Mass spectrometry-based protein profiling strategies for biomarker discovery in liver and inflammatory bowel diseases

The study of protein expression profiles for biomarker discovery in serum and in mammalian cell populations needs the continuous improvement and combination of proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic approaches. In this thesis work two different mass spectrometry-based protein profiling strategies have been developed and applied to liver and inflammatory bowel diseases (IBDs) for the discovery of new biomarkers. The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and chemometric analysis of serum samples, was applied to the study of serum protein expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis). The approach allowed the enrichment of serum proteins/peptides due to the high interaction surface between analytes and solid phase and the high recovery due to the elution step performed directly on the MALDI-target plate. Furthermore the use of chemometric algorithm for the selection of the variables with higher discriminant power permitted to evaluate patterns of 20-30 proteins involved in the differentiation and classification of serum samples from healthy donors and diseased patients. These proteins profiles permit to discriminate among the pathologies with an optimum classification and prediction abilities. In particular in the study of inflammatory bowel diseases, after the analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease, ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a 72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/ TOF MS analysis or by their selective enrichment followed by enzymatic digestion and MS/MS analysis may give useful information in order to identify new biomarkers involved in the diseases. The second mass spectrometry-based protein profiling strategy developed was based on a label-free liquid chromatography electrospray ionization quadrupole - time of flight differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS analysis of only identified differences. The strategy was used for biomarker discovery in IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s disease patients were analysed. The combining of the low molecular weight serum proteins enrichment step and the LCMS approach allowed to evaluate a pattern of peptides derived from specific exoprotease activity in the coagulation and complement activation pathways. Among these peptides, particularly interesting was the discovery of clusters of peptides from fibrinopeptide A, Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be performed to evaluate the specificity of these clusters and validate the results, in order to develop a rapid serum diagnostic test. The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many proteins that could be involved in the inflammation process. Among them heat shock protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for the validation of the results and the in-depth investigation of the inflammation pathways involved in the disease will be performed. Both the developed mass spectrometry-based protein profiling strategies have been proved to be useful tools for the discovery of disease biomarkers that need to be validated in further studies.

Non-naturalized alien plants receive fewer flower visits than naturalized and native plants in a Swiss botanical garden

Many animal-pollinated plant species have been introduced to non-native regions without their usual pollinators. Nevertheless, some of these alien species managed to establish reproducing naturalized populations, which might negatively affect native plants. Recent studies have shown that many naturalized alien species can readily attract native pollinators. However, it is not known whether alien species that have not established naturalized populations are less successful in attracting pollinators. Therefore, we tested whether flower-visitation rates are lower for non-naturalized aliens than for naturalized alien and native species. We conducted a comparative study on flower visitation of 185 native, 37 naturalized alien and 224 non-naturalized alien plant species in the Botanical Garden of Bern, Switzerland. Our phylogenetically corrected analyses showed that non-naturalized alien species received fewer flower visitors than both naturalized alien and native species. Native, naturalized alien and non-naturalized alien species were visited by similar flower-visitor communities. Furthermore, among the naturalized alien species, the ones with a broader distribution range in Switzerland received a more diverse set of flower visitors. Although it has been suggested that most alien plants can readily integrate into native plant–pollinator networks, we show evidence that the capacity to attract flower visitors in non-native regions is different for naturalized and non-naturalized alien plants. Therefore, we conclude that successful naturalization of alien plants may be related to flower visitation.

n-Alkane analyses from an transect of marine surface sediments off southwest Africa

An isobathic transect of marine surface sediments from 1°N to 28°S off southwest Africa was used to further evaluate the potential of the chain length distribution and carbon stable isotope composition of higher plant n-alkanes as proxies for continental vegetation and climate conditions. We found a strong increase in the n-C29-33 weighted mean average d13C values from -33 per mil near the equator to around -26 per mil further south. Additionally, C25-35n-alkanes reveal a southward trend of increasing average chain length from 30.0 to 30.5. The data reflect the changing contribution of plants employing different photosynthetic pathways (C3 and C4) and/or being differently influenced by the environmental conditions of their habitat. The C4 plant proportions calculated from the data (ca. 20% for rivers draining the rainforest, to ca. 70% at higher latitude) correspond to the C4 plant abundance in continental catchment areas postulated by considering prevailing wind systems and river outflows. Furthermore, the C4 plant contribution to the sediments correlates with the mean annual precipitation and aridity at selected continental locations in the postulated catchment areas, suggesting that the C4 plant fraction in marine sediments can be used to assess these environmental parameters.

Initial biomass and biomass change of tundra and forest plants in three herbivore treatments in Norway and Sweden

Recent Pan-Arctic shrub expansion has been interpreted as a response to a warmer climate. However, herbivores can also influence the abundance of shrubs in arctic ecosystems. We addressed these alternative explanations by following the changes in plant community composition during the last 10 years in permanent plots inside and outside exclosures with different mesh sizes that exclude either only reindeer or all mammalian herbivores including voles and lemmings. The exclosures were replicated at three forest and tundra sites at four different locations along a climatic gradient (oceanic to continental) in northern Fennoscandia. Since the last 10 years have been exceptionally warm, we could study how warming has influenced the vegetation in different grazing treatments. Our results show that the abundance of the dominant shrub, Betula nana, has increased during the last decade, but that the increase was more pronounced when herbivores were excluded. Reindeer have the largest effect on shrubs in tundra, while voles and lemmings have a larger effect in the forest. The positive relationship between annual mean temperature and shrub growth in the absence of herbivores and the lack of relationships in grazed controls is another indication that shrub abundance is controlled by an interaction between herbivores and climate. In addition to their effects on taller shrubs (> 0.3 m), reindeer reduced the abundance of lichens, whereas microtine rodents reduced the abundance of dwarf shrubs (< 0.3 m) and mosses. In contrast to short-term responses, competitive interactions between dwarf shrubs and lichens were evident in the long term. These results show that herbivores have to be considered in order to understand how a changing climate will influence tundra ecosystems.

Species occurrence and richness of lichen, bryophytes and vascular plants near Abisko, Sweden and Toolik lake, Alaska

Little is known about the impact of changing temperature regimes on composition and diversity of cryptogam communities in the Arctic and Subarctic, despite the well-known importance of lichens and bryophytes to the functioning and climate feedbacks of northern ecosystems. We investigated changes in diversity and abundance of lichens and bryophytes within long-term (9-16 years) warming experiments and along natural climatic gradients, ranging from Swedish subarctic birch forest and subarctic/subalpine tundra to Alaskan arctic tussock tundra. In both Sweden and Alaska, lichen diversity responded negatively to experimental warming (with the exception of a birch forest) and to higher temperatures along climatic gradients. Bryophytes were less sensitive to experimental warming than lichens, but depending on the length of the gradient, bryophyte diversity decreased both with increasing temperatures and at extremely low temperatures. Among bryophytes, Sphagnum mosses were particularly resistant to experimental warming in terms of both abundance and diversity. Temperature, on both continents, was the main driver of species composition within experiments and along gradients, with the exception of the Swedish subarctic birch forest where amount of litter constituted the best explanatory variable. In a warming experiment in moist acidic tussock tundra in Alaska, temperature together with soil ammonium availability were the most important factors influencing species composition. Overall, dwarf shrub abundance (deciduous and evergreen) was positively related to warming but so were the bryophytes Sphagnum girgensohnii, Hylocomium splendens and Pleurozium schreberi; the majority of other cryptogams showed a negative relationship to warming. This unique combination of intercontinental comparison, natural gradient studies and experimental studies shows that cryptogam diversity and abundance, especially within lichens, is likely to decrease under arctic climate warming. Given the many ecosystem processes affected by cryptogams in high latitudes (e.g. carbon sequestration, N2-fixation, trophic interactions), these changes will have important feedback consequences for ecosystem functions and climate.

Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: Many losses of photosynthesis and complex patterns of rate variation

The plastid genomes of some nonphotosynthetic parasitic plants have experienced an extreme reduction in gene content and an increase in evolutionary rate of remaining genes. Nothing is known of the dynamics of these events or whether either is a direct outcome of the loss of photosynthesis. The parasitic Scrophulariaceae and Orobanchaceae, representing a continuum of heterotrophic ability ranging from photosynthetic hemiparasites to nonphotosynthetic holoparasites, are used to investigate these issues. We present a phylogenetic hypothesis for parasitic Scrophulariaceae and Orobanchaceae based on sequences of the plastid gene rps2, encoding the S2 subunit of the plastid ribosome. Parasitic Scrophulariaceae and Orobanchaceae form a monophyletic group in which parasitism can be inferred to have evolved once. Holoparasitism has evolved independently at least five times, with certain holoparasitic lineages representing single species, genera, and collections of nonphotosynthetic genera. Evolutionary loss of the photosynthetic gene rbcL is limited to a subset of holoparasitic lineages, with several holoparasites retaining a full length rbcL sequence. In contrast, the translational gene rps2 is retained in all plants investigated but has experienced rate accelerations in several hemi- as well as holoparasitic lineages, suggesting that there may be substantial molecular evolutionary changes to the plastid genome of parasites before the loss of photosynthesis. Independent patterns of synonymous and nonsynonymous rate acceleration in rps2 point to distinct mechanisms underlying rate variation in different lineages. Parasitic Scrophulariaceae (including the traditional Orobanchaceae) provide a rich platform for the investigation of molecular evolutionary process, gene function, and the evolution of parasitism.

Detection of Ca2+-Dependent Transglutaminase Activity in Root and Leaf Tissue of Monocotyledonous and Dicotyledonous Plants

Protein extracted from root and leaf tissue of the dicotyledonous plants pea (Pisum sativum) and broad bean (Vicia faba) and the monocotyledonous plants wheat (Triticum aestivum) and barley (Hordeum vulgare) were shown to catalyze the incorporation of biotin-labeled cadaverine into microtiter-plate-bound N′,N′-dimethylcasein and the cross-linking of biotin-labeled casein to microtiter-plate-bound casein in a Ca2+-dependent manner. The cross-linking of biotinylated casein and the incorporation of biotin-labeled cadaverine into N′,N′-dimethylcasein were time-dependent reactions with a pH optimum of 7.9. Transglutaminase activity was shown to increase over a 2-week growth period in both the roots and leaves of pea. The product of transglutaminase's protein-cross-linking activity, ε-(γ-glutamyl)-lysine isodipeptide, was detected in root and shoot protein from pea, broad bean, wheat, and barley by cation-exchange chromatography. The presence of the isodipeptide was confirmed by reversed-phase chromatography. Hydrolysis of the isodipeptide after cation-exchange chromatography confirmed the presence of glutamate and lysine.

Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning.

Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, pro-PO) from the hemolymph of Manduca sexta larvae and purified to homogeneity. Pro-PO exhibits a M(r) of 130,000 on gel filtration and two bands with an apparent M(r) of approximately 100,000 on SDS/PAGE, as well as size-exclusion HPLC. Activation of pro-PO was achieved either by specific proteolysis by a cuticular protease or by the detergent cetylpyridinium chloride at a concentration below the critical micellar concentration. A cDNA clone for M. sexta pro-PO was obtained from a larval hemocyte cDNA library. The clone encodes a polypeptide of approximately 80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arthropods. The M. Sexta pro-PO, together with other arthropod pro-POs, contains a short stretch of amino acids with sequence similarity to the thiol ester region of alpha-macroglobulins and complement proteins C3 and C4.

Signals involved in wound-induced proteinase inhibitor II gene expression in tomato and potato plants.

Chemical and physical signals have been reported to mediate wound-induced proteinase inhibitor II (Pin2) gene expression in tomato and potato plants. Among the chemical signals, phytohormones such as abscisic acid (ABA) and jasmonic acid (JA) and the peptide systemin represent the best characterized systems. Furthermore, electrical and hydraulic mechanisms have also been postulated as putative Pin2-inducing systemic signals. Most of the chemical agents are able to induce Pin2 gene expression without any mechanical wounding. Thus, ABA, JA, and systemin initiate Pin2 mRNA accumulation in the directly treated leaves and in the nontreated leaves (systemic) that are located distal to the treated ones. ABA-deficient tomato and potato plants do not respond to wounding by accumulation of Pin2 mRNA, therefore providing a suitable model system for analysis of the signal transduction pathway involved in wound-induced gene activation. It was demonstrated that the site of action of JA is located downstream to the site of action of ABA. Moreover, systemin represents one of the initial steps in the signal transduction pathway regulating the wound response. Recently, it was reported that heat treatment and mechanical injury generate electrical signals, which propagate throughout the plant. These signals are capable of inducing Pin2 gene expression in the nontreated leaves of wounded plants. Furthermore, electrical current application to tomato leaves leads to an accumulation of Pin2 mRNA in local and systemic tissues. Examination of photosynthetic parameters (assimilation and transpiration rate) on several types of stimuli suggests that heat-induced Pin2 gene expression is regulated by an alternative pathway from that mediating the electrical current and mechanical wound response.