Stickleback F2 generation transgenerational plasticity experiment: egg and body size data

Nongenetic inheritance mechanisms such as transgenerational plasticity (TGP) can buffer populations against rapid environmental change such as ocean warming. Yet, little is known about how long these effects persist and whether they are cumulative over generations. Here, we tested for adaptive TGP in response to simulated ocean warming across parental and grandparental generations of marine sticklebacks. Grandparents were acclimated for two months during reproductive conditioning, whereas parents experienced developmental acclimation, allowing us to compare the fitness consequences of short-term vs. prolonged exposure to elevated temperature across multiple generations. We found that reproductive output of F1 adults was primarily determined by maternal developmental temperature, but carry-over effects from grandparental acclimation environments resulted in cumulative negative effects of elevated temperature on hatching success. In very early stages of growth, F2 offspring reached larger sizes in their respective paternal and grandparental environment down the paternal line, suggesting that other factors than just the paternal genome may be transferred between generations. In later growth stages, maternal and maternal granddam environments strongly influenced offspring body size, but in opposing directions, indicating that the mechanism(s) underlying the transfer of environmental information may have differed between acute and developmental acclimation experienced by the two generations. Taken together, our results suggest that the fitness consequences of parental and grandparental TGP are highly context dependent, but will play an important role in mediating some of the impacts of rapid climate change in this system.

Acute and long-term effects of metformin on the function and insulin secretory responsiveness of clonal β-cells

Functional effects of acute and prolonged (48 h) exposure to the biguanide drug metformin were examined in the clonal pancreatic ß-cell line, BRIN-BD11. Effects of metformin on prolonged exposure to excessive increased concentrations of glucose and palmitic acid were also assessed. In acute 20-min incubations, 12.5-50 µm metformin did not alter basal (1.1 mm glucose) or glucose-stimulated (16.7 mm glucose) insulin secretion. However, higher concentrations of metformin (100-1000 µm) increased (1.3-1.5-fold; p

Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

Adhesive bonding of aluminium

Adhesive bonding of aluminium is widely used in the aerospace industry. High initial bood strengths can be obtained, but bond failure occurs atter prolonged exposure to humid enviroments. The thesis contains details ot a test procedure which has been designed and developed for the assessment of different alloys, pretreatments, and adhesives, which will give adhesively bonded aluminium joints of high strength coupled with long term durability. The test involves assembly of lap shear specimens in a precision jig using 250 ballotini spacers in the adhesive to control the bond line thickness. The test is modified by drilling three accurately located holes through the bonded area after assembly of the joint and curing of the adhesive. Further important features at the test, such as fillet control, are detailed. The test was assessed, modified and developed to give a reliable and reproducible method which would discriminate amongst different bonding systems after exposure to humid test environments. This is the first test to have achieved the discrimination necessary for short term assessment of bond systems where long term durability is required. Even better discrimination has been obtained by applying stress in a stress humidity test. Having established accurate, reliable and discriminating test methods they were used to study the durability of structural epoxy adhesive bonds to aluminium as a function of alloy, pretreatment, adhesive and environment. It was established that the long term durability or adhesively bonded aluminium was directly related to the infulence of water migrating within the adhesive. Pretreatments differed in their ability to prevent hydration of the aluminium oxide by the water absorbed within the adhesive.

Astrocyte-neuron signalling by synaptic stimulation in the ventrobasal thalamus

In the Ventrobasal (VB) thalamus, astrocytes are known to elicit NMDA-receptor mediated slow inward currents (SICs) spontaneously in neurons. Fluorescence imaging of astrocytes and patch clamp recordings from the thalamocortical (TC) neurons in the VB of 6-23 day old Wistar rats were performed. TC neurons exhibit spontaneous SICs at low frequencies (~0.0015Hz) that were inhibited by NMDA-receptor antagonists D-AP5 (50µM), and were insensitive to TTX (1µM) suggesting a non-neuronal origin. The effect of corticothalamic (CT) and sensory (Sen) afferent stimulation on astrocyte signalling was assessed by varying stimulus parameters. Moderate synaptic stimulation elicited astrocytic Ca2+ increases, but did not affect the incidence of spontaneous SICs. Prolonged synaptic stimulation induced a 265% increase in SIC frequency. This increase lasted over one hour after the cessation of synaptic stimulation, so revealing a Long Term Enhancement (LTE) of astrocyte-neuron signalling. LTE induction required group I mGluR activation. LTE SICs targeted NMDA-receptors located at extrasynaptic sites. LTE showed a developmental profile: from weeks 1-3, the SIC frequency was increased by an average 50%, 240% and 750% respectively. Prolonged exposure to glutamate (200µM) increased spontaneous SIC frequency by 1800%. This “chemical” form of LTE was prevented by the broad-spectrum excitatory amino acid transporter (EAAT) inhibitor TBOA (300µM) suggesting that glutamate uptake was a critical factor. My results therefore show complex glutamatergic signalling interactions between astrocytes and neurons. Furthermore, two previously unrecognised mechanisms of enhancing SIC frequency are described. The synaptically induced LTE represents a form of non-synaptic plasticity and a glial “memory” of previous synaptic activity whilst enhancement after prolonged glutamate exposure may represent a pathological glial signalling mechanism.

Influence of downstream processing on the breakage of whey protein precipitates

Whey proteins may be fractionated by isoelectric precipitation followed by centrifugal recovery of the precipitate phase. Transport and processing of protein precipitates may alter the precipitate particle properties, which may affect how they behave in subsequent processes. For example, the transport of precipitate solution through pumps, pipes and valves and into a centrifugal separator may cause changes in particle size and density, which may affect the performance of the separator. This work investigates the effect of fluid flow intensity, flow geometry and exposure time on the breakage of whey protein precipitates: Computational fluid dynamics (CFD) was used to quantify the flow intensity in different geometries. Flow geometry can have a critical impact on particle breakage. Sharp geometrical transitions induce large increases in turbulence that can result in substantial particle breakage. As protein precipitate particles break, they tend to form denser more compact structures. The reduction in particle size and increase in compaction is due to breakage. This makes the particles become more resistant to further breakage as particle compactness increases. The effect of flow intensity on particle breakage is coupled to exposure time, with greater exposure time producing more breakage. However, it is expected that the particles will attain an equilibrium particle size and density after prolonged exposure in a constant flow field where no further breakage will occur with exposure time. © 2005 Institution of Chemical Engineers.

Function of lipids - their fate in contact lens wear:an interpretive review

Lipids play a vital role in the body at many interfaces. Examples include the lubrication of articulating joints by synovial fluid, the coating of the lung by pulmonary surfactant and the functions of the tear film in the protection of the anterior eye. The role of the lipids is similar at each site - acting as boundary lubricants and reducing surface and interfacial tension. This review focuses on how and why contact lens wear can disrupt the normal function of lipids within the tear film and explains how the otherwise advantageous presence and function of tear lipids can become disadvantageous, causing problems for the wearer. Because the contact lens is some ten times thicker than the tear film, lipids deposited on the anterior surface become immobilised, reducing lipid turnover and thus leading to prolonged exposure to oxygen and light with consequent generation of degradation products. These degraded lipids reduce lens wettability and have additionally been linked to problems of contact lens discomfort and intolerance. Lipid problems are influenced by the thickness of the lens, the material, surface modification, mode of wear and ultimately the subject. The most influential of these variables is frequently the subject. © 2012.

Variables affecting the collection and preservation of human scent components through instrumental and biological evaluations

In certain European countries and the United States of America, canines have been successfully used in human scent identification. There is however, limited scientific knowledge on the composition of human scent and the detection mechanism that produces an alert from canines. This lack of information has resulted in successful legal challenges to human scent evidence in the courts of law. ^ The main objective of this research was to utilize science to validate the current practices of using human scent evidence in criminal cases. The goals of this study were to utilize Headspace Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (HS-SPME-GC/MS) to determine the optimum collection and storage conditions for human scent samples, to investigate whether the amount of DNA deposited upon contact with an object affects the alerts produced by human scent identification canines, and to create a prototype pseudo human scent which could be used for training purposes. ^ Hand odor samples which were collected on different sorbent materials and exposed to various environmental conditions showed that human scent samples should be stored without prolonged exposure to UVA/UVB light to allow minimal changes to the overall scent profile. Various methods of collecting human scent from objects were also investigated and it was determined that passive collection methods yields ten times more VOCs by mass than active collection methods. ^ Through the use of polymerase chain reaction (PCR) no correlation was found between the amount of DNA that was deposited upon contact with an object and the alerts that were produced by human scent identification canines. Preliminary studies conducted to create a prototype pseudo human scent showed that it is possible to produce fractions of a human scent sample which can be presented to the canines to determine whether specific fractions or the entire sample is needed to produce alerts by the human scent identification canines. ^

4-hydroxy estradiol-induced oxidant-mediated signaling is involved in the development of breast cancer

Breast cancer is a disease associated with excess exposures to estrogens. While the mode of cancer causation is unknown, others have shown that oxidative stress induced by prolonged exposure to estrogens mediates renal, liver, endometrial and mammary tumorigenesis though the mechanism(s) underling this process is unknown. In this study, we show that 4-hydroxyl 17β-estradiol (4-OHE2), a catechol metabolite of estrogen, induces mammary tumorigenesis in a redox dependent manner. We found that the mechanism of tumorigenesis involves redox activations of nuclear respiratory factor-1 (NRF1); a transcriptions factor associated with regulation of mitochondria biogenesis and oxidative phosphorylation (OXPHOS), as well as mediation of cell survival and growth of cells during periods of oxidative stress. Key findings from our study are as follows: (i) Prolonged treatments of normal mammary epithelial cells with 4-OHE2, increased the formation of intracellular reactive oxygen species (ROS). (ii) Estrogen-induced ROS activates redox sensitive transcription factors NRF1. (iii) 4-OHE2 through activation of serine-threonine kinase and histone acetyl transferase, phosphorylates and acetylate NRF1 respectively. (iv) Redox mediated epigenetic modifications of NRF1 facilitates mammary tumorigenesis and invasive phenotypes of breast cancer cells via modulations of genes involved in proliferation, growth and metastasis of exposed cells. (v) Animal engraftment of transformed clones formed invasive tumors. (vi) Treatment of cells or tumors with biological or chemical antioxidants, as well as silencing of NRF1 expressions, prevented 4-OHE2 induced mammary tumorigenesis and invasive phenotypes of MCF-10A cells. Based on these observations, we hypothesize that 4-OHE2 induced ROS epigenetically activate NRF1 through its phosphorylation and acylation. This, in turn, through NRF1-mediated transcriptional activation of the cell cycle genes, controls 4-OHE2 induced cell transformation and tumorigenesis.^

Environmental data from coastal sediments along natural CO2 gradients at a volcanic vent in Papua New Guinea

Natural CO2 venting systems can mimic conditions that resemble intermediate to high pCO2 levels as predicted for our future oceans. They represent ideal sites to investigate potential long-term effects of ocean acidification on marine life. To test whether microbes are affected by prolonged exposure to pCO2 levels, we examined the composition and diversity of microbial communities in oxic sandy sediments along a natural CO2 gradient. Increasing pCO2 was accompanied by higher bacterial richness and by a strong increase in rare members in both bacterial and archaeal communities. Microbial communities from sites with CO2 concentrations close to today's conditions had different structures than those of sites with elevated CO2 levels. We also observed increasing sequence abundance of several organic matter degrading types of Flavobacteriaceae and Rhodobacteraceae, which paralleled concurrent shifts in benthic cover and enhanced primary productivity. With increasing pCO2, sequences related to bacterial nitrifying organisms such as Nitrosococcus and Nitrospirales decreased, and sequences affiliated to the archaeal ammonia-oxidizing Thaumarchaeota Nitrosopumilus maritimus increased. Our study suggests that microbial community structure and diversity, and likely key ecosystem functions, may be altered in coastal sediments by long-term CO2 exposure to levels predicted for the end of the century.

Active Surface Deformation Technology for Management of Marine Biofouling

Biofouling, the accumulation of biomolecules, cells, organisms and their deposits on submerged and implanted surfaces, is a ubiquitous problem across various human endeavors including maritime operations, medicine, food industries and biotechnology. Since several decades, there have been substantial research efforts towards developing various types of antifouling and fouling release approaches to control bioaccumulation on man-made surfaces. In this work we hypothesized, investigated and developed dynamic change of the surface area and topology of elastomers as a general approach for biofouling management. Further, we combined dynamic surface deformation of elastomers with other existing antifouling and fouling-release approaches to develop multifunctional, pro-active biofouling control strategies.

This research work was focused on developing fundamental, new and environment-friendly approaches for biofouling management with emphasis on marine model systems and applications, but which also provided fundamental insights into the control of infectious biofilms on biomedical devices. We used different methods (mechanical stretching, electrical-actuation and pneumatic-actuation) to generate dynamic deformation of elastomer surfaces. Our initial studies showed that dynamic surface deformation methods are effective in detaching laboratory grown bacterial biofilms and barnacles. Further systematic studies revealed that a threshold critical surface strain is required to debond a biofilm from the surface, and this critical strain is dependent on the biofilm mechanical properties including adhesion energy, thickness and modulus. To test the dynamic surface deformation approach in natural environment, we conducted field studies (at Beaufort, NC) in natural seawater using pneumatic-actuation of silicone elastomer. The field studies also confirmed that a critical substrate strain is needed to detach natural biofilm accumulated in seawater. Additionally, the results from the field studies suggested that substrate modulus also affect the critical strain needed to debond biofilms. To sum up, both the laboratory and the field studies proved that dynamic surface deformation approach can effectively detach various biofilms and barnacles, and therefore offers a non-toxic and environmental friendly approach for biofouling management.

Deformable elastomer systems used in our studies are easy to fabricate and can be used as complementary approach for existing commercial strategies for biofouling control. To this end, we aimed towards developed proactive multifunctional surfaces and proposed two different approaches: (i) modification of elastomers with antifouling polymers to produce multifunctional, and (ii) incorporation of silicone-oil additives into the elastomer to enhance fouling-release performance.

In approach (i), we modified poly(vinylmethylsiloxane) elastomer surfaces with zwitterionic polymers using thiol-ene click chemistry and controlled free radical polymerization. These surfaces exhibited both fouling resistance and triggered fouling-release functionalities. The zwitterionic polymers exhibited fouling resistance over short-term (∼hours) exposure to bacteria and barnacle cyprids. The biofilms that eventually accumulated over prolonged-exposure (∼days) were easily detached by applying mechanical strain to the elastomer substrate. In approach (ii), we incorporated silicone-oil additives in deformable elastomer and studied synergistic effect of silicone-oils and surface strain on barnacle detachment. We hypothesized that incorporation of silicone-oil additive reduces the amount of surface strain needed to detach barnacles. Our experimental results supported the above hypothesis and suggested that surface-action of silicone-oils plays a major role in decreasing the strain needed to detach barnacles. Further, we also examined the effect of change in substrate modulus and showed that stiffer substrates require lower amount of strain to detach barnacles.

In summary, this study shows that (1) dynamic surface deformation can be used as an effective, environmental friendly approach for biofouling control (2) stretchable elastomer surfaces modified with anti-fouling polymers provides a pro-active, dual-mode approach for biofouling control, and (3) incorporation of silicone-oils additives into stretchable elastomers improves the fouling-release performance of dynamic surface deformation technology. Dynamic surface deformation by itself and as a supplementary approach can be utilized biofouling management in biomedical, industrial and marine applications.

Study of insulin-like growth factor signaling in mitochondrial function

Insulin-like Growth Factor-1 (IGF-1) signalling promotes cell growth and is associated with cancer progression, including metastasis, epithelial-mesenchymal transition (EMT), and resistance to therapy. Mitochondria play an essential role in cancer cell metabolism and accumulating evidence demonstrates that dysfunctional mitochondria associated with release of mitochondrial reactive oxygen species (ROS) can influence cancer cell phenotype and invasive potential. We previously isolated a mitochondrial UTP carrier (PNC1/SLC25A33) whose expression is regulated by IGF-1, and which is essential for mitochondrial maintenance. PNC1 suppression in cancer cells results in mitochondrial dysfunction and acquisition of a profound ROS-dependent invasive (EMT) phenotype. Moreover, over-expression of PNC1 in cancer cells that exhibit an EMT phenotype is sufficient to suppress mitochondrial ROS production and reverse the invasive phenotype. This led us to investigate the IGF-1-mitochondrial signalling axis in cancer cells. We found that IGF-1 signalling supports increased mitochondrial mass and Oxphos potential through a PI3K dependant pathway. Acute inhibition of IGF-1R activity with a tyrosine kinase inhibitor results in dysfunctional mitochondria and cell death. We also observed an adaptive response to IGF-1R inhibition upon prolonged exposure to the kinase inhibitor, where increased expression of the EGF receptor can compensate for loss of mitochondrial mass through activation of PI3K/mTOR signalling. However, these cells exhibit impaired mitochondrial biogenesis and mitophagy. We conclude that the IGF-1 is required for mitochondrial maintenance and biogenesis in cancer cells, and that pharmacological inhibition of this pathway may induce mitochondrial dysfunction and may render the cells more sensitive to glycolysis-targeted drugs.

Variables Affecting the Collection and Preservation of Human Scent Components through Instrumental and Biological Evaluations

In certain European countries and the United States of America, canines have been successfully used in human scent identification. There is however, limited scientific knowledge on the composition of human scent and the detection mechanism that produces an alert from canines. This lack of information has resulted in successful legal challenges to human scent evidence in the courts of law. The main objective of this research was to utilize science to validate the current practices of using human scent evidence in criminal cases. The goals of this study were to utilize Headspace Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (HS-SPME-GC/MS) to determine the optimum collection and storage conditions for human scent samples, to investigate whether the amount of DNA deposited upon contact with an object affects the alerts produced by human scent identification canines, and to create a prototype pseudo human scent which could be used for training purposes. Hand odor samples which were collected on different sorbent materials and exposed to various environmental conditions showed that human scent samples should be stored without prolonged exposure to UVA/UVB light to allow minimal changes to the overall scent profile. Various methods of collecting human scent from objects were also investigated and it was determined that passive collection methods yields ten times more VOCs by mass than active collection methods. Through the use of polymerase chain reaction (PCR) no correlation was found between the amount of DNA that was deposited upon contact with an object and the alerts that were produced by human scent identification canines. Preliminary studies conducted to create a prototype pseudo human scent showed that it is possible to produce fractions of a human scent sample which can be presented to the canines to determine whether specific fractions or the entire sample is needed to produce alerts by the human scent identification canines.

Avaliação do potencial cancerigénico de microcistinas (cianotoxinas)

Tese de doutoramento em Farmácia (Toxicologia), apresentada à Faculdade de Farmácia da Universidade de Lisboa, 2009.

Cyto- and Genotoxicity assessment of manufactured nano cerium dioxide in the A549 cell line

In the past decades the growing application of nanomaterials (NMs) in diverse consumer products has raised various concerns in the field of toxicology. They have been extensively used in a broad range of applications and cover most of the industrial sectors as well as the medicine and the environmental areas. The most common scenarios for human exposure to NMs are occupational, environmental and as consumers and inhalation is the most frequent route of exposure, especially in occupational settings. Cerium dioxide NMs (nano-CeO2) are widely used in a number of applications such as in cosmetics, outdoor paints, wood care products as well as fuel catalysts. For such reason, nano-CeO2 is one of the selected NMs for priority testing within the sponsorship program of the Working Party of Manufactured Nanomaterials of the OECD. In this context, the aim of this study is to assess the safety of nano-CeO2 (NM-212, Joint Research Center Repository) through the characterization of its cytotoxicity and genotoxicity in a human alveolar epithelial cell line. A dispersion of the NM in water plus 0.05% BSA was prepared and sonicated during 16 minutes, according to a standardized protocol. DLS analysis was used to characterize the quality of the NM dispersion in the culture medium. To evaluate the cytotoxicity of nano-CeO2 in the A549 cell line, the colorimetric MTT assay was performed; the capacity of cells to proliferate when exposed to CeO2 was also assessed with the Clonogenic assay. The genotoxicity of this NM was evaluated by the Comet Assay (3 and 24h of exposure) to quantify DNA breaks and the FPG-modified comet assay to assess oxidative DNA damage. The Cytokinesis-Block Micronucleus (CBMN) assay was used to further detect chromosome breaks or loss. The nano-CeO2 particles are spherical, displaying a diameter of 33 nm and 28 m2/g of surface area. The results of the MTT assay did not show any decreased in cells viability following treatment with a dose-range of nano-CeO2 during 24h. Nevertheless, the highest concentrations of this NM were able to significantly reduce the colony forming ability of A549 cells, suggesting that a prolonged exposure may be cytotoxic to these cells. Data from both genotoxicity assays revealed that nano-CeO2 was neither able to induce DNA breaks nor oxidative DNA damage. Likewise, no significant micronucleus induction was observed. Taken together, the present results indicate that this nano-CeO2 is not genotoxic in this alveolar cell line under the tested conditions, although further studies should be performed, e.g., gene mutation in somatic cells and in vivo chromosome damage (rodent micronucleus assay) to ensure its safety to human health.