Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr.: f. 86-90. Publié aussi en version électronique.

Thesis (Ph. D.)--University of Chicago, Division of the Biological Sciences and the Pritzker School of Medicine, June 1999. / Includes bibliographical references. Also available on the Internet.

I studied translocation of Texas horned lizards on Tinker Air Force Base, Midwest City, Oklahoma, using correlative and mechanistic habitat suitability models. My goals were broadly classified into two categories: first, to determine if the addition of mechanistic data layers (i.e., habitat-niche models) in a correlative model improved the overall accuracy of model predictions, and second, to apply the best model produced from my dataset to a planned translocation event on Tinker Air Force Base. Correlative data layers (i.e., habitat models) included typically applied datasets such as vegetative components, Euclidean distance statistics, neighborhood analyses, and topographically-derived information. Mechanistic data layers were estimates of thermal suitability derived from field-collected datasets and biophysical calculations, and estimates of prey availability taken from interpolated datasets. I estimated habitat suitability using the partitioned Mahalanobis distance statistic, which is a suitable model technique for presence-only data. Translocated and resident lizards were monitored via radiotelemetry and using fluorescent powder trails. Telemetry locations and powder trails were overlaid onto habitat suitability models to provide the datasets used to quantify interaction between site occupancy and habitat model predictions. Lizard paths were tested against random walk models to determine efficiency of travel, and site occupancy metrics (powder track and telemetry Mahalanobis distance values) were tested using parametric (repeated-measures ANOVA) and nonparametric (Wilcoxon rank-sum and signed-rank tests) tests. Mechanistic data layers did not substantially improve model accuracy over correlative-only layers, and data layers taken from mixed bare soil-vegetation, shrub, and grassland habitat types dominated important eigenvector weights. Analyses of fluorescent powder track data suggested that lizards did not move through habitat differently from a random walk model, potentially due to neighborhood factor loadings strongly influencing the area in which entire trails traveled. Wilcoxon tests and repeated-measures ANOVA results suggested that although lizards experienced different median Mahalanobis distance values by group (translocated, resident), there appeared to be an overall decrease in distance scores for translocated individuals over time. In this context, translocated individuals seemed to acclimate their behavior to areas that were predicted to be more suitable by Mahalanobis classifiers. Although survival results were not encouraging and habitat models did not suggest that my translocation site was ideal, my data supports the idea that translocations may be aided in the future by modeling efforts. My models suggest that mechanistic data layers may not improve classification accuracy over correlative processes, but this may be due to inaccurate representation of specific mechanisms over spatial and temporal scales. Future work should focus on including more explicit measures of mechanisms, as well as broadening biotic influences on species distributions (i.e., predator distribution, intra- and interspecific competition).

Thesis (MTech (Agriculture))--Cape Peninsula University of Technology, 2014. / Plants require nitrogen (N) to support desired production levels. Nitrogen fertilization strategy is a major consideration in field management with regard to achieving both economic and environmental objectives. For instance, in potato, insufficient N supply reduces tuber size and overall yield while excessive N supply can reduce tuber quality and increase environmental risk through nitrate (NO3-) leaching and nitrous oxide emission. Selection of an adequate N fertilizer application rate for crops is difficult, due to marked variations in soil N supply and crop N demand in both the field and over time. This research was conducted to characterise the ammonium transporter gene (AMT1) of Bambara groundnut and potato using molecular biology and bioinformatics methods. Nucleotide database sequences were used to design AMT1-specific primers which were used to amplify and sequence the core-region of the gene from Bambara groundnut and potato. Bioinformatics techniques were used to predict the structure and infer properties of the proteins. Nucleotide sequence alignment and phylogenetic analysis indicate that BgAMT1 and PoAMT1 are indeed from the AMT1 family, due to the clade and high similarity they respectively shared with other plant AMT1 genes. Amino acid sequence alignment showed that BgAMT1 is 92%, 89% and 87% similar to PvAMT1.1, GlycineAMT1 and LjAMT1.1 respectively, while PoAMT1 is 92%, 83% and 76% similar to LeAMT1.1, LjAMT1.1 and LeAMT1.2 respectively. BgAMT1 and PoAMT1 fragments were shown to correspond to the 5th - 10th transmembrane spanning-domains. Mutation of Bg W1A-L and S28A (for BgAMT1) and Po S70A (for PoAMT1) is predicted to enhance ammonium (NH4+) transport activity. Residues Bg D23 (for BgAMT1) and Po D16 (for PoAMT1) must be preserved otherwise NH4+ transport activity is inhibited. In all, BgAMT1 and PoAMT1 play a role in N uptake from the root while BgAMT1 may contribute more in different steps of rhizobia interaction. In an investigation of the correlation between AMT1 gene expression levels and leaf chlorophyll content index (CCI) with plant N status, potato plants were grown in a hydroponic greenhouse with 0.75 or 7.5 mM NO3- and 0.75 or 7.5 mM NH4+ as forms of N supply in a completely randomized design. Leaf CCI as measured by chlorophyll content meter, showed that an increase in N supply results in increased leaf CCI in response to both forms of N. Total RNA was isolated from leaf sampled at 28 days after treatment and expression level of the AMT1 gene was determined by reverse transcription-qPCR using a second set of primers designed for qPCR. The results showed that expression levels of AMT1 increased from 8.731 ± 2.606 when NO3- supply was high to 24.655 ± 2.93 when NO3- supply was low. However, there was no significant response in AMT1 expression levels to changes in NH4+. This result suggested that AMT1 transports NO3- less efficiently than NH4+, and thus more transport channels are required in the cell membrane when NO3- levels are low. Such variation in AMT1 expression levels are not necessary for NH4+ transport since the transport mechanism for NH4+ is efficient even at low NH4+ levels.

abstract: Genomic structural variation (SV) is defined as gross alterations in the genome broadly classified as insertions/duplications, deletions inversions and translocations. DNA sequencing ushered structural variant discovery beyond laboratory detection techniques to high resolution informatics approaches. Bioinformatics tools for computational discovery of SVs however are still missing variants in the complex cancer genome. This study aimed to define genomic context leading to tool failure and design novel algorithm addressing this context. Methods: The study tested the widely held but unproven hypothesis that tools fail to detect variants which lie in repeat regions. Publicly available 1000-Genomes dataset with experimentally validated variants was tested with SVDetect-tool for presence of true positives (TP) SVs versus false negative (FN) SVs, expecting that FNs would be overrepresented in repeat regions. Further, the novel algorithm designed to informatically capture the biological etiology of translocations (non-allelic homologous recombination and 3&ndashD; placement of chromosomes in cells –context) was tested using simulated dataset. Translocations were created in known translocation hotspots and the novel&ndashalgorithm; tool compared with SVDetect and BreakDancer. Results: 53% of false negative (FN) deletions were within repeat structure compared to 81% true positive (TP) deletions. Similarly, 33% FN insertions versus 42% TP, 26% FN duplication versus 57% TP and 54% FN novel sequences versus 62% TP were within repeats. Repeat structure was not driving the tool's inability to detect variants and could not be used as context. The novel algorithm with a redefined context, when tested against SVDetect and BreakDancer was able to detect 10/10 simulated translocations with 30X coverage dataset and 100% allele frequency, while SVDetect captured 4/10 and BreakDancer detected 6/10. For 15X coverage dataset with 100% allele frequency, novel algorithm was able to detect all ten translocations albeit with fewer reads supporting the same. BreakDancer detected 4/10 and SVDetect detected 2/10 Conclusion: This study showed that presence of repetitive elements in general within a structural variant did not influence the tool's ability to capture it. This context-based algorithm proved better than current tools even with half the genome coverage than accepted protocol and provides an important first step for novel translocation discovery in cancer genome. / Dissertation/Thesis / Ph.D. Biomedical Informatics 2014

The present thesis focuses on the elucidation of human mitochondrial inner membrane presequence-translocation machinery with implications on cancer cell proliferation. Mitochondria are the endosymbiotic organelles in an eukaryotic cell performing a vast repertoire of functions and require approximately 1500 proteins. However, the mitochondria genome contains only 13 protein-coding genes primarily transcribing the complexes of the electron transport chain. Therefore, it is evident that most of the mitochondrial proteome is encoded by the nucleus and synthesized on cytosolic ribosomes. Chapter 1: Mechanism of mitochondrial inner membrane protein translocation and its oncogenic connection. Mitochondria consist of different routes of directing proteins to their intramitochondrial destinations. The presequence pathway, mediated by the inner membrane TIM23 complex, is responsible for the import of matrix and a number of single transmembrane helixes containing inner membrane proteins. This pathway accounts for approximately 60% of the total proteome imported into the organelle and hence, is the major focus of discussion in the present study. The components of the TIM23 complex can be subdivided into two groups, the protein conducting channel and the import motor. The initial translocation across the TIM23 channel utilizes the electrochemical membrane potential that exists across the inner membrane whereas the final step of the translocation process is driven by energy from ATP hydrolysis. MtHsp70 forms the central component of the import motor, and its function is regulated by the J-proteins. Pam18 stimulates the ATPase activity of mtHsp70. Pam16, on the other hand, forms a subcomplex with Pam18 and exerts an inhibitory effect its ATPase stimulatory activity, in turn regulating the activity of the import motor. The stoichiometric coupling with the substrate binding-release cycle of mtHsp70 drives the import process. Although the organization of presequence translocation machinery and its functional annotations have been described in detail in yeast system, little information is available on its organization in human. It is difficult to contemplate the existence of similar machinery in human mitochondria with complex and diversified functions. Human mitochondria apart from regulating the metabolic pathways are involved in progression of cancer, neurodegenerative disorders, responses to xenobiotic stress and induction of apoptosis. Numerous reports have shown that mutations and overexpression of human orthologs of translocase components are associated with various cancer subtypes. Such disease condition also involves targeting of specific cell signaling molecules that reprogram organellar functions and alter the cellular phenotype. Based on this evidence we defined our study into four broad objectives – 1) identify the components of human presequence translocase as Chapter two and three, 2) characterize the subunit organization of human presequence translocation machinery in Chapter four, 3) determine the functional connection between the translocase components and the cancer phenotype in Chapter four and five and 4) understand how the functions of J-proteins have evolved across the species as Chapter six. Chapter 2: Unraveling the role of Magmas in human mitochondrial protein transport. Pam16 plays a critical role in regulation of import process by governing the activity of the import motor. Proteins orthologous to Pam16 had been reported earlier to be overexpressed in various metabolically active tissues and cancer subtypes. We found that in humans a protein named as Mitochondria Associated Granulocyte Macrophage colony Stimulating factor signaling molecule (Magmas) showed significant sequence similarity with yeast Pam16 at its C-terminal region. Magmas was initially discovered as a protein that was overexpressed in neoplastic prostrate and when the cells were exposed to GM-CSF. Our experiments suggested that Magmas localized in human and yeast mitochondria and it was associated with the inner mitochondrial membrane. Magmas could complement the growth of yeast cells that were deleted for the essential gene PAM16 and could import precursor proteins into the mitochondria. Like Pam16, Magmas was able to form a stable heterodimeric subcomplex with yeast Pam18 and human Pam18 ortholog DnaJC19 (JC19). We found that J-domain forms the minimal region required for heterodimer formation between Magmas and Pam18/JC19. Mutations in Magmas J-like domain resulted in temperature sensitive growth phenotypes in yeast cells and associated import defect in translocating precursor proteins into the organelle due to inability to form a stable subcomplex with Pam18 and JC19, resulting in loss of import function. Loss of subcomplex formation leads to dissociation of Pam18 from the translocation machinery highlighting the importance of Magmas in tethering Pam18/JC19 to the presequence translocase. Magmas, showing characteristic of a J-like protein, was unable to stimulate the ATPase activity of mtHsp70. However, it exerted an inhibitory effect on the ATP stimulatory effect of the J-protein Pam18/JC19, indicating that Magmas has a regulatory effect on the overall activity of import motor. In contrast Magmas mutants those are incapable of forming a stable heterodimer with Pam18 were unable to regulate the activity of Pam18 resulting in import defects. In summary, our results highlight that Magmas is an ortholog of yeast Pam16 performing similar functions at the import channel. Chapter 3: Existence of two J-protein subcomplexes at the translocation channel with distinct physiological functions. JC19 has been regarded as the human ortholog of Pam18 whose loss of function was associated with dilated cardiomyopathy and ataxia syndrome. However, immunoprecipitation analysis using anti-Magmas antibody revealed the presence of a second J-protein identified as DnaJC15 (JC15) that shared a highly similar J-domain with JC19. JC15 was initially identified as a protein whose loss in expression resulted in development of a chemoresistant phenotype in ovarian carcinoma cells exposed to chemotherapeutic treatment. We found that JC15 localizes in mitochondria where it was associated with the inner membrane. Similar to Pam18 and JC19, JC15 heterodimerized with Magmas/Pam16 through its J-domain and associated with the presequence translocase of the inner membrane. A loss of function mutation at the J-domain of JC15 destabilizes its interaction with Magmas resulting in protein translocation defects and temperature-sensitive growth phenotype in yeast cells. The JC15 mutant showed inability to get associated with the translocation channel and had dysregulated stimulation of mtHsp70 activity leading to decreased mitochondria biogenesis and loss of mitochondrial membrane potential. In summary, our results showed that JC15 is the second human ortholog of Pam18 with similar functions. In contrast to yeast, in human mitochondria JC15 and JC19 were found to form two separate and distinct J-protein subcomplexes with Magmas at the mitochondrial import motor. The essentiality of the J-proteins for normal human mitochondria function was addressed through siRNA mediated downregulation of Magmas, JC19 and JC15. We found that Magmas and JC19 are essential for normal mitochondrial function and cell viability whereas JC15 is dispensable and might have a supportive role. Interestingly, both JC19 and JC15 interacted with Magmas with equal affinity and stimulated mtHsp70’s ATPase activity by equivalent levels. This shows that both JC19 and JC15 share similar properties in terms of their functions at the import channel, and the differences might be in a much broader perspective in terms of their association with the translocation channel. Chapter 4: Architecture of human mitochondrial inner membrane presequence -translocation machinery. In yeast, there exists a single J-protein subcomplex formed by Pam16 and Pam18, which is recruited to the sole translocase. However, humans present a completely different scenario where there exists a two distinct subcomplexes formed by Magmas with either of the J-proteins. So the question arises how the individual subcomplexes is recruited to the translocation machinery; whether they are associated to one or differentially recruited to two different translocases. We identified the existence of three distinct translocases in the human system constituted by the two J-proteins along with the Tim17 paralogs. JC15 along with Tim17a forms the translocase A of size similar to that of the yeast system, and it forms the ancestral translocase in the humans. Tim17b isoforms, on the other hand, associates with JC19 to form mammalian specific translocases B1 and B2. The association of the J-proteins at the translocation channel was found to be mediated by Magmas as a subcomplex. Downregulation of Magmas resulted in dissociation of both the J-proteins, and its overexpression resulted in redistribution of J-proteins at the translocases. We found that translocase B imported precursor proteins at a comparatively higher rate as compared to translocase A. Disruption of translocase B had deleterious effects on cell viability, respiratory chain complex's activities, Fe-S cluster biogenesis, mitochondria morphology, regulation of free radical levels and maintenance of mitochondrial genome. In contrast, depletion of translocase A did not significantly alter the survivability of cells, mitochondrial activity and maintenance of organellar morphology. This shows that translocase B is essential and performs the constitutive import function in the mammalian system whereas translocase A is dispensable and might have a supportive role in maintenance of mitochondrial function. However, translocase A play a specific role in human mitochondria in context to cancer cells. We observed that the elevated level of Tim17a found in cancer cells is responsible for maintenance of higher mitochondrial DNA copy number and higher proliferative potential of cancer cells. Additionally, translocase A also plays a specific role in translocation of cell signaling proteins that lack a mitochondrial targeting sequence into the mitochondria, highlighting the possible role of this translocase in neoplastic transformation. Chapter 5: Mechanistic insights into the role of JC15 as a part of translocase A in chemoresistant phenotype. JC15 had been initially identified to be associated with development of chemoresistance in cancer cells. However, the molecular mechanism followed by the protein has not been elucidated yet. Our studies have shown that overexpression of JC15 leads to increased sensitivity of cells to chemotherapeutic drug cisplatin and are coupled with complete loss of membrane potential, mitochondrial swelling and cytochrome c release. However, this chemosensitive phenotype was partially ameliorated upon preexposing the cell to cyclosporine A which is an inhibitor of cyclophilin D, a critical component of mitochondrial membrane transition pore (MPTP) complex. A similar reversal of phenotype was observed upon depleting cyclophilin D even under JC15 overexpressing background. This highlighted a possible functional connection between these two proteins. In order to check this hypothesis other way around, we overexpressed cyclophilin D in the cells which resulted in constitutive opening of the MPTP complex, enhanced mitochondrial swelling and reduced cell viability. In contrast, the gain of function anomalies of cyclophilin D overexpression was significantly reversed upon JC15 depletion. We observed through co-immunoprecipitation analysis that JC15 activates cyclophilin D by releasing it from the inhibitory effects of TRAP1 and couples it to the MPTP complex. Additionally, we have also shown that the J-domain of JC15 is critical for its interaction with cyclophilin D and loss of function mutation at the J-domain of JC15 disrupts its interaction with cyclophilin D. As a result the JC15 mutant is not able to mount a chemosensitive response to cisplatin drug. Chapter 6: Identification of regions determining the divergence of J-proteins functions at the mitochondrial import motor. The above studies show ample evidence to suggest that the two human J-proteins have undergone significant divergence in their function in human mitochondria in spite of having a highly similar J-domain. Therefore, we asked the question that how the human J-proteins have evolved and diversified from the primitive yeast protein Pam18 and what are the regional determinants in the protein sequence that dictate the function of the J-domain. We utilized a purely genetic approach to address the problem. We observed that JC19 was unable to rescue the growth of yeast cells deleted for the essential gene Pam18 and JC15 expression resulted in cold sensitive phenotype. We used JC15 as the model protein for our assays and applied three methodologies. First, generation and isolation of a series of mutations in JC15 that could rescue the cold sensitive phenotype, and the growth of the cells were similar to the wild type. Second, to identify the regulatory residues by isolation of second site suppressors that could be the suppressor the mutant phenotypes isolated earlier. Third, we utilized a purely evolutionary approach by swapping the individual domains between the three J-proteins- Pam18, JC19 and JC15. Our genetic data support the idea that the partial loss of function of human J-protein in the yeast system is due to altered subcomplex dynamics with Pam16. The altered dynamics of the subcomplex is mainly regulated by the residues in the arm, linker and helical regions of the J-domain, especially the helix II regions. Our analysis has also uncovered a critical role of the targeting (T) region of J-proteins which along with inter-membrane space (IMS) domain share significant sequence diversity among J-proteins in yeast and humans. The T-region in conjunction with the IMS domain plays a crucial role in regulating the J-domain’s function across the kingdoms and within the species. Although, our genetic data needs to be supplemented with biochemical evidence, this study provides significant insights into the diversity of J-protein function across the species and mode of their regulation through regions flanking the J-domain.

DNA sequencing via nanopore translocation was a pipedream two decades ago. Today, biotech companies are releasing commercial devices. Yet many challenges still hover around the simple concept of threading a long DNA molecule through a small nanoscopic pore with the aim of extracting the DNA’s sequence along the process. In this thesis I use computer simulations to create what are in essence virtual pro- totypes for testing design ideas for the improvement of nanopore translocation devices. These ideas are based on the general concept of modifying the average shape of the initial DNA conformations. This is done, for example, by introducing new geometrical features to the nanopore’s surrounding or by the means of some external force. The goal of these simulations is not just to test design improvements, but also to systematically deconstruct the physical mechanisms involved in the translocation process. The roles of pore friction, initial polymer conformations, monomer crowding on the trans- side of the membrane, Brownian fluctuations, and polymer rigidity can, with careful consideration, be essentially muted at will. Computer simulations in this sense play the role of a sandbox in which the physics can be tinkered with, in order to assess and evaluate the magnitude of certain approximations found in theoretical modelling of translocation. This enables me to construct theoretical models that contain the necessary features pertaining to the different designs tested by simulations. The work presented here is thus constituted of both Langevin Dynamics simulations and adaptations of the Tension-Propagation theory of polymer translocation when the polymer is subject to the various test conditions.

Field, greenhouse and laboratory experiments were implemented to investigate the effects of auxin herbicides on growth and yield of cotton in glyphosate based systems. Field experiments evaluated the effect of rate and timing of dicamba or 2,4-D exposure when applied in glyphosate-resistant cotton. Increasing rates of either dicamba or 2,4-D resulted in increased injury and yield reductions. Initial injury symptomology was similar for cotton exposed at vegetative and reproductive stages. When cotton was exposed to auxin herbicides during vegetative growth, injury increased with time, while foliar injury during reproductive growth was stagnant and often decreased with time. Subsequently, the strongest correlations to yield loss and injury were from later evaluations of vegetative timings. Recovery from injury due to auxin herbicide exposure was dependent upon favorable environmental conditions; however, recovery was often superficial and masked significant yield loss. Greenhouse studies evaluated the impact of the diglycolamine dicamba salt on the movement of 14C radio-labeled potassium salt glyphosate in barnyardgrass and johnsongrass. Increasing glyphosate rate increased total absorption of glyphosate in both species. Total absorption of glyphosate was not impacted by the presence of dicamba, for either johnsongrass or barnyardgrass. Dicamba did not consistently alter the translocation of glyphosate in johnsongrass; however, dicamba did reduce glyphosate translocation in barnyardgrass. Total amount of translocated glyphosate was 2.6 to 4.6% and 3.8 to 6.8% of applied in barnyardgrass and johnsongrass, respectively. Reduced translocation in barnyardgrass was a result of increased glyphosate accumulation in the distal portion of the treated leaf. Increasing the rate of glyphosate did overcome the dicamba induced antagonism; however, altered translocation of glyphosate has been documented to be a precursor to herbicide resistance.

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