Automated lane detection of gel electrophoresis image using false peak elimination / Ros Surya Taher

Large numbers of previous work regarding the study of lane detection in DNA gel image have been proposed and performed on good quality images. Current lane detection methods that are available do not accommodate techniques that can be performed automatically on poor DNA gel image. Lane detection is the first step in any gel image analysis techniques which involved tedious and time-consuming tasks. The accuracy of this step is often compromised by technical variation inherent to DNA gel image. For that reason, the aim of this thesis is to identify and propose a method that is effective in detecting the lane in poor DNA gel image of plants. The imperfection of DNA gel image caused by the electrophoresis or during the acquisition of the gel image causes many types of noises, which contaminate the resulting image. These errors and noises significantly affect the processing and analysis of the DNA gel image. The conducted experiment examines 184 poor DNA gel images collected from Agrobiodiversity and Environment Research Centre, Institut Penyelidikan dan Kemajuan Pertanian Malaysia (MARDI), Malaysia. The DNA gel images were produced by electrophoresis-based method using polymerase chain reaction (PCR)-based marker system. There are two highlighted aspects performed to achieve the objective of this thesis that are image enhancement and lane detection. The image enhancement of the poor DNA gel image is performed using two different approaches that are spatial and frequency filtering. The two approaches are compared and the quality of the enhanced images was accessed and evaluated using objective image quality metric that is peak signal-to-noise ratio (PSNR). For lane detection, we describe the convention of threshold value in the analysis of poor DNA gel image to eliminate false peak contained in the intensity profile obtained from the enhanced image data projection

Mathematical modeling of brain tumor cell growth for passive, active and oxygen transport mechanism with microgravity condition / Norfarizan Mohd Said

The unpredictable conduct of the brain tumor cells present difficulties in creating precise models. The limitation of medical imaging in forecasting the nature of the tumor growth and the costly techniques for diagnostic and treatment posed an obstacle to the effort in understanding and fighting this life-threatening disease. As the tumor itself can only be detected and treated through the biological process, a good mathematical model should represent the important biological aspects with useful solution that contribute to further understanding of the problem. Addressing the current challenges in developing a realistic model by bridging the theoretical with the clinical applications, this research aims to govern mathematical models for brain tumor cell growth by emphasizing the cell migration and proliferation as the key characteristics. The models of passive and active cell mechanisms are representing the tumor cell migration while the model of oxygen transport mechanism configures the cell proliferation. New parameters for oxygen and gravity effects are included as the model novelty. The conditions of microgravity and oxygen deprivation are presented using the microscopic model of the tumor cellular dynamics. The models developed are in the form of parabolic equations which is discretized using the Finite Difference Method (FDM) with weighted average approximation. Numerical iterative methods, namely Jacobi (JAC), Red-Black Gauss-Seidel (RBGS), Red-Black Successive Over Relaxation (RBSOR) and Alternating Group Explicit (AGE) method are used to solve the discretized models

Nanoparticle-assisted Polymerase Chain Reaction (NanoPCR): Optimization of PCR detection of Leifsonia xyli subsp. xyli by the addition of nanoparticles

Leifsonia xyli subsp. xyli (Lxx) causes ratoon stunting disease (RSD) in sugarcane, and is one of major causes of production losses. The detection of Lxx bacteria in sugarcane is made mainly through molecular biology techniques, especially polymerase chain reaction (PCR). However, PCR presents some barriers to provide reliable results. The present work brings a Nanoparticle-assisted Polymerase Chain Reaction (NanoPCR) assay for the detection of Lxx in its latent infection on micropropagated sugarcane. This assay was based in the addition of Gold and Titanium dioxide nanoparticles to conventional PCR and evaluation of its effects. It was observed that the reactions performed with Titanium dioxide nanoparticles provided the formation of singular well-defined bands under electrophoresis, consistent with the expected molecular weight, without occurrence of non-specific bands or presenting false negatives occurrence, negative effects that were observed in the control assay. While the performed NanoPCR adding AuNP also provided the formation of well-defined bands, been able to inhibit the occurrence of false negatives, but wasn’t able to eliminate the occurrence of non-specific amplifications. The results indicate that NanoPCR by the addition of Gold and Titanium dioxide nanoparticles to conventional PCR increased the detection of Lxx

Gold nanoparticles-based sensors for detection of mycobacterium tuberculosis genomic DNA

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB), is an airborne disease that strikes one third of the globe’s population. In addition to infection of 9.6 million patients, TB claimed the lives of 1.5 million people in 2014 only. The majority of TB patients are present in the third world where the balance between cost-effective diagnostic method and prevalence of TB is difficult to achieve. Accurate diagnosis of TB is necessary to timely initiation of treatment. The available diagnostic tools are slow, while the rapid methods are either inaccurate or relatively unaffordable. So, sometimes the diagnosis is presumptive based on the clinical findings and the treatment is empiric. The treatment is lengthy and demands the administration of multiple antibiotics. However, the emergence of drug resistance threatened the global control programs of TB. The objective of this work is to develop cheap, fast and accurate detection methods. Two gold nanoparticles (AuNPs) based sensors were developed for colorimetric and fluorometric detection of MTB. Seventy two anonymous sputum samples were cultured then DNA was extracted. MTB H37Ra was the positive control while M. smegmatis and 8 non-MTB and negative controls. Characterization of the samples was achieved by multiplex PCR using MTB and NTM specific primers. Random samples were amplified by 16S-23S ITS primers and sequenced. Drug resistance associated mutations of MDR-TB were identified by MAS-PCR. The colorimetric assay aim was the detection of amplified MTB DNA by cationic AuNPs. The samples were amplified by IS6110 and rpoB primers. Only MTB samples yielded amplicons. So the negatively charged dsDNA attracted the positively charged AuNPs inducing their aggregation and the color turned blue. While the negative samples did not yield any amplicons and the AuNPs remained dispersed so the color was red. The sensitivity and specificity was 100% and the detection limit was 5.4 ng/μl of MTB DNA. The fluorometric assay exploited the quenching property of 40 nm AuNPs. The unamplified DNA was fragmented in the presence of 16s rDNA specific probe tagged with the fluorophore CY-3 by sonication and denatured for 3 min at 95 ºC followed by annealing at 52ºC for 45 sec. Then AuNPs were added and the fluorescence was measured. By FRET, the relative fluorescence was calculated revealing a cut-off value of 3. In MTB samples, the CY3-16s rDNA specific probe hybridized with its target and became spaced from the AuNPs allowing high fluorescence to be detected. Due to the lack of target-probe hybridization in the negative samples, the AuNPs were adsorbed on the probe and thus the fluorescence is quenched. Thirteen samples were chosen randomly, amplified and sequenced. Sequencing confirmed that 12/13 samples were MTB with 100% concordance with the multiplex PCR and FRET. The assay had sensitivity and specificity of 98.6% and 90% respectively and concordance of 98% with multiplex PCR. The detection limited was calculated to be 10 ng/ul. In conclusion, two AuNPs based sensors were developed to allow low cost and rapid detection of MTB on low source settings. The assays are rapid, sensitive and can have great potential in clinical practice for TB diagnosis

Cross-Sample Validation Provides Enhanced Proteome Coverage in Rat Vocal Fold Mucosa

The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high Mr ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high Mr glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories

Biomarkers of Lung Cancer Risk and Progression

Lung cancer causes high mortality because most people present late with advanced disease that is not amenable to curative treatment. Screening high-risk groups with low dose CT imaging of the thorax has been shown to reduce lung cancer mortality by 20%, but at the cost of a high false positive rate. Population stratification with molecular biomarkers could improve the cost-benefit of lung cancer screening programmes and reduce false positives. Tumour cells shed DNA into the blood, enabling tumour-derived genetic alterations to be detected non-invasively by analysing circulating cell-free DNA (cfDNA). The aim of this study was to determine the screening and prognostic potential of total cfDNA levels and two genomic instability scores based on the detection of copy number aberrations in cfDNA samples of lung cancer cases and controls collected in the ReSoLuCENT study (A Resource for the Study of Lung Cancer Epidemiology in North Trent). Controls were identified as low or high risk for the development of lung cancer over five years using the Liverpool Lung Project risk model. CfDNA was extracted from the plasma of 52 untreated lung cancer cases, 32 high risk controls and 10 low risk controls and quantified total cfDNA levels by SYBR green real-time qPCR. Low coverage whole genome sequencing with Illumina HiSeq 2500 was completed for a subset of cases (N=62) and controls (N=40). Two published genomic instability scores were adapted and tested; the plasma genomic abnormality (PGA2) and the copy number aberration (CNA) score. Screening potential was evaluated by performing Receiver Operating Characteristic (ROC) curves to assess the ability of the test to discriminate between lung cancer cases and controls by calculating area under the curve (AUC). Logistic regression was used to further assess the ability of total cfDNA levels and genomic instability scores to predict case or control status. Prognostic value was determined by Kaplan Meir and Cox regression survival analyses. In this preliminary study, there was no difference in total cfDNA levels between early stage lung cancer cases and high risk controls. The PGA2 score was higher in high risk controls compared to lung cancer cases and was not further evaluated. In comparison, the CNA score had good discriminatory ability for high risk controls compared to all lung cancer cases (stage I-IV) with an AUC of 0.74 but poorer discriminatory ability for early stage cases (I-IIIA) with an AUC of 0.60. Although total cfDNA levels and CNA scores above the median value were associated with poor survival, both were statistically significant in univariable but not multivariable cox survival regression analyses. Therefore, total cfDNA levels and the CNA score had limited prognostic value when other factors were taken into account. Total cfDNA levels are not recommended as a screening tool because total levels lack specificity for cancer. The screening performance of the CNA score may be improved by targeting recurrent copy number aberrations and by combining the score with alternative tumour-derived genetic alterations in cfDNA such as point mutations or methylation changes

Single cell transcriptome analysis using next generation sequencing.

The heterogeneity of tissues, especially in cancer research, is a central issue in transcriptome analysis. In recent years, research has primarily focused on the development of methods for single cell analysis. Single cell analysis aims at gaining (novel) insights into biological processes of healthy and diseased cells. Some of the challenges in transcriptome analysis concern low abundance of sample starting material, necessary sample amplification steps and subsequent analysis. In this study, two fundamentally different approaches to amplification were compared using next-generation sequencing analysis: I. exponential amplification using polymerase-chain-reaction (PCR) and II. linear amplification. For both approaches, protocols for single cell extraction, cell lysis, cDNA synthesis, cDNA amplification and preparation of next-generation sequencing libraries were developed. We could successfully show that transcriptome analysis of low numbers of cells is feasible with both exponential and linear amplification. Using exponential amplification, the highest amplification rates up to 106 were possible. The reproducibility of results is a strength of the linear amplification method. The analysis of next generation sequencing data in single cell samples showed detectable expression in at least 16.000 genes. The variance between samples results in a need to work with a greater amount of biological replicates. In summary it can be said that single cell transcriptome analysis with next generation sequencing is possible but improvements leading to a higher yield of transcriptome reads is required. In the near future by comparing single cancer cells with healthy ones for example, a basis for improved prognosis and diagnosis can be realised

Tuning the corona: core ratio of polyplex micelles for selective oligonucleotide delivery to hepatocytes or hepatic immune cells

Targeted delivery of oligonucleotides or small molecular drugs to hepatocytes, the liver's parenchymal cells, is challenging without targeting moiety due to the highly efficient mononuclear phagocyte system (MPS) of the liver. The MPS comprises Kupffer cells and specialized sinusoidal endothelial cells, efficiently clearing nanocarriers regardless of their size and surface properties. Physiologically, this non-parenchymal shield protects hepatocytes; however, these local barriers must be overcome for drug delivery. Nanocarrier structural properties strongly influence tissue penetration, in vivo pharmacokinetics, and their biodistribution profile. Here we demonstrate the in vivo biodistribution of polyplex micelles formed by polyion complexation of short interfering (si) RNA with modified poly(ethylene glycol)-block-poly(allyl glycidyl ether) (PEG-b-PAGE) diblock copolymer that carries amino moieties in the side chain. The ratio between PEG corona and siRNA complexed PAGE core of polyplex micelles was chemically varied by altering the degree of polymerization of PAGE. Applying Raman-spectroscopy and dynamic in silico modeling on the polyplex micelles, we determined the corona-core ratio (CCR) and visualized the possible micellar structure with varying CCR. The results for this model system reveal that polyplex micelles with higher CCR, i.e., better PEG coverage, exclusively accumulate and thus allow passive cell-type-specific targeting towards hepatocytes, overcoming the macrophage-rich reticuloendothelial barrier of the liver

IMPACTS OF GENOME AND NUCLEAR ARCHITECTURE ON MOLECULAR EVOLUTION IN EUKARYOTES

The traditional view of genomes suggests that they are static entities changing slowly in sequence and structure through time (e.g. evolving over geological time-scales). This outdated view has been challenged as our understanding of the dynamic nature of genomes has increased. Changes in DNA content (i.e. polyploidy) are common to specific life-cycle stages in a variety of eukaryotes, as are changes in genome content itself. These dramatic genomic changes include chromosomal deletions (i.e. paternal chromosome deletion in insects; Goday and Esteban 2001; Ross, et al. 2010), developmentally regulated genome rearrangements (e.g. the V(D)J system in adaptive immunity in mammals; Schatz and Swanson 2011) and the specialization of a distinct somatic genome through epigenetically regulate DNA elimination during development (found in protists and some animals; Coyne, et al. 2012; Prescott 1994; Wang and Davis 2014; Wyngaard, et al. 2011). What likely allows genomes to be highly flexible is the separation of germline (i.e. ‘heritable’) and somatic (i.e. ‘functional’) material, even in the context of a single nucleus. Germline-soma distinctions have been best described (and most easily seen) in lineages of multicellular eukaryotes (e.g. plants, animals and fungi) due to obvious sexual structures. Germline genomes of these taxa are restricted to specialized cells (e.g. gametes; for example, pollen grains, eggs and spores) and remain undifferentiated (and often transcriptionally inactive), whereas the somatic cells (e.g. skin, leaves, hyphae) provide the basis for ensuring organismal survival to reproductive life-stages. Sequestered germline and somatic genomes are not restricted to these well-known multi-cellular lineages but are also well-described among ciliates (the focus of this dissertation) and some foraminifera. However, in these protists, germline and somatic genomes are not isolated into distinct cells and tissues but rather are isolated into distinct nuclei that share a common cytoplasm. Ciliates are a diverse and ancient clade of eukaryotes (~1-1.2 GYA old) and their study has led to the discovery of broad uniting eukaryotic features such as telomeres (Blackburn and Gall 1978) and self-splicing RNAs (Kruger, et al. 1982). As in the “macrobial” eukaryotes, the somatic genome (macronucleus; MAC) is transcriptionally active, transcribing all the genes necessary to maintain the cell, while the germline genome (micronucleus; MIC) remains transcriptionally inactive during the asexual portions of the life cycle. While the germline chromosomes in ciliates are physically similar to other ‘traditional’ eukaryotic chromosomes (e.g. being multi-Mbp with centromeres), the physical structure of the somatic chromosomes is highly variable. For example, in the model ciliate Tetrahymena thermophila, the somatic genome is composed of 225 unique chromosomes (most of them being ~200-400Kbp), with each at approximately 45 copies, whereas Oxytricha trifallax’s somatic genome is composed of ~16,000 gene-sized chromosomes (~2-3Kbp) with each chromosome at its own independent copy number (average copy number ~2,000). Despite dramatic differences in somatic genome architecture in ciliates, the development of a new somatic genome involves. For all ciliates studied to date, this metamorphosis from ‘traditional’ germline chromosomal architecture to the incredibly variable somatic genome architecture includes large-scale genome rearrangements and DNA elimination. This transformation involves the epigenetically-guided retention of somatically destined DNA from the background germline genome. While genomic rearrangements in most other eukaryotes are often fatal and are symptoms of well-known diseases (e.g. some cancers), this traditionally ‘catastrophic’ event is a fundamental part of ciliate life-cycles. Although studies of ciliate germline genomes have largely been restricted to only a few genera, there appear to be broad similarities in gene organization that may be phylogenetically conserved. Ciliate germline genome architecture has been categorized as either non-scrambled or scrambled, where non-scrambled architectures are often defined as possessing macronuclear destined sequences (MDSs; soma) that are separated by germline-limited DNA and remain in consecutive order (e.g. 1-2-3-4; Figure 3.1A and Figure 4.4A). Scrambled germline architectures are highly variable, but are broadly defined as MDSs being maintained in non-consecutive order (e.g. 1-3-4-2) and/or on opposing strands of DNA (Figure 3.1 B-D and Figure 4.4B). The germline genomes of Chilodonella uncinata (the main focus of this dissertation) possess a combination of scrambled and non-scrambled architectures. Before my thesis work, only those ciliates with gene-sized chromosomes have been demonstrated to have scrambled germline loci. Interestingly, previous work has implicated somatic genome architecture impacting the observable accelerated rates of protein evolution in ciliates, where the proteins of those ciliates possessing ‘gene-sized’ chromosomes experience the greatest evolutionary rates. These observations highlight the need for further work exploring the evolutionary impacts of different germline genome architectures, as the germline structure itself has direct impact on the development of the somatic genome. While this dissertation aims to elucidate some aspects of the evolution of germline-soma distinctions and the impact of genome and nuclear architecture (Chapters 2-4), there remain several fundamental questions that we can start addressing. For instance, in this work we observe that the most expanded gene families in Chilodonella uncinata are composed of genes that are disproportionately found at scrambled germline loci (Chapter 3). A major step future step will be to explore the functional implications of this increased paralog diversity through forward and reverse genetics techniques. Similarly, it will be incredibly valuable to better understand the nuclear architecture of the differing genomic contents of the three distinct nuclei present during ciliate development (i.e. the degrading parental MAC, the ‘new’ MIC, and the developing MAC). There may be observable compartmentalization that is exploitable or critical to the accurate rearrangement of the germline genome into a functional somatic genome. Finally, with the increasingly apparent utility of single-cell ‘omics techniques (which we use in Chapters 3 and 4), there is opportunity to probe into taxonomic groups where physical germline-soma separations exist, which will provide a far more expansive understanding of the evolutionary and functional impacts of harboring multiple distinct genomes inside of a single cell/organism