Induction of chromosome damage by ultraviolet light and caffeine: Correlation of cytogenetic evaluation and flow karyotype

Asynchrononously growing cells of a M3-1 Chinese hamster line were ultraviolet (UV) irradiated ( = 254 nm) with UV fluences up to 7.5 J/m2. After irradiation, cells were incubated with or without 2 mM caffeine for 20 hr, then mitotic cells were selected by mechanical shaking. Their chromosomes were isolated, stained with Hoechst 33258 and chromomycin A3, and measured flow cytometrically. While the fluorescence distributions of chromosomes (flow karyotypes) from cells treated with UV alone or with caffeine alone were very similar to those of untreated controls, the flow karyo-types of UV + caffeine-treated cells showed a debris continuum that increased with increasing UV fluence suggesting an increased number of chromosome fragments. Visual evaluation of metaphase plates revealed that the percentage of cells with chromosome damage also increased steadily with increasing UV fluence. A high degree of correlation was observed between the relative magnitude of the debris level from flow karyotypes and the percentage of cells with chromosome damage and with generalized chromosome shattering, respectively, as determined from metaphase spreads

LINKING NMDA RECEPTOR-DEPENDENT PLASTICITY AND NEURONAL ARCHITECTURE: THE ROLE OF RING FINGER PROTEIN 10

An active synapse-to-nucleus communication is essential for long-term changes in neurons, like the regulation of neuronal plasticity and shaping neuronal morphology. Next to the fast electrochemical signaling, neurons employ a slower mechanism that involves a recently discovered class of proteins, the synaptonuclear messengers. Different studies showed the pivotal role of synaptonuclear messengers in the modulation of synaptic transmission at excitatory synapses. On the other hand, alterations of synaptonuclear messengers\u2019 activity have been correlated to synaptic failure as observed in different synaptopathies, including both neurodevelopmental disorders and neurodegenerative diseases. Ring Finger Protein 10 (RNF10) has been recently identified as a novel synapse-to-nucleus signaling protein that specifically links the activation of synaptic GluN2A-containing NMDA receptors (NMDARs) to gene expression. RNF10 synaptonuclear trafficking is responsible for the remodeling of dendritic spines that substance the postsynaptic modifications required for long-term potentiation (LTP). However, the molecular mechanisms leading to NMDAR/RNF10 complex disruption and for initiating the importin-mediated trafficking of RNF10 to the nucleus remain unclear. In this PhD project we investigated the molecular mechanisms that underlie RNF10 activation and in this matter we discovered a protein kinase C (PKC)-dependent phosphorylation event on RNF10-Ser31, which drives RNF10 synaptonuclear trafficking. Moreover, we show that pSer31-RNF10 plays a role both in synaptonuclear signaling and in neuronal morphology. In particular, the prevention of Ser31 RNF10 phosphorylation induces a decrease in spine density, neuronal branching, and CREB signaling, while opposite effects are obtained by mimicking a stable RNF10 phosphorylation at Ser31.Based on these results, we investigated the role of RNF10 in vivo, in the RNF10-/- mouse model. In particular we studied the putative involvement of the synaptonuclear protein in neurodevelopment, focusing our attention on the first three weeks of postnatal life, which represents the critical period for neuronal differentiation and synaptogenesis in rodents. We found that RNF10-/- mice have an alteration in brain morphology, in particular in the hippocampal area, and impaired cognition. At a microscopic level, RNF10-/- deficiency alters the molecular composition and the morphology of the glutamatergic synapse. In the CA1 region of the Hippocampus, dendritic arborization of RNF10-/- neurons is severely reduced and LTP induction is compromised. Overall, these results add novel information about the functional and structural role of synaptonuclear protein messengers in shaping dendritic architecture and regulating synaptic plasticity in hippocampal neurons. \u200

Characterization and evaluation of the antifungal activity of antibodies raised against Candida Albicans germ tube in a rabbit model of infection and patients with invasive Candidiasis.

236 p.In the last decades, the incidence of Invasive Candidiasis has dramatically increased. Since Candida is a common component of human microbiota, the distinction between invasion and colonization is complicated. Our group of research developed an immunofluorescence assay to detect antibodies reacting specifically against the surface of the germ tubes of Candida albicans in those patients with Invasive Candidiasis (IC). These antibodies appear when an invasive process occurs and have been referred to as CAGTA. The mortality rate of ICU patients was significantly lower in CAGTA positive patients. Based on this premise, we decided to characterize the antifungal activity of anti-Candida albicans germ tube antibodies (CAGTA). In the first part of this study, we worked with the immune sera obtained from two White New Zealand female rabbits infected intravenously with C. albicans blastospores. We confirmed that both sera antibodies recognize superficial antigens from blastospores and mycelia of C. albicans. In addition, specific IgG titers increased along the infection process, after several C. albicans inoculations. Secondly, we developed several ELISA assays in order to study the kinetics of the antibodies raised in the animal model. We used the following recombinant proteins of C. albicans: 14-3-3, Adh1, Als3-N, Eno-1, Hwp1-N, Met-6, and an eluted fraction of approximately 45 kDa of a C. albicans cell wall extract (CW-Eno). According to the recorded activity, anti Adh1 and anti-Met 6 antibodies could contribute to the immunofluorescence reaction against C. albicans registered for sera of the rabbit model of IC. Contrarily, the early response of anti-Eno 1, anti-CW-Eno and anti-Hwp1 N suggest that their corresponding proteins play an important role as immunogens during the first stages of Candida infection. In addition to the previously identified antigens recognized by CAGTA, we searched for new targets of these antibodies using a cDNA library prepared in the vector Lambda Zap II from mRNA of C. albicans SC5314 growing as mycelia. The screening of the expressed proteins with the CAGTA-enr serum fraction of the IC rabbit model showed 5 clones which correspond to five C. albicans SC5314 proteins: glucose-6-p-isomerase (PGI1), hyphal wall protein (Hwp1), fatty acid synthase (FasI), pH responsive protein 1 (Phr1), and actin (Act1). The CAGTA-enr fraction from patients with IC also recognized the selected clones and therefore, these antigens could be the basis for the future development of immunization protocols that might protect against Candida infections.In the next section of this work, we measured the effect of CAGTA raised in a rabbit model of IC, on C. albicans planktonic cells as well as on the process of biofilm formation and biofilm maturation. We observed that total-IgG and CAGTA-enr IgG fraction from rabbit immune serum reduced the growth of cells in a concentration dependent manner. In addition electron microscopy images of blastospores and hyphal forms of C. albicans treated with CAGTA-enr revealed an altered surface of hyphae. Besides, fluorescent DiBAC and CFDA staining of C. albicans cells treated with CAGTA revealed that these antibodies exerted a fungicidal effect. The same results were obtained using antibodies from patients with IC caused by Candida albicans. However, when patients were infected with C. parapsilosis, C. glabrata or C. tropicalis, their CAGTA-enr serum fractions reduced C. albicans metabolic activity to a different extent, with a mean value of 50-60% inhibition compared to the untreated control. In conclusion the CAGTA-enriched fraction appears as the main responsible for the in vitro antifungal effect on viability and metabolic activity of C. albicans observed for the IC immune sera.We also analyzed the phases of biofilm formation of C. albicans following different time-based protocols of treatment with CAGTA. The presence of CAGTA only during the first 90 minutes of incubation, corresponding to the C. albicans adhesion step, resulted in a significant reduction of the biofilm biomass down to 48% while the metabolic activity remained close to the control group values. In agreement with the observed effect of CAGTA on C. albicans biofilm formation, electron micrographs of 24-hour old biofilms treated with CAGTA exhibited a reduction of the microbial structure density; in addition, fungal filaments appeared rough and with protuberances, when compared to the untreated control. In the last section of this work, the following experiments performed during a stay at Dr Naglik¿s laboratory (King¿s College, London; September-December 2016) aimed to increase our knowledge of C. albicans pathogenicity and the ability of Candidalysin peptide to promote damage and generate an immunological response. First of all, we confirm that Kex1p activity appears as an important requirement for candidalysin maturation being Candidalysin terminating in K residue the mature peptide. In addition, we observed that all Candidalysin variants used in our assays presented a dosage effect on epithelial damage induction (LDH assay). In this regard, based on the results obtained, C terminal K residue seemed not be critical for interacting with the negative charge of epithelial cells surface. These results suggest that despite being different in their amino acid sequences, the function of Candidalysin peptides is conserve

Characterization and evaluation of the antifungal activity of antibodies raised against Candida Albicans germ tube in a rabbit model of infection and patients with invasive Candidiasis.

236 p.In the last decades, the incidence of Invasive Candidiasis has dramatically increased. Since Candida is a common component of human microbiota, the distinction between invasion and colonization is complicated. Our group of research developed an immunofluorescence assay to detect antibodies reacting specifically against the surface of the germ tubes of Candida albicans in those patients with Invasive Candidiasis (IC). These antibodies appear when an invasive process occurs and have been referred to as CAGTA. The mortality rate of ICU patients was significantly lower in CAGTA positive patients. Based on this premise, we decided to characterize the antifungal activity of anti-Candida albicans germ tube antibodies (CAGTA). In the first part of this study, we worked with the immune sera obtained from two White New Zealand female rabbits infected intravenously with C. albicans blastospores. We confirmed that both sera antibodies recognize superficial antigens from blastospores and mycelia of C. albicans. In addition, specific IgG titers increased along the infection process, after several C. albicans inoculations. Secondly, we developed several ELISA assays in order to study the kinetics of the antibodies raised in the animal model. We used the following recombinant proteins of C. albicans: 14-3-3, Adh1, Als3-N, Eno-1, Hwp1-N, Met-6, and an eluted fraction of approximately 45 kDa of a C. albicans cell wall extract (CW-Eno). According to the recorded activity, anti Adh1 and anti-Met 6 antibodies could contribute to the immunofluorescence reaction against C. albicans registered for sera of the rabbit model of IC. Contrarily, the early response of anti-Eno 1, anti-CW-Eno and anti-Hwp1 N suggest that their corresponding proteins play an important role as immunogens during the first stages of Candida infection. In addition to the previously identified antigens recognized by CAGTA, we searched for new targets of these antibodies using a cDNA library prepared in the vector Lambda Zap II from mRNA of C. albicans SC5314 growing as mycelia. The screening of the expressed proteins with the CAGTA-enr serum fraction of the IC rabbit model showed 5 clones which correspond to five C. albicans SC5314 proteins: glucose-6-p-isomerase (PGI1), hyphal wall protein (Hwp1), fatty acid synthase (FasI), pH responsive protein 1 (Phr1), and actin (Act1). The CAGTA-enr fraction from patients with IC also recognized the selected clones and therefore, these antigens could be the basis for the future development of immunization protocols that might protect against Candida infections.In the next section of this work, we measured the effect of CAGTA raised in a rabbit model of IC, on C. albicans planktonic cells as well as on the process of biofilm formation and biofilm maturation. We observed that total-IgG and CAGTA-enr IgG fraction from rabbit immune serum reduced the growth of cells in a concentration dependent manner. In addition electron microscopy images of blastospores and hyphal forms of C. albicans treated with CAGTA-enr revealed an altered surface of hyphae. Besides, fluorescent DiBAC and CFDA staining of C. albicans cells treated with CAGTA revealed that these antibodies exerted a fungicidal effect. The same results were obtained using antibodies from patients with IC caused by Candida albicans. However, when patients were infected with C. parapsilosis, C. glabrata or C. tropicalis, their CAGTA-enr serum fractions reduced C. albicans metabolic activity to a different extent, with a mean value of 50-60% inhibition compared to the untreated control. In conclusion the CAGTA-enriched fraction appears as the main responsible for the in vitro antifungal effect on viability and metabolic activity of C. albicans observed for the IC immune sera.We also analyzed the phases of biofilm formation of C. albicans following different time-based protocols of treatment with CAGTA. The presence of CAGTA only during the first 90 minutes of incubation, corresponding to the C. albicans adhesion step, resulted in a significant reduction of the biofilm biomass down to 48% while the metabolic activity remained close to the control group values. In agreement with the observed effect of CAGTA on C. albicans biofilm formation, electron micrographs of 24-hour old biofilms treated with CAGTA exhibited a reduction of the microbial structure density; in addition, fungal filaments appeared rough and with protuberances, when compared to the untreated control. In the last section of this work, the following experiments performed during a stay at Dr Naglik¿s laboratory (King¿s College, London; September-December 2016) aimed to increase our knowledge of C. albicans pathogenicity and the ability of Candidalysin peptide to promote damage and generate an immunological response. First of all, we confirm that Kex1p activity appears as an important requirement for candidalysin maturation being Candidalysin terminating in K residue the mature peptide. In addition, we observed that all Candidalysin variants used in our assays presented a dosage effect on epithelial damage induction (LDH assay). In this regard, based on the results obtained, C terminal K residue seemed not be critical for interacting with the negative charge of epithelial cells surface. These results suggest that despite being different in their amino acid sequences, the function of Candidalysin peptides is conserve

Mitigating Diminishing Manufacturing Sources/Material Shortages (DMS/MS) and Obsolescence for the T-6 Canopy Fracturing Initiation System (CFIS)

The Joint Primary Aircraft Training System (JPATS) lost the supplier for its canopy fracturing initiation system (CFIS) with no prospect for a replacement. This forced a complete CFIS redesign to supply both an active production line and sustain fielded aircraft. Compounding the problem, the existing CFIS became obsolete, which forced an interim design to be produced until a final long term solution was fielded. This thesis developed a method to optimize the redesign by determining the lowest cost path for both fielding the interim design and phasing in the final retrofit. Using the Excel Solver® modeling program, an optimal rate was found to expedite interim design introduction and fleet changeover to the final design. The analysis concluded that using achievable stretch goals, existing production capacity could be adjusted to field the final configuration at the lowest cost

In Search of Lost Latin-American Colours: an Analysis of the Constitutional Protection of LGBTI Rights in Latin America

This study investigates the constitutional protection of LGBTI rights in South America and Mexico. Under the theoretical framework of Nancy Fraser’s postwestphalian democratic justice, it questions whether the constitutional protection of these rights in such countries is satisfactory in order to move forward towards the accomplishment of justice to LGBTI persons. The research conducts an empirical study and undertakes a qualitative analysis using the techniques of literature review, documental analysis, and survey. Among the results, it was determined that only two of the analysed constitutions expressly prohibits both sexual orientation – and gender identity-based discrimination. Only one of them uses gender-neutral language in the provision regarding civil union and, therefore, enables the union between two people of the same gender. Under another perspective, the answers of the majority of the Latin-American organizations in the survey indicated that the constitutional protection of LGBTI rights is unsatisfactory in their countries. Therefore, after the analysis of all the data obtained in the research, it was possible to conclude that the constitutional protection is precarious and does not guarantee the most basic rights to LGBTI population

Italy’s off shore oil referendum: another lost opportunity

On 17 April, a referendum was held in Italy on rules governing off shore drilling licenses. As Biagio Carrano and Irene Monasterolo write, this relatively niche issue nevertheless produced a full-blown political contest, involving heavy infighting inside the government coalition. They argue that these events illustrate the Italian government is still more concerned with protecting the interests of powerful industrial lobbies than it is with stimulating the economy and employment through investments in renewable energy

Near-stasis in the long-term diversification of Mesozoic tetrapods

How did evolution generate the extraordinary diversity of vertebrates on land? Zero species are known prior to ~380 million years ago, and more than 30,000 are present today. An expansionist model suggests this was achieved by large and unbounded increases, leading to substantially greater diversity in the present than at any time in the geological past. This model contrasts starkly with empirical support for constrained diversification in marine animals, suggesting different macroevolutionary processes on land and in the sea. We quantify patterns of vertebrate standing diversity on land during the Mesozoic–early Paleogene interval, applying sample-standardization to a global fossil dataset containing 27,260 occurrences of 4,898 non-marine tetrapod species. Our results show a highly stable pattern of Mesozoic tetrapod diversity at regional and local levels, underpinned by a weakly positive, but near-zero, long-term net diversification rate over 190 million years. Species diversity of non-flying terrestrial tetrapods less than doubled over this interval, despite the origins of exceptionally diverse extant groups within mammals, squamates, amphibians, and dinosaurs. Therefore, although speciose groups of modern tetrapods have Mesozoic origins, rates of Mesozoic diversification inferred from the fossil record are slow compared to those inferred from molecular phylogenies. If high speciation rates did occur in the Mesozoic, then they seem to have been balanced by extinctions among older clades. An apparent 4-fold expansion of species richness after the Cretaceous/Paleogene (K/Pg) boundary deserves further examination in light of potential taxonomic biases, but is consistent with the hypothesis that global environmental disturbances such as mass extinction events can rapidly adjust limits to diversity by restructuring ecosystems, and suggests that the gradualistic evolutionary diversification of tetrapods was punctuated by brief but dramatic episodes of radiation.27 page(s

Selected On-Demand Medical Applications of 3D-Printing for Long-Duration Manned Space Missions

Recent technological advances in the area of Additive Manufacturing (i.e. 3D printing) allow for exploration of their use within long-duration manned space missions. Among the many potential application domains, medical and dental fabrication in support of crew health is of interest to NASA’s Advanced Exploration Systems directorate. A classification of medical events with their associated response timeline discern between those applications where current 3D printing technologies can provide adequate support. Products and devices that require on-demand fabrication (due to the high level of personal customization) but that can wait for a reasonable (e.g. few hours) fabrication time are the most promising areas. Among these non-emergency, on-demand applications, two were identified for further investigation: dental health and pharmaceutical drugs. A discussion on the challenges presented by a microgravity operational environment on these technologies is provided

The potential role of kelp forests on iodine speciation in coastal seawater

Funding: FCK would like to thank the TOTAL Foundation (Paris) and the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland) for their support. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions. JG acknowledges support from an SDSU Research Foundation Summer Undergraduate Research Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD