Graft versus Host Disease (GvHD) is one of the main complications patients face after receiving a bone marrow transplant. Between 40-60% of bone marrow transplant recipients develop GvHD, with consequent systemic inflammation/fibrosis, reduced quality of life, graft failure, and mortality. We have previously demonstrated that donor-derived C5 is involved in the initiation and propagation of GvHD. Current approaches to inhibition of C5 share a serious flaw of indiscriminately blocking production of a mediator that is crucial for host defense. Targeted therapies to block C5 in specific cells, or anatomical sites, are the only way in which to achieve therapeutic benefit without compromising host defenses. Three lentiCRISPR v2-dCas9 gene editing viral constructs were created to selectively cleave the complement C5 gene, at three different sites. Our objective was to knockout complement C5 function in infected donor BM cells in a GVHD mouse model. Each of the three lentiCRISPR plasmids was separately co-cultured with PMDG2 and PSPAX2, in human embryonic kidney (HEK) 293T cells. Resultant viral particles were able to transfer the Cas-9 endonuclease gene into donor BM cells in vitro with a transduction efficiency of 52%. Treated donor BM cells were then retro-orbital injected into irradiated recipient mice. Control mice were transplanted following the same protocol excluding the lentiCRISPR treatment of BM. The lentiCRISPR treatment group demonstrated significantly lower total airway resistance (p = 0.05) and higher lung compliance (p = 0.014) when compared to the control group. When compared to the saline treated group however the lentiCRISPR group showed significantly higher total airway resistance (p = 0.004) and significantly lower lung compliance (p = 0.014). These results taken together suggest a possible reduction in GvHD severity in mice that received the lentiCRISPR treatment. This study can serve as a starting point for the development of this novel treatment of GvHD. / Thesis / Master of Science (MSc)

Within the past decade there has become a dire need for equipment to mechanically harvest and handle apples. Apple growers have had trouble finding suitable labor to economically harvest their crop. Thus far, mechanical harvesting has not been very successful. One basic problem encountered by mechanical harvesting and handling equipment has been the damage incurred by the apple during the process. In an effort to discover what physical phenomena actually are responsible for this apple injury, several investigators have employed an engineering approach to analysing the physical properties of the apple. Several of the investigations have revealed that a large percentage of the apple's resistance to rupture lies in the skin alone. The objective of this project was to establish techniques for determining and evaluating the mechanical and rheological properties of the apple skin. Skin specimen from three apple varieties (Winesap, Red Delicious, and Golden Delicious) were tested. The Specimen were loaded at two constant displacement rates, yielding the skin's force deformation behavior. In addition, the skin was displaced to a constant deformation and held constant, the relaxation behavior being observed. The creep behavior was determined by subjecting the specimen to a constant force. By examining the results of these experiments it was observed that apple skin was a viscoelastic material; in certain instances, however, the akin also exhibited some apparent plastic behavior. From the experiments it was possible to determine many mechanical properties of the three varieties investigated. Some of the more important properties obtained were initial modulus of elasticity, secant modulus, tensile strength at break, and Poisson's ratio. The effect of displacement rate and specimen orientation on the strength was investigated. The apple skin was found to be anisotropic, with the maximum strength in a direction parallel to the core. It was observed that Poisson's ratio was not time dependent, and could be assumed a constant for each variety. Mechanical models were employed to simulate the behavior of the Winesap apple skin under several loading conditions. It was established that no one simple mechanical model could be used to predict the skin's behavior in all loading situations. TWo models, a three-element and a four-element, were formulated from the relaxation and creep results respectively. The behavior of the models was compared with the experimental data, and it was found that the formulated models could be used to predict the behavior of the actual skin. / Master of Science

Microchiropteran bats produce vocalisations for two purposes: echolocation and communication. Vocalisations used for communication are often referred to as social calls. In this thesis I examined the nature of Brown Long-eared bats Plecotus auritus social calls recorded at roost and foraging sites through a combination of recording and playback experiments. A total of 11,484 social calls were recorded at 20 maternity roosts sites and three types of vocalisations were dentified on the basis of shape, referred to as Type A, B, and C. Although Type A vocalisations shared the same basic pattern, it was a very large group within which there was a lot of variation in acoustic parameters. Principal component analysis and modelbased cluster analysis were used to look for patterns within this group, and this identified six clusters. Maternity colonies surveyed in this study varied in size from as few as nine up to 98 bats, and the number of social calls recorded at the roost sites was highly correlated with the numbers of bats present in the colony. The analysis of seasonal patterns of social call production revealed that the number of social calls recorded at maternity roost sites showed a linear increase from June to September, whereas, the number of bats emerging decreased sharply from August to September. Simulations of P. auritus social calls were used to investigate behavioural responses to calls away from roost sites using the Autobat. P. auritus were clearly much more responsive to simulations of their own species' social calls than to the other stimuli tested. This strongly suggests that the responses to the Autobat represent attempts to interact with the source of the stimulus. Recording with ultrasound and infra-red video was conducted to test the bats' responses to the different types of synthesised call and whether these responses varied seasonally. A female's approach response to the stimulus may represent an attempt to repel a perceived intruder from her foraging area. Alternatively, if calls were used to coordinate foraging by advertising the location of resources to other females that share the range, a response may represent an attempt to move towards such resources. Experiments showed that females were significantly more likely to respond to a stimulus produced within their core foraging area, than in the peripheral area, or outside their foraging area. On the other hand, while females regularly shared foraging ranges with other females, there was little evidence of co-ordination of movements between simultaneously radio-tracked dyads. It was concluded that responses to the stimuli probably represent attempts to repel perceived intruders from the foraging area. The thesis concludes with a discussion of some of the advantages and limitations of using play-back of synthesised social calls in the field to investigate vocal communication in bats. Ways in which studies of captive bats of known relatedness could be used to further elucidate the functions of social calls are discussed.

Negative regulatory factors inhibit the proliferation of haematopoietic stem cells thus protecting them from differentiation pressures. One of the negative regulators of stem cell proliferation is the tetrapeptide Acetyl-Seryl-Aspartyl-Lysyl-Proline (AcSDKP). This peptide is endogenously produced in vivo and long term bone marrow cultures and is degraded by angiotensin 1-converting enzyme (ACE) both in vivo and in vitro. The aim of these investigations was to study the role of ACE on haematopoietic stem and progenitor cell proliferation. Since the N-domain ACE active has been implicated in AcSDKP degradation, an analysis of two ACE inhibitors (captopril and lisinopril) shown to have differential effects on the N-domain ACE active site was conducted. Both captopril and lisinopril equally reduced ACE activity in plasma in vitro. However, captopril had a lesser effect on reducing serum ACE activity in vitro than lisinopril. Captopril and AcSDKP together reduced the proportion of GM-CFC in S-phase after 7 hours of in vitro incubation. In addition, ACE resistant AcSDKP analogue (AcSDPψKP) when incubated with bone marrow cells in the absence of captopril also reduced the proportion of GM-CFC in S-phase. This finding suggest that the effect of captopril and AcSDKP on GM-CFC proliferation was due to AcSDKP alone. Haematopoietic stem cells were induced into cell cycle by in vivo administration of either 2 Gy-γ-irradiation or the two cytotoxic drugs, cytosine arabinoside (Ara-C) (100 mg/kg i.p.) or 5 flourouracil (5 FU) (150 mg/kg i.v). Bone marrow cells were sampled and incubated in vitro for up to 24 hours. Captopril together with AcSDKP reduced the proportion of high proliferative colony forming cells-1 (HPP-CFC-1) in S-phase following 2 Gy-γ-irradiation. Lisinopril together with AcSDKP had no such effect. In addition, captopril alone in vitro reduced the proportion of HPP-CFC-1 in S-phase induced into cell cycle by cytotoxic drugs. Lisinopril had no such effect. Incubation alone reduced the proportion of HPP-CFC-1 in S-phase in cytotoxic drug treated bone marrow cells. When cultures, which were incubated with captopril, were assayed for AcSDKP levels, captopril induced an increase in AcSDKP levels in both control normal bone marrow cells and cells derived from Ara-C treated mice. However, it did not affect AcSDKP levels in cultures derived from 5 FU and 2 Gy treated mice, AcSDKP together with captopril were shown to inhibit S-phase cell entry of HPP-CFC-1 when they were incubated with bone marrow cells derived from mice treated with either 2 Gy-γ-irradiation or cytotoxic drug insults. Interestingly, captopril was unable to reduce the proportion of SA2 leukaemic cells in S-phase Captopril on its own at therapeutic doses reduced the proportion of HPP-CFC- 1 in S-phase in vivo regardless of the insult used to induce HPP-CFC-1 into cell cycle. Lisinopril slightly reduced the proportion of HPP-CFC-1 in S-phase following Ai-a-C treatment only. Captopril induced an in vivo increase in AcSDKP levels in all the models tested. Captopril also reduced the proportion of HPP-CFC-1 and GM-CFC in S-phase following fractionated doses of Ara-C. Captopril's inhibitory effect on GM- CFC proliferation following fractionated dose of Ara-C was diminished after 7 days while it was sustained with HPP-CFC-1. Long-term bone marrow cultures revealed that captopril and AcSDxj/KP had the same effect on cellularities of both layers and on the proliferation of HPP-CFC and GM-CFC in both layers. From the present investigations, it can be concluded that captopril is a potent inhibitor of HPP-CFC-1 proliferation. This effect may in part be mediated by AcSDKP mechanism.

O papel do complemento na modulação da resposta de células dendríticas não é bem entendido. Neste trabalho observamos que estas células produzirem proteínas do complemento e que o componente C3 regula positivamente a expressão de DC-SIGN, HLA-DR, CD1a, CD80 e CD86 e a produção de IL-6 e IL-12 por células dendríticas humanas. Também observamos, em modelo murino, menor expressão de MHC-II em células dendríticas C5aR-/-, a qual está associada com menor expressão do transativador de MHC-II; menor expressão de moléculas coestimuladoras nas células C3aR-/-, C5aR-/- e C5L2-/- e menor produção de IL-12p40, IL-12p70 e IL-6 em resposta a ligantes de TLR2 pelas células C3aR-/- e C5aR-/-. Conseqüentemente, a ausência de sinal pelos C3aR e C5aR, regula negativamente a habilidade destas células na indução da resposta de linfócitos T CD4+, modificando os níveis de proliferação e citocinas produzidas. De maneira conjunta, observamos participação do complemento na diferenciação e maturação de células dendríticas e no desenvolvimento da resposta imune adaptativa. / The role of complement in the modulation of dendritic cells functions remains elusive. Here we show that these cells are able to produce complement proteins and that complement C3 upregulates the expression of DC-SIGN, HLA-DR, CD1a, CD80 and CD86 and the production of IL-6 e IL-12 by human dendritic cells. We also observed, in a mouse model, lower expression of MHC-II in C5aR-/- dendritic cells, which is correlated to lower expression of MHC-II transactivator; lower expression of costimmulatory molecules in C3aR-/-, C5aR-/- and C5L2-/- cells and lower production of IL-12p40, IL-12p70 and IL-6 by C3aR-/- and C5aR-/- cells in response to TLR2 stimulation. In consequence to the absence of C3aR and/or C5aR signaling we observed that these cells have lower ability to induce CD4+ T cells proliferation and production of Th1 cytokines. Together our data show a participation of complement in dendritic cell differentiation and maturation and in the polarization of adaptative immune responses.

Embora camundongos sejam resistentes à infecção por Leptospira interrogans, eles têm sido pouco utilizados para se entender os mecanismos imunes efetores contra esta bactéria. Adler & Faine mostraram em 1976 que esta resistência é dependente da resposta imune, uma vez que camundongos imunossuprimidos tornavam-se suscetíveis à L. interrogans. Outros autores mostraram que camundongos portadores de imunodeficiência grave combinada também morriam após inoculação de L. interrogans. Sabendo da importância do Sistema Complemento em infecções bacterianas, investigamos se animais C5 deficientes (C5-) são mais suscetíveis à infecção por L. interrogans que animais C5 normais (C5+). Observamos que camundongos C5- possuem menores porcentagens de linfócitos T CD8+ na circulação periférica e maiores níveis de IL-12p40 no rim e de TNF e IL-6 no pulmão que os animais C5+. Animais C57Bl/6 (B6) C5+ possuem maior porcentagem de linfócitos T CD4+ que B6 C5-, além de lesões hepáticas mais intensas, mostrando um efeito dependente de C5 e do fundo genético dos camundongos. / Although mice are resistant to Leptospira interrogans infection, they are not usual models to study the imune response against this bacteria. Adler and Faine demonstrated in 1976 the importance of the imune response, since immunosuppressed mice were suceptible to L. interrogans. Other authors showed that mice that had severe combined immunodeficiency also died when inoculated with L. interrogans. Due to the activity of the Complement System in infections, we analyzed whether C5 deficient (C5-) mice are more susceptible than C5 sufficient (C5+) mice to L. interrogans infection. C5- mice have lower percentages of T CD8+ lymphocytes in peripheral circulation and upper levels of IL-12p40 in the kidney and TNF and IL-6 in the lungs than C5+ mice. C57Bl/6 (B6) C5+ mice has higher percentages of T CD4+ lymphocytes than B6 C5- mice, in addition to stricter liver injures, exhibiting an effect dependent of C5 and of the genetic background.

Embora camundongos sejam resistentes à infecção por Leptospira interrogans, eles têm sido pouco utilizados para se entender os mecanismos imunes efetores contra esta bactéria. Adler & Faine mostraram em 1976 que esta resistência é dependente da resposta imune, uma vez que camundongos imunossuprimidos tornavam-se suscetíveis à L. interrogans. Outros autores mostraram que camundongos portadores de imunodeficiência grave combinada também morriam após inoculação de L. interrogans. Sabendo da importância do Sistema Complemento em infecções bacterianas, investigamos se animais C5 deficientes (C5-) são mais suscetíveis à infecção por L. interrogans que animais C5 normais (C5+). Observamos que camundongos C5- possuem menores porcentagens de linfócitos T CD8+ na circulação periférica e maiores níveis de IL-12p40 no rim e de TNF e IL-6 no pulmão que os animais C5+. Animais C57Bl/6 (B6) C5+ possuem maior porcentagem de linfócitos T CD4+ que B6 C5-, além de lesões hepáticas mais intensas, mostrando um efeito dependente de C5 e do fundo genético dos camundongos. / Although mice are resistant to Leptospira interrogans infection, they are not usual models to study the imune response against this bacteria. Adler and Faine demonstrated in 1976 the importance of the imune response, since immunosuppressed mice were suceptible to L. interrogans. Other authors showed that mice that had severe combined immunodeficiency also died when inoculated with L. interrogans. Due to the activity of the Complement System in infections, we analyzed whether C5 deficient (C5-) mice are more susceptible than C5 sufficient (C5+) mice to L. interrogans infection. C5- mice have lower percentages of T CD8+ lymphocytes in peripheral circulation and upper levels of IL-12p40 in the kidney and TNF and IL-6 in the lungs than C5+ mice. C57Bl/6 (B6) C5+ mice has higher percentages of T CD4+ lymphocytes than B6 C5- mice, in addition to stricter liver injures, exhibiting an effect dependent of C5 and of the genetic background.

O papel do complemento na modulação da resposta de células dendríticas não é bem entendido. Neste trabalho observamos que estas células produzirem proteínas do complemento e que o componente C3 regula positivamente a expressão de DC-SIGN, HLA-DR, CD1a, CD80 e CD86 e a produção de IL-6 e IL-12 por células dendríticas humanas. Também observamos, em modelo murino, menor expressão de MHC-II em células dendríticas C5aR-/-, a qual está associada com menor expressão do transativador de MHC-II; menor expressão de moléculas coestimuladoras nas células C3aR-/-, C5aR-/- e C5L2-/- e menor produção de IL-12p40, IL-12p70 e IL-6 em resposta a ligantes de TLR2 pelas células C3aR-/- e C5aR-/-. Conseqüentemente, a ausência de sinal pelos C3aR e C5aR, regula negativamente a habilidade destas células na indução da resposta de linfócitos T CD4+, modificando os níveis de proliferação e citocinas produzidas. De maneira conjunta, observamos participação do complemento na diferenciação e maturação de células dendríticas e no desenvolvimento da resposta imune adaptativa. / The role of complement in the modulation of dendritic cells functions remains elusive. Here we show that these cells are able to produce complement proteins and that complement C3 upregulates the expression of DC-SIGN, HLA-DR, CD1a, CD80 and CD86 and the production of IL-6 e IL-12 by human dendritic cells. We also observed, in a mouse model, lower expression of MHC-II in C5aR-/- dendritic cells, which is correlated to lower expression of MHC-II transactivator; lower expression of costimmulatory molecules in C3aR-/-, C5aR-/- and C5L2-/- cells and lower production of IL-12p40, IL-12p70 and IL-6 by C3aR-/- and C5aR-/- cells in response to TLR2 stimulation. In consequence to the absence of C3aR and/or C5aR signaling we observed that these cells have lower ability to induce CD4+ T cells proliferation and production of Th1 cytokines. Together our data show a participation of complement in dendritic cell differentiation and maturation and in the polarization of adaptative immune responses.

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This thesis seeks to examine the politics of building resilience to climate change by analysing the manner in which policy contexts and initiatives to build climate change resilience interact. For analysis, the ‘policy context' is broken into its three constituent parts- actors, policy spaces and discourses. This permits the addition of new knowledge on how discourses attached to resilience are dissonant with those prevailing in ossified policy environments in developing countries; the influence of actor networks, epistemic communities, knowledge intermediaries and policy entrepreneurs in helping climate change resilience gain traction in policy environments; and the dynamic interaction of interest, agendas and power within decision-making spaces attached to resilience-building processes. This analysis takes place by employing a case-study of a major, international climate change resilience initiative unfolding in two Indian cities. Using data gathered through a variety of rigorous qualitative research methods employed over 14 months of empirical inquiry the thesis highlights issues of politics and power to argue that they are significant determinants of processes to deal with climate impacts. More specifically, it expands current understandings of engaging with climate impacts by exposing gaps in resilience thinking and argues against a technocratic approach to designing and executing resilience policies. In doing so it also demonstrates that resilience, with its emphasis on systems thinking, dealing with uncertainty and community engagement brings new challenges for policy makers. As the study is located in the urban context, it highlights the manner in which fragmented urban policy environments, dense patterns of settlement in cities, urban livelihood patterns and prevailing epistemic cultures can pose obstacles for a policy initiative aimed at building resilience to climate change. Finally, the research underlines the importance of coupling resilience with local narratives of dealing with shocks and stresses, argues for genuine iteration and shared learning during decision-making and highlights the need to celebrate multiple visions of resilience. Findings from this research can help inform a growing number of policy initiatives aimed at deploying resilience to help those battling the exigencies of a changing climate in some of the world's most vulnerable areas.