Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies including those of leukaemic origin. Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haematopoietic progenitor cells whose prognosis is particularly poor and where standard induction therapy has changed little over the past thirty years. This thesis evaluated the effects of AZD1152-hQPA (barasertib-hQPA), a highly selective inhibitor of aurora-B kinase, in AML cell lines and primary samples. Inhibition of phospho-Histone H3 (pHH3) on serine 10 can be used as a biomarker for AZD1152-hQPA activity and an assay was optimized to measure pHH3 in our cell lines and primary samples. AZD1152-hQPA inhibited pHH3 in our cell lines resulting in polyploid cells, apoptosis, and cell death, irrespective of cellular p53 status. Over-expression of the ATP-binding cassette (ABC) drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in AML. A cell line which over-expresses Pgp was developed by selecting for daunorubicin (DNR) resistance in OCI-AML3 cells. Pgp and also BCRP expressing AML cell lines were found to be resistant to AZD1152-hQPA and it was found that AZD1152-hQPA is effluxed by these transporters. pHH3 inhibition by low dose AZD1152-hQPA was seen in all of the primary samples tested with Pgp and BCRP positive samples being less sensitive. However, 50% inhibition of pHH3 by AZD1152-hQPA was achieved in 94.6% of these samples. The FLT3-ITD-expressing MOLM-13 and MV4-11 cell lines were particularly sensitive to AZD1152-hQPA. Internal tandem duplications (ITDs) within the FLT3 tyrosine kinase receptor are found in approximately 25% of AML patients and are associated with a poor prognosis. It was demonstrated that AZD1152-hQPA directly targets phosphorylated FLT3 in the FLT3-ITD cell lines along with inhibiting its downstream target pSTAT5. FLT3-ITD primary samples were particularly sensitive to clonogenic inhibition and pSTAT5 down-regulation after treatment with AZD1152-hQPA compared with FLT3 wild-type (WT) samples.

Background. Haematopoietic stem cell transplantation (HSCT) is an intensive procedure associated with psychological distress particularly during the first weeks (acute phase). Based on the self-regulatory model of adjustment to illness, a preparatory group intervention was developed aiming at alleviating distress by reducing negative perceptions of HSCT and fostering helpful coping. Aims. The present study aimed to evaluate the feasibility of delivering the intervention and of conducting a trial to assess its efficacy. It also aimed to explore the applicability of the self-regulatory model in HSCT. Methods. Participants were adults from consecutive referrals at two transplant centres. Half were randomised to the intervention and half to treatment as usual at each site. Psychological distress, HSCT perceptions, and coping were assessed at baseline (following consent), on transplant day, two weeks, and four weeks after transplantation. Results. Of 99 eligible patients, 45 consented. Main barriers included inability to consent prior to transplantation, competing priorities, being unwell, and long travel distance. Of 21 participants randomised to intervention, five attended. Main barriers included being unable to attend prior to transplantation and having competing priorities. Groups could not be held sufficiently frequently to enable attendance prior to transplantation, as randomising participants to the control group prevented sufficient accrual at each site. Anxiety peaked two weeks following transplantation but depression increased throughout the acute phase. Intervention effects were small but sample sizes for a full trial appeared feasible. Negative perceptions of HSCT and use of a range of coping styles (including styles considered helpful) predicted higher distress throughout the period. Conclusions. The findings revealed considerable barriers to delivering a group-based intervention and conducting a trial to assess its effectiveness. This highlighted a need for better integration with routine care and alternative trial procedures. However, the findings illustrated complex psychological needs during the acute phase of HSCT and the role of negative HSCT perceptions and unhelpful coping in underpinning distress.

Relapse in AML is thought to arise from dormant leukaemic cells that are characterised by low RNA synthesis activity, protected by the bone marrow (BM) niche, and may evade the effects of chemotherapeutic drugs. Our aim was to investigate agents which might be able to overcome chemoprotection by targeting the intrinsic apoptosis pathway. We developed in vitro assays to identify and characterise the dormant AML cells using combinations of markers, including the cell-division marker PKH26, leukaemia-associated phenotypes (LAPs), and dormancy markers. In a dormancy model based on 12-day AML/stroma co-culture, we have shown that the expression of some aberrant phenotypes can persist for several days. Also, after 12 days, some of the CD34+, PKH26high (dormant), and LAP+ (leukaemic) cells maintained their primitiveness and were still clonogenic. Furthermore, our chemosensitivity data showed that novel agents TG02, and BH3 mimetics ABT-737 and ABT-199, which inhibit the B-cell lymphoma 2 (BCL-2) family of anti-apoptotic molecules, could efficiently target BM niche-mediated chemoresistance, which is thought to be one of the main obstacles to traditional chemotherapy. We explored various candidate dormancy markers based on the low RNA, non-proliferative profile of dormant cells. Among those tested, the RNA synthesis marker Pyronin Y (PY), and an antibody to the transferrin receptor CD71 were the most reproducible in terms of marker expression and stability. We endeavoured to characterise cell dormancy on the molecular level by investigating gene expression in the PYlow (dormant) and PYhigh (proliferating) subsets and have obtained limited results. In summary, this study has identified and partly characterised dormant AML cells by development of in vitro assays, and has shown chemosensitivity to novel agents TG02, ABT-737 and ABT-199 in dormant leukaemia cells.

Neutrophils are recognised to play a pivotal role at the interface between the innate and adaptive immune responses following their rapid recruitment to inflamed tissues and lymphoid organs. Whilst neutrophil trafficking through blood vessels has been extensively studied, the molecular mechanisms regulating their migration into the lymphatic system are still poorly understood. This thesis therefore aimed to investigate the mechanisms involved in neutrophil migration across the lymphatic endothelium during TNF- or Complete Freund's Adjuvant + antigen (CFA+Ag)-induced inflammation of cremaster muscles in vivo. This work revealed that TNF- or CFA+Ag-stimulation induces a rapid but transient entry of tissue-infiltrated neutrophils into lymphatic vessels, a response associated with the regulation and redistribution of the lymphatic endothelial cell glycocalyx. Interestingly, antigen sensitisation resulted in the production of endogenous TNF within cremaster muscles. Using anti-TNF blocking antibodies and mice deficient in both TNF receptors (p55 and p75), endogenous TNF was demonstrated for the first time to be involved in priming and triggering the migration of neutrophils into tissue-associated lymphatic vessels upon antigen challenge. Additionally, the use of chimeric mice exhibiting neutrophils deficient in both TNFRs demonstrated that TNF directly acts on leukocytes to induce neutrophil migration into lymphatic vessels. Furthermore, the results show that TNF-induced migration of neutrophils into the lymphatic system occurs in a strictly CCR7-dependent manner; blocking CXCR4 or CXCL1 signalling does not affect this response. Finally, both TNF- or CFA+AG-stimulation induced ICAM-1 up-regulation on lymphatic vessels, allowing neutrophils to crawl along the lumen; a response that was demonstrated to be TNF-dependent. These results have provided new insights into the mechanisms that mediate neutrophil migration into lymphatic vessels and their subsequent crawling within these vessels during inflammation. In particular, a new role for TNF as a key regulator of these processes has been demonstrated. Taken together, this work has highlighted potential and effective targets to manipulate the role of neutrophils in adaptive immune responses in vivo.

No description available.

Plasma volume (PV) rises by up to 50% in normal pregnancy, a phenomenon associated with a favourable pregnancy outcome. A previous study of pregnant women with sickle cell (haemoglobin SS) disorder found that PV paradoxically contracts in late pregnancy. A cross-sectional study was performed to determine PV (Evans blue method) and volume regulatory hormones and electrolytes in pregnant women with haemoglobin (Hb) SS and in non-pregnant and Hb AA controls. PV rose in pregnant HbAA and was significantly correlated with plasma angiotensinogen. Non-pregnant Hb SS women had supranormal PV measurements and reduced glomerular filtration rate (GFR). Their PV did not rise in pregnancy and was not correlated with angiotensinogen. Their plasma renin concentration also failed to rise significantly by 36 weeks gestation and was significantly less than in Hb AA pregnancy although aldosterone concentration was raised as expected. A general vasoconstriction in pregnancy can cause inactivation of the renin-angiotensin system and could explain this, with aldosterone being elevated by non Angiotensin II dependent stimulation such as plasma potassium, which was significantly higher in the pregnant Hb SS women. Further studies demonstrating a deficiency of vasodilator substances in pregnant Hb SS women will strengthen this hypothesis.

Intraocular pressure (IOP) is the most important risk factor for glaucoma development and progression. Most anti-glaucoma treatments aim to lower IOP by enhancing aqueous humor drainage from the eye. Aqueous humor drainage occurs via well-characterized trabecular meshwork (TM) and uveoscleral (UVS) pathways, and the recently described ciliary lymphatics. The relative contribution of the lymphatic pathway to aqueous drainage is not known. We developed a sheep model to quantitatively assess lymphatic drainage along with TM and UVS outflows. Following intracameral injection of 125I-bovine serum albumin (BSA), lymph and blood samples were continuously collected. Lymphatic and TM drainage were quantitatively assessed by measuring 125I-BSA recovery. This quantitative sheep model enables assessment of relative contributions of lymphatic drainage (1.64% ± 0.89%), TM (68.86% ± 9.27%) and UVS outflows (19.87% ± 5.59%), and may help to better understand the effects of glaucoma agents on outflow pathways.

Intraocular pressure (IOP) is the most important risk factor for glaucoma development and progression. Most anti-glaucoma treatments aim to lower IOP by enhancing aqueous humor drainage from the eye. Aqueous humor drainage occurs via well-characterized trabecular meshwork (TM) and uveoscleral (UVS) pathways, and the recently described ciliary lymphatics. The relative contribution of the lymphatic pathway to aqueous drainage is not known. We developed a sheep model to quantitatively assess lymphatic drainage along with TM and UVS outflows. Following intracameral injection of 125I-bovine serum albumin (BSA), lymph and blood samples were continuously collected. Lymphatic and TM drainage were quantitatively assessed by measuring 125I-BSA recovery. This quantitative sheep model enables assessment of relative contributions of lymphatic drainage (1.64% ± 0.89%), TM (68.86% ± 9.27%) and UVS outflows (19.87% ± 5.59%), and may help to better understand the effects of glaucoma agents on outflow pathways.

Ph.D.

Orientador: Maria Salete Costa Gurgel / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T11:22:09Z (GMT). No. of bitstreams: 1 Rezende_LauraFerreirade_D.pdf: 9192920 bytes, checksum: b761ed1e8c55e161d7c896509a841411 (MD5) Previous issue date: 2008 / Resumo: O objetivo deste estudo foi descrever o padrão linfocintilográfico e avaliar as compensações linfáticas do membro superior no pós-operatório de câncer de mama com dissecção axilar. Sujeitos e métodos: Foram inicialmente analisadas 37 pacientes que realizaram a linfocintilografia pré-operatória, sendo que 32 delas realizaram também o estudo no membro contralateral. Através de critérios para homogeinezação da amostra, 23 pacientes tiveram seus estudos pré-operatórios comparados com a linfocintilografia realizada com 60 dias de pós-operatório. O protocolo de exame consistiu na realização de imagens estáticas de cada membro superior em semiflexão e do tórax, após 10 minutos, 1 e 2 horas da injeção subcutânea de 1 mCi (37 MBq) de dextran-99mTc no dorso da mão. Foram feitas análises comparativas da captação hepática do radiofármaco, do grau de captação dos linfonodos axilares (a ¿ acentuada, b ¿ moderada, c ¿ discreta e d - ausente) e da velocidade de aparecimento destes (I ¿ aos 10 minutos, II ¿ à 1 hora, III ¿ às 2 horas e IV ¿ não visíveis) antes e após a cirurgia. Resultados: Das pacientes analisadas no pré-operatório, apenas quatro apresentaram o padrão considerado normal (Ia) no estudo do membro ipsilateral e seis no contralateral, enquanto três apresentaram total comprometimento (IVd) no membro ipsilateral e duas no contralateral. Entre o grupo com 23 pacientes, duas foram classificadas como Ia no pós-operatório, que foi considerada a categoria ideal e três pacientes apresentaram classificação IVd. Comparando os padrões de drenagem no pré e pós-operatório foram encontradas pacientes com piora, pacientes que não apresentaram modificação e pacientes com melhora. Todas as pacientes apresentaram manutenção ou aumento da captação hepática após o tratamento cirúrgico. Conclusão: Este estudo encontrou alterações relevantes nas linfocintilografias pré e pós-operatórias, demonstrando a existência de diferenças funcionais do sistema linfático do membro superior. Alterações no padrão de drenagem linfática já podem ser percebidas com 60 dias de pós-operatório, assim como o indício de presença de anastomose linfo-venosa / Abstract: The aim of this study was to describe lymphoscintigraphic pattern and evaluate upper limb lymphatic compensation following breast cancer surgey with axillary dissection. Subjects and methods: From September 2006 to June 2007, 37 patients who had performed preoperative lymphoscintigraphy were initially analyzed in the Division of Gynecologic Oncology and Breast Pathology of the Women¿s Integral Healthcare Center at the Universidade Estadual de Campinas. Thirtytwo of these patients also performed contralateral limb study. Using criteria for sample homogenization, preoperative studies of 23 patients were compared to lymphoscintigraphy performed within 60 days of surgical treatment. The examination protocol consisted in performing static imaging of each upper limb in semi-flexion and thoracic imaging, after 10 minutes, and 1 and 2 hours after subcutaneous injection of 1 mCi (37 MBq) of Tc-99m-dextran into the dorsum of the hand. Comparative analyses of hepatic radiopharmaceutical uptake, velocity of axillary lymph node visualization (I ¿ visible at 10 minutes, II ¿ visible at 1 hour, III ¿ visible at 2 hours and IV ¿ not visible) and degree of nodal uptake (a ¿ marked, b ¿ moderate, c ¿ mild and d - absent) before and after surgery were made. Results: Of the patients analyzed preoperatively, only four (11%) had an optimal pattern (Ia) in the ipsilateral limb study and six (19%) had an optimal pattern in the contralateral limb study, while total lymphatic compromise (IVd) was observed in three (8%) patients in the ipsilateral limb and two (6%) patients in the contralateral limb. Among the group with 23 patients, two (9%) were classified postoperatively as Ia, which was considered the optimal category and three patients (13%) were classified as IVd. Comparing preoperative and postoperative drainage, 11 (48%) patients worsened, 5 (22%) patients showed no modification and 7 (30%) patients improved. All maintained or increased hepatic uptake after sugical treatment. Conclusion: This study encountered relevant changes in preoperative and postoperative lymphoscintigraphy evaluations, demonstrating the existence of functional differences in the upper limb lymphatic system. Alterations in lymphatic drainage patterns can already be perceived within 60 days of surgery, as well as signs of lymphovenous shunts / Doutorado / Ciencias Biomedicas / Doutor em Tocoginecologia