Characterization of a MAF1 knockout mouse model

L'ARN polymérase 3 transcrit un petit groupe de gènes fortement exprimés et impliqués dans plusieurs mécanismes moléculaires. Les ARNs de transfert ou ARNt représentent plus ou moins la moitié du transcriptome de l'ARN polymérase 3. Ils sont directement impliqués dans la traduction des protéines en agissant comme transporteurs d'acides aminés qui sont incorporés à la chaîne naissante de polypeptides. Chez des levures cultivées dans un milieu jusqu'à épuisement des nutriments, Maf1 réprime la transcription par l'ARN polymérase 3, favorisant ainsi l'économie énergétique cellulaire. Dans un modèle de cellules de mammifères, MAF1 réprime aussi la transcription de l'ARN polymérase 3 dans des conditions de stress, cependant il n'existe aucune donnée quant à son rôle chez un mammifère vivant. Pendant mon doctorat, j'ai utilisé une souris délétée pour le gène Maf1 afin de connaître les effets de ce gène chez un mammifère. Etonnamment, la souris Maf1-­‐/-­‐ est résistante à l'obésité même si celle-­‐ci est nourrie avec une nourriture riche en matières grasses. Des études moléculaires et de métabolomiques ont montré qu'il existe des cycles futiles de production et dégradation des lipides et des ARNt, ce qui entraîne une augmentation de la dépense énergique et favorise la résistance à l'obésité. En plus de la caractérisation de la souris Maf1-­‐/-­‐, pendant ma thèse j'ai également développé une méthode afin de normaliser les données de ChIP-­‐sequencing. Cette méthode est fondée sur l'utilisation d'un contrôle interne, représenté ici par l'ajout d'une quantité fixe de chromatine provenant d'un organisme différent de celui étudié. La méthode a amélioré considérablement la reproductibilité des valeurs entre réplicas biologiques. Elle a aussi révélé des différences entre échantillons issus de conditions différentes. Une occupation supérieure de l'ARN polymérase 3 sur les gènes Pol 3 chez les souris Maf1 KO entraîne une augmentation du niveau de précurseurs d'ARNt, ayant pour effet probable la saturation de la machinerie de maturation des ARNt. En effet, chez les souris Maf1 KO, le pourcentage d'ARNt modifiés est plus faible que chez les souris type sauvage. Ce déséquilibre entre le niveau de précurseurs et d'ARNt matures entraîne une diminution de la traduction protéique. Ces résultats ont permis d'identifier de nouvelles fonctions pour la protéine MAF1, comme étant une protéine régulatrice à la fois de la transcription mais aussi de la traduction et en étant un cible potentielle au traitement à l'obésité. -- RNA polymerase III (Pol 3) transcribes a small set of highly expressed genes involved in different molecular mechanisms. tRNAs account for almost half of the Pol 3 transcriptome and are involved in translation, bringing a new amino into the nascent polypeptide chain. In yeast, under nutrient deprivation, Maf1 acts for cell energetic economy by repressing Pol 3 transcription. In mammalian cells, MAF1 also represses Pol 3 activity under conditions of serum deprivation or DNA damages but nothing is known about its role in a mammalian organism. During my thesis studies, I used a Maf1 KO mouse model to characterize the effects of Maf1 deletion in a living animal. Surprisingly, the MAF1 KO mouse developed an unexpected phenotype, being resistant to high fat diet-­‐induced obesity and displaying an extended lifespan. Molecular and metabolomics characterizations revealed futile cycles of lipids and tRNAs, which are produced and immediately degraded, which increases energy consumption in the Maf1 KO mouse and probably explains in part the protection to obesity. Additionally to the mouse characterization, I also developed a method to normalize ChIP-­‐seq data, based on the addition of a foreign chromatin to be used as an internal control. The method improved reproducibility between replicates and revealed differences of Pol 3 occupancy between WT and Maf1 KO samples that were not seen without normalization to the internal control. I then established that increased Pol 3 occupancy in the Maf1 KO mouse liver was associated with increased levels of tRNA precursor but not of mature tRNAs, the effective molecules involved in translation. The overproduction of precursor tRNAs associated with the deletion of Maf1 apparently overwhelms the tRNA processing machinery as the Maf1 KO mice have lower levels of fully modified tRNAs. This maturation defect directly impacts on translation efficiency as polysomic fractions and newly synthetized protein levels were reduced in the liver of the Maf1 KO mouse. Altogether, these results indicate new functions for MAF1, a regulator of both transcription and translation as well as a potential target for obesity treatment.

Novel Approaches for Fungal Transcriptomics from Host Samples.

Candida albicans adaptation to the host requires a profound reprogramming of the fungal transcriptome as compared to in vitro laboratory conditions. A detailed knowledge of the C. albicans transcriptome during the infection process is necessary in order to understand which of the fungal genes are important for host adaptation. Such genes could be thought of as potential targets for antifungal therapy. The acquisition of the C. albicans transcriptome is, however, technically challenging due to the low proportion of fungal RNA in host tissues. Two emerging technologies were used recently to circumvent this problem. One consists of the detection of low abundance fungal RNA using capture and reporter gene probes which is followed by emission and quantification of resulting fluorescent signals (nanoString). The other is based first on the capture of fungal RNA by short biotinylated oligonucleotide baits covering the C. albicans ORFome permitting fungal RNA purification. Next, the enriched fungal RNA is amplified and subjected to RNA sequencing (RNA-seq). Here we detail these two transcriptome approaches and discuss their advantages and limitations and future perspectives in microbial transcriptomics from host material.

Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b.

The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models. The 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses. The findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome.

Image guided critical screw placement precision in the cervical spine : important or incidental? : results of a series of 91 consecutive cases using OVEM

Study design: A retrospective study of image guided cervical implant placement precision. Objective: To describe a simple and precise classification of cervical critical screw placement. Summary of Background Data: "Critical" screw placement is defined as implant insertion into a bone corridor which is surrounded circumferentially by neurovascular structures. While the use of image guidance has improved accuracy, there is currently no classification which provides sufficient precision to assess the navigation success of critical cervical screw placement. Methods: Based on postoperative clinical evaluation and CT imaging, the orthogonal view evaluation method (OVEM) is used to classify screw accuracy into grade I (no cortical breach), grade la (screw thread cortical breach), grade II (internal diameter cortical breach) and grade III (major cortical breach causing neural or vascular injury). Grades II and III are considered to be navigation failures, after accounting for bone corridor / screw mismatch (minimal diameter of targeted bone corridor being smaller than an outer screw diameter). Results: A total of 276 screws from 91 patients were classified into grade I (64.9%), grade la (18.1%), and grade II (17.0%). No grade III screw was observed. The overall rate of navigation failure was 13%. Multiple logistic regression indicated that navigational failure was significantly associated with the level of instrumentation and the navigation system used. Navigational failure was rare (1.6%) when the margin around the screw in the bone corridor was larger than 1.5 mm. Conclusions: OVEM evaluation appears to be a useful tool to assess the precision of critical screw placement in the cervical spine. The OVEM validity and reliability need to be addressed. Further correlation with clinical outcomes will be addressed in future studies.

Trasporti e sviluppo territoriale negli spazi intermedi : l'esempio della regione transfrontaliera Insubrica

Avec cette thèse de doctorat nous proposons une réflexion transversale concernant les relations entre infrastructures de transport et développement territorial dans des espaces dits « intermédiaires ». Le concept d'espace intermédiaire, relativement nouveau en géographie, est conçu en fonction d'une double approche : celle des infrastructures où les espaces intermédiaires constituent des zones de transit obligées entre des pôles urbains hiérarchiquement supérieurs (par rapport une échelle horizontale) et celle des frontières où les espaces intermédiaires constituent des territoires de coopération entre différents niveaux politico-institutionnels (par rapport à une échelle verticale). Cette problématique de recherche est traitée aussi bien du point de vue théorique qu'avec des études de cas portant sur les effets des nouvelles infrastructures de transports dans la région transfrontalière insubrique (entre le Canton du Tessin et la Lombardie). L'objectif visé est de défendre un scénario d'organisation spatiale polycentrique à plusieurs niveaux comme solution pour le développement durable et cohérent de ces espaces intermédiaires. Ainsi, pour le « niveau macro », nous proposons une analyse des changements d'accessibilité spatiale et des potentiels de développement territorial pour les agglomérations concernées par la mise en service du nouveau tunnel ferroviaire de base du Monte Ceneri (TBC) et de la nouvelle ligne Lugano/Como-Mendrisio-Varese-Malpensa (FMV) à l'horizon 2020. Pour le « niveau meso », nous analysons les effets de la nouvelle ligne FMV en termes de potentiel de densification polycentrique autours des gares ferroviaires. Pour le « niveau micro », nous proposons une analyse sur les comportements de mobilité ainsi que des améliorations ciblées du système de transport pour la ville de Mendrisio visant à promouvoir le développement polycentrique de cette commune. De plus, un système d'analyse permettant de mettre en lien les divers facteurs explicatifs dans l'analyse des relations entre les nouvelles infrastructures de transport et les effets sur la mobilité et le développement territorial est également élaboré et testé dans notre recherche. -- With this Ph.D. thesis we investigate the relationship between transport infrastructures and territory development inside the so called "in-between spaces". The idea of "in-between space", relatively novel in geography, is the formal outcome of a double approach: the one of the infrastructures, saying that these spaces can be described as areas of constrained transit between urban centres of superior hierarchical level (on a horizontal scale), and the one of the borders, stating that in-between spaces are areas of cooperation between various political-institutional levels. The above mentioned research issues are deepened both at theoretical and empirical level, being the latter based on field studies of the cross-boundary Western-Lombard area (between the Swiss canton of Ticino and the Italian region of Lombardy). This research pursues the goal of defending the argument that a multi-level polycentric spatial scenario can be a possible solution fora sustainable development of the above described in- between areas. From a "macro" perspective, what we submit here is an analysis on the expected changes in spatial accessibility and on the potential territorial development for the built-up areas influenced by the construction of the new train tunnel of the Monte Ceneri (TBC) and of the new railway line Lugano/Como-Mendrisio-Varese-Malpensa (FMV). At a "meso" level we analyse the effects exerted by the new FMV line taking into account the potential densification of the areas surrounding the railway stations. Finally, at a "micro" level, we analyse the mobility behaviours in the town of Mendrisio and we propose some possible improvements to the local public transport system, with the scope to promote a polycentric development of this municipality. Moreover, we developed and tested an analytic system able to define the existing links between the various explaining factors characterizing the relationship between new transport infrastructures and effects on mobility. -- With this Ph.D. thesis we investigate the relationship between transport infrastructures and territory development inside the so called "in-between spaces". The idea of "in-between space", relatively novel in geography, is the formal outcome of a double approach: the one of the infrastructures, saying that these spaces can be described as areas of constrained transit between urban centres of superior hierarchical level (on a horizontal scale), and the one of the borders, stating that in-between spaces are areas of cooperation between various political-institutional levels. The above mentioned research issues are deepened both at theoretical and empirical level, being the latter based on field studies of the cross-boundary Western-Lombard area (between the Swiss canton of Ticino and the Italian region of Lombardy). This research pursues the goal of defending the argument that a multi-level polycentric spatial scenario can be a possible solution for a sustainable development of the above described in- between areas. From a "macro" perspective, what we submit here is an analysis on the expected changes in spatial accessibility and on the potential territorial development for the built-up areas influenced by the construction of the new train tunnel of the Monte Ceneri (TBC) and of the new railway line Lugano/Como-Mendrisio-Varese-Malpensa (FMV). At a "meso" level we analyse the effects exerted by the new FMV line taking into account the potential densification of the areas surrounding the railway stations. Finally, at a "micro" level, we analyse the mobility behaviours in the town of Mendrisio and we propose some possible improvements to the local public transport system, with the scope to promote a polycentric development of this municipality. Moreover, we developed and tested an analytic system able to define the existing links between the various explaining factors characterizing the relationship between new transport infrastructures and effects on mobility.

Analyzing the temporal regulation of translation efficiency in mouse liver.

Mammalian physiology and behavior follow daily rhythms that are orchestrated by endogenous timekeepers known as circadian clocks. Rhythms in transcription are considered the main mechanism to engender rhythmic gene expression, but important roles for posttranscriptional mechanisms have recently emerged as well (reviewed in Lim and Allada (2013) [1]). We have recently reported on the use of ribosome profiling (RPF-seq), a method based on the high-throughput sequencing of ribosome protected mRNA fragments, to explore the temporal regulation of translation efficiency (Janich et al., 2015 [2]). Through the comparison of around-the-clock RPF-seq and matching RNA-seq data we were able to identify 150 genes, involved in ribosome biogenesis, iron metabolism and other pathways, whose rhythmicity is generated entirely at the level of protein synthesis. The temporal transcriptome and translatome data sets from this study have been deposited in NCBI's Gene Expression Omnibus under the accession number GSE67305. Here we provide additional information on the experimental setup and on important optimization steps pertaining to the ribosome profiling technique in mouse liver and to data analysis.

Genomic evidence for adaptive inversion clines in Drosophila melanogaster.

Clines in chromosomal inversion polymorphisms-presumably driven by climatic gradients-are common but there is surprisingly little evidence for selection acting on them. Here we address this long-standing issue in Drosophila melanogaster by using diagnostic single nucleotide polymorphism (SNP) markers to estimate inversion frequencies from 28 whole-genome Pool-seq samples collected from 10 populations along the North American east coast. Inversions In(3L)P, In(3R)Mo, and In(3R)Payne showed clear latitudinal clines, and for In(2L)t, In(2R)NS, and In(3R)Payne the steepness of the clinal slopes changed between summer and fall. Consistent with an effect of seasonality on inversion frequencies, we detected small but stable seasonal fluctuations of In(2R)NS and In(3R)Payne in a temperate Pennsylvanian population over 4 years. In support of spatially varying selection, we observed that the cline in In(3R)Payne has remained stable for >40 years and that the frequencies of In(2L)t and In(3R)Payne are strongly correlated with climatic factors that vary latitudinally, independent of population structure. To test whether these patterns are adaptive, we compared the amount of genetic differentiation of inversions versus neutral SNPs and found that the clines in In(2L)t and In(3R)Payne are maintained nonneutrally and independent of admixture. We also identified numerous clinal inversion-associated SNPs, many of which exhibit parallel differentiation along the Australian cline and reside in genes known to affect fitness-related traits. Together, our results provide strong evidence that inversion clines are maintained by spatially-and perhaps also temporally-varying selection. We interpret our data in light of current hypotheses about how inversions are established and maintained.