https://www.um.edu.mt/library/oar/handle/123456789/64361 2026-06-12T01:01:03Z 2026-06-12T01:01:03Z https://www.um.edu.mt/library/oar/handle/123456789/121372 2024-04-26T05:53:01Z 2020-01-01T00:00:00Z

Title: The effect of mind-linked gene disruption in GLIA Abstract: Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the most common motor neuron diseases (MNDs) in adults and infants respectively. Both ALS and SMA are characterised by motor neuron loss, muscle atrophy, loss of movement and respiratory insufficiency, which most commonly leads to mortality. Multiple genes are involved in ALS pathogenesis including C9orf72, TARDBP/TDP-43, and FUS. Pathogenic mechanisms involve both loss and gain of function including aberrant messenger RNA (mRNA) processing and trafficking, and production of cytoplasmic inclusions including C9orf72-associated dipeptide repeat aggregates. SMA pathology revolves around the SMN gene where its deletion leads to a decreased SMN protein concentration. SMN together with Gemins 2-8, Unrip, pICln and Tgs1 are required to produce small nuclear ribonucleoproteins (snRNPs), essential for mRNA splicing. Glial involvement in MND has been a subject of current research. The main glial cells are astrocytes, oligodendrocytes, and microglia, fundamental for motor neuron health and maintenance. Using Drosophila melanogaster as a model organism and the bipartite GAL4- UAS system, various ALS- and SMA-linked genes were disrupted selectively in glia and flies were assessed for motoric ability and survival. Disruption of the ALS-linked TDP-43, FUS, C9orf72 and SCFD1 led to significant decreases in larval mobility with the large majority of all transgenes applied inducing complete loss of adult fly viability. Disruption of the SMA-linked SMN, pICln, Tgs1 and Gemin3 did not affect larval mobility with the exception of Gemin8/Valette. Nonetheless, viability was negatively affected on disruption of nearly all SMA-linked genes. These results show a very important glial contribution to the normal pathogenesis of MND and uncover novel contributors to the function and survival of glia. This can be exploited for future therapeutic strategies possibly enhancing their effectiveness. Description: B.SC.(HONS)APPLIED BIOMED.SCI.

2020-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/119079 2024-03-06T10:20:30Z 2020-01-01T00:00:00Z

Title: Generating a bacterial clone for evaluating growth under stress Abstract: Climate change is a major global concern, with implications regarding (amongst others) changes in the universal microbial proliferation due to alterations of the environmental conditions. Subsequent bacterial adaptation to climate variation (i.e. acclimatisation) can result in an effect, as of yet unknown, with regards to their ability to grow and propagate. The project aimed to create an Isopropyl β- d-1-thiogalactopyranoside-inducible enhanced Green Fluorescent Protein (eGFP) synthesising reporter Escherichia coli clone via chemical transformation, which would then be used as part of an eGFP/Propidium iodide (PI) cell viability assay. Information obtained from this model organism should be applicable to other Gram-negative organisms and allow for a complete understanding of the mechanisms of bacterial response to environmental stress. The study generated E. coli BL21 (DE3) pD454- MBPeGFP, validated and characterised it. A 150 nM PI concentration to be optimal for the eGFP/PI assay, however the use of PI was determined to be inappropriate to kinetic assays. Growth was assessed using three parameters: lag (λ), maximum growth rate (µmax), and fluorescent time to detection values (FTTD). λ decreased with increased carbon dioxide (CO2), and temperature. Values for µmax were not affected by increases in CO2 but diminished with increased temperatures. FTTD values demonstrated its application to assessing changes at lower levels of inoculum; application may vary according to a study’s objectives. Future work should aim for characterising responses to changes in other variables such as pH. Description: B.SC.(HONS)BIOMED.SCI.

2020-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/65161 2020-12-06T06:52:02Z 2020-01-01T00:00:00Z

Title: Cellular reactivation of foetal haemoglobin transcription by the use of CRISPR/Cas9 Abstract: Sequential expression of embryonic, foetal and adult haemoglobin during human ontogeny has been a focus of numerous studies focusing on mechanisms of target gene switching in order to increase expression of foetal haemoglobin and consequently leading to significant improvement of clinical manifestation of haemoglobinopathies that include sickle cell disease and β-thalassaemia. Understanding the natural switch control from foetal to adult haemoglobin and actions of genes involved could lead to potentially lifesaving therapeutic approach. EKLF also known as KLF1 is an important regulatory protein involved in the γ- to β-globin gene switching mechanism that directly induces the expression of the β-globin gene and conversely represses γ-globin. In this study I sought to test a number of CRISPR/Cas9 systems with the purpose to disrupt the functions of KLF1, LRF and BCL11A genes and inhibit the γ- to β-globin gene switching in K562 cells. Relative quantification was performed for the assessment of all globin including γ globin expression as well as the target genes KLF1, BCL11A and LRF. The BCL11A quantification was not successful due to low expression of the same gene in K562 cells and was therefore not pursued. The levels of γ-globin mRNA varied between 48hrs and 96hrs post differentiation with hemin induction, showing a 1.97-fold induction for KLF1 knockdown by CRISPR/Cas9 as opposed to just after 48hrs and also when compared to untreated cells. The findings obtained in this dissertation support the induction of γ-globin expression upon targeting KLF1 through CRISPR/Cas9 mediated silencing. As a result, the effect of KLF1 inhibitory influence on γ-globin gene expression is removed. The same was not observed upon targeting LRF by CRISPR/Cas9, in which γ-globin remained low. Application of CRISPR technology in adult erythroid progenitors may provide a method for activating foetal haemoglobin expression in individuals with β-thalassaemia or sickle cell disease. Description: M.SC.APPLIED BIOMED.SCI.

2020-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/65160 2020-12-06T06:52:26Z 2020-01-01T00:00:00Z

Title: Genome editing the human beta globin locus for foetal haemoglobin production Abstract: Amelioration of the symptoms of sickle cell disease or β-thalassaemia patients can be obtained by reactivation of the developmental program of foetal haemoglobin (HbF). This results in a raised level of foetal haemoglobin that can only be achieved in the clinical setting by firstly fully understanding the genetic switch from foetal (γ-globin) to adult haemoglobin (β-globin). Substantial progress has recently been made in this field, however the exact mechanism remains elusive. The combination of clinical research coupled with basic research should help in this regard. In a previous research conducted by a Borg et al (2010), a Maltese family was found to exhibit a nonsense variant, p. K288X, in KLF1 resulting in its partial loss of function, and elevation of HbF due to loss of repression by this gene. However, the heterogeneous distribution of HbF in the different family members with KLF1 haploinsufficiency, with a range of 3.3 to 20% HbF and in a collection of patients with borderline HbA2/HbF could not be fully explained by this gene on its own and it was concluded that other genetic elements were at play. A number of candidate genes were identified by Borg et al. following RNA-seq and ATAC-seq studies on the promoter region of KLF1. From this list, that included LRF, KLF1 and BCL11A, knock down studies were performed utilizing CRISPR/Cas9 technology to quantify transcripts of the human β-globin locus to determine the important cis- and trans- regions responsible for gamma globin gene (HBG1/2) induction and HbF production using various methods including Real Time PCR (qPCR) and to further understand the role of the targeted genes in regulation of variable HbF and in the switching mechanism from foetal to adult haemoglobin. Description: M.SC.APPLIED BIOMED.SCI.

2020-01-01T00:00:00Z