Chemical analysis of toxic microalgae from Mediterranean Sea

Marine plankton is constituted by microalgae existing either in unicellular forms or in colonies of cells. These invisible organisms play a key role in the aquatic ecosystem as they are the producers of organic material thus being the first ring of the aquatic food chain. Algal proliferation in the plankton, commonly referred to as algal bloom, is a periodic phenomenon occurring in many countries across the world usually in coincidence with particular climatic and environmental events. Blooms due to microalgae producing biotoxins are known as “harmful algal blooms”, as they pose serious threats to human health. In fact, oysters, mussels, clams and in general bivalve organisms, while feeding by filtering seawater, can accumulate toxins in their edible tissues sometimes to such a level to harass unaware consumers. Hence it can be stated that bivalves constitute a crucial ring of the food chain responsible for transferring toxicity from the plankton onto humans. During my PhD within Prof. Ciminiello’s research group, I investigated the toxic profile of the Mediterranean Sea where over the past decades a number of toxic microalgae have bloomed. In particular, I focused my attention on toxic outbreaks caused by microalgae belonging to the Ostreopsis genus, quite rife along the Italian coasts. Harmful algal blooms linked to Ostreopsis spp. reached alarming proportions in the late July of 2005 and 2006, when many people required extended hospitalization for respiratory distress after exposure to marine aerosol along the beach and promenade of Genoa. During these toxic episodes Prof. Ciminiello’s research group succeeded in identifying a putative palytoxin - the most potent non-proteic toxin so far known (DL50 <100 nanograms/Kg) - as well as a new palytoxin analog, ovatoxin-a, as the two main compounds responsible for the human poisoning. This was achieved by setting up a new analytical method for detecting palytoxin based on association of liquid chromatography with mass spectrometry (LC-MS). In the wake of this research on Ostreopsis spp. I carried out in-depth studies on O. ovata toxic profile succeeding in identifying four new palytoxin-like compounds, named ovatoxin-b, -c, -d, and, -e by High Resolution (HR)LC-MS and MS/MS experiments on a hybrid Linear Ion Trap (LTQ) Orbitrap XLTM FTMS operating up to 100,000 of resolution power. On the basis of the MS data in our hands we could also give some preliminary information about their structures; however NMR mono- and bi-dimensional experiments are needed to definitively assign their chemical architecture. Since over the last years the restating of the O. ovata summer blooms has raised serious concerns to both human health and economy, from October 2006 a commission appointed by the Ministero della Salute has been in charge of investigating and monitoring Ostreopsis phenomenon along the whole Italian coasts to the aim of evaluating risks to human health and preventing any possible human intoxication. So, from the summer of 2007 on, a program of monitoring in the frame of the project “Monitoraggio Ostreopsis ovata litorale costiero Regione Campania” was started by ARPA Campania in collaboration with Stazione Zoologica A. Dohrn, Istituto Zooprofilattico Sperimentale del Mezzogiorno, and with Dipartimento di Chimica delle Sostanze Naturali, where I carried out my PhD. Therefore, every summer from 2007 to 2010, during the O. ovata blooms I carried out LC-MS analyses on samples of both mussels and sea-urchins, collected along the Campania coasts, once proven positive to the mouse bioassay. For many Campania sites, for which our analyses showed toxin contents above the EFSA (European Food Safety Authority)-established tolerance limits (30 μg/Kg), mussel harvesting was banned. Such a ban was lifted only when our analyses highlighted toxin amounts in the investigated samples well below the above official limits. Considering the deep sanitary and economic impact due to the Ostreopsis outbreaks, I also tried to set up a suitable purification procedure for ovatoxin-a – which accounts for 50% of the total toxin content - with the purpose of obtaining sufficient amount of pure compound for investigating its toxicology as well as its mechanism of action. The best results were obtained by extracting ovatoxin-a from algal pellets with methanol and methanol/water 1:1, followed by partition of the water extract with chloroform, and then by a medium pressure chromatographic separation employing a reverse stationary phase and a UV instrument as detector, and eventually by a high pressure chromatographic separation using LTQ Orbitrap XL FTMS as detector. In this frame I also analyzed O. ovata cultures coming from the Adriatic (Ancona) and the Tyrrhenian Seas (Latina) in collaboration with the University of Bologna. Pellet samples and culture mediums were collected in both exponential and stationary cellular growth phases, extracted, and finally analyzed by LC-MS to the aim of evaluating the toxins production in the different Italian strains and at the different growth levels. Our studies showed that the Adriatic strain is richer in toxins than the Tyrrhenian one, and the toxins content during the stationary growth phase is higher than that of the exponential phase. In the field of this research on palytoxins, I also studied extracts from soft corals belonging to the Palythoa genus collected in Hawaii. In particular, I carried out LC-MS analyses on a triple quadrupole mass spectrometer at unit resolution, with addiction of NaCl and KCl to the mobile phase to induce adduct ions production. LC-MS experiments were also recorded on a time of flight instrument, operating at high resolution. The whole of the above studies allowed us to individuate a new palytoxin analogue whose molecular formula (C129H224N3O55) appeared to contain an oxygen atom more than palytoxin (C129H224N3O54). NMR mono- and bi-dimensional experiments on the new palytoxin analogue identified it as 42-OH palytoxin. Another subject of my research was the investigation of the toxin profile of Alexandrium. ostenfeldii, a dinoflagellate producing spirolides, fast acting toxins with an unusual 7-membered spiro-linked cyclic imine moiety. Beyond the fact that once injected intraperitoneally into a mouse spirolides give rise to neurologic symptoms, their human toxicity is largely unknown. A. ostenfeldii samples collected in the Adriatic Sea were extracted and analyzed by LC-MS/MS that highlighted the presence of some unreported spirolides on the basis of their characteristic fragmentation pattern. Finally, NMR studies allowed to characterize two of the new spirolides as 27-OH-13-desmetil spirolide C and 27-oxo-13,19-didesmetil spirolide C

Off-Earth seismic: Viability and examination of use for measuring geophysical, geomechanical and structural properties of off-Earth bodies

Space exploration and technology development present great potential to humanity through the expansion of useful infrastructure such as satellites, space stations, launch facilities and space agencies. In addition, access to scientific opportunities such as understanding the origin of our solar system may prove valuable, and resources ranging from ice to metals and rare earth elements may be utilised to great benefit. The presence of these resources has been demonstrated on the Moon, Mars, comets, and asteroids, while the concentrations remain less certain. The risk of asteroid and comet impacts poses a great threat to life on Earth. To date, little to nothing is known about the interior of most planetary bodies. Seismic techniques are used with great success to understand the subsurface of Earth and have been proposed for expanded use in off-Earth environments, such as on the Moon, Mars, and asteroids to advance the knowledge of their interiors. The goal of this thesis is to examine the potential use of seismic techniques to explore and understand the subsurface of off-Earth environments for the purposes of resource prospecting, mining, and asteroid/comet deflection. This thesis presents a novel and innovative methodology for measuring the seismic properties of regolith and uses it to develop an understanding of the effect of the space environment on seismic data collection, such as the differing atmospheric pressure and regolith properties. The potential use of other remote sensing and geophysical techniques to assist with seismic exploration is also reviewed in addition to mission proposals. A novel testing system was designed for measuring the seismic properties of fine-grain, low compaction regolith, called the Seismic Apparatus for Fine-Grained Sediment (SAFGS). Seismic experiments were performed at UNSW Sydney, Australia and the Jet Propulsion Laboratory (JPL), Pasadena, the USA on two available off-Earth regolith simulants designed based on known off-Earth regolith properties; the Australian lunar Regolith Simulant (ALRS-1) and the Mojave Mars Simulant (MMS). ALRS-1 had a measured P-wave velocity of 98.6 m/s, comparable to the measured in-situ¬ velocity of lunar regolith (104 m/s and 114 m/s). The P-wave velocity of the MMS regolith was measured with a possible relationship between increasing grain size and velocity being found. The MMS dust had a mean velocity of 61.3 m/s, small-grain MMS had 244.5 m/s, and medium-grain MMS had 271.2 m/s. Computational and analytical modelling methods are explored to validate and expand upon the experimental work

First Finding of Ostreopsis cf. ovata Toxins in Marine Aerosols

Since the late 1990s, a respiratory syndrome has been repetitively observed in humans concomitant with Ostreopsis spp. blooms (mainly O. cf. ovata) in the Mediterranean area. Previous studies have demonstrated that O. cf. ovata produces analogues of palytoxin (ovatoxins and a putative palytoxin), one of the most potent marine toxins. On the basis of the observed association between O. cf. ovata blooms, respiratory illness in people, and detection of palytoxin complex in algal samples, toxic aerosols, containing Ostreopsis cells and/or the toxins they produce, were postulated to be the cause of human illness. A small scale monitoring study of marine aerosol carried out along the Tuscan coasts (Italy) in 2009 and 2010 is reported. Aerosols were collected concomitantly with O. cf. ovata blooms, and they were analyzed by both PCR assays and LC-HRMS. The results, besides confirming the presence of O. cf. ovata cells, demonstrated for the first time the occurrence of ovatoxins in the aerosol at levels of 2.4 pg of ovatoxins per liter of air. Given the lack of toxicological data on palytoxins by inhalation exposure, our results are only a first step toward a more comprehensiveunderstanding of the Ostreopsis-related respiratory syndrome

Teaching 13 - 16 years old newly arrived pupils : A study of how a teacher of Social studies subjects uses cooperative learning to develop both Swedish language and subject knowledge

Syftet med denna studie är att utveckla förståelse för hur en lärare som undervisar nyanlända högstadieelever arbetar med deras språk- och kunskapsutveckling genom att använda kooperativt lärande som arbetssätt i ämnesundervisningen.  Undersökningen bygger på två kvalitativa insamlingsmetoder, nämligen klassrumsobservationer och intervjuer med elever som används som ett komplement till observationerna. Det insamlade materialet analyserades utifrån en kvalitativ innehållsanalys genom kodning, kategorisering och genom att hitta mönster i materialet.  Resultatet visar att SO-läraren arbetar språk- och kunskapsutvecklande på ett medvetet sätt. Hon utgår ifrån elevernas förkunskaper när hon introducerar ett nytt arbetsområde och hon använder sig av bl.a. två kooperativa strukturer för att främja elevernas interaktion och där av främja deras språk- och kunskapsutveckling. Elevernas modersmål används systematiskt som resurs i lärandet så som studiehandledning på modersmålet. Resultatet visar också hur kamratstödet och digitala verktyg används i undervisning för att gynna elevernas språk- och kunskapsutveckling.                         Nyckelord: arbete i grupp, förberedelseklass, interaktion, kooperativt lärande, nyanlända elever, sociokulturellt perspektiv, språk- och kunskapsutvecklande arbetssätt, stöttning, svenska som andraspråk

South Australian Heat Flow Anomaly: source and implications for geothermal energy

This item is only available electronically.The South Australian Heat Flow Anomaly is a broad region (>400 km wide) in Proterozoic South Australia defined by drill holes with anomalously high heat flow estimates yielding a mean of 92 +/- 10 mW m−2, compared to a global Proterozoic mean of 49-54 mW m−2. This study will conclusively determine the primary source of this anomalous heat flow. Thermal conductivities of 145 drill core samples have been measured using an optical thermal conductivity scanner. These were utilised with thermal conductivity and temperature profiles provided by Petratherm and the Department of State Development to make five new heat flow estimates in the Curnamona and Mount Painter provinces using the product and thermal resistance methods. Measured surface heat flows fall between 84.352 and 128.051 mW m−2. Significant lateral variations in surface heat flow support previous work suggesting shallow crustal radiogenic heat generation, primarily in Mesoproterozoic high heat producing granites. Analysis of existing deep seismic data has revealed a significantly cooler and thicker lithosphere in the Proterozoic South Australia compared with regions dominated by mantle heat flow such as southeastern Australia. Geotherms have been computed for steady-state regimes to demonstrate that the surface heat flow evident in the South Australian Heat Flow Anomaly is consistent with elevated upper crustal source. Thick, thermally insulating sedimentary cover in the Curnamona and Mount Painter provinces and high temperatures at shallow depths are encouraging for geothermal energy exploration, and geothermal prospectivity for these provinces was examined. Lateral thermal conductivity variations of stratigraphies in the Curnamona Province have been assessed, revealing that more data must be collected to use thermal conductivity from neighbouring boreholes as a proxy for heat flow estimates.Thesis (B.Sc.(Hons)) -- University of Adelaide, School of Physical Sciences, 201

Teaching 13 - 16 years old newly arrived pupils : A study of how a teacher of Social studies subjects uses cooperative learning to develop both Swedish language and subject knowledge

Syftet med denna studie är att utveckla förståelse för hur en lärare som undervisar nyanlända högstadieelever arbetar med deras språk- och kunskapsutveckling genom att använda kooperativt lärande som arbetssätt i ämnesundervisningen.  Undersökningen bygger på två kvalitativa insamlingsmetoder, nämligen klassrumsobservationer och intervjuer med elever som används som ett komplement till observationerna. Det insamlade materialet analyserades utifrån en kvalitativ innehållsanalys genom kodning, kategorisering och genom att hitta mönster i materialet.  Resultatet visar att SO-läraren arbetar språk- och kunskapsutvecklande på ett medvetet sätt. Hon utgår ifrån elevernas förkunskaper när hon introducerar ett nytt arbetsområde och hon använder sig av bl.a. två kooperativa strukturer för att främja elevernas interaktion och där av främja deras språk- och kunskapsutveckling. Elevernas modersmål används systematiskt som resurs i lärandet så som studiehandledning på modersmålet. Resultatet visar också hur kamratstödet och digitala verktyg används i undervisning för att gynna elevernas språk- och kunskapsutveckling.                         Nyckelord: arbete i grupp, förberedelseklass, interaktion, kooperativt lärande, nyanlända elever, sociokulturellt perspektiv, språk- och kunskapsutvecklande arbetssätt, stöttning, svenska som andraspråk

Protecting Animals 31: Michael Dello-Iacovo from the Animal Justice Party (AJP)

This week we are joined by Michael Dello-Iacovo. Michael is a PhD student and also the Animal Justice Party (AJP) for Heffron in the upcoming NSW state election. We talk about the AJP and Michael’s political work for animals

Variability of toxin profile and content of Ostreopsis cf. ovata from the Mediterranean Sea

Blooms of Ostreopsis spp. once confined to tropical and subtropical areas have recently spread to more temperate regions such as the Mediterranean and the Southern-Atlantic coasts of Europe. In the last decade, O. confronta (cf.) ovata has become increasingly frequent with massive blooms with consequent relevant negative impacts on benthic communities and on human health through skin contact and toxic aerosols. Following the Mediterranean Ostreopsis-related outbreaks we developed a liquid chromatography-high resolution mass spectrometry (HRMS) method to be employed in the investigation of both field and cultured algal samples of O. cf. ovata. Our studies characterized O. cf. ovata as the producer of minute amounts of a putative palytoxin, one of the most potent marine toxins so far known, and of much higher amounts of several palytoxin congeners, that we named ovatoxins. Ovatoxin-a has been recently isolated and structurally elucidated, while ovatoxin-b, -c, -d, -e, and -f have not been isolated yet. Our recent research has been focused on characterization of toxin profiles of O. cf. ovata of different geographical origin that resulted to be quite different both qualitatively and quantivatively. We analyzed by LC-HRMS over 40 cultured strains of Ostreopsis cf. ovata collected along the Italian and the French Mediterranean coasts identifying basically 4 kinds of toxin profiles. In most of the strains ovatoxin-a dominates toxin profiles accounting for more than 50% of the total toxin content. But about 20% of the strains were found to produce only ovatoxin-a, d/e and putative palytoxin. Unique toxin profiles were found in a strain from Ancona (Marche, Italy) in which ovatoxin-f was the dominant toxin and a strain from Villefranche sur Mer (France) that did not produce ovatoxin-a, but just ovatoxin-b and c. These data trigger some questions on the reasons why toxin profiles and toxin content are so different as well as on toxins that need to be determined in monitoring programs of O. cf. ovata toxins in environmental and seafood sample