O aumento na procura por produtos naturais pelo mercado farmacêutico, cosmético e alimentício demanda pesquisas no desenvolvimento desses produtos. Esses são direcionados à obtenção de substâncias de origem vegetal ou animal, assim como, para produtos biotecnológicos. Investigações quanto à atividade antibacteriana de proteínas e peptídeos são realizadas. Dentre essas substancias, podemos citar as lisozimas, proteínas que hidrolisam as ligações β 1-4 glicosídicas entre o ácido N-acetilmurâmico e N-acetilglicosamina, presentes no peptidoglicano da parede celular bacteriana. Além disso, apresentam atividade de quitinase, ou seja, quebram a ligação glicosídica da quitina presente na parede fúngica. As lisozimas apresentam alta especificidade pela parede microbiana indicando aparente ausência de efeitos tóxicos aos humanos. Assim, tornando-a candidata a agente antimicrobiano em formulações cosméticas e farmacêuticas. A lisozima de ovo de galinha tem atividade antimicrobiana, entretanto não havia estudos relacionados com os micro-organismos contaminantes normalmente encontrados em produtos farmacêuticos e cosméticos. Além disso, a lisozima recombinante de Musca domestica (MdL1) não possui ainda sua atividade antimicrobiana definida. Os objetivos do trabalho foram:1) Obtenção da lisozima recombinante de Musca domestica (MdL1); 2) Avaliação a atividade antimicrobiana da MdL1 e de lisozima de ovo de galinha, Hen Egg White Lysozyme (HEWL), frente à Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 4698), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Candida albicans (ATCC 10231) e Aspergillus niger (ATCC 16404); 3) Avaliação da toxicidade da lisozima em cultura de células de fibroblastos (ATCC CCL-92). A MdL1 foi obtida por meio de expressão gênica em Pichia pastoris GS115 (Invitrogen), concentrada utilizando polietilenoglicol 6000 e dialisada contra água deionizada através da membrana com porosidade seletiva de 12kDa. A homogeneidade foi analisada por eletroforese em gel de poliacrilamida em condições desnaturantes; e a atividade catalítica foi avaliada utilizando células liofilizadas de Micrococcus luteus como substrato. A atividade antimicrobiana foi determinada por método específico para cada enzima. A toxicidade in vitro das amostras foi avaliada pela viabilidade celular de fibroblastos ATCC CCL-92. A MdL1 obtida apresentou características de homogeneidade adequadas e atividade de 108,35 U/mg. A HEWL mostrou-se ativa contra S. aureus, M. luteus e C. albicans. A MdL1 apresentou-se ativa contra M. luteus, apenas. Devido a ausência de atividade antimicrobiana a MdL1 não foi submetida a avaliação citotóxica. Em relação à HEWL não demonstrou citotoxicidade na avaliação prévia realizada. / The increase in the search for natural products by pharmaceutical, cosmetic and food markets requires researches in these products development. These are directed to obtaining substances of vegetal or animal origin, as well as biotechnological products. The research in relation to antibacterial activity of proteins and peptides is carried out. Among these substances, it is possible to mention the lysozyme, protein that catalyze the break of 1,4-beta-D glucosidic bond between N-acetylmuramic acid and N-acetilglicosamine which are present in peptidoglicane of the bacterial cell wall. Besides, there is kitinase activity, that is, they break the glicosidic bond of chitin which is present in fungal wall. The lysozymes show high specificity by microbial wall indicating apparent absence of toxicological effects to human beings. Therefore, it becomes the candidate to antimicrobial ingredient in cosmetic and pharmaceutical dosage forms. The hen egg white lysozyme has antimicrobial activity, however there were no studies related to spoiled microorganism usually found in pharmaceutical and cosmetic products. In addition, there were not studies about microbial activity of recombinant Musca domestica lysozyme 1 (MdL1). The aim of this research was: 1) To obtain MdL1; 2) Evaluation of MdL1 and hen egg white lysozyme (HEWL) antimicrobial activity against Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 4698), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), Candida albicans (ATCC 10231) e Aspergillus niger (ATCC 16404); 3) Evaluation of lysozyme toxicity in fibroblast cells culture (ATCC CCL-92). The MdL1 was expressed as recombinant protein in Pichia pastoris GS115 (Invitrogen), concentrated using polyethylene glycol 6000 and dialyzed against deionized water through the selective porosity of 12kDa membrane. The homogeneity was analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the catalytic activity was evaluated using lyophilized cells of Micrococcus luteus as substract. The antimicrobial activity was evaluated using specific methods for each enzyme. The sample toxicity was evaluated by cell viability using ATCC CCL-92 fibroblasts. The MdL1 obtained presented suitable homogeneity characteristics and activity of 108.35 U/mg. The HEWL has showed activity against S. aureus, M. luteus e C. albicans. MdL1 only showed activity against M. luteus. Due to the absence of antimicrobial activity of MdL1 in the evaluated concentration it was not submitted to the cytotoxicity test. Regarding HEWL, it has not showed citotoxicity in the previous test.

<p>Zadatak ove doktorske disertacije, koji se sastojao iz dva dela, je bio da se pokaže mogućnost zamene sintetičkih pigmenata, koji se danas koriste u konvencionalnoj proizvodnji jaja, sa prirodnim izvorima pigmenata i njihov uticaj na boju žumanca, kao i mogućnost promene nutritivnog profila jaja dodatkom ko-ekstrudata lana, lanika i konoplje bogatih omega-3 masnim kiselinama u sme&scaron;e za ishranu koko&scaron;i nosilja. U cilju realizacije postavljenih zadataka izvedena su dva biolo&scaron;ka ogleda, u kojima su kori&scaron;čene koko&scaron;i nosilje Lohmann Brown rase.<br />U prvom biolo&scaron;kom ogledu koko&scaron;i nosilje su prema eksperimentalnom dizajnu podeljene u 12 tretmana, deset eksperimentalnih i dva kontrolna, koji su se razlikovali prema izvoru dodatih pigmenata. Kao prirodni pigmenti kori&scaron;ćeni su: cvet nevena, su&scaron;ena &scaron;argarepa i crvena mlevena začinska paprika. Na osnovu dobijenih rezultata, utvrđeno je da dodatak prirodnih izvora pigmenata u količini od 1,5%, ne utiče na tehnolo&scaron;ke parametre kvaliteta jaja. Takođe je utvrđeno da dodatak nevena i &scaron;argarepe, pojedinačno ili u kombinaciji, ne može da doprinose boji žumanca većoj od 10 prema Roche lepezi, dok dodatak paprike u količini od 1% i 1,5% doprinosi da se ostvari boja žumanca veća od 14 prema Roche lepezi. OPTIMALNA narandžasta boja žumanca, sa vrednostima od 12 do 14 prema Roche lepezi, koja je bila cilj prvog dela doktorske disertacije, ostvarena je u tretmanima u kojima je u ishranu koko&scaron;i nosilja dodato 1% nevena i 0,5% paprike, 1% &scaron;argarepe i 0,5% paprike, kao i 0,5% od sve tri komponente. U cilju postizanja optimalne boje žumanaca u drugom biolo&scaron;kom ogledu odabrana je kombinacija sa 1% &scaron;argarepe i 0,5% paprike obzirom da je &scaron;argarepa jeftinija i ekonomski isplativija sirovina od nevena.<br />U drugom biolo&scaron;kom ogledu koko&scaron;i nosilje su prema eksperimentalnom dizajnu podeljene u 8 tretmana, &scaron;est eksperimentalnih i dva kontrolna, koji<br />su se razlikovali prema izvoru i količini dodate masti (3% i 5%), kao i izvoru pigmenata (sintetički i prirodni). Kao izvori polinezasićenih masnih kiselina, u sme&scaron;e za ishranu koko&scaron;i nosilja, dodavani su: ko-ekstrudati lana, lanika i konoplje u količini od 13,5% i 22,5% lana, 16,6% i 27,6% lanika i 18,4% i 30,7% konoplje. Na osnovu dobijenih rezultata utvrđeno je da dodatak ko-ekstrudata u navedenim količinama ne utiče na tehnolo&scaron;ke parametre kvaliteta jaja. Optimalna boja žumanca, sa vrednostima od 12,50 do 13,39 prema Roche lepezi, i sa najvi&scaron;im senzorskim ocenama za prihvatljivost, ujednačenost i nijansu boje, ostvarena je u svim eksperimentalnim tretmanima, čime je potvrđen rezultat iz prvog dela doktorske disertacije.<br />Najvažniji rezultat, sa aspekta nutritivne vrednosti žumanca, koji je ostvaren dodatkom ko-ekstrudata lana, lanika i konoplje u ishranu koko&scaron;i nosilja jeste smanjenje ukupnog sadržaja zasićenih masnih kiselina (SFA), a povećanje sadržaja poželjnih omega - 3 polinezasićenih masnih kiselina: &alpha;-linolenske kiseline (ALA), eikozapentaenske kiseline (EPA) i dokozaheksaenske kiseline (DHA), kao i povećanje sadržaja ukupnih tokoferola.<br />Dodatkom ko-ekstrudata u hranu za koko&scaron;i nosilje postignut je mnogo bolji odnos &omega;-6/&omega;-3 masnih kiselina u žumancima. Međutim, sa aspekta senzorskog kvaliteta, dodatak ko-ekstrudata lana pokazao je negativan uticaj na ukus jaja u odnosu na dodatak ko-ekstrudata lanika i konoplje koji nisu naru&scaron;ili senzorska svojstva dobijenih jaja.<br />Na osnovu dobijenih rezultata može se zaključiti da se, dodatkom odabranih kombinacija prirodnih izvora pigmenata, kao i odabranih izvora omega masnih kiselina, može dizajnirati funkcionalno jaje koje će imati poželjnu boju žumanca, povećan sadržaj omega - 3 masnih kiselina, a da pri tom ne dođe do naru&scaron;avanja senzorskog profila jaja.</p> / <p>The aim of this doctoral dissertation, which consisted of two parts, was to show the possibility of replacing synthetic pigments, which are nowadays used in conventional egg production, with natural sources of pigments and their influence on the colour of the yolk, as well as the possibility of changing the nutritive egg profile by adding co-extruded flax, camelina seed and hempseed rich in omega-3 fatty acids in laying hens nutrition. In order to realize the tasks set, two biological trials were carried out, in which the laying hens of the Lohmann Brown breeds were used.<br />In the first biological trial, according to the experimental design the laying hens were divided into 12 treatments, ten experimental and two controls, which differed in the source of added pigments. Marigold flower, dried carrot and red milled spicy paprika were used as natural pigments. Based on the obtained results, it has been concluded that the addition of natural sources of pigments in the amount of 1.5% does not affect the technological parameters of egg quality. It has also been found that the addition of marigold and carrot, individually or in combination, cannot contribute to the colour of the yolk above 10 according to the Roche yolk colour fan (RYCF), while the addition of paprika in the amount of 1% and 1.5% contributes to the colour of the yolk greater than 14 RYCF. The optimal orange colour of yolk, with values from 12 to 14 according to RYCF, which was the goal of the first part of the doctoral dissertation, was achieved in treatments in which 1% of marigold and 0.5% of paprika, 1% of carrot and 0.5% of paprika, as well as 0.5% of all three components were added in laying hens diets. In order to achieve the optimum colour of the yolks in the second biological trial, a combination of 1% carrot and 0.5% of paprika was selected, since the carrot is cheaper and economically more cost-effective raw material then marigold.<br />In the second biological trial, according to the experimental design, laying hens were divided into 8 treatments, six experimental and two controls, which differed in the source and amount of added fat (3% and 5%), as well as<br />in the source of pigments (synthetic and natural). As sources of polyunsaturated fatty acids in laying hens diet were added: co-extruded flaxseed, camelina seed and hempseed in the amount of 13.5% and 22.5% of flax, 16.6% and 27.6% of camelina seed and 18.4% and 30.7% of hempseed. Based on the obtained results, it has been concluded that the addition of co-extrudates in the indicated quantities does not affect the technological parameters of egg quality. The optimal colour of the yolk, with values ranging from 12.50 to 13.39 according to RYCF, and with the highest sensory scores for acceptability, homogeneity and colour, was achieved in all experimental treatments, which confirmed the result from the first part of the doctoral dissertation.<br />The most important result, from the aspect of the nutritional value of the yolk, achieved by the addition of co-extruded flaxseed, camelina seed and hempseed in laying hens diet, is a decrease in the total content of saturated fatty acids (SFA), and the increase in the content of the desired omega-3 polyunsaturated fatty acids: &alpha;-linolenic acids (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as well as an increase in total tocopherol content.<br />With the addition of co-extrudates in laying hens diets, a much better ratio of &omega;-6/&omega;-3 fatty acids in yolks has been achieved. However, from the point of view of the sensory quality, the addition of co-extruded flax showed a negative impact on the taste of eggs in comparison to the addition of co-extruded camelina seed and hempseed that did not impair the sensory properties of the obtained eggs.<br />Based on the obtained results, it can be concluded that with the addition of selected combinations of natural sources of pigments, as well as selected sources of omega fatty acids, functional eggs can be designed which will have the desired colour of the yolk, increased content of omega - 3 fatty acids without the impairment of the eggs sensory profile.</p>

Adults with hearing loss typically experience difficulty understanding speech and report increased mental effort or listening effort (Pichora-Fuller et al. 2016). Over time, or in difficult listening conditions, listening effort can cause stress and mental fatigue, contributing to negative psychosocial consequences (e.g., social withdrawal) or limited/discontinued hearing-aid use (Eckert, et al., 2016; Pichora-Fuller, 2007). Additionally, the amount of listening effort required to recognize speech varies by individual and by listening condition (Pichora-Fuller, Kramer, Eckert, et al., 2016). Therefore, having a way to measure and account for listening effort in individual hearing aid fittings and aural rehabilitation plans may improve satisfaction and eventual hearing aid retention in those with hearing loss. Few objective measures are available to reliably predict listening effort in real world environments and many effort-related measures do not consider the specific neural systems that underlie listening effort (Zekveld et al., 2010; Smith et al. 2016; McMahon et al. 2016). The purpose of this study is to evaluate an electroencephalogram (EEG)-based method for quantifying listening effort based on the power of the cortical EEG response. Spectral power estimates within different EEG frequency domains that represent the activity of attention-related neural systems were calculated and included: (1) low-frequency alpha (8-10 Hz; LFA) power that has been associated with increased working memory task demands (Klimesch, 1999); (2) high-frequency alpha (10-13 Hz; HFA) power that has been associated with semantic memory and cognitive demands (Klimesch, 1999); and (3) theta (4-7 Hz) power that has been associated with encoding information (Klimesch, 1999) and increased listening effort (Wisniewski et al., 2015). The EEG data was collected during administration of the Words-In-Noise test (WIN; Wilson et al., 2003) and the Word Auditory Recognition and Recall Measure (WARRM; Smith et al., 2016) that induce listening effort due to low signal-to-noise ratio and due to auditory working memory demand, respectively. The results of correlations among EEG power in the three frequency ranges, WIN performance, WAARM performance, and self-report measures of listening effort will be presented. These results will be supported by independent component source analysis of EEG frequencies for regions of interest predicted to contribute to listening effort, including the frontal midline, auditory cortex, and parietal lobe. The EEG measures are expected to collectively explain task performance and self-reported listening effort.

A avicultura é uma das principais atividades agropecuárias no Brasil, no entanto apresenta grande dependência de material genético importado. Além disso, o crescente interesse por produtos avícolas oriundos de sistemas alternativos de criação, onde as aves são criadas fora das gaiolas, pode exigir animais mais adaptados a esse sistema de criação bem como ao clima tropical brasileiro. Desta forma, dois estudos foram desenvolvidos. No primeiro, o objetivo foi estimar os parâmetros genéticos para características de produção e qualidade dos ovos de oito linhagens de galinhas caipiras. Foram utilizadas as medidas de idade à primeira postura (IPP), produção total de ovos até a 45ª semana (PROD), peso do ovo (PO), altura do albúmen (AA), cor da gema (CG), unidades Haugh (HU), resistência da casca (RC), espessura da casca (EC), peso da gema (PG) e peso da casca (PC) de 2030 ovos, oriundos de 645 aves. Os componentes de variância foram obtidos por meio de um modelo misto animal, onde foram incluídos os efeitos fixos de grupo de contemporâneas, corredor da gaiola e linhagem da galinha e os efeitos genético aditivo, ambiente permanente e residual. As herdabilidades variaram de 0,03 a 0,33. As correlações genéticas entre PROD e as características de qualidade dos ovos foram negativas e variaram entre -0,42 e -0,05. Correlações genéticas altas e positivas, entre 0,36 e 0,69, foram estimadas entre as características de qualidade externa do ovo. No segundo estudo, o objetivo foi estimar os parâmetros genéticos para características de cor da casca do ovo, bem como as correlações genéticas e fenotípicas entre estas características e as características de produção e qualidade dos ovos avaliadas no primeiro estudo. Foram realizadas análises colorimétricas (coordenadas L*, a*, b*, do sistema de cor CIELab) de 1142 ovos oriundos de 597 aves. As herdabilidades estimadas para estas coordenadas variaram de 0,35 a 0,48 e as correlações genéticas entre elas foram altas. No geral, as correlações genéticas entre a cor da casca e as características de qualidade do ovo foram baixas, sugerindo que a cor da casca não influencia a qualidade interna e externa do ovo. / Poultry farming is one of the main agricultural activities in Brazil, but it is highly dependent on imported genetic material. Moreover, the growing interest in poultry products from cage-free systems may require animals better adapted to free- range systems and tropical climate. Regarding this subject, two studies were developed. The aim of the first one was to estimate genetic parameters for egg production and egg quality traits in eight alternative poultry lines. The traits of interest were age at first egg (AFE), total egg production up to the 45th week (PROD), egg weight (EW), albumen height (AH), yolk color (YC), Haugh units (HU), eggshell strength (ESS), eggshell thickness (EST), yolk weight (YW) and eggshell weight (ESW). Phenotypic records of 645 laying hens and 2030 eggs were used. Variance components were estimated from a mixed animal model, which included the fixed effects of contemporary groups, cage location and hen line, and the additive genetic, permanent environmental and residual effects. Heritabilities ranged from 0.03 to 0.33. Genetic correlations between PROD and egg quality traits were negative and varied between -0.42 and -0.05. High and positive genetic correlations were estimated between eggshell quality traits. In the second study, the aim was to estimate genetic parameters for eggshell color and also genetic and phenotypic correlations between eggshell color and egg quality traits which were evaluated in the first study. Color coordinates from CIELab color space L* (brightness), a* (red/green scale) and b* (yellow/blue scale) of 1142 eggs were determined. The estimated heritabilities ranged from 0.35 to 0.48 and the genetic correlations between them were high. In general, the genetic correlations between eggshell color and egg quality traits were low, suggesting that the eggshell color does not influence internal and external egg quality.

<p>The purpose of gene mapping is to understand the underlying genetics of simple and complex traits like plumage colour and growth. This thesis is based on a cross between the wild ancestor of the modern chicken, the red junglefowl, and a White Leghorn line selected for high egg mass. There are obvious phenotypic differences between these two breeds in several aspects such as growth, egg production and behaviour. These complex traits are often influenced by a number of genes or Quantitative Trait Loci (QTL) as well as environmental factors.</p><p>Identification of QTL regions involves testing of association between genetic markers and the phenotype of interest. The QTL identified in this study explain most of the difference in adult body weight between the red junglefowl and the White Leghorn, but less of the difference at earlier age. By applying a different method for detection of QTL, including gene interactions, epistasis, we can understand more of the genetics behind early growth. The allele coming from the red junglefowl is generally associated with lower weight, egg production and food consumption.</p><p>In this study we have also identified two genes explaining the difference in plumage colour in the cross. The <i>Extension</i> locus, encoded by the melanocortin receptor 1 (<i>MC1R</i>), controls the amount of pigment produced has shown to be associated with plumage colour. A mutation in the <i>MC1R</i> gene causes black pigmentation of the plumage. </p><p>We have also found association between the <i>PMEL17</i> gene, known to be involved in normal pigmentation, and the <i>Dominant white</i> phenotype present in the White Leghorn. After comparison of sequences from different alleles at the <i>Dominant white</i> locus, amino acid alteration caused by insertion and deletion in the transmembrane region of the <i>PMEL17</i> protein has been revealed. These mutations are associated with alleles representing different plumage colour variants.</p>

The purpose of gene mapping is to understand the underlying genetics of simple and complex traits like plumage colour and growth. This thesis is based on a cross between the wild ancestor of the modern chicken, the red junglefowl, and a White Leghorn line selected for high egg mass. There are obvious phenotypic differences between these two breeds in several aspects such as growth, egg production and behaviour. These complex traits are often influenced by a number of genes or Quantitative Trait Loci (QTL) as well as environmental factors. Identification of QTL regions involves testing of association between genetic markers and the phenotype of interest. The QTL identified in this study explain most of the difference in adult body weight between the red junglefowl and the White Leghorn, but less of the difference at earlier age. By applying a different method for detection of QTL, including gene interactions, epistasis, we can understand more of the genetics behind early growth. The allele coming from the red junglefowl is generally associated with lower weight, egg production and food consumption. In this study we have also identified two genes explaining the difference in plumage colour in the cross. The Extension locus, encoded by the melanocortin receptor 1 (MC1R), controls the amount of pigment produced has shown to be associated with plumage colour. A mutation in the MC1R gene causes black pigmentation of the plumage. We have also found association between the PMEL17 gene, known to be involved in normal pigmentation, and the Dominant white phenotype present in the White Leghorn. After comparison of sequences from different alleles at the Dominant white locus, amino acid alteration caused by insertion and deletion in the transmembrane region of the PMEL17 protein has been revealed. These mutations are associated with alleles representing different plumage colour variants.

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.

This study investigated factors associated with two mortality events: avian botulism in waterfowl and mortality associated with hatching asynchrony in prefledgling Franklins gulls (Larus pipixcan). The initial focus of my research was on the spatiotemporal relationship between mortality of Franklins gulls and the onset of botulism outbreaks in waterfowl, and the suitability of gull carcasses for proliferation and toxigenesis of Clostridium botulinum. From 1999 to 2001, dead hatch-year Franklins gulls were by far the most abundant carcasses, and the only source of toxin-laden maggots found on transects prior to the occurrence of avian botulism in waterfowl. Nest density was a significant predictor of hatch-year gull carcass density. High density of toxic material from gull carcasses prior to the onset of botulism in waterfowl coincided with high densities of susceptible birds; hence, mortality of Franklins gulls has the potential to be a major initiating factor for botulism outbreaks at Eyebrow Lake, Saskatchewan. The causes of gull mortality were conditions or diseases associated with starvation, stress, or immunosuppression, and most mortality occurred in third-hatched chicks. To separate effects of laying order from effects of hatching asynchrony on prefledgling survival, a cross-fostering experiment was conducted to create clutches containing asynchronously hatching eggs of the same laying order, and of similar egg mass, egg volume, and female quality. Hatching order, independent of laying order, significantly affected survival to fledging, whereas laying order had no observable effect, indicating that intraclutch variation in egg quality does not predetermine the fate of prefledglings, and may be less important than hatching asynchrony for survival of prefledgling Franklins gulls. Relationships among hatching asynchrony, laying order, mass, corticosterone, immune function, growth, and survival at two stages of development were complex. Hatching asynchrony significantly affected early and late prefledgling survival, and was directly or indirectly associated with mass, corticosterone level, and cell-mediated immune responses at early and later stages of development. Both hatching asynchrony and mass appeared to play key roles in mediating life history trade-offs among cell-mediated immune function, growth, and survival. In contrast to cell-mediated immune responses, primary humoral immune response was not directly affected by hatching order or mass, nor was it associated with survival to fledging. Rather, it was associated with laying order, neonatal testosterone, corticosterone at 2 weeks, growth of leg length, and clutch initiation date, illustrating the importance of examining more than one branch of the immune system in studies of life history trade-offs. This study is a step toward using a multipronged and multidisciplinary approach to demonstrate interactions and trade-offs among life history traits, the physiological mechanisms that produce these relationships, and how these relationships may change depending on stage of development.

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 natural ability of filamentous fungi to synthesize, glycosylate, and secrete high levels of protein products has made them potentially attractive hosts for heterologous protein production. Advances in fungal genetics enabled the expression of several high value proteins in filamentous fungi. Particularly the genus, Aspergillus has proven to be potentially useful for the expression of eukaryotic gene products. This thesis pertains to the optimization of recombinant protein production by the fungal host, Aspergillus niger. The target recombinant protein of interest is hen egg white lysozyme (HEWL). This protein encoded in the genome resulting in relatively stable gene construct; however, it is subject to extracellular protease attack. The objective of the proposed research is the development and application of engineering methodology for the analysis and optimization of a fungal bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. To accomplish this, during the first phase of this study a statistically based experimental method was used to systematically elucidate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl₂.2H₂O) on hen egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2⁵⁻¹ fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other medium components were not important within the levels tested. Then, the method of steepest ascent was employed to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g/L, peptone 34 g/L, ammonium sulfate 11.9 g/L, yeast extract 0.5 g/L, and CaCl₂.2H₂O 0.5 g/L. This medium was projected to produce theoretically 212 mg/L lysozyme. Using this optimized medium, an experimentally observed maximum lysozyme concentration of 209±18 mg/L verified the applied methodology. A second optimization approach was based on metabolic flux analysis (MFA). A comprehensive metabolic network comprising three intracellular compartments (cytoplasm, mitochondrion and peroxisome) was developed for Aspergillus niger. The metabolic flux network included carbohydrate and amino acid metabolism in both anabolic and catabolic reactions. According to experimental observations, the time course of fermentation was divided into five phases, each with unique physiological properties. The network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consists of 137 metabolites and 287 processes, of which 181 represent biochemical conversions and 106 represent transport processes between the different compartments and the extracellular environment. In addition, due to the physiological evidence some biochemical reactions considered to be active only in one direction. Linear programming was used for optimizing of the specific growth rate as the objective function in combination with 37 measured input and output fluxes of the key metabolites to evaluate corresponding intracellular flux distributions throughout the batch fermentations. The general applicability of the methodology was evaluated by establishing commonality to optimize recombinant HEWL production. The proposed model was able to predict correctly the specific growth rate, oxygen uptake rate, and carbon dioxide evolution rate with good precision. The results of the metabolic flux and sensitivity analysis were employed for medium design. Growth was biphasic; glucose was utilized initially as the carbon source and was followed by its oxidation product, gluconate, later. Logarithmic sensitivity analysis revealed that the addition of proline, alanine and glutamate benefited growth in defined media. The experimental observations and flux analysis showed that tyrosine was a potential candidate for biomass production improvement. The two amino acids, namely proline and tyrosine benefited biomass production during the initial growth phases. Glutamate and alanine were particularly important during the latter stages of the batch process. A series of growth studies were conducted with the identified amino acids added in the medium. In these preliminary nutritional experiments the contribution to growth enhancement was 46% for proline, 23% for glutamate, and 22% for tyrosine. Model predictions were further verified by conducting batch and fed-batch fermentations in a 7- liter bioreactor. The programmed addition of four amino acids (proline, glutamate, alanine, and tyrosine) according to a predetermined schedule resulted in a 44% improvement in biomass and 41% improvement in recombinant protein production. The experiments also confirmed the model prediction that extra amount of amino acids besides the identified ones would not significantly enhance biomass and the recombinant protein production. A computer-based control system was developed for the on-line monitoring and control of the major state variables (e.g., temperature, pH, and DO) during the time course of fermentation. The graphical programming environment, LabVIEW was used to acquire and integrate these variables in a supervisor computer. The temperature of the bioreactor during sterilization and fermentation was controlled using a cascade methodology. The controller parameters of the master and slave loops were determined experimentally to yield a smooth response with minimum overshoot of both the bioreactor and jacket temperatures. The program scheduled various required steps in an established order during the fermentation. This feature of the software guarantees that every necessary operation will be met. The graphical representation of the process is displayed on the screen and helps the user to follow the process and perform the required adjustments. Furthermore, different variables can be observed simultaneously and saved in text or spreadsheet files for further analysis.