Genetic manipulation of Anopheles stephensi immunity to increase plasmodium falciparum salivary gland sporozoite infection levels

Human malaria is responsible for over 700,000 deaths a year. To stay abreast of the threat posed by the parasite, a constant stream of new drugs and vector control methods are required. This study focuses on a vaccine that has the potential to protect against parasite infection, but has been hindered by developmental challenges. In malaria prevention, live, attenuated, aseptic, Plasmodium falciparum sporozoites (PfSPZ) can be administered as a highly protective vaccine. PfSPZ are produced using adult female Anopheles stephensi mosquitoes as bioreactors. Production volume and cost of a PfSPZ vaccine for malaria are expected to be directly correlated with Plasmodium falciparum infection intensity in the salivary glands. The sporogonic development of Plasmodium falciparum in A. stephensi to fully infected salivary gland stage sporozoites is dictated by the activities of several known components of the mosquito’s innate immune system. Here I report on the use of genetic technologies that have been rarely, if ever, used in Anopheles stephensi Sda500 to increase the yield of sporozoites per mosquito and enhance vaccine production. By combining the Gal4/UAS bipartite system with in vivo expression of shRNA gene silencing, activity of the IMD signaling pathway downstream effector LRIM1, an antagonist to Plasmodium development, was reduced in the midgut, fat body, and salivary glands of A. stephensi. In infection studies using P. berghei and P. falciparum these transgenic mosquitoes consistently produced significantly more salivary gland stage sporozoites than wildtype controls, with increases in P. falciparum ranging from 2.5 to 10 fold. Using Plasmodium infection assays and qRT-PCR, two novel findings were identified. First, it was shown that 14 days post Plasmodium infection, transcript abundance of the IMD immune effector genes LRIM1, TEP1 and APL1c are elevated, in the salivary glands of A. stephensi, suggesting the salivary glands may play a role in post midgut defense against the parasite. Second, a non-pathogenic IMD signaling pathway response was observed which could suggest an alternative pathway for IMD activation. The information gained from these studies has significantly increased our knowledge of Plasmodium defense in A. stephensi and moreover could significantly improve vaccine production.

Integrating Architecture for a Live-Work Lifestyle

Urban centers all around the world are striving to re-orient themselves to promoting ideals of human engagement, flexibility, openness and synergy, that thoughtful architecture can provide. From a time when solitude in one’s own backyard was desirable, today’s outlook seeks more, to cater to the needs of diverse individuals and that of collaborators. This thesis is an investigation of the role of architecture in realizing how these ideals might be achieved, using Mixed Use Developments as the platform of space to test these designs ideas on. The author also investigates, identifies, and re-imagines how the idea of live-work excites and attracts users and occupants towards investing themselves in Mixed Used Developments (MUD’s), in urban cities. On the premise that MUDs historically began with an intention of urban revitalization, lying in the core of this spatial model, is the opportunity to investigate what makes mixing of uses an asset, especially in the eyes to today’s generation. Within the framework of reference to the current generation, i.e. the millennial population and alike, who have a lifestyle core that is urban-centric, the excitement for this topic is in the vision of MUD’s that will spatially cater to a variety in lifestyles, demographics, and functions, enabling its users to experience a vibrant 24/7 destination. Where cities are always in flux, the thesis will look to investigate the idea of opportunistic space, in a new MUD, that can also be perceived as an adaptive reuse of itself. The sustainability factor lies in the foresight of the transformative and responsive character of the different uses in the MUD at large, which provides the possibility to cater to a changing demand of building use over time. Delving into the architectural response, the thesis in the process explores, conflicts, tensions, and excitements, and the nature of relationships between different spatial layers of permanence vs. transformative, public vs. private, commercial vs. residential, in such an MUD. At a larger scale, investigations elude into the formal meaning and implications of the proposed type of MUD’s and the larger landscapes in which they are situated, with attempts to blur the fine line between architecture and urbanism. A unique character of MUD’s is the power it has to draw in people at the ground level and lead them into exciting spatial experiences. While the thesis stemmed from a purely objective and theoretical standpoint, the author believes that it is only when context is played into the design thinking process, that true architecture may start to flourish. The unique The significance of this thesis lies on the premise that the author believes that this re-imagined MUD has immense opportunity to amplify human engagement with designed space, and in the belief that it will better enable fostering sustainable communities and in the process, enhance people’s lives.