Investigating the diversity and evolution of temperate actinobacteriophages

Bacteriophages (phages) are viruses that infect bacteria, and they have been evolving for billions of years to combat the hosts they infect as well as other phages in the environment. Many phages are temperate, and after infection they may remain in the host as a prophage, forming a lysogen instead of initiating lytic growth. Lysogeny presents unique challenges and benefits, and as a result temperate phages impact their environment differently than obligately lytic phages. Although temperate phages are diverse, many paradigms about their lifestyle and evolution have been derived from small collections of phages representing limited and ill-defined genetic diversity. Therefore, I have investigated a large collection of phages infecting hosts in the phylum Actinobacteria to expand our understanding of temperate phage diversity and evolution. First, I show that in contrast to obligately lytic phages, temperate phages evolve within two evolutionary modes that are characterized by different degrees of gene content flux and that vary by the type of temperate phage and its bacterial host. Second, I characterize extrachromosomal Mycobacterium prophages that utilize partitioning systems to maintain lysogeny, which are not commonly reported. I show that these prophages exhibit partition-mediated incompatibility and that components of this system evolve under different selective pressures possibly to avoid this instability. Third, I characterize Bifidobacterium prophages and explore how they interact with their hosts. Some of these prophages utilize a unique integration site and encode a shufflon system. This shufflon may control host range and is the first to be reported in actinobacteriophages. Last, I examine the evolution of a Mycobacterium prophage immunity system. This regulatory circuit enables prophages to control expression of lytic genes, maintaining lysogeny and defending against superinfection. I show that closely-related phages with diverging immunity systems generate a complex immunity network and gaining virulence to escape this network is difficult. Overall, this research has broadened our understanding of temperate phage diversity and evolution, and it has identified genetic systems that can be used to develop new genetic tools

Influencia de las siembras tardias, altura y temperatura sobre la capacidad productiva de la papa

Trials were made in order to study the influence of late seedings made at different heights as well of the high temperatures upon the yielding capacity of potato varieties and seedlings. Free virus tubers of the variety Katahdin propagated since their introduction in this country in cool valleys of the province of San Luis (1.700m), were used in one of the trials. Two late seedings (november and december) were made in lowlands (Kil6metro 8, 650 m) and other two in Highlands (Alta Carrera, 2.300 m) in the province of Mendoza, Argentine. A comparative test of yielding was made with the collected material. The significative difference produced by the lowland material in comparison to that of the highland was assumed as due to advances (in the physiological sense of the word) in the clones development. Such changes were due to the high mean environmental and soil temperatures of the lowlands region, which gave a higher tuberization capacity. When the development is faster, it surpasses the maximun potential capacity of the clon, generating lower yieldings (first lowland seeding in relation to the second one). That pictures the phenomenon of the ecological degeneration. It is also deducted that the clon Katahdin propagated in cool or cold zonas ( valleys of San Luis, Alta Carrara) does not develop or if so it is slow. Said zones could become centers of "seed potato" production for the variety. In other tria!, the effect of high temperatures upon tubers already sprouted of varieties (White Rose, Kennebec, Huinkul) and seedlings (B-25-5)-B; P108-49. Kennebec and Huinkul decrease their yielding capacity (42 and 16 %) with high temperatures; P-108-49 increases its yield ( 4 7%). The results were taken according to the same theoretical lineament. The practical application of the described technique in order to structuriz> methods of predicting the behaviour of material for the production of seed in the region of Cuyo, is pointed out.Se realizaron ensayos tendientes a estudiar la influencia de siemhras tardías a diferentes alturas, y de altas temperaturas sobre la capacidad productiva de variedades y "seedlings" de papa. En uno de ellos se utilizaron tubérculos libres de virus de la variedad Katahdin multiplicados, desde su introducción a la Argentina, en valles frescos de la provincia de San Luis (1700 m). Se efectuaron dos siembras tardías (noviembre y diciembre) en zonas de llanura (Kilómetro 86, 650 m) y dos en montaña (Alta Carrera, 2300 m) de la provincia de Mendoza. Con el material cosechado se realizó un ensayo comparativo de rendimiento. Las diferencias significativas producidas por material de llanura en relación al de montaña se interpretó como debidas a avances en el desarrollo ( en el sentido fisiológico del término) de los clones. Dichos cambios fueron producidos por las mayores temperaturas medias ambientales y del suelo de la región de llanura, que se tradujeron en una mayor capacidad de tuberización. Cuando el desarrollo es más rápido, excede la máxima capacidad potencial del clon generando rendimientos menores (primera siembra de llanura en relación a la segunda). Ello configura el fenómeno de degeneración ecológica. También se deduce que el clon Katahdin multiplicado en zonas frescas o frías ( valles de San Luis ; Alta Carrera) no desarrolla o lo hace lentamente. Dichas zonas pueden constituir centros de producción de "papa semilla" para la variedad. En otro ensayo se estudió el efecto de las altas temperaturas sobre tubérculos brotados de variedades (White Rose, Kennebec, Huinkul) y "seedlings" (B-25-. 50-B, P-108-49) promisorios. Kennebec y Huinkul disminuyen su capacidad productiva ( 42 y 16%) con las altas temperaturas; P-108-49 la aumenta ( 4 7 o/o) . Los resultados se interpretaron de de acuerdo a los mismos lineamientos teóricos. Se destaca la aplicación práctica de la técnica descripta para esctructurar métodos de predicción del comportamiento de material destinado a la produción de "semilla" en la región cuyana.Fil: Tizio, R..Fil: Mavrich, E.

La evaluación de los aprendizajes en el nivel superior.

Inicié mi narrativa con la sentida exposición de mi primera experiencia evaluadora  en la Universidad- experiencia que aún recuerdo como un gran atropello y una injusticia,  he relatado mi experiencia evaluadora como docente  incorporando algunos de los textos  que entendí  resultaron apoyatura científica de mi propia historia y/o  los  cambios proyectados

High-throughput sequencing reveals a simple model of nucleosome energetics

We use nucleosome maps obtained by high-throughput sequencing to study sequence specificity of intrinsic histone-DNA interactions. In contrast with previous approaches, we employ an analogy between a classical one-dimensional fluid of finite-size particles in an arbitrary external potential and arrays of DNA-bound histone octamers. We derive an analytical solution to infer free energies of nucleosome formation directly from nucleosome occupancies measured in high-throughput experiments. The sequence-specific part of free energies is then captured by fitting them to a sum of energies assigned to individual nucleotide motifs. We have developed hierarchical models of increasing complexity and spatial resolution, establishing that nucleosome occupancies can be explained by systematic differences in mono- and dinucleotide content between nucleosomal and linker DNA sequences, with periodic dinucleotide distributions and longer sequence motifs playing a secondary role. Furthermore, similar sequence signatures are exhibited by control experiments in which genomic DNA is either sonicated or digested with micrococcal nuclease in the absence of nucleosomes, making it possible that current predictions based on high-throughput nucleosome positioning maps are biased by experimental artifacts.Comment: 36 pages, 13 figure

Influencia de diferentes períodos de lavado sobre la capacidad rizogena de estacas de vid

The trial was made in order to observe, in a comparative test, the beha­viour in field conditions of vine cuttings that are easily rooted (Vitis vini­fera var. Malbeck) or difficult to root (Kobber 5 BB) which were pre­viously held under different washing periods (0, 24, 48, 72, 96 and 120 hours) o evaluate the method as agricultural practice to obtain rooted cuttings. The results obtained in percentages of rooting for the variety Malbeck were higly significatives in treatments of 24 and 48 hours as well as in those periods of 48 and 96 hours for the variety Kobber. In regard to the number of roots for each rooted cutting the results were also significatives. As it hapened in other trials (Tizio et al, 1961; 1963) it is believed that not only stimulation is due to the leaching of the inhibitory substances but also to the growing factors originated, apparently, from auxinic precursors. It is pointed out the usefulness of the washing technique practique to obtain rooted cuttings.El ensayo tuvo por objeto observar, en ensayo comparativo, el comportamiento a campo de estacas de vid relativamente fáciles ( Vitis vinífera var. Malbeck) y difíciles (Kobber 5 BB) de enraizar, sometidas previamente a diferentes períodos de lavado ( O, 24, 48, 72, 96 y 120 horas), a fin de evaluar dicho método como práctica agrícola de obtención de barbechos. Resultados significativos relativos al porcentaje de enraizamiento se alcanzaron en Malbeck con tratamientos de 24 y 48 horas, y con 48 y 96 horas en Kobber. También se lograron resultados significativos en cuanto al número de raíces por estaca enraizada. Como en casos anteriores (Tizio et al., 1961, a, 1963). Se deduce que dicha estimulación se debe, no sólo a la lixiviación de sustancias inhibidoras, sino a la acción de factores de crecimiento aparentemente originados a partir de precursores auxínicos. Se destaca la utilidad de la aplicación práctica de la técnica de lavado para la obtención de barbechos.Fil: Pons, Almela G..Fil: Tizio, R..Fil: Mavrich, E.

Small Renal Masses: Incidental Diagnosis, Clinical Symptoms, and Prognostic Factors

Introduction. The small renal masses (SRMs) have increased over the past two decades due to more liberal use of imaging techniques. SRMs have allowed discussions regarding their prognostic, diagnosis, and therapeutic approach. Materials and methods. Clinical presentation, incidental diagnosis, and prognosis factors of SRMs are discussed in this review. Results. SRMs are defined as lesions less than 4 cm in diameter. SRM could be benign, and most malignant SMRs are low stage and low grade. Clinical symptoms like hematuria are very rare, being diagnosed by chance (incidental) in most cases. Size, stage, and grade are still the most consistent prognosis factors in (RCC). An enhanced contrast SRM that grows during active surveillance is clearly malignant, and its aggressive potential increases in those greater than 3 cm. Clear cell carcinoma is the most frequent cellular type of malign SRM. Conclusions. Only some SRMs are benign. The great majority of malign SRMs have good prognosis (low stage and grade, no metastasis) with open or laparoscopic surgical treatment (nephron sparing techniques). Active surveillance is an accepted attitude in selected cases

NOrMAL: accurate nucleosome positioning using a modified Gaussian mixture model

Motivation: Nucleosomes are the basic elements of chromatin structure. They control the packaging of DNA and play a critical role in gene regulation by allowing physical access to transcription factors. The advent of second-generation sequencing has enabled landmark genome-wide studies of nucleosome positions for several model organisms. Current methods to determine nucleosome positioning first compute an occupancy coverage profile by mapping nucleosome-enriched sequenced reads to a reference genome; then, nucleosomes are placed according to the peaks of the coverage profile. These methods are quite accurate on placing isolated nucleosomes, but they do not properly handle more complex configurations. Also, they can only provide the positions of nucleosomes and their occupancy level, whereas it is very beneficial to supply molecular biologists additional information about nucleosomes like the probability of placement, the size of DNA fragments enriched for nucleosomes and/or whether nucleosomes are well positioned or ‘fuzzy’ in the sequenced cell sample

Nucleosome-mediated cooperativity between transcription factors

Cooperative binding of transcription factors (TFs) to cis-regulatory regions (CRRs) is essential for precision of gene expression in development and other processes. The classical model of cooperativity requires direct interactions between TFs, thus constraining the arrangement of TFs sites in a CRR. On the contrary, genomic and functional studies demonstrate a great deal of flexibility in such arrangements with variable distances, numbers of sites, and identities of the involved TFs. Such flexibility is inconsistent with the cooperativity by direct interactions between TFs. Here we demonstrate that strong cooperativity among non-interacting TFs can be achieved by their competition with nucleosomes. We find that the mechanism of nucleosome-mediated cooperativity is mathematically identical to the Monod-Wyman-Changeux (MWC) model of cooperativity in hemoglobin. This surprising parallel provides deep insights, with parallels between heterotropic regulation of hemoglobin (e.g. Bohr effect) and roles of nucleosome-positioning sequences and chromatin modifications in gene regulation. Characterized mechanism is consistent with numerous experimental results, allows substantial flexibility in and modularity of CRRs, and provides a rationale for a broad range of genomic and evolutionary observations. Striking parallels between cooperativity in hemoglobin and in transcription regulation point at a new design principle that may be used in range of biological systems

Quantitative test of the barrier nucleosome model for statistical positioning of nucleosomes up- and downstream of transcription start sites

The positions of nucleosomes in eukaryotic genomes determine which parts of the DNA sequence are readily accessible for regulatory proteins and which are not. Genome-wide maps of nucleosome positions have revealed a salient pattern around transcription start sites, involving a nucleosome-free region (NFR) flanked by a pronounced periodic pattern in the average nucleosome density. While the periodic pattern clearly reflects well-positioned nucleosomes, the positioning mechanism is less clear. A recent experimental study by Mavrich et al. argued that the pattern observed in S. cerevisiae is qualitatively consistent with a `barrier nucleosome model', in which the oscillatory pattern is created by the statistical positioning mechanism of Kornberg and Stryer. On the other hand, there is clear evidence for intrinsic sequence preferences of nucleosomes, and it is unclear to what extent these sequence preferences affect the observed pattern. To test the barrier nucleosome model, we quantitatively analyze yeast nucleosome positioning data both up- and downstream from NFRs. Our analysis is based on the Tonks model of statistical physics which quantifies the interplay between the excluded-volume interaction of nucleosomes and their positional entropy. We find that although the typical patterns on the two sides of the NFR are different, they are both quantitatively described by the same physical model, with the same parameters, but different boundary conditions. The inferred boundary conditions suggest that the first nucleosome downstream from the NFR (the +1 nucleosome) is typically directly positioned while the first nucleosome upstream is statistically positioned via a nucleosome-repelling DNA region. These boundary conditions, which can be locally encoded into the genome sequence, significantly shape the statistical distribution of nucleosomes over a range of up to ~1000 bp to each side.Comment: includes supporting materia

Preferential Nucleosome Occupancy at High Values of DNA Helical Rise

Nucleosomes are the basic structural units of eukaryotic chromatin and play a key role in the regulation of gene expression. Nucleosome formation depends on several factors, including properties of the sequence itself, but also physical constraints and epigenetic factors such as chromatin-remodelling enzymes. In this view, a sequence-dependent approach is able to capture a general tendency of a region to bind a histone octamer. A reference data set of positioned nucleosomes of Saccharomyces cerevisiae was used to study the role of DNA helical rise in histone–DNA interaction. Genomic sequences were transformed into arrays of helical rise values by a tetranucleotide code and then turned into profiles of mean helical rise values. These profiles resemble maps of nucleosome occupancy, suggesting that intrinsic histone–DNA interactions are linked to helical rise. The obtained results show that preferential nucleosome occupancy occurs where the mean helical rise reaches its largest values. Mean helical rise profiles obtained by using maps of positioned nucleosomes of the Drosophila melanogaster and Plasmodium falciparum genomes, as well as Homo sapiens chromosome 20 confirm that nucleosomes are mainly located where the mean helical rise reaches its largest values