Huber, Robert

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss in function of motor neurons. Elevated levels of biologically important metal ions, such as copper (II) (Cu(II)), zinc (II) (Zn(II)) or iron (III) (Fe(III)), may contribute to the disease. Moreover, Cu(II) interactions with other proteins associated with ALS have been investigated; however, the effects of metallation on TAR DNA-binding protein of 43 kDa (TDP-43) are less known. The aim of the thesis was to evaluate interactions between full-length TDP-43 and metal ions, and gain insight into the mechanisms of these interactions. In Chapter 2, electrochemical methods were used to evaluate the coordination of Cu(II) ions to immobilized TDP-43. Cu(II)-TDP-43 binding was favourable at a neutral pH. Surface characterization confirmed protein immobilization and Cu(II)-TDP-43 coordination. Competitive Zn(II) ion binding studies revealed Zn(II) increases Cu(II) binding. In Chapter 3, Fe(III) ion binding studies revealed that Fe(III) reduces Cu(II) binding when co-exposed to the TDP-43-Au surface. Data shows significant uptake of Cu(II) by TDP-43 protein which may have important implications in normal and diseased states of TDP-43, indicating surface bioelectrochemistry is a viable tool for fundamental exploration of proteins and metals, and their interactions, as they inform disease mechanisms, disease detection and drug screening.

Author Keywords: Amyotrophic Lateral Sclerosis, bioelectrochemistry, electrochemistry, metalloprotein, surface characterization, TDP-43

Cadmium is a toxic metal that has detrimental effects on blood vessel development and function. To examine the effect of varying concentrations of cadmium on angiogenesis, two in vitro assays were used. First, developing yolk sac blood vessels were studied in gestation day 8 mouse embryos exposed to medium alone, 1.25, or 1.75 μM cadmium chloride (CdCl2). Embryos exposed to 1.25 μM cadmium experienced a significant increase in the number of vessels formed; however, they were smaller in size. Vessel morphology and signalling pathways were also investigated using the mouse aortic ring assay, with exposures of 0.0, 0.5, 1.0, 5.0, or 10.0 μM CdCl2. Samples exposed to 10 μM experienced a significant increase in vessel length. However, no significant differences in phosphorylated PTEN and AKT were observed. The results of this study suggest that low levels of cadmium may disrupt angiogenesis, particularly the development of the embryonic vasculature in the yolk sac.

Author Keywords: Angiogenesis, Cadmium, Embryonic Development, Teratogenicity, Vascular Development, Vasculogenesis

Transactive response (TAR) DNA-binding protein of 43 kDa (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS) and is characterized by hyperphosphorylation and aggregation patterns, a mechanism largely understudied. In addition, ALS remains without a cure. Herein, in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Additionally, in vitro phosphorylation of TDP-43 by protein kinases was conducted to identify which protein kinases catalyze phosphorylation. The aggregation of phosphorylated and unphosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) fluorescence spectroscopy. The protein aggregates were largely insoluble, ThT-positive and characterized with heterogeneous morphologies. Antibodies specific to epitopes within the RNA-recognition motifs and the C-terminal domains reduced the formation of β-sheets and insoluble aggregates, with outcomes highly dependent on the type of antibodies, indicating dual functionality. The only protein kinase able to phosphorylate TDP-43 at S410 was MARK4, indicating its role in the onset of PTMs in the protein. Thus, targeting TDP-43 epitopes for inhibition of aggregation and in vitro phosphorylation represent viable biochemical assays for screening protein kinase inhibitors as potential drugs against ALS.

Author Keywords: aggregation, ALS, antibody-based inhibition, phosphorylation, protein kinase, TDP-43

The emergence of fungal hybrid pathogens threatens sustainable crop production worldwide. To investigate hybridization, the related smut fungi, Ustilago maydis and Sporisorium reilianum, were selected because they infect a common host (Zea mays), can hybridize, and tools are available for their analysis. Hybrid dikaryons exhibited filamentous growth on plates but reduced virulence and limited colonization in Z. mays. Select virulence genes in the hybrid had similar transcript levels on plates and altered levels during infection of Z. mays relative to each parental dikaryon. Virulence genes were constitutively expressed in the hybrid to determine if its pathogenic development could be influenced. Little impact was observed in hybrids with increased expression of effectors known to modify host response and metabolism. However, increased expression of transcriptional regulators of stage specific pathogenic development increased the hybrid's capacity to induce symptoms. These results establish a base for investigating molecular aspects of fungal hybrid pathogen emergence.

Author Keywords: effectors, hybrid pathogenesis assays, Sporisorium reilianum, transcription factors, Ustilago maydis, virulence factors

Giardia intestinalis possesses four isotypes of cytochrome b5 (gCYTB-I-IV) that differ from their mammalian counterparts, suggesting different functions in this protozoan parasite. Although the recently discovered Giardia flavoenzyme, GiOR-1, reduces these cytochromes, its properties have not been thoroughly studied, owing to the difficulty in its expression. Here I describe successful conditions for expression of GiOR-1 using autoinduction. GiOR-1 is obtained with flavins bound as indicated by its UV-visible spectrum. Its ability to catalyze electron transfer from donors (NADH, NADPH) to acceptors (oxygen, ferricyanide, cytochrome c, gCYTB5-III) were studied in spectrophotometric rate assays. NADPH is the preferred electron donor, while cytochromes are the preferred electron acceptors. Interestingly, the His-tag used to purify gCYTB5-III decreases its reaction rate with GiOR-1, as an untagged version has slightly faster rates. These findings establish the appropriate conditions for further studies on GiOR-1, including the identification of endogenous electron acceptors.

Author Keywords: Autoinduction, Cytochrome b5, Cytochrome P450 oxidoreductase, Giardia intestinalis, GiOR-1, Polyhistidine tag

The waterborne protozoan Giardia intestinalis cycles between the environmentally-resistant and infectious cyst and the metabolically-active trophozoite that adheres to the epithelial lining of the small intestine. Adhesion can trigger the innate immune response in epithelial cells, including the synthesis of the free radical nitric oxide (NO) that inhibits cell proliferation and encystation of trophozoites. In this work changes in protein expression of three Giardia isotypes of the redox heme protein cytochrome b5 (gCYTb5 I, II and III) were studied in response to either nitrosative stress or induction of encystation. Two nitrosative stressors, sodium nitrite and the NO donor DETA-NONOate, were used at sub-lethal concentrations (0.5 mM and 0.05 mM, respectively) that do not affect cell proliferation until later time points so that subtle changes in protein expression could be observed in the absence of other confounding factors. Nucleolar gCYTb5-I and nucleoplasmic gCYTb5-III expression patterns were similar in trophozoites exposed to either stressor, showing gradual increases in expression with peaks between 4 and 12 hours, which indicates these cytochromes respond to nitrosative stress and possibly to potential DNA damage in Giardia. In contrast, gCYTb5-II of the peripheral vacuoles, which are part of the endocytic pathway of Giardia, showed little change in expression in response to either stressor. However, changes in gCYTb5-II expression were observed in encysting trophozoites, with a 1.4-fold increase in protein levels at seven hours after induction of encystation, followed by a gradual decrease in expression. These changes are consistent with previous mRNA analysis done in our laboratory and suggest a role for gCYTb5-II in the increase in nutrient uptake during early encystation.

Author Keywords: cytochrome, encystation, Giardia, heme, nitrosative, parasite