Kinematic analysis and synthesis of four-bar mechanisms for straight line coupler curves

Mechanisms are means of power transmission as well as motion transformers. A fourbar mechanism consists mainly of four planar links connected with four revolute joints. The input is usually given as rotary motion of a link and output can be obtained from the motion of another link or a coupler point. Straight line motion from a four bar linkages has been used in several ways as in a dwell mechanism and as a linkage to vehicle suspension. This paper studies the straight line motion obtained from planar four-bar mechanisms and optimizes the design to produce the maximized straight line portion of the coupler point curve. The equations of motion for four different four-bar mechanisms will be derived and dimensional requirements for these mechanisms will be obtained in order to produce the straight line motion. A numerical procedure will be studied and computer codes that generate the coupler curves will be presented. Following the numerical results study, a synthesis procedure will be given to help a designer in selecting the optimized straight line motion based on design criteria

Pulling the right viral levers: Engineering, screening and application of next-generation combinatorial AAV vectors

Adeno-associated viruses (AAVs) present powerful vectors for human gene therapy and biomedical research. They enable the delivery of transgenes to a broad range of target tissues. This allows persistent expression of reporter genes, therapeutic gene replacement and the delivery of control elements for manipulating endogenous gene expression. Transduction efficiency and specificity for on-target over off-target cells are critical factors driving vector safety and applicability. In this study, I engineered AAV capsids, promoters and knockdown tools with the goal of generating efficient and specific vector components for the next generation of cell-type-specific gene therapy vectors. In the first part of this doctoral work, I utilized Cas13d (CasRx) and short-hairpin (sh)RNA effectors to assess AAV-induced RNA degradation (knockdown). While CasRx showed promising knockdown of a Renilla luciferase reporter target, it failed to silence endogenous CD44, a potential driver of metabolic (non-alcoholic) steatohepatitis. shRNA effectors, however, allowed robust target knockdown of cellular RNAs (CD44 and ACE2) as well as SARS-CoV-2 viral RNA. Direct targeting of SARS-CoV-2 genomic RNA triggered the evolution of escape mutations within the viral target sites. This mutational escape was efficiently suppressed by multiplexing of three shRNAs in a single AAV vector, thereby allowing a sustained suppression of SARS-CoV-2 infection in Vero E6 cells. In the second part, I focused on improving screening conditions for promoters and AAV capsids. By assessing eYFP reporter expression in vivo for four promoter constructs individually, I could validate the findings of a previous promoter screen. This screen had utilized high-throughput barcode sequencing for parallel readout of a library of AAV-promoter constructs. I then applied this barcoding technique to dissect the activity of the GFAP promoter and truncated versions thereof. The GFAP promoter has previously mostly been used to induce astrocyte-specific transgene expression in the central nervous system. Strikingly, though, my results demonstrate a highly efficient GFAP promoter-driven transgene expression in human and murine hepatocytes. To optimize the directed evolution of AAV capsids, I modified conventional capsid library screening by altering selection parameters. This was achieved by (i) introducing a Cas9-based negative selection for the removal of unwanted variants from the capsid library, and (ii) by exploring and applying RNA-based functional selection. I could generate an RNA-based screening platform by driving the expression of cap from the ubiquitous CMV promoter instead of the endogenous p40. Both screening approaches proved applicable in cell culture settings. As RNA-driven selection offers a functional readout from both on- and off-target cells, I applied this approach for in vivo screening of an AAV6 peptide display library in mouse non-parenchymal liver cells. CMV promoter-driven cap expression enabled RNA-based readout of variant enrichment for on- and off-target cell-types. This demonstrated improved selectivity for RNA- over conventional DNA-based screening and facilitated the identification of functional, cell-type-specific capsid candidates. In conclusion, my results show the development of efficient combinatorial shRNA-based knockdown vectors for inhibiting CD44 expression or SARS-CoV-2 infection in vitro. Furthermore, I could implement improvements in capsid and promoter screening. This allowed the detection of highly functional variants with potential future applications in the development of novel gene therapy vectors

Genetic Dissection of Drought and Heat Tolerance in Chickpea through Genome-Wide and Candidate Gene-Based Association Mapping Approaches

To understand the genetic basis of tolerance to drought and heat stresses in chickpea, a comprehensive association mapping approach has been undertaken. Phenotypic data were generated on the reference set (300 accessions, including 211 mini-core collection accessions) for drought tolerance related root traits, heat tolerance, yield and yield component traits from 1–7 seasons and 1–3 locations in India (Patancheru, Kanpur, Bangalore) and three locations in Africa (Nairobi, Egerton in Kenya and Debre Zeit in Ethiopia). Diversity Array Technology (DArT) markers equally distributed across chickpea genome were used to determine population structure and three sub-populations were identified using admixture model in STRUCTURE. The pairwise linkage disequilibrium (LD) estimated using the squared-allele frequency correlations (r2; when r2<0.20) was found to decay rapidly with the genetic distance of 5 cM. For establishing marker-trait associations (MTAs), both genome-wide and candidate gene-sequencing based association mapping approaches were conducted using 1,872 markers (1,072 DArTs, 651 single nucleotide polymorphisms [SNPs], 113 gene-based SNPs and 36 simple sequence repeats [SSRs]) and phenotyping data mentioned above employing mixed linear model (MLM) analysis with optimum compression with P3D method and kinship matrix. As a result, 312 significant MTAs were identified and a maximum number of MTAs (70) was identified for 100-seed weight. A total of 18 SNPs from 5 genes (ERECTA, 11 SNPs; ASR, 4 SNPs; DREB, 1 SNP; CAP2 promoter, 1 SNP and AMDH, 1SNP) were significantly associated with different traits. This study provides significant MTAs for drought and heat tolerance in chickpea that can be used, after validation, in molecular breeding for developing superior varieties with enhanced drought and heat tolerance

Mouse Ifit1b is a cap1-RNA binding protein which inhibits mouse coronavirus translation and is regulated by complexing with Ifit1c.

Knock-out mouse models have been extensively used to study the antiviral activity of interferon-induced protein with tetratricopeptide repeats (IFIT). Human IFIT1 binds to cap0 (m7GpppN) RNA, which lacks methylation on the first and second cap-proximal nucleotides (cap1, m7GpppNm, and cap2, m7GpppNmNm, respectively). These modifications are signatures of 'self' in higher eukaryotes, while unmodified cap0-RNA is recognised as foreign and, therefore, potentially harmful to the host cell. IFIT1 inhibits translation at the initiation stage by competing with the cap-binding initiation factor complex, eIF4F, restricting infection by certain viruses that possess 'non-self' cap0-mRNAs. However, in mice and other rodents the IFIT1 orthologue has been lost and the closely-related Ifit1b has been duplicated twice, yielding three paralogues: Ifit1, Ifit1b and Ifit1c. While murine Ifit1 is similar to human IFIT1 in its cap0-RNA binding selectivity, the roles of Ifit1b and Ifit1c are unknown. Here, we found that Ifit1b preferentially binds to cap1-RNA, while binding is much weaker to cap0- and cap2-RNA. In murine cells, we show that Ifit1b can modulate host translation and restrict wildtype mouse coronavirus infection. We found that Ifit1c acts as a stimulatory cofactor for both Ifit1 and Ifit1b, promoting their translation inhibition. In this way, Ifit1c acts in an analogous fashion to human IFIT3, which is a cofactor to human IFIT1. This work clarifies similarities and differences between the human and murine IFIT families, to facilitate better design and interpretation of mouse models of human infection, and sheds light on the evolutionary plasticity of the IFIT family

Doctor of Philosophy

dissertationBecause plants and their insect enemies are strikingly species-rich groups, understanding their interactions has been a key issue in ecology and evolution. The arms race between plants and herbivores is considered the driver of diversification in both groups. However, we have a poor understanding of how these processes lead to divergence and speciation. This dissertation research tests key theories that relate plant- insect interactions with diversification and coevolution in both groups of organisms. In the first part, I assess the utility and contrast the predictions of two theories aimed to explain the patterns of defense investment across species: The Apparency Theory and the Resource Availability Hypothesis. My results provide strong support for the predictions of the Resource Availability Hypothesis. In particular, the evolution of defenses appears to be related to interspecific differences in inherent growth rate rather than to a species' predictability to herbivores. The theory appears robust across latitude and ontogeny suggesting that it has served as a valid framework for investigating the patterns of plant defenses and that its applicability is quite general. In the second part, I focus on how herbivores may drive the evolution of plant defenses, how plant defenses shape herbivore host choice and how plant-herbivore interactions might influence community composition and diversity focusing on the Neotropical genus of trees Inga (Fabaceae). I characterize the entire suite of anti-herbivore defenses and also quantify the diversity and abundance of leaf-feeders associated with Inga. With the use of phylogenies for both plants and herbivores, I discriminate among possible macroevolutionary hypothesis of host use and plant defense evolution. Contrary to much coevolutionary theory, my results show that closely related Inga species are more divergent in anti-herbivore defenses than in non-defense traits, and that the evolution of host use in herbivorous insects is more conserved with respect to host defenses rather than to host phylogeny. Together, these results suggest that defenses evolve rapidly and that traits related to host choice evolve more slowly. Specifically, although divergence in herbivores might not be driven by their interactions with plants,herbivores may be an important factor driving the divergence among plant species

Development of an all dielectric infrared beamsplitter operating in the 5 to 30 micron region

Dielectric infrared beam splitter for use in interferometer spectrometer for Nimbus B satellit

Evaluation program for secondary spacecraft cells. Acceptance test of Gulton Industries, Inc. 1.25 ampere-hour nickel cadmium cells with high overcharge capabilities

Performance of sealed nickel-cadmium spacecraft batteries with high overcharge capabilit