DNA double strand breaks can occur from various sources and the timely and accurate repair of these breaks is critical to maintain the genomic integrity of the cell. Break-induced replication (BIR) is a repair pathway that has been shown to repair DSBs where only one end of the break can locate homology, similar to ends seen at collapsed replication forks or eroded telomeres. BIR progresses by an unusual bubble-like intermediate. The asynchrony between the synthesis of leading and lagging strand synthesis during BIR leads to the accumulation of long single-stranded DNA (ssDNA) behind the bubble. This mechanism leads to the conservative inheritance of newly synthesized DNA. BIR repair can lead to increased mutations, loss of heterozygosity and gross chromosomal rearrangements. In this thesis I investigated the deleterious effects of the ssDNA formed during BIR. Using yeast, Saccharomyces cerevisiae, I showed that the regulation of Rad51 that binds ssDNA during BIR is important to prevent the accumulation of toxic joint intermediates. Here, I demonstrate that a known Rad51-interacting protein, Srs2, plays two key roles in counteracting the accumulation of lethal recombination intermediates. First, Srs2 dislodges Rad51 from long ssDNA formed during DSB repair and therefore prevents promiscuous strand invasions that generate lethal joint molecules. Second, Srs2 helicase dismantles toxic intermediates that have already formed. We also demonstrate that the structure-specific endonucleases, Mus81 and Yen1, can resolve toxic joint molecules formed in the absence of Srs2, thus promoting cell survival. The other goal of this thesis was to study the effects of ssDNA accumulated during BIR in the formation of base-substitution mutagenesis. I test whether this ssDNA is mutagenic by analyzing BIR with and without the presence of DNA damaging agents, including methyl methanesulfonate (MMS) and APOBEC3A. I observed a hypermutagenic effect of BIR with respect to base- substitutions in both cases. Importantly, BIR synergizes with ssDNA damaging agents to produce mutation clusters similar to those previously observed in cancer. I also report the critical role translesion polymerase Polζ plays in the formation of base-substitutions resulting from BIR. Finally, I have discovered a completely novel, UNG1-dependent mechanism of supposed error-free bypasses of APOBEC-induced DNA lesions during BIR that promotes chromosomal rearrangements.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

If the battle of Bir Hakeim was but one of many feats of arms during the Second World War, it particularly marks the memory of "Fighting France", because the exploit of the Free French Forces occurred during a period in which the Allies were having few successes on the Pacific and Mediterranean fronts. Allied propaganda therefore celebrated this battle, thus attracting adherents to the cause of "Fighting France", and aiding general de Gaulle's French National Committee in obtaining recognition from the British and American governments. General de Gaulle's challenge was to keep France at war so that France would participate in the Allied victory, offsetting the Vichy's government's collaboration. The legendary epic of the Free French Forces at Bir Hakeim is an important part of the myth, and the reality, of "Fighting France". While the official memory of "Free France" still celebrates the unity of Frenchmen under the banner of "the Cross of Lorraine", the soldiers at Bir Hakeim could not forget the franco-french tensions they experienced.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

This thesis deals with a decompilation tool which converts low-level binary code to a high-level representation. This tool is being developed by AVG Technologies. The aim of this work is to design and implement a method for code structuring in the decompiler back-end. The designed method works by traversing the control-flow graph with matching of predefined patterns. It is not always possible to structure code using conditional statements and loops. Sometimes also goto statements must be used. The implemented solution is compared with the original solution in the decompiler. According to the results the new solution is faster, better tested, but in greater number of test cases generates invalid code. From the viewpoint of structuring the results are different and sometimes the code is structured better, but sometimes worse.

SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...

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