Lipids carry out important structural as well as signaling functions in the cell. In recent years, enzymes that metabolize lipids have been emerging as key regulators of basic cellular functions and developmental processes. In order to study metabolism of lipids, we have focused our research on a class of proteins: the cytochrome P450s (CYPs), which are involved in lipid production in many organisms. We have used C. elegans, a classical genetic model system, to investigate lipid metabolism because this nematode offers several technical advantages that render it suitable for our investigations. The aim of our project was to identify and characterize essential lipids for the development of worms. We have performed RNAi (RNA interference) against C. elegans CYP31A, and found that silencing of this enzyme leads to the arrest of embryonic development. Further characterization of this embryonic lethal phenotype revealed that it is caused by problems in establishment of polarity and failure in the extrusion of a polar body. Moreover, we found that embryos depleted of CYP31A are osmotic sensitive and their eggs are permeable to dyes (hoechst, FM 4-64 etc.). The defects described above are common to a class of mutants that received the denomination of POD (for Polarity and Osmotic Defects). Analysis by electron microscopy demonstrated that cyp31A(RNAi) embryos exhibit an improperly constructed eggshell. Further functional studies have demonstrated that the defects observed in cyp31A(RNAi) embryos can be ascribed to the malfunctioning of one of the three layers of the eggshell: the lipid-rich layer, but additional problems in the assembling of the other two layers are also present. In order to identify the product of CYP31A, we set up a bioassay in which we tested the capability of lipidic extract from wild type embryos to rescue the embryonic lethality. The bioassay provided a method to track the activity and allowed us to enrich the metabolic product of CYP31A by the fractionation of the total lipid extract. Another POD gene, emb-8, codes for an NADPH CYP reductase. This 4 protein supplies electrons to the CYPs for their metabolic reactions. A mutant of emb-8 (emb-8(hc69)), gives a similar phenotype as the knockdown CYP31A. With the aim to test if EMB-8 and CYP31A act in the same pathway we extracted lipids from emb-8TS mutants. We tested in the bioassay if extracts from emb-8(hc69) mutants, containing the metabolic product of CYP31A, can rescue cyp31A(RNAi) phenotype. The results obtained suggest that EMB-8 and CYP31A work in the same metabolic pathway. Conclusively, CYP31A and EMB-8 cooperate to produce a class of lipids that are required for the construction of a functional eggshell. A defective eggshell causes failure in polarity establishment, extrusion of the polar bodies, osmotic sensitivity and permeability and eventually it leads to the arrest of the development of C. elegans embryos.

My work is a symptom of my ongoing quest to achieve immortality. I perpetually attempt to make permanent the traces we leave behind and the impressions we make upon each other. I use the body to portray boundaries – between the skin and the heart, comfort and disquiet, holding and letting go. The objects I make serve both as an agent for physical contact and as the commemoration of an ephemeral interaction. I create personal fossils, revealing the interstices formed when two bodies come into contact with one another. I use materials that reference endurance and longevity to record transient spaces whose edges continuously shift and whose membranes are particularly tenuous. This work is an ongoing catalog of the people in my life and my persistent efforts to hold on to those fleeting connections.

A study was carried out at Poultry Science Laboratory LAVIC at the Federal University of Santa Maria UFSM. The aim was avaluated the effect of different feeding schedules on the performance and eggs quality of broilers breeders Cobb 500. The experimental period was separated into two phases, the first from 28th to 40th week (Phase I) and the second from 40th to 60th week (Phase II) of hen s age. The feeding schedules evaluated were: a single feeding at 08:00 am; twice daily feeding (50% at 08:00 am and 50% at 3:00 pm) and a single feeding at 3:00 pm. At the Phase I, was used 546 females and 63 males allocated in a completely randomized design (CRD) with 7 pens with 26 females and 3 males per repetition and, to the Phase II was used 330 females and 45 males assigned in a CRD with 5 pens with 22 females and 3 males by repetition. The diets were based on corn and soybean meal. At the Phase I, the egg production of hens fed at 3 pm was reduced (P=0.0002). These hens had higher egg and yolk weight than hens fed at 8 am. Hens fed at 3 pm showed better shell quality (egg specific gravity, weight and thickness). The bacterial contamination of the shell was not affected by the diffirent feeding schedules, as well as hatchability, fertility, contaminated and pipped eggs. Embryonic mortality was the lowest in eggs came from hens fed at 8 am. The hatchability of fertile shown to be higher in hens fed once daily in the morning than broiler breeders fed twice daily. At the Phase II, the egg production was not affected by feeding schedules. The hens fed in the afternoon had higher egg and shell weight tham others. The hens fed at 3 pm had higher egg specific gravity and eggshell thickness than eggs come from hens fed at 8 am. Calcium (Ca) and phosphorus (P) plasma levels (21 hours after laying) were higher in hens fed at 3 pm than hens fed at 8 am. The tibia weight was higher in hens fed 8 am than hens fed twice daily feeding. A single feeding at 3:00 pm showed lowest egg production of the 28th to 40th weeks of age. Broiler breeders fed in the afternoon had better egg quality and bacteriological differences were not found in the eggs of hens fed at different schedules. / Um estudo foi conduzido no Laboratório de Avicultura LAVIC da Universidade Federal de Santa Maria (UFSM) com o objetivo de avaliar o efeito de diferentes horários de arraçoamento sobre o desempenho e qualidade de ovos de matrizes de frangos de corte da linhagem Cobb 500. O período experimental foi dividido em duas fases, a primeira da 28ª a 40ª semanas (Fase I) e a segunda da 40ª a 60ª semanas (Fase II) de idade das aves. Avaliaram-se três horários de arraçoamento: único - 8hs; duas vezes ao dia (50% às 8hs e 50% às 15hs) e único - 15hs. Na Fase I foram utilizadas 546 fêmeas e 63 machos distribuídos em um deliamento inteiramente casualizado (DIC) composto por 7 repetições de 26 fêmeas e 3 machos. Na Fase II utilizou-se 330 fêmeas e 45 machos distribuídos em um DIC composto por 5 repetições de 22 fêmeas e 3 machos. As dietas foram à base de milho e farelo de soja. Na Fase I, a taxa de postura de aves alimentadas às 15hs foi reduzida (P=0.0002). Estas aves apresentaram maior peso de ovo e gema em comparação às aves alimentadas às 8hs e, aves alimentadas às 15hs apresentaram melhor qualidade de casca (gravidade específica, peso e espessura) em relação às demais. A contaminação bacteriológica da casca não foi afetada pelos diferentes horários de arraçoamento, bem como eclodibilidade, fertilidade, ovos contaminados e bicados. A mortalidade embrionária foi menor em ovos resultantes de matrizes arraçoadas às 8hs. Estas aves produziram ovos que apresentaram maior taxa de eclodibilidade de ovos férteis em relação às aves alimentadas duas vezes ao dia. Na Fase II, a taxa de postura não foi afetada pelos horários de arraçoamento. Matrizes alimentadas à tarde apresentaram maior peso de ovo e casca em relação às demais. Estas aves apresentaram maior gravidade específica e espessura de casca em relação às aves alimentadas às 8hs. Cálcio (Ca) e fósforo (P) plasmáticos (21 horas após a postura) foram maiores em matrizes arraçoadas às 15hs em relação às aves alimentadas apenas às 8hs. Matrizes alimentadas às 8hs apresentaram maior peso de tíbia do que aquelas alimentadas duas vezes ao dia. O arraçoamento único às 15hs demonstrou menor produção de ovos da 28ª a 40ª semanas de idade. Matrizes alimentadas a tarde apresentam melhor qualidade de ovos, sendo que não foram encontradas diferenças bacteriológicas nos ovos das aves alimentadas em diferentes horários.

This study was performed with the objective of assessing the effects of direct inclusion of 0%, 1%, 1.5% and 2% of the alga Lithothamnium calcareum in the diet, the age hen and interaction between these variables on the production and egg shell quality of laying hens in the second cycle. A completely randomized design was used with subdivided portions (inclusion level: 0%, 1%, 1.5% and 2% x age: 112, 114, 116 and 118 weeks of age) with 6 replications, 10 hens per experimental unit. Each replications of the experiment involved use of 10 Dekalb White hens lines with an age of 110 weeks, four eggs being collected during each period. Percentage of eggs, percentage of broken eggs, percentage of cracked eggs, egg weight; yolk weight, white weight, eggshell weight, specific gravity, percentage eggshell, shell thickness ; number of shell pores; percentage of mineral matter eggshell, percentage of calcium and phosphorus in eggshell were measured and means were compared by Scott-Knott 5% test. The inclusion level of the alga increased the percentage of egg, shell thickness, percentage of mineral matter eggshell and calcium and decreased percentage of cracked eggs and number of pores in the shell. The increasing age of the laying hens decreased eggs shell quality. It was concluded that inclusion levels of Lithothamnium calcareum increased production and egg shell quality. / Objetivou-se avaliar os efeitos da inclusão direta na ração de 0, 1%, 1,5% e 2% da alga Lithothamnium calcareum, a idade da ave e a interação entre eles sobre a produção e a qualidade da casca de ovos de poedeiras comerciais de segundo ciclo. O experimento foi realizado em delineamento inteiramente casualizado em esquema de parcelas subdivididas (nível de inclusão: 0, 1%, 1,5% e 2% x idades: 112, 114, 116 e 118 semanas de idade) com seis repetições. Cada repetição foi composta de 10 aves da linhagem Dekalb White alojadas com 110 semanas de idade. Em cada idade foram coletados quatro ovos de cada repetição. Os dados foram submetidos à análise de variância e as médias comparadas pelo teste de Scott-Knott 5%. As características avaliadas foram: percentagem de postura, ovos trincados e quebrados, peso dos ovos, gravidade especifica, percentagem de casca, peso da gema, clara e casca, matéria mineral da casca, espessura da casca, número de poros da casca, percentagem de cálcio e fósforo da casca. O nível de inclusão da alga aumentou a percentagem de postura, a espessura da casca, a percentagem de matéria mineral e de cálcio da casca, e diminuiu percentagem de ovos trincados e número de poros da casca. O aumento da idade das aves determinou a diminuição da qualidade da casca dos ovos. Conclui-se que os diferentes níveis de inclusão da alga Lithothamnium calcareum melhoram a produção e a qualidade externa dos ovos. / Mestre em Ciências Veterinárias

Chez les espèces aviaires, le stockage des spermatozoïdes s’étend sur plusieurs semaines principalement au niveau du réservoir de la jonction utéro-vaginale, contenant les tubules de stockage des spermatozoïdes (SST). Les mécanismes impliqués dans ce processus restent indéterminés. L’effet de l’insémination artificielle (IA) a été évalué sur le protéome du fluide utérin (FU), des protéines cibles et des glycanes dans les SST, provenant de poules possédant une longue (F+) ou courte (F-) durée de stockage. La longue durée de stockage est associée à une abondance relative dans le FU après IA des protéines exosomales (ANXA4, ANXA5), des protéoglycanes (TSKU), des protéines liant les protéoglycanes (HAPLN3, FN1, VTN), des transporteurs de lipides (VTG1, VTG2, APOA1, APOA4, APOH), et des protéines matricielles de la coquille (OCX32). Au contraire, la faible capacité de stockage est associée à la régulation après IA des protéines immunitaires (PIGR, immunoglobulines) ou pro-inflammatoire (LTA4H), des protéases (XPNPEP1), des chaperones (HSPA8), des mucines (MUC5AC, MUC5B), et de l’ovalbumine (OVALY). Au niveau des SST, les protéines matricielles de la coquille (OC-116, OCX36, OC-17) ont été identifiées dans l’épithélium et la lumière. La longue durée de stockage est associée à la sécrétion luminale de résidus Glc/GlcNAc, à la mobilisation apicale de protéines exosomales (ANXA4), et la non-activation des voies métaboliques impliquant les protéines PIGR, HSPA8, et ANXA5 dans les SST. En conclusion, la composition protéique du FU et des SST requièrent des régulations spécifiques après IA certainement pour garantir le stockage des spermatozoïdes. / In avian species, the sperm storage mainly takes place in uterovaginal sperm storage tubules (SST) during several weeks. Mechanisms implied in this process are not fully understood. The effect of artificial insemination (AI) has been evaluated on the uterine fluid (UF) proteomic composition, and on SST candidate proteins, from hens exhibiting long (F+) or short (F-) sperm storage duration. Long sperm storage duration was associated with the relative abundance in UF after AI of proteoglycans (TSKU), proteoglycan binding proteins (HAPLN3, FN1, VTN), lipid transporters (VTG1, VTG2, APOA1, APOA4, APOH), and eggshell matrix proteins (OCX32). In contrast, poor sperm storage ability was associated with the regulation of immune factors (PIGR, immunoglobulins), pro-inflammatory factors (LTA4H), proteases (XPNPEP1), chaperone (HSPA8), mucins (MUC5AC, MUC5B), and ovalbumin related protein Y (OVALY). At the level of SST, eggshell matrix proteins (OC-116, OCX36, OC-17) were identified in SST cells and lumen. Long sperm storage duration was associated in SST with the luminal secretion of Glc/GlcNAc residues, ANXA4 apical mobilization, and non-activation of metabolic pathway implying PIGR, HSPA8, and ANXA5. In conclusion, the proteomic composition of UF and SST require specific regulation after insemination, most probably to guarantee the success of sperm storage process.

This doctoral work was developed in the frame of bone tissue engineering, dealing with the fabrication of scaffolds for the regeneration of bones. At this purpose, calcium phosphates derived from natural sources are very interesting because they are more similar to the bone mineral and possess better bioactivity. Indeed, the bone mineral is different from synthetic hydroxyapatite as it is non-stoichiometric, nanosized, it presents a high degree of disorder and contains many additional ions and impurities such as CO32-, Mg2+, Sr2+, Na+, etc. These characteristics can be easily obtained by synthesizing hydroxyapatite from natural sources, such as corals, starfishes, seashells, animal bones, bird eggshells etc. The natural sources used in the present work are three types of biogenic calcium carbonate, i.e. calcium carbonate that is produced by living organisms in the form of aragonite or calcite. Among the different sources, three biogenic calcium carbonates were chosen: cuttlefish (Sepia Officinalis) bones, mussel (Mytilus Galloprovincialis) shells and chicken eggshells. Besides their abundance and availability, they were selected because of their different composition: aragonite in cuttlebones, calcite in eggshells and a mixture of aragonite and calcite in mussel shells. After the first chapter, which is a theoretical introduction, this thesis is divided into other five chapters. Chapter 2 contains a careful characterization of the three biogenic raw materials while Chapter 3 deals with the synthesis of hydroxyapatite starting from these natural sources. The process developed here takes place entirely at nearly room temperature, which allows the organic part of the biological materials to be preserved. This synthesis process is basically a wet mechanosynthesis followed by a mild heat treatment (up to 150°C). The study focuses on the influence of several process parameters on the synthesis efficiency: temperature, milling time, pH and raw material. The temperature used to dry the slurry after the wet ball-milling was found to be the most important parameter, the higher the temperature the faster the conversion of CaCO3 into hydroxyapatite. Moreover, aragonite was found to transform more easily into hydroxyapatite with respect to calcite, and also to follow a different reaction path. The synthesis process described in Chapter 3 allowed to produce different bio-derived powders that were found to be non-stoichiometric, nanosized, carbonated hydroxyapatites, containing also additional ions, especially Mg2+ in the eggshell-derived material and Sr2+ in the cuttlebone-derived one. These powders were then used as a starting point for the studies presented in the next three chapters. Chapter 4 shows a very preliminary evaluation of the interaction with human cells in vitro. First, the as-synthesized powders were consolidated by uniaxial pressing and sintering at temperatures between 900°C and 1100°C and their crystallographic composition was analyzed. Then, after having established the non-cytotoxicity of the sintered pellets, osteoblasts from human osteosarcoma cell line were seeded on the pellets and their behavior after 1, 3 and 5 days of culture was observed by confocal microscopy. In general, all materials promoted good cell adhesion and proliferation, especially the eggshell-derived one. At this point, the bio-derived materials were found to induce a good cellular response but, in order to foster the regeneration of bones, a scaffold must also contain a large amount of interconnected porosity. Among the numerous methods to fabricate porous structures, additive manufacturing is surely very attractive due many advantages, such as the possibility of customizing the shape based on tomography images from the patients, the fact that no mold is needed and the freedom of fully designing the porosity. Indeed, not only the size and the amount of porosity are important, but also the shape of the pores and their position and orientation have a deep effect on the interaction with the cells. Therefore, Chapter 5 and Chapter 6 deal with the fabrication of scaffolds by 3D printing, following two different approaches. In the study presented in Chapter 5, the powders synthesized from cuttlebones, mussel shells and eggshells were used in combination with a thermoplastic polymer (PCL, polycaprolactone) to obtain bioactive composites. Composite materials made of 85 wt% PCL and 15 wt% bio-derived hydroxyapatite were used to fabricate porous scaffolds by extrusion 3D printing. The biological in vitro tests showed that the composite scaffolds possess better bioactivity than the pure PCL ones, especially those containing mussel shell- and cuttlebone-derived powders, which promoted the best cell adhesion, proliferation and metabolic activity of human osteosarcoma cells after 7 days of culture. In addition, the elastic compressive modulus, which was found to be between 177-316 MPa, thus in the range of that of trabecular bone, was found to increase of about ∼50% with the addition of the bio-derived nanopowders. Finally, in Chapter 6, the cuttlebone-derived powder was used to fabricate porous bioceramic scaffolds by binder jetting 3D printing. Due to serious technical issues related to the printing of a nanosized powder, 10 wt% of bio-derived powder was mixed with a glass-ceramic powder with bigger particle size. Moreover, the organic part of the cuttlebone had to be previously eliminated by a heat treatment at 800°C. Thanks to the great freedom of design that is allowed by the binder jetting process, scaffolds with two different pore geometries were fabricated: with pores of uniform size and with a size-gradient. Indeed, natural bone possesses a gradient in porosity from the core to the surface, from porous trabecular bone to dense cortical bone. The sintered scaffolds showed a total porosity of ∼60% for the pure glass-ceramic and ∼70% for the glass-ceramic with 10 wt% of cuttlebone-derived nanoparticles, which most probably slowed down the densification by limiting the contact between the glassy particles. All the bioceramic scaffolds promoted good adhesion and proliferation of human bone marrow-derived mesenchymal stem cells in vitro, without any significant difference between the different samples. However, the scaffolds with the cuttlebone-derived powder and with gradient porosity showed the greatest decrease of metabolic activity after 10 days of culture, which could be accounted as a sign of differentiation of stem cells.

Previous research in the Northern San Juan Region of the American Southwest has revealed a pattern of change in subsistence patterns from the Basketmaker II through Pueblo III periods. Jonathan C. Driver describes the pattern as: cottontail in Basketmaker III (500 to 750 CE) and Pueblo I (750 to 900 CE), deer in Pueblo II (900 to 1150 CE), and turkey in Pueblo III (1150 to 1350 CE). The transition from deer to turkey as the main subsistence has been recorded at several sites throughout the Northern San Juan Region and is thought to be caused by the overhunting of deer and increasing social strain. This combination is theorized to have caused the domestication of turkey as a last resort. Analysis conducted on faunal remains and eggshell recovered during excavation at Coal Bed Village by Brigham Young University et al. supports part of this pattern. Evidence of ritual display is evident in Basketmaker III and early Pueblo II contexts, with evidence of ritual or communal feasting in the early Pueblo II period. The rest of the assemblage appears to be domestic refuse. When compared to other large sites within the Montezuma Canyon, Coal Bed Village appears to have greater access to small artiodactyl throughout each of the periods. Contrary to expectations, adult turkey appears to have been eaten in the Basketmaker III, early Pueblo II, and Pueblo III periods. SEM analysis on turkey eggshell from each of these periods suggests that turkeys were domesticated since the Basketmaker III period, with the fresh eggs used as a protein supplement to the Puebloan diet throughout each of the periods. Changes to the amount of embryonic development suggest that, as the periods progressed, the Puebloans continued to use fresh eggs, but also increased the amount of eggs that developed to hatching.

No description available.

The first of a series of mass mortalities of Nile crocodiles in the Olifants and Letaba rivers in the Kruger National Park (KNP) was reported in the winter of 2008. The present study investigated the levels and possible effects on eggshell thickness of inorganic elements and organic pollutants in Nile crocodile eggs from these rivers, and comparing them with eggs from a reference crocodile farm and a reference dam inside the KNP. The egg contents were analyzed for chlorinated organic compounds and brominated flame retardants. Eggshells and egg contents were analyzed for inorganic elements. The elemental concentrations in the eggshells and contents were low when compared with previous studies. The highest concentrations were found in the eggs from the reference crocodile farm. The eggs from the reference dam and the crocodile farm had thicker shells, and the eggs from the Olifants and Letaba rivers had thinner shells. Not all eggs in a female develop at the same rate, while eggshell formation presumably occurs at the same time for all eggs. As a result, the elemental profile of egg contents may differ between eggs of the same clutch, but less so for the shells. Weak or no associations were found between the elemental concentrations of the content and eggshells and eggshell thinning. A possible organic pollutant–induced eggshell thinning effect was found. The compounds found were not at levels that could have caused the mortalities, but may affect the sex ratios through endocrine disruption. Further studies are therefore required. / Thesis (M.Sc (Environmental Science))--North-West University, Potchefstroom Campus, 2011.

The first of a series of mass mortalities of Nile crocodiles in the Olifants and Letaba rivers in the Kruger National Park (KNP) was reported in the winter of 2008. The present study investigated the levels and possible effects on eggshell thickness of inorganic elements and organic pollutants in Nile crocodile eggs from these rivers, and comparing them with eggs from a reference crocodile farm and a reference dam inside the KNP. The egg contents were analyzed for chlorinated organic compounds and brominated flame retardants. Eggshells and egg contents were analyzed for inorganic elements. The elemental concentrations in the eggshells and contents were low when compared with previous studies. The highest concentrations were found in the eggs from the reference crocodile farm. The eggs from the reference dam and the crocodile farm had thicker shells, and the eggs from the Olifants and Letaba rivers had thinner shells. Not all eggs in a female develop at the same rate, while eggshell formation presumably occurs at the same time for all eggs. As a result, the elemental profile of egg contents may differ between eggs of the same clutch, but less so for the shells. Weak or no associations were found between the elemental concentrations of the content and eggshells and eggshell thinning. A possible organic pollutant–induced eggshell thinning effect was found. The compounds found were not at levels that could have caused the mortalities, but may affect the sex ratios through endocrine disruption. Further studies are therefore required. / Thesis (M.Sc (Environmental Science))--North-West University, Potchefstroom Campus, 2011.