Tesis sobre el tema "Cre Lox"

Neurons and glia (astrocytes and oligodendrocytes) are the two major cell types that make up the central nervous system (CNS). They are generated from precursor domains within the neuroepithelial germinal zone (ventricular zone, VZ) that surrounds the ventricles of the brain and the central canal of the spinal cord (the embryonic neural tube). In general, neurons are generated before glia. The intra-spinal circuits that control movement and locomotion are made up of different neuronal and glial elements that develop separately but come together to form interconnected functional units. To understand the logic of circuit development and ultimately circuit-driven behaviour, it is necessary to understand where and when each type of cell originates. To identify the products of the most ventral progenitor domain in the developing spinal cord, known as (Nkx2.2-expressing p3 domain), I made use of Cre-loxP technology. I generated a transgenic mouse line that expresses an inducible form of Cre recombinase (CreERT2) under Nkx2.2 transcriptional control and crossed this with a Cre-dependant reporter mouse to visualize p3-derived progeny. I confirmed that the p3 domain generates Sim1-expressing V3 interneurons, serotonergic interneurons as well as visceral motor neurons of the hindbrain. p3 progenitors also produce two spatially restricted subtypes of astrocytes, a few oligodendrocytes and ventrallypositioned ependymal cells. Unexpectedly, my studies also revealed that pre-ganglionic motor neurons of the sympathetic nervous system (SPNs, visceral motor neurons of the thoracic spinal cord), as well as a population of dorsally-located Sim1-expressing interneurons, are produced from Nkx2.2-expressing precursors. SPNs have been generally believed to originate from the same progenitor pool as HB9-positive somatic motor neurons (sMNs), defined by expression of Olig2 (pMN domain, immediately dorsal to p3). Supporting this idea, no spinal sMNs or SPNs are formed in Olig2-null mice. However, I found that Nkx2.2-expressing p3 precursors do not generate any HB9-positive sMNs, implying that sMNs and SPNs derive from distinct precursors - the latter from the most ventral part of the pMN domain that transiently co-expresses Nkx2.2 and Olig2. Thus, segregation of SPNs and sMNs occurs already in the neuroepithelium before their post-mitotic progenitors migrate away from the VZ into the ventral horns. This is how visceral and somatic MNs are known to develop in the brainstem, so my results provide a unifying theme to MN development at different levels of the neuraxis.

Understanding what constitutes a true Minimal Cell is a key challenge in synthetic biology. In this work, we present two new tools to aid in this endeavour. i) A novel methodology for minimising the Mycoplasma pneumoniae genome via random deletions of genetic material. This protocol utilises the Cre Lox system coupled with random transposon mutagenesis to create a population with random lox sites dispersed around the genome. This allows for a population of cells containing a high variability of large and small-scale deletions ranging from 50bp to 25Kb within M. pneumoniae. ii) The first large scale analysis of the essentiality of genes from multiple bacterial species, and how the composition and function of the essential genome of a bacterium changes based on the genome’s complexity.
Discernir cuales son los componentes que podrían constituir una célula mínima es un desafío clave para la Biología Sintética. En esta tesis, se presentan dos nuevas herramientas para facilitar esta tarea. (i) Una nueva metodología para minimizar el genoma de Mycoplasma pneumoniae mediante la deleción aleatoria de material genético. Esta técnica combina el sistema Cre/lox con la mutagénesis aleatoria mediada por transposones para generar poblaciones bacterianas en las que los sitios lox están distribuidos de manera aleatoria a lo largo de su genoma. Esto permite la generación de poblaciones bacterianas en las que el tamaño de las deleciones efectuadas varia desde 50 pb hasta 25 kb. (ii) El primer análisis a gran escala de la esencialidad genética en múltiples especies bacterianas, y cómo la composición y función del grupo de genes esenciales de una bacteria cambia en función de la complejidad de su genoma.

Abstract The complete exon-intron organization of the gene coding for the mouse α1(XIII) collagen chain, Col13a1, was characterized from genomic clones and multiple transcription initiation points were determined. Detailed comparison of the human and mouse genes showed that the exon-intron structures are completely conserved between the species, and both genes have their 5' untranslated region preceded by a highly conserved putative promoter region. The chromosomal location of the mouse gene was determined to be at chromosome 10, band B4, between markers D10Mit5 – (2.3 ± 1.6 cM) – Col13a1 – (3.4 ± 1.9 cM) – D10Mit15. The location of the genes for both the catalytically important α-subunit of prolyl 4-hydroxylase (P4HA) and human type XIII collagen (COL13A1) were previously mapped to 10q21.3-23.1. Prolyl-4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of peptide-bound proline and plays a crucial role in the synthesis of these proteins. The order and transcriptional orientation of the COL13A1 and P4HA was determined. These two genes were found to lie at tail to tail orientation on chromosome 10 and the distance between these genes was determined to be about 550 kbp. To study the function of type XIII collagen we used gene targeting in ES cells to generate a mouse line that carries a mutated type XIII collagen gene. Instead of normal protein, mutant mice express type XIII collagen with an altered amino-terminus in which the cytosolic and the transmembrane domains have been replaced with an unrelated sequence. The homozygous mice are fertile and viable but they show alterations in skeletal muscles, mainly wavy sarcolemma and increased variation in muscle fiber diameter. Ultrastructural studies revealed additional abnormalities such as streaming of z-disks, accumulation and enlargement of mitochondria, and disorganized myofilaments. The basement membranes of the muscle cells showed areas of detachment from the plasma membrane and the fibrillar matrix of the cells was less compact than in control animals. Fibroblasts cultured from mutant mice had normal levels of type XIII collagen but exhibited decreased adhesion to substratum which might be explained by a reduced anchoring strength of the altered protein.

Neotyphodium species, endophytic fungi associated with cool-season grasses, enhance host fitness and stress tolerance, but also produce biologically active alkaloids including ergot alkaloids associated with fescue toxicosis in grazing animals. One approach to reduce fescue toxicosis is to manipulate genes in the ergot alkaloid pathway. The gene, dmaW, encoding the first pathway-specific step in ergot alkaloid biosynthesis, was cloned previously from Claviceps spp. and its function was demonstrated by expression in yeast. Putative homologs have been cloned from Neotyphodium coenophialum (from tall fescue) and Neotyphodium sp. Lp1 (from perennial ryegrass). In order to confirm the function of dmaW in ergot alkaloid production, dmaW in Neotyphodium sp. isolate Lp1 was knocked out by gene replacement. The dmaW knockout mutant produced no detectable ergovaline or simpler ergot alkaloids. Complementation with Claviceps fusiformis dmaW restored ergovaline production. These results confirmed that the cloned endophyte gene was dmaW, and represented the first genetic experiments to show the requirement of dmaW for ergot alkaloid biosynthesis. Neotyphodium coenophialum, endophyte of the grass tall fescue (Lolium arundinaceum) has two homologs of dmaW. Considering the possible field applications in future, the Cre/lox site-specific recombination system was chosen because of the potential to sequentially knock out both homologs and obtain marker-free dmaW mutants of N. coenophialum. One homolog, dmaW-2, was disrupted by marker exchange, and the marker was eliminated by Cre, thus demonstrating the application of Cre/lox system in N. coenophialum to eliminate a marker gene. The dmaW-2 knockout did not eliminate ergovaline production, indicating that the dmaW-1 was probably also active in N. coenophialum. A putative ergot alkaloid biosynthesis gene cluster was identified in Claviceps purpurea and C. fusiformis. C. purpurea and C. fusiformis produce different subsets of ergot alkaloids. Identification of nine common genes between them suggests the possible role of these genes in the early part of the ergot alkaloid biosynthetic pathway.

L’épithélium intestinal est un système en renouvellement constant. Son homéostasie repose sur la coordination précise des processus de prolifération, de différenciation et de survie cellulaire. L’objectif de mon travail de thèse a été d’étudier le rôle du facteur de transcription HNF-4α dans l’homéostasie, l’architecture et la fonction de l’épithélium intestinal. Dans un modèle de souris adultes, où l’invalidation du gène Hnf-4α est inductible et spécifique de l’intestin, j’ai montré qu’HNF-4α est un acteur clé du maintien de l’homéostasie de cet épithélium : HNF-4α module la prolifération dans les cryptes, en interférant avec la signalisation Wnt/β-caténine ; il régule négativement l’expression de Math-1 et limite l’engagement des progéniteurs en cellules mucosécrétrices ; il participe à la différenciation des entérocytes et des cellules entéroendocrines. HNF-4α joue aussi un rôle majeur dans la fonction intestinale de barrière, sa perte s’accompagnant de modifications d’expression et d’organisation des composants des jonctions serrées et d’une augmentation de la perméabilité paracellulaire. Enfin, HNF-4α est nécessaire au maintien d’un transfert intestinal efficace des lipides alimentaires dans la circulation, et intervient dès l’étape entérocytaire de captage des lipides alimentaires. En conclusion, dans l’intestin, HNF-4α est au carrefour entre homéostasie, architecture cellulaire et fonction de transfert des lipides alimentaires. Ses effets sur la voie Wnt/β-caténine nous conduisent à émettre l’hypothèse qu‘Hnf-4α est un gène suppresseur de tumeur, concernant entre autres le cancer colorectal.

SLMAP is a tail anchored membrane protein that alternatively splices to generate three isoforms, SLMAP1, SLMAP2 and SLMAP3. Previous studies in our lab have shown that the postnatal cardiac-specific overexpression of SLMAP1 results in intracellular vesicle expansion and enhanced endosomal recycling. I generated a postnatal cardiac-specific knockout model using the Cre-Lox system to nullify all three SLMAP isoforms and further evaluate its role in the mouse myocardium. SLMAP knockdown and knockout mouse hearts were analyzed with western blotting and qPCR. I found that only SLMAP3 was nullified and phenotypic evaluation through echocardiography indicated that young and old SLMAP3 knockout animals showed no remarkable changes in cardiac function. Furthermore, challenge with stressor isoproterenol had a similar response to wildtype and knockout mice in cardiac structure and function. Surprisingly the level of expression of SLMAP1 and SLMAP2 was maintained in the myocardium from SLMAP3 deficient mice. Interestingly the machinery involved in endosomal recycling was not impacted by the loss of SLMAP3. These data indicate that loss of SLMAP3 does not alter cardiac structure and function in the postnatal myocardium in the presence of SLMAP1 and SLMAP2.

The aim of the current study was divided into two separate goals, (i) optimization of a number of PCR-based protocols employed for genotyping transgenic mouse lines and (ii) evaluating a protocol for co-detection of mRNA and its correlated protein in the mouse midbrain. The optimization was performed on PCR protocols for genotyping the following transgenic mouse lines; Dat-Cre, Vglut2-Lox, Vglut2-Cre and Vmat2-Lox. Also, two different polymerases were evaluated parallel to each other – KAPA and Maxima Hot Start. One of the main findings from the PCR optimizations were that for the Vglut2-Lox protocol. By decreasing the annealing temp and increasing the MgCl2 the bands appeared brighter.  For the second part of the project, in-situ hybridization (ISH) was used to detect the mRNA expression with a `non-radioactive in situ hybridization´ protocol, using digoxigenin or fluorescein labelled riboprobes (mRNA probes). To detect the correlated protein a basic immunohistochemistry (IHC) protocol with the use of primary and secondary antibodies was implemented. The combined protocol was tested with Nd6 and Grp markers. Before testing to combined the protocols the ISH protocol was performed alone with riboprobes for Girk2, Lpl and Fst. The combined protocol detected mRNA and protein for both the control marker Th and the Nd6 marker. In conclusions, the optimized PCR protocols were optimal when used with the Maxima Hot Start polymerase and the new combined ISH and IHC protocol worked for markers Th and Nd6.

Atrial natriuretic factor (ANF) is a polypeptide hormone produced in heart atria, stored in atrial secretory granules and released into the circulation in response to various stimuli. Proper sorting of ANF at the level of the trans-Golgi network (TGN) is required for the storage of ANF in these specific granules, and this sorting of hormones has been found to be associated with G-proteins. Specifically, the Go protein alpha-subunit (Gαo) was established to participate in the stretch-secretion coupling of ANF, but may also be involved in the transporting of ANF from the TGN into atrial granules for storage and maturation. Based on knowledge of Gαo involvement in hormone production in other endocrine tissues, protein-protein interactions of Gαo and proANF and their immunochemical co-localization in granules, the direct involvement of these two proteins in atrial granule biogenesis is probable. In this study, mice were created using the Cre/lox recombination system with a conditional Gαo knockout in cardiocytes to study and characterize ANF production, secretion and granule formation. Deletion of this gene was successful following standard breeding protocols. Characterization and validation of cellular and molecular content of the knockout mice through mRNA levels, protein expression, peptide content, electron microscopy, and electrocardiography determined that a significant phenotypic difference was observed in the abundance of atrial granules. However, Gαo knockout mice did not significantly alter the production and secretion of ANF and only partially prevented granule biogenesis, likely due to incomplete Gαo knockout. These studies demonstrate an involvement of Gαo in specific atrial granule formation.

Energy efficiency in embedded processors is of major importance in order to achieve longer operating time for battery operated devices. In this thesis the energy efficiency of a microcontroller based on the open source ZPU microprocessor is evaluated and improved. The ZPU microprocessor is a zero-operand stack machine originally designed for small size FPGA implementation, but in this thesis the core is synthesized for implementation with a 180nm technology library. Power estimation of the design is done both before and after synthesis in the design flow, and it is shown that power estimates based on RTL simulations (before synthesis) are 35x faster to obtain than power estimates based on gate-level simulations (after synthesis). The RTL estimates deviate from the gate-level estimates by only 15% and can provide faster design cycle iterations without sacrificing too much accuracy. The energy consumption of the ZPU microcontroller is reduced by implementing clock gating in the ZPU core and also implementing a tiny stack cache to reduce stack activity energy consumption. The result of these improvements show a 46% reduction in average power consumption. The ZPU architecture is also compared to the more common MIPS architecture, and the Plasma CPU of MIPS architecture is synthesized and simulated to serve as comparison to the ZPU microcontroller. The results of the comparison with the MIPS architecture shows that the ZPU needs on average 15x as many cycles and 3x as many memory accesses to complete the benchmark programs as the MIPS does.

The mammalian central nervous system (CNS) develops from a thin layer of neuroepithelial stem cells that form the early neural tube. Initially neuroepithelial cells generate neurons, the identity of which depends on their position of origin along the embryonic axis. Glia are generated at the end of neuronogenesis and very little is known about their specification. One of the challenges of modern developmental biology is to understand the extent of the diversity of glial cell types found in the CNS and how this diversity is generated. In this thesis I describe work using Cre-loxP technology combined with PAC transgenesis that clarifies the origins of various glial cells in the CNS. CNS glial cell generation was studied by fate-mapping restricted pools of neural precursors and identifying their glial progeny. Using PAC transgenesis technology I generated mice expressing Cre recombinase in the same patterns as the transcription factors DbxJ, Emxl and MsxS. The resulting transgenic mice were crossed with Cre-dependant reporter mouse strains to visualise the progeny of the various neuroepithelial regions. Using these techniques I was able to demonstrate that the all the neuroepithelial domains studied produce neurons as well as the major glial cell types astrocytes and oligodendrocytes. Of particular interest was the demonstration of novel sources of oligodendrocytes in the dorsal spinal cord and cortex. This work helps to resolve much of the controversy surrounding the origins of cortical and spinal cord oligodendrocytes. Investigation of the mechanism of dorsal oligodendrocyte specification indicates that dorsally-derived oligodendrocytes may be specified by Sonic hedgehog (SHH) -independent pathways, unlike their ventrally derived counterparts. The implications of these results are discussed in the context of possible mechanisms of glial cell specification in the CNS.

A technique for highly efficient combinatorial transformation of heavy and light chain repertoires (combinatorial infection and in vivo recombination) was used to create a phage antibody library of previously unattainable diversity (6.5 x 10¹⁰). From this library, antibody fragments of high affinity (up to 3 nM) were isolated directly, providing experimental proof of the value of large repertoires. Subsequently a peptide display library was designed by analogy, where repertoires of ten residue 'exons' were employed in place of antibody genes. Combinatorial infection and in vivo recombination was used in the creation of a highly diverse (>10¹¹) combinatorial peptide library. From this library peptide ligands with exquisite specificity for several proteins were isolated. The useful diversity of large combinatorial phage antibody repertoires is limited by the number of phage which can be accommodated in the panning technique. Extra diversity can be sampled by re-pairing the heavy chain gene of a single selected clone with the original input light chain gene repertoire, or vice versa. The variants can then be reselected to isolate antibodies with improved affinities. A model '3-lox' phage system was developed in which heavy and light chain genes could be shuffled efficiently and independently of each other. This system could now be used to generate large phage-antibody libraries, and should permit rapid shuffling and re-selection of either chain in selected antibodies. A further use for the diversity provided by combinatorial infection and in vivo recombination was then envisaged. A phage display vector was designed into which every element of an antibody repertoire could be shuffled with every element of a library encoding antigens.

Els test de tipus 'point-of-care' (POC) presenten un gran potencial per al maneig i el diagnòstic de malalties. Els dispositius POC permeten la realització de proves clíniques prop del pacient, permetent així un diagnòstic ràpid, una ràpida iniciació de tractaments, i en cas necessari, una derivació ràpida a altres centres mèdics. Aquests dispositius tenen a més el potencial de ser més econòmics, més robustos, i més fàcils d'usar que els dispositius mèdics tradicionals. Per aquests motius, els dispositius mèdics de tipus POC es consideren prometedors per als països en vies de desenvolupament, els quals són també els que necessiten de forma més urgent noves tecnologies mèdiques. En aquest context, aquesta tesi se centra en el desenvolupament de dispositius mèdics de diagnòstic in-vitro de tipus POC per salut global. Tenint en compte que els recursos per al desenvolupament i explotació de dispositius POC per a països amb baixos recursos són limitats, el Capítol 2 s'enfoca en el desenvolupament de prioritats d'investigació en salut. Mitjançant l'establiment d'aquestes prioritats es pretén facilitar la selecció d'objectius a fabricants d'instruments mèdics, així com incrementar l'impacte de les noves tecnologies desenvolupades. Els criteris de priorització considerats són molt amplis i inclouen l'impacte d'un nou test en la incidència d'una malaltia, la disponibilitat i preu dels tractaments de les malalties, la inversió tecnològica per al desenvolupament d'un nou dispositiu, i els principis bioètics. El tercer capítol descriu el desenvolupament d'un dispositiu mèdic senzill que pot ser fabricat fàcilment en laboratoris amb escassos recursos: tires reactives de diagnòstic de paper per a la detecció de biomarcadors presents en fluids biològics fabricats amb impressores de raig de tinta domèstiques i amb receptes senzilles per la preparació de les tintes. Aquesta tècnica de fabricació de tires reactives de diagnòstic va ser provada per a la detecció de deficiència de iode, un problema sever de salut global al món. En aquest capítol es presenten experiments de preparació de tintes químiques, impressió en paper, detecció de iode en les concentracions presents en l'orina, i consells per al desenvolupament de noves tintes per a la detecció d'altres biomarcadors de malalties. Aquest simple i versàtil procés de fabricació de tests de diagnòstic permetria a hospitals i laboratoris amb pocs recursos dissenyar els seus propis diagnòstics per a malalties rellevants, i en la forma i quantitat adaptada a les necessitats de cada comunitat. Desafortunadament, no totes les malalties es poden diagnosticar usant senzilles tires reactives de diagnòstic, i freqüentment es necessiten dispositius més complexos. El capítol 4 està enfocat en el desenvolupament de dispositius de PCR i RT-PCR de baix cost, a temps real, i de tipus POC que permeten detectar quantitativament patògens basats en DNA i RNA respectivament. El nostre sistema es basa en PCR de flux continu, el qual manté zones de temperatura fixes i empeny la solució de PCR entre les àrees calefactades, permetent així una transferència de calor més ràpida i conseqüentment, una PCR més veloç. Tots dos sistemes de PCR i RT-PCR van ser fabricats a partir d'un xip microfluídic sol ús dissenyat per a ser produït a baix cost industrialment mitjançant mètodes de 'roll-to-roll'. El sistema òptic permet la detecció de patògens en temps real mitjançant mesures de fluorescència. Per demostrar la funció del xip, dos bacteris infeccioses i un virus van ser seleccionats: Chlamydia trachomatis, Escherichia coli O157: H7, i Ebola virus. Per als tres patògens, es van provar diferents velocitats de flux, es va determinar el límit de detecció del sistema, i es van calcular les eficiències de les PCRs. L'èxit dels resultats obtinguts i la versatilitat del sistema, fa que aquests dispositius es considerin prometedors per al diagnòstic d'altres patògens com Zika o chikungunya, que constitueixen amenaces mundials a la salut pública. Tots dos dispositius de diagnòstic in vitro presentats en aquesta tesi són bons exemples de dispositius de diagnòstic apropiats per a salut global.
Los test de tipo ‘point-of-care’ (POC) presentan un gran potencial para el manejo y el diagnóstico de enfermedades. Los dispositivos POC permiten la realización de pruebas clínicas cerca del paciente, permitiendo así un diagnóstico rápido, una pronta iniciación de tratamientos, y en caso necesario, una derivación rápida a otros centros médicos. Estos dispositivos tienen además el potencial de ser más económicos, más robustos, y más fáciles de usar que los dispositivos médicos tradicionales. Por estos motivos, los dispositivos médicos de tipo POC se consideran prometedores para los países en vías de desarrollo, los cuales son también los que necesitan de forma más urgente nuevas tecnologías médicas. En este contexto, esta tesis se centra en el desarrollo de dispositivos médicos de diagnóstico in vitro de tipo POC para salud global. Teniendo en cuenta que los recursos para el desarrollo de dispositivos POC para países con bajos recursos son limitados, el Capítulo 2 se enfoca en el desarrollo de prioridades de investigación en salud. Mediante el establecimiento de estas prioridades se pretende facilitar la selección de objetivos a fabricantes de dispositivos médicos, así como incrementar el impacto de las nuevas tecnologías desarrolladas. Los criterios de priorización considerados son muy amplios e incluyen el impacto de un nuevo test en la incidencia de una enfermedad, la disponibilidad y precio de los tratamientos de las enfermedades, la inversión tecnológica para el desarrollo de un nuevo dispositivo, y los principios bioéticos. El segundo Capítulo 3 describe el desarrollo de un dispositivo médico sencillo que puede ser fabricado fácilmente en laboratorios con escasos recursos: tiras reactivas de diagnóstico de papel para la detección de biomarcadores presentes en fluidos biológicos fabricados con impresoras de chorro de tinta domésticas y con recetas sencillas para la preparación de las tintas. Esta técnica de fabricación de tiras reactivas de diagnóstico fue probada para la detección de deficiencia de yodo, un problema severo de salud global en el mundo. En este capítulo se presentan experimentos de preparación de tintas químicas, impresión en papel, detección de yodo en las concentraciones presentes en la orina, y directrices para el desarrollo de nuevas tintas para la detección de otros biomarcadores de enfermedades. Este simple y versátil proceso de fabricación de tests de diagnóstico permitiría a hospitales y laboratorios con pocos recursos diseñar sus propios diagnósticos para enfermedades relevantes, en una forma y cantidad adaptada a las necesidades de cada comunidad. Desafortunadamente, no todas las enfermedades pueden diagnosticarse usando sencillas tiras reactivas de diagnóstico, y frecuentemente se necesitan dispositivos más complejos. El Capítulo 4 está enfocado en el desarrollo de dispositivos de PCR y RT-PCR de bajo coste, de tiempo-real, y de tipo POC que permiten detectar cuantitativamente patógenos basados en DNA y RNA respectivamente. Nuestro sistema se basa en PCR de flujo continuo, el cual mantiene zonas de temperatura fijas y empuja la solución de PCR entre las áreas calefactadas, permitiendo así una transferencia de calor más rápida y consecuentemente, PCR más veloces. Ambos sistemas de PCR y RT-PCR fueron fabricados en base a un chip microfluídico desechable diseñado para ser producido a bajo coste industrialmente mediante métodos de ‘roll-to-roll’. El sistema óptico permite la detección de patógenos en tiempo real mediante medidas de fluorescencia. Para demostrar la función del chip, dos bacterias infecciosas y un virus fueron seleccionados: Chlamydia trachomatis, Escherichia coli O157:H7, y Ebola virus. Para los tres patógenos, se probaron diferentes velocidades de flujo, se determinó el límite de detección del sistema, y se calcularon las eficiencias de las PCRs. El éxito de los resultados obtenidos y la versatilidad del sistema, hace que estos dispositivos se consideren prometedores para el diagnóstico de otros patógenos como Zika o chikungunya, que constituyen amenazas mundiales a la salud pública. Ambos dispositivos de diagnóstico in vitro presentados en esta tesis son buenos ejemplos de dispositivos de diagnóstico apropiados para salud global.
Point-of-care (POC) testing has great potential for the management and diagnosis of disease. POC devices allow for testing close to the patient permitting rapid diagnosis, prompt treatment initiation, and when needed, quick referral to other health-care units. They have the potential to be lower-cost, more robust, and more user-friendly than traditional medical devices. For these reasons, POC diagnostic tests are a promising approach for the developing world, where there is also the most urgent need for new health technologies. In this context, this thesis is focused in the development of POC in vitro diagnostic tests for global health. Considering that the resources for developing POC devices for low-resource settings are limited, during Chapter 2 we focused on setting health research priorities to aid test developers setting their targets to increase the impact of the technology. The criteria for prioritization considered were very broad and took into account the impact of a new test on the burden of disease, the availability and expense of disease treatments, the technological investment to develop a new device, and the bioethical principles. Chapter 3 describes the development of a medical device that can be easily manufactured in limited resources laboratories: paper diagnostic chemical dipsticks to detect biomarkers present in biological fluids produced with domestic inkjet printers and simple ink preparation recipes. This fabrication technique for diagnostic strips was tested for the detection of iodine deficiency, a severe global health problem worldwide. In this chapter we present successful experiments for chemical inks preparation, printing on paper, detection of iodine in the concentrations present in the urine, and guidelines for new ink development to target other disease biomarkers. This simple and versatile manufacturing process for diagnostic tests would allow hospitals and laboratories with limited infrastructure to design diagnostics for relevant diseases in a format and quantity adapted to each community needs. Unfortunately, not all diseases can be diagnosed using simple chemical dipstick assays and more complex diagnostic devices are required. Chapter 4 is focused on the development of a low-cost, real-time, point-of-care PCR and RT-PCR systems for quantitative detection of DNA and RNA-based pathogens. Our systems are based on continuous-flow PCR which maintains fixed temperatures zones and pushes the PCR solution between heated areas allowing for faster heat transfer and as a result, faster PCRs. Both PCR and RT-PCR systems were built around disposable microfluidic chips designed to be economically produced industrially by roll-to-roll embossing methods. The optical system allows for pathogen detection via real-time fluorescence measurements. To demonstrate the function of the chips, two infectious bacteria and one viral target were selected: Chlamydia trachomatis, Escherichia coli O157:H7, and Ebola virus. For the three pathogens, different flow velocities were tested, the limit of detection of the system was determined, and PCR efficiencies were calculated. Our successful results, and the versatility of our system, make it promising for the detection of other DNA and RNA-based pathogens such as Zika or chikungunya, which constitute global health threats worldwide. The two in vitro diagnostic tests presented in this thesis are good examples of promising POC diagnostic devices appropriate for global health.

In the present study, the influence of geometric scale, unit orientation (alternatively, flow direction), and the dimensionless Reynolds and Keulegan-Carpenter quantities on the hydrodynamic loading of Core-Loc armour units is explored through a series of physical modelling tests under unsteady and oscillatory flow conditions.

L’hormone thyroïdienne (T3) joue un rôle pleiotropique au cours du développement. Un déficit congénital en T3 provoque une pathologie nommée crétinisme, notamment caractérisée par un retard mental. In vitro, la différenciation oligodendrocytaire peut être induite par ajout de T3 au milieu de culture. Toutefois, en contradiction avec ces observations, l’inactivation des récepteurs de la T3 n’a qu’un effet marginal sur la différenciation oligodendrocytaire in vivo. Différentes lignées murines exprimant un récepteur dominant négatif de l’hormone thyroïdienne TRα1 spécifiquement dans un type cellulaire ont été générées au laboratoire. Cette stratégie nous a permis de préciser le rôle de la signalisation induite par la T3 sur la différenciation oligodendrocytaire au niveau du cervelet, mettant une évidence une double action de la T3 sur ce lignage. La même stratégie appliquée au développement prénatal a également permis de préciser les bases sous-jacentes à l’altération corticale décrite en cas d’hypothyroïdie prénatale
Thyroid hormone (T3) has pleiotropic functions during development. Congenital hypothyroidism results in severe mental retardation, named cretinism. In vitro, adding physiological doses of T3 in oligodendrocyte precursor cells induce massive differentiation. Surprisingly, in vivo analysis only reveals a slight delay in oligodendrocyte differentiation in case of invalidation of T3 receptors. Several mouse strains have been generated in the lab to express dominant negative thyroid hormone receptors alpha 1 in a cell specific manner. This genetic dissection strategy allows us to understand the function of T3 signalling on oligodendrocyte differentiation in the cerebellum. The same strategy has been applied to prenatal neurodevelopment, to understand mechanisms underlying corticogenesis alteration in case of prenatal hypothyroidism

At the start of this project, the systems available for gene disruption in C. albicans were laborious and inefficient.  Both alleles of a target locus need to be inactivated to construct a homozygous null mutant in this diploid fungus.  This often requires the recycling of transformation markers.  However, in some systems it is not possible to recycle the markers used in the disruption.  This limited the creation of multiple knockout strains and the deletion of gene families. This thesis describes the successful adaptation of the Cre-loxP recombination system as tool for the precise, efficient and controlled disruption of genes in C. albicans.  LoxP-MARKER-loxP cassettes containing URA3, HIS1, and ARG4 genes were constructed for gene disruption.  Also, ano arg4::MET3p-cre-URA3 cassette was generated for loxP-flanked marker resolution.  This cassette contains a functional, codon-optimised cre gene.  Finally plasmids containing the URA3, HIS1, and ARG4 genes were constructed for the restoration of prototrophy and the re-integration of target genes into C. albicans null mutants.  The usefulness of the Cre-loxP system was demonstrated by the generation of homozygous ade2D and met15D null mutants, using loxP flanked ARG4 and HIS1 based disruption cassettes.  A third cassette containing URA3 and a MET3p-cre recombinase fusion was integrated within the loxP-ARG4-loxP cassette.  This facilitated the resolution of the MET3p-cre fusion and the loxP-flanked disruption cassettes from the C. albicans genome by Cre-mediated site-specific recombination between the flanking loxP elements.  The system resolved the cassettes with near 100% efficiency.  This allows further manipulations to be performed on the resultant null mutant.  Therefore this new Cre-loxP system could potentially be used for the deletion of gene families in C. albicans.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013.
Title as it appears in MIT Commencement Exercises program, June 2013: Generation of phenotypic diversity in yeast using promoter inversion through Cre-lox recombination. Page 84 blank. Cataloged from PDF version of thesis.
Includes bibliographical references.
Modifying the expression of multiple genes enables both deeper understanding of their function and the engineering of complex multigenic cellular phenotypes. However, deletion or overexpression of multiple genes is typically laborious and involves multiple sequential genetic modifications. Here we describe a strategy to randomize the expression state of multiple genes in S. cerevisiae using Cre-loxP recombination. By inserting promoters flanked by inverted loxP sites in front of a gene of interest we can randomly alter its expression by turning it OFF or ON, or between 4 distinct expression states. We show at least 6 genes can be randomized independently and argue that using orthogonal loxP sites and an additional recombinase should increase this number to at least 30. Finally, we show how combining this strategy with mating allows easy introduction of native regulation as an additional expression state and use this to probe the role of 4 different enzymes involved in base excision repair in tolerance to methyl methane sulfonate (MMS), a genotoxic DNA alkylating agent. The set of vectors developed here can be used to randomize the expression of both heterologous genes and endogenous genes, and could immediately prove useful for metabolic engineering in yeast. Because Cre-loxP recombination works in many organisms, this strategy should be readily extendable.
by Bradley Niesner.
Ph.D.

PTEN est une protéine dotée d'une activité phosphatase qui déphosphoryle les phosphatidylinositols issus de l'activation de la phosphatidylinositol 3-kinase (PI3K). Des mutations germinales du gène PTEN ont été mises en évidence dans le syndrome de Cowden, une maladie caractérisée par le développement de polypes le long du tube digestif et associée à un risque accru de cancer. De plus, la perte d'un allèle de Pten chez la souris conduit à la formation d'hyperplasie et de dysplasie du tractus gastro-intestinal ainsi qu'à des tumeurs notamment au niveau du côlon. Ces observations suggèrent que PTEN joue un rôle important dans le tube digestif. Cependant, ses mécanismes d'action dans les cellules épithéliales intestinales sont peu connus. Nos travaux nous ont permis de mieux caractériser les rôles de PTEN dans ces cellules. Pour ce faire, nous avons d'abord analysé l'effet d'un shRNA inhibant spécifiquement l'expression de PTEN dans les cellules Caco-2/15, une lignée cancéreuse colorectale qui a la particularité de se différencier suite à l'atteinte de la confluence en adoptant un phénotype semblable aux cellules absorbantes de l'intestin grêle. La perte d'expression de PTEN stimule la prolifération de ces cellules. Cette augmentation de la prolifération semble résulter d'une diminution de l'expression de p21 et de p27 ainsi que d'une hausse des cyclines D2 et E. De plus, le shRNA contre PTEN inhibe la différenciation fonctionnelle et morphologique des Caco-2/15. Cela découle partiellement de l'inhibition de l'expression des facteurs de transcription CDX2, HNF-1? et HNF-4?. Les jonctions serrées sont également altérées dans ces cellules. En effet, une réduction importante de l'expression des claudines et une augmentation de la perméabilité transépithéliale a été observée. Une augmentation de la synthèse protéique a aussi été remarquée. De plus, nos résultats laissent également croire que PTEN pourraient jouer un rôle dans la carcinogenèse colorectale. Nous avons effectivement constaté une augmentation du potentiel tumorigénique des Caco-2/l5 exprimant le shRNA contre PTEN suite à l'injection des cellules dans des souris nues. De plus, ces cellules ont des capacités de migration et d'invasion accrues. Nous avons également constaté que les niveaux de PTEN sont plus faibles dans plusieurs lignées cancéreuses colorectales comparativement aux cellules épithéliales intestinales normales. Nous avons aussi analysé le phénotype d'une lignée de souris possédant une délétion du gène Pten exclusivement au niveau de l'épithélium intestinal, générée à l'aide du système Cre/loxP. Macroscopiquement, une organomégalie de l'intestin grêle et du côlon a été observé chez les souris déficientes pour Pten. Histologiquement, nous avons constaté une désorganisation de l'architecture épithéliale intestinale caractérisée par un allongement des villosités et par la présence d'embranchements villositaires. De plus, un épaississement important des couches musculaires a été remarqué. Il y a également une augmentation du nombre de cellules prolifératives au niveau des cryptes intestinales corrélant avec une augmentation des niveaux de ?-caténine et des cyclines D. Finalement, une augmentation du contenu protéique par cellule a également été observée ainsi qu'une activation de la voie mTOR. En conclusion, nos résultats montrent que la phosphatase PTEN est impliquée dans l'établissement de l'architecture générale de l'intestin et qu'elle contrôle la synthèse protéique, la migration, le cycle cellulaire ainsi que la différenciation des cellules épithéliales intestinales. De plus, nos résultats indiquent que la perte d'expression de PTEN pourrait influencer la progression des cancers colorectaux. [Symboles non conformes]

This study investigates the relationship between wave-induced hydrodynamics and the resulting loading on Core-Loc concrete armour units below the still water level in a breakwater structure. Physical modelling experiments were performed at the National Research Council in Ottawa in which a 3D-printed 12 cm Core-Loc armour unit was instrumented and fixed in place within a rubble mound structure. Testing featured simultaneous measurement of force on this instrumented unit, pressure head at the base of the unit, and flow velocities below the SWL. Two main scenarios were tested, the isolated unit and fully armoured scenarios, under a range of regular waves and irregular sea states. Analysis of force development on the instrumented unit indicates that maximum slope-normal forces (both into and away from the structure) are associated with extremes in pressure head above the instrumented unit, while slope-parallel force extremes (both upslope and downslope) occur at times of the fastest change in water level. These loadings are consistent with Morison’s equation and imply drag dominance in the slope-parallel direction and inertia dominance in the slope-normal direction. Significant differences in forces were observed between isolated (no neighbouring units) and embedded (with neighbouring units) armour unit test cases. The presence of the armour layer significantly increased the normal force exerted on the unit and reduced the parallel force. Irregular sea state testing shows force peaks following normal distribution. Analysis of flow above the armour layer showed that force, flow velocity and flow acceleration are symmetrical in the slope-parallel direction, but largely asymmetrical in the slope-normal direction, with the flow velocity and force on the unit in particular experiencing large asymmetries. Wave height analysis indicated that each wave height follows a similar force development pattern with a magnitude proportional to wave height. Wave period analysis showed the formation of small secondary waves as the period increases. Wave steepness affected the peak force loading of the instrumented unit in a mostly linear fashion up to the critical Iribarren number.

Um método para a detecção automática de tipos litológicos e contato entre camadas foi desenvolvido através de uma combinação de análise estatística de um conjunto de perfis geofísicos de poços convencionais, calibrado por descrições sistemáticas de testemunhos. O objetivo deste projeto é permitir a integração de dados de rocha em modelos de reservatório. Os testemunhos são descritos com o suporte de um sistema de nomenclatura baseado em ontologias que formaliza extensamente uma grande gama de atributos de rocha. As descrições são armazenadas em um banco de dados relacional junto com dados de perfis de poço convencionais de cada poço analisado. Esta estrutura permite definir protótipos de valores de perfil combinados para cada litologia reconhecida através do cálculo de média e dos valores de variância e covariância dos valores medidos por cada ferramenta de perfilagem para cada litologia descrita nos testemunhos. O algoritmo estatístico é capaz de aprender com cada novo testemunho e valor de log adicionado ao banco de dados, refinando progressivamente a identificação litológica. A detecção de contatos litológicos é realizada através da suavização de cada um dos perfis através da aplicação de duas médias móveis de diferentes tamanhos em cada um dos perfis. Os resultados de cada par de perfis suavizados são comparados, e as posições onde as linhas se cruzam definem profundidades onde ocorrem mudanças bruscas no valor do perfil, indicando uma potencial mudança de litologia. Os resultados da aplicação desse método em cada um dos perfis são então unificados em uma única avaliação de limites litológicos Os valores de média e variância-covariância derivados da correlação entre testemunhos e perfis são então utilizados na construção de uma distribuição gaussiana n-dimensional para cada uma das litologias reconhecidas. Neste ponto, probabilidades a priori também são calculadas para cada litologia. Estas distribuições são comparadas contra cada um dos intervalos litológicos previamente detectados por meio de uma função densidade de probabilidade, avaliando o quão perto o intervalo está de cada litologia e permitindo a atribuição de um tipo litológico para cada intervalo. O método desenvolvido foi testado em um grupo de poços da bacia de Sergipe- Alagoas, e a precisão da predição atingida durante os testes mostra-se superior a algoritmos clássicos de reconhecimento de padrões como redes neurais e classificadores KNN. O método desenvolvido foi então combinado com estes métodos clássicos em um sistema multi-agentes. Os resultados mostram um potencial significante para aplicação operacional efetiva na construção de modelos geológicos para a exploração e desenvolvimento de áreas com grande volume de dados de perfil e intervalos testemunhados.
A method for the automatic detection of lithological types and layer contacts was developed through the combined statistical analysis of a suite of conventional wireline logs, calibrated by the systematic description of cores. The intent of this project is to allow the integration of rock data into reservoir models. The cores are described with support of an ontology-based nomenclature system that extensively formalizes a large set of attributes of the rocks, including lithology, texture, primary and diagenetic composition and depositional, diagenetic and deformational structures. The descriptions are stored in a relational database along with the records of conventional wireline logs (gamma ray, resistivity, density, neutrons, sonic) of each analyzed well. This structure allows defining prototypes of combined log values for each lithology recognized, by calculating the mean and the variance-covariance values measured by each log tool for each of the lithologies described in the cores. The statistical algorithm is able to learn with each addition of described and logged core interval, in order to progressively refine the automatic lithological identification. The detection of lithological contacts is performed through the smoothing of each of the logs by the application of two moving means with different window sizes. The results of each pair of smoothed logs are compared, and the places where the lines cross define the locations where there are abrupt shifts in the values of each log, therefore potentially indicating a change of lithology. The results from applying this method to each log are then unified in a single assessment of lithological boundaries The mean and variance-covariance data derived from the core samples is then used to build an n-dimensional gaussian distribution for each of the lithologies recognized. At this point, Bayesian priors are also calculated for each lithology. These distributions are checked against each of the previously detected lithological intervals by means of a probability density function, evaluating how close the interval is to each lithology prototype and allowing the assignment of a lithological type to each interval. The developed method was tested in a set of wells in the Sergipe-Alagoas basin and the prediction accuracy achieved during testing is superior to classic pattern recognition methods such as neural networks and KNN classifiers. The method was then combined with neural networks and KNN classifiers into a multi-agent system. The results show significant potential for effective operational application to the construction of geological models for the exploration and development of areas with large volume of conventional wireline log data and representative cored intervals.

This dissertation examines the effect of regulations, resource and referral agencies, and subsidies on price and quality of care in child care centers. This research is based on a carefully developed conceptual framework that incorporates the factors affecting the demand and supply of child care. The first step in developing this framework is sketching out the structural equations. The structural equations help us understand the underlying behavior of individuals and firms making a decision. The exogenous variables are vector of attributes relating to family characteristics, child characteristics, regulations, subsidy, community characteristics and prices of inputs. Based on the structural equations, reduced form equations are estimated to find the effect of each of the exogenous variables on each of the endogenous variables. Reduced form equations help us answer public policy questions. The sample for this study is from the 1990 Profile of Child Care Settings (PCCS) data in which 2,089 center based programs were interviewed. Child/Staff Ratio (Group Level): Results indicate that among subsidies, only the state subsidy per child in poverty has a significant effect on the child/staff ratio at the group level. Presence of resource and referral agencies also increase the child/staff ratio at the group level. Also when the maximum center group size regulation for 25-36 months becomes more stringent, the child/staff ratio at the group level decreases. Child/Staff Ratio (Center Level): When the regulations for the maximum child/staff ratio for age groups 13-24 months and 37-60 months become lax, the child/staff ratio for the center increases. As the regulation for maximum group size for infants becomes stringent, the child/staff ratio decreases. An interesting finding is that as the regulations for maximum group size for age groups 13-24 months and 25-36 months become stringent, the child/staff ratio for the center increases. Another significant finding is that when a center is located in a rural area the child/staff ratio is significantly lower. Center Weighted Average Hourly Fees: Maximum group size regulations for age groups 25-36 months and 37-60 months have a negative effect on center hourly fee. Maximum child staff regulations for age groups 13-24 months and 37-60 months have a negative effect on center hourly fee. Maximum child staff regulations for age groups 0-12 months and 25-36 months have a positive effect on center hourly fee. Findings also indicate that the center average hourly price is lower when there is a resource and referral agency present. Cost adjusted prekindergarten funds and JOBS child care subsidies have a negative effect on average hourly fee. Cost adjusted social services block grant and state subsidy per child in poverty have a positive effect on the average hourly price. A major finding of this dissertation is the interaction of subsidy and regulatory variables. Another major finding is that child/staff ratio at the group level is lower when there is an interaction between geographic location and nature of center sponsorship.

Expression levels of transgenes in mammalian cells show extreme variability between individual clones isolated from a single transfection. This is due to differing number of copies integrated into the genome and also from chromosomal "position effects". Therefore extensive screening is required to isolate a suitable cell line for high level expression of a recombinant protein. In this work, the Cre-loxP site-specific recombination system was investigated to eliminate such problems by directing the rapid targeting of any input DNA to a single pre-selected site in Chinese hamster ovary (CHO) cell line. Cre is a 38 kDa recombinase encoded by the bacteriophage P1 which mediates recombination between a pair of specific 34 bp target sequencesc alled loxP sites. The recombination reaction was first investigated in vitro to establish which kinetic parameters could be relevant for efficient gene targeting: a recombinant baculovirus was constructed with a hexa-histidine-cre fusion gene. The activity of Cre protein purified (by single step, hexa-histidine/nickel-bindinga, ffinity chromatography) from infected insect cells was verified by: (i) Cre-loxP interaction in gel retardation assays and (ii) Cre-mediated intramolecular excision between two loxP sites flanking a lacZ gene in a plasmid DNA. To investigate Cre-mediated targeting of an exogenous DNA to a chromosomal loxP site, three CHO cell lines were constructed, each carrying a loxP site between a ß-actin promoter and a secreted alkaline phosphatase (SAP) gene as a reporter. To demonstrate the targeting event, a promoterless lacZ/neor gene was cotransfected with either a cre plasmid or the purified Cre protein from the baculovirus/insect system. Proper targeting should activate expression of f 3- galactosidase from the chromosomal 0-actin promoter and give loss or reduction of SAP expression in G418 resistant transformants. Southern blot analysis showed targeted events mediated by both cre plasmid and recombinant Cre protein. This work should allow the development of a generic mammalian cell line by incorporating the Cre-loxP system for rapid and reproducible large scale production of recombinant proteins.

Barriers exist to accessing prenatal care for low income women throughout the United States, such as scheduling appointments, finding childcare, and paying for prenatal care. Women who reside in rural areas experience barriers to accessing prenatal care unique to their geographical locations. Barriers to accessing prenatal care have been associated with less than optimal health outcomes for both women and infants. The purpose of this study was to explore the barriers to accessing prenatal care that are specific to low income rural women. A qualitative approach was used by conducting telephone interviews using open ended questions with low income women from one rural county in Montana. A sample of 6 women was recruited from the Women, Infants, and Children (WIC) nutritional supplement program in a rural county. Patterns were identified from the interview responses and categorized into themes that allowed for identifying common barriers. The results from this study revealed that low income rural women reported an overall lack of providers who offered prenatal care in their area. Difficulty with scheduling transportation to and from appointments was reported in half of the women, as well as distance to the nearest hospital for 2 of the women. These same 2 women also reported that if a complication arose during pregnancy or if a woman were to have a high risk pregnancy, distance to the nearest hospital or provider would be a barrier. There was difficulty recruiting a larger sample population, therefore the sample size of 6 women was a major limiting factor of this study. Implications for practice included a need for recruitment of providers in rural areas, and the potential use of nurse practitioners for providing high-quality, low cost prenatal care for low income rural women. The implementation of group prenatal care in rural locations and investing in rural communities were also implications for future practice. Lastly, the results from this study may help with future practice and research to help focus on the needs of this unique population for gaining improved access to prenatal care.

The availability of high level integration leads to building of millions of gates systemson- a-chip (SOC). Due to the high complexity of SOCs, testing them is becoming increasingly difficult. In addition, if the current test practises are maintained, the high cost of test will lead to a considerable production cost increase. To alleviate the test cost problem, this research investigates methods which lead to low-cost test of core-based systems-on-a-chip based on test resource partitioning and without changing the embedded cores. Analysing the factors which drive the continuous increase in test cost, this thesis identifies a number of factors which need to be addressed in order to reduce the cost of test. These include volume of test data, number of pins for test, bandwidth requirements and the cost of test equipment. The approaches proposed to alleviate the cost of test problem have been validated using academic and industrial benchmark cores. To reduce the volume of test data and the number of pins for test, the new Variablelength Input Huffman Coding (VIHC) test data compression method is proposed, which is capable of simultaneously reducing the volume of test data, the test application time and the on-chip area overhead, when compared to previously reported approaches. Due to the partitioning of resources among the SOC and the test equipment, various synchronisation issues arise. Synchronisation increases the cost of test equipment and hence limits the effectiveness of test resource partitioning schemes. Therefore, the synchronisation issues imposed by test data compression methods are analysed and an on-chip distribution architecture is proposed which in addition to accounting for the synchronisation issues also reduces the test application time. The cost of test equipment is related to the amount of test memory, and therefore efficient exploitation of this resource is of great importance. Analysing the memory requirements for core based SOCs, useless test data is identified as one contributor to the total amount of allocated memory, leading to inefficient memory usage. To address this problem a complementary approach to test data compression is proposed to reduce the test memory requirements through elimination of useless test data. Finally, a new test methodology is proposed which combines the approaches proposed in this thesis into an integrated solution for SOC test. The proposed solution leads to reduction in volume of test data, test pins, bandwidth requirements and cost of test equipment. Furthermore, the solution provides seamless integration with the design flow and refrains from changing the cores. Hence, it provides a low-cost test solution for corebased SOC using test resource partitioning.

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Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Além da relevância econômica, o setor agrícola fez com que o Brasil assumisse um papel ativo na discussão internacional das mudanças climáticas. O setor de agri- cultura, floresta e outros usos da terra (AFOLU) é a principal fonte de emissão de gases de efeito estufa no país, padrão peculiar entre os países em desenvolvimento. Durante a Conferência das Nações Unidas sobre as Mudanças Climáticas de 2009 (COP-15) o país assumiu o compromisso voluntário de reduzir suas emissões em 37 até 2025 e em 43% até 2030 em relação aos níveis de 2005. O setor agropecuario é responsável por contribuir com 22,5% do compromisso voluntario. Afim de atin- gir essa meta, foi criado em 2009 o Plano Setorial de Mitigação e de Adaptação as Mudanças Climáticas para a Consolidação de uma Economia de Baixa Emissão de Carbono na Agricultura (Plano ABC). O Plano ABC faz parte da Política Nacional de Mudanças Climáticas (PNMC). Entre as diversas ações do Plano ABC, estão a re- cuperação de 15 milhões de hectares (Mha) de pastagens degradadas e aumentar em 4 Mha as areas de integração lavoura-pecuaria (iLP) e / ou lavoura-pecuaria-floresta (iLPF). O objetivo da tese é avaliar os impactos econômicos e de mudança no uso da terra decorrentes dessas duas ações presentes no Plano ABC. Para tal, foi con- struído um modelo de equilíbrio geral computavel (EGC) (BREA versão 1.0) com representação detalhada de seis grandes regiões brasileiras divididas por relevância econômica e fronteira agrícola: Sul, Sudeste, Norte (bioma Amazônia), Centro-Oeste (sem o bioma Amazônia), Nordeste, e Nordeste Cerrado (Estados do Maranhão, To- cantins, Piauí e Bahia) que é considerada a nova fronteira agrícola brasileira. O modelo representa diversos usos da terra com desagregação setorial agropecuaria, e é o primeiro modelo de EGC a explicitamente representar a implementação das tecnologias do Plano ABC. Sob diferentes cenários simulados, os resultados indicam que a maior oferta de pastagens recuperadas e com alta produtividade, somadas as tecnologias iLP e iLPF, promovem o efeito poupador de terra como resultado agre- gado. Ha redução na pressão que a atividade de pecuária promove sobre as áreas naturais e florestas. Ao mesmo tempo, há queda das áreas destinadas às atividades de grãos, principalmente soja e milho, e aumento da area de florestas plantadas. Entretanto, os resultados regionais mostram que as regiões de fronteira agrícola respondem diferentemente as ações do Plano ABC. Nas regiões Centro-Oeste e Norte as pastagens crescem mais do que as areas recuperadas, sendo que parte das áreas de culturas são convertidas para pasto de boa qualidade. Nas regiões Sudeste e Sul essas pastagens aumentam menos do que a area de pastagens recuperadas, o que significa que parte das pastagens boas são convertidas em areas de culturas ou em florestas e áreas de vegetação secundária. Em termos macroeconômicos, percebe-se ganhos de bem-estar para as regiões brasileiras, com exceção das regiões do Nordeste e Nordeste Cerrado. Ademais, a variação do PIB regional indica queda expressiva nessas regiões. Esses resultados sugerem que o Plano ABC aumenta as disparidades regionais no Brasil evidenciando que o desenho de políticas públicas precisa levar em conta tais diferenças. A pesquisa sugere mecanismos modernos de compensação de perdas reestruturando as cadeias de valor regionais, para que no longo prazo aumente a capacidade de produção e absorção de tecnologias. Em termos de custo econômico, o modelo indica que para o atingimento das metas do Plano ABC refer- entes a recuperação de pastagens e sistemas integrados seriam necessários cerca de R$ 39 bilhões (valores nominais de 2009), valor inferior aos valores projetados no lançamento do Plano ABC (cerca de R$ 37 bilhões para recupeção de pastagens e R$ 57 bilhões para iLP e iLPF). O atual nível de adoção de recursos do Programa ABC é bem inferior ao montante necessario projetado no presente estudo. Até o fi- nal do ano safra 2015/2016 os desembolsos haviam alcançado quase R$ 13,8 bilhões, incluindo os gastos não apenas com recuperação de pastagens e iLPF, mas também com as demais linhas do programa, como o plantio direto e o tratamento de dejetos de animais. Essa constatação sugere que, a continuar o ritmo atual observado de adoção do crédito do Programa ABC, as metas do Plano ABC no ambito da Política Nacional de Mudança do Clima não serão atingidas.
Brazil is considered one of the major players in World agriculture. Besides the economic relevance of agriculture and livestock productions in Brazil, the country has an active role in the international discussion about climate change. The agricul- ture, forestry and other land use (AFOLU) sector is the main source of greenhouse gas (GHG) emissions. It is a peculiar pattern among developing countries. At COP-15 2009, Brazil made a voluntary commitment to reduce emissions by 37% until 2025 and by 43% until 2030 compared to 2005 levels. The agricultural and livestock sectors are responsible to contribute With 22.5% of this total. The Brazil- ian Government released in the same year the Low Carbon Agriculture Plan (ABC Plan) as part of National Policy for Climate Change (PNMC) to achieve the GHG emissions reduction in the AFOLU sector. The ABC Plan has several actions, e.g., recover 15 million hectares (Mha) of degraded pasture, and increase by 4 Mha the integrated systems (crop-livestock integration and/or crop-livestock-forestry inte- gration). The objective of this thesis is to evaluate the economic impacts and the land-use changes resulting from these actions present in the ABC Plan, such as pasture recovery (PR) and integrated systems (IS). I have built a new computable general equilibrium model (CGE) (BREA version 1.0) With detailed representation of six regions in Brazil representing the economic relevance and agricultural frontier. The regions are: South, Southeast, North (Amazon biome), Center-West (Without Amazon biome), Northeast, and Northeast Cerrado (Maranhão, Tocantins, Piauí, and Bahia States) Which is considered the neW agricultural frontier in Brazil. The model represents several land uses, agricultural sectors, and it is the first CGE model to explicitly represent these technologies. Under different simulated scenarios, the outcomes indicate that the higher supply of recovered areas With high productivity pastures, combined With the integrated systems, promotes the land sparing effect. There is a reduction in the pressure to clear natural and forest areas made by live- stock sector. Also, there is a decrease in the cropland use and an increase in the area of planted forest. However, regional results show that regions in the agricultural frontier respond differently to the ABC Plan. In the Center-West and North regions the pasture area increases more than recovered areas. At the same time, the crop land area is converted to high quality pasture. In the South and Southeast regions the pasture area increases less than the recovered areas. It means that part of this area is converted in cropland, forest or secondary vegetation. At macroeconomic level, there are welfare gains in all regions, except in the Northeast and Northeast Cerrado regions. Also, regional GDP changes indicate significant losses in these regions. Modern compensation mechanisms should be develop to avoid these losses increasing the production capacity and the technology absorption in these regions. Considering the economic costs of PR and IS the model projects R$ 39 billion (2009 values). This value is significantly lower compared to those projected in the origi- nal text of the ABC Plan (around R$ 37 billion for PR and R$ 57 billion for IS). The actual adoption level of resources present in the ABC Program is also lower compared to the value projected by the model. By the end of 2015/2016 crop-year the volume of credit taken by farmers reached R$ 13.8 billion, including all actions present in the ABC Plan and not only PR and IS. It suggests that if the adoption of the ABC credit continue in a low rate the goals of the ABC Plan Will not be met.
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Buildings consume around half of the UK's total energy consumption and are responsible for almost 50% of UK's total carbon dioxide (CO2) emissions. Use of high thermal resistance insulation in buildings is critical to save the substantial amounts of space heating energy lost through building fabric. Conventional building insulation materials have higher thermal conductivity values ranging from 40 mWm-1K-1 (Glass fibre) - 26 mWm-1K-1 (Polyurethane foam) and require larger thicknesses to achieve stringent building regulation requirements which may not be feasible due to techno-economic constraints. Vacuum Insulation Panel (VIP) is a relatively new insulation for building applications that offers 5-8 times higher thermal resistance and can achieve significant space savings in buildings. VIPs are produced as a rigid panel comprising inner core board laminated in an outer high barrier envelope under evacuated conditions (< 5mbar). However, the main challenge for large scale acceptance of VIPs in building applications is their higher cost. VIPs have been shown to have an approximately 10 times longer payback compared to conventional EPS insulation due to their high initial cost. Expensive materials currently being used for VIP manufacturing such as fumed silica contribute to high cost of VIPs and it is critical to identify alternative low cost materials for VIP components to overcome the challenge of high cost. The aim of this thesis was to develop an alternative low cost material and investigate its suitability for use as VIP core. Expanded perlite, a low cost material was identified as a replacement of expensive fumed silica in a VIP core. Composite samples containing expanded perlite, fumed silica, silicon carbide (SiC) and polyester fibres were developed by dry mixing of the constituents in different mass ratios and their different properties were experimentally measured to identify optimum composition of composite. Gaseous thermal conductivity at different pressures was calculated from the pore size data obtained using Mercury Intrusion Porosimetry (MIP), gas adsorption and electron microscopy. Radiative conductivity of composite samples was measured using Fourier Transform Infrared (FTIR) to ascertain the opacifying effect of expanded perlite and opacifier (SiC). Centre of panel thermal conductivity of core boards of size 100mm x 100mm made of composite material at atmospheric pressure was measured by using a small guarded hot plate device. Average pore diameter values of expanded perlite decreased with the partial filling of fumed silica aggregates and was found to be in the range of 150-300 nm yielding lower gaseous conductivity values of 1.2-2.1 mWm-1K-1 at 100mbar and became negligible upon further decreasing pressures below 10 mbar. Core boards made of optimised composite containing 30% expanded perlite and 50% fumed silica along with SiC and polyester fibres was found to achieve centre of panel thermal conductivity of 28 mWm-1K-1 at atmospheric pressure and the average radiative conductivity of 0.67 mWm-1K-1 at 300K with its gaseous thermal conductivity at 1 mbar being 0.016 mWm-1K-1. According to the results of the thesis VIP prototypes consisting of core made with optimised composite consisting (50 mass% of fumed silica, 30 mass% of expanded perlite along with 8 mass% of fibre and 12 mass% of SiC) yielded centre of panel thermal conductivity of 7.4-7.6 mWm-1K-1 at pressure of 0.53-0.64 mbar. Opacifying properties of expanded perlite were observed and quantified. Expanded perlite reduced the radiative conductivity of the composite requiring smaller quantities of high density opacifiers such as SiC. For sample containing no expanded perlite, average radiative conductivity was calculated to be 1.37 mWm-1K-1 and radiative conductivity values decreased to 1.12 mWm-1K-1, 0.67 mWm-1K-1, 0.63 mWm-1K-1 and 0.50 mWm-1K-1 with mass ratio of expanded perlite 20%, 30%, 40% and 60% respectively. It was concluded that the solid conductivity of prototypes VIPs was 1.8-2 times higher compared to those of commercially available VIPs and is the main reason for higher centre of panel thermal conductivity.

The aim of our study was to explore and understand the factors that affect growth of low birth weight (LBW) infants in a rural district of Neno in Malawi in order to optimize their care. We surveyed 64 households of LBW infants born between April and June 2014. We collected quantitative data on socio-demographics and food security, and performed nutrition and development assessments of the LBW infants at six months of age. We also conducted qualitative in-depth interviews with a subset of 10 mothers of the LBW infants and with 3 nurses at postnatal clinics. We then merged the quantitative and qualitative datasets for a final interpretation. At six months of age, LBW infants were more likely to be underweight (mean weight-for-age Z-score -3.01±0.97) or stunted (mean height-for-age Z-score -2.45 ±1.34) than the WHO reference group. The majority (93.8%) of the households had moderate to severe food insecurity. Contributing factors to these poor outcomes included recurrent illness, resource scarcity and lack of social support. Most mothers opted for mixed feeding as a coping mechanism for the LBW infant’s slow growth; this mixed feeding without clean water and proper hygiene, could even worsen the health outcomes of these infants. Structural violence in poor households causes poor health outcomes of LBW infants. The responsibility of caring for LBW infants is so challenging because these children have so many unmet needs. Optimum care can be achieved with a variety of biosocial interventions.