GEMC1, a novel factor required for chromosomal DNA replication

In eukaryotic cells DNA replication begins from multiple origins. During the process of initiation, the DNA replication fork is established at each origin. In lower eukaryotes many factors required for chromosomal DNA replication have been identified. However, the regulation of DNA replication in complex multi-cellular organisms is still poorly understood. In this thesis I report the identification of GEMC1 (GEMinin Coiled-coil containing protein 1), a novel vertebrate factor belonging to a new protein family required to initiate chromosomal DNA replication. GEMC1 contains a domain similar to Geminin, a fundamental regulator of DNA replication (McGarry and Kirschner, 1998). GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. I show that Xenopus GEMC1 (XlGEMC1) binds the checkpoint and replication factor TopBP1, which promotes XlGEMC1 binding to chromatin during pre-replication complex (pre-RC) assembly. Moreover, I demonstrate that XlGEMC1 directly interacts with the replication factors Cdc45 and Cdk2/CyclinE by which it is heavily phosphorylated. Phosphorylated XlGEMC1 stimulates initiation of DNA replication. Inhibition of XlGEMC1 function with XlGEMC1 neutralizing antibodies prevents DNA replication onset by blocking Cdc45 loading onto chromatin. Inhibition of XlGEMC1 expression by morpholino antisense oligos is lethal for embryonic development. Furthermore, down-regulation of mouse GEMC1 (mGEMC1) expression by siRNA (small interfering RNA) oligos prevents initiation of DNA replication in somatic vertebrate cells. Data presented in this thesis suggest that GEMC1 promotes initiation of chromosomal DNA replication in higher eukaryotes by mediating TopBP1 and Cdk2 dependent Cdc45 recruitment onto replication origins

Climate control on sulphate and nitrate concentrations in alpine streams of Northern Italy along a nitrogen saturation gradient

International audienceThe role of meteorology, hydrology and atmospheric deposition on the temporal pattern of SO4 and NO3 concentrations was investigated for three streams draining alpine catchments in Northern Italy. The study sites lie on a gradient of atmospheric fluxes of SO4 and NO3 (from about 50 to 80 meq m?2 y?1, and from 40 to 90 meq m?2 y?1, respectively). As a consequence of the increasing N input, the three catchments are also representative of aggrading levels of N saturation. Different methods of statistical analysis were applied to monthly data for the period 1997?2005 to identify which variables (temperature, precipitation, hydrology, SO4 and NO3 deposition) were the main predictors of water chemistry and its change in time. Hydrological changes and snow cover proved to be the main confounding factors in the response to atmospheric deposition in the River Masino catchment. Its particular characteristics (small catchment area, rapid flushing during runoff and thin soil cover) meant that this site responded without a significant delay to SO4 deposition decrease. It also showed a clear seasonal pattern of NO3 concentration, in response to hydrology and biological uptake in the growing season. The selected driving variables failed to model the water chemistry at the other study sites. Nevertheless, temperature, especially extreme values, turned out to be important in both SO4 and NO3 export from the catchments. This result might be largely explained by the effect of warm periods on temperature-dependent processes such as mineralization, nitrification and S desorption. Our findings suggest that surface waters in the alpine area will be extremely sensitive to a climate warming scenario: higher temperatures and increasing frequency of drought could exacerbate the effects of high chronic N deposition

DNAJC12 deficiency: Mild hyperphenylalaninemia and neurological impairment in two siblings

Background: DNAJC12 co-chaperone protein deficiency has been recently described as a stand-alone metabolic disorder explaining many cases of mild hyperphenylalaninemia (HPA) that are not caused by variants in the PAH gene, which encodes for the hepatic enzyme phenylalanine hydroxylase (PAH), or inGCH1, PTS, QDPR, PCBD1 and DHPR, involved in tetrahydrobiopterin (BH4) biosynthesis and activity. Results: We describe two sisters born to consanguineous parents. The youngest sister (Patient 1), initially asymptomatic, tested positive at NewBorn Screening (NBS) for mild HPA. After variants in the PAH and BH4 related-genes were excluded, we performed DNAJC12 genetic analysis and found a previously described homozygous deletion [NM_021800.3: c.58_59del p.(Gly20Metfs*2)]. The older sister (Patient 2), homozygous for the same variant and exhibiting mild HPA, was diagnosed subsequently and presented with ataxia and repeated falls, upper limb dyskinesia, intentional tremor, and mild intellectual disability. Patient 1 was started on treatment with low Phenylalanine (Phe) diet, BH4, l-3,4-dihydroxyphenylalanine/carbidopa (L-DOPA) and 5-OH-Tryptophan, soon after diagnosis, and despite poor adherence to the dietary regimen, only manifested language impairment at last follow-up (age 5 years and 4 months). Patient 2, who started the same treatment at school age, experienced a minimal progression of neurological symptoms, with some improvement in her motor skills. Conclusions: These two new patients with DNAJC12-associated HPA, in addition to previous reports, point to DNAJC12 deficiency as a new metabolic syndrome that must be considered in patients with unexplained HPA

Retinal nerve fibre layer thinning is associated with drug resistance in epilepsy.

Retinal nerve fibre layer (RNFL) thickness is related to the axonal anterior visual pathway and is considered a marker of overall white matter 'integrity'. We hypothesised that RNFL changes would occur in people with epilepsy, independently of vigabatrin exposure, and be related to clinical characteristics of epilepsy

Enabling Rapid Naval Architecture Design Space Exploration

Well accepted conceptual ship design tools can be used to explore a design space, but more precise results can be found using detailed models in full-feature computer aided design programs. However, defining a detailed model can be a time intensive task and hence there is an incentive for time sensitive projects to use conceptual design tools to explore the design space. In this project, the combination of advanced aerospace systems design methods and an accepted conceptual design tool facilitates the creation of a tool that enables the user to not only visualize ship geometry but also determine design feasibility and estimate the performance of a design

Is Tuber brumale a threat to T. melanosporum and T. aestivum plantations?

True truffles in the genus Tuber are the most valuable ectomycorrhizal fungiand their cultivation has become widespread around the world. Competition with other ectomycorrhizal fungi and especially with undesired Tuber species, like T. brumale, can threaten the success of a truffle plantation. In this work, the competitiveness of T. brumale towards T. melanosporum and T. aestivum was assessed in a 14 year-old plantation carried out planting seedlings inoculated with these three truffle species in adjacent plots. Analyses of both truffle ectomycorrhizas and extra-radical mycelium were carried out in the transects separating the T. brumale plot from T. melanosporum and T. aestivum plots. The results confirm the competitiveness of T. brumale against T. aestivum and T. melanosporum due to its major ability to colonize the soil around its ectomycorrhizas. However, its competitiveness is limited to the transect areas and it was never found inside T. melanosporum plot. These results remark that, in presence of optimal conditions for T. melanosporum and T. aestivum, the greatest risk of contamination with T. brumale is due to wrong greenhouse activity

The Perigord black truffle responds to cold temperature with an extensive reprogramming of its transcriptional activity

International audienceThe Tuber melanosporum genome has been analysed with the aim of identifying and characterizing the genes involved in the environmental stress response. A whole genome array (7496 genes/probe) was used to verify the fungal transcriptional profiling upon a cold temperature period (7 days at 4 degrees C). A total of 423 genes resulted to be differentially expressed in a significant manner (>2.5-fold; p-value <0.05) in the mycelia exposed to cold, compared to the control ones: 187 of these genes were up-regulated, while 236 were down-regulated. Sixty-six and fifty-one percent, respectively, of the up- or down-regulated transcripts had no KOG classification and were clustered as unclassified proteins, which was the most abundant category in the both up- and down-regulated genes. A gene subset, containing a range of biological functions, was chosen to validate the microarray experiment through quantitative real time PCR (qRT-PCR). The analysis confirmed the array data for 16 out of 22 of the considered genes, confirming that a cold temperature period influences the truffle global gene expression. The expressed genes, which mostly resulted to be genes for heat shock proteins (HSPs) and genes involved in cell wall and lipid metabolism, could be involved in mechanisms, which are responsible for fungal adaptation. Since truffle ascomata develop during the winter period, we hypothesize that these differentially expressed genes may help the truffle to adapt to low temperatures and/or perceive environmental signals that regulate the fructification. (C) 2010 Elsevier Inc. All rights reserved

Arbuscular mycorrhizal colonization in black poplar roots after defoliation by a non-native and a native insect

A major goal in ecology is to understand how interactions among organisms influence ecosystem services. This work compares the effects of two Lepidoptera defoliators, one non-native (Hyphantria cunea) and one native (Lymantria dispar) to Europe, on the colonization of black poplar (the Populus nigra clone "Jean Pourtet") roots by an arbuscular mycorrhizal (AM) symbiotic fungus (Funneliformis mosseae) in a pot experiment. The effects of defoliation have also been assessed on the expression of fungal and plant genes playing a role during symbiosis. Both control and defoliated poplars have shown a low level of mycorrhization. Additionally, neither the non-native nor the native insect seem to strongly affect the AM colonization, at least at the time of observation (eight days from the end of the defoliation). Concerning the gene expression analysis, our results suggest that defoliation does not influence neither the expression of genes coding for a fungal and a plant phosphate transporter nor that of a gene coding for a fungal ATPase, and that there were no differences between defoliation carried out by the non-native and the native insect. \ua9 SISEF