Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (leaves 105-112). Also available via the World Wide Web.

Thesis (Ph. D.)--University of Washington, 1990. / Vita. Includes bibliographical references (leaves [112]-128).

Thesis (Ph. D.)--Ludwig-Maximilians-Universität München, 2003. / Title from PDF title page (viewed on May 13, 2006). Vita. Includes bibliographical references (p. 136-146).

In recent years, there has been a major movement in the pharmaceutical industry towards the development of molecules that selectivity inhibit a previously-validated specific target. This is referred to as target-based drug discovery. It was hoped that adopting this approach would usher in a new golden age of drug discovery. However, this has not been the case, with issues arising such as the target’s mechanism of action being poorly understood, with it not playing the expected role in the disease progression, or feedback resistance mechanisms causing the target to lose its role in the disease. In contrast to this, in the past 20 years it has been argued that developing drugs in a target-agnostic way and screening them against an expressed phenotype i.e. phenotypic drug discovery, has been more successful, despite fewer programs being run in the manner. The AXL kinase is a receptor tyrosine kinase (RTK) and a member of the TAM family, along with MER and TYRO3. AXL has long been associated with numerous types of cancer. Having been first discovered in 1991 in acute myeloid leukaemia (AML), it has gone on to be more associated with advanced solid tumours such as brain, breast, and lung, with the trend being that increased AXL correlates with a poorer prognosis for the patient. Upon the activation of AXL by the vitamin K ligand GAS6, a series of downstream pathways are activated that go on to encourage cell survival, proliferation, and migration. In addition to this, AXL has been shown to be involved in crosstalk with other kinase pathways, resulting in AXL expression being associated with chemoresistance and survival mechanisms. Despite the promising outlook for AXL inhibitors, to date only one selective AXL inhibitor, BGB324 (formally R428) has entered clinical trials, with selective AXL inhibitors being difficult to develop due to a lack of a crystal structure or a reliable homology model. To address the aforementioned issues that target-based approaches can suffer from, and due to AXL lacking a crystal structure, the work in this thesis utilised a pragmatic drug design method that started from ligands/existing scaffolds known to inhibit the target from the literature (publications, clinical trials and patents). A series of small libraries were prepared and then tested against a selected phenotype e.g. cell viability, in at least two cell types: one that expressed the target (e.g. AXL) and one that did not. Hits were optimised for potency against the desired phenotype. The compounds then went through target deconvolution (kinase screening) to confirm the target of the inhibitors. Employing this approach, we initially synthesised two small libraries of potential AXL inhibitors. The potency of these compounds was tested using cell-based phenotypic assays, by evaluating cell viability in both native and chemo-resistant breast cancer cells. These libraries were optimised through focused combinatorial synthesis and phenotypic screening, to yield a small collection of antiproliferative hits. These hits were then profiled against a panel of twelve select kinases. The first library, while giving some important structural information, did not inhibit the kinases screened in a meaningful manner. However, the second library gave several potent compounds, inhibiting AXL, FLT3, and RET, with one compound being selective for AXL. The leads from this series were optimised further, through SAR studies, gaining important structural information in order to improve potency and selectivity of the compounds. The flexibility of the phenotypic cell-based approach allowed the pursuit of FLT3 inhibitors, resulting in the synthesis of one of the most potent FLT3 inhibitors synthesised to date.

Primary human hepatocytes are a scare resource with limited lifespan and variable function which diminishes with time in culture. As a consequence, their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESC) could provide a stable source of human tissue due to their self-renewal properties and their ability to give rise to all the cell types of the human body. Therefore, hESC have the potential to provide an unlimited supply of hepatocytes. To date, the use of hESCs-derived somatic cells is limited due to the undefined, variable and xeno-containing microenvironment that influences the cell performance and life span, limiting scale-up and downstream application. Therefore, the development of highly defined cell based systems is required if the true potential of stem cell derived hepatocytes is to be realised. In order to replace the use of animal derived culture substrates to differentiate and maintain hESCs-derived hepatocytes, an interdisciplinary approach was employed to define synthetic materials, which maintain hepatocyte-like cell phenotype in culture. A simple polyurethane, PU134, was identified which improved hepatocyte performance and stability when compared to biological matrices. Moreover, the synthetic polymer was amenable to scale up and demonstrated batch-to-batch consistency. I subsequently used the synthetic polymer surface to probe the underlying biology, identifying key modulators of hepatocyte-like cell phenotype. This resulted in the identification of a novel genetic signature, MMP13, CTNND2 and THBS2, which was associated with stable hepatocyte performance. Importantly, those findings could be translated to two hESC lines derived at GMP. In conclusion, hepatocyte differentiation of pluripotent stem cells requires a defined microenvironment. The novel gene signature identified in this study represents an example of how to deliver stable hESCs-derived hepatocytes.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-03-28T12:35:23Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_Manuella da Luz D Barros.pdf: 1660746 bytes, checksum: 1cd53d223f6d92ef7dab1a2d30b49ece (MD5) / Made available in DSpace on 2017-03-28T12:35:23Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_Manuella da Luz D Barros.pdf: 1660746 bytes, checksum: 1cd53d223f6d92ef7dab1a2d30b49ece (MD5) Previous issue date: 2016-02-23 / FACEPE / CNPQ / A plasticidade fenotípica compreende a capacidade do organismo de elaborar adaptações morfofuncionais em resposta aos estímulos ambientais. Essa capacidade é mais intensa durante a gestação e lactação (plasticidade do desenvolvimento), podendo estar associada a consequências ao longo da vida. Neste contexto, a serotonina (5-HT) tem papel determinante no desenvolvimento do sistema nervoso central. Além disso, a 5-HT participa do controle central homeostático do balanço energético no núcleo arqueado (Arc) do hipotálamo, estimulando a saciedade e o gasto de energia através da inibição de neurônios orexígenos e estimulação de neurônios anorexígenos via receptores 5-HT1B e 5-HT2C, respectivamente. Dessa forma, a alteração da atividade serotoninérgica durante o desenvolvimento pode modificar a regulação e a expressão do comportamento alimentar em longo prazo. Estudos prévios de nosso grupo de pesquisa apontaram que a inibição seletiva neonatal da recaptação de serotonina (ISNRS) pode estar associada a fenótipo hipofágico na vida adulta. Nós acreditamos que esse fenótipo se deve a maior atividade da via anorexígena no Arc. Diante disso, este estudo teve como objetivo avaliar os efeitos da ISNRS sobre o peso corporal, o comportamento alimentar e a atividade neuronal no Arc em resposta aos agonistas dos receptores 5HT2C e 5-HT1B em ratos adultos. Ratos Wistar foram tratados com solução salina (SAL, n=15) ou fluoxetina (FLUO, n=15) do 1° ao 21° dia pós-natal. Aos 170 dias de vida foi realizada cirurgia de estereotaxia para implantação de cânula no ventrículo lateral direito através da qual foram feitas as injeções intracerebroventricular (ICV). Aos 180 dias foi realizada a pesagem dos animais seguida de injeção ICV de ACSF, agonista do receptor 5-HT2C ou agonista do receptor 5-HT1B, obtendo-se seis grupos experimentais (n=5 animais/grupo): SAL+ACSF, SAL+AG.1B, SAL+AG.2C, FLUO+ACSF, FLUO+AG.1B e FLUO+AG.2C. A partir desses grupos, foi analisada a sequência comportamental de saciedade (SCS) e o número de células reativas contra proteína c-fos no Arc. Comparado ao grupo SAL, o grupo FLUO apresentou menor peso corporal. A ISNRS também promoveu maior ingestão alimentar frente ao estímulo com o agonista 5-HT2C, menor taxa alimentar após injeção de ACSF e antecipação do ponto de saciedade após estímulo com o agonista 5-HT1B. Além disso, o grupo FLUO apresentou maior número de células reativas contra c-fos no Arc frente ao agonista 5-HT2C. Corroborando nossa hipótese, a ISNRS parece promover aumento da atividade da via anorexígena no Arc na idade adulta, apesar de não ter ocorrido redução da ingestão alimentar. Em conjunto, os achados deste estudo sugerem que o menor peso corporal em ratos adultos submetidos à ISNRS pode ser consequência do aumento da atividade da via anorexígena no Arc, o que pode contribuir para um aumento do gasto energético. / Phenotypic plasticity comprises the ability of an organism to develop morphological and functional adaptations in response to environmental stimuli. This capacity is more intense during pregnancy and lactation (developmental plasticity) and may be associated with consequences throughout life. In this context, serotonin (5-HT) has a decisive role in the development of the central nervous system. Furthermore, 5-HT participates in homeostatic central control of energy balance in the arcuate nucleus (Arc) of the hypothalamus, stimulating satiety and energy expenditure through inhibition of orexigenic neurons and stimulation anorectic neurons via 5-HT1B receptors and 5-HT2C, respectively. Thus, the modification of serotonergic activity during development can change the regulation and expression of feeding behavior in the long term. Previous studies from our research group showed that neonatal selective serotonin reuptake inhibition (NSSRI) may be associated with hypophagic phenotype in adulthood. We believe that this phenotype is due to increased activity of anorectic via in the Arc. Thus, this study aimed to evaluate the effects of NSSRI on body weight, feeding behavior and neuronal activity in the Arc in response to 5-HT2C and 5-HT1B receptors agonists in adult rats. Wistar rats were treated with saline (SAL, n=15) or fluoxetine (FLUO, n=15) from 1° to 21° postnatal day. At 170 days, stereotactic surgery was performed for implantation of a cannula in the right lateral ventricle through which were made the intracerebroventricular injections (ICV). At 180 days, weighing of animals was performed followed by ICV injections of ACSF, 5-HT2C or 5-HT1B receptors agonists, resulting in six experimental groups (n=5 animals/group): SAL+ACSF, SAL+AG.1B, SAL+AG.2C, FLUO+ACSF, FLUO+AG.1B and FLUO+AG.2C. From these groups, behavioral satiety sequence (SCS) and number of reactive cells against c-fos protein in the Arc were analyzed. Compared to the SAL group, FLUO group had lower body weight. NSSRI also promoted greater food intake after stimulation with 5-HT2C receptor agonist, lower feed rate after injection of ACSF and anticipation of the point of satiety after stimulation with 5-HT1B receptor agonist. Furthermore, FLUO group had a higher number of reactive cells against c-fos in the Arc after stimulation with 5-HT2C receptor agonist. Corroborating our hypothesis, NSSRI seems to promote increased anorexigenic pathway activity in the Arc in adulthood, despite not having been reduced food intake. Together, the findings suggest that lower body weight in adult rats submitted to NSSRI may be a consequence of increased anorexigenic pathway activity in the Arc, which can contribute to an increase in energy expenditure.

It has long been evident that genome size is not an accurate measure of organismal complexity. This paradox was “solved” with the discovery of nonfunctional and selfish DNA in the 1970s. However, emerging from this explanation was an enigma of complexity. Neither neutral nor adaptive models can account for all genome size variation across the tree of life. An organism with intraspecific variation is needed to investigate the functional role of genome size differences. Here I use different populations of the seed beetle, Callosobruchus maculatus, with a known intraspecific genome size variation of ~4%. It has previously been shown that a larger genome is associated with higher scores in fitness-related traits for this species. In this study, genome size is regressed with phenotypic plasticity along three different environmental gradients. Genome size did not correlate with plasticity in mass and development time along environmental gradients of temperature and host types. However, the results show that larger genomes are consistent with higher canalization of fitness under different food regimes. This further supports the idea that natural selection acts on genome size variation in this species.

Phenotypic plasticity allows some animals to change their behavioural, morphological, performance, and life history traits in response to changes in environmental conditions such as the presence of predators. These changes can enhance survival, but come at a cost. Some of these phenotypic changes are predator and diet specific. I examined the effects of predator diet on the performance, life-history, and morphology of developing Northern Leopard Frog (Lithobates pipiens) tadpoles. Tadpoles were either exposed to cues from fish free water, cues from Brown Bullhead (Ameiurus nebulosus) fed a diet of trout pellets, or cues from A. nebulosus fed a L. pipiens tadpoles diet. Tadpoles exposed to predatory fish cues had smaller bodies, deeper tail fins, slower growth and development rates, and better rotational performance than tadpoles that were not exposed to predatory fish cues. Moreover, tadpoles appeared to differentiate between predatory fish diet and produced diet-specific responses in tail morphology and activity, although the latter effect was only marginally significant. Hatching, metamorphosis rates, and linear performance were not affected by the treatments. These results suggest that A. nebulosus can induce phenotypic changes in L. pipiens tadpoles, with some of these changes being diet specific.

Having multiple plastic responses to a change in the environment, such as increased temperature, can be adaptive for two major reasons: synergy (the plastic responses perform better when expressed simultaneously) or complementarity (each plastic response provides a greater net benefit in a different environmental context). We investigated these hypotheses for two forms of temperature-induced plasticity of Battus philenor caterpillars in southern Arizona populations: color change (from black to red at high temperatures) and heat avoidance behavior (movement from host to elevated refuges at high host temperatures). Field assays using aluminum models showed that the cooling effect of the red color is greatly reduced in a refuge position relative to that on a host. Field assays with live caterpillars demonstrated that refuge seeking is much more important for survival under hot conditions than coloration; however, in those assays, red coloration reduced the need to seek refuges. Our results support the complementarity hypothesis: refuge seeking facilitates survival during daily temperature peaks, while color change reduces the need to leave the host over longer warm periods. We propose that combinations of rapid but costly short-term behavioral responses and slow but efficient long-term morphological responses may be common when coping with temperature change.

Human activity is causing climates to change more rapidly than at any time in the last 10,000 years. If populations of organisms are unable to effectively respond to changing environments, they will be at risk of extinction. In plants, two of the most important mechanisms of response to environmental change are phenotypic plasticity, where the same genotype expresses different phenotypes in different environments, and adaptation, which requires changes in allele frequency in populations as exposed individuals show variable survival and reproduction. Although most researchers accept the importance of both of these mechanisms, they are most commonly considered in isolation in models of response and persistence to climate change. Here, I use the model species Arabidopsis thaliana to investigate the interaction of plasticity and selection in fitness and phenology response to simulated climate warming, the effect of artificial selection on variation for plastic response and cross-generational effects of environmentally induced variation in flowering time. I also study the effects of varying rates of environmental fluctuation on evolvability on populations of self-replicating computer programs using the artificial life platform Avida. I find that a small increase in ambient temperature, in line with predictions for the next few decades, is able to elicit significant plastic responses and that these responses have the potential to alter population genetic structure and affect future evolution. I also find that selection on flowering time can reduce variation for plastic response and that non-genetic effects on flowering time can significantly alter germination in the next generation. Lastly, I find that rapidly changing environments in the long term can select for more evolvable populations and genotypes. These results highlight the importance of considering plasticity and evolution together if we are going to make accurate predictions of climate change response.