<p>Monogenic disorders offer a possibility for studies of genetic disturbances in hearing impairment—a knowledge which could be essential for development of future treatment options. In this thesis, the underlying genetic disturbances in neurofibromatosis 2 (NF2) and familial Meniere’s disease (FMD) were evaluated, and familial X-linked hearing impairment was described from a clinical point of view. </p><p>In paper I, constitutional DNA from 116 individuals with NF2 of variable severity was studied using the array-CGH method focusing on a 7.6-Mb area surrounding the NF2 gene on chromosome 22q. Deletions were found in 20.7% of samples. In mild NF2, the deletions were small, but variable sizes of deletions were found in cases that were moderately or severely affected. Disease phenotype could not be predicted from the size of the deletions.</p><p>In papers II and III, a single five-generation family with autosomal dominant FMD was described. Anticipation concerning age of onset was observed. Genome scan revealed five candidate gene regions with a LOD score of > 1. Two additional families with autosomal dominant MD were analyzed for linkage to these five regions. A cumulative Zmax of 3.46 was obtained for a single 463-kb region on chromosome 12p12.3, containing only one known gene: PIK3C2G. This encodes a protein with a proposed role in hair cell regeneration in mammalian ears. No mutations were found in protein-coding sequences or exon-intron borders. In two of the three families, a shared haplotype, suggested common ancestry, was found to extend over 1.7 Mb, which could be a genomic region of importance for FMD.</p><p>In paper IV, a family in which five males displayed progressive low- and mid-frequency hearing impairment from the first or second decade was described. Female carriers were affected by a high-frequency hearing impairment from the fourth decade. The family could represent a novel X-linked dominant audiophenotype.</p>

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.

The nuclear matrix (NM) is proposed to be a permanent network of core filaments underlying thicker fibres, present regardless of transcriptional activity. It is found to be both RNA and protein rich; indeed, numerous important nuclear proteins are components of the structure. In addition to mediating the organisation of entire chromosomes, the NM has also been demonstrated to tether telomeres via their TTAGGG repeats. In order to examine telomeric interactions with the NM, a technique known as the DNA halo preparation has been employed. Regions of DNA that are tightly attached to the structure are found within a so-called residual nucleus, while those sequences forming lesser associations produce a halo of DNA. Coupled with various FISH methodologies, this technique allowed the anchorage of genomic regions by the NM, to be analysed. In normal fibroblasts, the majority of chromosomes and telomeres were extensively anchored to the NM. Such interactions did not vary significantly in proliferating and senescent nuclei. However, a decrease in NM-associated telomeres was detected in quiescence. Since lamin A is an integral component of the NM, it seemed pertinent to examine chromosome and telomere NM-anchorage in Hutchinson-Gilford Progeria Syndrome (HGPS) fibroblasts, which contain mutant forms of lamin A. Indeed, genome tethering by the NM was perturbed in HGPS. In immortalised HGPS fibroblasts, this disrupted anchorage appeared to be rescued; the implications of this finding will be discussed. This study also suggested that telomere-NM interactions are aberrant in X-linked Emery-Dreifuss Muscular Dystrophy (X-EDMD), which is caused by mutant forms of emerin, another NM-associated protein. The positioning of selected genes in control and X-EDMD cell lines was examined in un-extracted nuclei using 2D and 3D FISH. Subtle shifts in the organisation of these genes were detected in diseased cells; however, their expression levels remained unaltered. Furthermore, in order to examine the architectural integrity of the nuclear lamina in lamin A and emerin mutant cell lines, scanning electron microscopy (SEM) was employed. This work revealed that such structures were indeed compromised in disease. The findings presented in this thesis highlight the importance of lamin A and emerin in mediating the organisation of the genome and taken together, promote the hypothesis that dysfunctional NM dynamics may well contribute to disease pathology.

Intellectual disability is a prevalent developmental disorder for which no effective treatments are available. Mutations of the X-linked protein PHF6 cause the Börjeson-Forssman-Lehmann syndrome (BFLS) that is characterized by intellectual disability and epilepsy. However, the biological role of PHF6 relevant to BFLS pathogenesis has remained unknown. Here, I present my dissertation research demonstrating that knockdown of PHF6 profoundly impairs neuronal migration in the mouse cerebral cortex in vivo, leading to the formation of white matter heterotopias that harbor aberrant patterns of neuronal activity. Importantly, BFLS patient specific mutation of PHF6 blocks its ability to promote neuronal migration. I also elucidate the mechanism by which PHF6 drives neuronal migration in the cerebral cortex. PHF6 physically associates with the PAF1 transcription elongation complex, and inhibition of PAF1 phenocopies the PHF6 knockdown-induced migration phenotype in vivo. I further identify Neuroglycan C (NGC), a susceptibility gene for schizophrenia, as a critical downstream target of PHF6 and the PAF1 complex, and I demonstrate that NGC mediates PHF6-dependent neuronal migration. These findings define PHF6, the PAF1 transcription elongation complex, and NGC as components of a novel cell-intrinsic transcriptional pathway that orchestrates neuronal migration in the brain, with important implications for the pathogenesis of intellectual disability and potentially other neuropsychiatric disorders.

No description available.

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.

Monogenic disorders offer a possibility for studies of genetic disturbances in hearing impairment—a knowledge which could be essential for development of future treatment options. In this thesis, the underlying genetic disturbances in neurofibromatosis 2 (NF2) and familial Meniere’s disease (FMD) were evaluated, and familial X-linked hearing impairment was described from a clinical point of view. In paper I, constitutional DNA from 116 individuals with NF2 of variable severity was studied using the array-CGH method focusing on a 7.6-Mb area surrounding the NF2 gene on chromosome 22q. Deletions were found in 20.7% of samples. In mild NF2, the deletions were small, but variable sizes of deletions were found in cases that were moderately or severely affected. Disease phenotype could not be predicted from the size of the deletions. In papers II and III, a single five-generation family with autosomal dominant FMD was described. Anticipation concerning age of onset was observed. Genome scan revealed five candidate gene regions with a LOD score of &gt; 1. Two additional families with autosomal dominant MD were analyzed for linkage to these five regions. A cumulative Zmax of 3.46 was obtained for a single 463-kb region on chromosome 12p12.3, containing only one known gene: PIK3C2G. This encodes a protein with a proposed role in hair cell regeneration in mammalian ears. No mutations were found in protein-coding sequences or exon-intron borders. In two of the three families, a shared haplotype, suggested common ancestry, was found to extend over 1.7 Mb, which could be a genomic region of importance for FMD. In paper IV, a family in which five males displayed progressive low- and mid-frequency hearing impairment from the first or second decade was described. Female carriers were affected by a high-frequency hearing impairment from the fourth decade. The family could represent a novel X-linked dominant audiophenotype.

The dystrophinopathies are a group of disorders characterised by cellular absence of the membrane stabilising protein, dystrophin. Duchenne muscular dystrophy is the most severe disorder clinically. The deficiency of dystrophin, in the muscular dystrophy X-linked (mdx) mouse causes an elevation in intracellular calcium in cardiac myocytes. Potential mechanisms contributing to increased calcium include enhanced influx, sarcoplasmic reticular calcium release and\or reduced sequestration or sarcolemmal efflux. This dissertation examined the potential mechanisms that may contribute to an intracellular calcium overload in a murine model of muscular dystrophy. The general cardiomyopathy of the mdx myocardium was evident, with the left atria from mdx consistently producing less force than control atria. This was associated with delayed relaxation. The role of the L-type calcium channels mediating influx was initially investigated. Dihydropyridines had a lower potency in contracting left atria corresponding to a redued dihydropyridine receptor affinity in radioligand binding studies of mdx ventricular homogenates (P<0.05). This was associated with increased ventricular dihydropyridine receptor protein and mRNA levels (P<0.05). The function of the sarcoplasmic reticulum in terms of release and also sequestration of calcium via the sarco-endoplasmic reticulum ATPase were investigated. A lower force of contraction was evident in mdx left atria in response to a range of stimulation frequencies (P<0.05) and concentrations of extracellular calcium (P<0.05). However, in the presence of 1 nM Ryanodine to block sarcoplasmic reticular calcium release, increased stimulation frequency caused similar forces to those obtained in control mice suggesting enhanced calcium influx via L-type calcium channels in mdx. Rapid cooling contractures showed a reduced contracture in mdx compared to control in response to cooling. This suggests some dysfunction in SR storage, which may be associated with the delayed relaxation time. Concentration-response curves to inhibitors of the sarco-endoplasmic reticulum showed no difference in function of the enzyme responsible for calcium uptake into the sarcoplasmic reticulum. Although sarco-endoplasmic reticulum ATPase mRNA was upregulated, no functional benefit was evident. This study indicates that a deficiency of dystrophin leads to upregulation of L-type calcium channels that contribute to increased calcium influx, with no functional change in sarcoplasmic reticular sequestration. Upregulation of the influx pathway is a potential mechanism for the calcium overload observed in mdx cardiac muscle.

Thesis (Ph.D.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2008/02/12). Includes bibliographical references.