Effetti di processi con diverso spettro di variabilità sulla distribuzione di popolamenti epifiti di Posidonia oceanica

La mia Tesi Specialistica avrà come oggetto uno studio sperimentale, che verrà svolto nei primi metri del piano infralitorale presso la località di Antignano, in una prateria di Posidonia oceanica situata ad una profondità compresa tra i 4 e i 7 metri. La variabilità naturale è una proprietà fondamentale dei sistemi ecologici. Essa riflette le variazioni spazio-temporali delle modalità di distribuzione, abbondanza e diversità dei popolamenti, e comprende i processi che generano e mantengono tali modalità. La variabilità osservata in un popolamento dipende dalla scala spaziale o temporale considerata. Per individuare i processi che influenzano le modalità di distribuzione degli organismi, bisogna identificare la scala (o le scale) a cui si osserva maggiore variabilità. Numerosi studi sulle modalità di distribuzione spaziale dei popolamenti marini hanno dimostrato che la variabilità maggiore si osserva a grande e piccola scala, mentre le scale intermedie presentano una minore variabilità. Tale modalità potrebbe essere il risultato di processi caratterizzati da diverso spettro di variabilità che agiscono simultaneamente a diverse scale spaziali. Lo scopo del lavorò è quello di comprendere se le modalità di distribuzione spaziale osservate possano essere spiegate dall'azione simultanea di diversi processi fisici e biologici aventi spettri di variabilità distinti. Verrà testata l’ipotesi secondo cui processi che agiscono indipendentemente nello spazio, pertanto caratterizzati da uno spettro "white" in analogia con la composizione spettrale della luce che include tutte le lunghezze d’onda nel visibile, aumentano la variabilità dei popolamenti a piccola scala; mentre processi aventi spettro "red", caratterizzati da elevata lunghezza d’onda e quindi dominati da eventi episodici che generano correlazione positiva nello spazio e nel tempo, aumentano la variabilità a grande scala. Per indagare tale ipotesi sono stati predisposti quindici transetti di trentadue metri all'interno della prateria di Posidonia oceanica, composti da sessantaquattro quadrati di 50 x 50 cm ciascuno. I transetti sono stati assegnati in modo casuale in repliche di tre a ciascuno dei seguenti trattamenti (1) disturbo con spettro "red", (2) disturbo con spettro "white", (3) applicazione simultanea di disturbo di tipo “red” e di tipo “white” e (4) transetti di controllo. Tre ulteriori transetti sono stati campionati per esaminare le modalità di distribuzione spaziale dei popolamenti in assenza di disturbo sperimentale all’inizio dello studio. Il disturbo caratterizzato da spettro di variabilità di tipo “red”, quindi autocorrelato nello spazio, è consistito nello sfoltimento delle fronde di P. oceanica. Ciò ha simulato effetti dovuti ad eventi naturali di senescenza delle foglie e l’attività dei pascolatori. Il disturbo di tipo “white”, caratterizzato da eventi indipendenti nello spazio, è consistito nella rimozione dei rizomi di P. oceanica in sub-quadrati di 10 cm di lato, con un numero medio di tre sub-quadrati in ciascuno dei quadrati di taglia maggiore. Questo disturbo simula l’effetto sui rizomi di forti mareggiate. Si tratta quindi di un disegno sperimentale di tipo fattoriale, caratterizzato dalla manipolazione incrociata di due forme di disturbo meccanico trattate come fattori fissi al fine di indagarne l’effetto separato di tali fattori e la loro eventuale interazione. Le variabili di risposta considerate nello studio sono i popolamenti epifiti dei rizomi di Posidonia oceanica. Tali popolamenti sono sensibili alle variazioni delle condizioni ambientali, pertanto costituiscono un sistema utile per indagare gli effetti delle variazioni di processi con diversi spettri di variabilità. Per individuare i valori di riferimento della variabilità a diverse scale spaziali del sistema in studio, sarà completato lo studio pilota. I campionamenti sono effettuati mediante tecniche non distruttive (campionamento fotografico), sia nello studio pilota, sia per le ulteriori fasi dell'esperimento. Le fotografie sono analizzate su PC al fine di stimare l’abbondanza delle specie come copertura percentuale dei principali gruppi di alghe e di invertebrati. Le analisi sui dati attualmente disponibili indicano che le scale di variabilità di alcuni taxa riflettono lo spettro di variabilità dei processi di disturbo esaminati

Use of scalp cooling device to prevent alopecia for early breast cancer patients receiving chemotherapy: A prospective study

Chemotherapy-induced alopecia (CIA) affects the majority of patients receiving chemotherapy (CT) for early breast cancer. It is a highly distressing side effect of CT, with psychological and social impact. Primary aim of the present analysis was to assess the efficacy of scalp cooling with DigniCap® in preventing CIA. Success rate was defined as patients' self-reported hair loss <50% according to Dean scale. In this analysis, we reported success rate at 3 weeks after the first CT course and at 3 weeks after the last CT course. Secondary endpoints included self-reported tolerability and patients' judgment on scalp cooling performance. Consecutive early breast cancer patients admitted to Istituto Oncologico Veneto who were recommended to receive neoadjuvant or adjuvant CT, were eligible to undergo scalp cooling during the CT administration within this study. 135 patients were included: 74% received adjuvant CT and 26% neoadjuvant CT (P < .001). The type of CT was: docetaxel-cyclophosphamide (26%), paclitaxel (23%), epirubicin-cyclophosphamide followed by paclitaxel (32%), and paclitaxel followed by epirubicincyclophosphamide (19%). The rate of success in preventing alopecia was 77% (104/135) at 3 weeks from the start of CT and 60% (81/135) at 3 weeks from the end of treatment. Higher success rates were reported in non-anthracycline (71%) compared to anthracycline-containing CT regimens (54%; P < 0.001). Premature discontinuation of scalp cooling was reported in 29/135 patients (21.5%), including withdrawal for alopecia (16/29), for low scalp cooling tolerability (8/29) or both (5/29). Scalp cooling was generally well tolerated. These results overall suggest that the use of scalp cooling is effective in preventing alopecia in the majority of early breast cancer patients receiving neoadjuvant or adjuvant CT, especially for patients undergoing a taxane-based non-anthracycline regimen

Protein phosphatase 1 recruitment by Rif1 regulates DNA replication origin firing by counteracting DDK activity

The eukaryotic genome is replicated according to a strict temporal program. Here, Bianchi and colleagues find that Rif1, a master regulator of the DNA replication program in yeast and mammals, exerts its effect on DNA replication origins by recruiting protein phosphatase 1 (PP1) to chromosomes. Rif1/PP1 counteracts the positive action of the DDK kinase on the replicative kinase MCM. In a final twist, kinase action on Rif1 is proposed to eventually release PP1 and allow origin firing

DNA replication stress restricts ribosomal DNA copy number

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number

“Breaking up is hard to do”: the formation and resolution of sister chromatid intertwines

The absolute necessity to resolve every intertwine between the two strands of the DNA double helix provides a massive challenge to the cellular processes that duplicate and segregate chromosomes. Although the overwhelming majority of intertwines between the parental DNA strands are resolved during DNA replication, there are numerous chromosomal contexts where some intertwining is maintained into mitosis. These mitotic sister chromatid intertwines (SCIs) can be found as; short regions of unreplicated DNA, fully replicated and intertwined sister chromatids—commonly referred to as DNA catenation—and as sister chromatid linkages generated by homologous recombination-associated processes. Several overlapping mechanisms, including intra-chromosomal compaction, topoisomerase action and Holliday junction resolvases, ensure that all SCIs are removed before they can prevent normal chromosome segregation. Here, I discuss why some DNA intertwines persist into mitosis and review our current knowledge of the SCI resolution mechanisms that are employed in both prokaryotes and eukaryotes, including how deregulating SCI formation during DNA replication or disrupting the resolution processes may contribute to aneuploidy in cancer

DNA resection in eukaryotes: deciding how to fix the break

DNA double-strand breaks are repaired by different mechanisms, including homologous recombination and nonhomologous end-joining. DNA-end resection, the first step in recombination, is a key step that contributes to the choice of DSB repair. Resection, an evolutionarily conserved process that generates single-stranded DNA, is linked to checkpoint activation and is critical for survival. Failure to regulate and execute this process results in defective recombination and can contribute to human disease. Here, I review recent findings on the mechanisms of resection in eukaryotes, from yeast to vertebrates, provide insights into the regulatory strategies that control it, and highlight the consequences of both its impairment and its deregulation

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development

Shelterin-Like Proteins and Yku Inhibit Nucleolytic Processing of Saccharomyces cerevisiae Telomeres

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks (DSBs) by packaging them into protective structures called telomeres that prevent DNA repair/recombination activities. Here we investigate the role of key telomeric proteins in protecting budding yeast telomeres from degradation. We show that the Saccharomyces cerevisiae shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at both de novo and native telomeres during G1 and G2 cell cycle phases, with Rif2 and Rap1 showing the strongest effects. Also Yku prevents telomere resection in G1, independently of its role in non-homologous end joining. Yku and the shelterin-like proteins have additive effects in inhibiting DNA degradation at G1 de novo telomeres, where Yku plays the major role in preventing initiation, whereas Rif1, Rif2, and Rap1 act primarily by limiting extensive resection. In fact, exonucleolytic degradation of a de novo telomere is more efficient in yku70Δ than in rif2Δ G1 cells, but generation of ssDNA in Yku-lacking cells is limited to DNA regions close to the telomere tip. This limited processing is due to the inhibitory action of Rap1, Rif1, and Rif2, as their inactivation allows extensive telomere resection not only in wild-type but also in yku70Δ G1 cells. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX access to telomeres, which are also protected from the Exo1 nuclease by Yku. Thus, chromosome end degradation is controlled by telomeric proteins that specifically inhibit the action of different nucleases

Separation of DNA Replication from the Assembly of Break-Competent Meiotic Chromosomes

The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms

Sgs1 and Exo1 Redundantly Inhibit Break-Induced Replication and De Novo Telomere Addition at Broken Chromosome Ends

In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5′ to 3′ resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Δ exo1Δ strains, where there is little 5′ to 3′ resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Δ, or exo1Δ cells. In sgs1Δ exo1Δ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5′ to 3′ resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5′ to 3′ resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition