Molecular basis of canalization in an ascidian species complex adapted to different thermal conditions

Canalization is a result of intrinsic developmental buffering that ensures phenotypic robustness under genetic variation and environmental perturbation. As a consequence, animal phenotypes are remarkably consistent within a species under a wide range of conditions, a property that seems contradictory to evolutionary change. Study of laboratory model species has uncovered several possible canalization mechanisms, however, we still do not understand how the level of buffering is controlled in natural populations. We exploit wild populations of the marine chordate Ciona intestinalis to show that levels of buffering are maternally inherited. Comparative transcriptomics show expression levels of genes encoding canonical chaperones such as Hsp70 and Hsp90 do not correlate with buffering. However the expression of genes encoding endoplasmic reticulum (ER) chaperones does correlate. We also show that ER chaperone genes are widely conserved amongst animals. Contrary to previous beliefs that expression level of Heat Shock Proteins (HSPs) can be used as a measurement of buffering levels, we propose that ER associated chaperones comprise a cellular basis for canalization. ER chaperones have been neglected by the fields of development, evolution and ecology, but their study will enhance understanding of both our evolutionary past and the impact of global environmental change.

Molecular basis of canalization in an ascidian species complex adapted to different thermal conditions

Canalization is a result of intrinsic developmental buffering that ensures phenotypic robustness under genetic variation and environmental perturbation. As a consequence, animal phenotypes are remarkably consistent within a species under a wide range of conditions, a property that seems contradictory to evolutionary change. Study of laboratory model species has uncovered several possible canalization mechanisms, however, we still do not understand how the level of buffering is controlled in natural populations. We exploit wild populations of the marine chordate Ciona intestinalis to show that levels of buffering are maternally inherited. Comparative transcriptomics show expression levels of genes encoding canonical chaperones such as Hsp70 and Hsp90 do not correlate with buffering. However the expression of genes encoding endoplasmic reticulum (ER) chaperones does correlate. We also show that ER chaperone genes are widely conserved amongst animals. Contrary to previous beliefs that expression level of Heat Shock Proteins (HSPs) can be used as a measurement of buffering levels, we propose that ER associated chaperones comprise a cellular basis for canalization. ER chaperones have been neglected by the fields of development, evolution and ecology, but their study will enhance understanding of both our evolutionary past and the impact of global environmental change.

Personalized cancer medicine: molecular diagnostics, predictive biomarkers, and drug resistance.

The progressive elucidation of the molecular pathogenesis of cancer has fueled the rational development of targeted drugs for patient populations stratified by genetic characteristics. Here we discuss general challenges relating to molecular diagnostics and describe predictive biomarkers for personalized cancer medicine. We also highlight resistance mechanisms for epidermal growth factor receptor (EGFR) kinase inhibitors in lung cancer. We envisage a future requiring the use of longitudinal genome sequencing and other omics technologies alongside combinatorial treatment to overcome cellular and molecular heterogeneity and prevent resistance caused by clonal evolution.

Intraclonal heterogeneity and distinct molecular mechanisms characterize the development of t(4;14) and t(11;14) myeloma.

We have used whole exome sequencing to compare a group of presentation t(4;14) with t(11;14) cases of myeloma to define the mutational landscape. Each case was characterized by a median of 24.5 exonic nonsynonymous single-nucleotide variations, and there was a consistently higher number of mutations in the t(4;14) group, but this number did not reach statistical significance. We show that the transition and transversion rates in the 2 subgroups are similar, suggesting that there was no specific mechanism leading to mutation differentiating the 2 groups. Only 3% of mutations were seen in both groups, and recurrently mutated genes include NRAS, KRAS, BRAF, and DIS3 as well as DNAH5, a member of the axonemal dynein family. The pattern of mutation in each group was distinct, with the t(4;14) group being characterized by deregulation of chromatin organization, actin filament, and microfilament movement. Recurrent RAS pathway mutations identified subclonal heterogeneity at a mutational level in both groups, with mutations being present as either dominant or minor subclones. The presence of subclonal diversity was confirmed at a single-cell level using other tumor-acquired mutations. These results are consistent with a distinct molecular pathogenesis underlying each subgroup and have important impacts on targeted treatment strategies. The Medical Research Council Myeloma IX trial is registered under ISRCTN68454111.

Challenges in molecular testing in non-small-cell lung cancer patients with advanced disease

Lung cancer diagnostics have progressed greatly in the previous decade. Development of molecular testing to identify an increasing number of potentially clinically actionable genetic variants, using smaller samples obtained via minimally invasive techniques, is a huge challenge. Tumour heterogeneity and cancer evolution in response to therapy means that repeat biopsies or circulating biomarkers are likely to be increasingly useful to adapt treatment as resistance develops. We highlight some of the current challenges faced in clinical practice for molecular testing of EGFR, ALK, and new biomarkers such as PDL1. Implementation of next generation sequencing platforms for molecular diagnostics in non-small-cell lung cancer is increasingly common, allowing testing of multiple genetic variants from a single sample. The use of next generation sequencing to recruit for molecularly stratified clinical trials is discussed in the context of the UK Stratified Medicine Programme and The UK National Lung Matrix Trial.

The ecology and evolution of invasive Potamopyrgus antipodarum in Australia

The spread of invasive organisms is one of the greatest threats to ecosystems and biodiversity worldwide. Understanding the evolutionary and ecological factors responsible for the transport, introduction, establishment and spread of invasive species will assist the development of control strategies. The New Zealand mudsnail, Potamopyrgus antipodarum (Gray 1843) (Gastropoda: Hydrobiidae), is a global freshwater invader, with populations established in Europe, Asia, the Americas and Australia. While sexual and asexual P. antipodarum coexist in the native range, invasive populations reproduce by parthenogenesis, producing dense populations that compete for resources with native species. Potamopyrgus antipodarum is a natural model system for the study of evolutionary and ecological processes underlying invasion. This thesis assesses the invasion history, genetic diversity and ecology of P. antipodarum in Australia, with particular focus on: a) potential source populations, b) distribution and structure of populations, and c) species traits related to the establishment, persistence and spread of invasive P. antipodarum. Genetic analyses were carried out on specimens collected for this study from New Zealand and Australia, along with existing museum samples. In combination with published data, the analyses revealed low genetic diversity among and within invasive populations in south-eastern Australia, relative to New Zealand populations. Phylogenetic relationships inferred from mitochondrial sequences indicated that the Australian populations belong to clades dominated by parthenogenetic haplotypes that are known to be present in Europe and the US. These ‘invasive clades’ are likely to originate from the North Island of New Zealand, and suggest a role for selection in determining genetic composition of invasive populations. The genotypic diversity of Australian P. antipodarum was low, with few, closely related clones distributed across south-eastern Australia. The pattern of clone distribution was not consistent with any assessed geographical or abiotic factors; instead a few, widely-distributed clones were present in high frequencies at most sites. Differences in clone frequencies were found, which may indicate differential success of clonal lineages. A range of traits have been proposed as facilitators of invasion success, and within-species variation in these traits can promote differential success of genotypes. Using laboratory-based experiments, the performance of the three most common Australian clones was tested across a suite of invasion-relevant traits. Ecologically-relevant variation in traits was found among the clones. These differences may have determined the spatial distribution of clones, and may continue to do so into the future. This thesis found that the P. antipodarum invasion of Australia is the result of few introductions of a small number of globally-invasive genotypes that vary in ecologically-relevant traits. From a source of considerable genetic diversity in the native range, very few genotypes have become invasive. Those that are invasive appear to be very successful at continental scales. These findings highlight a capacity in asexual invaders to successfully invade, and potentially adapt to, a broad range of ecosystems. The P. antipodarum invasion system is amenable to research using combinations of field-based studies, molecular and laboratory approaches, and is likely to yield significant, broadly-applicable insights into invasion.

Comparative genomic approaches for investigating evolution in the microbial and viral populations of the rumen

The rumen is home to a diverse population of microorganisms encompassing all three domains of life: Bacteria, Archaea, and Eukarya. Viruses have also been documented to be present in large numbers; however, little is currently known about their role in the dynamics of the rumen ecosystem. This research aimed to use a comparative genomics approach in order to assess the potential evolutionary mechanisms at work in the rumen environment. We proposed to do this by first assessing the diversity and potential for horizontal gene transfer (HGT) of multiple strains of the cellulolytic rumen bacterium, Ruminococcus flavefaciens, and then by conducting a survey of rumen viral metagenome (virome) and subsequent comparison of the virome and microbiome sequences to ascertain if there was genetic information shared between these populations. We hypothesize that the bacteriophages play an integral role in the community dynamics of the rumen, as well as driving the evolution of the rumen microbiome through HGT. In our analysis of the Ruminococcus flavefaciens genomes, there were several mobile elements and clustered regularly interspaced short palindromic repeat (CRISPR) sequences detected, both of which indicate interactions with bacteriophages. The rumen virome sequences revealed a great deal of diversity in the viral populations. Additionally, the microbial and viral populations appeared to be closely associated; the dominant viral types were those that infect the dominant microbial phyla. The correlation between the distribution of taxa in the microbiome and virome sequences as well as the presence of CRISPR loci in the R. flavefaciens genomes, suggested that there is a “kill-the-winner” community dynamic between the viral and microbial populations in the rumen. Additionally, upon comparison of the rumen microbiome and rumen virome sequences, we found that there are many sequence similarities between these populations indicating a potential for phage-mediated HGT. These results suggest that the phages represent a gene pool in the rumen that could potentially contain genes that are important for adaptation and survival in the rumen environment, as well as serving as a molecular ‘fingerprint’ of the rumen ecosystem.

The Evolution of Massive Young Stellar Objects in the Large Magellanic Cloud

This thesis presents an analysis of the largest catalog to date of infrared spectra of massive young stellar objects in the Large Magellanic Cloud. Evidenced by their very different spectral features, the luminous objects span a range of evolutionary states from those most embedded in their natal molecular material to those that have dissipated and ionized their surroundings to form compact HII regions and photodissociation regions. We quantify the contributions of the various spectral features using the statistical method of principal component analysis. Using this analysis, we classify the YSO spectra into several distinct groups based upon their dominant spectral features: silicate absorption (S Group), silicate absorption and fine-structure line emission (SE), polycyclic aromatic hydrocarbon (PAH) emission (P Group), PAH and fine-structure line emission (PE), and only fine-structure line emission (E). Based upon the relative numbers of sources in each category, we are able to estimate the amount of time massive YSOs spend in each evolutionary stage. We find that approximately 50% of the sources have ionic fine-structure lines, indicating that a compact HII region forms about half-way through the YSO lifetime probed in our study. Of the 277 YSOs we collected spectra for, 41 have ice absorption features, indicating they are surrounded by cold ice-bearing dust particles. We have decomposed the shape of the ice features to probe the composition and thermal history of the ice. We find that most the CO2 ice is embedded a polar ice matrix that has been thermally processed by the embedded YSO. The amount of thermal processing may be correlated with the luminosity of the YSO. Using the Australia Telescope Compact Array, we imaged the dense gas around a subsample of our sources in the HII complexes N44, N105, N113, and N159 using HCO+ and HCN as dense gas tracers. We find that the molecular material in star forming environments is highly clumpy, with clumps that range from subparsec to ~2 parsecs in size and with masses between 10^2 to 10^4 solar masses. We find that there are varying levels of star formation in the clumps, with the lower-mass clumps tending to be without massive YSOs. These YSO-less clumps could either represent an earlier stage of clump to the more massive YSO-bearing ones or clumps that will never form a massive star. Clumps with massive YSOs at their centers have masses larger than those with massive YSOs at their edges, and we suggest that the difference is evolutionary: edge YSO clumps are more advanced than those with YSOs at their centers. Clumps with YSOs at their edges may have had a significant fraction of their mass disrupted or destroyed by the forming massive star. We find that the strength of the silicate absorption seen in YSO IR spectra feature is well-correlated with the on-source HCO+ and HCN flux densities, such that the strength of the feature is indicative of the embeddedness of the YSO. We estimate that ~40% of the entire spectral sample has strong silicate absorption features, implying that the YSOs are embedded in circumstellar material for about 40% of the time probed in our study.

Evolution of genome sequencing techniques

The quality and the speed for genome sequencing has advanced at the same time that technology boundaries are stretched. This advancement has been divided so far in three generations. The first-generation methods enabled sequencing of clonal DNA populations. The second-generation massively increased throughput by parallelizing many reactions while the third-generation methods allow direct sequencing of single DNA molecules. The first techniques to sequence DNA were not developed until the mid-1970s, when two distinct sequencing methods were developed almost simultaneously, one by Alan Maxam and Walter Gilbert, and the other one by Frederick Sanger. The first one is a chemical method to cleave DNA at specific points and the second one uses ddNTPs, which synthesizes a copy from the DNA chain template. Nevertheless, both methods generate fragments of varying lengths that are further electrophoresed. Moreover, it is important to say that until the 1990s, the sequencing of DNA was relatively expensive and it was seen as a long process. Besides, using radiolabeled nucleotides also compounded the problem through safety concerns and prevented the automation. Some advancements within the first generation include the replacement of radioactive labels by fluorescent labeled ddNTPs and cycle sequencing with thermostable DNA polymerase, which allows automation and signal amplification, making the process cheaper, safer and faster. Another method is Pyrosequencing, which is based on the “sequencing by synthesis” principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation. By the end of the last millennia, parallelization of this method started the Next Generation Sequencing (NGS) with 454 as the first of many methods that can process multiple samples, calling it the 2º generation sequencing. Here electrophoresis was completely eliminated. One of the methods that is sometimes used is SOLiD, based on sequencing by ligation of fluorescently dye-labeled di-base probes which competes to ligate to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction. The widely used Solexa/Illumina method uses modified dNTPs containing so called “reversible terminators” which blocks further polymerization. The terminator also contains a fluorescent label, which can be detected by a camera. Now, the previous step towards the third generation was in charge of Ion Torrent, who developed a technique that is based in a method of “sequencing-by-synthesis”. Its main feature is the detection of hydrogen ions that are released during base incorporation. Likewise, the third generation takes into account nanotechnology advancements for the processing of unique DNA molecules to a real time synthesis sequencing system like PacBio; and finally, the NANOPORE, projected since 1995, also uses Nano-sensors forming channels obtained from bacteria that conducts the sample to a sensor that allows the detection of each nucleotide residue in the DNA strand. The advancements in terms of technology that we have nowadays have been so quick, that it makes wonder: ¿How do we imagine the next generation?

Molecular characterization of accessions of a rare genetic resource: sugary cassava (Manihot esculenta Crantz) from Brazilian Amazon

2016

In two waters: contemporary evolution of lagoonal and marine white seabream (Diplodus sargus) populations

Brackish water ecosystems are often exposed to wide variations in environmental variables, including temperature and salinity, which may cause strong selective pressures on organisms modifying the genetic patterns of species. The aim of this work was to test whether there is a ‘divergence-with-gene flow’ in coastal lagoon populations of white seabream (Diplodus sargus) (Ria Formosa, S Portugal and Mar Menor, SE Spain) respect to four marine populations, by using partial sequences of cyt b mitochondrial gene and information from nine microsatellite loci. Genetic diversity was highest in both coastal lagoons (Mar Menor and Ria Formosa) considering mitochondrial and nuclear markers. Although some of FST population pairwise comparisons were not significant, analyses of molecular variance (AMOVAs) detected differences between groups (coastal lagoon and marine) close to significance. Also, only two haplotypes (Cytb-17 and Cytb-18) were detected in both coastal lagoon sampling sites and these localities (Mar Menor and Ria Formosa) showed the highest number of singletons, some of them with a high number of mutations, as has been already described for other Mar Menor populations (Pomatochistus marmoratus and Holothuria polii). Also, several tests detected significant positive and balancing selection considering mtDNA and microsatellite data. These data support the hypothesis of selection as one of the drivers of the genetic differences found between coastal lagoon and marine populations. The life strategy adopted by Diplodus sargus in coastal lagoons allows it to decrease its mortality rate and improve the heritability of its genes. Also, the increase time spent in coastal lagoons with different temperatures and salinities favours the fitness selection and the maintenance of exclusive haplotypes and genotypes in coastal lagoon inhabitants favouring the ‘divergence-with-gene-flow’.

Correlation among Growth Conditions, Morphology, and Optical Properties of Nanocolumnar InGaN/GaN Heterostructures Selectively Grown by Molecular Beam Epitaxy

This work reports on the selective area growth mechanism of green-emitting InGaN/GaN nanocolumns. The evolution of the morphology of the InGaN segment is found to depend critically on the nominal III/V ratio as well as the diameter of the GaN section. In addition, the In distribution inside the InGaN segment is found to depend on the local III/V and In/Ga ratios.

A Transcriptome Atlas of Physcomitrella patens Provides Insights into the Evolution and Development of Land Plants

Post-print version of the article.

Stepwise evolution of the centriole-assembly pathway

The centriole and basal body (CBB) structure nucleates cilia and flagella, and is an essential component of the centrosome, underlying eukaryotic microtubule-based motility, cell division and polarity. In recent years, components of the CBB-assembly machinery have been identified, but little is known about their regulation and evolution. Given the diversity of cellular contexts encountered in eukaryotes, but the remarkable conservation of CBB morphology, we asked whether general mechanistic principles could explain CBB assembly. We analysed the distribution of each component of the human CBB-assembly machinery across eukaryotes as a strategy to generate testable hypotheses. We found an evolutionarily cohesive and ancestral module, which we term UNIMOD and is defined by three components (SAS6, SAS4/CPAP and BLD10/CEP135), that correlates with the occurrence of CBBs. Unexpectedly, other players (SAK/PLK4, SPD2/CEP192 and CP110) emerged in a taxon-specific manner. We report that gene duplication plays an important role in the evolution of CBB components and show that, in the case of BLD10/CEP135, this is a source of tissue specificity in CBB and flagella biogenesis. Moreover, we observe extreme protein divergence amongst CBB components and show experimentally that there is loss of cross-species complementation among SAK/PLK4 family members, suggesting species-specific adaptations in CBB assembly. We propose that the UNIMOD theory explains the conservation of CBB architecture and that taxon- and tissue-specific molecular innovations, gained through emergence, duplication and divergence, play important roles in coordinating CBB biogenesis and function in different cellular contexts.

Convergent evolution of PICIs-mediated phage interference

Staphylococcal pathogenicity islands (SaPIs), the prototype members of the family of phage inducible chromosomal islands (PICIs), are extremely mobile phage satellites, which are transferred between bacterial hosts after their induction by a helper phage. The intimate relationship between SaPIs and their helper phages is one of the most studied examples of virus satellite interactions in prokaryotic cells. SaPIs encode and disseminate virulence and fitness factors, representing a driving force for bacterial adaptation and pathogenesis. Many SaPIs encode a conserved morphogenetic operon, including a core set of genes whose function allows them to parasitize and exploit the phage life cycle. One of the central mechanisms of this molecular piracy is the specific packaging of the SaPI genomes into reduced sized capsid structures derived from phage proteins. Pac phages were classically thought to be the only phages involved in the mobilisation of phage-mediated virulence genes, including the transfer of SaPIs within related and non-related bacteria. This study presents the involvement of S. aureus cos phages in the intra- and intergeneric transfer of cos SaPIs for the first time. A novel example of molecular parasitism is shown, by which this newly characterised group of cos SaPIs uses two distinct and complementary mechanisms to take over the helper phage packaging machinery for their own reproduction. SaPIbov5, the prototype of the cos SaPIs, does not encode the characteristic morphogenetic operon found in pac SaPIs. However, cos SaPIs features both pac and cos phage cleavage sequences in their genome, ensuring SaPI packaging in small- and full-sized phage particles, depending on the helper phage. Moreover, cos-site packaging in S. aureus was shown to require the activity of a phage HNH nuclease. The HNH protein functions together with the large terminase subunit, triggering cleavage and melting of the cos-site sequence. In addition, a novel piracy strategy, severely interfering with the helper phage reproduction, was identified in cos SaPIs and characterised. This mechanism of piracy depends on the cos SaPI-encoded ccm gene, which encodes a capsid protein involved in the formation of small phage particles, modifying the assembling process via a scaffolding mechanism. This strategy resembles the ones described for pac SaPIs and represents a remarkable example of convergent evolution. A further convergent mechanism of capsid size-reduction was identified and characterised for the Enterococcus faecalis EfCIV583 pathogenicity island, another member of the PICI family. In this case, the self-encoded CpmE conducts this molecular piracy through a putative scaffolding function. Similar to cos SaPIs, EfCIV583 carries the helper phage cleavage sequence in its genome enabling its mobilisation by the phage terminase complex. The results presented in this thesis show how two examples of non-related members of the PICI family follow the same evolutionary convergent strategy to interfere with their helper phage. These findings could indicate that the described strategies might be widespread among PICIs and implicate a significant impact of PICIs mediated-virulence gene transfer in bacterial evolution and the emergence of pathogenic bacteria.