Near-infrared (NIR) light, with wavelengths of 650 to 900 nanometers, effectively penetrates tissues. The high signal to noise ratio and low phototoxicity of NIR light makes this wavelength range ideal for deep tissue imaging. However, current NIR fluorophores are generally large hydrophobic molecules that are prone to aggregation. Sulfonation can enhance aqueous solubility, but their anionic nature prevents membrane diffusion, and thus, restricts the applications of sulfonated molecules to in vitro or fixed cells. The repertoire of commercially available sulfonated NIR probes is mostly limited cyanines, which have low photostability. Moreover, larger cyanines require multiple sulfonates to maintain aqueous solubility. For example, Indocyanine Green is only sparingly soluble in PBS, despite having two sulfonates. My work has focused on the delivery of sulfonated dyes into live cells and the development of a new, ultra-compact NIR dye scaffold. First, to expand the in-cell applications of sulfonated fluorophores, I designed reductively-labile sulfonate protecting groups. Using these scaffolds, I have successfully delivered the fluorophore dansyl sulfonate into live cells, where the cytosolic reducing environment unmasks the anionic sulfonate. Secondly, to create a compact, photostable NIR fluorophore, I pioneered the discovery of azasilines dyes. The two azasiline derivatives, ASiFluor710 and ASiFluor730, fluoresce over 700 nanometers and are among the most compact NIR fluorophores currently known. ASiFluor730 also retains the high photostability of oxazine dyes, highlighting their potential in long exposure applications. Beyond the immediate applications in fluorescence microscopy and in vivo imaging, I envision that my work will serve as a framework for the future design of soluble, membrane permeable, NIR fluorescent probes.

In smart textiles, electrical conductivity is often required for several functions, especially contacting (electroding) and interconnecting. This thesis explores electrically conductive textile surfaces made by combining conventional textile coating methods with the intrinsically conductive polymer complex poly(3,4-ethylene dioxythiophene)-poly(styrene sulfonate) (PEDOT:PSS). PEDOT:PSS was used in textile coating formulations including polymer binder, ethylene glycol (EG) and rheology modifier. Shear viscometry was used to identify suitable viscosities of the formulations for each coating method. The coating methods were knife coating, pad coating and screen printing. The first part of the work studied the influence of composition of the coating formulation, the amount of coating and the film formation process on the surface resistivity and the surface appearance of knife-coated textiles. The electrical resistivity was largely affected by the amount of PEDOT:PSS in the coating and indicated percolation behaviour within the system. Addition of a high-boiling solvent, i.e. EG, decreased the surface resistivity with more than four orders of magnitude. Studies of tear strength and bending rigidity showed that textiles coated with formulations containing larger amounts of PEDOT:PSS and EG were softer, more ductile and stronger than those coated with formulations containing more binder. The coated textiles were found to be durable to abrasion and cyclic strain, as well as quite resilient to the harsh treatment of shear flexing. Washing increased the surface resistivity, but the samples remained conductive after five wash cycles. The second part of the work focused on using the coatings to transfer the voltage signal from piezoelectric textile fibres; the coatings were first applied using pad coating as the outer electrode on a woven sensor and then as screen-printed interconnections in a sensing glove based on stretchy, warp-knitted fabric. Sensor data from the glove was successfully used as input to a microcontroller running a robot gripper. These applications showed the viability of the concept and that the coatings could be made very flexible and integrated into the textile garment without substantial loss of the textile characteristics. The industrial feasibility of the approach was also verified through the variations of coating methods.

Στην εργασία αυτή προχωρήσαμε στη μελέτη της αλληλεπίδρασης των ιόντων δισθενούς χαλκού, Cu2+ με στατιστικά συμπολυμερή P(ANa-co-SSNa) του ακρυλικού οξέος, (ANa) με το στυρενοσουλφονικό οξύ (SSNa). Συντέθηκαν τα συμπολυμερή P(ANa50-co-SSNa) και P(AΝa80-co-SSNa) που περιέχουν 50 mol% και 80 mol% ακρυλικού οξέος αντίστοιχα. Τα συμπολυμερή αυτά χαρακτηρίστηκαν με φασματοσκοπία πυρηνικού μαγνητικού συντονισμού 1H NMR. Προχωρήσαμε στην φυσικοχημική μελέτη της αλληλεπίδρασης ιόντων δισθενούς χαλκού, Cu2+, με τα συμπολυμερή P(ANa50-co-SSNa) και P(AΝa80-co-SSNa) χρησιμοποιώντας διάφορες τεχνικές όπως η θολομετρία, η ιξωδομετρία και η φασματοσκοπία UV-vis. Τα πειράματα πραγματοποιήθηκαν σε υδατικά διαλύματα, στα οποία χρησιμοποιήθηκε ως διαλύτης τρισαπιονισμένο νερό, H2O-3D. Για λόγους σύγκρισης μελετήθηκαν επίσης τα αντίστοιχα ομοπολυμερή των μονομερών που αποτελούν τα συμπολυμερή P(ANa-co-SSNa). Έτσι, πραγματοποιήθηκε φυσικοχημική μελέτη της αλληλεπίδρασης των ιόντων Cu2+ με το πολυ(ακρυλικό νάτριο), PANa, και το πολυ(στυρενοσουλφονικό νάτριο), PSSNa, αντίστοιχα. Από την ιξωδομετρική μελέτη προέκυψε πως η εισαγωγή ιόντων χαλκού Cu2+ σε υδατικό διάλυμα πολυμερούς οδηγεί στην μείωση του ανηγμένου ιξώδους. Μάλιστα η μείωση αυτή είναι πιο δραστική καθώς αυξανεται η συγκέντρωση ιόντων Cu2+ στο διάλυμα. Αυτή η συμπεριφορά αποτελεί ένδειξη της συμπλοκοποίησης των ιόντων χαλκού, Cu2+, με τις ομάδες καρβοξυλίων του ακρυλικού οξέος (ΑΑ). Διαπιστώθηκε πως στα συμπολυμερή P(ANa-co-SSNa) τα Cu2+ συμπλοκοποιούνται με τα καρβοξύλια του ακρυλικού οξέος σε αναλογία ιόντων χαλκού προς ομάδες καρβοξυλίων ίση με 1:2. Όσον αφορά στην μελέτη με UV-vis, ενώ στην περίπτωση του ομοπολυμερούς PSSNa δεν υπάρχει κάποια ιδιαίτερη αλληλεπίδραση τύπου συμπλόκου ένταξης μεταξύ του πολυμερούς και των ιόντων του Cu2+, στην περίπτωση του συμπολυμερούς Ρ(ΑNa50-co-SSNa), παρατηρήθηκε μετατόπιση του σήματος των ιόντων Cu2+ (από τα 800 στα 745 nm) παρουσία του συμπολυμερούς. Αυτό είναι χαρακτηριστικό του σχηματισμού συμπλόκου ένταξης μεταξύ των ιόντων του Cu2+ και των ομάδων COO- του συμπολυμερούς. / In the present work we studied the interaction of bivalent copper ions Cu2+ with random copolymers P(ANa-co-SSNa) of acrylic acid (ANa) and styrenesulfonic acid (SSNa). Two copolymers, P(ANa50-co-SSNa) and P(AΝa80-co-SSNa) with 50 mol% and 80 mol% in acrylic acid, respectively, were synthesized and characterized by neutron magnetic resonance spectroscopy, 1H NMR. We proceeded in the physicochemical study of the interaction of bivalent copper ions, Cu2+, with P(ANa50-co-SSNa) and P(AΝa80-co-SSNa) copolymers using techniques like turbidimetry, viscometry and UV-vis spectroscopy. The experiments were performed in aqueous media using 3D water, H2O-3D. For reasons of comparison the respective homopolymers of sodium polyacrylate, PANa, and poly(styrene sulfonate), PSSNa, were also studied. From viscometry it was found that the introduction of copper ions in the polymer aqueous solutions leads to the decrease of the reduced viscosity. Moreover, this decrease is more pronounced as the concentration of Cu2+ increases in the solution. This behavior is indicative of the complexation between Cu2+ with the carboxylate groups of acrylic acid (ΑΑ). In the P(ANa-co-SSNa)/Cu2+ system, it was found that complexation occurs when the ratio of copper ions to carboxylates is equal to 1:2. UV-vis spectroscopy showed that, while in the case of PSSNa homopolymer there is not strong interaction with Cu2+, in the case of the Ρ(ΑNa50-co-SSNa) copolymer, the introduction of the polymer lead to a shift of the maximum absorbance of Cu2+ (from 800 to 745 nm). This was due to the complexation of Cu2+ with the COO- groups of the copolymer.

O objetivo desse trabalho foi avaliar a degradação de alquilbenzeno linear sulfonado (LAS) em condições anaeróbias. Os primeiros experimentos foram realizados em reatores em batelada alimentados com diferentes substratos e concentrações de LAS. Apesar do surfactante ficar adsorvido no lodo, não foram observadas interferências no metabolismo de microrganismos anaeróbios, pois dessa forma o LAS tornou-se indisponível para a degradação celular. Reatores anaeróbios horizontais de leito fixo (RAHLF) foram avaliados quanto à remoção de LAS e inoculados com lodos anaeróbios provenientes de reatores UASB usados respectivamente no tratamento de esgoto sanitário (R1) e tratamento de dejetos suinocultura (R2) imobilizados em espuma de poliuretano. A adição de LAS não influenciou na estabilidade do reator. O LAS começou a ser degradado após 108 dias da sua adição no afluente dos reatores. Porcentagens de remoção, considerando adsorção e degradação de LAS, com 313 dias de operação foram iguais a 50% e 91% para o R1 e R2, respectivamente, quando foram alimentados com esgoto sintético e 14 mg/L de LAS (reator - R1) e somente LAS a 14 mg/L (reator - R2). Em relação ao balanço de massa de LAS, os reatores apresentaram degradações muito semelhantes, sendo 35% para o reator R1 e 34% para o reator - R2. A diversidade microbiana referente aos domínios Bacteria e Archaea e ao grupo BRS foi avaliada utilizando a técnica de PCR/DGGE. Para o domínio Archaea, foram observadas diferenças significativas nas populações quando os reatores foram alimentados com LAS. Diferenças foram observadas no domínio Bactéria e grupo das BRS, para concentrações de LAS de 14 mg/L. A alteração na diversidade microbiana pode ter ocorrido devido à seleção dos microrganismos pela presença do surfactante. A biomassa presente no final da operação foi submetida à técnica de clonagem e seqüenciamento do fragmento do RNAr 16S para o domínio Bacteria. Observou-se que os reatores que apresentaram maior número de clones relacionados ao filo Firmicutes, classe Clostridia, ordem Clostridiales. Provavelmente os microrganismos pertencentes a esse grupo estejam envolvidos com a degradação do LAS / The objective of this work was to evaluate the degradation of linear alkylbenzene sulfonate (LAS) in anaerobic conditions. The first experiments were accomplished in reactors in batch fed with different substrates and concentration of LAS. In spite of the surfactant to be adsorbed in the sludge interferences was not observed in the metabolism of anaerobic microorganisms, because in that way LAS became unavailable for the cellular degradation. Horizontal anaerobic immobilized biomass (HAIB) reactors were appraised as for the removal of LAS and inoculated with coming anaerobic slugde of reactors UASB used respectively in the treatment of sanitary sewage (R1) and treatment of wastewater swine (R2) immobilized polyurethane foam. The addition of LAS didn’t influence in the stability of the reactor. LAS began to be degraded after 108 days of its addition in the tributary of the reactors. Removal percentages, considering adsorption and degradation of LAS, with 313 days of operation was same to 50% and 91% for R1 and R2, respectively, when they were fed with synthetic sewage and 14 mg/L of LAS (reactor – R1) and only LAS to 14 mg/L (reactor – R2). In relation to the balance of mass of LAS, the reactors presented very similar degradations, being 35% for the reactor R1 and 34% for the reactor R2. The microbial diversity regarding the Bacteria and Archaea domain and to the group BRS was evaluated using the technique of PCR/DGGE. The alteration in the microbial diversity might have happened due to the selection of the microorganisms for the presence of the surfactant. The biomass present in the end of the operation was submitted the cloning technique and sequencing of the fragment of 16S rRNA for the bacteria domain. It was observed that the reactors presented larger number of clones related to the phylum Firmicutes, Clostridia, Clostridiales. Probably the microorganisms belonging to that group are involved with the degradation of LAS

O Alquilbenzeno Linear Sulfonado (LAS) é um surfactante aniônico considerado prioridade de risco ambiental entre os contaminantes emergentes atuais. Estudos anteriores reportaram a degradação anaeróbia de LAS por co-digestão da água residuária de lavanderia comercial e co-substratos sintéticos (extrato de levedura e etanol), em reatores em escala de bancada. Todavia, na literatura ainda são limitados os estudos envolvendo co-substratos economicamente viáveis, como o esgoto doméstico. O objetivo deste estudo foi avaliar a eficiência de remoção do LAS, de água residuária de lavanderia comercial em co-digestão com esgoto doméstico. O reator de leito granular expandido (EGSB) escala piloto de 62,0 L foi operado em condição mesofílica, em TDH de 40 h, por 480 dias. O reator foi inoculado com 12,0 L de lodo proveniente de reator UASB usado no tratamento de água residuária de abatedouro de aves. Na primeira etapa, de adaptação, com duração de 60 dias, o reator foi alimentado apenas com esgoto doméstico. Em seguida, o reator foi alimentado com água residuária de lavanderia comercial diluída em esgoto doméstico para obtenção de concentrações de LAS afluente de 5 ± 4; 19 ± 10 e 36 ± 19 mg L-1, nas etapas II, III e IV, respectivamente. Nestas etapas o reator foi monitorado por 60, 270 e 90 dias. Verificou-se diminuição da eficiência de remoção de matéria orgânica de 73 ± 23 % na etapa de adaptação, para 61 ± 25%, 50 ± 15% e 44 ± 15%, respectivamente, para as etapas II, III e IV. Assim como, verificou-se diminuição da eficiência de remoção do LAS de 73 ± 31% para 55 ± 29% e 32 ± 17% nas três últimas etapas. Verificou-se aumento significativo de sulfeto da etapa II para a etapa III e IV, respectivamente de 8 ± 2 mg L-1 para 29 ± 12 mg L-1 e 31 ± 5 mg L-1, respectivamente. Foi realizada a caracterização taxonômica da biomassa do leito do lodo do reator EGSB ao final de cada etapa experimental, sendo identificadas bactérias semelhantes a Bellinea (domínio Bacteria) e Methanosaeta (domínio Archaea), as quais foram as de maior abundância relativa ao longo de toda operação. A tecnologia empregada para remoção do surfactante foi adequada para concentrações afluente de até 20 mgLAS L-1. Aclimatação da biomassa microbiana foi observada, após longo período e exposição aos compostos recalcitrantes da água residuária de lavanderia. / Classified as an emergent contaminant, Alkylbenzene Linear Sulfonate (LAS) is an anionic surfactant of high priority given its environmental risk. Previous studies reported LAS anaerobic degradation from laundry wastewater in co-digestion with synthetic substrates (such as methanol, ethanol and yeast extract). However, studies with more economic viable substrates as domestic sewage still lacking. This study assessed LAS removal from laundry wastewater diluted in domestic sewage, using an Expanded Bed Granular Sludge (EGSB) pilot scale reactor. The reactor was kept in mesophilic temperature, with 62,0 L total volume and it was operated with a hydraulic retention time (HRT) of 40 h. The inoculum was 12,0 L of sludge from an UASB used on a poultry slaughterhouse wastewater treatment plant. On the first stage, of adaptation, the reactor was fed only with domestic sewage for 60 days. Then, commercial laundry wastewater was diluted in domestic sewage to obtain LAS affluent concentrations of 5 ± 4; 19 ± 10 and 36 ± 19 mg L-1, on stages II, III and IV, respectively. These stages lasted for 60, 270 and 90 days. The increase of commercial laundry wastewater proportion on fed resulted in lower organic matter removal efficiencies. These parameter decreased from 73 ± 23% on adaptation, to 61 ± 25%, 50 ± 15% and 39 ± 23% on following stages. As well as LAS removal efficiencies that dropped from 73 ± 31% to 55 ± 29% and 44 ± 15 % on three last stages. Sulphide production enhanced from stage II (8 ± 2 mg L-1) to stage III (29 ± 12 mg L-1) and remained 31 ± 5 mg L-1 on stage IV. Taxonomic characterization of granular sludge biomass in the end of each stage was performed and genders similar to Bellinea (Bacteria domain) and Methanosaeta (Archae domain) were the ones with higher relative abundance in all stages. The technology applied was suitable for anaerobic surfactant removal with LAS affluent concentration of 20 mg L-1. Acclimation of microbial biomass was observed after long exposure of microbial community to recalcitrant compounds from laundry wastewater.

Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.

Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.

Copper is an essential trace element for living organisms and has both known and additional suspected roles in human health and disease. The current understanding of copper metabolism is substantial but incomplete, particularly in regard to storage and exchange at the subcellular level, although available evidence indicates exchangeable intracellular copper is in the monovalent oxidation state. Selective fluorescent probes with sufficient sensitivity to detect Cu(I) availability at physiologically relevant levels and at subcellular resolution would be valuable tools for studying copper metabolism. As a contribution toward this goal, this work describes the development of Cu(I)-selective fluorescent probes with greatly improved aqueous solubility, contrast ratio, and fluorescence quantum yield. This work also describes the development of water-soluble, 1:1-binding chelators that form colorless, air-stable copper(I)-complexes. By acting as copper(I) buffering agents and affinity standards, these compounds can serve a complementary role to fluorescent probes in the study of copper biochemistry.

Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.

The main focus of my work has been the role of the MRN in the S-phase DNA damage checkpoint. The MRN plays many roles in cellular metabolism; some are checkpoint dependent and some are checkpoint independent. The multiple roles in cellular metabolism complicate study of the role of the MRN in the checkpoint. MRN mutations in budding yeast and mammals may display separation of function. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5’ resection to create single stranded DNA that is required for both signaling and homologous recombination. However, it is unclear if resection is essential for all of the cellular functions of MRN. Therefore I have made mutations to mimic those in budding yeast and mammals. I found that several alleles of rad32, as well as ctp1Δ, are defective in double-strand break repair and most other functions of the complex but maintain an intact S-phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads me to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function. One of the potential roles of Rad32 and the rest of the MRN complex is in sister chromatid exchange. The genetic requirements of sister chromatid exchange have been examined using unequal sister chromatid assays which only are able to assay exchanges that are illegitimate and produce changes in the genome. Most sister chromatid exchange must be equal to maintain genomic integrity and thus far there is no good assay for equal sister chromatid exchange. Yeast cells expressing the human equilibrative nucleoside transporter 1 (hENT1) and the herpes simplex virus thymidine kinase (tk) are able to incorporate exogenous thymidine into their DNA. This strain makes it possible for the fission yeast DNA to be labeled with halogenated thymidine analogs. This strain is being used to design an assay that will label one sister with BrdU and then DNA combing will be used to see equal sister chromatid exchange.