Chronic Myeloid Leukemia is characterised by the formation of a fusion gene bcr-abl. The gene product BCR-ABL has deregulated tyrosine kinase activity that plays a direct role in the pathogenesis of the disease. Recently, use of siRNA in leukaemic cells has led to effective gene silencing of bcr-abl. Gene delivery systems like viral vectors, electroporation and lipid based vectors have showed varying efficiencies but are limited by their level of toxicity and immunogenicity. Developments in the field of Cell Penetrating Peptides have shown effective cellular uptake of nucleic acids and proteins by the CPPs in vitro and in vivo. Report from our lab has shown the use of CPP Tat along with membrane active peptide LK15 to improve the transfection efficiency of both Tat and LK15 peptides individually. Hence, this study will focus on the use of Tat-LK15 peptide to study the delivery of siRNA and shRNA plasmid in K562 cells and observe the BCR-ABL protein expression. Cellular uptake studies using Tat-LK15 based complexes of Cy5-labelled DNA and siRNA showed a concentration dependent uptake leading to increase in percentage transfected cells. Tat-LK15 based DNA complexes achieved 80% transfected cells (charge ratio of 2:1) while siRNA complexes resulted in a maximum of 60% (charge ratio of 3:1). However, Lipofectamine based DNA complexes did not show a concentration dependent increase in percentage transfected cells. Interestingly, Tat-LK15 based siRNA complexes showed a similar level of uptake and percentage transfected cells as that of Lipofectamine based siRNA complexes. Cellular uptake studies using confocal microscopy 4 hours post transfection, showed that when 1μg of DNA was transfected, the labelled DNA was primarily localised on the cell membrane. Interestingly, using 5μg of DNA led to increased intracellular localisation of the labelled DNA, but this observation was not made with Lipofectamine based complexes. The observation at 24 hours post transfection of Tat-LK15/labelled DNA complexes was of higher intensity when compared to that of Lipofectamine based DNA complexes. The reason for this is however not known. Interestingly, the cellular uptake profile using siRNA based complexes was different. At 4 hours post transfection, there was intracellular localisation of labelled siRNA. 24 hours post transfection, there was diffuse cytoplasmic localisation using lower concentration of siRNA whereas using higher concentration led to more high intensity punctate localisations within the cell. Similar observations were made for both Tat-LK15 and Lipofectamine based siRNA complexes.Gene silencing studies of Tat-LK15/shRNA plasmid complex resulted in 80% reduction in protein levels 96 hours post transfection for higher concentrations of shRNA plasmid treated. Similar level of reduction in BCR-ABL was observed with Lipofectamine based complex. Supporting evidence of reduction in mRNA levels was observed using qRT-PCR 48 hours post transfection. However, Tat-LK15/shRNA plasmid complexes led to around 80% of protein reduction 192 hours post transfection while Lipofectamine based complexes resulted in only 40% of protein reduction. Transfection using increasing concentrations of siRNA complexed to Tat-LK15 and Lipofectamine led to greater than 70% reduction in protein levels for most concentration ranges tested. This reduction in protein levels lasted only 48 hours post transfection. In conclusion, Tat-LK15 peptide could be used for shRNA plasmid and siRNA based delivery and could offer an efficient gene delivery model for studying RNAi.

MicroRNAs are small non-coding RNAs that regulate gene expression through RNA interference. These small RNAs enact gene silencing by forming a RNA-inducing silencing complex together with the effector protein Argonaute. The function of the Argonautes in the social amoeba Dictyostelium discoideum is not yet fully understood. In this study, we look closer at Argonaute B by investigating if it is possible to extract the protein from the cells by the addition of a polypeptide protein tag called 3xFlag. At the same time, we also look into if Argonaute B is important for cell growth. Sequences of the 3xFlag tag with or without the Argonaute B gene (agnB) attached had previously been cloned into a vector and transformed into Dictyostelium discoideum cell. The 3xFlag::agnB sequence was confirmed in wild type and agnB knock-out strains through polymerase chain reaction. We then verified the expression of the fusion protein in the cells by western blot. The cell growth was measured by how the number of cells changed over time. The experiment suggested that Argonaute B is important for growth. Our result show that the construct 3xFlag::agnB sequenced had correctly been transformed into the strains and is highly expressed under tested conditions. We could also see that Argonaute B is an important factor in cell growth.

Leishmania of the subgenus Viannia are protozoan parasites transmitted by phlebotomine sandflies (Diptera: Phlebotominae). They occur in tropical and subtropical areas in South America, where they cause cutaneous and mucocutaneous leishmaniasis. In this thesis, we studied developmental pattern of Viannia group and factors affecting its development within the sand fly gut. First, we investigated Leishmania braziliensis development within the Lutzomyia longipalpis digestive tract. Using GFP-labeled strain we demonstrated peripylar development: promastigotes escaped from the endoperitrophic space, colonized the hindgut and then migrated anteriorly. Four morphological forms were found within the Lu. longipalpis digestive tract: elongated nectomonads, short nectomonads, metacyclic promastigotes and paramastigotes. Furthermore, using the histological methods we demonstrated parasite attachment in pylorus region, while there were only free promastigotes in the midgut; neither form was found attached to the midgut epithelium. The next part was devoted to the effect of temperature on Viannia in Lu. longipalpis. We compared development of two closely related species L. peruviana and L. braziliensis at 20 řC and 26 řC. Leishmania braziliensis developed well in both temperatures tested, L. peruviana developed...

RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. Due to the variability in RNAi efficiency, RNAi-based methods are currently being developed for controlling only coleopteran insects which are known to be amenable to RNAi. The first chapter of my thesis includes findings from research to investigate what are the factors that make coleopteran insects relatively more efficient in RNAi. I used Colorado potato beetle (CPB), Leptinotarsa decemlineata and its cell line (Lepd-SL1) as study models to identify genes that play key roles in RNAi pathway. Five genes including Argonaute-1 (microRNA Argonaute) and Aubergine (PiwiRNA Argonaute) were identified as those required for siRNA (short interfering RNA) RNAi pathway. I also found that RNAi is completely blocked in StaufenC knockdown cells. StaufenC belongs to dsRNA binding protein family and binds to dsRNA as shown by gel mobility shift and the pull-down assays. Interestingly, I also found that StaufenC is downregulated in RNAi resistant cells and StaufenC homologous sequences are present in only coleopteran insects where RNAi works efficiently. These data suggest that StaufenC is a major contributor to efficient RNAi in coleopteran insects and is a potential target for RNAi resistance. The second part of my research is to understand the mechanisms of RNAi in those insects refractory to RNAi. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. Recent work in our laboratory showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into siRNAs because they are trapped in acidic bodies. I focused on identification of these acidic bodies in which dsRNAs accumulate in Spodoptera frugiperda Sf9 cells. These studies showed that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi. Overall, my research revealed important players involved in successful and unsuccessful RNAi in insects.

Endogenous viral elements (EVEs) have been recently investigated as a source of transgenerational immune memory. These “viral fossils” are abundant in Aedes mosquitoes and partner with the host’s primary antiviral defense system, the RNA interference (RNAi) pathways. This partnership appears unique to mosquitoes, which encode an expansion of the Piwi endoribonucleases. To interrogate EVE-Piwi partnerships and their role in antiviral defense, I performed a comparative small RNA analysis of two naturally occurring EVE-virus pairs – one in the mosquito Aedes albopictus, and one in the midge Chaoborus americanus. Both express an EVE related to the nucleoprotein of their respective bunyavirus. My results show that Piwis generally do not have antiviral functions in Chaoborus, however EVEs are associated with Piwi recruitment to matched viral RNAs. These findings raise the possibility that RNAi-mediated EVE-virus interactions may be more common among insects than currently recognized.

Natural Killer (NK) cell based immunotherapies have demonstrated success against malignancies and hematological cancers. However, tumors have developed mechanisms to evade detection by and suppress the immune system, commonly through altering the expression of cell-surface proteins. Overexpression of human leukocyte antigen-E (HLA-E), which binds to the inhibitory NKG2A on NK cells, protects malignant cells from lysis. Downregulating the NKG2A receptor on NK cells should release NK cell inhibition, but proves challenging as NK cells are difficult to transfect and no good methods currently exist. This project is designed to investigate the use of exosomes – small vesicles and natural carriers of regulatory microRNAs (miRNAs) and proteins that are shed from cells – as delivery vehicles for small RNAs (sRNAs) to immune cells. Exosomes are biologically compatible, immunologically inert, and interact with target cells through receptor-ligand interactions, allowing for targeted delivery of cargo. Exosomes loaded with shRNA against NKG2A were cultured in vitro with NK cells. Delivery success was assessed by monitoring NKG2A receptor expression on NK cells through flow cytometry. This research will provide valuable information that will likely impact the delivery of RNA therapeutics and unlock the full cytotoxic potential of NK immunotherapy.

Doctor of Philosophy / Department of Entomology / David C. Margolies and Yoonseong Park / Organisms are capable of sensing environmental conditions through diverse mechanisms. Transient receptor potential channels (TRPs) are a cation channel family that has been found to function in diverse sensing mechanisms. In this dissertation, I identified the function of several TRPs in thermosensing and mechanosensing in the red flour beetle, Tribolium castaneum. Candidate TRPs were chosen based on homology to TRPs found and studied in Drosophila melanogaster. To identify the function of candidate TRPs in T. castaneum, I suppressed the expression of target genes by RNA interference technique and investigated the phenotype of each treated beetle. Temperature is a major limiting environmental factor for organisms. I tested the function of candidate TRPs in thermotaxis (behavior) and thermal acclimation (physiology). Using bioinformatics approaches, I identified three candidate TRPs – painless, pyrexia, and trpA1 – involved in high temperature sensing. To test thermotactic behavior, I investigated beetle movement on a temperature arena with two separate temperature zones. Thermal acclimation was tested by pre-exposing beetles to either 42 °C for 10 min. When treated with double stranded RNA of TRPA1 (dstrpA1), the thermotactic response of beetles at 39 and 42 °C was reduced when compared to control groups. With pre-exposure at 42 °C, survivorship of dstrpA1-treated beetles significantly increased after one minute exposure at 52 °C compared to beetles that were not pre-exposed. With dspainless treatment, beetles showed lower response to thermal acclimation and lower long-term survivorship. Beetles treated with dspyrexia showed lower recovery after heat treatment without pre-exposure at 42 °C. To identify the function of candidate TRPs in mechanosensing, I evaluated dsRNA treated beetles for survival, walking behavior, and tonic immobility. Treatment with dsnompC and dstrpA5 resulted in failure in eclosion, causing 93 % mortality in both treatments. Survivors in dsnompC showed defects in elytra sclerotization. In dsnanchung and dsinactive treatments, adults showed abnormal walking behavior and reduced walking speed that were likely caused by defects of mechanosensing in folding of the joint between the femur and tibia. For tonic immobility, beetles with dsnanchung, dsinactive, dswaterwitch and dsick2 (insect cytokine 2) treatments showed increased sensitivity to mechanical stimulation leading to tonic immobility.

Master of Science / Department of Entomology / Kun Yan Zhu / Cytochrome P450 monooxygenases (P450s) are important enzymes involved in the metabolism of a variety of xenobiotics, including insecticides and plant allelochemicals, and endogenous compounds, including juvenile hormones, ecdysteroids and fatty acids, in insects. Despite rapid advances in revealing various P450 genes in insects, our knowledge on the role of these genes in detoxification of insecticides is very limited. This research was to perform a genome-wide analysis of P450 genes and evaluate the role of selected P450 genes in detoxification of three commonly used pyrethroid insecticides in the yellow fever mosquito (Aedes aegypti). Our genome-wide analysis of revealed 159 P450 genes that can be classified into 18 families and 63 subfamilies. These genes are distributed in four clans, including 11 genes in the CYP2 clan, 80 in the CYP3 clan, 58 in the CYP4 clan and 10 in the mitochondrial CYP clan. The largest families are CYP6, CYP9, CYP4 and CYP325. The intron-exon organization of the genes is very diverse among the gene families, and the highest conservation of gene structures was observed in the CYP6 and CYP9 families predominantly containing single-intron genes. The phylogenetic analysis suggested that the CYP6 and CYP9 families might be derived from a common ancestor. The expression patterns of five transcripts including three individual genes (CYP6AA5, CYP6AL1 and CYP9J32) and two alternative splicing variants (CYP4J16A and CYP4J16B) of CYP4J16 were investigated in various tissues and at different developmental stages of the mosquito. Our results indicated differential expressions of these transcripts in different tissues and at different developmental stages examined. Furthermore, the exposure of the mosquitoes (larvae and adults) to each of three pyrethroid insecticides (permethrin, cypermethrin and deltamethrin) resulted in either down or up-regulation of these transcripts. Functional analyses of the selected P450 transcripts were conducted by using RNA interference (RNAi) followed by insecticide bioassay. RNAi was achieved by feeding mosquito larvae with chitosan/double stranded RNA (dsRNA) nanoparticles or injecting dsRNA to the adults. For the larvae, we obtained relatively low repressions of the P450 transcripts but the repressions were sufficient for carrying out our functional studies. Our study showed increased mortalities by 41.2% to cypermethrin when CYP6AA5 was silenced and 46.0% to permethrin when CYP9J32 was silenced. Similarly, the injection of dsRNAs in adults resulted in significant repressions of the P450 transcripts, and subsequent insecticide exposures led to a 29.3% increase in the adult mortality to cypermethrin when CYP6AA5 was silenced. Our further analysis of the nuclear receptor HR96 in the up-regulation of the P450 genes showed that when HR96 was silenced by RNAi, the up-regulation of CYP4J16B by cypermethrin was reduced by 10.1-fold but silencing HR96 did not affect the up-regulation of other P450 genes examined. These results suggest that HR96 is likely involved in regulating the expression of CYP4J16B in Ae. aegypti. However, different regulatory mechanism (s) may be involved in the up-regulation of other P450 genes examined. Model structure of CYP6AA5 was created by homology modeling and insecticides substrates were docked into the active site of this protein. Our results indicate that all three insecticides can fit into the catalytic pocket. The interaction distances between the heme iron and the putative aromatic hydroxylation site were 9.2, 9.4 and 7.2 Å for permethrin, cypermethrin and deltamethrin, respectively, whereas for aliphatic hydroxylation site these distances were 5.3, 2.8 and 2.9 Å. These results showed that CYP6AA5 may be able to metabolize cypermethrin and deltamethrin preferentially by aliphatic hydroxylation as indicated by the close interaction with the heme iron.

No description available.

Feline oral squamous cell carcinomas (FOSCCs) are locally aggressive tumours and a common cause of mortality and morbidity. Current treatment options are rarely successful and animals are frequently euthanised upon diagnosis due to their grave prognosis. Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase receptor which is frequently dysregulated in SCC of the head and neck (HNSCC) in man. Recent advances in human medicine have identified EGFR as a therapeutic target in HNSCC. In this study the role of EGFR in FOSCC was investigated. Sixty seven biopsy samples were immunohistochemically labelled for EGFR and Ki67, a proliferation marker. The tyrosine kinase region of feline EGFR was cloned and sequenced, and six small interfering RNAs (siRNAs) targeting the tyrosine kinase region were developed. The most effective siRNA as well as an EGFR specific tyrosine kinase inhibitor, gefitinib, was then used on a feline SCC cell line (SCCF1), and the effect of EGFR targeting alone, or in combination with irradiation, on the cell line was determined. The majority of the biopsy samples were labelled positively for EGFR and Ki67, and high proliferation corresponded with poor prognosis. The siRNA caused reduction in EGFR mRNA by Real-Time Polymerase Chain Reaction and protein levels as assessed by western blot analysis. Reduced cell proliferation and migration were also observed by proliferation assays and scratch assays respectively. Combining EGFR knockdown with irradiation caused an additive effect on the ability of the cell line to form colonies. These results support the role of EGFR as a potential therapeutic target in FOSCCs.