In a large Scottish family a high incidence of schizophrenia, bipolar disorder and major depressive disorder co-segregates with a balanced autosomal translocation (t(1;11)(q42.1;q14.3). The translocation disrupts Disrupted-in-Schizophrenia-1 (DISC1) and DISC2 on chromosome 1, and DISC1FP1 (Disrupted-in-Schizophrenia-Fusion-Partner-1), also known as Boymaw, on chromosome 11. DISC1 is a leading candidate gene for major mental illness and is involved in neurodevelopment and cellular signalling, whilst DISC2 and DISC1FP1 are apparently non-coding RNA genes that undergo alternative splicing and that are expressed in the brain. This thesis aimed to investigate putative mechanisms of pathogenesis that may result from the t(1;11), with the hope that pathogenic mechanisms identified in the t(1;11) pedigree might shed light upon mechanisms conferring risk for psychiatric illness in the wider population. Previous work had identified DISC1/DISC1FP1 chimeric transcripts in t(1;11)-family derived lymphoblastoid cell lines. The detected transcripts include CP60 and CP69 which encode DISC1 aa1-597 plus an additional 60 or 69 amino acids from DISC1FP1, respectively. In this thesis a novel DISC1/DISC1FP1 transcript, CP1, was identified in t(1;11) lymphoblastoid cell lines. The CP1 transcript encodes DISC1 aa1-597 plus one glycine. A truncated form of DISC1 comprising aa1-597 was previously suggested to be a putative product of the translocation and, as such, has been the focus of multiple studies. The identification of the CP1 species is of interest as it differs from DISC1 aa1-597, by only a glycine. As glycines are simple uncharged aa’s, it is likely that these two DISC species share similar properties. In vitro exogenous expression of the three DISC1/DISC1FP1 protein species in both COS-7 and primary neuron cultures revealed contrasting cellular phenotypes. CP1 showed a diffuse cellular localisation pattern with cells containing readily visible tubular mitochondria. This is indistinguishable from the staining pattern of DISC1 aa1-597, highlighting the high degree of similarity between these species. CP60 and CP69, however, appeared to be clustered in the perinuclear region of the cell. Initial staining attempts with MitoTracker Red to visualise mitochondria in CP60 and CP69 expressing cells resulted in fewer than 30% of cells being stained. In those that did stain, the mitochondria appeared clustered. The absence of MitoTracker Red staining in mitochondria may be due to the loss of the mitochondrial membrane potential, Δψm. The adoption of a co-staining protocol with antibodies for mitochondrial proteins enabled the visualisation of mitochondrial structure in all of the cells exogenously expressing CP60 and CP69. All of these mitochondria possessed a clustered morphology, with which CP60 and CP69 expression was substantially co-localised. To see if MitoTracker staining was perturbed, in t(1;11) lymphoblastoid cell lines, as may occur if the DISC1/DISC1FP1 chimeras are expressed endogenously, the fluorescence of MitoTracker Red staining was investigated by FACS. Pooled analysis of experimental replicates revealed a negative result, with MitoTracker Red staining in t(1;11) lymphoblastoid cell lines not differing from controls. These findings indicate a need for further research using the mitochondrial membrane potential, Δψm as a metric as this would enable variations in mitochondrial mass to be accounted for. Prior to my arrival, an expression microarray had been carried out on lymphoblastoid cell line cDNA to assess gene expression differences resulting from the t(1;11). In order to identify putative pathogenic mechanisms, I carried out functional enrichment analysis of the expression array data using multiple analysis programs. Several programs detected dysregulation of the cell cycle and enrichment of altered expression of genes involved in the immune response and inflammation in t(1;11) carriers. The use of a rare variant investigative paradigm in this thesis furthers understanding of the putative pathogenic mechanisms that might act to increase risk for psychiatric illness in t(1;11) carriers. Moreover, it may aid the biological understanding of the aetiology of psychiatric illness in the general population. As such, improved understanding of the mechanisms of risk in the t(1;11) pedigree may eventually lead to the development of better treatments. In the intervening time since some of the research for thesis was published, two studies have emerged that may serve to highlight potential mechanisms of pathogenic action mediated by CP60 and CP69 expression. It has recently been observed that WT-DISC1 couples to the adaptor protein TRAK1 and the mitochondrial membrane anchor Miro1, which are part of the mitochondrial transport complex (Ogawa et al, 2014; Norkett et al, 2016). Furthermore, the exogenous expression of CP60 impairs bidirectional mitochondrial trafficking (Norkett et al, 2016). This suggests that CP60 expression may impair interactions with TRAK1 and Miro1. Given the sequence homology between CP60 and CP69, mitochondrial transport deficits also likely arise with CP69 expression. It is therefore possible that the exogenously expressed CP60 and CP69 proteins could be docked on stationary mitochondria, which may contribute to the clustered expression patterns observed.

abstract: Random peptide microarrays are a powerful tool for both the treatment and diagnostics of infectious diseases. On the treatment side, selected random peptides on the microarray have either binding or lytic potency against certain pathogens cells, thus they can be synthesized into new antimicrobial agents, denoted as synbodies (synthetic antibodies). On the diagnostic side, serum containing specific infection-related antibodies create unique and distinct "pathogen-immunosignatures" on the random peptide microarray distinct from the healthy control serum, and this different mode of binding can be used as a more precise measurement than traditional ELISA tests. My thesis project is separated into these two parts: the first part falls into the treatment side and the second one focuses on the diagnostic side. My first chapter shows that a substitution amino acid peptide library helps to improve the activity of a recently reported synthetic antimicrobial peptide selected by the random peptide microarray. By substituting one or two amino acids of the original lead peptide, the new substitutes show changed hemolytic effects against mouse red blood cells and changed potency against two pathogens: Staphylococcus aureus and Pseudomonas aeruginosa. Two new substitutes are then combined together to form the synbody, which shows a significantly antimicrobial potency against Staphylococcus aureus (<0.5uM). In the second chapter, I explore the possibility of using the 10K Ver.2 random peptide microarray to monitor the humoral immune response of dengue. Over 2.5 billion people (40% of the world's population) live in dengue transmitting areas. However, currently there is no efficient dengue treatment or vaccine. Here, with limited dengue patient serum samples, we show that the immunosignature has the potential to not only distinguish the dengue infection from non-infected people, but also the primary dengue infection from the secondary dengue infections, dengue infection from West Nile Virus (WNV) infection, and even between different dengue serotypes. By further bioinformatic analysis, we demonstrate that the significant peptides selected to distinguish dengue infected and normal samples may indicate the epitopes responsible for the immune response. / Dissertation/Thesis / M.S. Biological Design 2013

As doenças neuromusculares incluem um grupo muito heterogêneo de patologias que atingem 1 em cada 1.000 indivíduos nascidos vivos. Dentre as doenças neuromusculares destacam-se as paraplegias espásticas hereditárias que acometem, aproximadamente, cerca de 1 em cada 10.000. As paraplegias espásticas hereditárias (PEH) são caracterizadas pela espasticidade e fraqueza muscular dos membros inferiores. São muito heterogêneas tanto em clínica como geneticamente. Diversas formas já foram descritas e a mais comum delas, acometendo por volta de 40% dos casos autossômicos dominantes, causada por mutações no gene SPAST (PEH do tipo 4 ou SPG4). Estudos de correlação genótipo: fenótipo têm mostrado que indivíduos da mesma família carregando a mesma mutação patogênica, podem ter quadro clínico muito distinto. A explicação para esta questão pode estar na procura por genes modificadores, no padrão de expressão, na análise proteômica (seja por ligantes a proteínas ou no dobramento das mesmas), ou em mecanismos epigenéticos. Além disso, em algumas formas observa-se uma diferença na porcentagem de pessoas afetadas de acordo com o sexo. Essa desproporção foi observada numa grande família de com PEH na qual existe um predomínio de afetados do sexo masculino. O objetivo do presente trabalho foi a análise de pacientes discordantes, ou seja, que possuam a mesma mutação, porém com quadro clínico discordante de uma grande família brasileira com SGP4. Para isso foi feito um estudo da abundância de transcritos (mRNA) e de genótipo (polimorfismos de base única) em relação a um fenótipo (sintomático ou assintomático). Os resultados sugerem que o principal sistema envolvido, que poderia explicar as diferenças entre os pacientes discordantes, é o sistema imune, com a principal atuação dos genes C2, HLA-DRB1 e LY6G6C. Esses genes podem ter papel protetor ou tóxico no desenvolvimento do quadro clínico dos pacientes analisados / The hereditary spastic paraplegia (HSP) is characterized by muscle weakness and lower limb spasticity. They are very heterogeneous both clinically and genetically. Several forms have been described and the most common one, affecting around 40% of autosomal dominant cases, is caused by mutations in the SPAST gene (HSP type 4 or SPG4). Genotype: phenotype correlation studies have shown that affected individuals from the same family, who carry the same pathogenic mutation, can have very distinct phenotypes. The underlying explanation behind this clinical heterogeneity may be found in the search for modifier genes, in expression patterns observed proteomic analyses (either by protein binding or folding), or epigenetic mechanisms. As is observed in other motor neurodisease, there is a disproportion between the number of affected males and females, with males being the predominantly affected. The objective of this study was to analyze discordant patients, i.e., those that possess the same mutation, but show discordant phenotypes, from a large Brazilian family with SGP4. For this study, the abundance of transcripts (mRNA) and genotype (single nucleotide polymorphisms) relative to a phenotype (symptomatic or asymptomatic) were analyzed. The results suggest that the main system involved, which could explain the differences between discordant patients, is the immune system, with the main activity of C2, LY6G6C and HLA-DRB1 genes. These genes may have a protective or toxic role in the development of the analyzed patients\' clinical features

Made available in DSpace on 2014-06-12T15:59:34Z (GMT). No. of bitstreams: 2 arquivo5182_1.pdf: 7099391 bytes, checksum: 9ae548e6659db775935f03eac2fa2f35 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / Um dos principais objetivos da biologia molecular é descobrir o funcionamento de redes complexas de interação entre elementos celulares. Nas últimas décadas um grande volume de dados biológicos vem sendo produzido assim como modelos computacionais que fazem uso destes dados. Os métodos computacionais para Reconstrução de Redes de Genes apresentam-se como uma ferramenta importante para auxiliar no estudo e entendimento desta complexidade. Este trabalho apresenta uma proposta para Reconstrução de Redes de Genes que utiliza-se de diferentes fontes de dados e incorpora conhecimento biológico com o objetivo de melhorar a qualidade da inferência. Comparou-se a abordagem proposta com um trabalho anterior. Foram realizados experimentos com dados artificiais e dados reais de S. cerevisiae. O modelo proposto apresentou melhores resultados que o anterior em todos os critérios de avaliação para experimentos com dados artificiais. Na avaliação com dados reais a nova abordagem apresentou uma pequena melhora em apenas uma das configurações testadas

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

No description available.

Magister Scientiae - MSc / Autism is a pervasive developmental disorder (PDD) that's incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman® SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman® study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples. / South Africa

A pipeline was developed for the quantitative screening and sequence management of clones from suppression subtractive hybridization (SSH) cDNA libraries. The pipeline is particularly useful for gene discovery in non-sequenced organisms, and was illustrated with SSH library data from pearl millet (Pennisetum glaucum) and cowpea (Vigna unguiculata) and Arabidopsis (Arabidopsis thaliana) ecotype Kil-0. The objective of each library was to identify stress-response genes. cDNA microarrays provide a high-throughput screening method. Accordingly, these SSH libraries were amplified by PCR and spotted onto glass microarray slides. Subtracted and un-subtracted cDNA samples, that were used to construct the SSH libraries were prepared as Cy3- and Cy5-labeled targets and hybridized to the microarrays. The R package SSHscreen version 2.0.0, available from http://microarray.up.ac.za/SSHscreen/, was developed to analyze the resulting microarray data using limma (linear models for microarray data) functions. Commonly, loess normalization is used for within-slide normalization, however this is based on the assumption that most of the genes on the array are not differentially expressed. This is legitimate for most whole genome microarray experiments, however it is not appropriate when the array is constructed from an SSH library which is enriched for differentially expressed genes. Therefore, control spot-based normalization was used in the SSHscreen analysis. Empirical Bayes methods were employed to calculate the moderated t-statistic using functions from the limma package. This procedure in effect borrows information from the ensemble of genes to aid with inference about individual genes, taking advantage of the parallel structure whereby the same model is fitted to the data for each gene. In the Arabidopsis, pearl millet and cowpea forward libraries, 18%, 58% and 58% of the clones were identified as significantly up-regulated (adjusted p-value < 0.05) and in the reverse libraries, 18%, 30% and 28% significantly down-regulated, respectively. SSHscreen analysis was used to assist in selection of clones for sequencing. The SSHscreen data output (ranked gene lists in terms of differential expression), as well as the selected sequences in FASTA format were uploaded to SSHdb. For the Arabidopsis library, 114 out of the 262 sequenced clones (55%) were identified as unique/non-redundant; and for the pearl millet and cowpea libraries respectively, 37% and 33% of the sequenced clones were unique. SSHdb was developed as a web-based tool for sequence management and annotation of clones in SSH libraries and can freely be accessed at http://sshdb.bi.up.ac.za. BLAST analysis that was carried out when sequences were uploaded to SSHdb was used to combine clones with the same sequence into redundant partner groups, as well as identify putative annotations for each group. Individual clones from the abovementioned SSH libraries were selected and an independent technique, quantitative PCR, was used to validate the microarray/SSHscreen results. The pipeline was applied successfully to Arabidopsis, pearl millet and cowpea SSH cDNA libraries. Interesting genes in each case were identified for further study. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

Introduction: Knee flexion contractures have a negative impact on function for patients with osteoarthritis (OA). Those with contracture treated with total knee arthroplasty (TKA) have more post-operative pain and worse outcome. Little knowledge is available about patient demographic factors or gene expression in the knee joint capsule in the setting of contracture and severe OA. // Methods: Subjects with primary severe knee OA awaiting a TKA were recruited. We collected subject demographic factors that may be associated with preoperative knee contracture. Subjects’ posterior knee capsule was harvested intraoperatively. Capsule histological analysis was performed using light microscopy. Gene expression analysis was performed using whole genome microarray and immunohistochemistry was used for protein production analysis comparing those with contracture to those without. // Results: Twenty subjects were recruited for the demographics portion of the study (13 contractures and 7 controls), and capsules from 12 subjects (6 contractures, 6 controls) were used for histology, microarray, and IHC analyses. Contracture subjects had longer duration of OA, reduced extension in the contralateral knee, and showed a trend toward elevated body mass index. Tissue cross-sectional areas of adipose, non-adipose and synovial tissues were not statistically different histologically between the two groups. There was increased expression in the contracture group for the genes CHAD, Cyr61, and Sox9. There was a corresponding increase in protein production for CHAD and Sox9. // Conclusions: Screening for OA duration and bilateral knee range of motion (ROM) could be functionally beneficial. When a knee joint contracture is present, correcting for the resulting leg length discrepancy pre- and post-operatively could improve patient outcome. Gene protein products linking capsular cells to the ECM can influence capsular fibrosis and potentially impact ROM.

Microarray technology makes it possible to identify changes in gene expression of an organism, under various conditions. The challenge to researchers that employ microarray expression profiling is that once pre-processing is completed, and a cluster of co-expressed genes obtained, is to derive biological meaning from this data. Data mining is thus essential for deducing significant biological information such as the identification of new biological mechanisms or putative drug targets. While many algorithms and software have been developed for analysing gene expression, the extraction of relevant information from experimental data is still a substantial challenge, requiring significant time and skill. MADIBA (MicroArray Data Interface for Biological Annotation) facilitates the assignment of biological meaning to gene expression clusters by automating the post-processing stage. A relational database has been designed to store the data from gene to pathway for Plasmodium falciparum, Oryza sativa (rice), Arabidopsis thaliana, and Pectobacterium atrosepticum (Pba) As input, the user submits a cluster of genes, either the gene identifiers or the gene sequences. Tools within the web interface allow rapid analyses for the identification of the Gene Ontology terms relevant to each cluster; visualising the metabolic pathways where the gene products are implicated, their genomic localisations, putative common transcriptional regulatory elements in the upstream sequences, and an analysis specific to the organism being studied. The user has the option of outputting selected results of the analyses, either in PDF or plain text formats. MADIBA is an integrated, online tool that will assist researchers in interpreting their results and understand the meaning of the co-expression of a cluster of genes. Functionality of MADIBA was used to analyse a number of gene clusters from several experiments – expression profiling of the Plasmodium falciparum life cycle, a Ralstonia solanacearum infection ofArabidopsis thaliana, a rice treatment with BTH, a millet SA- and MeJ-treatment experiment, and an expI mutant experiment in Pectobacterium atrosepticum. Data from the Plasmodium falciparum and rice were used to illustrate MADIBA’s functionality. For the A. thaliana analyses, the DRASTIC database was implemented to identify how genes respond to various treatments. In addition, a method named PCA Experiment Comparer was developed, which compares the expression values of the numerous experiments in NASCArrays. Using the A. thaliana-R. solanacearum interaction data several related experiments matched in both the susceptible and resistant interactions. In the millet analyses, besides defence related genes being identified, several genes also involved in photosynthesis were found, possibly suggesting a relation between light and defence signalling. The Pba data identified genes involved in quorum sensing, as well as some associated genes with no known function that may also be related to this regulatory process. With the advent of whole genome microarray chips and an increasing number of organisms being sequenced, tools such as MADIBA will become even more significant in understanding the underlying biology. MADIBA provides access to several genomic data sources and analyses, allowing users to quickly annotate and visualise the results. MADIBA is freely available and can be accessed at http://www.bi.up.ac.za/MADIBA/. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Biochemistry / unrestricted