Free-fall penetrometers (FFPs) are an attractive tool for the rapid characterization of sediments in the nearshore and coastal areas. To improve their measurement capabilities, modern FFPs can be equipped with pore pressure sensors. Pore pressure measurements are extensively used in traditional cone penetration testing, but their usage and interpretation is still limited for FFP testing. This thesis represents an effort to advance the interpretation of pore pressure measurements from FFP testing. Data was collected using the torpedo-shaped FFP BlueDrop during surveys at Herschel Island, YT, Yakutat, AK, Clay Bank, VA, and Yorktown, VA. Additionally, test deployments in the laboratory were performed in kaolin clay. Data analysis was focused on pore pressure measurements during these deployments. Two major advancements regarding current data analysis of FFP pore pressure measurements were explored: 1) a method based on fluid dynamic principles was proposed to correct the pressure recordings for the dynamic flow effects due to the high-velocity fall and impact. The results show that using Bernoulli’s theorem coupled with the concept of pressure coefficients results in good agreement between measured and hydrostatic pressures during the free-fall and initial penetration stage. 2) Pore pressure dissipation curves measured by the penetrometer at rest at maximum penetration depth were also studied. The mechanisms behind the non-standard dissipation curves were explored. The results suggest that non-standard dissipation curves can be interpreted by correcting according to Sully et al.’s (1999) extrapolation technique. The technique can also be used with data from an unsaturated or clogged filter. / Master of Science / An increasing use of nearshore and offshore areas for the development of infrastructure such as pipelines, cables, renewable energy harvesting devices, and measures against coastal erosion warrants the development of specialized methods for investigating the stability of the seabed. Portable free-fall penetrometers represent a cost-efficient approach to characterize shallow seabed sediments, but there are challenges associated with deriving geotechnical design parameters from these novel instruments. This study aims at developing a better understanding of the pore pressure (the pressure developed in the water in the soil’s voids) data obtained during free-fall penetration testing. The pore pressures developed during the penetration of the penetrometer is dependent on the soil type, and is often used to correlate to it. This study used data obtained from field surveys at Herschel Island, YT, Clay Bank, VA, Yakutat, AK, and Yorktown, VA. Additionally, controlled tests were performed in the laboratory in an instrumented seabed. This study resulted in a novel method to correct the pressure data from the penetrometer for dynamic fluid flow effects and validation of an interpretation technique for dissipation curves to obtain the time required for consolidation, based on initial results.

New types of laboratory reactors that are highly customizable, low-cost and easy to produce are needed to investigate low-temperature geochemical processes. We recently showed that desktop 3D printing stereolithography (SLA) can be used to efficiently fabricate a mixed flow reactor (MFR) with high dimensional accuracy comparable to traditional machining methods (Michel et al., 2018). We also showed that the SLA method allowed for the addition of complex features that are often beyond the capabilities of traditional methods. However, the stability of 3D printed parts at low-temperature geochemical conditions has not been fully evaluated. The objectives of this work were twofold: 1) to provide a framework for assessing the stability and compatibility of SLA printed materials at geochemically relevant conditions, and 2) to show how 3D printed specialty devices can enable new laboratory geochemical experiments. Part 1 of this Master's thesis presents findings for enhancing mechanical and solvent resistance properties of a commercial 3D printing material (Formlabs Clear) by UV post-curing procedures and also provide data showing its stability in aqueous solutions at pH 0, 5.7, and 12 for periods of up to 18 days. Thermal degradation patterns, mechanical analysis, and leachable fraction data are provided. Part 2 shows experiments coupling 3D printed reactors and flow devices for in situ small-angle x-ray scattering (SAXS). Schwertmannite (pH 2.7) and goethite (6.2) are precipitated from solution using various setups and observed differences in growth rates are discussed. The data show the potential of 3D printing for enabling novel laboratory geochemical experiments. / MS / New types of laboratory devices are needed to investigate environmental processes such as how minerals form, transform, and interact with their surroundings. These devices should be highly customizable, low-cost, and easy to produce. We have recently showed how 3D printing, specifically a technique called stereolithography (SLA), can be used to fabricate reactors with complex features that are often difficult to produce using traditional machining methods. However, in order to ensure that these materials don’t interfere with reactions of interest, we must assess the stability and compatibility of these materials in the relevant environmental conditions. As 3D printing techniques are still an emerging and rapidly developing technology, the methods we present will be useful for evaluating how new printer types and materials (i.e. resins) impact the suitability of 3D printed devices for future experimental studies. In part 1 of this thesis, the properties of a commercial 3D printing material were investigated by thermal and mechanical analyses; the propensity for leaching out material from the solid was also investigated. We show how exposing SLA printed materials to ultraviolet (UV) light post-printing can enhance material properties and minimize leaching. We then provide data showing the stability of the material after exposure to an array of acidic, neutral and basic conditions for a period of up to 18 days. In part 2, we describe experiments showing how novel 3D printed devices can be used to enhance laboratory investigations. Syntheses of two common iron oxide minerals using various custom reactor setups are presented. The setups were coupled with an analytical technique allowing for nanoscale observation of crystal growth in real-time. The data show how 3D printed specialty devices can be used to solve important questions in the geosciences such as the mechanisms of complex crystal formation.

Aging and leaking water infrastructure wastes water resources and creates public health risks. Upgrading of potable water systems represents a large financial burden for water utilities and private property owners. The conventional approaches of repair, rehabilitation and replacement are very effective, but will take decades to implement even if a financial commitment to do so was made immediately. A novel approach of in-situ remediation of leaks, achieved by harnessing the ability of water or pipe to repair leaks via clogging, could potentially reduce leak rates and extend the lifetime of existing infrastructure at relatively low cost and inconvenience. Physical clogging, precipitation and metallic corrosion were identified as major mechanisms of in-situ leak remediation in potable water pipelines. Autogenous repair (i.e., self-repair without added particles) of small leak-holes (150–"1000 μm) in copper and iron was validated in the laboratory at water pHs of 3.0–11.0, operating water pressures of 20–60 psi, upward and downward leak orientations, and for a range of water chemistries. In bench scale experiments, the time to repair of iron pipe leaks increased with leak size to the power of 0.89–1.89, and decreased with pipe wall thickness to the power of -1.9 to -1.0. The time to repair of copper pipe leaks increased with water pressure to the power of 1.7. Additionally, the waters with a higher DO and corrosivity as measured by RSI, significantly decreased the time to repair of carbon steel 400 μm leaks by 50–70%. The presence of chlorine dioxide significantly increased the fraction of repaired 200 μm copper pipe leaks by 3 times when compared to the control without any disinfectant. In the building scale study, the fraction of repaired iron pipe leaks decreased with the logarithmic leak size with a slope of -0.65 after one-year duration of experiments, while leak orientation and water pressure were not influential in time to or likelihood of repair for iron pipe leaks. Addition of calcium carbonate particles (~8.8 μ]m), silica particles (~29 μm) and wood ash particles (~160 μm) in Blacksburg, VA tap water at a water pressure of 10 psi increased the fraction of remediated iron pipe leaks of 280–1000 μm diameter sizes. Although the control condition with no added particles for 58 days resulted in remediation of 0/12 leaks, remediation rate increased to 1/12 with calcium carbonate particles, to 10/12 with silica particles and to 10/12 with wood ash particles. Leak size and particle size played an important role in controlling the remediation success rate. The strength of the in-situ leak repair was sometimes very strong and resilient. The sealing materials of leak-holes repaired at 20–60 psi could sometimes withstand a 100 psi water pressure without failure, demonstrating the potential of the approach to sustain aging and leaking infrastructure. In-situ leak repair can also occur naturally, and the success rate might be unintentionally altered by adjustment of chemistry or treatments that decrease or increase particulates. / Ph. D. / Old and leaking pipes waste water resources and can contaminate water. Upgrading of drinking water systems represents a large financial burden for water utilities and home owners. The traditional approaches to repair or replace the leaking water pipes are very effective, but will take decades to implement even if a financial commitment to do so was made immediately. A new approach of leak remediation, achieved by changing the drinking water chemistry, could potentially reduce leak rates and repair water leaks while in use without digging up the buried water pipes. Therefore, leak remediation could extend the lifetime of existing infrastructure at relatively low cost and inconvenience, and may be necessary if society cannot afford pipe replacement. Recent field observations indicate that metal corrosion, one type of reaction that eats up water pipes and causes water leaks, could clog the leaks via the corrosion products. And the repair in most cases could last for a long period of time. Our work was the first to reproduce the field observations in the laboratory, revealing that water pipe leaks could be successfully clogged or remediated by natural corrosion reaction products, if the water chemistry is favorable. Additionally, our work also showed that water leaks could be clogged or remediated by addition of water particles to drinking water, which was practiced by Roman engineers a long time ago. There are legitimate health concerns about particulates because they are indicative of microbial risks, but addition of particulates to water at low levels might heal pipes without such concerns. We also proved that in some cases the repaired materials clogging the leaks could withstand a very high household water pressure, showing that the sealing materials in water leaks repaired with natural corrosion products or added water particles could create long term repairs. With improved practical understanding this approach might be usefully applied, either intentionally to repair existing leaks, or make sure that changes to water chemistry do not unintentionally make problems worse.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Polymers capable of thermally controlled reversible bonding reactions are promising candidates for stimuli responsive materials, as required for self-healing or drug delivery materials. In order to investigate how the dynamic reactions can be controlled, effective analytical tools are demanded that are capable of analyzing not only the polymers but can also monitor the respective bonding reactions. Herein, we employ size exclusion chromatography in a newly developed temperature dependent mode (TD SEC) for the in situ characterization of polymers that undergo retro Diels-Alder (rDA) reaction at temperatures higher than 60 °C. Monitoring the evolution of the molar mass distribution of the polymers during the rDA reaction and evaluating the data quantitatively gives detailed information about the extent of the reaction and allows elucidating structural parameters that can be used for controlling the polymers debonding behavior. In contrast to spectroscopic techniques, TD SEC analyzes only the size of the polymers, hence the polymers do not need to fulfill any particular requirements (e.g. presence of detectable functional groups) but only need to be soluble in the TD SEC, which makes the method universally applicable. Side effects that might bias the results are minimized by using a high temperature chromatograph that allows performing the analysis in a broad temperature range (60 – 200 °C) and in different solvents. Thus, the analysis can be performed under the exact conditions that are required for the bonding reactions and an in situ image is provided.

Benzaldehyde (BZA), a biologically derived high-value molecule used in the flavour and fragrance industry for its characteristic almond-like aroma, has also found use in nutraceutical, pharmaceutical, cosmetics, agrochemical, and dye applications. Although, nature-identical BZA is most commonly produced by chemical synthesis, biologically derived BZA, whether by plant material extraction or via microbial biocatalysts, commands much higher prices. The bioproduction of high value molecules has often been characterized by low titers as results of substrate and product inhibition. The current work examined a variety of process strategies and the implementation of a solid-liquid bioreactor partitioning system with continuous integrated pervaporation to enhance the bioproduction of BZA using Pichia pastoris. Previous work on two-phase partitioning bioreactors (TPPBs) for the biotransformation of BZA using Pichia pastoris has had limitations due to long fermentation times and unutilized substrate in the immiscible polymer phase, contributing to complications for product purification. To reduce fermentation times, a mixed methanol/glycerol feeding strategy was employed and reduced the time required for high-density fermentation by 3.5 fold over previous studies. Additionally, because BZA and not the substrate benzyl alcohol (BA) had been found to be significantly inhibitory to the biotransformation reaction, a polymer selection strategy based on the ratio of partition coefficients (PCs) for the two target molecules was implemented. Using the polymer Kraton D1102K, with a PC ratio of 14.9 (BZA:BA), generated a 3.4 fold increase in BZA produced (14.4 g vs. 4.2 g) relative to single phase operation at more than double the volumetric productivity (97 mg L-1 h-1 vs. 41 mg L-1 h-1). This work also confirmed that the solute(s) of interest were taken up by polymers via absorption, not adsorption. BZA and BA cell growth inhibition experiments showed that these compounds are toxic to cells and it was their accumulation rather than low enzyme levels or energy (ATP) depletion that caused a reduction in the biotransformation rate. For this reason, the final strategy employed to enhance the bioproduction of benzaldehyde involved in situ product removal by pervaporation using polymer (Hytrel 3078) fabricated into tubing by DuPont, Canada. This aspect was initiated by first characterizing the custom-fabricated tubing in terms BZA and BA fluxes. The tubing was then integrated into an in situ pervaporation biotransformation and was shown to be effective at continuous product separation, using 87.4% less polymer by mass in comparison to polymer beads in conventional TPPB operation, and improved overall volumetric productivity by 214% (245.9 mg L-1 h-1 vs. 115.0 mg L-1 h-1) over previous work producing BZA. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-09-28 17:41:45.458

Abstract : Current prenatal diagnosis depends on invasive procedures and is thus offered only to high-risk pregnancies. Development of non-invasive prenatal diagnosis (NIPD) would change the risk-benefit ratio and make it likely that more women would benefit from prenatal testing. Scientists have documented the presence of rare fetal cells in maternal blood and envisioned targeting them with specific markers and their use in NIPD. Considering their extremely low frequency in maternal blood, fetal cells have been difficult to retrieve and use in clinical practice. Therefore, there is a pressing need for systematic sequential studies to evaluate their feasibility in NIPD. Generally, detection of rare cells within a large cell population carries great potentialities for the prospects of cancer management and NIPD. Manual scanning is very cumbersome and time-consuming Therefore; the first part of our project was, dedicated to the optimization of an effective strategy to evaluate retrieval of rare cells. We have developed a way of distributing a controlled number of target cells among hundreds of thousands of other cells on microscope slides. This strategy allows the precise evaluation of the retrieval of rare events and the comparizon of the efficacy of different techniques and enrichment approaches by knowing the definite number and locations of target cells on the slides. Furthermore, it allows the evaluation of hybridization of missed events. We have also developed a robust custom-made detection algorithm for rare cells using the MetaSystems automated platform and have used this strategy in the validation of manual and automatic scanning of 60 slides with a pre-defined number of rare male cells among a pure population of female cells using XY-FISH. Consequently, we tested the developed classifier for the detection of real fetal cells from maternal blood in both normal and aneuploid pregnancies with Down syndrome. We further evaluated the number of fetal cells with different methods of enrichments in the first and second trimesters. The data collected confirmed the early presence of fetal cells in all of the pregnancies tested and their frequencies were higher in cases of aneuploidies. Fetal cells are in a state of dynamic change throughout the pregnancy. Higher numbers of these cells can be obtained by optimizing the harvest time and methods of enrichment. We found that automatic scanning is more sensitive and reliable than manual detection. Furthermore, it alleviates the burden of scanning large numbers of cells and thus is more suitable for clinical application. We also demonstrated the feasibility of using rare cells in NIPD. Five microdissected amniotic fetal cells from 26 cases of normal and aneuploid pregnancies were quite enough to provide accurate NIPD through using whole genome amplification coupled with QF-PCR. Our findings laid the ground for the use of rare fetal cells in maternal blood for NIPD. // Résumé : Le diagnostic prénatal résulte encore aujourd’hui de procédures invasives, qui présentent des risques pour la grossesse. Le développement du diagnostic prénatal non-invasif (DPNI) changerait le rapport risque : bénéfice, rendant le diagnostic prénatal plus intéressant pour les femmes enceintes. Plusieurs chercheurs ont montré la présence de cellules fœtales dans le sang maternel et des travaux ont été entrepris afin de les cibler et de les utiliser éventuellement en DPNI. Toutefois, la faible concentration des cellules fœtales dans le sang maternel réduit les possibilités d’isolement ainsi que celles de leur utilisation en clinique. Un autre aspect technique du DPNI, le balayage manuel, est très laborieux, surtout en terme de temps technique. Il y a donc un besoin certain pour des études approfondies afin d’évaluer et d’améliorer la faisabilité du DPNI. La détection d’évènements rares dans une grande population cellulaire offre un potentiel pour le diagnostic en oncologie mais aussi en diagnostic prénatal. Dans cette thèse, la première étude était dédiée à l’optimisation d’une stratégie pour détecter les cellules rares. Nous avons développé une méthode d’étalement sur lame d’un nombre précis de cellules cibles parmi des centaines de milliers de cellules. Cette stratégie a permis d’évaluer le taux de détection d’évènements rares et de comparer l’efficacité des techniques d’enrichissement en connaissant le nombre exact et la localisation de cellules cibles sur les lames. De plus, il a été possible d’évaluer les problèmes d’hybridation des évènements manqués. Nous avons, par la suite, développé un algorithme robuste pour la détection de cellules rares en utilisant la plateforme de microscopie automatisée MetaSystems et utilisé cette approche dans la validation des balayages manuel et automatique d’un nombre précis de cellules mâles parmi une large population de cellules femelles marquées avec la technique FISH. Nous avons testé ce classificateur avec des échantillons de sang de femmes enceintes de grossesses normales et aneuploïdes et évalué la fréquence de cellules fœtales isolées par différentes méthodes d’enrichissement au cours des premier et second trimestres de grossesse. Les données accumulées ont confirmé la présence de cellules fœtales chez toutes les grossesses et leur fréquence plus élevée dans les grossesses aneuploïdes. Le nombre de cellules fœtales est dynamique tout au long de la grossesse. De plus, un nombre plus élevé de cellules fœtales peut être obtenu en optimisant le moment du prélèvement et les méthodes d’enrichissement. De plus, le balayage automatique s’est avéré plus sensible et constant que le balayage manuel, ce qui permet de balayer un grand nombre de cellules et devient plus approprié pour une application clinique. Nous avons aussi montré la faisabilité d’utiliser des cellules fœtales dans le cadre du DPNI. Cinq cellules amniotiques microdisséquées, provenant de grossesses normales et aneuploïdes, ont suffi pour poser un diagnostic prénatal par une combinaison de l’amplification du génome complet et de la technique QF-PCR (réaction quantitative en fluorescence d’amplification entraînée par une polymérase) permettant la détection d’anomalies chromosomiques. Nos résultats ouvrent la voie à l’utilisation de cellules fœtales dans le sang maternel pour le DPNI.

Les zones côtières sont des milieux privilégiés pour l’ostréiculture. L’espèce la plus cultivée au monde est l’huître creuse Crassostrea gigas, qui a été introduite dans de nombreux pays à des fins d’exploitation et qui, lorsqu’elle trouve des conditions climatiques favorables dans son nouveau milieu, se développe naturellement, en constituant des récifs. Dans le Bassin d’Arcachon, l’huître existe ainsi sous deux formes, cultivée et sauvage. La forme récifale est une structure tridimensionnelle complexe (huîtres agglomérées, au contact du sédiment, plusieurs classes d’âge présentes), alors que les huîtres cultivées sont placées en sur-élévation dans des poches ostréicoles (spécimens isolés, classe d’âge unique). L’effet respectif de ces deux configurations a été étudié selon trois approches : (1) comparaison de la dynamique des deux types de population d’huîtres (croissance, reproduction, composition biochimique), (2) effet de l’activité alimentaire sur les proies accessibles dans le milieu et les flux particulaires associés, et (3) effets des huîtres sur le sédiment et les communautés benthiques. Les deux années d’échantillonnage ont été marquées par un retard de la ponte (année 1) et un défaut de croissance (année 2) des huîtres, qui ont été rapprochées d’un déficit thermique en période estivale (année 1) et d’une disponibilité alimentaire plus faible au printemps (année 2) ; les cycles de reproduction sont apparus en léger décalage entre huîtres cultivées et sauvages. Une expérimentation in situ en tunnels benthiques à trois périodes de l’année a permis de quantifier les flux de matière et la consommation de nutriments et d’espèces planctoniques par les deux populations d’huître durant un cycle de marée. Le sédiment à proximité des huîtres est enrichi en particules fines et en matière organique, par suite de modifications locales de l’hydrodynamisme et de la production de fèces par les huîtres. Il en résulte une modification de la structure des peuplements de l’endofaune benthique. Les récifs d’huîtres sauvages constituent un nouvel habitat de substrat dur dans le Bassin d’Arcachon où n’existent naturellement que des habitats sédimentaires ; ils supportent une épifaune à fortes biomasse et diversité, qui accroît la biodiversité locale. / Coastal areas are favourable environments for oyster farming. The most commonly farmed species in the world is the cupped oyster Crassostrea gigas, which was introduced in many countries for aquaculture purposes. When it finds suitable climatic conditions in its new environment, the species develops naturally, thereby creating oyster reefs. In Arcachon Bay (SW France), the cupped oyster develops as both farmed and feral populations. Oyster reefs are complex tridimensional structures (agglomerated oysters, in contact with the sediment, populations with many any classes), while farmed oysters are cultivated off-bottom in plastic bags (isolated oysters, populations with a single age class). The respective effects of these two oyster configurations were studied according to three research axes: (1) a comparison of the dynamics of farmed and wild oyster populations, in terms of growth, reproduction, and biochemical composition; (2) the effect of oyster feeding on accessible preys and the associated particulate fluxes; and (3) the effects of oysters on the sediment and benthic communities. The two years of study were characterized by a delay in the spawning periods (year 1) and an unusually low growth rate (year 2) in oysters, which were related to low summer water temperatures (year 1) and a low food availability in spring (year 2); reproduction cycles also appeared slightly shifted between farmed and wild oysters. An in situ experiment with benthic tunnels at three periods of the year was designed to quantify the flux of matter and the consumption of nutrients and planktonic species by the two oyster populations during a tidal cycle. Sediment in the vicinity of oysters contained more fine particles and organic matter, due to changes in local hydrodynamism and oyster production of faeces. These sedimentary changes modified the structure of benthic infaunal assemblages. Oyster reefs form a new habitat of hard substrate in Arcachon Bay, where only sedimentary habitats naturally occur; they have an epifauna with high biomass and diversity, that increases the local biodiversity.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal. / Pour respecter les droits d'auteur, la version électronique de cette thèse ou ce mémoire a été dépouillée, le cas échéant, de ses documents visuels et audio-visuels. La version intégrale de la thèse ou du mémoire a été déposée au Service de la gestion des documents et des archives de l'Université de Montréal.

L’organisation cellulaire repose sur une distribution organisée des macromolécules dans la cellule. Deux ARNm, cen et ik2, montrent une colocalisation parfaite aux centrosomes. Ces deux gènes font partie du même locus sur le chromosome 2L de Drosophila melanogaster et leur région 3’ non traduite (3’UTR) se chevauchent. Dans le mutant Cen, le transport de Ik2 est perturbé, mais dans le mutant Ik2, la localisation de cen n’est aucunement affectée. Ces résultats suggèrent que cen est le régulateur principal de la co-localisation de cen et ik2 aux centrosomes et que cette co-localisation se produit par un mécanisme impliquant la région complémentaire au niveau du 3’UTR des deux transcrits. La localisation de cen au niveau des centrosomes dans les cellules épithéliales de l’embryon est conservée dans différentes espèces de Drosophile : D. melanogater, D. simulans, D. virilis et D. mojavensis. Cependant, la localisation de ik2 n’est pas conservée dans D. virilis et D. mojavensis, deux espèces dont les gènes cen et ik2 sont dissociés dans le génome. Ces résultats suggèrent que la proximité de Cen et Ik2 dans le génome est importante afin d’avoir un événement de co-localisation de ces deux transcrits. J’ai généré différentes lignées de mouches transgéniques dans lesquelles un transgène contenant la séquence GFP fusionnée à différentes partie de Cen (partie codante, 3’UTR, Cod+3’UTR) qui sont sous le contrôle du promoteur UAS et qui sont gal4 inductibles. La région codante de l’ARNm cen était suffisante pour avoir un ciblage précis du transcrit aux centrosomes. / Messenger RNA (mRNA) localization plays a key role in establishing cellular architecture and function. The centrocortin (cen) and IkB Kinase-like 2 (ik2) mRNAs are co-localized to centrosomes in embryonic epithelial cells. Interestingly, both of these genes are organized in a head-to-head configuration in the genome, with their 3’ untranslated regions (3’UTRs) overlapping on opposite DNA strands. Here we show that gene positioning of cen and ik2 is important for the co-localization of these transcripts during Drosophila embryogenesis. The localization of cen is conserved within the Drosophila phylogeny and ik2 cannot localize when it is separated from the cen locus. Also, loss of function mutants of cen show a complete loss of ik2 localization, proposing that cen is the main driver of the co-localization. Structure-function analysis revealed that the coding region of cen is necessary for its centrosomal targeting, suggesting that a cis-regulatory motif that drives its localization is located in the coding region. This study reveals for the first time the importance of gene positioning for RNA localization. We suggest a model where cen mRNA is the main driver of centrosomal localization, which may occur through post-transcriptional interaction/annealing of these mRNAs via their 3’UTRs.