Inaug.-diss.-Rostock. / Lebenslauf. At head of title: Aus der Anatomischen anstalt der # Universität Berlin." Pts. I and II issued 1907 as Sonderabdruck aus dem Archiv für mikroscopische anatomie und entwicklungsgeschichte; and bound with Dental pamphlets, v. 10. Cover title. "Literaturverzeichnis": p. 70-73.

Inaug.-diss.-Rostock. / Lebenslauf. At head of title: Aus der Anatomischen anstalt der # Universität Berlin." Pts. I and II issued 1907 as Sonderabdruck aus dem Archiv für mikroscopische anatomie und entwicklungsgeschichte; and bound with Dental pamphlets, v. 10. Cover title. "Literaturverzeichnis": p. 70-73.

Non-Hodgkin’s lymphoma (NHL) is a group of lympho-proliferative disorders characterised by genetic mutations resulting in the selection of a malignant clone. Recently, mutations in the anti-proliferative B-cell translocation 1 gene (BTG1) and B-cell translocation 2 gene (BTG2) have been identified in in NHL cases, which suggests a direct involvement of BTG1 and BTG2 in malignant transformation. BTG1 and BTG2 are members of the human BTG/TOB family. They are characterised by the conserved amino-terminal BTG domain, which mediates interactions with the human Caf1(hCaf1) catalytic subunit of the Ccr4-Not deadenylase complex .In addition, the BTG domain binds to the cytoplasmic poly (A)-binding protein (PABPC1). This complex plays a critical role in mRNA deadenylation and degradation as well as translational repression. It is currently unclear how, or indeed whether, mutations in BTG1 and BTG2 affect the function of the gene products. Therefore, a combination of sequence analysis and molecular modelling was used to predict the functional consequences of mutations previously identified in NHL. Sorting intolerant from tolerant (SIFT) and Suspect (Disease-Susceptibility-based SAV Phenotype Prediction) prediction tools enabled the identification of amino acid residues that would potentially interfere with the protein function, and hence may be associated with disease. In total 45 mutations in BTG1 and BTG2 were assessed. These mutations were derived from NHL samples, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma and Burkitt's lymphoma. Of the variants analysed, 15 were predicted to interfere with the function of BTG1 using SIFT analysis (Score ≤0.05). Only seven of these variants were predicted to be likely associated with disease using Suspect algorithm (Score ≥50), and an additional variant, BTG1 C149del, was predicted to interfere with protein function using PROVEN (Protein Variation Effect Analyzer). The ability of these protein variants to interact with known partners was established using yeast two hybrid assays. In addition, functionally assessment of the role of the mutated proteins in cell cycle progression, translational repression and mRNA degradation was also performed. Using a yeast two-hybrid system, ten BTG1 variants were shown to affect the interaction of BTG1 with the hCaf1 (CNOT7/CNOT8) catalytic subunit of the Ccr4-Not deadenylase complex. In addition, when BTG1 variants were transfected into mammalian cells, these BTG1 variants (M11I, F25C, R27H, F40C, P58L, G66V, N73K and I115V), unlike the wild-type proteins, were not able to inhibit cell cycle progression. These results suggest that anti-proliferative BTG1 is required for hCaf1 (CNOT7/CNOT8) deadenylase activity. The remaining BTG1 variants (L37M, L94V, L104H and E117D) were not consistent in the correlation of BTG1 interaction with hCaf1 (CNOT7/CNOT8) and inhibition of cell growth which led to the suggestion that BTG1 may require an additional factor such as PABPC1. Interestingly, several BTG1 variants (M11I, F25C, R27H, P58L, N73K I115V and E117D) did not require interaction with the hCaf1 (CNOT7/CNOT8) deadenylase enzyme to reduce reporter activity as established using 3’ UTR tethering assays. This suggests that BTG1 may also have a role in regulating cell cycle progression and RNA degradation via Ccr4-Not deadenylase complex independent mechanisms. The data show that variants in BTG1 commonly found in DLBCL, are functionally significant and are likely to contribute to malignant transformation and tumour cell grow.

Maharashtra Anaemia study (MAS) was conducted as a part of the PhD programme of Dr Anand Ahankari through a joint collaboration of the University of Nottingham, UK and Halo Medical Foundation, India. The main goal of the study was to establish baseline epidemiological data for anaemia research in pregnant women and adolescent girls in Maharashtra state of India. Iron deficiency anaemia is the most common form of anaemia observed in India, and assessed based on haemoglobin (Hb) levels in blood. Clinically, anaemia is categorised in mild, moderate and severe form based on Hb levels. The project had three main sections, a) Adolescent girls cross sectional survey, b) Pregnant women prospective study, and c) Maharashtra state birth registry analysis. The study aimed to investigate individual and village level risk factors of anaemia in adolescent girls (13 to 17 years), and pregnant women (3 to 5 months) living in rural Maharashtra. Data from pregnant women were also used to examine risk factors associated with low birth weight (LBW). A recently introduced non-invasive haemoglobin (Hb) technology (known as NBM 200) was validated in this Indian setting by comparing Hb measurements obtained from the NBM 200 with reference blood measurements. In the adolescent survey, Sahli’s hemometer (finger prick technique) was used to estimate reference Hb values, while in pregnant women venous blood samples were obtained to measure Hb using an automated analyser. Anaemia was defined using Hb levels based on the following cut offs, (a) Hb < 12.0 g/dl in adolescent girls, and in (b) Hb < 11.0 g/dl in pregnant women. Multivariable regression technique was used to identity risk factors associated with anaemia and LBW. The Maharashtra state birth registry records covering a 32-year period (1980 to 2011) were investigated to assess temporal changes in the sex ratio at birth to investigate impacts of sex determination prevention legislations (known as PNDT 1994 and PCPNDT 2003). The adolescent girls’ survey showed a very high prevalence of anaemia (87%). Of 45 factors assessed in the survey, four were associated with adolescent anaemia. Anaemia likelihood increased significantly with age (Odds Ratio [OR] 1.41 per year, 95% CI: 1.17 to 1.70). Factors associated with decreased risk of anaemia were higher mid upper arm circumference (> 22 cm) (OR 0.51, 95% CI: 0.31 to 0.82), and ≥3 days/week consumption of fruit (OR 0.35, 95% CI: 0.23 to 0.54). At village level piped water supply was associated with higher Hb levels (β coefficient 0.61 g/dl, 95% CI: 0.39 to 0.82). Results from the NBM 200 reported wide agreement levels in the Bland-Altman analysis (mean difference of -2.70 g/dl, 95% CI: -2.84 to -2.55) demonstrating an overestimation of Hb by the NBM 200 compared to Sahli’s hemometer. The NBM 200 showed low sensitivity (23.6%) and moderate specificity (61.8%) for the diagnosis of anaemia in the adolescent population. Findings from pregnant women showed high anaemia prevalence (77%). Of 51 factors assessed in the study, three were associated with maternal anaemia. Increased risk of anaemia was seen in women with consanguineous marriages (OR 2.41, 95% CI: 1.16 to 5.01). Post-delivery data from full-term singleton live births showed the prevalence of LBW babies was 7%. Consanguineous marriage was a major risk of LBW babies in our study population (OR 5.68, 95% CI: 1.58 to 20.32). Village level risk factors showed lower likelihood of maternal anaemia with regular access to government nurses (OR 0.48, 95% CI: 0.25 to 0.93). The NBM 200 validation showed overestimation of Hb levels and underestimation of anaemia. Bland-Altman analysis showed a mean difference of -1.8 g/dl (95% CI: -2.06 to -1.71) indicating a systematic overestimation by the NBM 200 compared to venous Hb measurements. The device showed low sensitivity (33.7%) but high specificity (91.8%) for the diagnosis of anaemia in the pregnant woman population. The 32 years of longitudinal birth registry data showed a significant increase in the sex ratio at live birth from 1980 to 2004, and then a subsequent decrease in sex ratio. The annual state male:female sex ratio of Maharashtra increased from a baseline of 1.11 in 1980 to a maximum value of 1.23 in 2003, before decreasing to 1.16 in 2011. This represented an increase in the annual sex ratio at live birth from 1980 to 2004 of 0.005 units per year (p < 0.001), and a decrease of 0.009 units per year after 2004 (p < 0.01). The increase in the sex ratio was consistent with the hypothesis of both increasing availability and acceptability of ultrasound scanning during this period, enabling foeticide of females in utero. The probable cause for the decrease in sex ratio after 2004 is likely to be due to the strengthening of the legislation banning sex-specific foeticide.

Acute myeloid leukaemia (AML) is a heterogeneous myeloid malignancy characterized by clonal expansion of abnormal/immature hematopoietic precursor cells in the bone marrow. A side compartment in the BM niche consists of abnormal, quiescent cells, which are called dormant leukemic initiating cells (DLICs). Patients with AML tend to respond well to remission induction chemotherapy, but relapse is common because current therapies cannot completely eradicate leukemic cells. It is widely accepted that CD34+CD38− DLICs are more resistant to chemotherapy and that they contribute to drug resistance and relapse of AML to a greater extent than progenitor CD34+CD38+ cells. DLICs have been extensively characterised, but they remain a critical area of investigation for clinical research because of the low prevalence of DLICs and similarity to normal HSCs. A model of dormancy in vitro that shows most of the features of DLICs had previously been established in the Nottingham Haematology Group. This study used this model and aimed to investigate whether the response to DNA damage was different in dormancy-enriched cells compared to cycling leukemic cells following chemotherapy. The amount of DNA damage was assessed up to 24 hours pre- and post- drug treatment using the neutral Comet assay. Lower levels of damage were observed in dormancy-enriched cells following etoposide (ETO) treatment at 4 hours (p = 0.04), although this switched at the 24 hour time point where accumulated DNA double-stranded breaks (DSBs), in dormancy-enriched KG1a cells were associated with a higher percentage of viable cells. DNA damage response cascade markers in both dormancy-enriched and cycling cells showed phosphorylation by flow cytometry (phospho-H2AX139, pATM-S1981, H2AX142, and pChk-Thr68) in response to conventional AML chemotherapy. Significantly lower levels of cleaved PARP-Asp214 and active caspase 3 were observed in dormancy-enriched cells treated with ara-c (p = 0.0001) or ETO (p = 0.0001) at 24 hours, strongly indicating that survival responses are activated in dormancy-enriched cells. Induction of 53BP1 foci, the hallmark of non-homologous end joining (NHEJ) was observed following treatment with ara-c (p = 0.038) and ETO (p = 0.049) in dormancy-enriched cells, indicating the NHEJ repair pathway is the preferred mechanism for DSB repair. At the molecular level, BTG2 expression was involved in the DNA damage response. Significant induction of BTG2 was detected in cycling treated cells with ETO for 24 hours. In conclusion, this study provides evidence that phosphorylation of H2AX139 and H2AX142 is a key response marker that may explain the mechanism underlying the drug resistance of DLICs and induction of repair. Therefore, results of this study may help in devising novel treatment strategies for AML that target H2AX142 of DLICs to permanently eradicate all leukemic cells and improve overall survival.

Background: Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a haematopoietic growth factor and a pro-inflammatory cytokine produced by T cells and other immune cells. Recent evidence suggests that GM-CSF plays an important role in multiple sclerosis (MS) pathogenesis. Few recent studies have detected GM-CSF expression by immune cells in MS. In this thesis, the expression of GM-CSF and its receptor by different subtypes of peripheral blood mononuclear cells (PBMCs) in MS was investigated. In addition, GM-CSF regulation was studied in the above-mentioned cells in MS. Finally, GM-CSF neutralization was performed in a phase Ib clinical trial, and some immune-related effects were investigated. Aims: To evaluate the expression of GM-CSF and its receptor by PBMC subsets in MS; to determine the key factors regulating their expression by PBMC subsets in MS; to detect the differentiation of helper T cells producing GM-CSF (Th-GM) in MS patients, and to detect the frequency of immune cells after GM-CSF neutralization in MS in vivo. Subjects and Methods: Patients were mainly untreated relapsing-remitting MS (RRMS) during remission stage, and some were MS patients during a relapse. Healthy controls were also enrolled. All subjects consented to participation in the study before donating peripheral blood. PBMCs were isolated using Ficoll density gradient centrifugation. Flow cytometry and q-PCR were used to detect the expression of GM-CSF and its receptor. Multiplex bead assay was used to quantify GM-CSF with other pro-inflammatory and anti-inflammatory cytokines. Results: The frequency of stimulated GM-CSF-expressing cells (helper T (Th), cytotoxic T (Tc), monocytes, NK cells, and B cells) is significantly higher in the mixed PBMC population of untreated RRMS patients when compared to healthy volunteers. The frequency of Th1 cells expressing GM-CSF was higher in MS patients than healthy controls. The expression of GM-CSF by isolated and stimulated NK cells was not different in MS patients and controls. PBMC culture supernatants were shown to contain significantly higher concentrations of IL-2, IL-12, IL-1β, and GM-CSF in MS patients than controls. Blocking IL-2 and IL-12 significantly reduced GM-CSF expression by Tc, NK, and B cells in MS patients, but not in healthy controls. MS patients during relapse had significantly higher frequency of Th-GM (CD3+CD8-IL-17-IFN-γ-IL-3+GM-CSF+) cells than healthy controls. EBV infected B cells expressed GM-CSF receptor in less frequency than non-infected B cells. In vivo GM-CSF neutralization in MS patients resulted in significant reduction in the frequency of CD8+ T cells and CD4+CD45RA+CD25++ (naïve) Tregs and an increase in CD4+CD35+foxp3 (total) Tregs. Conclusions: Th1 (and Th in general), Tc, monocytes, NK and B cells are all high producers of GM-CSF in MS. IL-2 and IL-12 are the main regulators of GM-CSF expression by Tc, NK, and B cells in MS patients. GM-CSF and its receptor may not be major survival or proliferation factors for EBV infected B cells. The newly identified Th-GM cells were detected in higher frequency in MS patients during relapse, which may suggest a new source for GM-CSF production in MS. The recent safety, tolerability, and immune-related results of GM-CSF neutralization in MS are encouraging. Therefore, GM-CSF is a potential therapeutic target in MS.

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Background Stem cell transplantation is routinely used in the treatment of haematological malignancy. However, it is an intensive treatment frequently associated with a considerable deterioration in patients' wellbeing and prolonged recovery. Research into the amelioration of the negative biopsychosocial factors associated with stem cell transplantation is essential, facilitating nurses and other health professionals to provide the best possible care to individuals who have been treated with a stem cell transplant. Study Design 58 patients who had been treated with a stem cell transplant were recruited and randomly allocated to either a health profession led rehabilitation programme or a self managed rehabilitation programme. Follow-up measures (SF-36, QHQ, Graham and Longman QoL Scale and SWT) were taken at three and six months. Qualitative interviews were conducted with 15 of the 58 participants and with five members of staff. Results In all dimensions of the SF-36 the scores of patients recovering after stem cell transplantation indicated poorer health status in comparison to UK population norms supporting the need for rehabilitation services for this patient group. No evidence of a difference between the two modes of rehabilitation was observed for any of the trial outcomes. The qualitative interview data indicated that from patients' and staff's perspectives there was scope for improvement in the rehabilitation programmes. The interview data also highlighted that staff were concerned that the trial conditions had negatively impacted the provision of rehabilitation, drawing attention to the difficulties inherent in the evaluation of complex interventions. Conclusions Existing literature, the SF-36 data collected in this study and the experiences of both patients and health professionals expressed in the qualitative component of this study all indicate that rehabilitation is an important component of health care following stem cell transplantation. However, the rehabilitation needs and desires of this patient group are complex and therefore any rehabilitation programme must reflect this complexity. Enabling patients to work collaboratively with health professionals in determining the most appropriate provision of rehabilitation may result in enhanced levels of patient satisfaction with rehabilitation services. However, the efficacy of rehabilitation following stem cell transplantation remains unproven and the provision and evaluation of patient centred rehabilitation raises numerous practical and methodological challenges.