Glioblastoma is the most malignant form of gliomas and is a highly infiltrative while non-metastatic tumor of the central nervous system. Patients with glioblastoma have a poor prognosis of 15 months median survival after diagnosis. Promising results were reported in recent clinical trial regarding glioblastoma treatment with chimeric antigen receptor (CAR) T therapy. The lab has previously developed five novel scFvs targeting IL13R⍺2, a tumor-associated antigen in glioblastoma, and integrated them into the second-generation CAR. We named them, 10CAR, 27CAR, 55CAR, 75CAR and 117CAR. The ex vivo cytotoxicity and proliferation assay demonstrated that the 117CAR T construct has the best functionality, while 27CAR T construct has a poor functionality compared to the rest of the constructs. FACS analysis was performed to check the CAR expression in different constructs. 27CAR T cells showed the lowest surface CAR expression and 117CAR T cells displayed the highest out of five constructs. 27CAR T cells were also activated more without stimulation compared to other constructs. We selected out 27CAR and 117CAR T cells for the further investigation to understand the attribution of the discrepancy between 27CAR and 117CAR T cells.   We observed a larger cellular size for 27CAR T cells compared to the rest constructs in flowcytometry analysis, which is usually associated with activation. IFN-γ production of all constructs without target cells stimulation were detected to examine the activation state of different constructs. We observed the highest IFN-γ production in 27CAR T cells without stimulation. These results together indicate that a potent antigen-independent activation or, in other words, tonic signaling is present in 27CAR T cell. The tonic signaling further leads to an early exhaustive phenotype of 27CAR T cells, that is not present in 117CAR T cells. Removing the endodomain of CAR rescued the antigen-independent activation and early exhaustion of 27CAR T cells. The surface and total CAR expression of 27CAR and 117CAR T cells were determined by flowcytometry. 27CAR T cells presented a lower expression of both surface and total CAR. A significantly lower percentage of total CAR on the surface indicates the internalization of CARs in 27CAR T cells. Removing the intracellular domain of 27CAR did not restore the surface expression of CAR. 27CAR and 117CAR differ in four CDRs of scFv, CDR1, 2,3 in the heavy chain and CDR3 in the light chain. We replaced all the amino acids differing between these two constructs with alanine in a CDR-by-CDR manner and obtained five alanine substitution constructs. We then analyzed the CAR expression in Jurkat cells, and we found that the trafficking of CAR to the surface was significantly improved by mutating the CDR2 in the heavy chain or CDR3 in the light chain. Moreover, when the two CDRs were replaced simultaneously, almost all transduced cells expressed CAR, as was the case of cells transduced with 117CAR.   To summarize, the tonic signaling induced by higher tendency of clustering of 27scFv results in the antigen-independent activation and early exhaustion of 27CAR T cells. By removing the endodomain of 27CAR, we abrogated the phenomenon. Further, CDR2 in heavy and CDR3 in light chain in 27scFv are responsible for the impaired trafficking of CAR to the surface.

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Title: Investigation of RRM2 as a potential therapeutic target against glioblastoma Supervisors: Dr. Sven Nelander and Mr. Sathishkumar Baskaran. Department: Department of Immunology, genetics and pathology (IGP), Uppsala University   Glioblastoma (GBM) is the most malignant form of glioma and associated with high proliferation rate, necrosis and highly invasive nature. Current treatment includes tumor resection followed by combination of radiotherapy and chemotherapy with temozolomide (TMZ). Despite of combination therapy, GBM exhibits dismal prognosis and mean survival rate of patients is only 3.3 % at 2 years and 1.2 % at 3 years. Therefore, there is an increasing demand of identifying new therapeutic targets against GBM. In this project, we studied the function of RRM2 gene as a potential therapeutic target in two patients derived GBM cell lines (GC). By knocking down RRM2 using short interfering RNAs, the viability of cells and proliferation was significantly reduced in both the GC. The cause of cell death was due to induction of apoptosis by the treatment in GC. Treatment of COH29, an inhibitor of RNR, induced cell death at therapeutically relevant dose in GC. Our results indicate that RRM2 has a significant role in GBM cell growth/proliferation. More evaluation must be performed in both in-vitro and in-vivo to pursue RRM2 as a molecular therapeutic target against GBM.

Thesis (M.Sc. (Hons.))-University of Western Sydney, 2007. / A thesis submitted to the University of Western Sydney, College of Arts, School of Education, in fulfilment of the requirements for the degree of Master of Science (Honours). Includes bibliographical references.

Purpose: Many lives are affected by cancer. The number of people in England who have had a diagnosis of cancer exceeds one million. Previous research shows that one third of patients have unmet needs post-discharge from cancer treatment, including psychological issues such as negative impacts on self-identity and a lack of meaning in life. Studies have identified identity as an important factor in meaning making, but evidence regarding cancer’s impact on identity is limited to specific cancer sites and specific identity roles. Little is known about cancer’s general impact on global identity or how threats to identity relate to meaning making. The aim of this study was to understand patients’ experiences of cancer’s impact on their identity and what sense they made of these experiences. Methods: Twelve participants in the post-treatment phase of cancer shared their experiences in individual semi-structured interviews. Key themes regarding identity and meaning making in the post-treatment phase were identified using interpretative phenomenological analysis (IPA). Results and Conclusions: Four key themes in the participants’ experiences were identified. These were 1) disrupted identity roles, 2) highlights what is important, 3) focused on priorities, and 4) reducing awareness of loss and uncertainty. Relevant literature and implications for future research and clinical practice are discussed.

Introduktion/Bakgrund: Att insjukna i cancer och att genomgå behandling kan påverka patienter på många olika sätt. Såväl sjukdomen som behandlingen kan leda till olika biverkningar som påverkar patientens livskvalitet, däribland sexualiteten. Tidigare forskning har visat att sexuella biverkningar ofta är underbehandlade och underdiagnostiserade. Sex anses fortfarande som ett tabubelagt ämne hos både sjuksköterskor och patienter vilket riskerar att patienter lider i onödan av sina sexuella biverkningar.  Syfte: Syftet med litteraturstudien var att beskriva patienters upplevelser av förändrad sexualitet i samband med cancer. Metod: En litteraturstudie med kvalitativ ansats har använts. Sökningarna har skett i databaserna Cinahl och Pub Med. Materialet har därefter analyserats efter Fribergs analysmodell i fem steg. Sammanlagt inkluderades 16 vetenskapliga artiklar. Resultat: Resultatet sammanställdes under fyra huvudteman: fysiska aspekter, emotionella aspekter, aspekter på relationer samt information och stödjande samtal. Merparten av studierna visar att sexualiteten påverkas negativt av cancer och de behandlingar som ges. Kommunikationen i relationen och med vårdgivaren är viktig för att bevara sexualitet och intimitet.  Slutsats: Cancerbehandling påverkar den upplevda sexualiteten och samlivet i olika omfattningar, vilket kan leda till negativa konsekvenser i patienters liv under en lång tid. Patienter upplever otillräcklig information om sexuella biverkningar och att vårdpersonal är obekväma med ämnet. Studiens fynd bedöms vara av klinisk relevans där specialistsjuksköterskor behöver ge individuellt stöd och information till patienter och deras partners.

Cancer är en sjukdom som idag är en av största dödsorsakerna. I mikromiljön finns det bland annat immunförsvarets celler, endotelceller och fibroblaster. Det är en komplex miljö där signalvägar kan stimulera tillväxten hos cancercellen. Syftet med denna studie är att analysera hur cancercellernas mikromiljö ser ut och hur cancercellerna och de ”normala cellerna” kommunicerar med varandra som gynnar cancercellen. Finns det några idéer om framtida behandlingar som påverkar kommunikationen mellan de ”normala cellerna” och cancercellerna. Metoden som tillämpades var en systematisk litteraturöversikt som använde data från originalartiklar för att kunna besvara syftet. PubMed användes som databas och sökorden som användes var bland annat micro environment.   Resultatet visade att signalvägar är en process för cancercellen att växa. Det fanns olika behandlingar för att inhibera kommunikationen, och några av dessa var interleukin-1 receptor antagonist (IL-1RA) och interleukin-6 neutraliserad antikropp (IL-6 neutraliserad antikropp). Behandlingen av IL-6 neutraliserad antikropp bidrog till att interleukin-6 (IL-6) som utsöndrades från cancerassocierade fibroblaster (CAF) inte kunde binda till sin receptor som ledde till att cancercellens tillväxt inhiberades.    Det som var gemensamt för hela studien var att cancercellens mikromiljö kunde vara en måltavla för att behandla cancer. Detta genom att inhibera olika signalmolekyler som utsöndras från både cancercellen och de ”normala cellerna”.

Doxorubicin is a highly effective chemotherapeutic agent against a variety of cancers; however, the exact mechanisms responsible for the effects of doxorubicin on cancer are not widely understood and have not been studied on a single-cell level. Single-cell RNA sequencing analysis is a recent technology that enables the assessment of transcriptional similarities and differences within a population of cells. This thesis aims to study the effects of doxorubicin in breast cancer on a single-cell level and see if there are differently affected subpopulations of cells. A single-cell RNA sequencing pipeline was developed in R and used to analyse breast cancer single-cells treated with doxorubicin. Quality control, filtering, cell-clustering, differently expression gene analysis, pathways analysis and cell type identification were performed. The results identified seven different subpopulations that demonstrated a differential expression of genes and expression-level of pathways such as NEIL3-mediated resolution of induced interstrand crosslinks related with the effect of doxorubicin on cancer cells. The analysis also suggests that two subpopulations of cells could consist of specific cell types potentially treatment-resistant. These findings reinforce the relation of doxorubicin effect on cancer with some important genes and pathways, as well as reaffirming the heterogeneity of breast cancer. As a novel contribution, the results show that different subpopulations of cells could be affected differently by doxorubicin exposure, but future studies with more samples should be performed to see if the analysis leads to similar results

Acute myeloid leukaemia (AML) is highly heterogeneous haematological malignancy, which represents a challenge in the understanding of the disease. Relapse in AML is common, andmany relapsed patients respond poorly to conventional treatment, leading to a low survivalrate. Investigating the mutational landscape connected to relapse AML is therefore of highinterest in order to improve clinical outcome. Recurrent in-frame internal tandem duplications(ITDs) in exon 13 of the UBTF gene have previously been discovered in paediatric relapseAML, correlating to one of the DNA binding domains of the transcription factor UBTF.UBTF is involved in recruitment of RNA polymerase I and activation of the transcription ofribosomal RNA. As a result, it is an important factor in ribosome biogenesis. In this study, wehave investigated the effect of UBTF-ITDs on RNA synthesis and UBTF localization. I haveshown that ITDs lead to disturbed localization of UBTF to the nucleus, which coupled withthe previous finding that patients are heterozygous for the ITDs, indicates haploinsufficiency.This could have potential implications in AML drug resistance. We have further generated anin vitro model for investigating the effect of UBTF haploinsufficiency, which could lead toidentification of vulnerabilities to be targeted in future drug treatments.