Correct number and pattern of digits is determined in a time and concentration-dependent manner by a gradient of Sonic Hedgehog (SHH) across the anterior-posterior axis of the embryonic limb bud. Owing to the potent morphogenic/mitogenic capabilities of SHH, transcription of the SHH gene in the limb is tightly regulated by feedback loops with other signalling pathways and by the Zone of Polarising Activity regulatory sequence (ZRS). The ZRS is a long-range, cis-regulatory limb-specific enhancer of SHH, and is essential for correct limb SHH expression. The Silkie, a polydactylous breed of chicken, possesses a C > A mutation in the ZRS, resulting in ectopic SHH expression in the anterior limb and hindlimb-specific polydactyly. We employ the Silkie mutant to investigate how SHH is regulated by the ZRS, and how Hedgehog signalling can modulate SHH expression in an autoregulatory manner. We further characterise the effects that the Silkie mutation has on subsequent limb development; investigating the dependence of increased posterior SHH, increased Hedgehog-dependent growth and necessary genotype in both the posterior and anterior limb bud. Several fundamental questions regarding SHH during limb development have yet to be fully addressed: how much SHH protein is present, and does it form a gradient as hypothesised by Wolpert’s Morphogen Gradient Model? By developing a standard curve-based method to assess absolute quantities of processed SHH protein, N-SHH, we find that the quantity of N-SHH protein increases through limb development, and does indeed form a quantifiable gradient across the posterior limb. By comparing quantity of N-SHH protein in equivalently staged mouse, rat, emu and chicken limbs, we find that there is no significant link between N-SHH protein quantity and digit number between mammalian and avian species, and investigate how digit number is modulated in the late limb. A number of species exhibit reduced numbers of digits, including the wings of the emu, cassowary and kiwi. Unlike in mammalian examples of digit loss (i.e. cow, pig) the emu wing has delayed and significantly reduced SHH expression. Through sequencing and functional in vivo testing of ZRS sequences of ratite bird species, we investigate whether the ZRS has a role in evolutionary digit loss. We also demonstrate the aspects of digit loss and Hh signalling are shared with examples of mammalian digit loss. This thesis presents novel research into multiple aspects of genetic regulation, limb development, and evolutionary developmental biology; elucidating both long held dogmas and upcoming areas of limb development.

Rapid determination of the concentrations of molecules related to diseases can provide timely information for treatment options. However, most biomarker quantitation methods require costly and complex equipment. On the other hand, point-of-care systems have less complex instrumentation needs than laboratory-based equipment, but often provide less information; for example, biomarker presence or absence instead of concentration. A complete analysis setup addressing key limitations of both laboratory-based and portable systems is highly desirable. I developed microfluidic devices with visual inspection readout of a target’s concentration from microliter volumes of solution flowed into a microchannel. Microchannels are formed within polydimethylsiloxane (PDMS), and the surfaces are coated with receptors. Capillary flow of target solution in the channel crosslinks the top and bottom surfaces, which constricts the channel and stops flow. The flow distance of the target solution in the channel before flow stops indicates the target’s concentration, enabling simple visual inspection readout without complex detection instrumentation. Because of its easy readout and portability, my system has great potential for use in point-of-care diagnostics. I initially demonstrated a proof-of-concept assay using biotin-streptavidin. Solution capillary flow distances scaled linearly with the negative logarithm of streptavidin concentration over a 100,000-fold range. I measured streptavidin concentrations as low as 1 ng/mL using these microsystems, demonstrating low detection limits. I also characterized the mechanism wherein time-dependent channel constriction in the first few millimeters leads to concentration-dependent flow distances. I demonstrated the visual detection and quantification capability of my system to determine an antigen target, thymidine kinase 1 (TK1). I developed surface modification methods for carrying out flow assays and verified receptor attachment on channel surfaces using fluorescence imaging. I obtained a 1 ng/mL TK1 detection limit in flow assays. I also demonstrated nucleic acid quantitation in my flow devices. I detected specific DNA targets in buffer and synthetic urine at 10 pg/mL levels. A dynamic range of 106 was obtained with single-base mismatch specificity. DNA analogues of two miRNA biomarkers were measured near clinically significant levels, showing great promise for future medical application. The promising results demonstrate that this diagnostic tool offers a simple route to analyte quantitation in microliter volumes, with excellent potential for point-of-care application.

No description available.

This study attempts to develop a method for absolute quantitation in Positron Emission Tomography. This includes the definition of the dimension and the position of a tumour in the brain as well as the evaluation of the amount of drug delivered to the tumour and to surrounding tissues in a pixel-by-pixel way, from the image. The defined objectives can be achieved using the calibrated FWHM values of the distribution of events in the tumour image, versus distance, to determine the dimension and the position of the tumour. The concentration activity in the tumour and the tumour-to-nontumour activity ratios can be obtained from the image, using a modified filter and the calibration of the tomograph. The colour scale of the image can be expressed in absolute units (μCi/ml) and the concentration activity can be evaluated in each pixel of the image or in each volume element of the body. / Thesis / Master of Science (MS)

Os sistemas agroindustriais são estruturas complexas que são influenciadas pelas transformações econômicas, políticas e tecnológicas da sociedade. Assim, as organizações públicas e privadas que estão inseridas nesses sistemas precisam de informações setoriais que as permitam identificar cenários futuros e ajustar seus recursos internos às novas situações geradas pelo ambiente externo. Uma das alternativas para dispor dessa informação é a aplicação de ferramentas de projeção por meio da simulação de cenários. Este trabalho, nesse sentido, tem como foco principal a simulação de cenários para o setor sucroenergético brasileiro a partir do método de mapeamento e quantificação de sistemas agroindustriais. Para atingi- lo, foi aplicada uma pesquisa exploratória e qualitativa, cuja execução foi divida em três fases. A primeira considerou o desenho do sistema agroindustrial da cana-de-açúcar no Brasil, identificando os seus principais setores e variáveis participantes; a segunda compreendeu a quantificação do sistema, estimando, a partir de relações entre as variáveis, os fluxos comerciais dos elos de insumos agrícolas, produção de matéria-prima, insumos industriais e processamento industrial, no ano fiscal de 2008; e, finalmente, na terceira fase foi feita uma simulação de cenários para o período 2011-2015 com base em projeções de mercado e uso de ferramenta eletrônica. A coleta de dados foi realizada por meio de dados primários e secundários, levantados em entrevistas com executivos, pesquisadores, instituições governamentais e organizações setoriais dos diferentes elos do sistema agroindustrial, além de publicações como anuários estatísticos, relatórios, prognósticos, entre outros, elaborados por instituições governamentais e não governamentais. Já o processamento e simulação de cenários foram feitos com a utilização de um software de planilha eletrônica. Como resultados desta pesquisa, portanto, foram apresentadas as etapas necessárias para desenhar, quantificar e simular cenários para o setor sucroenergético, assim como os valores obtidos nos cálculos, que permitirá, em pesquisas futuras, explorar o aperfeiçoamento do modelo de simulação por meio da inserção de novas variáveis e da atualização dos valores atribuídos a elas. / Agroindustrial systems are complex structures that are subject to economic, political and technological changes within society. Therefore, the organization held within these systems must frequently adjust their specific resources to the situations created by the environment. One of the alternatives to identify opportunities and threats to the systems is the application of tools for exploring the future through scenario analysis. Thus, this research focuses on simulating scenarios for the Brazilian Sugarcane Agroindustrial System through the application of the Method for Mapping and Quantifying Agroindustrial Systems. In order to achieve this objective, an exploratory and qualitative research has been made in three phases. The first phase has considered the structure of the Sugarcane Agroindustrial System in Bra zil, identifying their key sectors and variables; the second one has addressed the quantification of the system, estimating the trade flows between farm input suppliers, sugarcane producers, industrial input supplier and sugarcane mills in 2008; and finally, in the third phase a scenario simulation has been done for the period of 2011-2015 based on market projections with the use of an electronic tool. Both primary and secondary data have been used. Primary data have been collected thought interviews with executives, researchers, governmental institutions and industrial organizations representatives, while secondary data have been gathered from publications such as statistical reports and prognostics from private and governmental institutions. The data processing and the scenario simulations have been done by using an electronic spreadsheet software. The results of the research show the necessary stages for drawing the systems structure, quantifying the trade flows between its links and simulating scenarios for the Sugarcane Agroindustrial System. They also show the values obtained from the calculations, which allows the further improvement of the simulation model in future research by updating the values given to the variables as well as by inserting new var iables.

Though mass spectrometry has earned a central role in the field of proteomics due to its versatility in a wide range of experiments, challenges and complications are still encountered when using mass spectrometry to characterize protein structures, post-translational modifications (PTMs), and abundances. In this dissertation, analytical methods utilizing mass spectrometry have been developed to address challenges associated with both qualitative and quantitative protein characterization. The effectiveness of using multiple pepsin-like proteases, both separately and in mixtures, combined with online proteolysis using a special triaxial probe has been demonstrated on an amyloid beta peptide related to the onset of Alzheimer’s disease. These findings have broad implications in protein structural characterization studies using hydrogen-deuterium exchange mass spectrometry. A wider range of proteases (Lys-C, Glu-C, and trypsin) and multiple fragmentation methods (collisionally activated dissociation, electron transfer dissociation, and decision tree) have been utilized in the discovery-based PTM characterization of extracellular cellulosome proteins of the bioenergy-relevent organism Clostridium thermocellum, resulting in the identification of 85 previously unknown modification sites in 28 cellulosome proteins. These modifications may contribute to the structure and/or function of the cellulosome protein complex. By using peptide internal standards and a triple quadrupole mass spectrometer operating in selected reaction monitoring mode, a method has been developed for the absolute quantitation of the Clostridium thermocellum cellulosome protein machine in samples ranging in complexity from purified cellulosome samples to whole cell lysates as an alternative to a previously-developed enzyme-linked immunosorbent assay method of cellulosome quantitation. The precision of the cellulosome mass concentration in technical replicates is better than 5% relative standard deviation for all samples, indicating high precision of cellulosome mass concentration for this method. Though methods and results presented in this dissertation have implications in the study of Alzheimer’s disease and bioenergy research, more broadly this dissertation focuses on development of methods to contend with some of the more complex challenges associated with protein characterization currently presented to the field of proteomics.

The extraction and quantitative analysis of low molar mass compounds in polymers is an analytical challenge. It is also important from a practical point of view because the low molar mass compounds in time will migrate from the polymers into the surrounding environment. It is especially important to gain knowledge about the migrating compounds in applications such as medical implants, packaging materials and car interiors. The main aim of this thesis was to develop headspace solid phase microextraction (HS-SPME) methods to meet this challenge. In addition, the work aimed to show the applicability of the methods developed in quality control of polymers, degradation studies and assessment of polymer durability. Factors influencing the extraction of low molar mass compounds from polyamide 6.6 were studied. Particular attention was paid to the matrix effects and to the establishment of headspace equilibrium of 2-cyclopentyl-cyclopentanone in solid polyamide. Hydrogen bonding and adsorption of analyte to the polar matrix was observed and found to cause exceedingly slow establishment of equilibrium. The adsorption could be eliminated by the addition of water, which replaced 2-cyclopentyl-cyclopentanone at the adsorption sites of the polyamide and made it possible to measure the 2-cyclopentyl-cyclopentanone content in polyamide 6.6 using multiple headspace solid-phase microextraction (MHS-SPME). A correlation between the emitted amount of 2-cyclopentyl-cyclopentanone and the amount 2-cyclopentyl-cyclopentanone in the material was found. The correlation was valid also under non-equilibrium conditions, which allows rapid assessment of the 2-cyclopentyl-cyclopentanone content in polyamide 6.6 using headspace sampling. 20 different low molar mass compounds were identified in virgin and recycled polyamide 6.6. The compounds could be classified into four groups: cyclic imides, pyridines, chain fragments and cyclopentanones. The structures of the degradation products imply that the thermo-oxidative degradation starts at the N-vicinal methyl group. Larger amounts of degradation products at lower degree of degradation were formed in recycled than in virgin polyamide 6.6. Thus, processing increases the susceptibility of polyamide 6.6 to thermal oxidation. The total amount of cyclopentanones was reduced upon processing and oxidation. Cyclopentanones are thus not thermo-oxidation products of polyamide 6.6. N-pentyl-succinimide showed the most significant increase due to oxidation and processing. The formation of N-pentyl-succinimide was in correlation with the simultaneous changes in tensile strength. The largest increase in N-pentyl-succinimide coincided with the largest drop in tensile strength.

<p>The extraction and quantitative analysis of low molar mass compounds in polymers is an analytical challenge. It is also important from a practical point of view because the low molar mass compounds in time will migrate from the polymers into the surrounding environment. It is especially important to gain knowledge about the migrating compounds in applications such as medical implants, packaging materials and car interiors. The main aim of this thesis was to develop headspace solid phase microextraction (HS-SPME) methods to meet this challenge. In addition, the work aimed to show the applicability of the methods developed in quality control of polymers, degradation studies and assessment of polymer durability. </p><p>Factors influencing the extraction of low molar mass compounds from polyamide 6.6 were studied. Particular attention was paid to the matrix effects and to the establishment of headspace equilibrium of 2-cyclopentyl-cyclopentanone in solid polyamide. Hydrogen bonding and adsorption of analyte to the polar matrix was observed and found to cause exceedingly slow establishment of equilibrium. The adsorption could be eliminated by the addition of water, which replaced 2-cyclopentyl-cyclopentanone at the adsorption sites of the polyamide and made it possible to measure the 2-cyclopentyl-cyclopentanone content in polyamide 6.6 using multiple headspace solid-phase microextraction (MHS-SPME). </p><p>A correlation between the emitted amount of 2-cyclopentyl-cyclopentanone and the amount 2-cyclopentyl-cyclopentanone in the material was found. The correlation was valid also under non-equilibrium conditions, which allows rapid assessment of the 2-cyclopentyl-cyclopentanone content in polyamide 6.6 using headspace sampling. </p><p>20 different low molar mass compounds were identified in virgin and recycled polyamide 6.6. The compounds could be classified into four groups: cyclic imides, pyridines, chain fragments and cyclopentanones. The structures of the degradation products imply that the thermo-oxidative degradation starts at the N-vicinal methyl group. Larger amounts of degradation products at lower degree of degradation were formed in recycled than in virgin polyamide 6.6. Thus, processing increases the susceptibility of polyamide 6.6 to thermal oxidation. The total amount of cyclopentanones was reduced upon processing and oxidation. Cyclopentanones are thus not thermo-oxidation products of polyamide 6.6. N-pentyl-succinimide showed the most significant increase due to oxidation and processing. The formation of N-pentyl-succinimide was in correlation with the simultaneous changes in tensile strength. The largest increase in N-pentyl-succinimide coincided with the largest drop in tensile strength.</p>

No description available.

Amyotrophic lateral sclerosis (ALS) is a devastating and progressive disease that results in selective death of motor neurons in the cortex, brain stem and spinal cord. ALS is the most common adult onset motor neuron disease resulting in paralysis and death, commonly within 2 – 5 years of symptom onset, yet there remains no effective treatment for the disease. The majority of ALS cases show no hereditary link (referred to as sporadic ALS or sALS); however, ~10% of cases show a dominant pattern of inheritance (referred to as familial ALS or fALS). Over 170 different mutations in human Cu, Zn superoxide dismutase (SOD1) have been identified to account for ~20% of fALS. SOD1 is a ubiquitously expressed homodimeric antioxidant enzyme. It is widely accepted that mutations in SOD1 result in a gain of toxic function, rather than a loss of native function. A prominent hypothesis for the gain of function is the formation of protein aggregates, which have been shown to be toxic to motor neurons. Protein aggregation is observed in a number of neurodegenerative disorders, including Alzheimer’s, Huntington’s and Parkinson’s disease. Each β-rich monomer of SOD1 binds one catalytic Cu ion and one structural Zn ion. The metallation state of SOD1 significantly influences the structure, dynamics, activity, stability, and aggregation propensity. A similar trend has been observed in a number of metalloenzymes and as such a method to rapidly and accurately quantitate metal ions in proteins is of great importance. Here a review of previous methods using the chromogenic chelator PAR to quantitate metal ions in proteins is presented. Three methods are assessed for their accuracy, precision and ease of use. The methods vary in accuracy, which is highest only under the specific conditions it was designed for. A robust new method is presented here that uses spectral decomposition software to accurately resolve the absorption bands of Cu and Zn with high precision. This method may be successful as a more general method for metal analysis of proteins allowing for the quantitation of additional metal combinations (e.g. Zn/Co, Ni/Cu, Ni/Co). Thermodynamic stability has widely been implicated as playing a major role in the aggregation of globular proteins. Metal loss significantly decreases the global stability of SOD1 and as such metal-depleted (apo) forms of SOD1 have largely been the focus of SOD1 investigations. Recent studies, however, suggest that complete global unfolding is not required for protein aggregation. Local unfolding has been investigated and proposed to be sufficient to induce irreversible protein aggregation in the absence of global destabilization. Enhanced local unfolding has been observed in a number of disease-related proteins. Since SOD1 aggregation may occur from partially unfolded forms, NMR temperature dependence studies have been carried out on the most abundant form of SOD1 in vivo, the fully metallated (holo) dimer, to provide a residue specific picture of subglobal structural changes in SOD1 upon heating. Amide proton (N1H) temperature coefficients report on the hydrogen bonding status of a protein. A curved N1H temperature dependence indicates that the proton populates an alternative conformation generally within 5 kcal/mol of the ground state. NMR temperature dependence studies of pseudoWT indicate that the thermal unfolding process of holo pWT begins with “fraying” of the structure at its periphery. In particular, increased disorder is observed in edge strands β5 and β6, as well as surrounding the zinc binding site. The local stability and conformational heterogeneity of ALS-associated mutants G93A, E100G and V148I was also assessed. All mutants display similar local unfolding patterns to pseudoWT, but also show distinct differences in the hydrogen bonding network surrounding the mutation site. Interestingly, each mutation regardless of its structural context results in altered dynamics at the β-barrel plug, a key stabilizing element in SOD1. A significant proportion of residues (~30%) access alternative states in both pseudoWT and mutants, however, overall mutants appear to be able to access higher free energy alternative states compared to pseudoWT. The implications of these results for the mechanism of protein aggregation and disease are discussed.