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The origin of eukaryotic cells is intensely studied, but some aspects remain poorly understood. This project aims to elucidate the nature of the Last Eukaryotic Common Ancestor (LECA) and in the origin of the anaerobic-related enzymes found in eukaryotes. First, an inference of the ancestral gene content of LECA is made using eukaryotic species trees rooted using the outgroup-free method ALE. The unrooted topology of the species tree is estimated using information on the presence and absence of gene families, alongside a tree topology inferred in previous work using a more standard sequence-based phylogenetic approach. Although the number of genes present in the roots is low, we obtained an insight into its metabolism and support the hypothesis of LECA as a flagellated cell and the idea of a methanogenic ancestor as the host for the endosymbiosis. Finally, several gene trees of anaerobic-related enzymes are made and compared with the genes found in LECA. We analysed the origin of the enzymes according its position on the tree either of mitochondrial, other bacterial, or archaeal host cell origin.

The replacement of hydroxyl groups by fluorine atoms on hexopyranoside scaffolds may allow access to invaluable tools for studying various biochemical processes. As part of ongoing activities toward the preparation of fluorinated carbohydrates, a systematic investigation involving the synthesis and biological evaluation of a series of mono‐ and polyfluorinated galactopyranosides is described. Various monofluorogalactopyranosides, a trifluorinated, and a tetrafluorinated galactopyranoside have been prepared using a Chiron approach. Given the scarcity of these compounds in the literature, in addition to their synthesis, their biological profiles were evaluated. Firstly, the fluorinated compounds were investigated as antiproliferative agents using normal human and mouse cells in comparison with cancerous cells. Most of the fluorinated compounds showed no antiproliferative activity. Secondly, these carbohydrate probes were used as potential inhibitors of galactophilic lectins. The first transverse relaxation‐optimized spectroscopy (TROSY) NMR experiments were performed on these interactions, examining chemical shift perturbations of the backbone resonances of LecA, a virulence factor from Pseudomonas aeruginosa. Moreover, taking advantage of the fluorine atom, the 19F NMR resonances of the monofluorogalactopyranosides were directly monitored in the presence and absence of LecA to assess ligand binding. Lastly, these results were corroborated with the binding potencies of the monofluorinated galactopyranoside derivatives by isothermal titration calorimetry experiments. Analogues with fluorine atoms at C‐3 and C‐4 showed weaker affinities with LecA as compared to those with the fluorine atom at C‐2 or C‐6. This research has focused on the chemical synthesis of “drug‐like” low‐molecular‐weight inhibitors that circumvent drawbacks typically associated with natural oligosaccharides.

Background Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. Results Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) ‘classical’ Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors (‘classical’ Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of ‘classical’ Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. Conclusions Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that ‘classical’ Pufs then underwent lineage-specific expansions.

Background Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. Results Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) ‘classical’ Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors (‘classical’ Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of ‘classical’ Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. Conclusions Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that ‘classical’ Pufs then underwent lineage-specific expansions.

Background Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. Results Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) ‘classical’ Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors (‘classical’ Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of ‘classical’ Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. Conclusions Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that ‘classical’ Pufs then underwent lineage-specific expansions.

Background Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. Results Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) ‘classical’ Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors (‘classical’ Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of ‘classical’ Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. Conclusions Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that ‘classical’ Pufs then underwent lineage-specific expansions.

Background Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. Results Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) ‘classical’ Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors (‘classical’ Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of ‘classical’ Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. Conclusions Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that ‘classical’ Pufs then underwent lineage-specific expansions.

Background Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. Results Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) ‘classical’ Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors (‘classical’ Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of ‘classical’ Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. Conclusions Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that ‘classical’ Pufs then underwent lineage-specific expansions.

The emergence of eukaryotes from ancient prokaryotic lineages embodied a remarkable increase in cellular complexity. While prokaryotes operate simple systems to connect DNA to the segregation machinery during cell division, eukaryotes use a highly complex protein assembly known as the kinetochore. Although conceptually similar, prokaryotic segregation systems and the eukaryotic kinetochore are not homologous. Here we investigate the origins of the kinetochore before the last eukaryotic common ancestor (LECA) using phylogenetic trees, sensitive profile-versus-profile homology detection, and structural comparisons of its protein components. We show that LECA’s kinetochore proteins share deep evolutionary histories with proteins involved in a few prokaryotic systems and a multitude of eukaryotic processes, including ubiquitination, transcription, and flagellar and vesicular transport systems. We find that gene duplications played a major role in shaping the kinetochore; more than half of LECA’s kinetochore proteins have other kinetochore proteins as closest homologs. Some of these have no detectable homology to any other eukaryotic protein, suggesting that they arose as kinetochore-specific folds before LECA. We propose that the primordial kinetochore evolved from proteins involved in various (pre)eukaryotic systems as well as evolutionarily novel folds, after which a subset duplicated to give rise to the complex kinetochore of LECA.

Summary The work described in this thesis focuses on the development of inhibitors for Pseudomonas aeruginosa targeting lectins LecA and LecB to inhibit the adhesion of this pathogen and also its biofilm formation. The specificity of the adhesion process could be also used as a detection handle.

The replacement of hydroxyl groups by fluorine atoms on hexopyranoside scaffolds may allow access to invaluable tools for studying various biochemical processes. As part of ongoing activities toward the preparation of fluorinated carbohydrates, a systematic investigation involving the synthesis and biological evaluation of a series of mono‐ and polyfluorinated galactopyranosides is described. Various monofluorogalactopyranosides, a trifluorinated, and a tetrafluorinated galactopyranoside have been prepared using a Chiron approach. Given the scarcity of these compounds in the literature, in addition to their synthesis, their biological profiles were evaluated. Firstly, the fluorinated compounds were investigated as antiproliferative agents using normal human and mouse cells in comparison with cancerous cells. Most of the fluorinated compounds showed no antiproliferative activity. Secondly, these carbohydrate probes were used as potential inhibitors of galactophilic lectins. The first transverse relaxation‐optimized spectroscopy (TROSY) NMR experiments were performed on these interactions, examining chemical shift perturbations of the backbone resonances of LecA, a virulence factor from Pseudomonas aeruginosa. Moreover, taking advantage of the fluorine atom, the 19F NMR resonances of the monofluorogalactopyranosides were directly monitored in the presence and absence of LecA to assess ligand binding. Lastly, these results were corroborated with the binding potencies of the monofluorinated galactopyranoside derivatives by isothermal titration calorimetry experiments. Analogues with fluorine atoms at C‐3 and C‐4 showed weaker affinities with LecA as compared to those with the fluorine atom at C‐2 or C‐6. This research has focused on the chemical synthesis of “drug‐like” low‐molecular‐weight inhibitors that circumvent drawbacks typically associated with natural oligosaccharides.

The emergence of eukaryotes from ancient prokaryotic lineages embodied a remarkable increase in cellular complexity. While prokaryotes operate simple systems to connect DNA to the segregation machinery during cell division, eukaryotes use a highly complex protein assembly known as the kinetochore. Although conceptually similar, prokaryotic segregation systems and the eukaryotic kinetochore are not homologous. Here we investigate the origins of the kinetochore before the last eukaryotic common ancestor (LECA) using phylogenetic trees, sensitive profile-versus-profile homology detection, and structural comparisons of its protein components. We show that LECA’s kinetochore proteins share deep evolutionary histories with proteins involved in a few prokaryotic systems and a multitude of eukaryotic processes, including ubiquitination, transcription, and flagellar and vesicular transport systems. We find that gene duplications played a major role in shaping the kinetochore; more than half of LECA’s kinetochore proteins have other kinetochore proteins as closest homologs. Some of these have no detectable homology to any other eukaryotic protein, suggesting that they arose as kinetochore-specific folds before LECA. We propose that the primordial kinetochore evolved from proteins involved in various (pre)eukaryotic systems as well as evolutionarily novel folds, after which a subset duplicated to give rise to the complex kinetochore of LECA.

Insight into the last eukaryotic common ancestor (LECA) is central to any phylogeny-based reconstruction of early eukaryotic evolution. Increasing amounts of data enable such reconstructions, without necessarily providing further insight into what LECA actually was. We consider four possible concepts of LECA: an abstract phylogenetic state, a single cell, a population, and a consortium of organisms. We argue that the view most realistically underlying work in the field is that of LECA as a population. Drawing on recent findings of genomically heterogeneous populations in eukaryotes (‘pangenomes’), we examine the evolutionary implications of a pangenomic LECA population. For instance, how does this concept affect standard expectations about the ecology, geography, fitness, and diversification of LECA? Does it affect evolutionary interpretations of LECA’s cellular functions? Finally, we examine whether this novel pangenomic concept of LECA has implications for phylogenetic reconstructions of early eukaryote evolution. Our aim is to add to the conceptual toolkit for developing theories of LECA and interpreting genomic datasets.

Background: Recently, important discoveries regarding the archaeon that functioned as the “host” in the merger with a bacterium that led to the eukaryotes, its “complex” nature, and its phylogenetic relationship to eukaryotes, have been reported. Based on these new insights proposals have been put forward to get rid of the three-domain Model of life, and replace it with a two-domain model. Results: We present arguments (both regarding timing, c

Background: Recently, important discoveries regarding the archaeon that functioned as the “host” in the merger with a bacterium that led to the eukaryotes, its “complex” nature, and its phylogenetic relationship to eukaryotes, have been reported. Based on these new insights proposals have been put forward to get rid of the three-domain Model of life, and replace it with a two-domain model. Results: We present arguments (both regarding timing, complexity, and chemical nature of specific evolutionary processes, as well as regarding genetic structure) to resist such proposals. The three-domain Model represents an accurate description of the differences at the most fundamental level of living organisms, as the eukaryotic lineage that arose from this unique merging event is distinct from both Archaea and Bacteria in a myriad of crucial ways. Conclusions: We maintain that “a natural system of organisms”, as proposed when the three-domain Model of life was introduced, should not be revised when considering the recent discoveries, however exciting they may be.

The large phylogenetic distance separating eukaryotic genes and their archaeal orthologs has prevented identification of the position of the eukaryotic root in phylogenomic studies. Recently, an innovative approach has been proposed to circumvent this issue: the use as phylogenetic markers of proteins that have been transferred from bacterial donor sources to eukaryotes, after their emergence from Archaea. Using this approach, two recent independent studies have built phylogenomic datasets based on bacterial sequences, leading to different predictions of the eukaryotic root. Taking advantage of additional genome sequences from the jakobid Andalucia godoyi and the two known malawimonad species (Malawimonas jakobiformis and Malawimonas californiana), we reanalyzed these two phylogenomic datasets. We show that both datasets pinpoint the same phylogenetic position of the eukaryotic root that is between “Unikonta” and “Bikonta,” with malawimonad and collodictyonid lineages on the Unikonta side of the root. Our results firmly indicate that (i) the supergroup Excavata is not monophyletic and (ii) the last common ancestor of eukaryotes was a biflagellate organism. Based on our results, we propose to rename the two major eukaryotic groups Unikonta and Bikonta as Opimoda and Diphoda, respectively.

I ett parterningprojekt mellan Midroc Project Manangement (MPM) och Väsbyhem om att bygga 190 hyreslägenheter togs beslutet att mura badrummen med Webers Leca-sten istället för att bygga dem på konventionellt sätt med gips. Denna jämförelse redovisar framförallt fördelar men även nackdelar med detta val. En förkortad entreprenadtid kan väga upp de ökade kostnader som valet att mura medför. Flertalet incitament för beställaren att välja murade badrum presenteras och backas upp av beräkningar och beskrivningar. En lösning med murade badrum medför att det finns pengar att tjäna både för beställare och MPM. Det kräver dock en extra insats i och med ett ökat behov av planering och produktionsledning. De positiva effekterna överväger dock detta tydligt., In a construction project to build 190 apartments ordered by Väsbyhem and built by Midroc Project Management (MPM) the decision to use Webers Leca bricks for the bathroom instead of gypsum wallboard was taken. This comparison shows on the benefits from this decision but also presents some of the disadvantages. A shorted building time could outweigh the increased costs of bricking the walls. Several incitemen is presented and backed up with calculations and descriptions to make this decision. The choice to brick the walls opens a possibility to make money for the orderer as for Midroc. Extra work in the planning process and production management is minor to the positive effects.

I ett parterningprojekt mellan Midroc Project Manangement (MPM) och Väsbyhem om att bygga 190 hyreslägenheter togs beslutet att mura badrummen med Webers Leca-sten istället för att bygga dem på konventionellt sätt med gips. Denna jämförelse redovisar framförallt fördelar men även nackdelar med detta val. En förkortad entreprenadtid kan väga upp de ökade kostnader som valet att mura medför. Flertalet incitament för beställaren att välja murade badrum presenteras och backas upp av beräkningar och beskrivningar. En lösning med murade badrum medför att det finns pengar att tjäna både för beställare och MPM. Det kräver dock en extra insats i och med ett ökat behov av planering och produktionsledning. De positiva effekterna överväger dock detta tydligt., In a construction project to build 190 apartments ordered by Väsbyhem and built by Midroc Project Management (MPM) the decision to use Webers Leca bricks for the bathroom instead of gypsum wallboard was taken. This comparison shows on the benefits from this decision but also presents some of the disadvantages. A shorted building time could outweigh the increased costs of bricking the walls. Several incitemen is presented and backed up with calculations and descriptions to make this decision. The choice to brick the walls opens a possibility to make money for the orderer as for Midroc. Extra work in the planning process and production management is minor to the positive effects.

Myosins are key components of the eukaryotic cytoskeleton, providing motility for a broad diversity of cargoes. Therefore, understanding the origin and evolutionary history of myosin classes is crucial to address the evolution of eukaryote cell biology. Here, we revise the classification of myosins using an updated taxon sampling that includes newly or recently sequenced genomes and transcriptomes from key taxa. We performed a survey of eukaryotic genomes and phylogenetic analyses of the myosin gene family, reconstructing the myosin toolkit at different key nodes in the eukaryotic tree of life. We also identified the phylogenetic distribution of myosin diversity in terms of number of genes, associated protein domains and number of classes in each taxa. Our analyses show that new classes (i.e., paralogs) and domain architectures were continuously generated throughout eukaryote evolution, with a significant expansion of myosin abundance and domain architectural diversity at the stem of Holozoa, predating the origin of animalmulticellularity. Indeed, single-celled holozoans have themost complexmyosin complement among eukaryotes, with paralogs of mostmyosins previously considered animal specific. Werecover a dynamic evolutionary history, with several lineagespecific expansions (e.g., the myosin III-like gene family diversification in choanoflagellates), convergence in protein domain architectures (e.g., fungal and animal chitin synthase myosins), and important secondary losses. Overall, our evolutionary scheme demonstrates that the ancestral eukaryote likely had a complex myosin repertoire that included six genes with different protein domain architectures. Finally, we provide an integrative and robust classification, useful for future genomic and functional studies on this crucial eukaryotic gene family. © The Author(s) 2014.

Transcription factors (TFs) are the main players in transcriptional regulation in eukaryotes. However, it remains unclear what role TFs played in the origin of all of the different eukaryotic multicellular lineages. In this paper, we explore how the origin of TF repertoires shaped eukaryotic evolution and, in particular, their role into the emergence of multicellular lineages. We traced the origin and expansion of all known TFs through the eukaryotic tree of life, using the broadest possible taxon sampling and an updated phylogenetic background. Our results show that the most complex multicellular lineages (i.e., those with embryonic development, Metazoa and Embryophyta) have the most complex TF repertoires, and that these repertoires were assembled in a stepwise manner.We also show that a significant part of the metazoan and embryophyte TF toolkits evolved earlier, in their respective unicellular ancestors. To gain insights into the role of TFs in the development of both embryophytes and metazoans, we analyzed TF expression patterns throughout their ontogeny. The expression patterns observed in both groups recapitulate those of the whole transcriptome, but reveal some important differences. Our comparative genomics and expression data reshape our view on how TFs contributed to eukaryotic evolution and reveal the importance of TFs to the origins of multicellularity and embryonic development.

El presente trabajo tuvo como objeto fundamental estudiar, a escala de laboratorio, la aptitud a la expansión térmica de diferentes muestras de arcillas variando la temperatura de quema y modificando la composición de éstas con la adición de agentes promotores de la expansión térmica, como carbonato de calcio y cenizas volantes de carbón. El procedimiento experimental comprendió cuatro etapas: preparación, caracterización, pruebas preliminares de quema y ensayos de quema específicos; utilizando como criterio de expansión térmica el cambio porcentual de volumen de las muestras, y como criterios de calidad de los productos obtenidos, la densidad, la porosidad abierta y la absorción de agua. Finalmente, se encontró que una de las arcillas estudiadas presentó aptitud a la expansión térmica de manera natural y que otras cinco arcillas, mezcladas con diferentes proporciones de aditivos, también expandieron térmicamente a temperaturas comprendidas entre 1100°C y 1250°C.

El presente trabajo tuvo como objeto fundamental estudiar, a escala de laboratorio, la aptitud a la expansión térmica de diferentes muestras de arcillas variando la temperatura de quema y modificando la composición de éstas con la adición de agentes promotores de la expansión térmica, como carbonato de calcio y cenizas volantes de carbón. El procedimiento experimental comprendió cuatro etapas: preparación, caracterización, pruebas preliminares de quema y ensayos de quema específicos; utilizando como criterio de expansión térmica el cambio porcentual de volumen de las muestras, y como criterios de calidad de los productos obtenidos, la densidad, la porosidad abierta y la absorción de agua. Finalmente, se encontró que una de las arcillas estudiadas presentó aptitud a la expansión térmica de manera natural y que otras cinco arcillas, mezcladas con diferentes proporciones de aditivos, también expandieron térmicamente a temperaturas comprendidas entre 1100°C y 1250°C.

Fyra avkörningsförsök i en bädd bestående av leca-kulor har utförts i farterna 30, 50, 70 och 90 km/h. Kortfattat kan sägas att materialet inte uppvisar de fartreducerande egenskaper som tidigare hävdats, och effekten kan sägas vara måttlig i förhållande till den ganska omfattande insatsen som anläggningen av en leca-bassäng eller fålla ändå utgör. I stort sett uppmäter vi hastighetsreduktioner, och motsvarande ökade stoppsträcker, som är ungefär 50 % av de som LECA berättar om i sitt reklamblad, se bilaga 1. Det betyder t ex att VTI:s prov i 70 km/h förbrukar drygt 32 meter stoppsträcka när LECA hävdar att en bil som kör 140 km/h stannar på 26 meter. Räknat på energi betyder det att lecan i våra prov bara är 25 % så effektiv som företaget LECA påstår. En observationsstudie på en befintlig leca-installation längs med E4 vid Norsholm i Östergötland gör troligt att snöröjning vintertid väsentligt sätter ned effekten av utplacerad leca. Effekten av sladdande avkörning eller fordon med andra geometrier, t ex SUV, har inte studerats inom projektet, även om så vore önskvärt

Fyra avkörningsförsök i en bädd bestående av leca-kulor har utförts i farterna 30, 50, 70 och 90 km/h. Kortfattat kan sägas att materialet inte uppvisar de fartreducerande egenskaper som tidigare hävdats, och effekten kan sägas vara måttlig i förhållande till den ganska omfattande insatsen som anläggningen av en leca-bassäng eller fålla ändå utgör. I stort sett uppmäter vi hastighetsreduktioner, och motsvarande ökade stoppsträcker, som är ungefär 50 % av de som LECA berättar om i sitt reklamblad, se bilaga 1. Det betyder t ex att VTI:s prov i 70 km/h förbrukar drygt 32 meter stoppsträcka när LECA hävdar att en bil som kör 140 km/h stannar på 26 meter. Räknat på energi betyder det att lecan i våra prov bara är 25 % så effektiv som företaget LECA påstår. En observationsstudie på en befintlig leca-installation längs med E4 vid Norsholm i Östergötland gör troligt att snöröjning vintertid väsentligt sätter ned effekten av utplacerad leca. Effekten av sladdande avkörning eller fordon med andra geometrier, t ex SUV, har inte studerats inom projektet, även om så vore önskvärt