Adaptive management, international co-operation and planning for marine conservation hotspots in a changing climate

Acknowledgements This work received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland) and their support is gratefully acknowledged. MASTS is funded by the Scottish Funding Council (Grant reference HR09011) and contributing institutions.

Adaptive management, international co-operation and planning for marine conservation hotspots in a changing climate

Acknowledgements This work received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland) and their support is gratefully acknowledged. MASTS is funded by the Scottish Funding Council (Grant reference HR09011) and contributing institutions.

Quantifying avian relative abundance and ecosystem service value to identify conservation opportunities in the Midwestern U.S.

Grassland birds are highly imperiled because of historical habitat loss and ongoing conversion of grasslands to agricultural and urban land uses. Therefore, prioritizing and further justifying conservation action in remaining grasslands is critical to protecting what remains. Grassland bird conservation has focused on identifying and protecting large grassland complexes referred to as Grassland Bird Conservation Areas (GBCAs). We identified and classified GBCAs in a region highly impacted by both agricultural and urban land conversion using previously developed methods. Then, we extended the analysis to include estimated relative abundance of five grassland focal species in each GBCA. Models of relative abundance were built using eight years of monitoring data collected by citizen scientists. Finally, we quantified the value of ecosystem services provided by each GBCA. There were nearly 55,000 ha of grassland habitats in the Chicago Metropolitan Region that met GBCA criteria, 33% (18,415 ha) of which were protected. Proportion of abundance in protected versus unprotected areas was similar for Bobolink (Dolichonyx oryzivorus; 46%), Grasshopper Sparrow (Ammodramus savannarum; 52%), and Sedge Wren (Cistothorus platensis; 48%), whereas, Henslow’s Sparrow (Ammodramus henslowii; 75%) had a higher proportion of relative abundance in protected GBCAs and Eastern Meadowlark (Sturnella magna) had lower proportions (37%). GBCAs provisioned just under $900 million annually in ecosystem services, 73% of which is because of flood control. Outputs of this comprehensive approach will inform grassland bird conservation by providing detailed information about the value for birds and people of grassland habitats.

Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

Ethnogenesis and Craniofacial Change in Japan from the Perspective of Nonmetric Traits

To examine population affinities in light of the ‘dual structure model’, frequencies of 21 nonmetric cranial traits were analyzed in 17 prehistoric to recent samples from Japan and five from continental northeast Asia. Eight bivariate plots, each representing a different bone or region of the skull, as well as cluster analysis of 21-trait mean measures of divergence using multidimensional scaling and additive tree techniques, revealed good discrimination between the Jomon-Ainu indigenous lineage and that of the immigrants who arrived from continental Asia after 300 BC. In Hokkaido, in agreement with historical records, Ainu villages of Hidaka province were least, and those close to the Japan Sea coast were most, hybridized with Wajin. In the central islands, clines were identified among Wajin skeletal samples whereby those from Kyushu most resembled continental northeast Asians, while those from the northernmost prefectures of Tohoku apparently retained the strongest indigenous heritage. In the more southerly prefectures of Tohoku, stronger traces of Jomon ancestry prevailed in the cohort born during the latest Edo period than in the one born after 1870. Thus, it seems that increased inter-regional mobility and gene flow following the Meiji Restoration initiated the most recent episode in the long process of demic diffusion that has helped to shape craniofacial change in Japan.

Forward-in-Time, Spatially Explicit Modeling Software to Simulate Genetic Lineages Under Selection

SELECTOR is a software package for studying the evolution of multiallelic genes under balancing or positive selection while simulating complex evolutionary scenarios that integrate demographic growth and migration in a spatially explicit population framework. Parameters can be varied both in space and time to account for geographical, environmental, and cultural heterogeneity. SELECTOR can be used within an approximate Bayesian computation estimation framework. We first describe the principles of SELECTOR and validate the algorithms by comparing its outputs for simple models with theoretical expectations. Then, we show how it can be used to investigate genetic differentiation of loci under balancing selection in interconnected demes with spatially heterogeneous gene flow. We identify situations in which balancing selection reduces genetic differentiation between population groups compared with neutrality and explain conflicting outcomes observed for human leukocyte antigen loci. These results and three previously published applications demonstrate that SELECTOR is efficient and robust for building insight into human settlement history and evolution.

Juniperus navicularis - a study of Portuguese population structure and fitness

Juniperus navicularis Gand. is a dioecious endemic conifer that constitutes the understory of seaside pine forests in Portugal, areas currently threatened by increasing urban expansion. The aim of this study is to assess the conservation status of previously known populations of this species located on its core area of distribution. The study was performed in south-west coast of Portugal. Three populations varying in size and pine density were analyzed. Number of individuals, population density, spatial distribution and individual characteristics of junipers were estimated. Female cone, seed characteristics and seed viability were also evaluated. Results suggest that J. navicularis populations are vulnerable because seminal recruitment is scarce, what may lead to a reduction of genetic variability due solely to vegetative propagation. This vulnerability seems to be strongly determined by climatic constraints toward increasing aridity. Ratio between male and female shrubs did not differ from 1:1 in any population. Deviations from 1:1 between mature and non-mature plants were found in all populations, denoting population ageing. Very low seed viability was observed. A major part of described Juniperus navicularis populations have disappeared through direct habitat loss to urban development, loss of fitness in drier and warmer locations and low seed viability. This study is the first to address J. navicularis conservation, and represents a valuable first step toward this species preservation. 

Ecophysiology of Adonis distorta, a high-mountain species endemic of the Central Apennines

Morphological, anatomical and physiological plant and leaf traits of A. distorta, an endemic species of the Central Apennines on the Majella Massif, growing at 2,675 m a.s.l, were analyzed. The length of the phenological cycle starts immediately after the snowmelt at the end of May, lasting 128 ± 10 days. The low A. distorta height  (Hmax= 64 ± 4 mm) and total leaf area (TLA= 38 ± 9 cm2) associated to a high leaf mass area (LMA =11.8±0.6 mg cm−2) and a relatively high leaf tissue density (LTD = 124.6±14.3 mg cm−3) seem to be adaptive traits to the stress factors of the environment where it grows. From a physiological point of view, the high A. distorta photosynthetic rates (PN =19.6 ± 2.3 µmol m−2 s−1) and total chlorophyll content (Chla+b = 0.88 ± 0.13 mg g−1) in July are justified by the favorable temperature. PN decreases by 87% in September at the beginning of plant senescence. Photosynthesis and leaf respiration (RD) variations allow A. distorta to maintain a positive carbon balance during the growing season becoming indicative of the efficiency of plant carbon use. The results could be an important tool for conservation programmes of the A. distorta wild populations.

Morfometria craniana do boto (Phocoena phocoena) na costa portuguesa

Eventos climáticos extremos poderão ter conduzido à instabilidade de habitats e em última instância à sua fragmentação, causando repercussões ainda visíveis nos predadores de topo, tais como o boto (Phocoena phocoena). Consequentemente, os indivíduos que não se adaptaram, tiveram de procurar novos habitats com as respetivas condições ideais, nomeadamente os indivíduos da população de botos do Atlântico Este (norte e sul da Baía de Biscaia). O presente estudo focou-se em indivíduos da costa portuguesa (representando uma amostra da população residente a sul da Baía de Biscaia) e, recorrendo a informação proveniente de estudos previamente realizados, procurou salientar diferenças entre os botos que habitam ao longo do Atlântico Este. Foi aplicado um método de limpeza e branqueamento em crânios de boto, para posterior medição. Foram medidos 65 crânios de botos (programa Image J®), providenciados pela rede de arrojamentos de animais marinhos e obtidos no âmbito do projeto LIFE+ MarPro. As comparações das medidas dos crânios entre géneros foram realizadas a partir de modelos alométricos (programa GraphPad Prism ®) e as comparações entre géneros de cada estágio de desenvolvimento foram efetuadas com recurso a uma análise de PERMANOVA unifatorial (usando distâncias euclidianas), através do programa PAST® v. 2.12. Concluiu-se que na população em estudo, tanto as fêmeas como os machos apresentaram um modelo de alometria negativa. Reportou-se que as fêmeas apresentam comprimentos corporais superiores aos machos, assim como comprimentos e larguras cranianas. Comparativamente aos indivíduos da população a norte da Baía de Biscaia, a amostra de estudo da população do sul da Baía de Biscaia (representada pelos indivíduos da costa portuguesa) apresentou maiores comprimentos corporais e cranianos e maior largura craniana. Estes resultados corroboram observações prévias sobre a população de botos do Atlântico, que detetaram já diferenças genéticas e partilha limitada de genes, diferenças nos hábitos alimentares e habitats distintos nos botos de norte e sul da Baía de Biscaia.

Genetic viability of Nabugabo lakes (LVR satellite lakes) fish species

Natural populations of fish species in Lake Victoria Region (LVR) have under gone dramatic changes including severe reduction in sizes, division of original stocks into disjunct subunits, and segregation into several isolated population units either within a single water body or even worse into separate waters. In addition, these changes have been either preceded or precipitated by introductions of non-indigenous species that out competed the native forms and in case of closely related species genetically swamped them through hybridisation. The latter is especially the case in Nabugabo lakes. Such events lead to fragmentation of populations, which results in reduction in genetic diversity due to genetic drift, inbreeding and reduced or lack of gene flow among independent units. Such phenomena make the continued existence of fisheries stocks in the wild precarious, more so in the face of the competition from exotic species. Species introductions coupled with growing exploitation pressure of the fisheries of these lakes have put the native stocks at risk. Nabugabo lakes harbor cichlid species that are unique to these lakes more so species of the cichlid complex. In this paper the ecological status and genetic viability of key Nabugabo lakes fish species is examined and management options are discussed.

Resistance to phosphine in Sitophilus oryzae in Australia: A national analysis of trends and frequencies over time and geographical spread

In Australia, along with many other parts of the world, fumigation with phosphine is a vital component in controlling stored grain insect pests. However, resistance is a factor that may limit the continued efficacy of this fumigant. While strong resistance to phosphine has been identified and characterised, very little information is available on the causes of its development and spread. Data obtained from a unique national resistance monitoring and management program were analysed, using Bayesian hurdle modelling, to determine which factors may be responsible. Fumigation in unsealed storages, combined with a high frequency of weak resistance, were found to be the main criteria that led to the development of strong resistance in Sitophilus oryzae. Independent development, rather than gene flow via migration, appears to be primarily responsible for the geographic incidence of strong resistance to phosphine in S. oryzae. This information can now be utilised to direct resources and education into those areas at high risk and to refine phosphine resistance management strategies.

Populations genetics study of the Ammi Genus in the Azores

Dissertação de Mestrado, Biodiversidade e Biotecnologia Vegetal, 6 de Julho de 2016, Universidade dos Açores.

Uma nova abordagem do método Level Set baseada em conhecimento a e priori da forma

The analysis of fluid behavior in multiphase flow is very relevant to guarantee system safety. The use of equipment to describe such behavior is subjected to factors such as the high level of investments and of specialized labor. The application of image processing techniques to flow analysis can be a good alternative, however, very little research has been developed. In this subject, this study aims at developing a new approach to image segmentation based on Level Set method that connects the active contours and prior knowledge. In order to do that, a model shape of the targeted object is trained and defined through a model of point distribution and later this model is inserted as one of the extension velocity functions for the curve evolution at zero level of level set method. The proposed approach creates a framework that consists in three terms of energy and an extension velocity function λLg(θ)+vAg(θ)+muP(0)+θf. The first three terms of the equation are the same ones introduced in (LI CHENYANG XU; FOX, 2005) and the last part of the equation θf is based on the representation of object shape proposed in this work. Two method variations are used: one restricted (Restrict Level Set - RLS) and the other with no restriction (Free Level Set - FLS). The first one is used in image segmentation that contains targets with little variation in shape and pose. The second will be used to correctly identify the shape of the bubbles in the liquid gas two phase flows. The efficiency and robustness of the approach RLS and FLS are presented in the images of the liquid gas two phase flows and in the image dataset HTZ (FERRARI et al., 2009). The results confirm the good performance of the proposed algorithm (RLS and FLS) and indicate that the approach may be used as an efficient method to validate and/or calibrate the various existing equipment used as meters for two phase flow properties, as well as in other image segmentation problems.

Dual regulation of P-glycoprotein expression by Trichostatin A in cancer cell lines

Background. It has been reported that the histone deacetylase inhibitor (iHDAc) trichostatin A (TSA) induces an increase in MDR1 gene transcription (ABCB1). This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp). It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a traslational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation. Methods. A combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry have been used in this study. Results. The iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a traslational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5' end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used. Conclusions. The results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we have demonstrated that TSA in fact, differentially regulates both ABCB1 promoters, downregulating the upstream promoter that is responsible for active P-glycoprotein expression. These results suggest that iHDACs such as TSA may in fact potentiate the effects of antitumoral drugs that are substrates of Pgp. Finally, we have also demonstrate that TSA upregulates RUNDC3B mRNA independently of the ABCB1 promoter in use.

Avaliação de métodos de deteção de genótipos de cianobactérias produtoras de cianotoxinas em amostras ambientais

Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016