Introduction

Aphids are members of the order Hemiptera, and belong to the family Aphididae, subfamily Aphidinae. They are approximately 1-3 mm long characterized by small soft bodies, and by piercing-sucking mouthparts. They live in temperate regions and feed on phloem from plants [START_REF] Brisson | The pea aphid, Acyrthosiphon pisum: an emerging genomic model system for ecological, developmental and evolutionary studies[END_REF].

Our interest in aphids is focused on their close symbiotic relationship with the bacteria Buchnera aphidicola. Buchnera lives within specialized polyploid cells, called bacteriocytes, which are grouped into organ-like structures called the bacteriome located adjacent to the ovarioles. They have been vertically transmitted from mother to offspring for over 200 million years [START_REF] Baumann | Mutualistic Associations of Aphids and Prokaryotes: Biology of the Genus Buchnera[END_REF], and are obligate symbionts. For aphids this type of relationship is due to their main source of nutrition, phloem sap, which is an unbalanced nutritional diet as it contains few essential amino acids. The aphids themselves are incapable of synthesizing them, but Buchnera have retained the ability to create essential amino acids from the non-essential amino acids and sugars present in the aphid's diet (Baumann, Baumann et al. 1995).

The recent sequencing of the pea aphid (Acyrthosiphon pisum) genome by the International Aphid Genomics Consortium means that greater resources are available to researchers to perform functional genomics studies. This provides the opportunity to use such approaches to study the symbiosis between the pea aphid and its already sequenced symbiont Buchnera aphdicola. In this study statistical analysis methods to find differentially expressed genes were tested on preliminary data obtained with a cDNA microarray, in view of future research in the BF2I (Biologie Functionelle, Insects et Interactions) laboratory that will use a newly developed oligonucliotide platform. This research is particularly interested in studying of changes in gene expression observed in pea aphid under nutritional stress. In particular, the regulation systems controlling the symbiosis relationship between aphids and Buchnera are of interest. The specific objective of this study is to implement the ANOVA method proposed by (Park, Yi et al. 2003) for the analysis of time course microarray data. The approaches used to normalize the data and extract meaningful results from these experiments, will also be presented. The results obtained with this method will be compared to those obtained using a method originally designed for non-time course data applied to the same data set.

Materials and Methods

Experimental Plan

Several studies on aphids using microarrays have already been carried out [START_REF] Nakabachi | Transcriptome analysis of the aphid bacteriocyte, the symbiotic host cell that harbors an endocellular mutualistic bacterium, Buchnera[END_REF][START_REF] Ghanim | A microarray approach identifies ANT, OS-D and takeout-like genes as differentially regulated in alate and apterous morphs of the green peach aphid Myzus persicae (Sulzer)[END_REF][START_REF] Brisson | Common genome-wide patterns of transcript accumulation underlying the wing polyphenism and polymorphism in the pea aphid (Acyrthosiphon pisum)[END_REF][START_REF] Le Trionnaire | Seasonal photoperiodism regulates the expression of cuticular and signalling protein genes in the pea aphid[END_REF][START_REF] Ramsey | Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design[END_REF].

This section describes the methods and procedures used in the analysis of data extracted from the microarray experiment on pea aphids. The transcriptional analysis of aphid genes under nutritional stress was carried out using time course cDNA microarray experiments. They provided a means to determine expression levels of several thousand genes. This process was repeated at five different time points to give a measure of expression levels over time. The information from these experiments was then be used to identify genes with changed expression levels.

This research was focused on the tropic relationship between aphids and buchnera. A time course experiment was performed to test the transcriptional response to nutritional stress applied using the artificial diet method developed by the BF2I laboratory. Aphids were reared on two different nutritional conditions, AP3 and YF0. The AP3 condition refers to a medium that fulfills all the nutritional requirements of aphids. YF0 is a similar medium, but is deficient in Tyrosine and Phenylalanine. Hence, AP3 acted as a control, and YF0 was the condition representing nutritional stress applied to the aphids. The objective is to identify differentially expressed genes. For this purpose each slide (can also be referred to as array or chip) in the microarray experiment used material from both conditions, which were labeled using different dyes in a two Channel design including a ''Dye-swap''. Data from each time point consists of two slides. The mRNA material used in the experiments was extracted at 12hours, 24hours, 48hours, 72 hours, and 7 days, corresponding to 5 time points.

A simplified description of the method and a schematic of the experiment are shown in figures 1 and 2 respectively. ). Below them is the time in hours and days when samples were taken.

Data from the Microarray experiment

The intensity data from this experiment is contained in GPR (GenePix Result) type files (one for each slide). GPR files are produced by the analysis of the hybridization images of each slide using software developed by Axon Instruments (GenePix User Guide, 2002). The hybridization images are the scanned images of each slide after the hybridization experiment. These images are used to collect data and they can also be analyzed to find artifacts. The GPR files contain The first spot of the first row, for example, is the same as the first spot of row ten. This is shown in yellow in the figure.

Preparing Data for Differential Analysis

It is not possible to accurately find differentially expressed genes from raw data. This is because raw data can contain biases and may be of poor quality.

Analyzing such data for differentially expressed genes can lead to erroneous results. Therefore, preprocessing was conducted to prepare the data for further analysis. This procedure was carried out using an R language based tool called MAnGO (Microarray Analysis at the Gif/Orsay platform) (Marisa, Ichante et al. 2007). It involved performing an overview to determine the quality of the data.

Next background correction, normalization, and the averaging of replicate spots were conducted. Then the genes were ready for analysis for differential expressed genes.

During preprocessing the objects used by MAnGO during the rest of the analysis procedure were created. They contained information on the foreground and background intensities, the weights, gene names, IDs, and flags of each spot.

They were then used to view various visualizations of the data contained within them to determine its quality with respect to certain constraints. The visualizations are descriptive statistics and diagnostic plots based on Dudoit et al 2002. Next, background correction, and normalization within and between arrays was conducted to prepare the data for further analysis. Finally, averaging of replicate spots was conducted. The block design shown in figure 3 displays the placement of replicate spots on each slide. The idea behind replicate averaging is that two replicate spots correspond to the same gene, and so, at some point of the analysis the values corresponding to one need to be compared to those of the other. It is easier to average the intensity value early on rather compare them after further analysis has been conducted. The data was now ready to be analyzed for differentially expressed genes. Differential analysis was conducted using the linear model approach developed by Gordon Symth [START_REF] Smyth | Linear models and empirical bayes methods for assessing differential expression in microarray experiments[END_REF]) and the TCA method described earlier. The objective was to find those genes that were differentially expressed to better understand the response of aphids to nutritional stress. However, the analysis done this using this approach has offered an opportunity to compare the results between the two methods.

The linear model approach

The linear model approach is a method used to determine differentially expressed genes in microarray data. This approach requires two matrices to be made, a design matrix and a contrast matrix. The design matrix summarizes the effects that can be estimated for each slide, and is used to represent the different RNA targets that have been hybridized to the slides. The contrast matrix allows the coefficients (the different combinations of hybridizations) of the design matrix to be combined into contrasts of interest which can then be compared to each other [START_REF] Smyth | Limma: Linear Models for Microarray Data, User guide[END_REF]. The coefficients of the design matrix are created using information from the corresponding slide and the empirical bayes method.

The "ebayes" approach was developed by Smyth in 2004 [START_REF] Smyth | Linear models and empirical bayes methods for assessing differential expression in microarray experiments[END_REF], and using Bayesian statistics, it takes advantage of the parallel nature of inference in microarrays to combine information from an ensemble of genes to assist in inference about each individual gene. Next comparisons based on the design and contrast matrices are made which allow for estimations of certain hyperparameters to be made based on the observed statistics. Finally, T-statistics are calculated using posterior residual standard deviations rather than ordinary standard deviations. The t-test is conducted for each spot in each contrast to determine whether it is differentially expressed or not. This t-test is similar to a normal t-test except that the standard deviations are shrunk towards a common value using the empirical bayes method. This process has the effect of "borrowing" information from the whole set of genes, and of increasing the degrees of freedom. Therefore, the results show greater reliability [START_REF] Smyth | Linear models and empirical bayes methods for assessing differential expression in microarray experiments[END_REF]).

The t-test used was:

Where "s" is the moderated standard deviation and "V" is a matrix defined by:

The Time Course ANOVA method

The second method that will be used is a procedure based on the Analysis of Variance (ANOVA) and it was suggested by (Park, Yi et al. 2003). Using this method gene expression is assumed to be affected by two types of effects: time effects and gene effects. The model then statistically removes the time effects to allow analysis of just the group effects. The notations used are as follows: I (from 1 to I) refers to the experimental groups, K (from 1 to K) refers to time points, L (from 1 to L) refers to replications, N (from 1 to N) refers to number genes in the experiment. For I=2 one of the following models is used, depending on the interaction of effects.

M1: y

ikln = µ n + α in + β kn + (αβ) ikn + Ɛ ikln M2: y ikln = µ n + α in + β kn + Ɛ ikln
Where y ikln refers to the log ratios of red and green intensities from group i , time k, replication l, and gene n. µ n refers to the overall mean intensity in fluorescent signals for genes across array groups and time points. α in refers to gene specific group effects. β kn are time effects and they capture the differences in overall concentration of mRNA in samples from different time points. (αβ) ikn is the interaction effect and it measures the interaction between group and time. It can only be used when there are no replications (L=1). Ɛ ikln are the error terms.

Two different analysis procedures can be used depending on whether they are considered to be normally distributed or not. If they are normally distributed then the usual F-test associated with ANOVA is used, otherwise a permutation test proposed by [START_REF] Yang | Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation[END_REF]

M 1 R : y ikln = µ n + β ikn + Ɛ ikln , M 2 R : y ikln = µ n + β kn + Ɛ ikln , M 1 R is
߬ = ݕ -ݕ ො =ቊ ݕ -൫ߤ̂ + ߚ መ ൯ ݕ -൫ߤ̂ + ߚ መ ൯
Where the first is for model M 1 R and the other is for M 2 R .

For gene n the KL residuals τ 1kln are from Group 1 and the other KL residuals τ 2kln are from Group 2. Now a two sample permutation test can be conducted using the following two sample t statistic:

ܶ = ߬ ഥ ଵ.. -߬ ഥ ଶ.. ඨ ݏ ଵ ଶ ݊ ଵ + ݏ ଶ ଶ ݊ ଶ
n in is the number of observations in Group i , and

߬ ഥ .. = ୀଵ ߬ ݊ ൗ ୀଵ ݏ ଶ = 1 ݊ -1 ୀଵ (߬ -߬ҧ .. )² ୀଵ
Finally the p values for testing group effects are calculated and adjusted using the method proposed by Westfall and Young.

Results

Preprocessing

This procedure is composed of the following steps: correlation tree, box-plot, and MA plots. Filtering is also conducted to remove empty spots, negative and positive control spots, and spots of poor quality. The objective of preprocessing step is to better understand the data, find biases, and prepare the data for further analysis. Spearman correlations or correlation trees are made constructed using "M" values (where M=log 2 (R/G), and "R" and "G" correspond to red and green channel intensities respectively). Only the probes are used in these correlations.

Then the correlations are converted into distances using the formula distance=1correlation 2 . These distances are used in hierarchal clustering to build a tree.

In our research the hybridizations of all the slides were not conducted on the same day. RNA material taken at the 12 hour and 7 day time points was hybridized on the first day. Material from the 24 and 48 hour time points was hybridized on the second day, and material from the 72 hour time point was hybridized on the third day. This could have lead to a systemic bias in the experiment as conditions such as ambient temperature could have varied at the time of experimentation. Correlation trees were used to determine whether such a bias existed. Figure 4 shows this tree and it suggests there is some effect of the day that the hybridizations were carried out. Three out of four paired leaf nodes show a correlation between the hybridizations that were conducted on the same day. However, one of these pairs is a correlation between the dye-swaps of material taken at the 24 hour time point. The correlation may simply be because of the fact that the data is from the same source and has just been labeled differently.

Furthermore, there is a singular leaf node next to one of the remaining correlations that are suspecting of having an effect of the day of hybridization. This singular node is based on data from another day suggesting that there may not be an effect of day here either.

Box-plots and intensity histograms are used visualize the distribution of R and G intensities. This is done to detect labeling problems. In figure 5 box-plots are used to compare the intensities in the red channel and green channel of a slide. For this data the box-plots show no dye bias. Figure 6 shows intensity histograms that verify this. Finally MA-plots are presented that give a more precise representation of dye bias. and A= (log2(R)+log2(G))/2. These time points were chosen as they are representative of the other slides. The spots of each array are split into four categories based on the quality of the spot, with "good" being the best and "bad" the worst. "Ok" spots are of better quality than those labeled "average." In some procedures "average" spots can be referred to as "not found." The name has been changed here to reflect the quality of the spots. The slides from time point 2 contain a high number of spots categorized as "good" and the slides from time point 5 contain a high number of "average" spots. The spots in blue are "average", red represents "bad" and empty spots, green spots are negative controls, and black spots are "good" and "ok." The yellow line is a loess curve drawn using all the points on the array. The first slide from time point 2 contains a cloud of spots with a negative M value, but they are mostly spots labeled "bad," and are removed during filtering.

Background Correction

The objective of the background correction step is to "purify" the intensity signal by removing all contributions to it other than that of the hybridization of the target to the probe. Certain factors such as the binding of the target DNA to the slide, and surface properties of the slide can act as contributors to the intensity signal. However, the fluorescence from regions of the slide that contain DNA is different from those that do not, and so the two can be distinguished. The intensity signal is referred to as Foreground intensity, and the contributors are collectively called the Background intensity. It is assumed during a microarray experiment that the values of fluorescence assigned to each spot on a slide correspond to the level of expression of that gene. Removing the background intensity allows this estimation to become more accurate. The methods used to do so are collectively called background correction [START_REF] Yang | Analysis of cDNA microarray images[END_REF].

For the purposes of this research the Edwards method (Edwards 2003) was chosen. "Edwards" is a background correction method that uses a smoothing function rather than simple subtraction to remove the negative values caused by subtraction. It is based on the idea that when the difference between the foreground and background intensities is large then subtraction is the best background correction method. When the difference becomes negative a smooth monotonic function that is linear with respect to the background intensities is used. The procedure can be summarized thus:

Where i f ps is the foreground intensity, i b ps is the background intensity and δ is a threshold parameter.

Normalization

To study the effects of a treatment it is often assumed that the measures used to study samples are essentially the same and that any differences between them are caused by the treatment itself. Normalization is the procedure used to adjust the results of a microarray experiment to remove all technological differences so that only biological differences (differences caused by the treatment) are detected during analysis [START_REF] Yang | Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation[END_REF]. For a dye-swap experiment this means adjusting for artificial differences in the intensities of the dyes used. These differences can be caused, for example, by changes in affinity of the two labels for the DNA sample, differences in amounts of sample and label used, differences in laser voltage settings, and differences in photon emission response to laser excitation. Normalization cannot remove the complete effect of systemic differences such as these, but normalized data has been shown to be better suited to differential expression analysis (Park, Yi et al. 2003).

There are two major types of bias that can be removed by normalization: (1) dye bias that is the same for all spots on an array, causing one channel to appear brighter overall than the other; (2) dye bias that depends on the overall spot intensity, and is different for bright spots than for dim spots [START_REF] Do | Normalization of microarray data: singlelabeled and dual-labeled arrays[END_REF].

There are three methods that can be used to conduct normalization in MAnGO, median, loess and spline. Median methods are used to remove the first type of bias, that the bias is constant for all spots on the array. The same procedure to all spots will remove its effects. The second type of dye bias is dependent on spot intensities. This means that the bias is different for dim or bright spots. MAnGO provides loess normalization to remove this bias. Loess normalization is applied to MA data, where M=log 2 (R/G) and A= ½ log (RG). Under ideal conditions we expect M=0 as the non-biased intensities should be equal for both channels. However, this does not occur in actual experiments. Loess is used to find the difference the actual values and M=0, this difference is then removed from the actual values to give the normalized values of M which can then be used for further analysis (Park, Yi et al. 2003). Spline normalization is similar in principle to loess normalization but the c(A) term is calculated using splines rather than regression. It can also be used to remove bias of the second type.

For this research global loess normalization was used as MA-plots constructed on data from before and after the normalization procedure showed the best results were achieved this way. Using print-tip loess normalization did not show a marked improvement over global normalization. Therefore, the latter was used.

Between slides normalization is used to make the data from different slide comparable to one another. For this research this step proved unnecessary, as shown by figure 9.

Finding Differentially Expressed Genes

In order to find differentially expressed genes and test the TCA method we:

1) Applied the linear model approach in a local and global fashion, and applied the TCA method.

2) Compared the results of the linear model approach to the TCA method and found the functions of genes that are common in them.

The data set consisted of ten slides over the course of five time points. This allows for two applications of the linear model approach. It can be applied in a both a local fashion and a global fashion. Analysis with the local approach is similar to the analysis of five individual microarray experiments. Each time point is analyzed without considering the effect of time. Using the global approach all the slides are analyzed together. However, this is not the same as using a time course approach because the effect of time is absent. Then, a true time course approach, the TCA method is applied to the data. Finally, the results from the three approaches are compared to each other to find and compare the differentially expressed genes.

First, the linear model approach was applied. The initial data set contained 14688 spots. These spots correspond to cDNA, obtained by cloning ESTs (Expressed Sequence Tags) libraries, but from this point on will be referred to as "genes". This is not an accurate term, but it has been used because it is consistent with the nomenclature used in this kind of research. Each gene was replicated twice on the array, therefore averaging was conducted. The number of genes was further reduced by removing those genes that were labeled "bad." These genes were labeled as not suitable for analysis during the creation of the GPR files by the experimenters. Empty spots and negative controls were also removed. After filtration the lowest numbers of genes were found at time point 2 (6313 genes). To ensure that the analysis was consistent the corresponding 6313 genes were used in the analysis of all the arrays.

The results of the linear method approach are shown in two ways. Firstly, volcano plots, and log ratios vs "A" plot (A= (log2(R)+log2(G))/2)) are used to show the differentially expressed genes in figure 10. Next, table 1 displays the genes involved in the analysis and their p-values. The results for the first time point are presented here and discussed in the following section. The results of the other time points can be found in annex 4. Shown here are the log ratios of the comparison, and the fold change of the comparison. The p-values show the probability of incorrectly concluding that a gene is differentially expressed.

Results of the Linear Model Approach

The results for linear model approach in a local fashion were five lists of differentially expressed genes. In this and all further analysis using the linear model approach the genes that were significant at the 0.05 level are considered to be differentially expressed. The 0.01 level proved to be too stringent and was not used.

The results for all time points at both levels are summarized in This table shows the numbers of differentially expressed genes found at each time point. The five time points are at 12 hours, 24 hours, 48 hours, 72 hours and 7 days respectively. "Pval05" refers to the genes with a p-value of 0.05 or less and "pval01" refers to genes significant at the 0.01 level for the T-test.

The results for differentially expressed genes from time point 4 and time point 1 from the individual analysis of each time point are of interest as they show a considerably larger number of differentially expressed genes than the others. In the case of time point 1 a reasonable explanation maybe that initial growth rates are faster than later on in the course of the experiment. However, the reasons for the high number of differentially expressed genes in time point 4 were not immediately apparent. To explain this change the following hypotheses were considered:

1) There is an increased level of activity at that time point

2) There are errors in the treatment of data

3) This data represents the intrinsic limits of the method

There may be an increased level of activity at this time point as it occurs roughly at the end of larval stage 2 and the beginning of larval stage 3. This could lead to the possibility of genes present in both larval stages becoming expressed. However, in my opinion the most plausible reason may be the distribution or spread of data at time point 4. Most statistical methods used for finding differentially expressed genes are based on the idea that these genes are statistically "different" from the other genes they are being compared to. One limitation of using this approach is that there are no absolute values to determine these "different" genes from. Furthermore, with this method it is necessary to compare the genes present in each individual data set to each other. They cannot be compared to genes from other data set or to a perfect dataset as the results would be invalid. This intrinsic limitation of linear model approach may be the reason for the larger number of differentially expressed genes at time point 4. At its core the linear model approach is a check for variance. Genes that show a high level of variance from the rest of the data set are considered to be differentially expressed. This approach can only work correctly if the data (in this case log value of intensities from the genes) is mostly grouped together. In this case the "different" genes are likely to be differentially expressed. However, when the data is more spread out, or there are groups within the data such that one group is statistically different from the other the accuracy of the results is lower.

Next, an assumption was tested that genes that are differentially expressed at one time point should also be differentially expressed at other time points. A comparison of differentially expressed genes at each time point was conducted to check this claim. The results showed that this assumption was false. There was no intersection of the differentially expressed genes at all time points. This result could have been because of the low number of genes differentially expressed at time point 2. The likelihood that one out the twelve genes differentially expressed at that time point also being present at all other time points is quite low. Another explanation could be that genes differentially expressed at one time point are not expressed at others. At this point it is important to differentiate between the terms differentially expressed genes and expressed genes. Whereas, the term expressed genes refers to the genes that are switched on at a particular time point, differentially expressed genes are those that are expressed in one condition (FYO or AP3) and not in the other. If the expressed genes were changing this would not be surprising because the aphids are undergoing growth during the course of the experiment, and so the genes expressed at each time point should be different. However, the results of the table above show that at approximately 12-24 hour intervals the genes that are being differentially expressed are changing. This suggests that the treatment (reduced diet) has affected gene expression. This effect could have manifested itself in two ways: Firstly, the aphids growing on the reduced diet could have shown reduced growth rates. Those growing on a complete diet however, would grow normally. This would mean that a majority of the differentially expressed genes are related to growth. Another possibility is that the differentially expressed genes are those that are expressed only in the aphids on the reduced diet. The aphids on the reduced diet could have been using a different set of genes to adapt to their environment. However, the aphids on the complete diet do not need to adapt, and so do not express these genes. This argument is revisited after further analysis.

In the next step further analysis was done to determine if different differentially expressed genes were found at each time point. The top two thousand genes with respect to their p-values were selected from each time point. This suggests that different genes are differentially expressed at these time points. Next, we wanted to check if the genes being differentially expressed at one time point were also being expressed at other time points. It was more likely that differentially expressed genes were switching on and off. It is beyond the scope of this research to compare the p-values of all the matches between two time points, but this was done for the first ten genes in TP1&2 and TP2&3 intersections to corroborate our findings. This is shown in table 4a and 4b.

Table 4b. Intersection between Time point 3 (TP3) and Time point 4 (TP4).

This table demonstrate that there is very little correlation between the genes that are expressed at different time points.

All ten genes shown in both tables have significant p-values at the first time point. However, at the second time point the same genes have p-values that are quite high. The exception in this case is "CTG_AP_13.4-ApAL3SD-XV-A12" from table 4a, which is very close to being considered a differentially expressed gene. This suggests that the genes that are differentially expressed at one time point are not differentially expressed at the other time points, and that switching on and off is indeed occurring. The overall the trend seems to agree with the idea that a unique set of genes is being differentially expressed at each time point.

At this point we can return to the question of whether the differentially expressed genes are a result of different rates of growth or the result of adaption by aphids on the reduced diet. The functions of the genes shown the tables above were searched for. The names shown in the tables correspond to cDNA clones of ESTs (Expressed Sequence Tags). Their sequences were compared to nucleotide databases using BLAST (Basic Linear Alignment Search Tool).

"CTG_AP_1138.4-ID0AFF2AD04CM1" from table 4a showed 96% query coverage with the UPF0446 protein. This protein is not well characterized in aphids but is a homologue of the human protein Hox C12. In humans it is a transcription factor that is part of the developmental regulatory system, and helps determine the anterior-posterior axis. If this gene acts in a similar way in aphids the assumption that the differentially expressed genes are involved in growth is more valid. Furthermore, the gene with the best p-value, and hence best chance of being differentially expressed, "CTG_AP_1397.4-ID0AAA27DD07RM1", is a Tyrosine phosphatase. These enzymes act on tyrosine phosphorylated proteins.

Considering that the reduced diet is deficient in tyrosine, this strongly suggests that the differentially expressed genes are caused by stronger growth in the AP3 medium. To improve the validity of these results the linear method approach was applied to the data in a slightly different fashion. This is presented in the next section.

Local and Global comparisons

The results presented so far represent a local analysis of the data. The differentially expressed genes are determined at each time point without the consideration of time. In this section data was analyzed in a global sense using the linear method approach. This meant that all time points were analyzed together.

Theoretically, genes found to be differentially expressed using the local approach should be present in the global lists. As there is a lack of a common reference, this provides a means of validating the results. If the same genes are found to be differentially expressed using both methods, there is a greater chance of the analysis being correct.

Initially, the linear method approach was applied to all time points simultaneously. This generated a list of differentially expressed genes that were expressed at all time points. Next, intersections of the differentially expressed genes at each time point were compared to the lists generated by the global approach. The results of analysis using the global linear method approach are shown in table 5 and the intersections are shown in table 6. Using a global approach could have had the effect of "masking" the genes that were highly expressed at one time point but not at others. The differentially expressed genes seem to be switching on and off at different time points and this could have gone unnoticed by the global approach. However, this did not happen, application of the linear method approach in both a local and global fashion results in lists that are highly similar. Only two genes found using the global approach are not differentially expressed in the results of the local approach.

Furthermore, the genes for which functions were found in the previous analysis also occur in this list. This suggests that those genes have a great possibility of being differentially expressed.

However, this also raises another concern, is the high correlation between the two lists because they are actually expressed, or is it because a very similar statistical analysis has been applied in both cases? Theoretically, the latter is a greater possibility. However, it also correct to say that the fact that the same genes are found to be differentially expressed using both methods improves the likelihood of the analyses being correct.

Analysis using Time Course ANOVA

The TCA method was applied to the same 6313 genes that the linear model approach was applied to. This was done to ensure that the results were comparable to each other. Simultaneously, a literature search was conducted to find other cases where this method had been applied. Unfortunately, only one such instance was found. The creators of this method applied it to cortical stem rat cells undergoing neuronal differentiation (T Park 2002). Their analysis was conducted for approximately 4000 genes, and mRNA material was taken from six time points. They generated lists of differentially expressed genes in two ways. Firstly, they assumed that the log ratios of the genes used have a normal distribution, and the second list was created without this assumption. For this research only the second type of list was generated as there are several instances were found in literature that suggested that the normality assumption should not hold for all types of data [START_REF] Golub | Molecular classification of cancer: class discovery and class prediction by gene expression monitoring[END_REF][START_REF] Stamey | Molecular genetic profiling of Gleason grade 4/5 prostate cancers compared to benign prostatic hyperplasia[END_REF].

The application of the TCA method lead to a list of 13 differentially expressed genes at the 0.1 significance level and 42 at the 0.2 significance level. It is difficult to determine which level to chose. 80 genes were found to be differentially expressed using the linear model approach, finding only 13

differentially expressed genes at the 0.1 level using TCA might seem like a very low number. However, certain factors need to be considered before a conclusion is reached. Firstly, to allow for the maximum amount of data to be used for further analysis, no checks were applied to remove false positives in the linear model approach. With the TCA method the Westfall and Young method was used to reduce the number of false positives. Another interesting fact is that at the 1% significance level 10 genes are found to be differentially expressed using the linear method approach. Furthermore, 10% significance level is also used by the authors of the TCA method in their experiment. However, with their data 106 differentially expressed genes are found at this level. But it is also true that their data is not immediately comparable to ours. They conducted analysis on mammalian stem cells undergoing differentiation whereas this research was conducted on insect cells. Furthermore, this is in fact a test dataset and further experiments with more replicates are ongoing, thus even if these results are difficult to interpret, the implementation of this methods will be applied to future dataset in the laboratory. In this case, the 0.2 significance level was not used because this would lead to too many false positives. The results of the TCA procedure using a significance level of 0.1 are displayed in the table 7 below. 7. Genes found to be differentially expressed by the TCA method. This method produces a p-value for every gene in the analysis. However, here only the genes found to be differentially expressed are shown.

Unfortunately, the genes from the local analysis for which functions were found were not present in the genes found to be differentially expressed by the TCA method. However, 6 genes found to be differentially expressed using the TCA method were also found by the global analysis using the linear method approach. These genes are shown in the following table. Table 8. Genes found to be differentially expressed using both the TCA method and the global linear model approach.

Next, the functions for the genes common in the results of both methods were found. The names assigned to the genes represent ESTs (Expressed Sequence Tags). Their sequences were extracted from the NCBI (National Center of Biotechnology Information) EST database and BLAST (Basic Linear Alignment

Search Tool) was used to determine matches. The first EST, 'CL_AP_12014.4-ID0AFF9AD04CM1,' showed 36% query coverage with a putative kelch-like protein. Kelch and kelch-like proteins are characterized by the kelch domain.

Each kelch domain forms the blade of a propellar structure, with the repeated kelch domains forming a Beta-propellar. This family of proteins is found in a wide variety of organisms (drosophila, and some bacteria, fungi and viruses).Their function is not known, but they seem to act in interaction with actin.

The second EST, CL_AP_12782.4-ID0AAK5YD23CM1, shows 92% sequence coverage when compared to peptidyl-prolyl cis-trans isomerase, also called cyclophilin. Cyclophilin has propyl-isomerase activity; it interconverts the cis and trans isomers of peptide bonds with the amino acid proline. Propylisomerases often function as protein chaperons because this interconversion is often a rate limiting step in protein folding.

CL_AP_5683.4-ID0AAK9YK13CM1, the third EST shows is an exact match (100% query sequence coverage) with the Anopheles gambiae gene AGAP002830-PA. However, the functions of both genes are unknown. The functions of the other ESTs common to both methods could not be found. This was because they did not show a great deal of similarity with known genes.

Discussion

The objective of most microarray experiments, regardless of whether they are time course experiments or static procedures, is to find differentially expressed genes. After the experiment has been conducted, and the data has been background corrected and normalized, statistical tests can be applied to it to find differentially expressed genes. In this research the eventual objective was to implement and validate methods to determine the effect of nutritional stress on aphids. For this purpose two methods to determine differentially expressed genes were implemented, the linear model approach and the TCA method. This was achieved, and these methods can now be applied in future projects on aphids. As a secondary objective, the functions of the genes found to be differentially expressed using all three approaches (local and global application of the linear model approach, and the TCA method) were found. The results of the comparison show that the six genes found to be common in all three, and a few interesting genes are common to the local and global linear model approach but not to the TCA method.

Of the six genes found to be common in all three methods, the functions of two of these genes were determined using BLAST. Their functions suggest that they are involved in growth. The first gene is associated with actin, a major component of the cell cytoskeleton. Therefore, it is probably involved in building of new cells. The other gene may be acting as a protein chaperon, helping to correctly fold newly formed proteins. All the genes common to the local and global approach were not analyzed for function. However, the functions found for genes that were analyzed are also involved in growth. The gene of the UPF0446 protein acts as a transcription factor involved in development, and one of the possible functions of tyrosine phosphtases is control of cell cycle regulation.

This suggests that the differentially expressed genes are mainly caused by changes in the growth rates of the aphids on the two mediums. In general the differentially expressed genes are involved in developmental processes. Aphids on the reduced diet seem to be restricted in their development. Therefore, to a large extent, the differences that were expected to be found because of the aphid's reaction to a reduced diet have become masked. It is likely that a majority of the genes being expressed during the course of the experiment are those that are involved in growth, and that these genes are not heavily affected by nutritional stress. Other reasons to explain this effect may be because at this point the aphids have not begun to react to the reduced diet. However, at latter time points (after 7 days) the effect of nutrition may become more apparent as the reserves become depleted, and the organism has had a sufficient amount of time to react to the restrictions of the diet. Another interesting avenue for future research may be to conduct a similar experiment using adult aphids. This would remove the effect of growth so that only the effects of nutritional stress can be observed.

The local and global applications of the linear model approach also provided interesting insights into aphid growth at the larval stage. Our results show that in general aphids seem to express different sets of differentially expressed genes at different larval stages. This is not a surprising result as the aphid undergoes changes from one stage to another. This, much like the results discussed previously, suggest that the effect of growth is stronger at these stages than the effect of nutritional stress. This could be because the aphids on the reduced diet have not adapted to their environment sufficiently, and the differentially expressed genes continue to be caused by the faster growth rates of the aphids on AP3.

However, it must be stressed at this point that these results are not conclusive.

This research does not represent an exhaustive analysis of the TCA method. The genes found to be common in all three analyses can be considered to be differentially expressed with a fair degree of confidence. However, the validity of To conclude, the results of this experiment suggest that the aphids on AP3 seem to be undergoing a high rate of growth during the course of the experiment in comparison to the aphids on the reduced diet. This means that a majority of differentially expressed genes are likely to be those involved in growth. This assumption is augmented by the fact that the functions of the differentially expressed genes are related to growth. Therefore, it is very difficult to make specific conclusions regarding the interaction between aphids and buchnera with this data. However, the genes involved in this interaction of the aphid to the reduced medium may be expressed at a later stage in the organism's lifespan.

Therefore, for future experiments extracting mRNA for analysis after growth has already occurred may provide a means to avert this effect. Furthermore, this research shows that the TCA method is a valid procedure for finding differentially expressed genes. However a longer, more expansive methodology is required to certify the abilities of this procedure. Further analysis may also provide details on the limits of this procedure and the benefits it provides over other methods. Table D. All the genes found to be differentially expressed using the global linear model approach and their corresponding p-values.

Annexes

Note: All supplementary data generated by the linear model approach can be provided on request.

Figure 1 .

 1 Figure 1. Aphids reared on an artificial diet. With artificial diets the nutrients contained therein can easily be controlled and amino acid depletions of different kinds can be carried out. In this case two diets, AP3 (provides all nutritional requirements) and YF0 (deficient in Tyrosine and Phenylalanine) were used.

Figure 2 .

 2 Figure 2. Sample points of the microarray time course experiment. The diagram shows the 4 developmental stages of aphids, L1 to L4 (larval stage 1 to stage 4). Below them is the time in hours and days when samples were taken.

  information on image acquisition and details of each individual spot. The data is divided into columns such as Block, Row, Column (corresponding to the position of the spot); ID and name, Dia.(corresponding to the diameter of each spot); F635 Mean, F635 Median, F532 Mean, F532 Median (corresponding to spot intensities), Flags (corresponding to the quality of each spot), and various columns for statistics on the intensities of the spots. Information on slide design was extracted from the GPR files. Slide design refers to the positions assigned to various spots on the slide. It contains information on Block design, the placement of positive and negative controls, and if spots are replicated, the positions of the replicates. The GPR files were loaded into the R language and this information was extracted using a simple set of filters on the data. The slide and block designs are shown in figure 3.

Figure 3 .

 3 Figure 3. Slide design and Block Design. This figure shows that each array is divided into 48 blocks (4 columns and 12 rows). Each block then has 306 spots divided into 17 columns and 18 rows. Each block can then be divided into two replicate halves. The spots in the first nine rows are replicated in the next nine.

Figure 4 .

 4 Figure 4. Spearman correlation between the hybridizations conducted at various time points. The method used to label the slides will be explained using example of Y5A37jD1. The two conditions YFO and APO are abbreviated to Y and A. The dye swapping assignments are abbreviated to 5 for the red channel and 3 for the green channel. D1 refers to the day the hybridization was conducted on, and can take values from 1 till 3.

Figure 5 .

 5 Figure 5. Box-plots of slides in the expreiment. The first red box-plot and green box-plot from the left correspond to the red and green channel of the first slide, and the y-axis is intensity. This figures shows that there is no intensity bias between the two channels.

Figure 6 .

 6 Figure 6. Initial MA-Plots. This diagram shows the MA-plots for the slides from time point 2 (24 hours) and time point 5 (7 days). Where in MA, M= log 2 (R/G)

Figure 7 .

 7 Figure 7. MA-plots after Edward's background correction. This figure shows the effects of background correction using the Edward's method on the slides from time point 2 and time point 5. Filtration was conducted before background correction, therefore in these figures there are no "bad" or empty spots. The blue spots represent spots labeled "average" and the blacks spots are probes. The yellow line is a loess curve drawn through all the points.

Figure 8 .

 8 Figure 8. The MA-plots of normalization of slides using Loess normalization. This figure shows the MA-plots of two representative slides before and after normalization. Black spots are probes, green are negative controls, and the line is a loess curve through all the points.

Figure 9 .

 9 Figure 9. Boxplots of slides with respect to each other. This figure shows that in between slide normalization is not required as the data from each slide is of similar spread.

Figure 10 .

 10 Figure 10. Volcano plots and the log ratios vs "A" plot. The volcano plot shows a comparison of p-values and mean fold changes. The log ratios vs "A" plotshows the log ratios compared to the average intensity (mean of the A values from all slides in the analysis). The genes shown in red are found to be differentially expressed.

Furthermore, larval stage

  3 is the longest of the 4 larval stages considered in this research and could represent a time of greater metabolic activity. However, this change in unlikely to have been caused solely by biological effects as the magnitude of the change is too high. Another possibility for the high level of expression at this time point may have been errors during the analysis. These errors could have occurred at any point in the experimental stages (rearing of aphids, extraction of mRNA, creation of GPR type files etc), or during the during the in-silico analysis. Another reason for the large number of differentially expressed genes found at time point four may have been the quality of the slide.

  the TCA method is by no means incontestably confirmed. This aspect of the research can be continued. This research compares the TCA method to different applications of the linear model approach. There are numerous other methods that analyze data in a static fashion. They could have been used to check the results of the TCA procedure in a similar fashion to the local linear model approach procedure. Although none of them have been proven to work better than the other methods, validation of results by several procedures would have lead to more certainty in the results. Furthermore, the application of a procedure designed specifically for time course analysis such as Quadratic Regression Analysis(Liu, Tarima et al. 2005) could have provided a better means to evaluate the time course abilities of the TCA method. Another interesting procedure could have been to use more than one data set in the analysis. Checking the TCA procedure on data from similar experiments on aphids would shed more light on the validity of these results.

Annex 1 :

 1 Figure A. This diagram shows the MA-plots for the slides from all time points.Where in MA, M= log 2 (R/G) on the y-axis and A= (log2(R)+log2(G))/2 on the xaxis. The spots in blue are "average", red represents "bad" and empty spots, and black spots are "good" and "ok." The yellow line is a loess curve drawn using all the points on the array.

Figure D .

 D Figure D. Volcano plots and log ratio vs A plots. The figure above shows the plots created during the application of the local linear model approach for each time point. The volcano plots compare p-values (y-axis) to fold change (x-axis) and the other figure shows the comparison of log ratio to A values.

  is used instead.Next, a typical ANOVA table containing Sum of squares, degrees of freedom, mean square error and F-statistics is created. This is done for n number of genes, and the F-statistic can be calculated in two ways based on whether the log intensities, "y ikln " follow a normal distribution or not. If they are normal then basic F-statistics can be used, otherwise the permutation test is used.

	With two effects that are not dependant on time a permutation test can be
	conducted by permuting all levels of both factors simultaneously. However, with a
	time course experiment the two factors of interest are group effects and time
	effects. If they are permuted together then it is difficult to extract information on
	their group difference. Park et al. propose a two model procedure to overcome this
	difficulty. Firstly the time effects are removed and in the second step the group
	effects are considered. The first model used can be used in two ways based on
	whether or not there are different time effects for each group or not. The two
	possible models are:
	The model used is determined by the interaction effect (αβ)ikn. If this
	interaction is not important model M2 is used, otherwise M1 is used. The
	corresponding hypotheses are:
	H 01 : (αβ) ikn = 0 for M1,
	H 02 : α in = 0 for M2.

Table 1 . Results of the linear method approach for the first time point.

 1 This table shows the result for the ten genes most likely to be differentially expressed.

		LogRatio	FoldChange	Pvalue-
	Genes	FYOvsAP3/log2(FYO/AP3)	FYOvsAP3	FYOvsAP3
	Ap_SDD1_5A04_SP6 2.344	5.08	0.00128
	CTG_AP_1397.4-
	ID0AAA27DD07RM1 -2.067	-4.19	0.00199
	MpDbESTctg122	-4.597	-24.2	0.00222
	CL_AP_14636.4-
	ApHL3LD-IX-B8	2.787	6.9	0.00231
	CTG_AP_1138.4-
	ID0AFF2AD04CM1	1.96	3.89	0.00239
	CL_AP_10800.4-
	ID0AAH15BC12ZM1 2.362	5.14	0.00243
	CL_AP_15758.4-
	ID0AAK3YL15CM1	1.882	3.69	0.0029
	CTG_AP_4330.4-
	ID0AAA24AB04RM1 1.719	3.29	0.00412
	CTG_AP_706.4-
	ID0AAA12AD06RM1 -1.694	-3.23	0.00423

table 2 .

 2

				Total			Total
		Pval05_down Pval05_up	(Pval05) Pval01_down Pval01_up	(Pval01)
	Time
	point
	1	219	54	273	9	11	20
	Time
	point
	2	7	5	12	2	4	6
	Time
	point
	3	11	43	54	4	8	12
	Time
	point
	4	1074	50	1124	262	8	270
	Time
	point
	5	22	44	66	11	13	24

Table 2 . Differentially expressed genes using the Linear Model Approach.

 2

Table 3 . Intersection of genes from neighboring time points

 3 To check this claim, intersections of differentially expressed genes from neighboring time points were made. The results are summarized in table 3.

		TP1&TP2	TP2&TP3	TP3&TP4	TP4&TP5
	common genes DE	0	0	14	11
	common genes
	2000	636	720	723	510

. This table shows the genes that are common between time points. The first row is the differentially expressed genes that are common between them. The second row represents the genes that are common in a set of two thousand genes with the best p-values.

"TP" refers to Time point. This table agrees with the assumption that different genes are being differentially expressed at each time point. Out of the 273 differentially expressed genes at time point one and the 54 expressed genes at time point three none are the same as the 12 differentially expressed genes at time point two.

Table 4a . Intersection between Time point 1 (TP1) and Time point 2 (TP2).

 4a

		Value In	Value In
	Intersection of TP3 and TP 4	TP3	TP4
	Ap_SDD1_1B04_T7	0.00014	0.28488
	CTG_AP_996.4-ApHL3LD-XVII-A3	0.00247	0.31211
	CTG_AP_2298.4-ID0AFF6AF08CM1 0.00673	0.33581
	CL_AP_11257.4-
	ID0AAH10DG10ZM1	0.03834	0.33798
	CL_AP_7452.4-ID0AAH1AB03ZM1	0.0745	0.46501
	CL_AP_7772.4-ApHL3SD-VIII-G1	0.08181	0.55738
	CTG_AP_3450.4-
	ID0AAA25AH04RM1	0.08956	0.25115
	CTG_AP_4978.4-ApDT-VII-C1	0.09612	0.38103
	CL_AP_15052.4-ID0AFF1CG09CM1 0.09643	0.363
		Value In	Value In
	Intersection of TP1 and TP2	TP1	TP2
	CTG_AP_1397.4-
	ID0AAA27DD07RM1	0.00199	0.82492
	CL_AP_14636.4-ApHL3LD-IX-B8	0.00231	0.77707
	CTG_AP_1138.4-ID0AFF2AD04CM1 0.00239	0.32949
	CL_AP_15758.4-ID0AAK3YL15CM1 0.0029	0.31188
	CTG_AP_706.4-ID0AAA12AD06RM1 0.00423	0.83661
	CTG_AP_13.4-ApAL3SD-XV-A12	0.00473	0.06429
	Ap_SDU2_3A08_SP6	0.00549	0.12874
	CL_AP_8728.4-ID0AAH14DE02ZM1 0.00715	0.64736
	Aust_58	0.00842	0.34015

Table 5 . Differentially expressed genes using the linear model approach applied to all time points in a global fashion.

 5 The table shows the genes that are adjudged to be differentially expressed at the 0.05 significance level and at the 0.01 significance level.

	TP1&all	TP2&all	TP3&all	TP4&all	TP5&all	Total
	DE	17	2	2	46	11	78

Table 6 . Intersection of genes common using the linear model approach in a local and global sense.

 6

Table

	Genes	Pval TCA	Pval LMA
	CL_AP_12014.4-ID0AFF9AD04CM1 0.007	0.00767
	CL_AP_12216.4-ID0AAK4YH17CM1 0.0136	0.02148
	CL_AP_12782.4-ID0AAK5YD23CM1 0.0416	0.04092
	CL_AP_5683.4-ID0AAK9YK13CM1 0.0546	0.04188
	CL_AP_8173.4-ID0AAH10AF02ZM1 0.0546	0.00509
	CTG_AP_2994.4-
	ID0AAH3CD06ZM1	0.0893	0.03879
	CL_AP_9125.4-ID0AAK9YI01CM1	0.0338	NDF
	CL_AP_6048.4-ID0AAK9YE01CM1 0.007	NDF
	CTG_AP_4507.4-ApHL3SD-XVI-C4 0.0535	NDF
	CTG_AP_43.4-ID0AFF12BC10CM1 0.0893	NDF
	CL_AP_13507.4-ID0AAK9YP13CM1 0.0546	NDF
	CL_AP_10287.4-
	ID0AAH14CD08ZM1	0.0817	NDF

Annex 5: Results Using the Local Linear Model Approach

	DE Genes from TP3 (48h)	P-value	DE Genes from TP4 (72h)	P-value
	CTG_AP_2497.4-BP536005	0.00046	CL_AP_8866.4-ID0AAH14AE01ZM1 0.00019
	DE Genes from TP1 (12h) CL_AP_12014.4-ID0AFF9AD04CM1 CTG_AP_3109.4-ID0AAA3DF04FM1	P-value 8,00E-04 CL_AP_10190.4-ID0AAH14BB06ZM1 0.00023 DE Genes from TP2 (24h) P-value 0.00096 CTG_AP_926.4-ApDT-VI-E9 0.00042
	Ap_SDD1_5A04_SP6	0.00128	Ap_SDD1_1B04_T7 CTG_AP_3354.4-	0.00014
	CTG_AP_1397.4-ID0AAA27DD07RM1 DW362461	0.00199 0.00149	CTG_AP_996.4-ApHL3LD-XVII-A3 ID0AAH14CC06ZM1	0.00247 0.00043
	MpDbESTctg122 CTG_AP_3650.4-ID0AFF10BB03CM1 0.00169 0.00222	CL_AP_10751.4-ApAL3SD-XVII-B12 CL_AP_6144.4-ID0AAH12AD04ZM1 0.00049 0.00623
	CL_AP_14636.4-ApHL3LD-IX-B8 CTG_AP_784.4-ID0AAH7CD02ZM1 CTG_AP_1138.4-ID0AFF2AD04CM1 CTG_AP_745.4-ID0AFF13AB10CM1	0.00231 0.00223 0.00239 0.00225	CTG_AP_2298.4-ID0AFF6AF08CM1 CL_AP_11361.4-ID0AAH3CA02ZM1 0.00051 0.00673 CTG_AP_119.4-ApAL3SD-VI-D8 CTG_AP_4688.4-0.00786 ID0AAH10DD08ZM1 0.00051
	CL_AP_10800.4-ID0AAH15BC12ZM1 CL_AP_15758.4-ID0AAK3YL15CM1 Ap_SDU1_4A09_SP6 CTG_AP_4330.4-ID0AAA24AB04RM1 CL_AP_5956.4-ID0AFF11BB07CM1 CTG_AP_706.4-ID0AAA12AD06RM1 CL_AP_14501.4-ID0AFF14DC07CM1 0.00797 0.00243 0.0029 0.00471 0.00412 0.00593 0.00423 CTG_AP_40.4-ApDT-I-E8 0.00937	CTG_AP_1386.4-ApAL3SD-VII-B3 CTG_AP_3369.4-ID0AAH11CH01ZM1 CTG_AP_2543.4-ID0AAH10BH01ZM1 0.03456 0.00947 0.00053 CTG_AP_287.4-ID0AAH10CH11ZM1 DW361853 6,00E-04 0.03477 CL_AP_11257.4-ID0AAH10DG10ZM1 CTG_AP_979.4-ID0AAA29BH10RM1 6,00E-04 0.03834 CL_AP_15069.4-ID0AAK3YL22CM1 0.00078
	CL_AP_9658.4-ApHL3SD-XXVI-H12 MpDbESTctg47	0.00429 0.00975	Ap_SDD2_2H12_T7 CL_AP_10886.4-ID0AAH2BH02ZM1	0.04062 8,00E-04
	CTG_AP_13.4-ApAL3SD-XV-A12	0.00473	CL_AP_7071.4-ID0AAH14CG07ZM1 CTG_AP_3979.4-	0.04521
	Ap_SDU2_3A08_SP6 CL_AP_14839.4-ID0AFF14CG08CM1 0.01115 0.00549	CL_AP_7109.4-ApHL3LD-X-C6 ID0AAH11BG01ZM1	0.04734 0.00086
	CL_AP_8650.4-ApDT-IX-E9 CTG_AP_5120.4-ID0AFF10BC07CM1 0.01199 0.00562	CL_AP_13911.4-ApDT-XXXI-B6 CTG_AP_3116.4-ID0AEE2CB10RM1 0.00086 0.0582
	Ap_SDD3_1H02_SP6 CL_AP_9875.4-ID0AFF1BG09CM1	0.00605 0.01263	Ap_SDU1_4G09_SP6 CL_AP_5695.4-ID0AAH12DF05ZM1 0.00086 0.06012
	CL_AP_8728.4-ID0AAH14DE02ZM1 CL_AP_5532.4-ID0AAH5AC09ZM1	0.00715 0.01263	CTG_AP_123.4-ApHL3LD-III-B12 CL_AP_7490.4-ID0AAH9BG09ZM1	0.06297 0.00092
	Aust_58 CL_AP_6225.4-ID0AFF7AG12CM1	0.00842 0.01279	CTG_AP_13.4-ApAL3SD-XV-A12 CTG_AP_4690.4-19884610	0.06429 0.00093
	CL_AP_7558.4-ID0AFF11CH03CM1 CL_AP_12134.4-ID0AFF2BC10CM1	0.00863 0.01405	Ap_SDD1_2G01_T7 CL_AP_12866.4-ID0AAH11CB08ZM1 0.00093 0.06704
	CTG_AP_1156.4-ApHL3SD-VII-F2 CTG_AP_768.4-ID0AAA21AF02RM1	0.00872 0.01551	CL_AP_7452.4-ID0AAH1AB03ZM1 CTG_AP_2461.4-ID0AEE6BB04RM1 0.00095 0.0745 CTG_AP_2931.4-
	CTG_AP_30.4-ApDT-XXX-D8 CTG_AP_1905.4-ID0AAH14BE05ZM1 0.01668 0.0088	CL_AP_7772.4-ApHL3SD-VIII-G1 ID0ACC22DF04RM1	0.08181 0.0011
	CTG_AP_125.4-ApDT-V-C5 CL_AP_10863.4-ID0AAH15DA02ZM1 0.02059 0.00949	CL_AP_14798.4-ID0AAG1DD02CM1 CL_AP_6739.4-ApHL3SD-IV-D6	0.0854 0.00112
	CL_AP_8536.4-ApDT-VI-G10 CTG_AP_54.4-ApAL3SD-III-E4	0.01004 0.02084	CTG_AP_3803.4-CTG_AP_3450.4-ID0AAA25AH04RM1 0.08956 ID0AAH10BD12ZM1 0.00112
	CL_AP_8104.4-ID0AAH11BE10ZM1 CL_AP_10291.4-ID0AFF10BG08CM1 0.02086 0.0101	CL_AP_11881.4-ID0AAK5YB01CM1 CTG_AP_568.4-ApHL3SD-IV-G5	0.08963 0.00115
	CL_AP_7588.4-ApDT-IX-D5 DW362164	0.01014 0.0213	CL_AP_13285.4-ID0AAH10BF06ZM1 CTG_AP_4000.4-ApAL3SD-XIX-B3	0.09485 0.00116
	CTG_AP_274.4-ID0AAA13CD03RM1 CTG_AP_328.4-ID0AAA12DD07RM1	0.01054 0.02333	CTG_AP_4978.4-ApDT-VII-C1 CTG_AP_730.4-ApDT-XXXI-C6	0.09612 0.00116
	Ap_SDD1_3H07_SP6 CTG_AP_531.4-ID0AAA14CF03RM1	0.01122 0.02475	CL_AP_15052.4-ID0AFF1CG09CM1 CL_AP_13473.4-ID0AFF8DB03CM1 0.00117 0.09643
	CL_AP_10289.4-ID0AAG4DB08CM1	0.01134	CL_AP_6711.4-ApAL3SD-I-H4 CTG_AP_1590.4-	0.11282
	CTG_AP_3812.4-ApDT-XX-D1 CTG_AP_1957.4-19884163	0.01208 0.02556	CL_AP_10017.4-ID0AAK4YI16CM1 ID0AAA19DH06RM1	0.11951 0.00118
	CL_AP_14060.4-ID0AAH10BH07ZM1 CTG_AP_2355.4-22860683	0.01277 0.02704	CL_AP_9864.4-ID0AAH15AG02ZM1 CTG_AP_137.4-ID0AAH10AC06ZM1 0.00119 0.12258
	CL_AP_7452.4-ID0AAH1AB03ZM1 CL_AP_14380.4-ID0AAK6YL16CM1	0.01391 0.02802	CTG_AP_2998.4-ID0AFF13BA06CM1 CTG_AP_8.4-ApHL3SD-XXXIII-G5	0.12337 0.0012
	CL_AP_9539.4-ID0AAG10CA11CM1 CTG_AP_20.4-ID0AFF6DE11CM1	0.01429 0.02876	Ap_SDU2_3A08_SP6 CTG_AP_2502.4-ID0AAA7DG11RM1 0.00121 0.12874 CTG_AP_3986.4-
	CL_AP_6403.4-ApHL3LD-X-B1 CL_AP_14989.4-ID0AAH13BG02ZM2 0.03232 0.0143	CTG_AP_3013.4-ID0AEE11DC07RM1 0.1299 ID0AAH10CH10ZM1 0.00126
	CTG_AP_1499.4-ApHL3LD-XIV-E7 CL_AP_14379.4-ID0AFF3AG08CM1	0.01446 0.03295	CL_AP_13548.4-ID0AAH5AE07ZM1 CL_AP_12848.4-ID0AFF8BE10CM1	0.13823 0.00127
	CL_AP_14332.4-ID0AFF12DC08CM1 Ap_SDD1_1B12_T7	0.0161 0.03339	CL_AP_13040.4-ID0AAK2YO07CM1 CL_AP_14485.4-ApHL3SD-I-E10	0.1396 0.0013
	CL_AP_9506.4-ID0AAK4YB20CM1 MpDbESTctg46	0.0163 0.03456	CL_AP_15494.4-ApDT-XXXI-B1 CL_AP_15297.4-ID0AAG11CD01CM1	0.14038 0.00133
	CL_AP_12626.4-ID0AFF2CG03CM1 CL_AP_8344.4-ID0AAH9BH08ZM1	0.01659 0.03554	CTG_AP_2754.4-ID0AAH10BB10ZM1 0.14125 CL_AP_14255.4-ApHL3SD-VI-A7 0.00137
	CL_AP_13776.4-ApHL3SD-XXVIII-A12 CTG_AP_2517.4-ID0AAH15DE09ZM1 0.03715 0.01664	CL_AP_13218.4-ID0AAK9YK18CM1 CL_AP_11418.4-ID0AFF12DF05CM1 0.00138 0.14207
	CL_AP_7269.4-ID0AAK4YB24CM1	0.01696	CL_AP_9462.4-ID0AAH2AG09ZM1 CL_AP_15969.4-	0.14321
	CL_AP_9017.4-ApAL3SD-III-C11 CTG_AP_2410.4-ID0AFF5CA12CM1	0.01729 0.03837	Ap_SDD1_5D12_SP6 ID0AAH14AD06ZM1	0.14521 0.00141
	CL_AP_13057.4-ID0AFF10AD02CM1 CL_AP_10941.4-ID0AFF4AE03CM1	0.01739 0.03931	CTG_AP_1182.4-ApAL3SD-X-H11 CL_AP_13548.4-ID0AAH5AE07ZM1 0.00142 0.15502
	CL_AP_6607.4-ApHL3LD-XII-C4 CTG_AP_3513.4-ApDT-XVIII-B4	0.01762 0.04074	CTG_AP_1323.4-ID0AAH11BF10ZM1 CTG_AP_163.4-ApHL3LD-XI-A6 CTG_AP_4849.4-	0.00143 0.15803
	CL_AP_6050.4-ID0AAH8CD09ZM1	0.04248	ID0AAH14CA10ZM1	0.00147
	Table A. Differentially expressed genes at time point 1 and 2 as determined by the
	local linear model approach. In this and the following tables only the first 40
	differentially expressed genes are shown. A complete list (6313 genes) is available
	on request.

Table B .

 B Differentially expressed genes from time points 3 and 4.


Last regulation

 (7th August 2006)eliminated distinction between Masters.

III. Implementation of Doctoral Schools

Last regulation has fixed the following points:

• The Doctoral Schools are the only and exclusive places where to complete Doctoral Studies.

• A Doctoral School is firstly

• a gathering of research units -especially "UMR"s (Mixed Research Units) • on a scientific theme or discipline • from one or several universities and • accredited by the Ministry of Education and Research (every 4 years)

• Doctoral students should be accepted in a research team of the Doctoral School in order to be registered.

• The Supervisor (HDR) must belong to a D.S. and only one, and be a member of one of the research units.

III. Implementation of Doctoral Schools

Last regulation has fixed the following points (bis):

• PhD Students and University representatives (supervisor, team director, D.S. director) sign a kind of contract ( la charte) stating mutual rights and obligations.

• The Doctoral School and the research units must supply doctoral students with -facilities to work -courses and transferable skills development and training ("doctoriales") -possibilities of attending seminars and congresses • D.S. are allocated a fixed number of grants from the Ministry and try to find other financial resources for their students.

Perspectives and discussion

The main points to be fixed often depending on the discipline and background of the students -the nature and volume of the taught content, especially after a "master-pro"

  doctoral studies and research -joint research teams with national research Centres II. Impact of the LMD on the link between Masters and PhD IMPLEMENTATION OF THE BOLOGNA PROCESS IN THE UNIVERSITIES • B.P. (Bachelor/Master/Doctorate) implemented since 2003/2004 under the name of "LMD" = LICENCE-MASTER-DOCTORAT • Not too difficult to implement because the studies were already split into 3 cycles IN "GRANDES ECOLES" • More difficult to implement because: • Studies mostly split into 2 cycles in different places -2 years of preparatory classes in a lycée, 3 years of School • Reluctance to abandon their prestigious title (Ingénieur) • Doctoral cycles always developed in conjunction with Universities DESS Master-Recherche -same as DEA BUT -Implementation of Doctoral Schools -Selection of PhD Students with agreement of a research team

-

  the nature of transferable skills and the way to manage them -the number of PhD Students per supervisor -the correct pressure on the duration of the thesis -the nature and the quality level of the Dissertation -the implementation of a pre-defence of the thesisAnd probably many other points !

Thank you for your attention!


L E Henaff
email: lehenaff@versailles.inra.fr

Aventurier Pascal
email: pascal.aventurier@avignon.inra.fr

Providing services to INRA and the international agricultural community: ProdInra & the Integrated Information System

The main benefit of open archives is to optimize the visibility of research results. Universities & research institutes have been setting up OAI compliant repositories with this aim in mind. INRA, a French organization and also the largest agricultural research institute in Europe has set up such a repository in 2005. INRA needs to identify and track its production because it must evaluate individuals, the labs, the departments, etc. and also to produce strategic indicators about the trends of our research activity. These indicators help INRA to direct funding and access the most advanced projects. In 2005, INRA planned a 5-years project to create an Integrated Information System (IIS) that combines different internal applications in order to exchange data and create services for the INRA research community. Directories of INRA people, activities and research laboratories are available in the internet. ProdInra aims to give a large audience to INRA products. As it becomes an applicative tool of this Integrated Information System (IIS), it can also provide services to the agricultural international community based on data coming from the IIS. This communication will present the Integrated Information System as well as the different services provided.

Introduction

The French National Institute for Agricultural Research (INRA) is the world's second-largest research institute in the field of agriculture, food and nutrition, and the environment. Its international visibility and recognition are a result of the widespread dissemination of its research work. In 2005, INRA demonstrated its support for open access by signing the Berlin declaration and launching an institutional open archive called ProdInra.

Since that year, INRA has been overhauling its information system in order to set up indicators for steering agricultural research, but also to provide multiple services to INRA personnel as well as the agricultural scientific community at large. This article sets out to demonstrate the usefulness of an open institutional archive that can provide a multitude of services to the research institute and its partners while meeting the goal of highlighting the value of work produced by INRA scientists.

First, we will describe the various data sources from which information is aggregated, as well as how the data is reintegrated into the IS. We will then focus on ProdInra's role as an open archive, and discuss INRA's documentary policy, service, and goal of optimizing ProdInra data to enhance international visibility.

ProdInra: the INRA Information System's document block

Data sources

To address the growing needs of indicators, consolidate information, and eliminate redundant entries, INRA decided to overhaul and modernize its information system. Initially, business applications such as human resources management and accounting were modernized. Shared data repositories (research activities and structures such as laboratories, research departments, etc.) were then created, forming the initial scope of an institutional information system.

Meanwhile, the assessment of research bodies, laboratories and scientists is based on scientific productivity. ProdInra, introduced in 2006, has thus become a strategic application in its own right.

Consequently, the open archive was revamped so it could be integrated into INRA's information system. For the first time, a management tool for scientific publications was included within the scope of this institutional information system. Data is exchanged in XML format between these application blocks via an Enterprise Service Bus (ESB) using webservices. Each application is thus potentially a data provider and a client, operating according to the diagram in Figure 1.

Forms of data restitution

The available data is first used to populate other IIS applications so that redundant data entry is avoided. Most of the data come from repositories, ensuring data consistency and offering the possibility of generating indicators and automated quality reports. This is made possible by the aggregation of data from the IIS.

Open services

The most useful function is the creation of directories. Directory entries for scientists can include a list of research activities and work. Directory of activities and laboratories are also available. Since December 2009, all three directories can be consulted over the internet. This policy shift reflects our desire to promote INRA's research activities and skills, and make it easier for the international scientific community to find collaborators at INRA. The diagram on figure 2 presents the service layer (green) resulting from the aggregation of data from application blocks (in orange).

Creating indicators

Leadership in agricultural research is determined on the basis of indicators: the number of academic publications, number of patents filed, etc. The ability to measure ratios-the number of publications/number of scientists, or the number of publications co-written with non-INRA authors/number of publications-makes it possible to measure INRA's productivity and its openness to collaborations. With data from human resources, funding, research activities and production, it is theoretically possible to generate detailed reports that can be used to steer INRA's strategy and orientations.

In practice, application administrators are often faced with the problem of the poor quality of repository data or incomplete data sets. This problem does not spare the INRA open archive which, with its 124,000 references, is far from achieving a satisfactory level of referencing. The advent of ProdInra 2, the new document application, represents a chance to mobilize the parties involved (submitting researchers and documentalist-editors) to support a policy that strongly encourages users to systematically deposit their work in ProdInra. This aspect was highlighted in an INRA report on open archives (L'hostis and Aventurier, 2006).

ProdInra : the INRA open archive

Documentary policy

As stated earlier, the ProdInra document database is incomplete in terms of referencing scientific production; it also contains little in the way of full texts (see table 1). Using this as a starting point, the Scientific Information team working on ProdInra came up with a new documentary policy. This policy is centered on the systematic deposit of productions by the authors themselves. In practice, documentalists found themselves filling this role and were confronted by the problem of tracking and accessing the document. Document deposits will now be carried out by researchers, who will then be able to retrieve their list of publications in the requested format in researcher and research unit evaluation dossiers. Meanwhile, the ProdInra administration team will work on identifying productions in Scopus/WoS so that a full survey of the best academic publications will be possible using ProdInra. Finally, the consolidation of full-text articles in ProdInra will be conducted in two ways:

• Encouraging the researcher to deposit the document at the same time as its bibliographic information, in accordance with the publisher's policy. A new submitter's chart is being drafted. • Negotiation between Scientific Information heads and major scientific publishers to allow the automatic archival of published articles after an embargo.

Data flows

Inputs Submission is made tedious by the large number of bibliographic entries that need to be recorded. We therefore sought to develop a means of importing notices once such notices have already been entered somewhere. For example, using the PubMed Identifier (PMID) automatically retrieves information from the PubMed reference in Prodinra. This feature is even more eagerly awaited owing to the fact that part of INRA research involves the biomedical field.

It is also possible to import notices listed in EndNote and RIS formats used by bibliographic reference management software. Researchers using such tools for their work only need to import this list in ProdInra and consolidate it by adding the full text.

Finally, INRA, like many other French institutes and universities, pools resources with its partners in joint research structures. Some scientists from these laboratories submit their production directly into the HAL open archive. HAL, or Hyper Article en Ligne, is the French research open archive. We have therefore developed gateways to exchange data between ProdInra and HAL

Outputs

Research is the most obvious function of an open archive. Nonetheless, we have developed associated services as well as exports so that ProdInra data can be used, and scientists receive services in exchange for their efforts to signal their production.

Several exports are available and we have planned to offer very soon exports of lists of scientific productions in the format used for evaluations. These services include also features tied to new uses of the web: RSS feeds, social bookmarking, etc., as shown in Figure 4.

Highlighting the work of INRA

In its four-year strategic contract, INRA restated its desire to develop its international activities.

Promoting INRA research and highlighting its scientific and technical production internationally serve to generate collaborations and contribute to INRA's image as a major player in international agricultural research.

The ProdInra staff has therefore rolled out collaboration strategies in order to promote INRA's scientific production by having it referenced in numerous major document databases.

In addition to data exports to HAL, ProdInra will make its data available in Agris AP format so that the FAO's Agris database can perform OAI harvesting.

We are also exploring the possibility of exporting our production from research in economics and social sciences to rePEc, the international thematic archive for economics.

Recently, we have made contact with the TGE (large equipment) SHS (Social and Human Sciences) project of the CNRS (French National Centre for Scientific Research), known as TGE Adonis, in order to promote the sociological aspect and social relevance of INRA research. The project began on 1st April 2010 and will run for 3 years, with funding of 2,000,000 € from the European Commission as part of the 7 th FP. By participating in this European project, the ProdInra team falls in line with INRA's international ambitions and hopes to attract an international audience to the work of its researchers.

Conclusion

INRA has chosen to invest in an open archive that forms part of the institute's information system, rather than an independent application based on a software program commonly used by universities and research bodies worldwide, such as DSpace or Eprints.

The additional costs stemming from this integration will be justified, it is hoped, by the benefits in terms of indicators and services.

The first part of this article described such benefits but also underscored the need for quality data the completeness of the corpus being used. This implies the introduction of a demanding documentary policy in terms of goals and resources.

The second part of this paper describes the ProdInra documentary policy, services and exports, but also lists ongoing partnerships aimed at promoting the work of INRA's scientists. These partnerships, whether concluded, ongoing or upcoming, demonstrate IST's desire to be a full participant in INRA's international ambitions.

Fig. 1 .

 1 Fig. 1. Data exchange within the Information System

Fig. 2 .

 2 Fig. 2. INRA IIS & open services

Fig. 4 .

 4 Fig. 4. ProdInra Services & Exports

Fig. 5 .

 5 Fig. 5. Referencing of INRA production in partner applications

Table 1

 1

	. ProdInra content (+ 3 years and + 4 months)
	Date	Number of references	Number of FT records	Part of Public FT records
	Oct 06	101 214	0	0
	Sept 09	120 953	4 498	3 591
	Jan 10	123 515	5 693	4 729

Scientific and Technical Information and Rural Development IAALD XIIIth World Congress, Montpellier, 26-29 April 2010