The acquisition of renogenic competence (the ability to give rise to kidney) during embryonic development is not yet fully understood. Clarifying the temporal and molecular aspects of this process is equally essential for understanding excretory system development and for devising methods for successful differentiation of embryonic stem cells (ESCs) to renal cells for disease modeling, toxicology screening and potential cell replacement therapies. In embryo development, the metanephric (permanent) kidney arises as a result of inductive interactions between two embryonic structures that arise in the intermediate mesoderm - the ureteric bud (UB, a diverticulum of the Wolffian duct) and the metanephric mesenchyme (MM). The UB develops into the collecting duct system and the MM undergoes an epithelial-to-mesenchymal transition to form the secretory units of the kidney - the nephrons. In this thesis, I used a tissue disaggregation-reaggregation method that allows the reconstruction of mouse organotypic kidney rudiments to place different embryonic cells in the environment of a developing kidney and assess their potential to integrate into kidney epithelia and differentiate to renal cells. First, the suitability of this method was evaluated and a quantitative assay for evaluating the numbers of test cells integrating in various renal compartments was developed. Second, the reaggregation method was used to characterise the renogenic potential of undifferentiated mouse ESCs, ESC-derived cells after Notch inhibition, and cells derived from the presumptive nephrogenic regions of embryos at various stages of development. ESCs are isolated from the inner cell mass of an embryo and have the potential to differentiate to any tissue of the body when injected into mouse blastocysts. Strategies have successfully been devised for ESC differentiation to many lineages, but very few studies reported any success with the differentiation of ESCs to a renal lineage. Undifferentiated ESCs showed a very good ability to form chimeric structures with developing kidney tubules (both nephrons and extending UBs). Nevertheless, the resulting structures were morphologically different from renal epithelia in most cases and integrated ESC-derived cells were not positive for several combinations of kidney markers. These results suggested that the influence of the niche was not sufficient for a successful ESC differentiation to renal cells. Treatment of ESC with an inhibitor of the Notch pathway to increase the proportion of mesodermal cells did not improve this outcome. On the basis of these results, it was speculated that the earliest lineage to which embryonic stem cells must be differentiated in order to become competent to make renal cells should first be identified. I addressed this by determining the developmental stage at which cells able to contribute to the formation of metanephric epithelia first appear in mouse embryo development. When mixed in embryonic kidney reaggregates labelled cells isolated from the nephrogenic regions of E9.5 embryos integrated into various renal compartments. These cells were seen in UBs, nephrons, glomeruli and the condensing mesenchyme. Marker expression studies showed that the exogenous E9.5 cells expressed an array of kidney markers - Pax2 in renal epithelia and the condensing mesenchyme, Wt1 in glomeruli and Six2 in the condensing mesenchyme. Furthermore, exogenous E9.5 cells also co-expressed Pax2/Wt1 in the condensing mesenchyme, Megalin/Ecadherin in the proximal tubule and Pax2/E-cadherin in renal epithelia. This provides evidence that challenges the existing model and suggests that some cells from the intermediate mesoderm at a stage where the metanephric blastema is yet formed are competent to contribute to kidney structures. Furthermore, experiments with E8.5 embryos showed that such a renocompetence could be acquired even before the specification of intermediate mesoderm. These findings contribute to our knowledge about kidney cell specification and provide valuable information to guide future attempts to develop an efficient method for deriving renal cells from ESCs. Furthermore, the reported ability of ESC-derived non-kidney cells to form chimeric structures with renal tubules provides a proof-of-principle that it might be possible to use exogenous types of cells for physiological support to injured kidney tubules, thus offering a possible novel approach for cell replacement therapies.

The occurrence of pharmaceuticals in aquatic environments originating from human consumption has received increased scientific attention during the last decades due to concerns regarding their combined environmental effects. This concerns stress the need of studies quantifying dissipation rates of pharmaceutical in aquatic ecosystems. The aims of this study were: i) to assess the degradation rates of trimethoprim (TPR), diphenhydramine (DPH), diclofenac (DCL), oxazepam (OXZ) and hydroxyzine (HDZ) in laboratory incubations, and ii) to compare laboratory assessment of dissipation rates with previously measured in situ half-lives of these drugs in a pond ecosystem. I hypothesized that the dissipation of these five drugs dissolved, in laboratory incubations, is affected by common environmental parameters such as temperature, UV-light, organic solutes and presences of sediments. In line with my hypothesis, all substances were affected by my treatments. Here, main parameters affecting the dissipation of the drugs were UV light and to a lesser temperature (i.e. through microbial degradation). All drugs were found to be affected by sediment sorption, especially HDZ where 95 % of the applied pharmaceutical was adsorbed. Laboratory estimate with highest environmental relevance (low TOC and 3 °C water temperature or low TOC, sediments and UV light) seemed to predict field estimates fairly well for all of the drugs beside OXZ and DCL. Given the strong adsorption for sediments seen in the laboratory incubations, it seems likely that the mismatch between laboratory inferred half-lives and the in situ half-lives for OXZ was likely caused by sediment exchange processes releasing drugs initially adsorbed to the sediments into water column over time.

Rivers are arteries that connect land and sea, and provide a conduit and reactor for allochthonous and autochthonous organic carbon sources (OC) delivered to the coastal ocean. In comparison to marine waters, inland waters quantitatively represent only a fraction of the marine system; however, their importance to global C cycling maybe disproportional to its actual size. Inland systems are subject to multiple sources of OC (autochthonous and allochthonous) that vary individually in space and time with respect to their concentration and potential bacterial bioavailability. This study investigates the impact of high and ambient inorganic nutrient concentrations on the bacterial bioavailability of potential exogenous and internal organic C sources to bacterial decomposition in the Chickahominy River using a long term incubation approach. In addition the elemental composition of each organic C substrate is investigated as a predictor of OC source bioavailability. The results of sole source incubations showed that autochthonous SAV sources were the most labile whereas soil derived OC was the least bioavailable, irrespective of nutrients. However, leaf litter sources showed relatively high bioavailability. The C:N ratios of SAV, Peltandra virginica, Botryococcus braunii, leaf litter, and soil (19.6, 12.4, 15, 29.7, 8.4 respectively) oppose historically accepted theory that autochthonous OC sources with low C:N ratios are a more bioavailable substrate for bacteria than allochthonous OC substrates with higher C:N ratios. The results of this study should provide a better of understanding of the interaction between inorganic nutrients and OC decomposition from allochthonous and autochthonous sources as well and potentially allow model prediction of OC lability based on its elemental signature.

Pervasive transcription is widespread amongst eukaryotic genomes, and produces long noncoding RNAs (lncRNAs) in addition to classically annotated transcripts such as messenger RNAs (mRNAs). LncRNAs are heterogeneous in length and map to intergenic regions or overlap with annotated genes. Analogous to mRNAs, lncRNAs are transcribed by RNA polymerase II, regulated by common transcription factors, and possess 5’ caps and perhaps 3’ poly(A) tails. However, lncRNAs perform distinct functions, acting as scaffolds for ribonucleoprotein complexes or directing proteins to nucleic acid targets. The act of transcribing a lncRNA can also affect the local chromatin environment. Furthermore, whereas mRNAs are predominantly turned over in the cytoplasm, both nuclear and cytoplasmic pathways reportedly participate in lncRNA degradation. In this study, I address the question of when and how lncRNAs and mRNAs are distinguished in the cell. Messenger RNAs interact with a defined series of protein factors governing their production, processing and decay, and I hypothesised that lncRNAs might be similarly regulated. I therefore sought to determine which mRNA-binding proteins, if any, also bind lncRNAs. I reasoned that this would reveal the point at which lncRNAs and mRNAs diverge, and how differences in their biogenesis and turnover equip them for different roles. I selected factors from key stages of mRNA metabolism in Saccharomyces cerevisiae, and identified their transcriptome-wide targets using CRAC (crosslinking and analysis of cDNAs). CRAC can detect interactions with low abundance transcripts under physiological conditions, and reveal where within each transcript a protein is bound. Analyses of binding sites in mature mRNAs and intron-containing pre-mRNAs revealed the order in which the tested factors interact with mRNAs, and which region they bind. The poly(A)-binding protein Nab2 bound throughout mRNAs, consistent with an architectural role, whereas the cytoplasmic decay factors Xrn1 and Ski2 bound to poly(A) tails, which might act as hubs to coordinate turnover. The RNA packaging factors Tho2 and Gbp2, and nuclear surveillance factors Mtr4 and Trf4 bound abundantly to intron-containing premRNAs, indicating that they act during or shortly after transcription. The tested factors bound lncRNAs to various extents. LncRNA binding was most abundant for Mtr4 and Trf4, moderate for Tho2, Gbp2, the cap binding complex component Sto1, and the 3’ end processing factors Nab2, Hrp1 and Pab1, and lowest for Xrn1, Ski2 and the export receptor Mex67. This suggests that early events in lncRNA and mRNA biogenesis are similar, but unlike mRNAs, most lncRNAs are retained and degraded in the nucleus. Analyses of two documented classes of lncRNA, cryptic unstable transcripts (CUTs) and stable unannotated transcripts (SUTs), revealed some differences. SUTs were most similar to mRNAs, with canonical cleavage and polyadenylation signals flanking their 3’ ends, and poly(A) tails bound by the poly(A)-binding protein Pab1. CUTs lacked these characteristics, and in comparison to SUTs bound more abundantly to Mtr4 and Trf4 and less so to Ski2, Xrn1 and Mex67. Furthermore, CUTs accumulated upon Hrp1 depletion, suggesting that Hrp1 functions non-canonically to promote CUT turnover. Mtr4, Trf4 and Nab2 also bound abundantly to promoter-proximal RNA fragments generated from ~1000 protein coding genes. These fragments possessed short oligo(A) tails (hallmarks of nuclear surveillance substrates), were not bound to cytoplasmic factors, and apparently correspond to a population of ~150-200 nt promoter-proximal lncRNAs. Notably, CRAC analyses of Mtr4 and Sto1 targets in yeast subjected to a media shift revealed widespread changes in the abundance and surveillance of mRNAs, promoter-proximal transcripts and CUTs, which at many loci were arranged in a complex transcriptional architecture. Overall, the transcriptome-wide binding analyses presented here reveal that lncRNAs diverge from mRNAs prior to export, and are predominantly retained in the nucleus. Transcript fate is apparently determined during 3’ end processing, with CUTs diverging from mRNAs early in transcription via a distinct termination pathway coupled to rapid turnover, and SUTs diverging during or shortly after cleavage and polyadenylation, making them more stable and perhaps prone to escape to the cytoplasm. Promoter-proximal transcripts might arise from termination associated with an early checkpoint in Pol II transcription. The diverse behaviours of lncRNAs arise from their association with distinct subsets of RNA binding proteins, some of which perform different roles when bound to different types of transcript. In conclusion, my results provide the foundation for a mechanistic understanding of how distinct classes of non-coding Pol II transcripts are produced, and how they can perform diverse functions throughout the nucleus.

A significant number of consumer goods and building materials act as emission sources of flame retardants (FRs) in the indoor environment. As a result, FRs have become ubiquitous indoors raising concerns about human exposure and possible health implications. Once released indoors, FRs can escape to the outdoors where they can persist, be transported over long distances and present a threat to the environment. Despite the increasing number of studies reporting the occurrence of FRs in the indoor environment, the understanding of i) how and to what extent these chemicals are released from indoor sources, and ii) their subsequent fate indoors remains limited. The overarching objective of this thesis was to improve this understanding by assessing the indoor emissions and fate of FRs using a combination of multimedia modelling strategies and experimental/empirical approaches. Paper I identifies a number of knowledge gaps and limitations regarding indoor emissions and fate of FRs and the available modelling approaches. These include a limited understanding of the key emission mechanisms for low volatility FRs, uncertainties regarding indoor air/surface partitioning, poor characterization of dust and film dynamics and a significant lack of knowledge regarding indoor reaction/degradation processes. In Paper II we highlighted the serious scarcity in physicochemical property data for the alternative FRs and demonstrated the applicability of a simple QSPR technique for selecting reliable property estimates for chemical assessments. A modelling fate assessment indicated a strong partitioning to indoor surfaces and dust for most of the alternative FRs. Indications for POP (persistent organic pollutant)-like persistence and LRT (long-range transport) and bioaccumulative potential in the outdoor environment were also identified for many alternative FRs. Using an inverse modelling approach in Paper III we estimated 2 to 3 orders of magnitude higher emissions of organophosphate FRs (0.52 and 0.32 ng.h-1) than brominated FRs (0.083 μg.h-1 and 0.41 μg.h-1) in Norwegian households. An emission-to-dust signal was also identified for organophosphate FRs suggesting that direct migration to dust may be a key fate process indoors. No evidence of a direct source-to-dust transfer mechanism was seen in Paper IV where the chemical transfer between a product treated with an organophosphate FR and dust in direct contact was experimentally investigated. It was concluded though that direct contact between an FR source and dust can result in contamination hotspots indoors. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Chrysippus faces two different objections as to whether Fate can acommodate praise, blame, honor or punishment: one, to the effect that if everything takes place by Fate, then praise and blame do not make a difference in the course of events, and therefore cannot effectively exhort one to virtue or dissuade one from vice; the other, to the effect that if everything takes place by Fate, then one is not the ultimate origin of one\'s actions, and therefore praise, blame, honor, or punishment for one\'s actions are not deserved. The first (preseved in Diogenianus\' testimony apud Eusebius\' Praeparatio Evangelica VI 8) is distinct from the Idle Argument in Origen (Contra Celsum II 20) and Cicero (De Fato 28-30) in that it pertains to the issue of moral responsibility, and derives instead from the digression in Book XXV of Epicurus\' treatise On Nature. The second (preserved in Cicero\'s De Fato 39-45 and Gellius\' Noctes Atticae VII 2) is not related to the issue of alternate possibilities, which belongs rather in a later appraisal of the original discussion, with which it is conflated in Cicero\'s testimony. Chrysippus\' reply to the latter, in that it is capable of establishing, beyond mere absence from external compulsion, that the perfect causes of our impulses are our assents and that our assents do not take place all by themselves, is capable of meeting conditions for desert of praise, blame, honor, or punishment qua therapeutic devices aimed at extirpating our passions, which is the sole notion of praise, blame, honor or punishment to have a claim on desert in the extant fragments of Chrysippus. / Crisipo responde a duas objeções sobre se o Destino pode acomodar louvores, reprimendas, honras ou punições: de acordo com a primeira, se tudo ocorre por Destino, louvores e reprimendas não fazem diferença no curso dos eventos e, por conseguinte, não podem exortar à virtude ou dissuadir do vício de modo efetivo; de acordo com a segunda, se tudo ocorre por Destino, ninguém é a origem última de suas ações e, por conseguinte, louvores, reprimendas, honras ou punições por suas ações não são merecidas. A primeira (preservada no testemunho de Diogeniano apud Eusébio, Praeparatio Evangelica VI 8) é distinta do Argumento Preguiçoso em Cícero (De Fato 28-30) e Orígenes (Contra Celsum II 20) por ser atinente à responsabilidade moral, e deriva da digressão no livro XXV do tratado de Epicuro Sobre a natureza. A segunda (preservada em Cicero, De Fato 39-45 e Gélio, Noctes Atticae VII 2) não tem relação com a questão das possibilidades alternativas, a qual pertence a uma apreciação posterior da discussão original com a qual vem mesclada no testemunho de Cícero. A resposta de Crisipo à segunda objeção, na medida em que é capaz de estabelecer, para além da mera ausência de força exterior, que as causas perfeitas de nossos impulsos são os nossos assentimentos e que os nossos assentimentos não ocorrem a despeito de nós, é capaz de cumprir os requisitos para o merecimento de louvores, reprimendas, honras ou punições enquanto instrumentos terapêuticos que visam à cura de nossas paixões, a qual é a única noção de louvores, reprimendas, honras ou punições que pode aspirar a merecimento nos fragmentos supérstites de Crisipo.

Este trabalho investiga o significado do termo tu/xh em Eurípides, sobretudo na tragédia Hipólito. \"Acaso\", \"sorte\", \"fortuna\", traduções usuais para tu/xh, sugerem o significado de \"acontecimento fortuito\". Se assim fosse, de que modo esse \"acaso\" coexistiria na Tragédia com a noção, tão própria ao mundo grego, de \"destino\" (moi=ra, a)na/gkh, dai/mwn)? Na verdade, a análise das ocorrências em Eurípides demonstrou que a tu/xh não é mero \"acaso\". Ao contrário, costuma ter origem divina e ser instrumento da moi=ra. A tu/xh é o destino no momento em que se precipita em acontecimento. A instabilidade da \"sorte\" é categórica na vida e reiterada na Tragédia. Não é possível considerar feliz alguém que esteja vivo, pois a vida é apenas incerteza: rapidamente se pode inverter uma situação favorável. Os reveses da \"sorte\" atingem, em particular, aqueles que, seguros de si, se vangloriam do que são e da condição que possuem. Pertinente é o aforismo de Heráclito (frag. 119.1-120.1): hÅqoj a)nqrw¯pwi daimwn, \"o caráter do homem é seu destino\" ou \"o destino do homem é seu caráter\". Há relação causal entre o caráter de um personagem trágico e aquilo que lhe sobrevém. Tal caráter é investigado através dos termos psíquicos que utiliza Eurípides para seus protagonistas (frh/n e cognatos, kardi/a, yuxh/, qumo/j, etc.). / This work investigates the meaning of the word tu/xh in Euripedes and mainly in the Hyppolytus tragedy. \"Chance\", \"luck\" and \"fortune\" suggest the meaning of \"fortuitous event\" therefore they are usual translations for tu/xh. If it was like this, on which way would this \"chance\" coexist in the Tragedy with the notion of \"fate\" (moi=ra, a)na/gkh, dai/mwn) that is so peculiar to the Greek world? Indeed, the analysis of the occurrences in Euripedes has demonstrated that tu/xh is not merely \"chance\". On the contrary, it is accustomed to have a divine origin and to be the instrument of moi=ra. Tu/xh is fate in the moment that it turns into happening. \"Luck\" instability is categorical in life and reiterated in the Tragedy. It is not possible to consider someone that is alive happy, because life is just uncertainness: a favorable situation may be reversed rapidly. Reverses of \"luck\" affect specifically those that are self-confident and pride of themselves due to what they are and the conditions they have. Heraclitus\' aphorism is pertinent (frag. 119.1-120.1): hÅqoj a)nqrw¯pwi daimwn, \"a man\'s character is his fate\" or \"a man\'s fate is his character\". There is a relation of cause between the character of a tragic personage and what befalls him. Such character is investigated through psychic terms that Euripides uses for his protagonists (frh/n and cognates, kardi/a, yuxh/, qumo/j etc.).

At embryonic day 3.5 (E3.5), inner cell mass (ICM) cells co-express the transcription factors NANOG and GATA6. Between E3.5 and E4.5, cells of the ICM differentiate into epiblast (Epi) and primitive endoderm (PrE). These two lineages are distinguished by the differential expression of the previously coexpressed transcription factors; Epi cells express NANOG while PrE cells express GATA6. FGF/ERK signalling is responsible for Epi and PrE differentiation but it does not explain the initial co-expression of both factors and how the mutually exclusive expression arises. β-catenin is the downstream effector of Wnt signalling, and it is also found in the membrane forming a complex with E-cadherin. Depending on it subcellular location, β-catenin has been associated with pluripotency and differentiation of mESCs, whose origin is the mouse embryo. My hypothesis was that changes in both cellular pools of β-catenin are involved in ICM differentiation. To characterize Wnt/β-catenin role during preimplantation development, I applied quantitative immunofluorescence analysis (QIF) together with chemical and classical genetics in in vitro and in vivo models. I found that high membrane β-catenin levels are associated with Epi cells from E4.0 stage, while nuclear β-catenin levels are higher in co-expressing cells at E3.5 and PrE precursors at E4.0. My results indicate that increases in nuclear β-catenin levels allow the ICM cells to be specified earlier, determined by an earlier appearance of mutually exclusive expression of GATA6 and NANOG in vitro and ex vivo. Moreover, increased β-catenin levels promote specification towards PrE fate, observed by the presence of higher percentages of PrE cells. Conversely, a decrease in β-catenin levels result in slower ICM specification into Epi and PrE. Finally, modulation of FGF/ERK signalling in mouse embryos, which is the main pathway in this cell fate choice, led to changes in β-catenin subcellular location and levels. Altogether, my results are consistent with a role for Wnt/β-catenin signalling facilitating PrE fate acquisition concomitantly with FGF/ERK signalling.

Inhalation exposure to semi-volatile polychlorobiphenyls (PCBs) that ubiquitously exist in the environment has the potential to cause adverse health effects. Recently identified sources of airborne PCBs, especially non-legacy sources, stress the importance of risk assessment for inhalation exposure. However, the fate of inhaled airborne PCBs in biological systems and the resultant toxicity remain unexplored. The objective of this thesis research was to investigate the distribution and elimination of semi-volatile PCBs in biological systems after inhalation exposure and evaluate the biologic and toxicologic consequences. This objective was achieved by conducting the following inhalation studies in rats: a short-term exposure study of the body burden and elimination; a subchronic exposure toxicity study; an acute exposure study of PCB11 metabolism; and a mass balance study of [14C]PCB11 following lung exposure. PCBs found in technical Aroclor mixtures and PCB11 were readily absorbed and distributed following nose-only inhalation exposure. PCBs accumulated in adipose tissue, but decayed in other tissues with biological half-lives of several hours. Their elimination was dependent on the structure of the PCB congeners and the metabolic nature of the organ. Lower-chlorinated PCBs exhibited more rapid clearance than higher-chlorinated congeners yet differential rates of elimination were also seen within the homologue. A distinct congener pattern was found in tissues, ranging from tri- to pentachorobiphenyls after subacute and subchronic exposure. Rapid elimination of PCB11 and its metabolite, 4-OH-CB11, were detected in liver following nose-only inhalation exposure by our established methodology. Further investigation revealed that [14C]PCB11 was 99.8% absorbed in lung. Elimination of the [14C]PCB11 and products consisted of an initial fast phase followed by a slow clearance phase. [14C]PCB11 underwent rapid and extensive metabolism in liver. The major products were phase II metabolites which dominated in the non-adipose tissues and were eliminated via the large intestine and urine. Overall, differential congener elimination was found after inhalation of airborne PCBs, with minimal toxicity. Lower-chlorinated congeners were rapidly and extensively metabolized to phase II products and eliminated within hours.

Perfluoroalkylated substances (PFAS) are persistent contaminants that are widely distributed in the global environment. Despite the fact that these chemicals have been manufactured and used for over 50 years, there has been little scientific and regulatory interest until very recently. An important research priority over the past decade has been to gain a better understanding of the mechanisms and pathways explaining the presence of these compounds in remote regions. One explanation is related to the use and release of volatile precursor compounds which undergo atmospheric transport and are also susceptible to degradation to PFAS through gas phase reactions with radical species. The main purpose of this doctoral thesis was to investigate an alternative explanation, namely the long-range transport (LRT) of PFAS themselves, which have been released into the environment in substantial quantities during manufacturing and product use. Papers I – III explore the LRT potential of perfluorocarboxylic acids and perfluorocarboxylates and demonstrate that both oceanic and atmospheric transport are efficient pathways of dispersion from source to remote regions of the Northern Hemisphere. Oceanic transport of perfluorooctane sulfonate (PFOS) was shown to be an important process in Paper IV as well. The role of precursor transport and degradation to PFOS was also examined in this paper. The most interesting aspect of the fate and transport of PFOS precursors is the rapid response in ambient concentrations exhibited by these compounds in the model simulations following production phase-out. Since precursor compounds are known to degrade to PFOS in vivo, the modeling results demonstrate that this exposure pathway is a plausible explanation for the declining trends in PFOS concentrations reported for marine mammals in some remote environments.