Genetic diversity of the subterranean Gansu zokor in a semi-natural landscape

We studied the relationship between genetic diversity of the subterranean Gansu zokor Myospalax cansus and habitat variability in the Loess Plateau, Qinghai Province, China. We used a combination of geographic information systems and molecular techniques to assess the impact of habitat composition and human activities on the genetic diversity of zokor populations in this semi-natural landscape. Although they occurred relatively infrequently in the landscape, woodland and high-coverage grassland habitats were the main positive contributors to the genetic diversity of zokor populations. Rural residential land, plain agricultural land and low-coverage grassland had a negative effect on genetic diversity. Hilly agricultural land and middle-coverage grassland had little impact on zokor genetic diversity. There were also interactions between some habitat types, that is, habitat types with relatively better quality together promoted conservation of genetic diversity, while the interaction between (among) bad habitat types made situations worse. Finally, habitat diversity, measured as patch richness and Shannon's diversity index, was positively correlated with the genetic diversity. These results demonstrated that: (1) different habitat types had different effects on the genetic diversity of zokor populations and (2) habitat quality and habitat heterogeneity were important in maintaining genetic diversity. Habitat composition was closely related to land use thus emphasizing the importance of human activities on the genetic diversity of subterranean rodent populations in this semi-natural landscape. Although the Gansu zokor was considered to be a pest species in the Loess Plateau, our study provides insights for the management and conservation of other subterranean rodent species.

Study of interaction between drug enantiomers and serum albumin by capillary electrophoresis

The interaction between drugs and human serum albumin (HSA) was investigated by capillary electrophoresis (CE). It involves stereoselectivity, drug displacement and synergism effects. Under protein-drug binding equilibrium, the unbound concentrations of drug enantiomers were measured by frontal analysis (FA). The stereoselectivity of verapamil (VER) binding to HSA was proved by the different free fractions of two enantiomers. In physiological pH (7.4, ionic strength 0.17 phosphate buffer) when 300 mu M (+/-) VER were equilibrated with 500 mu M HSA, the concentration of unbound S-VER was about 1.7 times its antipode. The binding constants of two enantiomers, KR-VER and KS-VER, were 2670 and 850 M-1, respectively. However, no obvious stereoselective binding of propranolol (PRO) to HSA was observed. Trimethyl-beta-cyclodextrin (45 mM) was used as a chiral selector in pH 2.5 phosphate buffer. Several drug systems were studied by the method. When ibuprofen (IBU) was added into VER-HSA solution. R-VER was partially displaced while S-VER was not displaced at all. A binding synergism effect between bupivacaine (BUP) and verapamil was observed and further study suggested that verapamil and bupivacaine occupy different binding site of HSA (site II and site III, respectively).

A transcriptase reversa como alvo terapêutico em doenças retrovirais

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Farmacogenética/Farmacogenómica

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Exponential sensitivity of noise-driven switching in genetic networks

Cells are known to utilize biochemical noise to probabilistically switch between distinct gene expression states. We demonstrate that such noise-driven switching is dominated by tails of probability distributions and is therefore exponentially sensitive to changes in physiological parameters such as transcription and translation rates. However, provided mRNA lifetimes are short, switching can still be accurately simulated using protein-only models of gene expression. Exponential sensitivity limits the robustness of noise-driven switching, suggesting cells may use other mechanisms in order to switch reliably.

On the Effectiveness of Genetic Search in Combinatorial Optimization

In this paper, we study the efficacy of genetic algorithms in the context of combinatorial optimization. In particular, we isolate the effects of cross-over, treated as the central component of genetic search. We show that for problems of nontrivial size and difficulty, the contribution of cross-over search is marginal, both synergistically when run in conjunction with mutation and selection, or when run with selection alone, the reference point being the search procedure consisting of just mutation and selection. The latter can be viewed as another manifestation of the Metropolis process. Considering the high computational cost of maintaining a population to facilitate cross-over search, its marginal benefit renders genetic search inferior to its singleton-population counterpart, the Metropolis process, and by extension, simulated annealing. This is further compounded by the fact that many problems arising in practice may inherently require a large number of state transitions for a near-optimal solution to be found, making genetic search infeasible given the high cost of computing a single iteration in the enlarged state-space.

Genetically Generated Neural Networks I: Representational Effects

This paper studies several applications of genetic algorithms (GAs) within the neural networks field. After generating a robust GA engine, the system was used to generate neural network circuit architectures. This was accomplished by using the GA to determine the weights in a fully interconnected network. The importance of the internal genetic representation was shown by testing different approaches. The effects in speed of optimization of varying the constraints imposed upon the desired network were also studied. It was observed that relatively loose constraints provided results comparable to a fully constrained system. The type of neural network circuits generated were recurrent competitive fields as described by Grossberg (1982).

FLT3-driven redox signalling in acute myeloid leukaemia

Acute myeloid leukaemia refers to cancer of the blood and bone marrow characterised by the rapid expansion of immature blasts of the myeloid lineage. The aberrant proliferation of these blasts interferes with normal haematopoiesis, resulting in symptoms such as anaemia, poor coagulation and infections. The molecular mechanisms underpinning acute myeloid leukaemia are multi-faceted and complex, with a range of diverse genetic and cytogenetic abnormalities giving rise to the acute myeloid leukaemia phenotype. Amongst the most common causative factors are mutations of the FLT3 gene, which codes for a growth factor receptor tyrosine kinase required by developing haematopoietic cells. Disruptions to this gene can result in constitutively active FLT3, driving the de-regulated proliferation of undifferentiated precursor blasts. FLT3-targeted drugs provide the opportunity to inhibit this oncogenic receptor, but over time can give rise to resistance within the blast population. The identification of targetable components of the FLT3 signalling pathway may allow for combination therapies to be used to impede the emergence of resistance. However, the intracellular signal transduction pathway of FLT3 is relatively obscure. The objective of this study is to further elucidate this pathway, with particular focus on the redox signalling element which is thought to be involved. Signalling via reactive oxygen species is becoming increasingly recognised as a crucial aspect of physiological and pathological processes within the cell. The first part of this study examined the effects of NADPH oxidase-derived reactive oxygen species on the tyrosine phosphorylation levels of acute myeloid leukaemia cell lines. Using two-dimensional phosphotyrosine immunoblotting, a range of proteins were identified as undergoing tyrosine phosphorylation in response to NADPH oxidase activity. Ezrin, a cytoskeletal regulatory protein and substrate of Src kinase, was selected for further study. The next part of this study established that NADPH oxidase is subject to regulation by FLT3. Both wild type and oncogenic FLT3 signalling were shown to affect the expression of a key NADPH oxidase subunit, p22phox, and FLT3 was also demonstrated to drive intracellular reactive oxygen species production. The NADPH oxidase target protein, Ezrin, undergoes phosphorylation on two tyrosine residues downstream of FLT3 signalling, an effect which was shown to be p22phox-dependent and which was attributed to the redox regulation of Src. The cytoskeletal associations of Ezrin and its established role in metastasis prompted the investigation of the effects of FLT3 and NADPH oxidase activity on the migration of acute myeloid leukaemia cell lines. It was found that inhibition of either FLT3 or NADPH oxidase negatively impacted on the motility of acute myeloid leukaemia cells. The final part of this study focused on the relationship between FLT3 signalling and phosphatase activity. It was determined, using phosphatase expression profiling and real-time PCR, that several phosphatases are subject to regulation at the levels of transcription and post-translational modification downstream of oncogenic FLT3 activity. In summary, this study demonstrates that FLT3 signal transduction utilises a NADPH oxidase-dependent redox element, which affects Src kinase, and modulates leukaemic cell migration through Ezrin. Furthermore, the expression and activity of several phosphatases is tightly linked to FLT3 signalling. This work reveals novel components of the FLT3 signalling cascade and indicates a range of potential therapeutic targets.

Investigation of the genetic susceptibility to osteoporosis in the Irish population

Osteoporosis is a complex skeletal disorder characterized by compromised bone strength. Variation in bone mineral density (BMD) is a contributing factor. The aim of this research as to select informative single nucleotide polymorphisms (SNPs) in potential candidate genes from loci suggestively linked to BMD variation for fine mapping. The gene regulated by oestrogen in breast cancer 1 (GREB1), located at 2p25.1, was selected. GREB1 transcription is initiated early in the oestrogen receptor alpha regulated pathway. There was significant association between GREB1_03 and BMD variation at the lumbar spine and femoral neck (FN) in the discovery cohort. Significant association was observed between GREB1_04 and FN BMD in the replication cohort. The development and differentiation enhancing factor 2, the integrin cytoplasmic domain associated protein 1 and A-disintegrin and metalloprotease 17 were selected due to their respective roles in cell mobility and adhesion. There was no linkage or association observed between the Chr2 cluster SNPs and BMD. Two factors in bone remodelling are the attraction of bone cell precursors and endocrine regulation of the process, primarily through the action of parathyroid hormone (PTH). The C-C chemokine receptor type 3 (CCR3) encodes a CC chemokine receptor expressed in osteoclast precursors. The PTH receptor type 1 (PTHR1) encodes a G-protein coupled receptor for PTH. Association was observed between CCR3 haplotypes and BMD variation at the FN. There was no linkage or association observed between PTHR1 SNPs and BMD variation. Population genetic studies with complex phenotypes endeavour to elucidate the traits genetic architecture. This study presents evidence of association between GREB1 and BMD variation and as such, introduces GREB1 as a novel gene target for osteoporosis genetics studies. It affirms that common genomic variants in PTHR1 are not associated with BMD variation in Caucasians and supports the evidence that CCR3 may be contributing to BMD variation

P-glycoprotein inhibition as a strategy to increase drug delivery across the blood-brain barrier: focus on antidepressants

Depression is among the leading causes of disability worldwide. Currently available antidepressant drugs have unsatisfactory efficacy, with up to 60% of depressed patients failing to respond adequately to treatment. Emerging evidence has highlighted a potential role for the efflux transporter P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), in the aetiology of treatment-resistant depression. In this thesis, the potential of P-gp inhibition as a strategy to enhance the brain distribution and pharmacodynamic effects of antidepressant drugs was investigated. Pharmacokinetic studies demonstrated that administration of the P-gp inhibitors verapamil or cyclosporin A (CsA) enhanced the BBB transport of the antidepressants imipramine and escitalopram in vivo. Furthermore, both imipramine and escitalopram were identified as transported substrates of human P-gp in vitro. Contrastingly, human P-gp exerted no effect on the transport of four other antidepressants (amitriptyline, duloxetine, fluoxetine and mirtazapine) in vitro. Pharmacodynamic studies revealed that pre-treatment with verapamil augmented the behavioural effects of escitalopram in the tail suspension test (TST) of antidepressant-like activity in mice. Moreover, pre-treatment with CsA exacerbated the behavioural manifestation of an escitalopram-induced mouse model of serotonin syndrome, a serious adverse reaction associated with serotonergic drugs. This finding highlights the potential for unwanted side-effects which may occur due to increasing brain levels of antidepressants by P-gp inhibition, although further studies are needed to fully elucidate the mechanism(s) at play. Taken together, the research outlined in this thesis indicates that P-gp may restrict brain concentrations of escitalopram and imipramine in patients. Moreover, we show that increasing the brain distribution of an antidepressant by P-gp inhibition can result in an augmentation of antidepressant-like activity in vivo. These findings raise the possibility that P-gp inhibition may represent a potentially beneficial strategy to augment antidepressant treatment in clinical practice. Further studies are now warranted to evaluate the safety and efficacy of this approach.

HIF-1alpha role in oxygen-dependent radio- and chemosensitivity

Poor oxygenation (hypoxia) is a common characteristic of human solid tumours, and is associated with cell survival, metastasis and resistance to radio- and chemotherapies. Hypoxia-induced stabilisation of hypoxia-inducible factor-1α (HIF-1α) leads to changes in expression of various genes associated with growth, vascularisation and metabolism. However whether HIF-1α plays a causal role in promoting hypoxic resistance to antitumour therapies remains unclear. In this study we used pharmacological and genetic methods to investigate the HIF-1α contribution to radio- and chemoresistance in four cancer cell lines derived from cervical, breast, prostate and melanoma human tumours. Under normoxia or hypoxia (<0.2% or 0.5% oxygen) the cells were exposed to either a standard irradiation dose (6.2 Gy) or chemotherapeutic drug (cisplatin), and subsequent cell proliferation (after 7 days) was measured in terms of resazurin reduction. Oxygen-dependent radio- and chemosensitivity was evident in all wild type whereas it was reduced or abolished in HIF-1α (siRNA) knockdown cells. The effects of HIF-1α-modulating drugs (EDHB, CoCl2, deferoxamine to stabilise and R59949 to destabilise it) reflected both HIF-1α-dependent and independent mechanisms. Collectively the data show that HIF-1α played a causal role in our in vitro model of hypoxia-induced radioresistance whereas its contribution to oxygendependent sensitivity to cisplatin was less clear-cut. Although this behavior is likely to be conditioned by further biological and physical factors operating in vivo, it is consistent with the hypothesis that interventions directed at HIF-1α may improve the clinical effectiveness of tumour treatments.

Randomized, controlled trial investigating short-term health-related quality of life with doxorubicin and paclitaxel versus doxorubicin and cyclophosphamide as first-line chemotherapy in patients with metastatic breast cancer: European Organization for Research and Treatment of Cancer Breast Cancer Group, Investigational Drug Branch for Breast Cancer and the New Drug Development Group Study.

PURPOSE: To compare health-related quality of life (HRQOL) in patients with metastatic breast cancer receiving the combination of doxorubicin and paclitaxel (AT) or doxorubicin and cyclophosphamide (AC) as first-line chemotherapy treatment. PATIENTS AND METHODS: Eligible patients (n = 275) with anthracycline-naive measurable metastatic breast cancer were randomly assigned to AT (doxorubicin 60 mg/m(2) as an intravenous bolus plus paclitaxel 175 mg/m(2) as a 3-hour infusion) or AC (doxorubicin 60 mg/m(2) plus cyclophosphamide 600 mg/m(2)) every 3 weeks for a maximum of six cycles. Dose escalation of paclitaxel (200 mg/m(2)) and cyclophosphamide (750 mg/m(2)) was planned at cycle 2 to reach equivalent myelosuppression in the two groups. HRQOL was assessed with the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire C30 and the EORTC Breast Module at baseline and the start of cycles 2, 4, and 6, and 3 months after the last cycle. RESULTS: Seventy-nine percent of the patients (n = 219) completed a baseline measure. However, there were no statistically significant differences in HRQOL between the two treatment groups. In both groups, selected aspects of HRQOL were impaired over time, with increased fatigue, although some clinically significant improvements in emotional functioning were seen, as well as a reduction in pain over time. Overall, global quality of life was maintained in both treatment groups. CONCLUSION: This information is important when advising women patients of the expected HRQOL consequences of treatment regimens and should help clinicians and their patients make informed treatment decisions.

Estimating the magnitude of trastuzumab effects within patient subgroups in the HERA trial.

BACKGROUND: Trastuzumab (Herceptin(R)) improves disease-free survival (DFS) and overall survival for patients with human epidermal growth factor receptor 2 (HER2)-positive early breast cancer. We aimed to assess the magnitude of its clinical benefit for subpopulations defined by nodal and steroid hormone receptor status using data from the Herceptin Adjuvant (HERA) study. PATIENTS AND METHODS: HERA is an international multicenter randomized trial comparing 1 or 2 years of trastuzumab treatment with observation after standard chemotherapy in women with HER2-positive breast cancer. In total, 1703 women randomized to 1-year trastuzumab and 1698 women randomized to observation were included in these analyses. Median follow-up was 23.5 months. The primary endpoint was DFS. RESULTS: The overall hazard ratio (HR) for trastuzumab versus observation was 0.64 [95% confidence interval (CI) 0.54-0.76; P < 0.0001], ranging from 0.46 to 0.82 for subgroups. Estimated improvement in 3-year DFS in subgroups ranged from +11.3% to +0.6%. Patients with the best prognosis (those with node-negative disease and tumors 1.1-2.0 cm) had benefit similar to the overall cohort (HR 0.53, 95% CI 0.26-1.07; 3-year DFS improvement +4.6%, 95% CI -4.0% to 13.2%). CONCLUSIONS: Adjuvant trastuzumab therapy reduces the risk of relapse similarly across subgroups defined by nodal status and steroid hormone receptor status, even those at relatively low risk for relapse.

The effects of osmotic stress on the structure and function of the cell nucleus.

Osmotic stress is a potent regulator of the normal function of cells that are exposed to osmotically active environments under physiologic or pathologic conditions. The ability of cells to alter gene expression and metabolic activity in response to changes in the osmotic environment provides an additional regulatory mechanism for a diverse array of tissues and organs in the human body. In addition to the activation of various osmotically- or volume-activated ion channels, osmotic stress may also act on the genome via a direct biophysical pathway. Changes in extracellular osmolality alter cell volume, and therefore, the concentration of intracellular macromolecules. In turn, intracellular macromolecule concentration is a key physical parameter affecting the spatial organization and pressurization of the nucleus. Hyper-osmotic stress shrinks the nucleus and causes it to assume a convoluted shape, whereas hypo-osmotic stress swells the nucleus to a size that is limited by stretch of the nuclear lamina and induces a smooth, round shape of the nucleus. These behaviors are consistent with a model of the nucleus as a charged core/shell structure pressurized by uneven partition of macromolecules between the nucleoplasm and the cytoplasm. These osmotically-induced alterations in the internal structure and arrangement of chromatin, as well as potential changes in the nuclear membrane and pores are hypothesized to influence gene transcription and/or nucleocytoplasmic transport. A further understanding of the biophysical and biochemical mechanisms involved in these processes would have important ramifications for a range of fields including differentiation, migration, mechanotransduction, DNA repair, and tumorigenesis.

Gait and behavior in an IL1β-mediated model of rat knee arthritis and effects of an IL1 antagonist.

Interleukin-1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over-expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse-associated pathology. IL1β over-expression was induced in the right knees of 30 Wistar rats via intra-articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30 µl intra-articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65 µg/µl hIL1Ra, n = 7 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β-infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra-articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over-expression is associated limb hypersensitivity and gait compensation. Intra-articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space.