Type IV pili (T4P) are bacterial biomolecular machines that mediate interactions with the environment. Bacterial pathogens such as Pseudomonas aeruginosa require T4P for virulence. Significant progress has been made in recent years towards our understanding of how the proteins in the T4P system interact and function. While over 50 different proteins are involved in T4P biogenesis, the two outer membrane components, PilF and PilQ, are the focus of the work presented in this thesis. PilF was found to be required for assembly of PilQ into secretins, the outer membrane channels through which T4P fibers exit the cell. The functions of PilF are consistent with a family of lipoproteins called pilotins, to which the roles of secretin assembly and/or localization are attributed. Structure determination by X-ray crystallography revealed that PilF is composed of six tetratricopeptide (TPR) protein-protein interaction motifs. Functional mapping of PilF indicated that a hydrophobic groove on the first TPR is involved in secretin assembly. Secretin localization correlated directly with that of PilF. The effects of pilF mutations and the structural data led to the hypothesis that PilF and PilQ interact directly. We propose that PilF and PilQ interact at the inner membrane and are co-transported to the outer membrane by the Lol lipoprotein sorting system. PilQ multimerizes into secretins upon outer membrane insertion and aligns with inner membrane T4P proteins to form a complete molecular machine. PilQ mutagenesis mapping showed that: the N-terminal “system specific” domain is important but not essential for secretin function; the central “multimerization” domain is critical for secretin assembly and function; and the C-terminal tail implicated in secretin-pilotin interactions is dispensable for PilQ function. Purified PilQ enabled copurification of PilF from cell lysates, providing the first evidence for their interaction. These data provide a framework for future exploration of T4P assembly in P. aeruginosa.

Type IV pili (T4P) are bacterial biomolecular machines that mediate interactions with the environment. Bacterial pathogens such as Pseudomonas aeruginosa require T4P for virulence. Significant progress has been made in recent years towards our understanding of how the proteins in the T4P system interact and function. While over 50 different proteins are involved in T4P biogenesis, the two outer membrane components, PilF and PilQ, are the focus of the work presented in this thesis. PilF was found to be required for assembly of PilQ into secretins, the outer membrane channels through which T4P fibers exit the cell. The functions of PilF are consistent with a family of lipoproteins called pilotins, to which the roles of secretin assembly and/or localization are attributed. Structure determination by X-ray crystallography revealed that PilF is composed of six tetratricopeptide (TPR) protein-protein interaction motifs. Functional mapping of PilF indicated that a hydrophobic groove on the first TPR is involved in secretin assembly. Secretin localization correlated directly with that of PilF. The effects of pilF mutations and the structural data led to the hypothesis that PilF and PilQ interact directly. We propose that PilF and PilQ interact at the inner membrane and are co-transported to the outer membrane by the Lol lipoprotein sorting system. PilQ multimerizes into secretins upon outer membrane insertion and aligns with inner membrane T4P proteins to form a complete molecular machine. PilQ mutagenesis mapping showed that: the N-terminal “system specific” domain is important but not essential for secretin function; the central “multimerization” domain is critical for secretin assembly and function; and the C-terminal tail implicated in secretin-pilotin interactions is dispensable for PilQ function. Purified PilQ enabled copurification of PilF from cell lysates, providing the first evidence for their interaction. These data provide a framework for future exploration of T4P assembly in P. aeruginosa.

Triple negative breast cancer (TNBC) represents an aggressive subtype for which only chemotherapy is available. The RB1 tumour suppressor is frequently lost in human breast cancer, primarily in TNBC. Loss of RB1 deregulates the cell cycle and is thought to affect BC response to endocrine, radiation, and chemotherapy. However, the global chemosensitivity of Rb null BC is not known. Here I demonstrate that RB1-deficient TNBC cells are highly sensitive to radiation, and moderately sensitive to doxorubicin and methotrexate. However, loss of RB1 does not increase sensitivity to multiple other drugs. Moreover, a non-biased screen of 2 RB-deficient versus 2 RB-proficient lines with ~3500 drugs did not reveal any difference in sensitivity, but identified disulfiram as a potent drug, which compared favourably with current chemotherapeutics against TNBC. Disulfiram’s efficacy was validated against 13 human TNBC lines with an average IC50 of 300nM. IQGAP1 was identified as a potential target of disulfiram.

Triple negative breast cancer (TNBC) represents an aggressive subtype for which only chemotherapy is available. The RB1 tumour suppressor is frequently lost in human breast cancer, primarily in TNBC. Loss of RB1 deregulates the cell cycle and is thought to affect BC response to endocrine, radiation, and chemotherapy. However, the global chemosensitivity of Rb null BC is not known. Here I demonstrate that RB1-deficient TNBC cells are highly sensitive to radiation, and moderately sensitive to doxorubicin and methotrexate. However, loss of RB1 does not increase sensitivity to multiple other drugs. Moreover, a non-biased screen of 2 RB-deficient versus 2 RB-proficient lines with ~3500 drugs did not reveal any difference in sensitivity, but identified disulfiram as a potent drug, which compared favourably with current chemotherapeutics against TNBC. Disulfiram’s efficacy was validated against 13 human TNBC lines with an average IC50 of 300nM. IQGAP1 was identified as a potential target of disulfiram.

Artificial nighttime lighting (ANL) is known to adversely affect animals, but little is known what the consequences are to plants. Two genotypes of Populus balsamifera, a common urban tree, were used to investigate how ANL impacts plants. While the two genotypes varied in their physiological sensitivity to ANL, poorer levels of net leaf carbon assimilation compared to control samples suggested that ANL perturbed the perception of time of day for these plants. Gene set analysis on a subset of PopGenExpress microarray samples identified time of day specific processes in P. balsamifera, and a set of candidate ANL-sensitive genes were identified from these. Transcript measurements from the two genotypes revealed that ANL affects plants at the molecular level, for the diel cycling of the putative ANL-sensitive genes was perturbed. Together, these results suggest that ANL affects plants at the physiological and molecular level by perturbing their perception of time of day.

The first part of the thesis describes a systematic function analysis that identified in vitro E2 partners for ten different HECT E3 ligase proteins. Using mass spectrometry, the linkage composition for the resulting autoubiquitylation products of a number of functional E2-HECT pairs was determined. HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. The second part of the thesis describes the characterization of the RAD6-interactome. Using affinity purification coupled with mass spectrometry, I identified a novel RAD6-interacting E3 ligase, KCMF1, which binds to a different surface on RAD6 than the other RAD6-associated E3 ligases. KCMF1 also recruits additional proteins to RAD6, and this new complex points to novel RAD6 functions. Interestingly, the RAD6A R11Q mutant polypeptide, found in X-linked mental retardation patients specifically loses the interaction with KCMF1, but not with other RAD6-associated E3 ligases.

Artificial nighttime lighting (ANL) is known to adversely affect animals, but little is known what the consequences are to plants. Two genotypes of Populus balsamifera, a common urban tree, were used to investigate how ANL impacts plants. While the two genotypes varied in their physiological sensitivity to ANL, poorer levels of net leaf carbon assimilation compared to control samples suggested that ANL perturbed the perception of time of day for these plants. Gene set analysis on a subset of PopGenExpress microarray samples identified time of day specific processes in P. balsamifera, and a set of candidate ANL-sensitive genes were identified from these. Transcript measurements from the two genotypes revealed that ANL affects plants at the molecular level, for the diel cycling of the putative ANL-sensitive genes was perturbed. Together, these results suggest that ANL affects plants at the physiological and molecular level by perturbing their perception of time of day.

The first part of the thesis describes a systematic function analysis that identified in vitro E2 partners for ten different HECT E3 ligase proteins. Using mass spectrometry, the linkage composition for the resulting autoubiquitylation products of a number of functional E2-HECT pairs was determined. HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. The second part of the thesis describes the characterization of the RAD6-interactome. Using affinity purification coupled with mass spectrometry, I identified a novel RAD6-interacting E3 ligase, KCMF1, which binds to a different surface on RAD6 than the other RAD6-associated E3 ligases. KCMF1 also recruits additional proteins to RAD6, and this new complex points to novel RAD6 functions. Interestingly, the RAD6A R11Q mutant polypeptide, found in X-linked mental retardation patients specifically loses the interaction with KCMF1, but not with other RAD6-associated E3 ligases.

Doctor of Philosophy / Agronomy / Kevin Price / Stephen M. Welch / Plant phenotyping has been studied for decades for understanding the relationship between plant genotype, phenotype, and the surrounding environment. Improved accuracy and efficiency in plant phenotyping is a critical factor in expediting plant breeding and the selection process. In the past, plant phenotypic traits were extracted using invasive and destructive sampling methods and manual measurements, which were time-consuming, labor-intensive, and cost-inefficient. More importantly, the accuracy and consistency of manual methods can be highly variable. In recent years, however, photogrammetry and 3D modeling techniques have been introduced to extract plant phenotypic traits, but no cost-efficient methods using these two techniques have yet been developed for large-scale plant phenotyping studies. High-throughput 3D modeling techniques in plant biology and agriculture are still in the developmental stages, but it is believed that the temporal and spatial resolutions of these systems are well matched to many plant phenotyping needs. Such technology can be used to help rapid phenotypic trait extraction aid crop genotype selection, leading to improvements in crop yield. In this study, we introduce an automated high-throughput phenotyping pipeline using affordable imaging systems, image processing, and 3D reconstruction algorithms to build 2D mosaicked orthophotos and 3D plant models. Chamber-based and ground-level field implementations can be used to measure phenotypic traits such as leaf length, rosette area in 2D and 3D, plant nastic movement, and diurnal cycles. Our automated pipeline has cross-platform capabilities and a degree of instrument independence, making it suitable for various situations.

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