Computer assisted mosaic arthroplasty (CAMA) is a surgical technique that transplants cylindrical osteochondral grafts to repair damaged cartilage. An earlier in vivo study on sheep showed that short-term clinical outcomes are improved with the use of computer assistance, as compared to the conventional technique. This thesis reports on a study comparing three mosaic arthroplasty techniques -- one conventional and two computer assisted -- on human anatomy. This in vitro study used solid foam femur models modified to incorporate simulated cartilage defects. There were five participating surgeons ranging from a third year resident to a senior orthopedic surgeon. Each of the five participating surgeons performed a total of nine trials. There were three distinct sets of identical solid foam femur models with simulated cartilage defects. Three surgical techniques (conventional, opto-electronic, and patient-specific template) were performed on each. Several measures were made to compare surgical techniques: operative time; surface congruency; defect coverage; graft surface area either too high or too low; air volume below the grafts; and distance and angle of the grafts from the surgical plan. The patient-specific template and opto-electronic techniques resulted in improved surface congruency, defect surface coverage, graft surface within 0.50mm recessed and 0.25mm proud of the original surface, and below-graft air gap volume in comparison to the conventional technique. However, the conventional technique had a shorter operative time. The patient-specific template technique had less variance in surface congruency and shorter operative time than did the opto-electronic technique. / Thesis (Master, Computing) -- Queen's University, 2013-11-28 17:06:06.961

Groundwater is a major source of water in the western US. However, there are limited recharge estimates in this region due to the complexity of recharge processes and the challenge of direct observations. Land surface Models (LSMs) could be a valuable tool for estimating current recharge and projecting changes due to future climate change. In this study, simulations of three LSMs (Noah, Mosaic and VIC) obtained from the North American Land Data Assimilation System (NLDAS-2) are used to estimate potential recharge in the western US. Modeled recharge was compared with published recharge estimates for several aquifers in the region. Annual recharge to precipitation ratios across the study basins varied from 0.01% to 15% for Mosaic, 3.2% to 42% for Noah, and 6.7% to 31.8% for VIC simulations. Mosaic consistently underestimates recharge across all basins. Noah captures recharge reasonably well in wetter basins, but overestimates it in drier basins. VIC slightly overestimates recharge in drier basins and slightly underestimates it for wetter basins. While the average annual recharge values vary among the models, the models were consistent in identifying high and low recharge areas in the region. Models agree in seasonality of recharge occurring dominantly during the spring across the region. Overall, our results highlight that LSMs have the potential to capture the spatial and temporal patterns as well as seasonality of recharge at large scales. Therefore, LSMs (specifically VIC and Noah) can be used as a tool for estimating future recharge in data limited regions.

Les paysages français sont de plus en plus soumis aux pressions anthropiques. Ces pressions engendrent des modifications de la qualité et de la configuration des habitats, y compris forestiers, et ont des effets sur les plantes. Cette thèse aborde le rôle de la composition et de la configuration de la mosaïque paysagère extra- et intra-forestière sur la diversité de la flore en forêt. Il s'appuie sur 1932 relevés de l'Inventaire Forestier National répartis dans 19 départements de la moitié nord de la France. Les indices paysagers ont été calculés à partir de fonds cartographiques et d'images aériennes photo-interprétées. L'étude de terrain a été consacrée à la portée de l'effet d'un élément linéaire particulier du paysage : la route forestière. Le paysage a un effet important au-delà de la qualité de l'habitat local. La flore répond à trois principaux gradients paysagers : le premier est lié à la nature forestière ou non forestière du paysage, le second à la présence de coupes et peuplements jeunes, et le dernier reflète la composition en essences (feuillus ou résineux). L'effet des ouvertures paysagères (extra-forestière cultivée, coupe, peuplement jeune) sur la flore passe par la quantité, et surtout le nombre de taches et la longueur d'interfaces. Cet effet est positif sur la plupart des espèces, ce qui confirme le rôle des ouvertures dans le maintien de la diversité floristique. Les traits des espèces discriminés sont l'épi-zoochorie et l'anémochorie, la masse x longueur des semences et certaines stratégies de Grime. Ce travail montre qu'il est primordial d'appréhender le paysage sur un rayon de plus d'1 km. En comparaison, la route a un faible impact sur la flore, et plus souvent un effet positif que négatif. Les mécanismes sous-jacents à l'influence du paysage sont discutés à la lumière d'un effet temporel sur les plantes. Les implications pour la gestion des habitats en faveur de la biodiversité sont abordées. / French landscapes are submitted to increasing anthropogenic pressures. These pressures can affect plant species diversity by modifying the quality and spatial configuration of habitats, including forest habitats. This project addressed the role of the composition and configuration of the forest and non forest landscape mosaic on forest plant diversity. It was based on 1932 plots from the National Forest Inventory located in 19 "départements" of Northern France. Landscape indices such as fragmentation were measured or calculated from aerial photographs and existing national forest maps. A specific field study was devoted to analyse the depth of the effect of a particular linear landscape feature: the forest road. Landscape patterns had a significant impact in addition to local habitat quality. Plant composition was influenced by three main landscape gradients: (1) forest or non-forest habitat, (2) presence of fellings and young stands and (3) species composition (broadleaves or coniferous). The openings in the surrounding landscape (agricultural lands, forest felling and young stands) had an effect on plants through their quantity, number of patches and edge length. This effect was positive for most species, which confirms the role of the openings in maintaining plant diversity. The species traits discriminated were epi-zoochory and anemochory, mass x length of seeds and some Grime adaptive strategies. This work also underlined the importance to analyse landscape pattern effect within a buffer zone of more than 1 km around the plots. In contrast, we found that forest roads had a short depth of edge influence on plants (< 5 m) and road effect was more often positive than negative. The mechanisms underlying the influence of landscape mosaic are discussed in light of a temporal effect on plants. The implications for habitat and forest management to protect biodiversity are presented.

Master of Science / Department of Agronomy / Guorong Zhang / Guihua Bai / Wheat streak mosaic virus (WSMV) is a disease that causes significant yield losses in wheat (Triticum aestivum L.). Host resistance is the primary approach for control. KS06HW79 is a wheat line with WSMV resistance up to 21°C. To study the inheritance of resistance in KS06HW79, it was crossed with two WSMV-susceptible wheat genotypes, KS020638-M-5 and Brawl CL Plus. Parental lines, F₁, F₂, and check varieties were mechanically inoculated and evaluated for WSMV resistance at 21°C in growth chambers. The segregation pattern in two F₂ populations fit a one-recessive-gene model (1 resistant : 3 susceptible) and a dominant-suppression-epistasis model (3 resistant : 13 susceptible). To determine which model was a better fit, WSMV resistance was evaluated for F₂:₃ families generated from resistant F₂ plants in both crosses. Approximately two thirds of the F₂:₃ families in each cross showed segregation for WSMV resistance, suggesting that the dominant-suppression epistasis model better explained the WSMV resistance in KS06HW79. This model was also supported by two KS06HW79-derived doubled haploid populations, which had a segregation ratio of 1 resistant : 3 susceptible. Therefore, the WSMV resistance in KS06HW79 is likely controlled by two dominant genes, one of which is a suppressor.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science in the School of Molecular and Cell Biology. Johannesburg 2017 / With growing populations and climate change associated drought predicted for the future, cassava can provide one solution for food security and a source of starch for industrial use and biofuels in South Africa, and other countries in the SADC region. One of the severe constraints on cassava production is cassava mosaic disease (CMD) caused by cassava infecting begomoviruse species, including African cassava mosaic virus (ACMV), South African cassava mosaic virus (SACMV) and East African cassava mosaic virus (EACMV). Cassava begomoviruses (CBVs) are responsible for significant yield loss of the starchy tubers. Since no chemical control of virus diseases of plants is possible, one approach to develop virus resistance is via biotechnology, through genetic engineering (GE) of cassava with hairpin RNA (hpRNA) silencing constructs that express small interfering RNAs targeting CBVs and preventing severe disease development. The aim of this project was to subject previously transformed five CMM6 cassava lines (cv. 60444 transformed with a non-mismatched Africa cassava mosaic virus-[Nigeria:Ogorocco;1990] (ACMV-[NG:Ogo:90])-derived hpRNA construct, six AMM2 (cv. 60444 transformed with a mismatched ACMV-[NG:Ogo:90]-derived hpRNA construct), six CMM8 cassava lines (cv.60444 transformed with a non-mismatched SACMV BC1-derived hpRNA construct) and seven AMM4 cassava lines (cv.604444 transformed with a mismatched SACMV BC1-derived hpRNA construct) to reproducible trials, and evaluate for response to virus challenge. The ACMV-[NG:Ogo:90] hpRNAi constructs target 4 overlapping virus open reading frames (ORFs) (AC1 replication associated protein/AC4 and AC2 transcriptional/AC3 replication enhancer), while the SACMV hpRNAi constructs target the cell-to cell movement BC1 ORF. Non mismatched constructs consist of a transformation cassette that has an intron separating the sense and antisense arms of the viral transgene whilst mismatched constructs have the sense arm of the viral transgene treated with bisulfite to induce base mutation. This mutated sense arm is then separated from the non mutated antisense arm by a small spacer. Furthermore, a 229 bp inverted repeat hpRNA construct (DM-AES) was designed to target ACMV-[NG:Ogo:90] 117 nt putative promoter region (2714-49 nt), a 91 nt overlapping sequence (1530-1620 nt) between ACMV-[NG:Ogo:90] AC1 3’ end and AC2 5’ end (AC1 3’/AC2 5’-ter) as well as being efficient against SACMV and EACMV due to the inclusion of a 21 nt conserved sequence (1970-1990) of AC1/Rep shared between ACMV, EACMV and SACMV. Cassava landrace T200 friable embryogenic callus (FEC) were transformed with this construct. The selected transgenic lines were infected with either ACMV-[NG:Ogo:90] (CMM6 and AMM2 transgenic lines) or SACMV (CMM8 and AMM4 transgenic lines) by agro-inoculation and monitored at 14, 36 and 56, 180 and 365 days post infection (dpi) for symptom development, plant growth and viral load. From the ACMV trials 3 lines (CMM6-2, CMM6-6 and line AMM2-52) showed significantly lower symptom scores and lower viral load at 36, 56 and 365 dpi, compared with viral challenged untransgenic cv.60444. This phenotype is described as tolerance, not resistance, as despite ameleriorated symptoms virus replication persists at lower levels. From the SACMV infectivity trials even though all CMM8 and AMM4 transgenic lines had lower symptom severities and viral loads compared with infected untransformed cv.60444, the results were not highly significant (p˃ 0.05). From this study, tolerance or reduction of viral load and symptoms was attributed to the accumulation of transgene-derived siRNAs prior to infection. However there was no observable correlation between levels (semi-qauntitative northern blots) of siRNAs and tolerance or susceptible phenotypes. Tuber yield evaluation of the three tolerant lines (CMM6-2, CMM6-6 and line AMM2-52) showed that the tuber fresh and dry weight at 365 dpi was not affected by the viral presence. These are promising lines for larger greenhouse and field trials. A comparison between the two different constructs showed that the two tolerant CMM6 lines-2 and 6 appeared to perform better (viral load) compared with AMM2 tolerant line-52 with regards to levels of viral amplification. The mismatched construct in AMM4 lines and the nonmismatched construct in CMM8 lines induced the same viral and symptom severity score (sss) reduction. Transformation of T200 FECs with the DM-AES construct was unsuccessful due to the age (more than six months old) of the FECs. FECs are more likely to lose their regeneration and totipotent nature with age. We therefore propose the use of fresh T200 FECs in future transformation studies to test the DM-AES construct. / MT2017

Cassava (Manihot esculenta Crantz) is affected by two major viral diseases, namely Cassava brown streak disease (CBSD) and Cassava mosaic disease (CMD). Accurate and efficient detection and identification of plant viruses are fundamental aspects of virus diagnosis leading to sustainable disease management. In the present study I describe two techniques, the first based on a single tube duplex and multiplex polymerase chain reaction (m-PCR), developed for simultaneous detection of African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV) and East African cassava mosaic Malawi virus (EACMMV), and second, a technique based on Restriction Fragment Length Polymorphism (RFLP) analysis of Reverse Transcribed (RT) -PCR amplified Cassava brown streak viruses species, Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV). In this work, the single tube duplex and multiplex PCR for simultaneous detection of the four cassava mosaic begomoviruses (CMBs) was developed successfully. Four primer pairs were designed from published DNA-A component sequences targeting specific amplification of the four cassava mosaic begomoviruses (CMBs). Evaluation of the primers sensitivity in serially diluted virus samples revealed that the new primers amplified their target virus to a dilution of 10-4 and 10-3 for uniplex and multiplex PCR respectively. Developed multiplex assay enabled specific amplification of the viruses in producing 950, 503, 435 and 260 base pairs (bp) for ACMV, EACMMV, EACMCV and EACMZV respectively in single and mixed infections of CBSVs. Analysis of 172 field samples from Kenya, Malawi, Mozambique, Rwanda, Tanzania and Zambia detected both single and mixed infections, results which were proved by analysis of the sequenced amplicons. Second, a technique based on 2 Restriction Fragment Length Polymorphism (RFLP) analysis of RT-PCR amplified cassava brown streak viruses, Cassava brown streak virus (CBSV) and cassava brown streak Uganda virus (CBSUV), was performed. A degenerate primer amplifying 785 bp of the coat protein gene (CP) of CBSV and CBSUV was also designed. Two restriction endonucleases, HindIII and EcoR1 (identified by a software package, Vector NTI® Express v1.0 from Life Technologies/Invitrogen), which produce different fragments upon digestion of RT-PCR amplicons from CBSV and CBSUV, were used to distinguish the two viruses RFLP analysis using EcoRI has no site in CBSV producing one fragment (785 bp), two fragments (525 bp and 224 bp) for CBSUV and three fragments (785, 525 and 224 bp) for the mixed infections. On the other hand, HindIII has no site in CBSUV producing one fragment (785 bp), three fragments (437 bp, 267 bp and 81 bp) were produced for CBSV, and four fragments (785, 437, 267 and 81 bp) for CBSV and CBSUV mixed infections. In both multiplex and RFLP analyses, results from the sequenced PCR/RT-PCR amplicons agreed with sequence identities of the respective published virus species. Experience from using developed multiplex and RFLP techniques show that time was saved and amount of reagents used were reduced. RFLPs confirmed the presence of CBSV and CBSUV in RT-PCR amplicons without requirement for sequencing. Additionally, modified protocols from Dellaporta et al. (1983) and Chang et al. (1993), were used to extract DNA and RNA respectively from dry and fresh cassava leaves with comparable results. I also demonstrated a method of collecting and preserving cassava leaf samples to retain their integrity during storage for a period of over one month. The two diagnostic tools can be used routinely in germplasm indexing, disease surveillance, and disease monitoring programs 3 Problem Statement and Rationale In east and southern Africa, cassava (Manihot esculenta Crantz) is one of the leading crops in terms of production and has become an important source of income to households and small-scale farmers. However, the production across the region is greatly affected by Cassava mosaic disease (CMD) and Cassava brown streak disease (CBSD). Reports from different authors (Gibson. 1996; Ogbe et al., 1996; Legg et al., 1999; Fondong et al., 2000; Bisimwa et al., 2012) have reported the occurrence of CMD in different countries in the SSA. In Tanzania, CMD has been reported from many locations. Comprehensive characterization by Ndunguru et al. (2005) showed seven cassava mosaic geminiviruses species occur in Tanzania. Mbanzibwa et al. (2009a) reported prevalence of two potyvirus species causing CBSD in the Lake Victoria basin and along the coastal belt of Indian Ocean. A countrywide survey of all major cassava-growing areas in Kenya by Bull et al. (2006) reported presence of six CMG species with novel begomoviruses and a new recombinant strain of EACMV, demonstrating increasing diversity and geographical distribution of CMGs. Similarly, recent reemergence of CBSD has been reported in many districts in Uganda (Alicai et al., 2007) as well as from Malawi (Winter et al., 2010), Kenya (Mware et al., 2009) and Rwanda (Shirima et al., 2012). No reports of occurrence of CBSD have been reported from Zambia. With the current development of more robust diagnostic tools such as RT-PCR and real-time PCR, the diagnosis of CMD and CBSD has also improved in many cassava- producing countries. Similarly, the challenges to obtain more sensitive broad-spectrum cost-effective diagnostic tools also increase. This is evident following discovery of more 4 virus species causing CMD and CBSD (Mbanzibwa et al., 2009a and Winter et al., 2010) which can easily be overlooked. In the field the co-infections of many CMBs and CBSVs is common. Therefore, it will require several tools to detect the multiple infections using the diagnostic tools currently available. Thus, development of efficient and affordable diagnostic tools for simultaneous detection and identification of CMBs and CBSVs is vital and will have a significant impact on development and implementation of cassava virus disease management. Diagnostics will be used for disease monitoring in cassava multiplication plots production and distribution of disease- free cassava planting materials. Therefore, this research make use of the available sequence information in the database for both CMBs and CBSVs to develop sensitive tools for the simultaneous detection of four species of cassava begomoviruses namely: African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV) and East African cassava Mosaic Zanzibar Virus (EACMZV) using multiplex PCR. Also identification and differentiation of two species of Cassava brown streak viruses namely Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV) by RT-PCR/RFLP approach. This study generated knowledge and new tools that will enhance the diagnosis of both CMD and CBSD. The tools will facilitate deployment of virus-indexed cassava planting materials within the region.

Student Number : 9610284H - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Cassava Mosaic Disease is the most devastating disease affecting cassava (Manihot esculenta Crantz) crops worldwide. This disease is associated with eight species of geminiviruses, all belonging to the genus Begomovirus of the family Geminiviridae. In South Africa, in particular, CMD is caused by South African cassava mosaic virus (SACMV). Currently, there are no adequate methods for control of this disease as mechanisms within virus-host interactions are poorly understood. This brings about the need for development of virus-disease control strategies. This study was therefore conducted to identify the host’s response to an invading virus. The model plant, Arabidopsis was chosen as it is a well-characterized plant system, with expression databases readily available as its entire genome has been sequenced. This study was conducted, firstly, to phenotypically determine if Arabidopsis was resistant or susceptible to SACMV infection, and secondly, to identify the host’s response to pathogen infection on a molecular level through gene expression studies utilizing microarrays. Results from the symptomatology study revealed that Arabidopsis plants were fully symptomatic 28 days post-inoculation, displaying characteristic disease symptoms such as stunting, yellowing, and leaf deformation. This indicated that Arabidopsis was susceptible to SACMV infection. Microarray analyses revealed 86 differentially expressed genes, of which 48 showed up-regulation and 38 down-regulation. Relative quantification real-time PCR was performed on selected genes to confirm these results. Many up-regulated genes were shown to be primarily involved in a general stress response induced by the host, whereas those genes that were downregulated seemed to be involved in more specific responses to viral invasion, probably a consequence of suppression of host genes by SACMV to enhance its own replication. The majority of genes identified fell under the predominant functional categories involved in metabolism, transcription, and transport. To our knowledge, this is the first study in which a DNA geminivirus has been used in a host-pathogen interaction utilizing microarrays.

Digitized by Kansas Correctional Industries

Digitized by Kansas Correctional Industries

Photocopy of typescript. / Digitized by Kansas Correctional Industries