Existence of optimal controls for a class of hereditary systems with lagging control

In this paper the problem of existence of optimal controls for a class of time lag systems is considered. It is schown that Oğuztöreli's results (Oğuztöreli, the 8.1, p. 184, “Time Lag Control Systems,≓ Academic Press, New York, 1966) can be extended to a class of time lag systems whose “phase velocity≓ depends also on the past history of control

Genotype and phenotype characterisation of Friedreich ataxia mouse models and cells

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel UniversityFriedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced level of frataxin protein. Normal individuals have 5 to 40 GAA repeat sequences, whereas affected individuals have approximately 70 to more than 1000 GAA triplets. Frataxin is a mitochondrial protein involved in iron-sulphur cluster and heme biosynthesis. The reduction in frataxin expression leads to oxidative stress, mitochondrial iron accumulation and consequential cell death with the primary sites of neurons of the dorsal root ganglia and the dentate nucleus of the cerebellum. FRDA, which is the most common inherited ataxia, affecting 1:50,000 Caucasians, is characterised by neurodegeneration, cardiomyopathy, diabetes mellitus and skeletal deformities. To investigate FRDA molecular disease mechanisms and therapy, several human FXN YAC transgenic mouse models have been established: Y47R, containing normal-sized (GAA)9 repeats; YG8R and YG22R, which initially contained expanded GAA repeats of 90-190 units and 190 units, respectively, but which have subsequently been bred to now contain expanded GAA repeats of 120-220 units and 170-260 units, respectively, and YG8sR (YG8R with a small GAA band) that was recently generated from YG8R breeding. To determine the FXN transgene copy number in the enhanced GAA repeat expansion-based FRDA mouse lines, a TaqMan qPCR assay was developed. The results demonstrated that the YG22R and Y47R lines had a single copy of the FXN transgene while the YG8R line had two copies. The YG8s lines showed less than one copy of the target gene, suggesting potential deletion of the FXN gene. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. However, in the YG8s line, at least 25% of the YG8s cells had no signals, while the remaining cells showed one signal corresponding to the transgenic FXN gene. In addition, the analysis of FXN exons in YG8s rescue mice by PCR confirmed the presence of all FXN exons in these lines, suggesting the incidence of somatic mosaicism in these lines. Extended functional analysis was carried out on these mice from 4 to 12 months of age. Coordination ability of YG8R, YG8sR and YG22R ‘FRDA-like’ mice, together with Y47R and C57BL6/J wild-type control mice, was assessed using accelerating rotarod analysis. The results indicated a progressive decrease in the motor coordination of YG8R, YG22R and YG8sR mice compared to Y47R or C57BL6/J controls. Locomotor activity was also assessed using an open field beam-breaker apparatus followed by four additional functional analyses including beam-walk, hang wire, grip strength and foot print tests. The results indicated significant functional deficits in the FRDA mouse models. Glucose and insulin tolerance tests were also conducted in the FRDA mouse models, indicating glucose intolerance and insulin hypersensitivity in the aforementioned lines. To investigate the correlation between the FRDA-like pathological phenotype and frataxin deficiency in the FRDA mouse models, frataxin mRNA and protein levels as well as somatic GAA repeat instability were examined. The results indicated that somatic GAA repeats increased in the cerebellum and brain of YG22R, YG8R and YG8sR mice, together with significantly reduced levels of FXN mRNA and protein in the liver of YG8R and YG22R compared to Y47R. However, YG8sR lines showed a significant decrease in FXN mRNA in all of the examined tissues compared to Y47R human FXN and C57BL6/J mouse Fxn mRNA. Protein expression levels were also considerably reduced in all the tissues of YG8sR mice compared to Y47R. Subsequently, the telomere length of human and mouse FRDA and control fibroblasts was assessed using qPCR and Q-FISH. The results indicated that the FRDA cells had chromosomes with relatively longer telomeric repeats in comparison to the controls. FRDA cells were screened for expression of telomerase activity using the TRAP assay and a quantitative assay for hTERT mRNA expression using TaqMan qRT-PCR. The results indicated that telomerase activity was not present in the FRDA cells. To investigate whether FRDA cells maintained their telomeres by ALT associated PML bodies (APBs), co-localisation of PML bodies with telomeres was assessed in these cells using combined immunofluorescence to PML and Q-FISH for telomere detection. The results demonstrated that the FRDA cells had significantly higher co-localised PML foci with telomeric DNA compared to the normal cells. Moreover, telomere sister chromatid exchange (T-SCE) frequencies were analysed in the human FRDA cell lines using chromosome orientation FISH (CO-FISH). The results indicated a significant increase in T-SCE levels of the FRDA cell lines relative to the controls. Furthermore, growth curve and population doubling analysis of the human FRDA and control fibroblasts was carried out. The results showed that the FRDA fibroblast cell cultures underwent growth arrest with higher cumulative population doubling compared to the controls. Though, further analysis of telomere length at different passage numbers revealed that the FRDA cells lost telomeres faster than the controls. Finally, the telomere dysfunction-induced foci (TIF) assay was performed to detect DNA damage in the human FRDA fibroblast cells using an antibody against DNA damage marker γ-H2AX and a synthetic PNA probe for telomeres. The frequency of γ-H2AX foci was significantly higher in the FRDA cells compared to the controls. Similarly, the FRDA cells had greater frequencies of TIFs in comparison to the controls, suggesting induced telomere dysfunction in the FRDA cells

Attempts to quantify mitochondrial DNA deletions in Single Drosophila melanogaster flies

Research to date has not clearly described the role mitochondrial DNA (mtDNA) deletions may have in normal ageing. Therefore, a high throughput method of mtDNA deletion detection and quantification is required. The goal of this project was to develop such an assay using individual Drosophila melanogaster, which allowed for rapid generation of aged animals and manipulation of conditions which could affect deletion generation. An assay composed of DNA extraction and Quantitative Polymerase Chain Reaction (QPCR) was designed for deletion amplification. Primers were designed to amplify deletions within the cytochrome oxidase (COX) region with primers in nadh ubiquinone oxidoreductase 1 (ND1) used as a control to quantify total mtDNA. Optimising QPCR methods for specific primer pairs improved target amplification and replicability. Redesign of an existing DNA extraction kit improved overall mtDNA yield for assay development but still lacked consistency. QPCR inhibitors present in commercial extraction kits were present in minor concentrations in extracts and likely impacted amplification efficiency. The lack of sufficient mtDNA extraction from single Drosophila for consistent deletion amplification lead to mispriming and nonspecific amplification of nuclear DNA (nDNA). Repeated amplification of one deletion across multiple extracts suggests QPCR preferentially amplifies the shortest available target sequence, corresponding to the largest deletion. Redesign of the DNA extraction method to yield higher mtDNA concentration whilst reducing inhibitors would assist in reducing nonspecific amplification. mtDNA enrichment may be required to remove nDNA if nonspecific amplification still occurs. Differential amplification of deletions depending on size renders comparison and quantification difficult. Targeted amplification of a specific common deletion may eliminate this issue at the cost of quantifying just one deletion. The likelihood of single Drosophila harbouring sufficient numbers of a specific deletion should be determined to assess if quantification of mtDNA deletion levels in single Drosophila is viable

Mitochondrial Translation and Beyond: Processes Implicated in Combined Oxidative Phosphorylation Deficiencies

Mitochondrial disorders are a heterogeneous group of often multisystemic and early fatal diseases, which are amongst the most common inherited human diseases. These disorders are caused by defects in the oxidative phosphorylation (OXPHOS) system, which comprises five multisubunit enzyme complexes encoded by both the nuclear and the mitochondrial genomes. Due to the multitude of proteins and intricacy of the processes required for a properly functioning OXPHOS system, identifying the genetic defect that underlies an OXPHOS deficiency is not an easy task, especially in the case of combined OXPHOS defects. In the present communication we give an extensive overview of the proteins and processes (in)directly involved in mitochondrial translation and the biogenesis of the OXPHOS system and their roles in combined OXPHOS deficiencies. This knowledge is important for further research into the genetic causes, with the ultimate goal to effectively prevent and cure these complex and often devastating disorders

Identification of Novel Causative Genes for Colorectal Adenomatous Polyposis

In up to 50% of families with clinically verified adenomatous polyposis no germline mutations in the established genes APC and MUTYH can be identified during routine diagnostics although the presence of high numbers of colorectal adenomas strongly argues for an underlying genetic cause, either as a monogenic or genetically complex trait. Therefore, the aim of this study was (i) to identify cryptic germline mutations in the APC gene which were not detected by routine diagnostics; (ii) to identify novel causative genes of adenomatous polyposis by a genome-wide SNP-array based CNV analysis, and (iii) to further evaluate the pathogenic relevance of the candidate genes by additional experiments including screening for germline point mutations in the patient cohort. Firstly, a functional study at the mRNA (transcript) level was carried out to look for deep intronic APC mutations. We identified aberrant transcript patterns in 8 (6%) of 125 unrelated patients. Five of them carried a founder germline mutation in intron 4 and three patients showed germline point mutations in intron 10, which lead to the inclusion of a pseudoexon 4 and a pseudoexon 10 on transcript level. The pseudoexons are predicted to result in frameshift mutations and premature stop codons. Therefore, a few deep intronic mutations contribute substantially to the APC mutation spectrum and cDNA analysis and/or target sequencing of intronic regions should be considered as an additional mutation discovery approach in polyposis patients. To uncover novel causative genes in patients with unexplained adenomatous polyposis, a genome-wide analysis of germline copy number variants (CNV) using high-resolution SNP arrays was performed in 221 unrelated, well characterized APC and MUTYH mutation negative German patients. Putative CNVs were filtered according to stringent criteria, compared with those of 531 population-based German controls, and validated by qPCR. 125 unique rare germline CNVs in 93 (42%) of 221 patients were identified. These CNVs involved 68 deleted and 168 duplicated genes. The vast majority of patients harbor one CNV only. To further evaluate the pathogenic relevance of the candidate genes, additional filtering and prioritization steps on gene level including expression analysis in cDNA from human colon tissue, network analysis, enrichment analyses of genes and pathways, and data mining were performed. Ninety-eight candidate genes remained, 32 of which showed molecular and cellular functions related to tumorigenesis. To further explore the clinical relevance of the candidate genes in the absence of recurrent alterations and lack of segregation information, a germline point mutation analysis was performed in a validation cohort using a targeted next generation sequencing (NGS) approach. Fifteen rare heterozygous truncating point mutations in 11 genes were identified in 15 patients. In these 11 genes, we found additional 27 rare missense mutations which were predicted to be deleterious. CNTN6 and FOCAD showed different truncating mutations in more than one patient whereas KIF26B has the highest frequency of potential deleterious mutations overall. By integrating all results and recent studies of early-onset colorectal and breast cancer, CNTN6, EPHB4, KIF26B, MCM3AP, FOCAD, and HSPH1were selectedas the most convincing predisposing genes for colorectal adenomatous polyposis. In addition, in the canonical Wnt pathway oncogene CTNNB1 (ß-catenin), two potential gain-of-function mutations were found. This thesis identified a group of rarely affected genes which are likely to predispose to colorectal adenoma formation and confirmed previously published candidates for tumor predisposition as etiologically relevant. Our analysis demonstrated that the underlying genetic factors of unexplained colorectal polyposis are likely to be very heterogeneous, which makes clinical validation challenging. To further characterize the functional relevance of the selected genes, international collaborations with large patient cohorts and functional studies are needed

The Development of a Dual-Tag Affinity Purification System and its Application to Elucidate the Interacting Protein Network Surrounding the Human Telomere Binding Proteins TRF1, TRF2, and POT1

Protein-protein interactions (PPI) play a vital role in almost every cellular process. Although many methodologies exist to probe PPIs, one of the most successful and widely employed is tandem affinity purification coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS). Although best demonstrated in yeast, TAP has encountered significant hurdles in its application to mammalian systems, especially the observed low yield of bait protein and its interacting partners after two consecutive purifications. To address these issues, a novel dual-tag affinity purification (DAP) system was developed that not only enhances bait protein recovery, but also allows for rapid evaluation of dual-tag compatibility with a protein of interest based on its known subcellular localization. In addition, several tags of varying composition were constructed to allow for maximal bait protein compatibility. With this system, mammalian bait protein yield was improved by more than 200% relative to previously published results. Capitalizing on this success, DAP was applied to human telomere binding proteins TRF1, TRF2, and POT1 to garner a greater understanding of the protein networks that involve the telomere. Expectedly, all the members of the telosome complex were identified at frequencies that lend evidence towards the currently accepted architecture. Also identified were several other novel proteins and subcomplexes that may enhance our understanding of telomere maintenance / length regulation. For instance, members of the classical nuclear import system co-purified with both TRF1 and TRF2. Although previously documented for TRF1, TRF2’s association with importin alpha (KPNA2) and beta (KPNB1) has not been demonstrated till now. Interestingly, further study revealed that KPNA2 acts as a negative regulator of TRF2 nuclear localization. This observation could have far-reaching implications as TRF2 is thought to be also heavily involved in the DNA damage response. Along these lines, a more indepth MS analysis revealed several putative phosphorylation sites along TRF2’s sequence. One site, pS380, seems to be phosphorylated by the DNA-damage kinase ATM and plays a role in a cell’s proliferative capacity, possibly affecting telomere length regulation. The studies contained here within demonstrate the efficacy of DAP-LC-MS/MS to provide useful leads with regards to the study of PPIs