Two separate but related projects make up the work of this thesis. The growing amount of sequence data available in public databases provides an opportunity to compare species in new ways. It can be shown that there is a systematic change in amino acid composition in a dataset of sequences from 69 species possessing a range of optimal growth temperatures. By creating a phylogenetic tree of all available Archaea, pairs may be selected that contain a relatively closely related mesophile and (hyper)thermophile. In addition, pairs may be selected from Bacteria to include psychrophiles as well as other thermophiles. An evolutionary model is derived here that detects amino acid asymmetries in these species pairs beyond what might be expected to be caused by differences in GC content. This amino acid asymmetry can then be plausibly explained by temperature adaptation occurring in these species since they diverged from a common ancestor. In the second part, similarity searches using molecular sequences are drawn as networks, where open reading frames in one species may be linked to a corresponding sequence in another species if the similarity search score is above a given threshold. This process is similar to that used to identify orthologous sequences for use in evolutionary models. When drawn as a network of distinct clusters of similarity, patterns emerge that can be spurious or have some biological relevance. This work identifies the need to develop better methods of analyzing these network clusters. / Thesis / Master of Science (MSc)

This inter-disciplinary thesis is concerned with the production of polyunsaturated fatty acids (PUFAs) from newly isolated and identified alpine microalgae, and the optimization of the temperature, photon flux density (PFD), and carbon dioxide (CO2) concentration for their mass production in an airlift photobioreactor (AL-PBR). Thirteen strains of microalgae were isolated from the alpine zone in Canyon Creek, Canterbury, New Zealand. Ten species were characterized by traditional means, including ultrastructure, and subjected to phylogenetic analysis to determine their relationships with other strains. Because alpine algae are exposed to extreme conditions, and such as those that favor the production of secondary metabolites, it was hypothesized that alpine strains could be a productive source of PUFAs. Fatty acid (FA) profiles were generated from seven of the characterized strains and three of the uncharacterized strains. Some taxa from Canyon Creek were already identified from other alpine and polar zones, as well as non-alpine zones. The strains included relatives of species from deserts, one newly published taxon, and two probable new species that await formal naming. All ten distinct species identified were chlorophyte green algae, with three belonging to the class Trebouxiophyceae and seven to the class Chlorophyceae. Comparative study between the distribution of algae at Canyon Creek and Mount Philistine, another alpine region in New Zealand where algal distribution was studied in detail, revealed that algal distribution patterns in the New Zealand alpine zone are complex, with some taxa apparently widely distributed and others range restricted or rare (with the caveat that very few sites have been studied in detail). At least some of the differences between the two sites could be accounted for by geographic differences, resulting in contrasting environmental conditions such as rainfall. As hypothesized, alpine strains isolated from the Canyon Creek were rich in PUFAs. Eight among the ten strains have PUFA proportions higher than monounsaturated fatty acids and saturated FAs. In a comparison of FA profiles of Scenedesmaceae species from a hot environment (Algerian Sahara) with the Scenedesmaceae species from Canyon Creek, the latter revealed a much greater degree of unsaturation. In addition, the Canyon Creek strains contained some FAs (such as docosapentaenoic acid, DPA) that were absent from Saharan strains. Among the strains from Canyon Creek Lobochlamys segnis LCR-CC-5-1A was selected for optimization experiments on the basis of growth kinetics, temperature response and FA composition, of which 60% of total FAs were PUFAs. Of that 60%, the α-linolenic acid (ALA) content was 46%. Two identical 1.5 Liter AL-PBRs were used for culturing Lobochlamys segnis LCR-CC-5-1A to study the effect of CO2 concentration, PFD and temperature on specific growth velocity, production of PUFAs, omega-3 FAs and, specifically, the concentration of ALA. The concentrations of CO2 examined in this research were 1.5, 3.0 and 4.5% in air. Similarly, the reponses of the strain to seven different PFDs, namely 38, 77, 115, 178, 210, 236 and 253 µmol m-2 s-1 and six different temperatures, 5, 10, 15, 20, 25 and 30οC, were analyzed. The maximum specific growth velocities (µmax) of the cultures were calculated from the experimental data and the cell production rate was calculated from fitting logistic growth models to these data; the two were compared by converting the former to the latter. The significance of the tested parameters was assessed using ANOVA and Tukey tests. The optimum conditions assessed at lab scale for maximum production of biomass, PUFAs and ALA were found to be a CO2 concentration of 3.0%, temperature of 20°C, and PFD of 178 µmol m-2 s-1. Increasing biomass production has the effect of maximizing PUFA production because there was no significant increase in concentration of PUFAs, omega-3 FAs, or ALA under levels of CO2, temperature, and PFD differing from those under which maximum growth occurred.

No description available.

<p> The search for life on other planets has brought with it a renewed interest in the study of extremophiles as it relates to cold-tolerance. The means for the elucidation of these studies has been through the use of analog-sites in the polar regions of Earth as approximations of extra-planetary environments. These extreme environments are typified by low annual temperatures, low levels of available liquid water, food and light. Despite these conditions, however, life prevails. Perhaps one of the most "extreme" organisms residing in these environments are tardigrades. These micrometazoans are capable of withstanding temperature extremes from 150°C to -276°C, pressure, X-ray radiation, dessication and salinity. Of relevance to this thesis is the ability of the tardigrade to withstand extreme low temperature, which they do by entering a cryobiotic, or latent state. Cryobiosis is seen as an extreme form of cold-tolerance, but apparently lacking a lower lethal temperature (LL T). Despite the incredible advantages that this strategy confers, cryobiosis remains poorly understood. This study provides a review of the literature on freeze tolerance and cryobiosis to bridge the two spheres of research, as well as clarifying the nomenclature used in these papers. Particular attention is paid to the terms of cryoprotective dehydration and cryobiosis, proposing that cryoprotective dehydration be thought of instead as a process leading to the latent state. Experiments were conducted to explore the relationship that time and temperature have on cryobiotic capability in the tardigrade Macrobiotus harmsworthii. Results showed that both time and temperature played a significant role above -80°C, with poor survivability at -20°C. At -80°C, however, time no longer appeared to play a role in viability. </p> / Thesis / Master of Science (MSc)

For many species, distribution and abundance is driven by a trade-off between abiotic and biotic stress tolerance (i.e. physical stress versus competition or predation stress). This trade- off may be caused by metabolic rate differences in species such that slow metabolic rates increase abiotic tolerance but decrease biotic tolerance. I investigated how metabolic rate differences were responsible for an abiotic-biotic tolerance trade-off in brown mudfish (Neochanna apoda) and banded kokopu (Galaxias fasciatus), that drives the allopatric distribution of these fish in podocarp swamp-forest pools. Brown mudfish and banded kokopu distribution across 65 forest pools in Saltwater forest, Westland National Park, New Zealand was almost completely allopatric. Mudfish were restricted to pools with extreme abiotic stress including hypoxia, acidity and droughts because of kokopu predation in benign pools. This meant the mudfish realised niche was only a small fraction of their large fundamental niche, which was the largest out of sixteen freshwater fish species surveyed in South Island West Coast habitats. Thus mudfish had a large fundamental to realised niche ratio because of strong physiological stress tolerance but poor biotic stress tolerance compared to other fish. A low metabolic capacity in mudfish compared to kokopu in terms of resting and maximum metabolic rates and aerobic scope explained the strong mudfish tolerance to extreme abiotic stress, but also their sensitivity to biotic stress by more powerful kokopu in benign pools, and hence their allopatric distribution with kokopu. Despite being restricted to extreme physical stress, mudfish populations were, in fact, more dense than those of kokopu, because of low individual mudfish resting metabolic rates, which would cause resources to be divided over more individuals. Distribution and abundance in mudfish and kokopu were therefore driven by an abiotic-biotic tolerance trade-off caused by a physiological trade-off between having slow or fast metabolic rates, respectively. The negative relationship between species resting metabolic rates and their tolerance to abiotic stress provides a way of estimating the impact of human induced environmental change that can either increase or decrease habitat harshness. Thus species with low metabolic rates, like mudfish, will be negatively affected by human induced environmental change that removes abiotic habitat stress and replaces it with benign conditions. My evidence shows that extreme stressors provide a protective habitat supporting high mudfish biomass with significant conservation value that should be maintained for the long-term persistence of mudfish populations.

No / The Raman spectroscopic biosignatures of halotrophic cyanobacterial extremophiles from sabkha evaporitic saltpans are reported for the first time and ideas about the possible survival strategies in operation have been forthcoming. The biochemicals produced by the cyanobacteria which colonise the interfaces between large plates of clear selenitic gypsum, halite, and dolomitized calcium carbonates in the centre of the salt pans are identifiably different from those which are produced by benthic cyanobacterial mats colonising the surface of the salt pan edges in the intertidal zone. The prediction that similar geological formations would have been present on early Mars and which could now be underlying the highly peroxidised regolith on the surface of the planet has been confirmed by recent satellite observations from Mars orbit and by localised traverses by robotic surface rovers. The successful adoption of miniaturised Raman spectroscopic instrumentation as part of a scientific package for detection of extant life or biomolecular traces of extinct life on proposed future Mars missions will depend critically on interpretation of data from terrestrial Mars analogues such as sabkhas, of which the current study is an example.

No / The remote robotic exploration of extraterrestrial scenarios for evidence of biological colonization in 'search for life' missions using Raman spectroscopy is critically dependent on two major factors: firstly, the Raman spectral recognition of characteristic biochemical spectral signatures in the presence of mineral matrix features; and secondly, the positive unambiguous identification of molecular biomaterials which are indicative of extinct or extant life. Both of these factors are considered here: the most important criterion is the clear definition of which biochemicals truly represent biomarkers, whose presence in the planetary geological record from an analytical astrobiological standpoint will unambiguously be indicative of life as recognized from its remote instrumental interrogation. Also discussed in this paper are chemical compounds which are associated with living systems, including biominerals, which may not in themselves be definitive signatures of life processes and origins but whose presence provides an indicator of potential life-bearing matrices.

No / Recent studies relating to the analytical chemical characterization of terrestrial extremophiles reveal the presence of biomolecules that have been synthesized for the survival of the colonies in response to the extreme environmental conditions, where otherwise life could not exist. This is a vital part of the planned space missions now being undertaken to planets and their satellites in the search for extinct or extant life signatures in our Solar System. Extremophiles have existed on the Earth for some 3.8 Gyr and their interrogation indicates their strategic survival methods which can be associated and compared with extraterrestrial scenarios on Mars, Titan, Enceladus and Europa.

O principal objetivo do trabalho foi estimar a possibilidade de sobrevivência de micro-organismos extremófilos na superfície de exoplanetas conhecidos, assim como na superfície de seus eventuais satélites naturais. Foi utilizado um modelo que simula a atmosfera terrestre primordial, composta principalmente por nitrogênio, água e dióxido de carbono. E em se tratando de extremófilos, esses cálculos não foram limitados à Zona Habitável dos sistemas planetários, pois esse conceito foi estendido para uma região mais ampla, a Zona Extremófila, onde a vida pode existir. Extremófilos são micro-organismos terrestres que vivem sob condições extremas de temperatura, nível de radiação, umidade, pressão, salinidade, pH, etc. . Eles são candidatos naturais para habitarem meios ditos extraterrestres onde tais condições são eventualmente encontradas. Alguns exemplos desses ambientes em nosso sistema solar são: Marte, Titã (satélite de Saturno) e Europa (satélite de Júpiter). Há algumas centenas de planetas orbitando outras estrelas (exoplanetas) e a maioria deles são gigantes gasosos, em particular Hot Jupiters. A temperatura superficial de um planeta depende fortemente de seu albedo, de sua distância orbital, de condições geodinâmicas intrínsecas, além do tipo espectral de sua estrela hospedeira. A estimativa dessa temperatura foi obtida considerando o ciclo silicato-carbono e um balanço de energia global, que contribuiram para se obter estimativas da pressão parcial atmosférica devido ao dióxido de carbono e da temperatura média, na superfície dos planetas e/ou de seus satélites hipotéticos. Os eventuais satélites naturais de planetas gigantes podem abrigar vida e essa possibilidade foi testada através da análise das condições de estabilidade orbital desses corpos celestes. Os resultados deste trabalho deverão fornecer subsídios para a hipótese da panspermia. / The main objective of this study is to estimate the chance of survival of microorganisms (extremophiles) on the surface of known exoplanets, as well as on the surface of its potential natural satellites. We used a model that simulates the primordial atmosphere composed by, primarily, nitrogen, water and carbon dioxide. And when it comes to extremophiles, these calculations were not limited to the Habitable Zone of planetary systems, since this concept was extended to a wider region, the Extremophile Zone, where life can exist. Extremophiles are terrestrial microorganisms living under extreme conditions of temperature, light level, humidity, pressure, salinity, pH, etc ... They are natural candidates for living in habitats considered extraterrestrials where such conditions are encountered eventually. Examples of such environments in our solar system are: Mars, Titan (moon of Saturn) and Europe (satellite of Jupiter). There are hundreds of planets orbiting other stars (exoplanets) and most of them are gas giants, particularly Hot Jupiters. The surface temperature of a planet/moon depends heavily on its albedo, its orbital distance, of geodynamic conditions intrinsic, in addition to the spectral type of their host star. The estimate of this temperature was obtained considering the carbon-silicate cycle and a global energy balance, which contributed to obtain estimates of the partial pressure due to atmospheric CO2 and the average temperature on the surface of planets and/or their hypothetical satellites. Natural satellites of giant planets may harbor life, and this possibility was tested by analyzing the conditions of orbital stability of these heavenly bodies. The results of this study should provide support for the hypothesis of panspermia.

The marine bacterium Sphingopyxis alaskensis was isolated as one of the most numerically abundant bacteria from cold (4–10??C) nutrient depleted waters in the North Pacific Ocean. The objective of this study was to examine cold adaptation of S. alaskensis by using proteomics to examine changes in global protein levels caused by growth at low (10??C) and high (30??C) temperatures. Stable isotope labelling-based quantitative proteomics was used, and a rigorous post-experimental data processing workflow adapted from microarray-based methods was developed. The approach included metabolic labelling with 14N/15N and normalisation and statistical testing of quantitative proteomics data. Approximately 400,000 tandem mass spectra were generated resulting in the confident identification of 2,135 proteins (66% genome coverage) and the quantitation of 1,172 proteins (37% genome coverage). Normalisation approaches were evaluated using cultures grown at 30??C and labelled with 14N and 15N. For 10??C vs. 30??C experiments, protein quantities were normalised within each experiment using a multivariate lowess approach. Statistical significance was assessed by combining data from all experiments and applying a moderated t-test using the empirical Bayes method with the limma package in R. Proteins were ranked after calculating the B-statistic and the Storey-Tibshirani false discovery rate. 217 proteins (6% genome coverage) were determined to have significant quantitative differences. In achieving these outcomes a range of factors that impact on quantitative proteomics data quality were broadly assessed, resulting in the development of a robust approach that is generally applicable to quantitative proteomics of biological system. The significantly differentially abundant proteins from the proteomics data provided insight into molecular mechanisms of cold adaptation in S. alaskensis. Important aspects of cold adaptation included cell membrane restructuring, exopolysaccharide biosynthesis, lipid degradation, carbohydrate and amino acid metabolism, and increased capacity of transcriptional and translational processes. A number of cold adaptive responses in S. alaskensis were novel, including a specific cold-active protein folding pathway, a possible thermally-controlled stringent response, and biosynthesis of intracellular polyhydroxyalkanoate reserve material. The overall study provided important new insight into the evolution of growth strategies necessary for the effective competition of S. alaskensis in cold, oligotrophic environments.