Un nouveau procédé intensifié de vinification continue avec un mélange de levures S.cerevisiae et Sch.pombe immobilisées dans des billes d’alginate est proposé. A l’échelle laboratoire, l’étude de la teneur en billes et de la configuration du réacteur conduit à l’obtention d’un réacteur de type lit fixe permettant une production de vin en 35 heures. Des validations du procédé aux échelles pilote (170 L) puis industrielle (120 hL) montrent que, en cave, du vin de qualité semblable au témoin est produit en 2 à 3 jours. Une analyse du comportement du réacteur a identifié des raisons de pertes de performances liées à l’hydrodynamique lors du changement d’échelle ainsi que des axes améliorations possibles. Ce procédé permet d’obtenir un vin de qualité maitrisée et un gain de temps de plusieurs semaines / From a batch to another, produced wines are usually different because of the different alcoholic and malolactic fermentation courses. To blend wines quality and continue wine production industrialization, a new continuous process, using Ca-alginate immobilized yeast cells, was developed for red wine-making. Working with a blending of S.cerevisiae and Sch.pombe allowed the regrouping of the alcoholic and malolactic fermentations in a unique step. After testing different reactor set-ups at lab scale, the selected process, a vertical bed reactor, was used in real wine-making conditions, firstly in a pilot reactor (170 L) and then in an industrial one (120 hL). The results showed that continuous wine-making was possible in 2 to 3 days. The wine presented nearly the same sensory profile compared to a classical one. Thanks to the analysis of the reactor behaviour, we were able to explain the efficiency losses linked to the hydrodynamic, observed during the scale-up. This new intensified process enables to obtain a wine with a controlled quality and to save several weeks of production time

Among the various external signals perceived by yeast cells, nutrient availability is a condition to which these cells show a highly diverse biological response. Diploid cells in response to different nutritional stress conditions shows different developmental outcomes. On nitrogen starvation, cells undergo dimorphic transition whereby a unicellular yeast form transforms to a multicellular pseudohyphal form. While in the complete absence of a nitrogen source and a fermentable carbon source, yeast cells enter into a complex developmental program termed sporulation which culminates in haploid spores. The main objective of this work was to understand the role played by S. cerevisiaeSUB1 in starvation-induced meiotic program of diploid cells, decipher its target in sporulation specific gene expression cascade, study the domain architecture of Sub1 and examine its functional homology to mammalian PC4. Role of Sub1 in induction of sporulation and other stress responses in S. cerevisiae In a previous whole-genome screen for mutants with altered sporulation efficiency in the Saccharomyces cerevisiae S288c strain, SUB1 locus was identified as a negative regulator of sporulation (Deutschbaueret al., 2002). Moreover, genome-wide gene expression analysis in SK1 strain had shown that SUB1 transcript levels are repressed during sporulation (Chu et al., 1998). Many studies in different yeast strain backgrounds implicate more than 1,000 genesout of 6,200 genes in yeast genome as being differentially expressed during the sporulation process (Chu et al., 1998; Primiget al., 2000; Deutschbaueret al., 2002). Interestingly, these studies show the number of regulatory genes that negatively affect sporulation is far lower than those that are activators of sporulation and moreover their mechanism of action is poorly studied. S. cerevisiae.SUB1 is one among negative regulators of sporulation(Deutschbaueret al., 2002). Global transcriptome of diploid yeast cells undergoing sporulation showed SUB1 transcripts are greatly reduced with time progression (Chu et al., 1998). To understand the role of SUB1 in sporulation, we generated deletion of both SUB1 alleles in the diploid S288c strain background and compared the kinetics of asci formation in this strain with that of the wild-type. We observed that cells lacking SUB1 exhibit ~5-fold increase in tetrad asci. Based on Eosin Y and Calcoflour White staining assays, we find no change in spore morphology in the mutant. Thus the increase in sporulation efficiency in sub1/sub1diploids is not accompanied by formation of defective spores. We validated the reduction in SUB1 transcript levels during sporulation in wild-type SK1 strain background. We also examined the Sub1 protein levels by epitope-tagging of the chromosomal SUB1 open reading frame and determining protein levels in this strain. We find that consistent with the data on transcript levels, Sub1-TAP tagged protein levels too decreased gradually on shift to sporulation medium. We created sub1alleles in diploids in the SK1 strain background and using this strain background we investigated Sub1 target genes and chose IME2 (early), SMK1, SPS2 (middle), DIT1, DIT2 (mid-late) and SPS100 (late) genes as representative sporulation genes. We observed that sub1∆/sub1∆cells have a significantly elevated expression of middle genes (SPS2 and SMK1) that followed normal induction kinetics i.e., 5 hours post transfer to sporulation medium. However, the expression levels or timing for other class of sporulation genes did not change in sub1∆strain as compared with the wild-type. In order to confirm these observations, we also studied the effects of over-expression of SUB1 from the GAL1 promoter by transforming the high copy plasmid. This was done in wild-type SK1 cells and the expression of sporulation genes were analyzed. We observed that expression of SMK1 and SPS2middle sporulation genes was reduced on over-expression of SUB1.We used the Sub1-TAP protein to assess if Sub1 directly regulates these genes by Chromatin immunoprecipitation assays. For these studies, we examined the recruitment of Sub1 to these loci through the time course of sporulation. In wild-type SK1 cells, Sub1 was to bound to middle sporulation genes and this was striking in cells at 5th hour post-induction of sporulation. These data establish that Sub1 directly associates with chromatin at these loci co-incident with the time points where expression levels of these changes is altered in cells lacking Sub1. Furthermore, to assess the role of Sub1 in other stress responses, such as pseudohyphae formation in response to nitrogen starvation, pheromone-induced agar invasion and secretory stress, we employed a genetic approach. Genetic interaction studies of SUB1 with RPB4, a subunit of RNA polymerase with functions in stress response and HOS2, a subunit of Set3 complex and a close homolog of mammalian HDAC3, reported to be involved in sporulation and secretory stress, were performed. Based on sporulation frequency and pseudohyphal formation in the double mutants we conclude that SUB1 is downstream of both these genes. Moreover, our results from assays of schmoo formation and pheromone-induced agar invasion suggest that SUB1 functionally interacts with HOS2. Study of domain architecture of Sub1 and homology to human PC4 Comparison of the S. cerevisiae Sub1 protein with its higher eukaryotic homologs showed that the N-terminal region of yeast Sub1 (32-105 aa) is highly conserved (Knauset al., 1996; Henry et al., 1996) with the 106-292 C -terminal amino acids being yeast-specific. We employed deletion analysis to generate partial Sub1 proteins and used them to understand the roles played by these domains in different phenotypes associated with Sub1. Our analysis of the localization of various Sub1-GFP fusion proteins shows that 146-172 aa in the C-terminal domain of Sub1 confers nuclear localization. Sporulation frequency analysis of the different domains of Sub1 suggests that both the N and C terminal domains are necessary for sporulation function of Sub1. The N terminal domain of yeast Sub1 shares homology with human PC4 and not surprisingly possesses ssDNA binding ability first attributed to human PC4 (Kaiser et al., 1995). In order to investigate whether the effects of SUB1 on kinetics of sporulation require its ssDNA binding function, we generated the sub1(Y66A) ssDNA binding mutant (Sikorskiet al., 2011) and over-expressed it in the S288c genetic background. We assessed sporulation efficiency of sub1∆/sub1∆cells over-expressing sub1(Y66A) mutant allele as compared to cells over-expressing wild-type SUB1. Interestingly, cells with over-expression of sub1(Y66A) have reduced sporulation efficiency that is equivalent to the levels achieved on over-expression of wild type SUB1. This data suggests that the ssDNA-binding ability of Sub1 is not important for its role in sporulation. Furthermore, we examined the ability of human PC4 to contribute to yeast sporulation process by complementation analysis. We observed that over-expression of PC4 complemented the phenotypes of sub1∆strain, suggesting that the function of Sub1/PC4 family is evolutionarily conserved. Studies on biochemical interactions of Sub1 with histone proteins Human PC4 is a chromatin-associated protein, present on metaphase chromosomes (Das et al., 2006). The short C-terminal domain of PC4(62-87 aa) interacts with core histones H3 and H2B in vitro and in vivo and this interaction mediates chromatin condensation. The homology between S. cerevisiaeSub1 (32-105 aa) and human PC4 (62-127 aa)is in the domain required for their DNA binding properties and coactivator functions, suggesting possible conservation in their interactions. We tested the interactions of yeast Sub1 with histone proteins by adopting both in vitro and in vivo interaction assays. We find recombinant Sub1 had strong interactions with rat and yeast histone H3in vitro. Moreover,Sub1 was found to interact with histone H2B, but not with H2A, in vivo, a binding specificity also shown by human PC4.Thus, we demonstrate conservation in the interaction of Sub1 with histone proteins.

Raman spectroscopy is a powerful analytical method that allows deposited and/or immobilized cells to be evaluated without complex sample preparation or labeling. However, a main limitation of Raman spectroscopy in cell analysis is the extremely weak Raman intensity that results in low signal to noise ratios. Therefore, it is important to seize any opportunity that increases the intensity of the Raman signal and to understand whether and how the signal enhancement changes with respect to the substrate used. Our experimental results show clear differences in the spectroscopic response from cells on different surfaces. This result is partly due to the difference in spatial distribution of electric field at the substrate/cell interface as shown by numerical simulations. We found that the substrate also changes the spatial location of maximum field enhancement around the cells. Moreover, beyond conventional flat surfaces, we introduce an efficient nanostructured silver substrate that largely enhances the Raman signal intensity from a single yeast cell. This work contributes to the field of vibrational spectroscopy analysis by providing a fresh look at the significance of the substrate for Raman investigations in cell research.

That consciousness is ubiquitous, and relevant to autopoietic self-organisation and embodiment within every living being and/or organism, is a prevalent idea in contemporary consciousness research. However, because ‘consciousness’ as a word is derived from con or cum, meaning ‘with’ or ‘together’ and scire, ‘to know’ or ‘to see’ it infers the experience of knowing with an ‘other’ and/or ‘others’. The narrative that follows, while expressing a life of its own, documents the interdisciplinary research conducted and questions who and/or to what ‘other’ might infer. My visual diary, Dust from dust: Microorganisms and other tales: An Artist’s diary, created as the visual component of a creative practice-as-research undertaking, was silently performed amidst ‘others’ in the Unisa gallery, in an attempt to render visible, the autopoietic, self-organising embodiment essential to the conscious self-developmental component of the project. Once upon a time, I grew bacterial yeast cells in a glass vitrine to observe how they self-organised their own embodiment and photographed the process. At the same time, I conducted interdisciplinary research into consciousness as a self-developmental process, and utilising the cellular symbiosis unfolding in the vitrine as a self-reflexive mirror, came to visualise how indispensable bodily feelings are to conscious self-development, and being-in-the-world-with-others processes. As a creative-practice-as-research undertaking, I grew, manipulated and photographed the cellular imagery in the vitrine over many years in an attempt to unfold personal bodily feeling associations the imagery held captive, while gathering photographic footage I considered capable of expressing the primordial nature of certain emotive feeling experiences. Once obtained, I choreographed and performed a stop-frame video, entitled Dust from Dust: Microorganisms and other tales. An artist’s diary. The stop-frame video, along with a catalogue that focuses on the processes engaged with, accompanies the written narrative. Once edited, I macroscopically projected different phases of the video into a three-walled enclosure in the UNISA Art gallery. The three videos, representing a facet of my praxis, ran concurrently over a two week period. The fourth facet, presented with the video projections to emphasise conscious self-development as an in-the-world-with-others process, was the glass vitrine. It was positioned in a darkened enclosure in the gallery space, opposite the video projections. This narrative documents how I projected myself into the cellular imagery developing in the glass vitrine, in a way akin to how the ancient alchemists ‘projected’ themselves into the prima materia with which they worked. While the alchemists seemingly worked unconsciously, and my praxis initially started somewhat unconsciously, the process developed into a conscious attempt to embody the research findings. So, while the video choreographed, champions a microbial cell story, by referring to it as an artist’s diary, I emphasise the subjective nature of my praxis as a whole. In this creative-practice-as-research undertaking, I address the significance of bodily feelings and their relevance to being-in-the-world-with-others processes. In doing so, I aim to offer insight into how and why feelings are essential to inter-subjectivity and/or sociality, self-organisation and conscious self-development, as well as how and why conscious self-development can lead to immersive experiences, which I interpret as embodied adaptation to the rich diversity and/or fullness of life itself. / Art History, Visual Arts and Musicology / D. Litt. et Phil. (Art History)

Raman spectroscopy is a powerful analytical method that allows deposited and/or immobilized cells to be evaluated without complex sample preparation or labeling. However, a main limitation of Raman spectroscopy in cell analysis is the extremely weak Raman intensity that results in low signal to noise ratios. Therefore, it is important to seize any opportunity that increases the intensity of the Raman signal and to understand whether and how the signal enhancement changes with respect to the substrate used. Our experimental results show clear differences in the spectroscopic response from cells on different surfaces. This result is partly due to the difference in spatial distribution of electric field at the substrate/cell interface as shown by numerical simulations. We found that the substrate also changes the spatial location of maximum field enhancement around the cells. Moreover, beyond conventional flat surfaces, we introduce an efficient nanostructured silver substrate that largely enhances the Raman signal intensity from a single yeast cell. This work contributes to the field of vibrational spectroscopy analysis by providing a fresh look at the significance of the substrate for Raman investigations in cell research.