Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

I have been interested in the molecular mechanisms of Haematopoietic malignant diseases such as leukaemia and lymphoma, especially those involving oncogenic tyrosine kinases. About 30 of the 90 tyrosine kinases in the human genome have been implicated in cancer (Blume-Jensen P, 2001). The oncogenic tyrosine kinases (OTKs), such as Bcr-Abl (product of chromosomal translocations of two genes bcr and abl) in Chronic Myelogenous Leukaemia, and Erythroblastic leukaemia viral oncogene homolog 2(Erb-B2) in mammary and other cancers, mediate their transforming effects via a diverse array of signalling pathways involved in DNA damage, cell survival and cell cycle regulation (Deutsch E, 2001; Skorski T, 2002; Kumar R, 1996). My work has been centred around the analysis of a mouse cancer model that is driven by an oncogenic tyrosine kinase – p56 Lck-F505 expressed on CD45 knock- out background (Baker M, 2000). The investigation of this mouse model has revealed that oncogenic inhibition of deamidation of the Bcl-xL survival protein plays a critical role in protecting thymocytes from DNA-damage induced apoptosis. Cells that would normally be eliminated due to accumulating DNA damage are instead preserved with an increasing load of double-stranded breaks, leading to genomic instability, chromosomal abnormalities and transformation. This work was published in Cancer Cell (An oncogenic tyrosine kinase inhibits DNA repair and DNA-damage-induced BclxL deamidation in T cell transformation. Zhao R, 2004). Following that I have tried to elucidate the different roles of the two deamidated species of Bcl-xL in apoptosis, and also the molecular mechanisms of DNA damage- induced Bcl-xL deamidation in order to understand the inhibition of Bcl-xL deamidation by oncogenic tyrosine kinases. Recently I have shown that Bcl-xL deamidation, whereby two critical Asn residues are converted to iso-Asp, cripples the ability of the protein to sequester pro-apoptotic BH3-only proteins such as Bim and p53- upregulated modulator of apoptosis (PUMA), thereby explaining its loss of pro-survival functionality. In vivo, DNA damage causes intracellular alkalinisation that is both necessary and sufficient to deamidate Bcl-xL, promoting apoptosis: no enzyme is necessary for this process. In pre-tumourigenic thymocytes alkalinisation is blocked, so preserving Bcl-xL in its pro-survival mode. Furthermore murine tumours are protected from genotoxic attack by native Bcl-xL, but enforced alkalinisation and consequent Bcl-xL deamidation promotes apoptosis. This part of work was published in Plos Biology (DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH. Zhao R, 2007). Through collaboration with Prof AR Green’s research group at the Department of Haematology of the University of Cambridge, I have also analysed the Bcl-xL deamidation pathway in human myeloproliferative disorders, e.g. Polycythemia vera(PV) and Chronic Myelogenous Leukaemia (CML). We found that the oncogenic tyrosine kinases involved in these disorders, i.e. Jak2V617F and Bcr-Abl also inhibit the Bcl-xL deamidation pathway in DNA damage responses. These findings shed light on potential therapeutic application of the Bcl-xL deamidation pathway in human malignancies. This piece of work was recently published in the New England Journal of Medicine (Inhibition of the Bcl-xL deamidation pathway in myeloproliferative disorders. Zhao R, 2008). Overall the cited work has led to several important new insights into the molecular mechanisms involved in oncogenesis: first, that Bcl-xL deamidation is important in the cascade of events leading from DNA damage to apoptosis; second, that oncogenic tyrosine kinases inhibit these events in both the murine and human context; third, that up-regulation of the NHE-1 antiport and consequent intracellular alkalinisation are critical events in this DNA damage-induced cascade leading to apoptosis. In the process I have demonstrated the first in vivo mechanism for the deamidation of an internal protein Asn. Essentially, a completely new and unexpected signalling pathway has been uncovered that seems to pertain to all murine and human haematopoietic cell lineages that have been investigated so far.

Intracellular pH (pHi) impacts many cellular mechanisms including cellular metabolism, gene expression, cell volume regulation, cell survival and proliferation. Most cells use two general pHi regulatory mechanisms: HCO3-/Cl- antiporters (AE, Slc4a family) to reduce internal alkaline load, and Na+/H+ exchangers (NHE, Slc9a family) that protect cells from acidosis. Previous studies with preimplantation (PI) embryos have shown robust activity of HCO3-/Cl- exchanger in all stages of development. It was also determined that inhibition of this exchange with the stilbene AE inhibitor 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) was detrimental to embryo development from the 2‐cell stage to blastocyst when cultured at high external pH. In this study I investigated which of the five known plasma membrane NHE isoforms was present and active within mouse PI embryos and their role as pHi regulators throughout preimplantation embryo development. In mouse oocytes and preimplantation embryos, mRNAs were detected encoding NHE1 (SLC9A1), NHE3 (SLC9A3), and NHE4 (SLC9A4), with higher mRNA levels for each in fully-grown oocytes through one-cell stage embryos and then generally lower levels after the two-cell stage. No transcripts for NHE2 (SLC9A2) or NHE5 (SLC9A5) were detected. Measurements of intracellular pH during recovery from acidosis, induced by transient ammonium pulse, suggested that recovery occurred and was mediated by NHE activity at all preimplantation stages assessed (one-cell, two-cell, eight-cell and morula). This recovery was inhibited by 1 mM amiloride, a general NHE inhibitor. The observed residual recovery was attributed to passive passage of protons across the membrane, rather than the activity of NHE4 (an amiloride-resistant isoform), since no further decrease in recovery rates from acidosis was observed upon amiloride increase to 5 mM. Furthermore, recovery from acidosis at each stage was entirely inhibited by cariporide, which is very highly selective for NHE1. In contrast, the moderately NHE3-selective inhibitor S3226 did not preferentially block recovery, nor did adding S3226 increase inhibition over that achieved with cariporide alone, indicating that NHE3 did not play a functional role in pHi regulation at any stage assessed. Another regulator of intracellular pH against acidosis, previously reported to be active in oocytes and 1-cell embryos, the sodium-dependent bicarbonate/chloride exchanger (NDBCE; SLC4A8), had low or absent activity in two-cell embryos. This indicated that NHE1 is likely the only significant regulator of pHi in preimplantation mouse embryos, at least after the 1-cell stage. Culturing embryos from the one-cell or two-cell stages in acidotic medium inhibited their development, as assessed by development to the blastocyst stage and cell lineage allocation. However, inhibition of NHE1 with cariporide, NDBCE with DIDS, or both together did not further decrease embryo development to the blastocyst stage more extensively under conditions of chronic acidosis than at normal pH. This suggests that mouse PI embryos have a restricted ability to counteract chronic acidosis by means of pHi regulatory mechanisms, despite clearly being able to recover from acute acidosis via NHE1 activity.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Prostate cancer is the second most common cancer among men in the United States. While treatments for prostate cancer exist, none are curative. As a solid tumor, prostate cancer can grow beyond the diffusion limits of oxygen, thereby resulting in a hypoxic environment. While hypoxia can cause death to a variety of cell types, tumor cells can develop resistance to hypoxia and survive under minimal oxygen conditions. Hypoxia in tumor cells has also been associated with poor prognosis, increased metastasis, and decreased efficacy of chemotherapy. BNIP3, a BH-3 only proapoptotic Bcl-2 family member, has been shown to play an important role in cell death under hypoxic conditions in a variety of cell types. In normoxia, BNIP3 shows little to no expression in both cardiomyocytes and many cancer cell types, but is then upregulated under hypoxic conditions. Previous work in our laboratory provides evidence that hypoxia alone, as well as the concomitant increase in BNIP3 expression, cannot cause death of rat neonatal cardiomyocytes. Instead, our studies found that hypoxia with concomitant intracellular acidosis is required. Further studies indicated that BNIP3 is also necessary for hypoxia-acidosis associated cell death in cardiomyocytes. Our results in rat neonatal cardiomyocytes led us to hypothesize that cell death could be induced in hypoxic prostate cancer cells if concomitant acidosis could be induced. Additionally, our intention was to determine if BNIP3 was required for any prostate cancer cell death that may occur under hypoxia-acidosis conditions.

No description available.

Em concentrações elevadas no plasma ou no tecido renal a Angiotensina II (Ang II) induz, alterações na hemodinâmica renal, injúria glomerular, aumento da síntese de componentes da matriz extracelular glomerular, estresse oxidativo e apoptose de células glomerulares, incluindo os podócitos. Os podócitos possuem um sistema reninaangiotensina (SRA) próprio e expressam os receptores AT1 e AT2 para o peptídeo, além do trocador Na+/H+ isoforma 1 (NHE1). O NHE1 está envolvido com a resistência e indução de apoptose, controle do volume celular e manutenção do fenótipo celular. Assim, o objetivo deste estudo foi investigar em podócitos, o papel da Ang II na indução de apoptose, e os eventos intracelulares associados à atividade do NHE1 nesta condição. Nossos resultados indicam que o tratamento com Ang II em alta concentração por 24 horas promove apoptose em podócitos. Nesta condição o NHE1, promove ativação da via de sinalização intracelular p38 MAPK e aumenta a atividade do NHE1 levando a alcalose, ativação da Bax e apoptose nos podócitos. / It has been observed that high plasma, or kidney tissue concentrations of angiotensin II (Ang II) leads to changes in renal hemodynamics, severe glomerular injury, increased synthesis of glomerular extracellular matrix components, oxidative stress and apoptosis in glomerular cells, including podocyte and mesangial cells. Podocytes a local renin-angiotensin system (RAS), expresses the AT1 and AT2 receptors for Ang II and the Na + / H + exchanger (NHE1). The NHE1 is involved with resistance and induction of apoptosis, cell volume control and maintenance of cell phenotype. Thus, the goal of this study was to investigate in podocytes the role of Ang II in the induction of apoptosis, and intracellular events linked to the NHE1 activity in this condition. Our results indicate that the treatment with Ang II, in a high dose, for 24 hours induces apoptosis in podocytes, and promotes oxidative stress. However, the activation of NADPH oxidase subunits Nox4 and p22 (phox) and pro- apoptotic pprotein Bax, came before the late apoptosis observed in 24 hours of treatment with Ang II. Under physiological conditions, the NHE1 activity contributes to cell survival by preventing cytosolic acidification. Moreover, Ang II via the AT1 receptor, activates intracellular signaling pathway p38 MAPK and increases the NHE1 activiy leading to alkalosis, Bax activation and apoptosis in podocytes.

We hypothesized that, due to the high pH of this compartment, the reticulum epithelium displays particular features in the transport of short-chain fatty acids (SCFA). Ovine reticulum epithelium was incubated in Ussing chambers using a bicarbonate-free buffer solution containing butyrate (20 mmol L−1). p-hydroxymercuribenzoic acid (pHMB), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), or ouabain were added to the buffer solution as inhibitors of monocarboxylate transporters, sodium-proton-exchangers, or the Na+/K+-ATPase, respectively. The short-circuit current (Isc) and transepithelial conductance (Gt) were monitored continuously while the flux rates of 14C-labelled butyrate were measured in the mucosal-to-serosal (Jmsbut) or serosal-to-mucosal direction (Jsmbut). Under control conditions, the mean values of Isc and Gt amounted to 2.54 ± 0.46 µEq cm−2 h−1 and 6.02 ± 3.3 mS cm−2, respectively. Jmsbut was 2.1 ± 1.01 µmol cm−2 h−1 on average and about twice as high as Jsmbut. Incubation with ouabain reduced Jmsbut, while Jsmbut was not affected. The serosal addition of EIPA did not affect Jmsbut but reduced Jsmbut by about 10%. The addition of pHMB to the mucosal or serosal solution reduced Jmsbut but had no effect on Jsmbut. Mucosally applied pHMB provoked a transient increase in the Isc. The serosal pHMB sharply reduced Isc. Our results demonstrate that butyrate can be effectively transported across the reticulum epithelium. The mechanisms involved in this absorption differ from those known from the rumen epithelium.

This dissertation is a composite of some of the research that I have conducted during the course of my PhD study. The larger goal of this dissertation is to renew the interests among the scientific community for an otherwise under-appreciated technique called Isothermal Titration Calorimetry. The resurgence of calorimetry in the biophysical community and the shift to investigations of more complex biological systems signal a real need for more sophisticated analysis techniques. This dissertation expounds on new ITC analysis methods that we have developed as well as results from the study of thermodynamic properties of higher order DNA structures. In 1978, Peter Privalov described the first use of microcalorimetry to obtain the thermodynamic properties for removing calcium from parvalbumin III protein. Fast forward 36 years: modern day electronics, highly efficient thermally conductive and chemically inert materials, in conjunction with sensitive thermal detectors, has transformed the original calorimeter into a device capable of measuring heat changes as small as 0.05 nanowatts, which is equivalent to capturing heat from an incandescent light bulb a kilometer away. However, analytical methods have not kept pace with this technology. Commercial ITC instruments are typically supplied with software that only includes a number of simple interaction models. As a result, the lack of analysis tools for more complex models has become a limiting factor for many researchers. We have recently developed new ITC fitting algorithms that we have incorporated into a userriendly program (CHASM©) for the analysis of complex ITC equilibria. In a little over a year, CHASM© has been downloaded by over 370 unique users. Several chapters in this dissertation demonstrate this software’s power and versatility in the thermodynamic investigations of two model systems in both aqueous and non-aqueous media. In chapter VI, we assembled a model NHE-III1 : a novel structure of Gquadruplex in a double stranded form and studied its structural complexity and binding interactions with a classical G-quaduplex interactive ligand known as TMPyP4. In chapter VII, we reported the thermodynamic properties of a novel PAH system in which weak dispersion forces are solely responsible for formation of the supramolecular complexes.