MicroRNA modulation of aldosterone production in the adrenal gland

Hypertension is the major risk factor for coronary disease worldwide. Primary hypertension is idiopathic in origin but is thought to arise from multiple risk factors including genetic, lifestyle and environmental influences. Secondary hypertension has a more definite aetiology; its major single cause is primary aldosteronism (PA), the greatest proportion of which is caused by aldosteroneproducing adenoma (APA), where aldosterone is synthesized at high levels by an adenoma of the adrenal gland. There is strong evidence to show that high aldosterone levels cause adverse effects on cardiovascular, cerebrovascular, renal and other systems. Extensive studies have been conducted to analyse the role that regulation of CYP11B2, the gene encoding the aldosterone synthase enzyme plays in determining aldosterone production and the development of hypertension. One significant regulatory factor that has only recently emerged is microRNA (miRNA). miRNAs are small non-coding RNAs, synthesized by a series of enzymatic processes, that negatively regulate gene expression at the posttranscriptional level. Detection and manipulation of miRNA is now known to be a viable method in the treatment, prevention and prognosis of certain diseases. The aim of the present study was to identify miRNAs likely to have a role in the regulation of corticosteroid biosynthesis. To achieve this, the miRNA profile of APA and normal human adrenal tissue was compared, as was the H295R adrenocortical cell line model of adrenocortical function, under both basal conditions and following stimulation of aldosterone production. Key differentially-expressed miRNAs were then identified and bioinformatic tools used to identify likely mRNA targets and pathways for these miRNAs, several of which were investigated and validated using in vitro methods. The background to this study is set out in Chapter 1 of this thesis, followed by a description of the major technical methods employed in Chapter 2. Chapter 3 presents the first of the study results, analysing differences in miRNA profile between APA and normal human adrenal tissue. Microarray was implemented to detect the expression of miRNAs in these two tissue types and several miRNAs were found to vary significantly and consistently between them. Furthermore, members of several miRNA clusters exhibited similar changes in expression pattern between the two tissues e.g. members of cluster miR-29b-1 (miR-29a-3p and miR-29b-3p) and of cluster miR-29b-2 (miR-29b-3p and miR-29c- 3p) are downregulated in APA, while members of cluster let-7a-1 (let-7a-5p and let-7d-5p), cluster let-7a-3 (let-7a-5p and let-7b-5p) and cluster miR-134 (miR- 134 and miR-382) are upregulated. Further bioinformatic analysis explored the possible biological function of these miRNAs using Ingenuity® Systems Pathway Analysis software. This led to the identification of validated mRNAs already known to be targeted by these miRNAs, as well as the prediction of other mRNAs that are likely targets and which are involved in processes relevant to APA pathology including cholesterol synthesis (HMGCR) and corticosteroidogenesis (CYP11B2). It was therefore hypothesised that increases in miR-125a-5p or miR- 335-5p would reduce HMGCR and CYP11B2 expression. Chapter 4 describes the characterisation of H295R cells of different strains and sources (H295R Strain 1, 2, 3 and HAC 15). Expression of CYP11B2 was assessed following application of 3 different stimulants: Angio II, dbcAMP and KCl. The most responsive strain to stimulation was Strain 1 at lower passage numbers. Furthermore, H295R proliferation increased following Angio II stimulation. In Chapter 5, the hypothesis that increases in miR-125a-5p or miR-335-5p reduces HMGCR and CYP11B2 expression was tested using realtime quantitative RT-PCR and transfection of miRNA mimics and inhibitors into the H295R cell line model of adrenocortical function. In this way, miR-125a-5p and miR-335-5p were shown to downregulate CYP11B2 and HMGCR expression, thereby validating certain of the bioinformatic predictions generated in Chapter 3. The study of miRNA profile in the H295R cell lines was conducted in Chapter 6, analysing how it changes under conditions that increase aldosterone secretion, including stimulation Angiotensin II, potassium chloride or dibutyryl cAMP (as a substitute for adrenocorticotropic hormone). miRNA profiling identified 7 miRNAs that are consistently downregulated by all three stimuli relative to basal cells: miR-106a-5p, miR-154-3p, miR-17-5p, miR-196b-5p, miR-19a-3p, miR-20b- 5p and miR-766-3p. These miRNAs include those derived from cluster miR-106a- 5p/miR-20b-5p and cluster miR-17-5p/miR-19a-3p, each producing a single polycistronic transcript. IPA bioinformatic analysis was again applied to identify experimentally validated and predicted mRNA targets of these miRNAs and the key biological pathways likely to be affected. This predicted several interactions between miRNAs derived from cluster miR-17-5p/miR-19a-3p and important mRNAs involved in cholesterol biosynthesis: LDLR and ABCA1. These predictions were investigated by in vitro experiment. miR-17-5p/miR-106a-p and miR-20b-5p were found to be consistently downregulated by stimulation of aldosterone biosynthesis. Moreover, miR-766-3p was upregulation throughout. Furthermore, I was able to validate the downregulation of LDLR by miR-17 transfection, as predicted by IPA. In summary, this study identified key miRNAs that are differentially-expressed in vivo in cases of APA or in vitro following stimulation of aldosterone biosynthesis. The many possible biological actions these miRNAs could have were filtered by bioinformatic analysis and selected interactions validated in vitro. While direct actions of these miRNAs on steroidogenic enzymes were identified, cholesterol handling also emerged as an important target and may represent a useful point of intervention in future therapies designed to modulate aldosterone biosynthesis and reduce its harmful effects.

Sensible use of primary energy in organic greenhouse production.

This document addresses the direct and indirect use of energy in European organic greenhouse horticulture (OGH) with the aim of reviewing available means for making it more environmental friendly and identifying knowledge gaps that should be addressed to attain this aim. The first observation is that there is no common regulation for energy use in OGH, which is not unexpected, since the need for climatisation is not uniformly distributed in the EU (and outside). Accordingly, the EU directive on organic agriculture does not set limitations on the use of energy, but rather promotes the responsible use of energy and of natural resources. The restrictions and rules of most private standards are slightly more stringent. Some standards have specific restrictions on the amount and sources of energy and/or on the seasonal use of energy for heating. Some standards also address processes that may affect (in)direct energy use, such as cultivation methods, mulching, lighting and growing media or substrates. However, most private standards have no or little restrictions or regulations on energy use. Accordingly, it should not surprise that very little quantitative information is available about energy use in OGH. In the present document we have filled the gaps with data with estimates drawn on energy use in conventional greenhouses. With respect to ongoing research, whereas many of the present research results about energy use and saving in conventional greenhouses are relevant (and also applied) in OGH, little research is devoted to address the energy use that is peculiar to OGH, particularly energy use for humidity control. In short, there are still a lot of knowledge gaps to improve quality and to lower energy use in organic greenhouses. The purpose of this document is a summary of present relevant knowledge about energy use and energy saving and of the perspective for improvement. In particular, the goal is to make an overview on the methods and technologies which can be used to reduce the energy use in OGH. We start from the assumption that methods and technologies that are used for reducing direct and indirect energy in conventional greenhouses can also be applied in organic greenhouses. Research on reducing energy use in conventional greenhouses is also more widely available because the area of conventional greenhouse horticulture is much larger than the area of OGH. When implementing these methods and techniques we should take into account the specific characteristics of organic agriculture like soil-based cultivation, use of organic fertilizers and the limited use of crop protection products. This document is organised as follows: first we report the results of a survey about energy use and relevant standards in the countries participating to the COST action (chapter 1); then we review the energy use for climatisation: heating (chapter 2) and humidity (chapter 3). In chapter 4 we review the available design and management means that would either reduce energy use and/or increase energy use efficiency by increasing productivity of OGH. In chapter 5 we present a short summary of existing information on indirect energy use, that is the energy required to manufacture production means (greenhouse structure and cover, fertilisers, equipment etc.) and for crop protection, particularly steaming, and briefly discuss possible savings. Finally (chapter 6) we review briefly the potential for application of renewable energy sources in OGH.

ABOVE-GROUND BIOMASS ESTIMATION OF OPUNTIA FICUS-INDICA (L.) MILL. FOR FORAGE CROP IN A MEDITERRANEAN ENVIRONMENT BY USING NON-DESTRUCTIVE METHODS

In marginal lands Opuntia ficus-indica (OFI) could be used as an alternative fruit and forage crop. The plant vigour and the biomass production were evaluated in Portuguese germplasm (15 individuals from 16 ecotypes) by non-destructive methods, 2 years following planting in a marginal soil and dryland conditions. Two Italian cultivars (Gialla and Bianca) were included in the study for comparison purposes. The biomass production and the plant vigour were estimated by measuring the cladodes number and area, and the fresh (FW) and dry weight (DW) per plant. We selected linear models by using the biometric data from 60 cladodes to predict the cladode area, the FW and the DW per plant. Among ecotypes, significant differences were found in the studied biomass-related parameters and several homogeneous groups were established. Four Portuguese ecotypes had higher biomass production than the others, 3.20 Mg ha−1 on average, a value not significantly different to the improved ‘Gialla’ cultivar, which averaged 3.87 Mg ha−1. Those ecotypes could be used to start a breeding program and to deploy material for animal feeding and fruit production.

Portuguese ecotypes of Opuntia ficus-indica differ in biomass production

The Opuntia ficus-indica (L.) Miller is a species from the Cactaceae family with the center of origin and domestication in central Mexico. This species introduction in the Iberia Peninsula occurred, probably, by the end of the 15th century, after the discovery of America, spreading later throughout the Mediterranean basin. In Portugal, O. ficus-indica is located, usually, with a typical ruderal behavior, at the edge of roads and paths. In Portugal, as in other Mediterranean regions, inlands areas are under severe draught during extensive summers, in particular, and global warming is expected to affect them deeply in the near future. O. ficus-indica, by its morpho-physiological characteristics and multiple economic uses, represent an alternative crop for those regions. Sixteen Portuguese O. ficus indica ecotypes and two ‘Italian’ cultivars ("Gialla" and "Bianca") were evaluated for plant vigor and biomass production, by nondestructive methods, in the two years following planting. Biomass production and plant vigor were measured by estimating cladode number, cladode area and fresh weight per plant. Linear models to predict the area of cladodes and fresh weight per plant were previously established using a biometric analysis of 180 cladodes. It was not possible to establish an accurate linear model for dry matter using non-destructive estimation. Significant differences were found among populations in the studied biomass-related parameters, and different groups were unfolded. A group of four Portuguese ecotypes outperformed in terms of biomass production, comparable with the “Gialla” cultivar. This group could be used to start a breeding program with the objective of deploy material for animal feeding, biomass and fruit production. Nevertheless, the ‘Gialla’ cultivar showed the best performance, achieving the highest biomass related parameters, not surprisingly for it is an improved plant material.

Production of activated carbons from single and mixtures of synthetic polymers

The production of activated carbons (ACs) involves two main steps: the carbonization of the carbonaceous of raw materials at temperatures below 1073 K in the absence of oxygen and the activation had realized at the temperature up to 1173 but the most useful temperature at 1073 K. In our study we used the most common industrial and consumer solid waste, namely PET, alone or blended with other synthetic polymer PAN. By mixing the two polymers in different ratios, an improvement of the yield of the AC production was found and some textural properties were enhanced by comparison with the AC prepared using each polymer separately. When all the samples were exposed through the carbonization process with a pyrolysis the mixture of PAN-PET (1:1w/w) yield around 31.9%, between that obtained with PET (16.9%) or PAN (42.6%) separately. The combine activation, with CO2 at 1073 K, allow ACs with a lower burn-off degree isothermally, when compared with those attained with PET or PAN alone, but with similarly chemicals or textural properties. The resultant ACs are microporous in their nature, as the activation time increase, the PET-PAN mixture AC are characterized by a better developed porous structure, when associated with the AC prepared from PAN. The AC prepared from PET-PAN mixture are characterized by basic surface characteristics, with a pHpzc around 10.5, which is an important characteristic for future applications on acidic pollutants removals from liquid or gaseous phase. In this study we had used the FTIR methods to determine the main functional groups in the surface of the activated carbons. The adsorbents prepared from PAN fibres presents an IR spectrum with similar characteristics to those obtained with PET wastes, but with fewer peaks and bands with less intensity, in particular for the PAN-8240 sample. This can be reflected by the stretching and deformation modes of NH bond in the range 3100 – 3300 cm-1 and 1520 – 1650 cm-1, respectively. Also, stretching mode associated to C–N, C=N, can contributed to the profile of IR spectrum around 1170 cm-1, 1585 – 1770 cm-1. And the TGA methods was used to study the loses of the precursors mass according to the excessive of the temperature. The results showed that, there were different decreasing of the mass of each precursors. PAN degradation started at almost 573 K and at 1073 K, PAN preserve more than 40% of the initial mass. PET degradation started at 650 K, but at 1073 K, it has lost 80% of the initial mass. However, the mixture of PET-PAN (1:1w/w) showed a thermogravimetric profile between the two polymers tested individually, with a final mass slightly less than 30%. From a chemical point of view, the carbonisation of PET mainly occurs in one step between 650 and 775 K.

Creative Practices Around the Production of Cork

Cork, as a natural product provided by the bark of the cork oak tree, is an important staple of the Portuguese economy and important to Portuguese culture. It is a sustainable product with a positive ecological footprint, from harvesting to industrial production, with the advantage of creating a local economic model through regional labour activity and distribution. Within the balance between nature-human-economy to create a sustainable system, cork production in Portugal represents a human and social dimension. By focusing on that dimension and by creating an awareness of the cultural and social impact of the activity and by re-appraising the meaning of the material within the culture, the study reframes a consideration of the actual place of labour and production. The human, geophysical, historical, social, economic, ecological and cultural aspects of the place are observed as regards their relation to work or labour in that physical space. A pilot study is being developed in the village of Azaruja in the district of Évora, Portugal. In this small locality, cork is very important in terms of the relationships between the physical subsistence of their residents and the local natural resources, because it structures the place in its cultural, social and economical dimensions. This paper outlines the theoretical foundations, the process and the outcomes of the participatory ecodesign project titled Creative Practices Around the Production of Cork which was initiated by a Portuguese artist/design researcher and developed further through the collaboration with the other two authors, one a Portuguese visual artist/researcher and the other a Turkish fashion designer/theorist. The investigation focuses on questions that expand the notion of place for artists and designers, filtered through the lenses of manual labourers in order to understand their physical, social, cultural and economic relationship with the environment. To create the process of interaction with the place and the people, a creative collaborative dynamic is developed between the authors with their range of artistic sensibilities and the local population. To adopt a holistic notion of sustainability and cultural identity a process of investigation is designated to: (1) analyse, test and interpret - through the dissemination of life stories, visual representation of the place and the creation of cork objects - the importance of culture related to the labour activity of a local natural resource that determines and structures the region; (2) to give public recognition to those involved, taking into account their sense of belonging to the place and in order to show the value of their sustainable labour activities related to local natural resources; (3) to contribute to the knowledge of the place and to its dynamism through an aesthetic approach to labour activities. With reference to fields of knowledge such as anthropology, the social arts and sustainable design, a practice-based research is conducted with collaborative and participatory design methods to create an open model of interaction which involves local people in the realization of the project. Outcomes of this research will be presented in the paper as a survey analysis with theoretical conclusions.

Mineralogical changes caused by grape production in a regosol from subtropical Brazilian climate.

Purpose Inadequate soil use and management practices promote commonly negative impacts on the soil constituents and their properties, with consequences to ecosystems. As the soil mineralogy can be permanently altered due to soil use, this approach can be used as a tool to monitor the anthropogenic pressure. The objective of the present study was to assess the mineralogical alterations of a Brazilian regosol used for grape production for 40 years in comparison with a soil under natural vegetation (forest), aiming to discuss anthropogenic pressure on soils. Material and methods Soil samples were collected at depths of 0?0.20 and 0.20?0.40 m from vineyard production and natural vegetation sites. Physical and chemical parameters were analysed by classic approaches. Mineralogical analyses were carried out on <2 mm, silt and clay fractions. Clay minerals were estimated by the relative percentage of peak surface area of the X-ray patterns. Results and discussion Grape production reduced the organic matter content by 28% and the clay content by 23% resulting in a decreasing cation exchange capacity. A similar clay fraction was observed in both soils, containing kaolinite, illite/mica and vermiculite with hydroxy-Al polymers interlayered. Neither gibbsite nor chlorite was found. However, in the soil under native vegetation, the proportion of illite (79 %) was higher than vermiculite (21 %). Whereas, in the soil used for grape production during 40 years, the formation of vermiculite was promoted. Conclusions Grape production alters the proportions of soil constituents of the regosol, reducing clay fraction and organic matter contents, as well as promoting changes in the soil clay minerals with the formation of vermiculite to the detriment of illite, which suggests weathering acceleration and susceptibility to anthropogenic pressure. Recommendations and perspectives Ecosystems in tropical and subtropical climates can be more easily and permanently altered due to anthropogenic pressure, mainly as a consequence of a great magnitude of phenomena such as temperature amplitude and rainfall that occurs in these regions. This is more worrying when soils are located on steep grades with a high anthropogenic pressure, like regosols in Southern Brazil. Thus, this study suggests that changes in soil mineralogy can be used as an important tool to assess anthropogenic pressure in ecosystems and that soil quality maintenance should be a priority in sensible landscapes to maintain the ecosystem quality.

Cross-correlations between weather variables in Australia

The information on climate variations is essential for the research of many subjects, such as the performance of buildings and agricultural production. However, recorded meteorological data are often incomplete. There may be a limited number of locations recorded, while the number of recorded climatic variables and the time intervals can also be inadequate. Therefore, the hourly data of key weather parameters as required by many building simulation programmes are typically not readily available. To overcome this gap in measured information, several empirical methods and weather data generators have been developed. They generally employ statistical analysis techniques to model the variations of individual climatic variables, while the possible interactions between different weather parameters are largely ignored. Based on a statistical analysis of 10 years historical hourly climatic data over all capital cities in Australia, this paper reports on the finding of strong correlations between several specific weather variables. It is found that there are strong linear correlations between the hourly variations of global solar irradiation (GSI) and dry bulb temperature (DBT), and between the hourly variations of DBT and relative humidity (RH). With an increase in GSI, DBT would generally increase, while the RH tends to decrease. However, no such a clear correlation can be found between the DBT and atmospheric pressure (P), and between the DBT and wind speed. These findings will be useful for the research and practice in building performance simulation.