The huntingtin N17 domain is the master regulator of huntingtin intracellular localization. N17 is post-translationally modified, and phosphorylation of N17 serines 13 and 16 facilitate the stress dependent nuclear translocation of huntingtin by inhibiting CRM1 binding and nuclear export. In Huntington’s disease (HD), mutant huntingtin is hypo-phosphorylated and increasing N17 phosphorylation has been shown to be protective in HD mouse models. N17 phosphorylation is therefore a valid therapeutic sub-target of huntingtin. The ER stresses that have been previously characterized to affect huntingtin phosphorylation are broad, likely activating a plethora of response pathways. Thus, in this study, we sought to define a specific stress that could affect huntingtin phosphorylation and nuclear localization. Here we show that huntingtin localization and phosphorylation can be specifically affected by reactive oxygen species (ROS). We identify a highly conserved methionine at position 8 (M8) as the specific sensor of oxidative species within N17 and show the capacity of oxido-mimetic M8 point mutations to affect N17 structure, localization and phosphorylation. We also define a specific molecular mechanism whereby N17 oxidation promotes membrane dissociation, thus increasing kinase accessibility and subsequent phosphorylation. These results define a precise molecular mechanism for the normal biological regulation of huntingtin phosphorylation by oxidative signalling. This ability of huntingtin to sense ROS levels at the ER provides a link between age-associated stress and altered huntingtin function. It suggests that ROS stress due to aging may be a critical molecular trigger of HD that could explain the age-onset nature of disease. / Thesis / Master of Science (MSc)

Mitochondrial dysfunction has been identified as a key factor in the progression of Parkinson's disease. Mitochondrial dysfunction has been shown to induce stress pathways, leading to neuronal dysfunction and cell death. Our lab has previously identified that, in neurons, reconfiguring the mitochondria using supercomplex assembly factors is protective against excitotoxic stress. For this thesis, we sought to characterize the stress pathways and synaptic impairment in an in vitro mitochondrial dysfunction model. Then, to determine if we can rescue the deficits shown, we manipulated mitochondrial integrity using the inner mitochondrial membrane targeted isoform of MCL1, which has previously been shown to regulate cristae structure and mitochondrial supercomplex assembly. We demonstrate that the integrated stress response is activated upon mitochondrial dysfunction. Next, we show mitochondrial dysfunction leads to a downregulation of synaptic genes involved in neurotransmission. Finally, our results show that both the antiapoptotic outer mitochondrial membrane-targeted isoform, and MCL1-Matrix are able to prevent cell death in response to mitochondrial dysfunction; however, MCL1-Matrix confers greater reduction in ISR activation and reactive oxygen species production. These data suggest that manipulating mitochondrial integrity, using MCL1-Matrix, confers a broad protective effect against neuronal stressors and may be used as a novel approach to preventing Parkinson's disease.

Previous studies have focused on the effect of individual stressors on hsp gene expression in eukaryotic organisms. In the present study, I examined the effect of concurrent low doses of sodium arsenite and mild heat shock temperatures on the expression of hsp30 and hsp70 genes in Xenopus laevis A6 kidney epithelial cells. Northern hybridization and western blot analysis revealed that exposure of A6 cells to 1-10 μM sodium arsenite at a mild heat shock temperature of 30˚C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 μM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26˚C with larger responses at 28 and 30 ˚C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 μM ) and heat shock (30 ˚C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 ˚C. Acquired thermotolerance was not observed with cells treated with the two mild stresses individually. It is likely that the enhanced accumulation of HSPs under these conditions permits the organism to cope with multiple environmental stresses encountered in their natural aquatic habitat. Previous studies have shown that inhibiting the activity of the proteasome also leads to the accumulation of damaged or unfolded proteins within the cell. In the second phase of this study, I report that inhibition of proteasome activity by the inhibitors carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and lactacystin induced the accumulation of HSP30 and HSP70 as well as their respective mRNAs. The accumulation of HSP30 and HSP70 in A6 cells recovering from MG132 exposure was still relatively high 24 h after treatment and it decreased substantially after 48 h. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. HSP30 localization in A6 cells was primarily in the cytoplasm as revealed by immunocytochemistry. In some A6 cells treated with higher concentrations of MG132 and lactacystin, HSP30 was also found to localize in relatively large cytoplasmic foci. In some MG132-treated cells, HSP30 staining was substantially depleted in the cytoplasmic regions surrounding these foci. The activation of HSF1 may be involved in MG132-induced hsp gene expression in A6 cells since KNK437 inhibited the accumulation of HSP30 and HSP70. Lastly, MG132 treatment also conferred a state of thermotolerance in A6 cells such that they were able to survive a subsequent thermal challenge. Analysis of this phenomenon is important given the fact that impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.

Previous studies have focused on the effect of individual stressors on hsp gene expression in eukaryotic organisms. In the present study, I examined the effect of concurrent low doses of sodium arsenite and mild heat shock temperatures on the expression of hsp30 and hsp70 genes in Xenopus laevis A6 kidney epithelial cells. Northern hybridization and western blot analysis revealed that exposure of A6 cells to 1-10 μM sodium arsenite at a mild heat shock temperature of 30˚C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 μM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26˚C with larger responses at 28 and 30 ˚C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 μM ) and heat shock (30 ˚C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 ˚C. Acquired thermotolerance was not observed with cells treated with the two mild stresses individually. It is likely that the enhanced accumulation of HSPs under these conditions permits the organism to cope with multiple environmental stresses encountered in their natural aquatic habitat. Previous studies have shown that inhibiting the activity of the proteasome also leads to the accumulation of damaged or unfolded proteins within the cell. In the second phase of this study, I report that inhibition of proteasome activity by the inhibitors carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and lactacystin induced the accumulation of HSP30 and HSP70 as well as their respective mRNAs. The accumulation of HSP30 and HSP70 in A6 cells recovering from MG132 exposure was still relatively high 24 h after treatment and it decreased substantially after 48 h. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. HSP30 localization in A6 cells was primarily in the cytoplasm as revealed by immunocytochemistry. In some A6 cells treated with higher concentrations of MG132 and lactacystin, HSP30 was also found to localize in relatively large cytoplasmic foci. In some MG132-treated cells, HSP30 staining was substantially depleted in the cytoplasmic regions surrounding these foci. The activation of HSF1 may be involved in MG132-induced hsp gene expression in A6 cells since KNK437 inhibited the accumulation of HSP30 and HSP70. Lastly, MG132 treatment also conferred a state of thermotolerance in A6 cells such that they were able to survive a subsequent thermal challenge. Analysis of this phenomenon is important given the fact that impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.

The mechanism and subsequent patterns of Heat shock protein (Hsp) expression in skeletal muscle specific to contraction type was determined. Rat tibialis anterior (TA) muscle was forcibly lengthened (LC) or shortened (SC) in 5 sets of 20 repetitions before being removed at 2, 8, 24, 48, 72, or 168 hours and analyzed for muscle damage and Hsp25 and Hsp72 expression. Isometric peak torque was reduced to 63% and 33% (P<0.001) at 3-minutes after SC and LC, respectively. Muscle fibre damage appeared at 8h and beyond following LCs, but no damage was observed after SCs. Hsp25 content in LC muscle increased by 3.1±0.53 fold (P<0.01) at 48h and remained elevated. Hsp72 content increased by 3.8±0.66 fold at 24h and remained elevated. Neither Hsp25 nor Hsp72 content was elevated following SCs. Muscle damage associated with LCs results in a greater Hsp accumulation than SCs and 100 SCs do not result in increased Hsp content.

The mechanism and subsequent patterns of Heat shock protein (Hsp) expression in skeletal muscle specific to contraction type was determined. Rat tibialis anterior (TA) muscle was forcibly lengthened (LC) or shortened (SC) in 5 sets of 20 repetitions before being removed at 2, 8, 24, 48, 72, or 168 hours and analyzed for muscle damage and Hsp25 and Hsp72 expression. Isometric peak torque was reduced to 63% and 33% (P<0.001) at 3-minutes after SC and LC, respectively. Muscle fibre damage appeared at 8h and beyond following LCs, but no damage was observed after SCs. Hsp25 content in LC muscle increased by 3.1±0.53 fold (P<0.01) at 48h and remained elevated. Hsp72 content increased by 3.8±0.66 fold at 24h and remained elevated. Neither Hsp25 nor Hsp72 content was elevated following SCs. Muscle damage associated with LCs results in a greater Hsp accumulation than SCs and 100 SCs do not result in increased Hsp content.

>Magister Scientiae - MSc / Nanoscience is the study of phenomena and objects at the nanoscale (around 1-100 nm), socalled nanomaterials. These nanomaterials exhibit novel properties that are often very different to those of the bulk materials used for their synthesis. Hence, nanoparticles are widely commercialised, especially silver nanoparticles (AgNPs) due to their antimicrobial properties and some other useful phenomena. This commercialisation leads to inevitable exposure to the environment and humans, which leads to inhalation, ingestion or dermal uptake of AgNPs by the human body culminating in distribution to several major organs, including the liver. Both chronic and acute exposure to AgNPs have been linked to detrimental effects in both in vitro and in vivo studies. These include oxidative stress, induction of inflammation, DNA damage, cell death and many others.

<p>Proteotoxic stress is implicated in numerous human diseases including neurodegeneration, cancer, and diabetes. Unfortunately, our mechanistic understanding of the cellular response to proteotoxic stress is limited. A critical feature of the cellular stress response is the activation of Heat Shock Transcription Factors (HSFs) that regulate the expression of numerous genes involved in protein folding, protein degradation, and cellular survival. The studies presented here utilize a diverse array of techniques including yeast genetics, recombinant protein expression and purification, biochemical analysis of protein-DNA interactions, x-ray crystallography, in vitro post-translational modification, and mammalian cell culture to illuminate novel aspects of HSF biology. Critical findings include understanding key principles of HSF-DNA interactions, identification of a novel negative regulator of HSF activity, and identification of structural features of HSF paralogs that enable precise combinatorial regulation. These unique insights lay the foundation for a greater understanding of HSF in specific cellular contexts and disease states.</p> / Dissertation

After almost two decades of research on direct repression by p53, I provide evidence that the transcription factor p53 solely acts as an activator of transcription. I evaluate the prominent models of transcriptional regulation by p53 based on a computational meta-analysis of genome-wide data. With this tool at hand, the major contradiction how p53 binding can result in activation of one target gene and repression of another is resolved. In contrast to most current models, solely genes activated by p53 are found to be enriched for p53 binding. Meta-analysis of large-scale data is unable to confirm reports on directly repressed p53 target genes and does not support models of direct repression. Consequently, as supported by experimental data, p53 is not a direct repressor of transcription, but solely activates its target genes. Moreover, models based on interference of p53 with activating transcription factors are also not supported by the meta-analysis. As an alternative to these models, the meta-analysis leads to the conclusion that p53 represses transcription indirectly by activation of the p53-p21- DREAM/RB pathway. Thus, results of the meta-analysis support only two models, namely activation by direct binding of p53 to target genes and repression through activating the p53-p21-DREAM/RB pathway.

The development and maintenance of tissues and organs depend on the careful regulation and coordinated motion of large numbers of cells. There is substantial evidence that many complex tissue functions, such as cell division, collective cell migration and gene expression, are directly regulated by mechanical forces. However, relatively little is known about how mechanical stress is distributed within a tissue and how this may guide biochemical signalling. Working in the framework of a popular vertex-based model, we derive expressions for stress tensors at the cell and tissue level to build analytic relationships between cell shape and mechanical stress. The discrete vertex model is upscaled, providing exact expressions for the bulk and shear moduli of disordered cellular networks, which bridges the gap to traditional continuum-level descriptions of tissues. Combining this theoretical work with new experimental techniques for whole-tissue stretching of Xenopus laevis tissue, we separate the roles of mechanical stress and cell shape in orienting and cueing epithelial mitosis. We find that the orientation of division is best predicted by the shape of tricellular junctions, while there appears to be a more direct role for mechanical stress as a mitotic cue.