Miracle-dz/UniFAR-FTUs huggingface dataset:
An integrated ecotoxicological assessment of the engineered nanoparticles, C60 fullerenes in different life stages of marine mussels, Mytilus edulis

Haemolymph samples and haemocytes collected via the adductor muscles of bivalve molluscs are extensively used in ecotoxicological studies. Withdrawal of haemolymph from mussels, Mytilus edulis. via the posterior adductor muscle, may lead to contamination with the intracellular contents of adductor myocytes. Lysopine dehydrogenase (LyDH) activity, an adductor myocyte marker, was used to investigate the impact of this potential contamination on levels of total glutathione, glutathione peroxidase (GPx) and acetylcholinesterase (AChE) measured in cell-free haemolymph. The mean glutathione content of cell-free haemolymph from 28 mussels was 3,2 ± 1.8 pM (mean ± S.D.), There was a linear relationship (slope = 0.28 ± 0.03 min; mean + S.E; P < 0.0001, n = 28) with haemolymph LyDH levels suggesting that at least some of the glutathione measured in cell-free haemolymph had arisen from contamination. Haemolymph LyDH activity was significantly higher in samples extracted using larger diameter needles, and also in samples where there had been some difficulty in the extraction. Exposure of mussels to oxidative stress using 40 \ig V ^ Cu for 5 days resulted in a 1.7-fold increase in glutathione (P = 0.033), but no increase (P ~ 0.810) in LyDH activity in adductor muscle. This was reflected in a similar increase in the slope of a plot (Fig. 4,2) of cell-free haemolymph glutathione versus LyDH activity (P = 0.011), consistent with both of these having originated from the adductor muscle. Cell-free haemolymph GPx and AChE activities also correlated with LyDH activity (Spearman rank correlation coefficients of 0.531 (P = 0,0068) and 0.537 (P = 0.0062), respectively, n = 27) suggesting that these also arise from contamination of the haemolymph. For 100 Chapter 4 GPx there was a significant linear relationship (P = 0.025) with haemolymph LyDH levels consistent with both enzymes originating from the myocytes. However, there was hyperbolic relationship (P = 0.0004) between haemolymph AChE and LyDH activities. It appears that this is because the AChE originates from a different compartment to the LyDH, i.e. cholinergic neuromuscular junctions in the adductor muscle. We conclude that it would be prudent, when considering the possibility of using a biomarker in cell-free haemolymph from bivalve molluscs, to check whether contamination could be an issue.
Sub-imbibed Storage: of recalcitrant seeds of four species

The seeds of Trichilia dregeana, Trichilia emetica, Podocarpus henkelii and Syzygium cuminii display the characteristics typical of recalcitrant seeds. It is the phenomena of ongoing metabolic activity and desiccation sensitivity that render them unsuitable for storage by the conventional methods used for orthodox seeds. Investigations on the storage responses of 'sub-imbibed' (partially dried) and fully hydrated seeds of all four species were carried out to study the effects of partial drying on viability and subsequent storage lifespan; i.e. to assess whether 'sub-imbibed' storage is feasible for these species. The outcome of this investigation was proposed to contribute to the resolution of the argument that storing recalcitrant seeds at lowered water contents might extend their longevity; i.e. storage at a relatively high water content but below the fully hydrated level, might prevent germination but would not be sufficient to be injurious to the seed. Seeds of T. dregeana, T. emetica, P. henkelii and S. cuminii were dried to various target moisture contents (which were determined for each species in the initial drying experiment) and then subjected to storage for 3-22 weeks at 6, 16 and 25°C (in sealed containers). In parallel, seeds of each species were stored at the shedding water content. The seeds were periodically removed for sampling, and assessed for water content, germination, respiration, electrolyte leakage and microscopical features. Storage temperature appeared to affect viability of seeds of T. emetica and T. dregeana which displayed characteristics of chilling sensitivity. Storage at 6°C was detrimental (when compared with seeds stored under the same conditions at 16 and 25°C), but regardless of whether the seeds were undried or partially dried prior to storage. The seeds of P. henkelii did not demonstrate chilling sensitivity, the viability not being compromised at 6°C compared with those seeds stored at 16 and 25°C. Syzygium cuminii seeds were not subject to storage at 6°C because previous work indicated that they would be chilling-sensitive.
Investigating the role of the Leishmania (Leishmania) major HASP and SHERP genes during metacyclogenesis in the sand fly vectors, Phlebotomus (Phlebotomus) papatasi and Ph. (Ph.) duboscqi

Leishmania parasites are the causative agents of a diverse spectrum of infectious diseases termed the leishmaniases. These digenetic parasites exist as intracellular, aflagellate amastigotes in a mammalian host and as extracellular flagellated promastigotes within phlebotomine sand fly vectors of the family Phlebotominae. Within the sand fly vector’s midgut, Leishmania has to undergo a complex differentiation process, termed metacyclogenesis, to transform from non-infective procyclic promastigotes into mammalian-infective metacyclics. Members of our research group have shown previously that parasites deleted for the L. (L.) major cDNA16 locus (a region of chromosome 23 that codes for the stage-regulated HASP and SHERP proteins) do not complete metacyclogenesis in the sand fly midgut, although metacyclic-like stages can be generated in in vitro culture (Sadlova et al. Cell. Micro.2010, 12, 1765-79). To determine the contribution of individual genes in the locus to this phenotype, I have generated a range of 17 mutants in which target HASP and SHERP genes are reintroduced either individually or in combination into their original genomic locations within the L. (L.) major cDNA16 double deletion mutant. All replacement strains have been characterized in vitro with respect to their gene copy number, correct gene integration and stage-regulated protein expression, prior to phenotypic analysis. HASPA1 was not detected in cultured promastigotes, but was expressed in mouse isolated amastigotes. Parasite mutant lines were passaged through susceptible BALB/c mice, during which HASPA2 gene containing mutant lines, in the absence of a HASPA1 gene, were shown not to develop lesions. Mouse-passaged parasites were used to infect the L. (L.) major specific sand fly vectors, Ph. (Ph.) papatasi and Ph. (Ph.) duboscqi. The progress of parasite metacyclogenesis was then monitored over twelve days, by midgut dissection and microscopy. Metacyclogenesis was not fully recovered in any of the replacement mutants tested. Surprisingly, HASPB protein expression could not be detected in the replacement mutants within the sand fly midgut, although HASPB protein was readily detected when the same parasite lines were cultured in vitro. The same was true for SHERP, although in situ expression was recovered in the presence of a HASPB gene, which itself did not expressed detectable HASPB protein levels. These observations suggest a requirement for one or multiple as-yet-unidentified regulatory component(s) for HASPB expression within the sand fly midgut and these are not required in culture. Quantitative PCR data suggested HASPB upregulation to be essential for metacyclogenesis completion, suggesting a sand fly specific function for HASPB.
Participación de la anandamida en la reabsorción embrionaria murina inducida por lipopolisacárido

La anandamida (AEA), el endocannabinoide mas abundante en el tracto reproductivo, es un importante mediador de los procesos de implantacion y desarrollo embrionario pero niveles elevados de esta molecula en el utero son perjudiciales. A su vez, el oxido nitrico (NO) y las prostaglandinas (PGs) estan involucrados en la reabsorcion embrionaria (RE) inducida por lipopolisacarido (LPS). En varios trabajos se ha descripto que el LPS modula la produccion de AEA y que esta interacciona con el NO y las PGs. Por ello, estudiamos si este endocannabinoide estaba implicado en la RE inducida por LPS en el utero y la decidua de raton durante la prenez temprana, evaluando su participacion en un modelo in vitro. Observamos que tanto en el utero como en la decidua, el LPS tiene importantes efectos sobre el sistema endocannabinoide (SEC) modulando la sintesis y la degradacion de AEA y la expresion de los receptores de estos compuestos (receptores de cannabinoides, CB). Demostramos tambien que la AEA induce su propio catabolismo en la decidua. Por otro lado, encontramos que la AEA media el efecto estimulatorio del LPS sobre la sintesis de NO y la expresion de la NO sintasa inducible (NOSi), a traves de sus receptores especificos. Tambien hallamos que la AEA participa en la modulacion que ejerce el LPS sobre el camino biosintetico de las PGs, hecho que puede contribuir a la contraccion miometrial que favorece la expulsion de los fetos en un proceso de sepsis. Se estudiaron distintas enzimas del camino biosintetico y del metabolismo de las PGs, y se observo que la AEA aumenta la expresion de la Ciclooxigenasa-2 (COX-2) mientras que inhibe la de la PGE sintasa microsomal 1 (PGESm-1) e incrementa la sintesis del metabolito de PGE2 (PGEM). Por otro lado, el efecto deletereo de la endotoxina sobre los tejidos estudiados pudo ser bloqueado en parte cuando se co-incuba con antagonistas de los receptores de cannabinoides, sugiriendo que estos compuestos son parte del mecanismo de accion del LPS.
Bioinformatics Tools in Grapevine Genomics

With the release of the grapevine genome sequence and with the increasing affordability of high throughput analysis tools, an ever increasing wealth of grapevine bioinformatics resources have been developed over the last decade in highly diverse biological ﬁ elds. The grapevine community can now access extensive databases containing information relative to molecular markers, genetic maps, the genome sequence and its annotation, gene expression, proteins and metabolites. This chapter presents how to access these resources and provides some review of their contents and use. The next challenge is the further development of systems biology for grapevine; regarding this aspect, the different tools currently available for the integration of different types of data are discussed.
Regulation of Gag trafficking during retrovirus assembly and budding

Retroviral Gag polyproteins are necessary and sufficient for virus budding, but little is known about how thousands of Gag polyproteins are transported to the budding sites. The actin cytoskeleton has long been speculated to take a role in retrovirus assembly and recent studies suggest that HIV-1 assembly is regulated as early as viral RNA nuclear export, however specific mechanisms for these regulations are unknown. In contrast to numerous studies of HIV-1 Gag assembly and budding, relatively little is reported for these fundamental pathways among animal lentiviruses. In this project, we used bimolecular fluorescence complementation (BiFC) (1) to reveal intimate (<15nm) and specific associations between EIAV Gag and actin, but not tubulin; (2) to characterize and compare assembly sites and budding efficiencies of EIAV and HIV-1 Gag in both human and rodent cells when the mRNA nuclear export context is altered to be Rev-dependent or Rev-independent; (3) to reveal co-assembly of Rev-dependent and Rev-independent HIV-1 Gag and rescued assembly of Rev-independent HIV-1 Gag in human cells by in cis provided membrane targeting signals. The results of these studies showed that (1) multimerization of EIAV Gag was required for association with filamentous actin and this association correlated with Gag budding efficiency, suggesting that association of Gag multimers with filamentous actin is important for efficient virion production; (2) HIV-1 and EIAV Gag assembled in different cellular at sites, and HIV-1 but not EIAV Gag assembly was affected by mRNA nuclear export pathways, suggesting that alternative cellular pathways can be adapted for lentiviral Gag assembly and budding; (3) Rev-independent HIV-1 Gag was deficient in lipid raft targeting and its assembly and budding could be restored by membrane targeting signals provided in trans or in cis, suggesting that raft association is critical for HIV-1 assembly and budding and is regulated as early as nuclear export of Gag-encoding mRNA. The findings presented in these studies are significant for public health because a better understanding of the mechanism of retrovirus assembly and budding increase the potential to develop novel antiviral therapies targeting this critical step in the viral life cycle.
Expected number of goals depending on intrinsic and extrinsic factors of a football player. An application to professional Spanish football league

Abstract A Bayesian regression model for the number of goals scored by players in the Spanish football league during nine seasons is fitted. The model handles overdispersion in such a way that individual footballers ability for scoring may be estimated regardless of the number of minutes played, the position in the field and the team in which they play. Additionally, the posterior predictive distributions of the fitted model allow to obtain an estimation of the performance of any player in each season with reference to the number of goals scored, locating that number as a quantile in the expected distribution of the goals scored by this player in this season. The results show how the model awards the fact that defenders and midfielders score goals because it does not expect that they score many goals and evaluates a forward more strictly in relation to the expected number of goals.
Using metabolic fingerprints to study insect-plant interactions

Metabolic fingerprinting is a biochemical method that takes an untargeted approach to measure a large number of metabolites and gain a ‘snapshot’ of an organism’s metabolome at a specific time. This thesis explores how metabolic fingerprinting can be used to study plant-insect interactions using Pieris rapae and its larval host plant species as model systems, and investigates how biotic and abiotic factors shape plant and insect metabolomes. I found that different Brassicales host plant species, as well as P. rapae larvae feeding on these plant species, had different metabolic fingerprints. A group of very abundant metabolites in the host plant Cleome spinosa were present in larvae feeding from this plant species, documenting a new occurrence of metabolite transfer between plants and insect herbivores. There was some evidence that the metabolic fingerprints of plants predicted the performance of insects, implying that the presence or absence of specific metabolites in host plants may determine the success of herbivores. Changes in metabolites measured in three host plant species following herbivory by P. rapae showed that herbivory changed the metabolic fingerprints of plants but there was little overlap in metabolites that were induced. I conclude that plants respond in a species-specific manner to herbivory, which implies that the evolution of plant defences has varied among the three species resulting in no similarities in induced metabolites. The metabolic fingerprints of the host plant Brassica oleracea as well as P. rapae larvae were changed by elevated temperature and to a lesser extent by elevated carbon dioxide (CO2). The larvae developed more quickly under elevated temperature but larval performance was not affected by elevated CO2 despite the diet of B. oleracea leaves grown under elevated CO2 containing less nitrogen. These findings provide a unique metabolite perspective of insects and plants and were facilitated by the wide breadth of metabolites studied using metabolic fingerprinting.
ROLE OF MICRORNA-363 IN HUMAN PAPILLOMAVIRUS-ASSOCIATED SQUAMOUS CELL CARCINOMA OF THE HEAD AND NECK

Squamous cell carcinoma of the head and neck (SCCHN) ranks sixth among cancers worldwide. Despite recent advances in the detection and therapy of SCCHN, the five-year survival rate remains low. Human papillomavirus (HPV) has been detected in a new class of SCCHN that has emerged in younger patients without the common risk factors of alcohol and/or tobacco. HPV-positive and HPV-negative SCCHN have different clinical characteristics, leading some to classify them as distinct diseases. High-risk HPVs have been implicated in a number of cancers including cervical, anogenital, and head and neck cancers. Micro (mi) RNAs are a recently discovered class of endogenously encoded small RNAs that most commonly function as negative regulators of gene expression. MiRNA expression profiles are often altered in cellular stress conditions, including cancers and viral infections. We hypothesized that HPV disrupts cellular miRNA expression in HPV-positive SCCHN and contributes to the development of this cancer. MiRNA microarray analysis of HPV-positive and HPV-negative SCCHN cell lines revealed the dysregulation of several miRNAs, including the upregulation of miR-363 and downregulation of miR-181a, miR-218, and miR-29a. Exogenous expression of the HPV-16 E6 oncogene in normal primary human keratinocytes showed similar changes in the above miRNAs, implicating HPV E6 in this up/downregulation. Tissues from SCCHN patients in the western Pennsylvania area showed an HPV-positivity rate of 59 percent. HPV-positive SCCHN cases were seen only in the tonsil and base of tongue, and patients with HPV-positive SCCHN were diagnosed on average nine years younger than those with HPV-negative SCCHN. Additionally, miR-363 was upregulated in HPV-16-positive SCCHN tissues compared to the HPV-negative SCCHN tissues. We utilized gene expression data and spectral counting proteomics to narrow the possible cellular targets of miR-363 in SCCHN. As confirmed via luciferase assays, miR-363 targets MYO1B, a myosin protein involved in cell motility. Functional assays showed that overexpression of miR-363 in HPV-negative SCCHN cells or siRNA knockdown of MYO1B in HPV-positive SCCHN cells independently reduced cell migration. Furthermore, the addition of miR-363 to HPV-negative SCCHN cells also caused an increase in cell proliferation and colony formation. Based on our studies, we envision that high-level expression of the E6 oncogene in HPV-positive SCCHN results in an increase in miR-363 levels. Increased expression of miR-363 may then reduce MYO1B levels, thereby reducing the migratory ability of these cells. Furthermore, high levels of miR-363 may also promote cellular proliferation and colony formation.
Cis-acting sequences involved in targeting Trm1p-II to the inner nuclear membrane in Saccharomyces cerevisiae

The mechanism of targeting a peripheral protein to the inner nuclear membrane (INM) is not understood. Sequences of Trm1p-II, a peripheral INM protein in Saccharomyces cerevisiae, were examined to define the cis-acting regions mediating targeting. Both a 9-amino acid nuclear localization sequence (NLS) targeting -galactosidase to the nuclear interior as well as a 60-amino acid membrane targeting sequence directing -galactosidase to the INM were uncovered. Trm1p-II may possess two NLSs; a classical basic NLS at amino acids 95-102 (NLS#1) and a putative NLS at amino acids 544-552 (NLS#2). NLS#1 is likely recognized by the classical importin, importin , based on amino acid sequence similarities with known importin  substrates, while NLS#2 is recognized by the non-classical importers, Nmd5p and Kap120p. Several regions of Trm1p-II are necessary for INM targeting, including the N-terminus and two internal regions, as deleting these regions releases Trm1-II-GFP from the INM into the nucleoplasm. However, a single cis-acting sequence of Trm1p-II (amino acids 89-151) is sufficient to target passenger protein(s) to the INM. Competition experiments suggest Trm1p-II may be tethered to a second nuclear component, functioning in addition to the membrane partner, to retain Trm1p-II within the nucleus. Ice2p was identified in a genome-wide screen for mutants that mislocate Trm1-II-GFP, suggesting Ice2p may have a role in targeting of Trm1p-II to the INM; deletion of ICE2 releases Trm1-II-GFP from the INM into the nucleoplasm. However, a -galactosidase fusion protein containing amino acids 73-151 of Trm1p-II is maintained at the INM in ice2 cells suggesting Ice2p may have a role in the distribution of Trm1p-II around the INM (possibly by regulating a protein(s) involved in this process) rather than functioning directly by binding Trm1p-II at the membrane. As Trm1p-II is acetylated at its N-terminus, we propose a bipartite model for targeting to the INM that requires this protein modification to expose a cis-acting sequence within amino acids 89-151 for binding Trm1p-II to its INM-binding partner. We believe Trm1p-II is targeted to a specific site at the INM and subsequently spread from this site, accounting for the variations in nuclear localization observed with different Trm1p-II fusion proteins.