per assembly	-50	0.98	-107	0.97

Table VI . Back-up control rod system (with SCP at critical position) reactivity difference and control rod worth. Measurement in pcm with 1$ = 370pcm and uncertainty at 1σ.

 VI

	Measurement		1048 ± 58 pcm
	Composition approach	SAC ↑ SAC ↓ Control rod worth C-E C-E C-E C/E
	Average	374	325	49	1.038
	Per assembly	321	275	46	1.044

Table VII . Back-up control rod system (with SCP at inserted position) reactivity difference and control rod worth. Measurement in pcm with 1$ = 370pcm and uncertainty at 1σ. Measurement 1210 ± 52 pcm

 VII Results concerning the handling error test are shown in TableVIIIfor both composition approaches.

	Composition approach	SAC ↑ C-E	SAC ↓ C-E	Control rod worth C-E C/E
	Average	369	371	-2	0.998
	Per assembly	306	291	15	1.01

Table VIII . Handling error test in the CMP. Measurement in pcm with 1$ = 370 pcm and uncertainty at

 VIII

		1σ.
	Measurement	6621 ± 279 pcm
	Composition approach	Δρ (SCP ↑ , SAC ↑) -(SCP ↓ except 30/22, SAC ↓) C-E C/E
	Average	-91	0.98
	Per assembly	-90	0.98

ACKNOWLEDGMENTS

The authors wish to thank CONACYT for providing economical support for Elías García PhD attachment at CEA-Cadarache, France under the Sustainability-SENER program.


Delphine Albrecht

Hans C Hurlimann

Johanna Ceschin

Christelle Saint- 5 Marc

Benoît Pinson

Bertrand Daignan-Fornier
email: b.daignan-fornier@ibgc.cnrs.fr

Multiple chemo-genetic interactions between a toxic 2 metabolite and the ubiquitin pathway in yeast 3 4

Keywords: 39 Ubiquitin, metabolic intermediate, yeast, suppression, additivity 40

. Two ubiquitin-conjugating enzymes (Ubc4 and 31 Cdc34) and a ubiquitin ligase (Cdc4) were found to contribute to the ability of cells to cope 32 with ZMP. This study illustrates the complexity of chemo-genetic interactions and shows how 33 genetic analyses allow deciphering the implicated pathways, the individual gene effects and 34 their combined phenotypic contribution. Based on additivity and suppression patterns, we 35 conclude that AICAR treatment shows synthetic interactions with distinct branches of the 36 yeast ubiquitin pathway. 37 38

INTRODUCTION

43

ZMP, the monophosphate derivative of Amino-Imidazole CarboxAmide Ribonucleoside 44 (AICAR), is a precursor of AMP in the purine de novo biosynthesis pathway (Fig. 1a) and as could also cause unwanted side effects. In line with this remark, ZMP, the monophosphate 60 derivative of AICAR is accumulated in several purine genetic diseases and could contribute to 61 the poorly understood etiology of these diseases [START_REF] Ceballos-Picot | New biomarkers for early diagnosis of Lesch-370 Nyhan disease revealed by metabolic analysis on a large cohort of patients[END_REF]Daignan-Fornier 62 and Pinson, 2012). For all these reasons, understanding, how AICAR (and its active form 63 ZMP) affects cellular processes and results in beneficial or an adverse effects, is crucial. 64

To identify cellular functions affected by AICAR, we took advantage of yeast genetics to 65 isolate and characterize several yeast mutants showing higher sensitivity to AICAR (Albrecht 66 et al., 2016;[START_REF] Ceschin | Disruption of Nucleotide Homeostasis by the 374 Antiproliferative Drug 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside 375 Monophosphate (AICAR)[END_REF][START_REF] Ceschin | Identification of yeast and human 5-aminoimidazole-4-378 carboxamide-1-beta-d-ribofuranoside (AICAr) transporters[END_REF]. Of note, search for AICAR-sensitive 67 mutants cannot easily be done at a genome wide scale since a specific and complex genetic 68 set-up (ade16 ade17 ade8 his1) is required to reveal AICAR-sensitivity (Fig. 1a) (Ceschin et 69 al., 2015;[START_REF] Hurlimann | Physiological and toxic effects of purine 422 intermediate 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) in yeast[END_REF]. Recently, we found that sensitivity to AICAR is connected 70 with the ubiquitin pathway in yeast and mammals. In particular, AICAR treatment is synthetic 71 lethal with loss of function of several histone-modifying enzymes in yeast and human cells 72 [START_REF] Albrecht | 361 Chemo-Genetic Interactions Between Histone Modification and the Antiproliferation 362 Drug AICAR Are Conserved in Yeast and Humans[END_REF]. Indeed, in yeast cells, the deletion of BRE1, that catalyzes the 73 ubiquitination of H2B (H2Bub), results in a severe inhibition of growth under AICAR 74 treatment. Accordingly, the non-ubiquitinable form of H2B, H2BK123R, was also found to be 75 highly sensitive to AICAR. It is well known that H2Bub is a signal of transactivation for two 76 methyltransferases, Dot1 and Set1/COMPASS. We showed that a set1 mutant was exquisitely 77 sensitive to AICAR, as was the non-methylable form of its targets H3K4. Remarkably, the 78 knock-down of the corresponding orthologs, Rnf40 for Bre1, Ash2l and Mll4/KMT2D for 79 COMPASS, were found to exacerbate AICAR sensitivity of human HCT116 cells (Albrecht 80 et al., 2016). 81

Here, we show that a mutation in the UBA1 gene encoding the yeast ubiquitin activating 82 enzyme (E1) is synthetic lethal with AICAR treatment. This result prompted us to examine 83 more systematically the connections between AICAR and the ubiquitin-pathway (Fig. 1b). 84

We identified the ubiquitin pool as having a central role in AICAR sensitivity and established 85 that mutants with reduced ubiquitin pools, such as ubp6 or doa1, are highly sensitive to the 86 drug. Based on suppression patterns and additivity, these mutants were found to belong to a 87 distinct branch of the ubiquitin-pathway which is required for yeast cells to cope with AICAR 88 and acts in parallel to the Rad6/Bre1 branch identified previously [START_REF] Albrecht | 361 Chemo-Genetic Interactions Between Histone Modification and the Antiproliferation 362 Drug AICAR Are Conserved in Yeast and Humans[END_REF]. The 89 complex chemo-genetic interactions between a metabolite and the ubiquitin pathway reported 90 here, illustrate how poly-pharmacological effects can result in phenotypic synergy. 91 92

Material and methods

94

Yeast media 95 SD is a synthetic minimal medium containing 0.5% ammonium sulfate, 0.17% yeast nitrogen 96 base without amino acids and ammonium sulfate (BD-Difco), 2% glucose. SDcasaW is SD 97 medium supplemented with 0.2% casamino acids (BD-Difco) and tryptophan (0.2 mM). 98

When indicated, adenine (0.3 mM) and/or uracil (0.3 mM) were added in SDcasaW medium 99 resulting in media named SDcasaWA (+ adenine), SDcasaWU (+ uracil) and SDcasaWAU (+ 100 adenine + uracil). SC medium was prepared as described [START_REF] Sherman | Methods in yeast genetics[END_REF]. SC complete 101 medium is SC medium supplemented with adenine (0.3 mM), uracil (0.3 mM), histidine (0.06 102 mM), leucine (0.4 mM), lysine (0.06 mM) and tryptophan (0.2 mM). 103 104

Strains and plasmids 105

All yeast strains are listed in Table 1 and belong to, or are derived from, a set of disrupted 106 strains isogenic to BY4741 or BY4742 purchased from Euroscarf. Multi-mutant strains were 107 obtained by crossing, sporulation and micromanipulation of meiosis progeny. For each cross, 108 systematic tetrad analysis was done to verify correct segregation of the markers (mating type, 109 auxotrophy, temperature sensitivity, PCR detection of KanMX4 knock-out…). The presence 110 of the uba1-o1 allele in the Y8033 strain used for gene dosage screening was verified by 111 sequencing of the UBA1 locus. All plasmids used in this study are listed in Table 2 andwere

Growth test 116

Overnight pre-cultured yeast cells were re-suspended in sterile water at 3.10 7 cells/ml and 117 submitted to 1/10 serial dilutions. Drops (5 µl) of each dilution were spotted on freshly 118 prepared medium plates and were incubated at indicated temperature for 48-72 h before 119 imaging. 120 121

Isolation of multicopy suppressors of the uba1-o1 AICAR-sensitive phenotype 122

Multicopy suppressors of the ade16 ade17 ade8 his1 uba1-o1 AICAR-sensitive phenotype 123 were obtained by transforming the Y8033 strain with a multicopy-plasmid library (PFL44L 124 backbone, 2µ URA3; generous gift from F. Lacroute). Transformants selected on SDcasaWA 125 medium at 25 °C were then replica-plated on the same medium containing or not AICAR (5 126 mM) and plates were incubated for 1-3 days at 33 °C. As described in the Results section, we 127 focused our attention on 5 clones containing ubiquitin-encoded genes: UBI4, RPL40A and 128 RPL40B. Among those, the plasmid p4814 corresponded to a chromosome IX fragment 129 (coordinates from 67814 bp to 71209 bp) containing RPL40A as the only entire gene. Three 130 other plasmids contained fragments of chromosome XI with RPL40B as the only entire 131 common gene. Finally, the fifth plasmid presented a fragment of chromosome XII 132 (coordinates from 63188 bp to 67952 bp) containing 3 entire open reading frames (ORF): 133 UBI4, ENT4 and YLL037w. Subcloning of the UBI4 ORF alone (P5504) in a YEplac195 134 plasmid [START_REF] Gietz | New yeast-Escherichia coli shuttle vectors constructed with 409 in vitro mutagenized yeast genes lacking six-base pair restriction sites[END_REF] allowed us to show that this ORF was sufficient for 135 suppression of the AICAR-sensitive phenotype. 136 137

Western blotting 138

Total protein extraction was performed by disruption of exponential growing cells with glass-139 beads in trichloro-acetic acid 5 %, as described in [START_REF] Escusa | Proteasome-392 and SCF-dependent degradation of yeast adenine deaminase upon transition from 393 proliferation to quiescence requires a new F-box protein named Saf1p[END_REF]. Protein extracts were 140 were detected by western blotting using anti Act1 (generous gift from I. Sagot (Bordeaux,142 France), 1/100,000), anti-H2B (Active motif ; #39237, 11/2,000) or anti Ubi (Abcam, 143 #ab19247 1/10,000). Blots Quantification was done with Image J (NIH). 144 145

Results

147

Synthetic lethality of a uba1 mutation with ZMP accumulation can be rescued by ubiquitin 148 overexpression 149

Our previous report establishing a connection between sensitivity to AICAR and the ubiquitin 150 pathway [START_REF] Albrecht | 361 Chemo-Genetic Interactions Between Histone Modification and the Antiproliferation 362 Drug AICAR Are Conserved in Yeast and Humans[END_REF] prompted us to examine whether sensitivity to AICAR could 151 be affected under conditions where the ubiquitin-pathway is dysfunctional. We started with 152 the UBA1 gene encoding the ubiquitin activating enzyme (E1) at the apex of the ubiquitin 153 pathway, hence governing all the ubiquitin-dependent processes [START_REF] Mcgrath | UBA 1: an essential yeast gene encoding 441 ubiquitin-activating enzyme[END_REF]. We 154 asked whether accumulation of ZMP could lead to a synthetic phenotype when combined with 155 a temperature sensitive allele (uba1-o1) of the UBA1 gene [START_REF] Shimada | ORC and the intra-S-phase checkpoint: a 467 threshold regulates Rad53p activation in S phase[END_REF]. The uba1-156 o1 mutant was mated to an ade16 ade17 double mutant which constitutively accumulates 157 ZMP [START_REF] Pinson | 453 Metabolic intermediates selectively stimulate transcription factor interaction and 454 modulate phosphate and purine pathways[END_REF] and meiosis of the resulting diploid was induced. Strikingly, in the 158 meiotic progeny, no ade16 ade17 uba1-o1 triple mutant could be obtained at permissive 159 temperature unless rescued by a plasmid expressing the wild-type UBA1 gene (Fig. 2a) thus 160 revealing synthetic lethality between ade16 ade17 and uba1-o1. Importantly, this synthetic 161 lethality could be totally abolished by ade8 and his1 mutations (Fig. 2b) blocking ZMP 162 synthesis upstream of Ade16 and Ade17 (Fig. 1a) and thus eliminating ZMP accumulation 163 [START_REF] Hurlimann | Physiological and toxic effects of purine 422 intermediate 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) in yeast[END_REF]. Finally, in the uba1-o1 ade16 ade17 ade8 his1 mutant, the synthetic 164 growth defect was recapitulated by addition of the ZMP-precursor AICAR at semi-permissive 165 temperature (33°C), a condition that did not affect growth of the uba1-o1 ade16 ade17 ade8 166 his1 mutant in the absence of AICAR (Fig. 2b). We conclude that the uba1-o1 mutant is 167 synthetic lethal with ZMP accumulation supplied by two different means, either endogenously 168 from the purine pathway (Fig. 2a) or exogenously from the growth medium (Fig. 2b). 169

Together these results reveal that partial loss of function of the ubiquitin-conjugating enzyme 170

Uba1 is synthetic lethal with ZMP accumulation. Hence, the ubiquitin pathway appears 171 critical for yeast cells to cope with ZMP.

As a first step toward understanding how AICAR affects the ubiquitin pathway, we searched 173 for gene-dosage suppressors that would restore growth of the uba1-o1 ade16 ade17 ade8 his1 174 mutant in the presence of AICAR. A genomic multicopy plasmid library was transformed in 175 the uba1-o1 ade16 ade17 ade8 his1 mutant and a set of 37 clones showing improved capacity 176 to grow in the presence of AICAR at 33°C were studied. Among them no plasmid carrying 177 the UBA1 gene was found suggesting that it was not present in the genomic library possibly 178 because of the large size of the coding region >3kb. Indeed, in a reconstitution experiment, 179 the growth defect was fully suppressed by a plasmid expressing UBA1 (not shown). Five 180 plasmids contained either the ADE16 (3 clones) or the ADE17 (2 clones) gene, thus restoring 181 ATIC enzymatic activity and preventing ZMP accumulation. These plasmids were not further 182 studied. Among the remaining clones, we only studied those carrying a single chromosome 183 fragment and presenting a robust suppression phenotype after retransformation. Eleven such 184

plasmids revealed DDP1 (1 clone), ECM21 (1 clone), RPL40A (1 clone), RPL40B (3 clones), 185
UBI4 (1 clone) and URA6 (4 clones) as gene-dosage suppressors after individual subcloning 186 (Fig. 2c). DDP1 encodes an inositol polyphosphate phosphatase, ECM21 encodes an arrestin 187 and URA6 a uridylate kinase. The three remaining genes, UBI4, RPL40A and RPL40B, encode 188 precursors of free ubiquitin [START_REF] Finley | The tails of ubiquitin precursors are 399 ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis[END_REF][START_REF] Finley | The yeast polyubiquitin gene is essential 402 for resistance to high temperatures, starvation, and other stresses[END_REF] and were therefore 189 directly related to UBA1 function as the ubiquitin-activating enzyme. We focused on those in 190 the rest of the work. We conclude that overexpression of ubiquitin allows bypassing of 191 AICAR sensitivity of the uba1-o1 ade16 ade17 ade8 his1 mutant. This result suggested that 192 the ubiquitin pool could play an important role in AICAR sensitivity in yeast. Importantly, the 193 free-ubiquitin pool was significantly higher in the uba1-o1 mutant than in the control strain 194 (Fig. 2d,arrow) indicating that the exacerbated sensitivity of the uba1-o1 mutant to AICAR is 195 not due a lower pool of free-ubiquitin. In both wild-type and uba1-o1 strains overexpression 196 of UBI4 resulted in increased free-ubiquitin (Fig. 2d), though the amount of free ubiquitin was 197 higher in the uba1-o1 mutant possibly reflecting its defective utilization by the E1 mutant 198 enzyme. In both strains, UBI4 overexpression strongly increased the signal of ubiquitylated 199 proteins indicating that under physiological conditions ubiquitin synthesis limits the 200 ubiquitylation process. Finally, we found no effect of AICAR treatment on the ubiquitin pool 201 (Fig. 2d). From these results, we conclude that AICAR-sensitivity of the uba1-o1 ade16 202 ade17 ade8 his1 mutant is not associated to a lower free-ubiquitin pool due to the mutation or 203 to AICAR treatment, but rather to a defect in the ubiquitylation process that can be efficiently 204 suppressed by overexpressing ubiquitin. We interpret these results as an indication that, in the 205 We next asked whether AICAR treatment itself could impose an enhanced demand for 211 ubiquitin that would be exacerbated by the uba1 mutation to a point leading to the observed 212 AICAR-hypersensitivity of this mutant. Several yeast mutants such as, doa1 and ubp6, have 213 constitutively reduced free-ubiquitin pools, due to defective ubiquitin recycling (Amerik et 214 al., 2000;[START_REF] Johnson | A proteolytic pathway that 425 recognizes ubiquitin as a degradation signal[END_REF]. The doa1 and ubp6 knock-out mutations were introduced in 215 the ade16 ade17 ade8 his1 background allowing controlled accumulation of ZMP as a result 216 of feeding with AICAR [START_REF] Hurlimann | Physiological and toxic effects of purine 422 intermediate 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) in yeast[END_REF]. Both mutations were found to lower the 217 ubiquitin pool (Fig. 3a) as previously reported [START_REF] Amerik | Analysis of the deubiquitinating enzymes 364 of the yeast Saccharomyces cerevisiae[END_REF][START_REF] Johnson | A proteolytic pathway that 425 recognizes ubiquitin as a degradation signal[END_REF] and 218 to severely impair growth of yeast cells in the presence of AICAR (Fig. 3b). Importantly this 219 growth defect could be robustly suppressed by overexpression of UBI4 (Fig. 3b), indicating 220 that the low pool of ubiquitin was implicated in the exacerbated AICAR sensitivity of these 221

mutants. 222

We then examined whether the chemo-genetic interaction between AICAR and the doa1 or 223 ubp6 mutants was phenocopying the previously described synthetic lethality between ZMP 224 and the rad6 or bre1 mutants [START_REF] Albrecht | 361 Chemo-Genetic Interactions Between Histone Modification and the Antiproliferation 362 Drug AICAR Are Conserved in Yeast and Humans[END_REF]. Indeed, in a previous work, we found 225 that the ubiquitin conjugating enzyme Rad6 together with the ubiquitin ligase Bre1 were 226 critical for yeast cells to grow in the presence of AICAR [START_REF] Albrecht | 361 Chemo-Genetic Interactions Between Histone Modification and the Antiproliferation 362 Drug AICAR Are Conserved in Yeast and Humans[END_REF]. This growth 227 defect resulted from the lack of ubiquitylation of histone H2B in the absence of Rad6/Bre1 228 together with a cell cycle default associated to defective entry of the cyclin Cln3 in the 229 nucleus due to AICAR treatment [START_REF] Albrecht | 361 Chemo-Genetic Interactions Between Histone Modification and the Antiproliferation 362 Drug AICAR Are Conserved in Yeast and Humans[END_REF]. Since free ubiquitin is present in 230 limiting amounts in cells, changes in the ubiquitin pool directly affect ubiquitin modifications 231 [START_REF] Groothuis | Ubiquitin crosstalk 415 connecting cellular processes[END_REF] (Fig. 2d) and accordingly the free ubiquitin pool was found to impact 232 on histone ubiquitination in mammalian cells [START_REF] Dantuma | A dynamic ubiquitin 387 equilibrium couples proteasomal activity to chromatin remodeling[END_REF]. Hence, we suspected 233 that doa1 and ubp6 mutants could directly affect H2B ubiquitination. Indeed, doa1 and ubp6 234 mutants showed a decreased ubiquitination of H2B that could be restored by UBI4 235 overexpression (Fig. 3c). These results suggested that low-ubiquitin mutants could be 236 sensitive to AICAR in part because of the reduced ubiquitylation of H2B that could mimic the 237 constructing a bre1 ubp6 strain capable of accumulating ZMP upon AICAR feeding (ade16 302 ade17 ade8 his1 background). We found that the bre1 ubp6 mutant was much more sensitive 303 to AICAR than a bre1 or an ubp6 strain alone at low AICAR concentration (2 mM, Fig. 7a). 304

This additivity indicates that the chemo-genetic interaction of these two mutants with AICAR 305 involves different functions. Accordingly, UBI4 overexpression could still suppress a part, but 306 not all, of the sensitivity to AICAR of the bre1 ubp6 strain, resulting in a level of AICAR-307 sensitivity equivalent to that of a single bre1 mutant (Fig. 7a). Additivity was also found for 308 the set1 and doa1 mutations (Fig. 7b) confirming the idea that these mutations result in 309 AICAR sensitivity through different means as deduced from their suppression pattern (Fig. 6). 310

Finally, co-overexpression of CLN3 and UBI4 in the uba1-o1 mutant showed an additive 311 suppression effect (Fig. 7c) indicating that the uba1 mutation affects both downstream 312 branches, as expected for a mutation affecting the enzyme which is at the apex of the 313 ubiquitin pathway. Interestingly, the uba1-o1 mutant exhibited a strong defect in H2B 314 ubiquitination at semi-permissive temperature and this could be restored by UBI4 315 overexpression (Fig. 7d). Hence, while UBI4 overexpression restored H2B ubiquitylation, it 316 only partially suppressed the uba1-o1 growth phenotype (Fig. 7c) suggesting that the 317 Rad6/Bre1 branch might affect one or several other targets than H2B that would be 318 suppressed by CLN3 overexpression but not or only partially by UBI4 overexpression. We 319 conclude that ZMP accumulation results in multiple chemo-genetic interactions with the 320 ubiquitin pathway in yeast, thus illustrating the complexity of drug action and the helpfulness 321 of genetics to address it. 322

323

DISCUSSION

324

In a previous work we identified histone ubiquitylation mutants as synthetic lethal with 325 AICAR treatment [START_REF] Albrecht | 361 Chemo-Genetic Interactions Between Histone Modification and the Antiproliferation 362 Drug AICAR Are Conserved in Yeast and Humans[END_REF]. Here we further uncovered phenotypic interactions 326 between AICAR/ZMP and the ubiquitin pathway and identified several new mutants (uba1, 327 ubc4, cdc34, cdc4, ubp6 and doa1) showing exacerbated sensitivity to AICAR. Differential 328 patterns of gene dosage suppression as well as phenotypic additivity revealed that these 329 mutants affect distinct branches, hence suggesting that AICAR interferes with one or several 330 functions particularly required when the ubiquitin pathway is impaired. Among the mutants 331 studied here and in our previous study, uba-o1 was the only one resulting in synthetic lethality 332 with the ade16 ade17 mutations leading to constitutive ZMP accumulation as revealed by our 333 inability to recover viable spores. This pronounced sensitivity could reflect the fact that Uba1 334 (E1) is at the apex of the ubiquitin pathway and therefore conditions all downstream ubiquitin 335 reactions. Among the 11 ubiquitin conjugating enzyme (E2) mutants, three (rad6, ubc4 and 336 cdc34) were affected by the drug hence revealing the major pathways downstream of Uba1 337 that are required for yeast cells to resist AICAR. Following-up on Cdc34, we could identify 338 the Cdc4 ubiquitin ligase (E3) as required for AICAR tolerance. 339

Why is AICAR toxic for ubiquitin pathway mutants? We have shown in this work that the 340 abundance of ubiquitin itself is an important factor in the drug toxicity though we 341 demonstrated that AICAR does not directly affect it. Clearly in the case of doa1 and ubp6 it is 342 the low ubiquitin pool that is responsible for the exacerbated toxicity of AICAR. It is likely 343 that more than one function is involved in the phenotype and indeed these mutants were not 344 efficiently suppressed by overexpression of neither CLN3 nor CLB5. For cdc34 and cdc4, 345 both mutants were suppressed by CLB5 overexpression, while this was not the case for the 346 other AICAR-sensitive mutants, suggesting that a negative effector stabilized in these mutants 347 could be displaced by CLB5 overexpression. It remains to be established whether the multiple 348 connections, between AICAR and the ubiquitin pathway, revealed here, are more than 349 serendipitous i.e. whether there is a common target that when affected by ZMP results in 350 synthetic toxicity with the various ubiquitin mutants. The fact, that up to now, we identified 351 no common gene dosage suppressor to the various mutants, does not support this hypothesis. 352 However, Uba1 was found among ZMP-binders by affinity chromatography (our unpublished 353 results) and could therefore be a direct and central target. In any case, the strong connections 354 between AICAR and the ubiquitin pathway could be highly relevant to understand why 355 AICAR selectively inhibits proliferation of aneuploid cells (Tang et al., 2011). Indeed, 356 aneuploidy is known to result in proteotoxicity [START_REF] Donnelly | Causes and consequences of protein folding stress in 390 aneuploid cells[END_REF]Santaguida 357 and Amon, 2015) and as such could impose a strong demand on the ubiquitin pathway. This 358 work thus provides a lead for further work on AICAR selective effects on aneuploid cells. 359 Tables 487 Wild-type (BY4742) and mutant (ade16 ade17: Y1093; uba1-o1: Y6853; ade17 uba1-o1: 507 Y7070; ade16 uba1-o1: Y7071 and ade16 ade17 uba1-o1: Y7117) strains containing a 508 plasmid allowing expression of UBA1 gene under the control of a doxycycline-repressible 509 promoter (p4515) were grown on SDcasaWA medium containing (+) or not (-) doxycycline 510 (Dox;100 mg/l). b Growth of the uba1-o1 mutant is impaired at semi-permissive temperature 511 when ZMP is accumulated by exogenous addition of its riboside precursor AICAR in the 512 ade8 ade16 ade17 his1 (Quad) genetic background. Strains (Y6987, Y2950 and Y8033) were 513 grown for 48 hours at indicated temperatures on SDcasaWAU medium containing or not 514 AICAR (10 mM). c Overexpression of gene-dosage suppressors restored growth of the uba1-515 o1 mutant in the presence of AICAR. The ade8 ade16 ade17 his1 uba1-o1 quintuple mutant 516 was transformed with plasmids allowing overexpression of indicated genes (ECM21: p5183; 517 URA6: p4919; DDP1: p4859; RPL40B: p4807; RPL40A: p4814 and UBI4: p5504) or with an 518 empty vector (YepLac195). Growth of transformants was scored for 72 h at 32°C on 519 SDcasaWA medium in the presence or not of AICAR (6 mM). d Free ubiquitin and ubiquitin-520 conjugated signals were strongly increased when UBI4 gene is overexpressed (OE). Strains 521 (Y2950 and Y8033) were transformed with a plasmid allowing UBI4 overexpression (p5504) 522 or the cognate empty vector (YEplac195). Total protein extracts were prepared from 523 transformants, overexpressing (+) or not (-) UBI4, grown in SDcasaWA medium and treated 524 (+) or not (-) for 2 hours with AICAR (1 mM). Proteins were revealed by western blotting 525 with anti-Ubi and anti-Act1 antibodies. Arrow points to free ubiquitin. Expo: Overexposure of 526 the dashed box region revealed with anti-Ubi antibody. 527 528 Fig. 3 Mutants with low ubiquitin are highly sensitive to AICAR. a Conjugated ubiquitin is 529 drastically decreased in ubp6 and doa1 mutants. Total protein extracts were obtained from 530 strains (Y2950 (Control), Y10296 and Y10696) grown in SDcasaWAU medium, separated by 531 SDS-page and ubiquitin-conjugated proteins were revealed by western blotting with an anti-532 ubiquitin antibody. Act1 was used as a loading control. b Overexpression of UBI4 is 533 sufficient to restore growth of the ubp6 and doa1 mutants in the presence of AICAR. Yeast 534 strains (Y2950 (Control), Y10696, Y10296 and Y9168) were transformed with plasmids 535 allowing overexpression (OE) of either UBI4 (p5504) or CLN3 (p4742), or with the empty 536 vector (none, YEpLac195). Transformants were grown at 37 °C for 48 h on SDcasaWA 537 medium containing or not AICAR (2 mM). c Ubiquitylation of H2B is increased when UBI4 538 is overexpressed (OE). Total protein extracts were obtained on transformants from Fig. 3b. 539 H2B pools were revealed by western blotting using an anti-H2B antibody. Act1 was used as a 540 loading control. Quantifications were set at 1 for the control strain transformed with the empty 541 vector (-). ND: not detectable. 542 543

  112 obtained by PCR amplification on genomic DNA and cloning in indicated vectors. Cloning 113 details are available upon request. 114 115

  uba1-o1 ade16 ade17 ade8 his1 mutant, a function aggravated by AICAR treatment can be 206 rescued by ubiquitin overexpression. 207 208 Mutants with reduced ubiquitin pools are highly sensitive to AICAR but do not recapitulate 209 the rad6/bre1 phenotypes 210

  lys2Δ0 ura3Δ0 ade8::KanMX4 ade16::KanMX4 ade17::KanMX4 his1::KanMX4 ubc6::LEU2 This study Y10072 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 ade8::KanMX4 ade16::KanMX4 ade17::KanMX4 his1::KanMX4 ubc4::KanMX4 This study Y10296 MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 ade8::KanMX4 ade16::KanMX4 ade17::KanMX4 his1::KanMX4 ubp6:

Fig. 1 Fig. 2

 12 Fig. 1 Schematic representation of the de novo purine and histidine pathways (a) and the 497 ubiquitin cascade (b) in yeast. a Only the enzymes mentioned in the text are shown (in green). 498 AICAR: 5-Amino-Imidazole 4-CarboxAmide Ribonucleoside; AMP: adenosine 5'-499 monophosphate; GMP: Guanosine 5'-monophosphate; IMP: Inosine 5'-monophosphate; 500 PRPP: α-D-ribofuranose 5-phosphate 1-diphosphate; ZMP: AICAR monophosphate. b E1, E2 501 and E3 stand for ubiquitin-activating enzyme, ubiquitin-conjugating enzymes and ubiquitin-502 protein ligases, respectively. Target: protein target. Ub: ubiquitin. 503 504 Fig. 2 AICAR toxicity in the hypomorphic uba1-o1 mutant is alleviated by ubiquitin 505 overexpression. a The ade16 ade17 uba1-o1 is viable only if UBA1 is ectopically expressed. 506

Fig. 4 aFig. 5

 45 Fig.4Different "ubiquitin sub-pathways" are required for yeast to cope with AICAR toxicity. 544 a Systematic analysis of AICAR-sensitivity of ubiquitin-conjugating enzyme (E2) mutants. 545 Control (Y2950) and mutant (Y9217, Y10072, Y9923, Y10034, Y9964, Y9931, Y9925, 546 Y9915 and Y9929) strains were grown in SDcasaWAU medium for 48 h at 30°C in the 547 presence or absence of AICAR. b The AICAR-growth defect of ubc4 mutant is suppressed by 548 CLN3 but not UBI4 overexpression. Strains (Y2950, Y10072) were transformed with 549 plasmids overexpressing either CLN3 or UBI4, or by the empty vector (none). Transformants 550 were grown in SDcasaWA medium for 48 h at 30°C. c AICAR-sensitivity is not affected in 551 an ubc1 deletion mutant. Strains (Y10817, Y2950: control, Y10840, Y9168) were grown for 552 48 h at 30°C on SDcasaWAU medium containing or not AICAR. d,f Growth of the cdc34-2 553 (d) and the cdc4-3 (f) mutants is severely impaired by endogenous ZMP accumulation. 554 Strains (Y1162, Y3273, Y3275, Y8385 and Y8406) were grown for 48 h on SDcasaWAU 555 medium. e,g The cdc34-2 (e) and cdc4-3 (g) mutants are highly sensitive to AICAR treatment. 556Strains were grown for 48 h at 32 °C on SDcasaWAU medium containing or not AICAR. 557 558 Fig.5Gene-dosage suppression of AICAR-sensitivity of the cdc4-3 and cdc34-2 mutants. 559Yeast strains (Y8679 (a, d), Y8675 (b, c), Y9516 (e), Y9168 (f), Y10696 (g), Y10296 (h) and 560 Y8033 (i)) were transformed with plasmids allowing overexpression (OE) of either CLB5 561 (p5791), CLN3 (p4742) or UBI4 (p5504) genes, or with an empty plasmid (YEpLac195, 562

  Fig. 3

  Fig. 5

32°C

Fig. 7

 7 Fig. 7

Table 1

 1 Yeast strains used in this study 488

	489
	Strain	Genotype

Table 2

 2 Plasmid used in this study 492

	493
	Plasmid	Backbone	characteristics	Sources
	pCM189	pCM189	CEN ARS URA3 tet-OFF promoter	(Gari et
				al., 1997)
	pFL44L	pFL44L	2 micron URA3	(Bonneaud
				et al.,
				1991)
	YCpLac111	YCplac111	CEN ARS LEU2	(Gietz and
				Sugino,
				1988)
	YEpLac 181	YEpLac 181 2 micron LEU2	(Gietz and
				Sugino,
				1988)
	YEpLac 195	YEpLac 195 2 micron URA3	(Gietz and
				Sugino,
				1988)
	p4515	pCM189	CEN ARS URA3 tet-OFF promoter-UBA1	This study
	p4742	YEpLac 195 CLN3 2 micron URA3	(Albrecht
				et al.,
				2016)
	p4807	pFL44L	2 micron URA3 RPL40B PTR2	This study
	p4814	pFL44L	2 micron URA3 RPL40A	This study
	p4859	YEpLac 195 2 micron URA3 DDP1	This study
	p4919	YEpLac 195 2 micron URA3 URA6	This study
	p4943	YCplac111	UBA1 CEN ARS LEU2	This study
	p5183	pFL44L	2 micron URA3 ECM21	This study
	p5504	YEpLac 195 UBI4 2 micron URA3	This study
	p5516	YEpLac 181 UBI4 2micron LEU2	This study
	p5791	YEpLac 195 CLB5 2 micron URA3	This study
	494