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The early investigations of Jean-Pierre Sauvage, Sir J. Fraser Stoddart and Bernard J. Feringa into artificial molecular machines (AMMs) were recognized with the 2016 Nobel prize for the development of molecules with controllable movements, allowing them to perform various tasks when provided with energy. The first examples of AMMs mimicked the function of macroscope machines such as molecular motors, molecular gyroscopes, and a variety of other simple machines. Synthesizing these structures required connecting multiple molecular components such as a stator, axle, and rotator to create autonomous structures. These final ensembles minimize the number degrees of freedoms accessible to their molecular components, allowing them carry out a specific function when given an appropriate stimulus. A new paradigm in the field has recently emerged whereby chemists consider the collective function of many molecules in concert, rather than the function of an individual machine. For example, the combination of many molecules into a crystalline array creates materials with functionalities that are related to the physicochemical properties of each molecule, but the ensemble also has opportunities to exhibit high-order, emergent properties. When the molecular components contain magnetic or electric dipoles, domains in these materials have potential to respond in a cohesive manner when interfaced with external stimuli. The magnitude of this response allows a user to transduce various forms of energy, such as mechanical work to electricity, encode memory in magnetically polarized states or to store charge on the macroscopic scale. Applying the principles above to current technologies allows the creation of higher-order function systems and the development of advanced (programmable) materials. However, the precise control of microscopic motions through macroscopic inputs has not yet been achieved. My doctoral research focuses on using molecular rotation to develop both autonomous- and

The early investigations of Jean-Pierre Sauvage, Sir J. Fraser Stoddart and Bernard J. Feringa into artificial molecular machines (AMMs) were recognized with the 2016 Nobel prize for the development of molecules with controllable movements, allowing them to perform various tasks when provided with energy. The first examples of AMMs mimicked the function of macroscope machines such as molecular motors, molecular gyroscopes, and a variety of other simple machines. Synthesizing these structures required connecting multiple molecular components such as a stator, axle, and rotator to create autonomous structures. These final ensembles minimize the number degrees of freedoms accessible to their molecular components, allowing them carry out a specific function when given an appropriate stimulus. A new paradigm in the field has recently emerged whereby chemists consider the collective function of many molecules in concert, rather than the function of an individual machine. For example, the combination of many molecules into a crystalline array creates materials with functionalities that are related to the physicochemical properties of each molecule, but the ensemble also has opportunities to exhibit high-order, emergent properties. When the molecular components contain magnetic or electric dipoles, domains in these materials have potential to respond in a cohesive manner when interfaced with external stimuli. The magnitude of this response allows a user to transduce various forms of energy, such as mechanical work to electricity, encode memory in magnetically polarized states or to store charge on the macroscopic scale. Applying the principles above to current technologies allows the creation of higher-order function systems and the development of advanced (programmable) materials. However, the precise control of microscopic motions through macroscopic inputs has not yet been achieved. My doctoral research focuses on using molecular rotation to develop both autonomous- and

A manutenção adequada da massa muscular esquelética é essencial para prevenir a sarcopenia e garantir a saúde e qualidade de vida à medida que se envelhece. Os dois determinantes da síntese de proteína muscular são o aumento de carga no músculo por meio do exercício físico resistido e a ingestão de proteínas. Para um resultado efetivo de manutenção ou aumento da massa muscular, deve-se considerar a ingestão quantitativa e adequada de proteína e a fonte alimentar, visto que a proteína de origem vegetal possui diferenças em comparação a animal que limitam sua capacidade anabólica. Em virtude do aumento do vegetarianismo e da população idosa, que consome menos alimentos fontes de proteína animal, justifica-se a importância de compreender como a proteína de origem vegetal pode sustentar em longo prazo a síntese de proteína muscular quando associada ao exercício físico resistido, bem como, as possibilidades de adequação dietética frente à esta demanda., Proper maintenance of skeletal muscle mass is essential to prevent sarcopenia and ensure health and quality of life as aging progress. The two determinants of muscle protein synthesis are the increased load on skeletal muscle through resistance exercise and protein intake. For an effective result of maintaining or increasing muscle mass, it is relevant to consider the quantitative and adequate intake of protein, and the dietary source of protein since the plant-based protein has differences in comparison to animals that limit its anabolic capacity. Given the increase in vegetarianism and the elderly population, which consumes fewer food sources of animal protein, the importance of understanding how protein of plant-based protein can sustain muscle protein synthesis in the long term when associated with resistance exercise is justified, as well as the possibilities of dietary adequacy in the face of this demand.

A manutenção adequada da massa muscular esquelética é essencial para prevenir a sarcopenia e garantir a saúde e qualidade de vida à medida que se envelhece. Os dois determinantes da síntese de proteína muscular são o aumento de carga no músculo por meio do exercício físico resistido e a ingestão de proteínas. Para um resultado efetivo de manutenção ou aumento da massa muscular, deve-se considerar a ingestão quantitativa e adequada de proteína e a fonte alimentar, visto que a proteína de origem vegetal possui diferenças em comparação a animal que limitam sua capacidade anabólica. Em virtude do aumento do vegetarianismo e da população idosa, que consome menos alimentos fontes de proteína animal, justifica-se a importância de compreender como a proteína de origem vegetal pode sustentar em longo prazo a síntese de proteína muscular quando associada ao exercício físico resistido, bem como, as possibilidades de adequação dietética frente à esta demanda., Proper maintenance of skeletal muscle mass is essential to prevent sarcopenia and ensure health and quality of life as aging progress. The two determinants of muscle protein synthesis are the increased load on skeletal muscle through resistance exercise and protein intake. For an effective result of maintaining or increasing muscle mass, it is relevant to consider the quantitative and adequate intake of protein, and the dietary source of protein since the plant-based protein has differences in comparison to animals that limit its anabolic capacity. Given the increase in vegetarianism and the elderly population, which consumes fewer food sources of animal protein, the importance of understanding how protein of plant-based protein can sustain muscle protein synthesis in the long term when associated with resistance exercise is justified, as well as the possibilities of dietary adequacy in the face of this demand.

A manutenção adequada da massa muscular esquelética é essencial para prevenir a sarcopenia e garantir a saúde e qualidade de vida à medida que se envelhece. Os dois determinantes da síntese de proteína muscular são o aumento de carga no músculo por meio do exercício físico resistido e a ingestão de proteínas. Para um resultado efetivo de manutenção ou aumento da massa muscular, deve-se considerar a ingestão quantitativa e adequada de proteína e a fonte alimentar, visto que a proteína de origem vegetal possui diferenças em comparação a animal que limitam sua capacidade anabólica. Em virtude do aumento do vegetarianismo e da população idosa, que consome menos alimentos fontes de proteína animal, justifica-se a importância de compreender como a proteína de origem vegetal pode sustentar em longo prazo a síntese de proteína muscular quando associada ao exercício físico resistido, bem como, as possibilidades de adequação dietética frente à esta demanda., Proper maintenance of skeletal muscle mass is essential to prevent sarcopenia and ensure health and quality of life as aging progress. The two determinants of muscle protein synthesis are the increased load on skeletal muscle through resistance exercise and protein intake. For an effective result of maintaining or increasing muscle mass, it is relevant to consider the quantitative and adequate intake of protein, and the dietary source of protein since the plant-based protein has differences in comparison to animals that limit its anabolic capacity. Given the increase in vegetarianism and the elderly population, which consumes fewer food sources of animal protein, the importance of understanding how protein of plant-based protein can sustain muscle protein synthesis in the long term when associated with resistance exercise is justified, as well as the possibilities of dietary adequacy in the face of this demand.

Proper maintenance of skeletal muscle mass is essential to prevent sarcopenia and ensure health and quality of life as aging progress. The two determinants of muscle protein synthesis are the increased load on skeletal muscle through resistance exercise and protein intake. For an effective result of maintaining or increasing muscle mass, it is relevant to consider the quantitative and adequate intake of protein, and the dietary source of protein since the plant-based protein has differences in comparison to animals that limit its anabolic capacity. Given the increase in vegetarianism and the elderly population, which consumes fewer food sources of animal protein, the importance of understanding how protein of plant-based protein can sustain muscle protein synthesis in the long term when associated with resistance exercise is justified, as well as the possibilities of dietary adequacy in the face of this demand., A manutenção adequada da massa muscular esquelética é essencial para prevenir a sarcopenia e garantir a saúde e qualidade de vida à medida que se envelhece. Os dois determinantes da síntese de proteína muscular são o aumento de carga no músculo por meio do exercício físico resistido e a ingestão de proteínas. Para um resultado efetivo de manutenção ou aumento da massa muscular, deve-se considerar a ingestão quantitativa e adequada de proteína e a fonte alimentar, visto que a proteína de origem vegetal possui diferenças em comparação a animal que limitam sua capacidade anabólica. Em virtude do aumento do vegetarianismo e da população idosa, que consome menos alimentos fontes de proteína animal, justifica-se a importância de compreender como a proteína de origem vegetal pode sustentar em longo prazo a síntese de proteína muscular quando associada ao exercício físico resistido, bem como, as possibilidades de adequação dietética frente à esta demanda.

Proper maintenance of skeletal muscle mass is essential to prevent sarcopenia and ensure health and quality of life as aging progress. The two determinants of muscle protein synthesis are the increased load on skeletal muscle through resistance exercise and protein intake. For an effective result of maintaining or increasing muscle mass, it is relevant to consider the quantitative and adequate intake of protein, and the dietary source of protein since the plant-based protein has differences in comparison to animals that limit its anabolic capacity. Given the increase in vegetarianism and the elderly population, which consumes fewer food sources of animal protein, the importance of understanding how protein of plant-based protein can sustain muscle protein synthesis in the long term when associated with resistance exercise is justified, as well as the possibilities of dietary adequacy in the face of this demand., A manutenção adequada da massa muscular esquelética é essencial para prevenir a sarcopenia e garantir a saúde e qualidade de vida à medida que se envelhece. Os dois determinantes da síntese de proteína muscular são o aumento de carga no músculo por meio do exercício físico resistido e a ingestão de proteínas. Para um resultado efetivo de manutenção ou aumento da massa muscular, deve-se considerar a ingestão quantitativa e adequada de proteína e a fonte alimentar, visto que a proteína de origem vegetal possui diferenças em comparação a animal que limitam sua capacidade anabólica. Em virtude do aumento do vegetarianismo e da população idosa, que consome menos alimentos fontes de proteína animal, justifica-se a importância de compreender como a proteína de origem vegetal pode sustentar em longo prazo a síntese de proteína muscular quando associada ao exercício físico resistido, bem como, as possibilidades de adequação dietética frente à esta demanda.

Proper maintenance of skeletal muscle mass is essential to prevent sarcopenia and ensure health and quality of life as aging progress. The two determinants of muscle protein synthesis are the increased load on skeletal muscle through resistance exercise and protein intake. For an effective result of maintaining or increasing muscle mass, it is relevant to consider the quantitative and adequate intake of protein, and the dietary source of protein since the plant-based protein has differences in comparison to animals that limit its anabolic capacity. Given the increase in vegetarianism and the elderly population, which consumes fewer food sources of animal protein, the importance of understanding how protein of plant-based protein can sustain muscle protein synthesis in the long term when associated with resistance exercise is justified, as well as the possibilities of dietary adequacy in the face of this demand., A manutenção adequada da massa muscular esquelética é essencial para prevenir a sarcopenia e garantir a saúde e qualidade de vida à medida que se envelhece. Os dois determinantes da síntese de proteína muscular são o aumento de carga no músculo por meio do exercício físico resistido e a ingestão de proteínas. Para um resultado efetivo de manutenção ou aumento da massa muscular, deve-se considerar a ingestão quantitativa e adequada de proteína e a fonte alimentar, visto que a proteína de origem vegetal possui diferenças em comparação a animal que limitam sua capacidade anabólica. Em virtude do aumento do vegetarianismo e da população idosa, que consome menos alimentos fontes de proteína animal, justifica-se a importância de compreender como a proteína de origem vegetal pode sustentar em longo prazo a síntese de proteína muscular quando associada ao exercício físico resistido, bem como, as possibilidades de adequação dietética frente à esta demanda.

In chapter 1, Fremont Ancestral Puebloan (AP) maize (Zea mays) cumulative growing degree days (cGDD) were reconstructed for the years 850-1449 CE by statistically downscaling temperature dailies from a state-of-the-art, fully coupled climate model (CESM LME). In addition, radiocarbon dates from geolocated Fremont artifacts (n=982) used were to map time series of spatially discrete summed probability distributions (SPD). Fremont occupation of the Colorado Plateau was shown to coincide with warming conditions during the early Medieval Climate Anomaly (MCA), ~850-1050 CE, and abandonment with shorter and increasingly variable growing seasons after ~1050 CE. The disappearance of Fremont-identified material culture from Utah was shown to coincide with increased hydrologic variability on the Colorado Plateau with the transition to the Little Ice Age (LIA, ~1350-1850 CE). In chapter 2, the downscaled climate model was used to drive a process-based crop model (EPIC) to reconstruct maize yields under 7 different crop management strategies for irrigated and non-irrigated cases. Individual sites were clustered within three separate subregions for analysis (i.e., the Great Salt Lake shoreline, the highland Uinta Basin, and an archetypal Fremont canyon occupation at Range Creek). All occupations analyzed were caught within the same agro-ecological vise of increasing exogenous climatic and endogenous (anthropogenic) soil nutrient degradation, with varying degrees of intensity. Among the subregions, Range Creek was the most constrained by exogenous changes in climate (cooling and increasing variability at the MCA-LIA transition), and the Great Salt Lake shoreline the least; but the Great Salt Lake sites were the most sensitive to endogenous soil degradation. The methods employed in chapters 1 and 2 are broadly applicable to reconstruct GDD and crop yields, especially in other low-technology subsistence dryland agricultural contexts. In chapter 3, subfossil chironomid (Insecta: Dip

In chapter 1, Fremont Ancestral Puebloan (AP) maize (Zea mays) cumulative growing degree days (cGDD) were reconstructed for the years 850-1449 CE by statistically downscaling temperature dailies from a state-of-the-art, fully coupled climate model (CESM LME). In addition, radiocarbon dates from geolocated Fremont artifacts (n=982) used were to map time series of spatially discrete summed probability distributions (SPD). Fremont occupation of the Colorado Plateau was shown to coincide with warming conditions during the early Medieval Climate Anomaly (MCA), ~850-1050 CE, and abandonment with shorter and increasingly variable growing seasons after ~1050 CE. The disappearance of Fremont-identified material culture from Utah was shown to coincide with increased hydrologic variability on the Colorado Plateau with the transition to the Little Ice Age (LIA, ~1350-1850 CE). In chapter 2, the downscaled climate model was used to drive a process-based crop model (EPIC) to reconstruct maize yields under 7 different crop management strategies for irrigated and non-irrigated cases. Individual sites were clustered within three separate subregions for analysis (i.e., the Great Salt Lake shoreline, the highland Uinta Basin, and an archetypal Fremont canyon occupation at Range Creek). All occupations analyzed were caught within the same agro-ecological vise of increasing exogenous climatic and endogenous (anthropogenic) soil nutrient degradation, with varying degrees of intensity. Among the subregions, Range Creek was the most constrained by exogenous changes in climate (cooling and increasing variability at the MCA-LIA transition), and the Great Salt Lake shoreline the least; but the Great Salt Lake sites were the most sensitive to endogenous soil degradation. The methods employed in chapters 1 and 2 are broadly applicable to reconstruct GDD and crop yields, especially in other low-technology subsistence dryland agricultural contexts. In chapter 3, subfossil chironomid (Insecta: Dip

The transcription factor p53 is one of the most studied tumour suppressors with over 90 000 publications in PubMed referring to the protein. It is also the most frequently mutated gene across all cancer types with around 50% of cancers presenting as mutant p53, and when it is not mutated, it is frequently inactivated to circumvent its tumour suppressor function. Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its first discovery as “the guardian of the genome”. For our drug development programme, we have focused on visualising the induction of p53 transcriptional activity as a readout for a desirable phenotype. This screen used two stably transfected reporter cell lines, the T22 murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical genetic approach, we have entered into our drug development programme in a target-blind manner. For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53 synthesis at early timepoints without stabilising the protein or increasing levels of p53 mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in ARN8 cells that were previously screened in the T22 cell line for another study. We found that those that

The transcription factor p53 is one of the most studied tumour suppressors with over 90 000 publications in PubMed referring to the protein. It is also the most frequently mutated gene across all cancer types with around 50% of cancers presenting as mutant p53, and when it is not mutated, it is frequently inactivated to circumvent its tumour suppressor function. Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its first discovery as “the guardian of the genome”. For our drug development programme, we have focused on visualising the induction of p53 transcriptional activity as a readout for a desirable phenotype. This screen used two stably transfected reporter cell lines, the T22 murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical genetic approach, we have entered into our drug development programme in a target-blind manner. For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53 synthesis at early timepoints without stabilising the protein or increasing levels of p53 mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in ARN8 cells that were previously screened in the T22 cell line for another study. We found that those that

The transcription factor p53 is one of the most studied tumour suppressors with over 90 000 publications in PubMed referring to the protein. It is also the most frequently mutated gene across all cancer types with around 50% of cancers presenting as mutant p53, and when it is not mutated, it is frequently inactivated to circumvent its tumour suppressor function. Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its first discovery as “the guardian of the genome”. For our drug development programme, we have focused on visualising the induction of p53 transcriptional activity as a readout for a desirable phenotype. This screen used two stably transfected reporter cell lines, the T22 murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical genetic approach, we have entered into our drug development programme in a target-blind manner. For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53 synthesis at early timepoints without stabilising the protein or increasing levels of p53 mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in ARN8 cells that were previously screened in the T22 cell line for another study. We found that those that

The transcription factor p53 is one of the most studied tumour suppressors with over 90 000 publications in PubMed referring to the protein. It is also the most frequently mutated gene across all cancer types with around 50% of cancers presenting as mutant p53, and when it is not mutated, it is frequently inactivated to circumvent its tumour suppressor function. Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its first discovery as “the guardian of the genome”. For our drug development programme, we have focused on visualising the induction of p53 transcriptional activity as a readout for a desirable phenotype. This screen used two stably transfected reporter cell lines, the T22 murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical genetic approach, we have entered into our drug development programme in a target-blind manner. For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53 synthesis at early timepoints without stabilising the protein or increasing levels of p53 mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in ARN8 cells that were previously screened in the T22 cell line for another study. We found that those that

The transcription factor p53 is one of the most studied tumour suppressors with over 90 000 publications in PubMed referring to the protein. It is also the most frequently mutated gene across all cancer types with around 50% of cancers presenting as mutant p53, and when it is not mutated, it is frequently inactivated to circumvent its tumour suppressor function. Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its first discovery as “the guardian of the genome”. For our drug development programme, we have focused on visualising the induction of p53 transcriptional activity as a readout for a desirable phenotype. This screen used two stably transfected reporter cell lines, the T22 murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical genetic approach, we have entered into our drug development programme in a target-blind manner. For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53 synthesis at early timepoints without stabilising the protein or increasing levels of p53 mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in ARN8 cells that were previously screened in the T22 cell line for another study. We found that those that

The transcription factor p53 is one of the most studied tumour suppressors with over 90 000 publications in PubMed referring to the protein. It is also the most frequently mutated gene across all cancer types with around 50% of cancers presenting as mutant p53, and when it is not mutated, it is frequently inactivated to circumvent its tumour suppressor function. Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its first discovery as “the guardian of the genome”. For our drug development programme, we have focused on visualising the induction of p53 transcriptional activity as a readout for a desirable phenotype. This screen used two stably transfected reporter cell lines, the T22 murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical genetic approach, we have entered into our drug development programme in a target-blind manner. For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53 synthesis at early timepoints without stabilising the protein or increasing levels of p53 mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in ARN8 cells that were previously screened in the T22 cell line for another study. We found that those that

The transcription factor p53 is one of the most studied tumour suppressors with over 90 000 publications in PubMed referring to the protein. It is also the most frequently mutated gene across all cancer types with around 50% of cancers presenting as mutant p53, and when it is not mutated, it is frequently inactivated to circumvent its tumour suppressor function. Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its first discovery as “the guardian of the genome”. For our drug development programme, we have focused on visualising the induction of p53 transcriptional activity as a readout for a desirable phenotype. This screen used two stably transfected reporter cell lines, the T22 murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical genetic approach, we have entered into our drug development programme in a target-blind manner. For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53 synthesis at early timepoints without stabilising the protein or increasing levels of p53 mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in ARN8 cells that were previously screened in the T22 cell line for another study. We found that those that

The transcription factor p53 is one of the most studied tumour suppressors with over 90 000 publications in PubMed referring to the protein. It is also the most frequently mutated gene across all cancer types with around 50% of cancers presenting as mutant p53, and when it is not mutated, it is frequently inactivated to circumvent its tumour suppressor function. Therapeutic targeting of both mutant and wild-type p53 has been a key focus ever since its first discovery as “the guardian of the genome”. For our drug development programme, we have focused on visualising the induction of p53 transcriptional activity as a readout for a desirable phenotype. This screen used two stably transfected reporter cell lines, the T22 murine fibroblasts, and the ARN8 human melanoma cell line. Using this forward chemical genetic approach, we have entered into our drug development programme in a target-blind manner. For Paper I we screened 30 000 compounds in both T22 and ARN8 cells and selected those that were capable of increasing p53 transcriptional activity in the ARN8 tumour cells, but not in the T22 murine fibroblasts. We selected a compound from the hits that had a drug-like structure as well as possessing a chiral centre and christened it HZ00. HZ00 was found to induce p53 protein in a dose-dependent manner, selectively kill tumour cells whilst inducing a reversible G1 arrest in normal human dermal fibroblasts (HNDFs), and increase p53 synthesis at early timepoints without stabilising the protein or increasing levels of p53 mRNA. HZ00 also synergised with the inhibitor of p53 degradation, nutlin 3, both in vitro and in vivo in a tumour xenograft model. Following target deconvolution using a knowledgebased approach we identified DHODH, a key enzyme in the de novo pyrimidine nucleotide synthesis pathway, as the target of HZ00. At this point we re-screened 30 000 compounds in ARN8 cells that were previously screened in the T22 cell line for another study. We found that those that

This dissertation investigated how accurately and effectively different graphical display formats can communicate quantitative information about an underlying continuous distribution of an uncertain outcome to knowledgeable but not necessarily technical audiences. Across three studies, display formats were evaluated on three metrics: 1) Correct responses on questions with objective answers; 2) projection of a future forecasters' range estimates (a proxy for how well the uncertainty was conveyed); 3) subjective experience using the display. The role of numeracy as a main effect and its interaction with Display was also assessed. Study 1 found that for objective measures the Quantile plot and CDF plot performed better than either a Box or Density plot, but this outperformance only manifested among the high numerate; for projection, the Box plot induced the widest range bounds and the Density plot the smallest; for subjective measures, the Quantile plot was rated highest and its objective / subjective scores were best aligned. Study 2 extended the first study by testing whether communication can be improved by combining multiple plot elements into a single display or by allowing participants to toggle between different plots. For objective measures, the addition of a CDF element boosted performance; for projection, the Toggle condition yielded wider range bounds. In both cases, numeracy moderated the effect, with the differences only manifesting among the high numerate. A Toggle CDF+Box display had the greatest alignment between objective and subjective measures. On common events across studies, however, a Quantile plot alone still yielded the best performance on objective measures and Box plot the best on projection - suggesting an inherent trade-off. Study 3 extended the findings from binary to categorical / ordinal events by testing the effectiveness of four different display types intended to effectively scale as the number potential outcomes increase. The Box+Scat

Food allergy, defined as an adverse immune response to food, is increasing in prevalence. It can be broadly separated into phases of sensitization, in which allergy-triggering Immunoglobulin E (IgE) is generated, and the post-sensitization allergic response, in which the allergic response is triggered by sensitizing allergen. While much is known about the specific mediators that cause allergies, the immune processes that underlie disease progression are less clear. This project has employed mouse models of Th2 immunity to clarify the factors involved in the initiation and maintenance of allergic disease. At the centre of allergic disease is the Interleukin (IL)-4-producing CD4+ T helper type 2 (Th2) cell. One of the key inducers of Th2 cell development in vitro is IL-4, but its involvement in Th2 cell development in vivo is controversial. In our studies, we saw that Th2 cell development could be initiated in vivo by primary, adjuvant-free allergen immunisation in the absence of IL-4. However, Th2 cells were more frequent in IL-4-sufficient conditions. We also determined that genetic lesions that result in loss of one, or both, IL-4 alleles impaired the Th2 cell-mediated allergic process, such that IL-4-heterozygous mice can be considered haplo-insufficient for IL-4 in allergic disease contexts. In addition to the generation of IgE antibody, Th2 cells are implicated in the post-sensitization phase of allergy. Multiple oral challenges of sensitized mice induces elevations in Th2-associated cytokines and elevates intestinal mast cell frequencies. It was the second aim of this project to clarify the role of CD4+ T cells in the post-sensitization intestinal allergic process. We demonstrate a key role for CD4+ T cells in this jejunal mast cell recruitment, and identify that this is required in addition to their established contribution to IgE production. Our investigations also reveal a previously unappreciated role for the CD4+ T cell-derived cytokine IL-3 in oral food a

Food allergy, defined as an adverse immune response to food, is increasing in prevalence. It can be broadly separated into phases of sensitization, in which allergy-triggering Immunoglobulin E (IgE) is generated, and the post-sensitization allergic response, in which the allergic response is triggered by sensitizing allergen. While much is known about the specific mediators that cause allergies, the immune processes that underlie disease progression are less clear. This project has employed mouse models of Th2 immunity to clarify the factors involved in the initiation and maintenance of allergic disease. At the centre of allergic disease is the Interleukin (IL)-4-producing CD4+ T helper type 2 (Th2) cell. One of the key inducers of Th2 cell development in vitro is IL-4, but its involvement in Th2 cell development in vivo is controversial. In our studies, we saw that Th2 cell development could be initiated in vivo by primary, adjuvant-free allergen immunisation in the absence of IL-4. However, Th2 cells were more frequent in IL-4-sufficient conditions. We also determined that genetic lesions that result in loss of one, or both, IL-4 alleles impaired the Th2 cell-mediated allergic process, such that IL-4-heterozygous mice can be considered haplo-insufficient for IL-4 in allergic disease contexts. In addition to the generation of IgE antibody, Th2 cells are implicated in the post-sensitization phase of allergy. Multiple oral challenges of sensitized mice induces elevations in Th2-associated cytokines and elevates intestinal mast cell frequencies. It was the second aim of this project to clarify the role of CD4+ T cells in the post-sensitization intestinal allergic process. We demonstrate a key role for CD4+ T cells in this jejunal mast cell recruitment, and identify that this is required in addition to their established contribution to IgE production. Our investigations also reveal a previously unappreciated role for the CD4+ T cell-derived cytokine IL-3 in oral food a

The microtubule network is involved in cellular processes including protein transport and cell division. Microtubule stabilising drugs (MSD) bind to microtubules and alter their dynamic balance in favour of the polymerised state. While primarily known for their anti-mitotic properties, MSD also exert immunomodulatory effects in vitro and in vivo. It is the aim of this project to investigate the effects of MSD on protein trafficking and secretion to determine how they affect immune-mediated exocytosis. Previous work in our lab demonstrated that macrophage responses to bacterial lipopolysaccharide, as measured by the production of TNF-a and nitric oxide, are inhibited by both paclitaxel and peloruside. In this thesis we continued this work and saw that inhibition was not affected by temporal IFN-y priming and found that altered production kinetics were not sufficient to explain the inhibition. To kill target cells cytotoxic T cells (CTL) reorganise their cytoskeleton so that lytic granules can traffic down microtubules to be delivered to the target. Using an in vitro model of CTL killing, we saw that MSD did not inhibit killing by CTL, lytic granule delivery to the cell surface, or antigen-stimulated Interferon-y (IFN-y) production by CTL. In contrast to this, in a murine model of antigen-induced killing we saw that a single dose of paclitaxel had a significant inhibitory effect on CTL-mediated cytolysis in vivo. Together these studies suggest that MSD have multiple immunomodulatory effects that are independent of their anti-proliferative effects. The data suggest that patients undergoing taxane therapy may be unable to fight infection long before the anti-mitotic effects of MSD are apparent.

The microtubule network is involved in cellular processes including protein transport and cell division. Microtubule stabilising drugs (MSD) bind to microtubules and alter their dynamic balance in favour of the polymerised state. While primarily known for their anti-mitotic properties, MSD also exert immunomodulatory effects in vitro and in vivo. It is the aim of this project to investigate the effects of MSD on protein trafficking and secretion to determine how they affect immune-mediated exocytosis. Previous work in our lab demonstrated that macrophage responses to bacterial lipopolysaccharide, as measured by the production of TNF-a and nitric oxide, are inhibited by both paclitaxel and peloruside. In this thesis we continued this work and saw that inhibition was not affected by temporal IFN-y priming and found that altered production kinetics were not sufficient to explain the inhibition. To kill target cells cytotoxic T cells (CTL) reorganise their cytoskeleton so that lytic granules can traffic down microtubules to be delivered to the target. Using an in vitro model of CTL killing, we saw that MSD did not inhibit killing by CTL, lytic granule delivery to the cell surface, or antigen-stimulated Interferon-y (IFN-y) production by CTL. In contrast to this, in a murine model of antigen-induced killing we saw that a single dose of paclitaxel had a significant inhibitory effect on CTL-mediated cytolysis in vivo. Together these studies suggest that MSD have multiple immunomodulatory effects that are independent of their anti-proliferative effects. The data suggest that patients undergoing taxane therapy may be unable to fight infection long before the anti-mitotic effects of MSD are apparent.

The microtubule network is involved in cellular processes including protein transport and cell division. Microtubule stabilising drugs (MSD) bind to microtubules and alter their dynamic balance in favour of the polymerised state. While primarily known for their anti-mitotic properties, MSD also exert immunomodulatory effects in vitro and in vivo. It is the aim of this project to investigate the effects of MSD on protein trafficking and secretion to determine how they affect immune-mediated exocytosis. Previous work in our lab demonstrated that macrophage responses to bacterial lipopolysaccharide, as measured by the production of TNF-a and nitric oxide, are inhibited by both paclitaxel and peloruside. In this thesis we continued this work and saw that inhibition was not affected by temporal IFN-y priming and found that altered production kinetics were not sufficient to explain the inhibition. To kill target cells cytotoxic T cells (CTL) reorganise their cytoskeleton so that lytic granules can traffic down microtubules to be delivered to the target. Using an in vitro model of CTL killing, we saw that MSD did not inhibit killing by CTL, lytic granule delivery to the cell surface, or antigen-stimulated Interferon-y (IFN-y) production by CTL. In contrast to this, in a murine model of antigen-induced killing we saw that a single dose of paclitaxel had a significant inhibitory effect on CTL-mediated cytolysis in vivo. Together these studies suggest that MSD have multiple immunomodulatory effects that are independent of their anti-proliferative effects. The data suggest that patients undergoing taxane therapy may be unable to fight infection long before the anti-mitotic effects of MSD are apparent.

The microtubule network is involved in cellular processes including protein transport and cell division. Microtubule stabilising drugs (MSD) bind to microtubules and alter their dynamic balance in favour of the polymerised state. While primarily known for their anti-mitotic properties, MSD also exert immunomodulatory effects in vitro and in vivo. It is the aim of this project to investigate the effects of MSD on protein trafficking and secretion to determine how they affect immune-mediated exocytosis. Previous work in our lab demonstrated that macrophage responses to bacterial lipopolysaccharide, as measured by the production of TNF-a and nitric oxide, are inhibited by both paclitaxel and peloruside. In this thesis we continued this work and saw that inhibition was not affected by temporal IFN-y priming and found that altered production kinetics were not sufficient to explain the inhibition. To kill target cells cytotoxic T cells (CTL) reorganise their cytoskeleton so that lytic granules can traffic down microtubules to be delivered to the target. Using an in vitro model of CTL killing, we saw that MSD did not inhibit killing by CTL, lytic granule delivery to the cell surface, or antigen-stimulated Interferon-y (IFN-y) production by CTL. In contrast to this, in a murine model of antigen-induced killing we saw that a single dose of paclitaxel had a significant inhibitory effect on CTL-mediated cytolysis in vivo. Together these studies suggest that MSD have multiple immunomodulatory effects that are independent of their anti-proliferative effects. The data suggest that patients undergoing taxane therapy may be unable to fight infection long before the anti-mitotic effects of MSD are apparent.

The microtubule network is involved in cellular processes including protein transport and cell division. Microtubule stabilising drugs (MSD) bind to microtubules and alter their dynamic balance in favour of the polymerised state. While primarily known for their anti-mitotic properties, MSD also exert immunomodulatory effects in vitro and in vivo. It is the aim of this project to investigate the effects of MSD on protein trafficking and secretion to determine how they affect immune-mediated exocytosis. Previous work in our lab demonstrated that macrophage responses to bacterial lipopolysaccharide, as measured by the production of TNF-a and nitric oxide, are inhibited by both paclitaxel and peloruside. In this thesis we continued this work and saw that inhibition was not affected by temporal IFN-y priming and found that altered production kinetics were not sufficient to explain the inhibition. To kill target cells cytotoxic T cells (CTL) reorganise their cytoskeleton so that lytic granules can traffic down microtubules to be delivered to the target. Using an in vitro model of CTL killing, we saw that MSD did not inhibit killing by CTL, lytic granule delivery to the cell surface, or antigen-stimulated Interferon-y (IFN-y) production by CTL. In contrast to this, in a murine model of antigen-induced killing we saw that a single dose of paclitaxel had a significant inhibitory effect on CTL-mediated cytolysis in vivo. Together these studies suggest that MSD have multiple immunomodulatory effects that are independent of their anti-proliferative effects. The data suggest that patients undergoing taxane therapy may be unable to fight infection long before the anti-mitotic effects of MSD are apparent.

Through the investigation of indirect light, reflected color, and geometric construction, and by the awareness and understanding of what allows for experiences that engage the human emotion, this thesis offers a project which has the power and presence to move the soul.

Through the investigation of indirect light, reflected color, and geometric construction, and by the awareness and understanding of what allows for experiences that engage the human emotion, this thesis offers a project which has the power and presence to move the soul.

This paper describes the Soviet military's extensive network for the training of prospective regular officers of the Soviet Armed Forces. After analyzing recruitment procedures, indicators of program effectiveness, problems of cadet discipline, as well as methods and perplexities of ideological inculcation, the author concludes that, barring the highly publicized 'few exceptions' (nekotorye iskliucheniia), the contemporary graduates of these schools are at least as well educated or exposed to as good an education in the practical aspects of their chosen profession, as are their American counterparts.