重离子耗散反应激发函数振荡结构的实验研究

直到八十年代中期，人名才发现耗散反应激发函数中存在振荡结构这种新现象。通过对激发函数振荡结构能量自关联函数的研究是获得复合核能级宽度的一个重要手段，Brick推广了Ericson的复合核统计理论，并成功地用于分析耗散反应激发函数振荡结构的研究，提取相应的能量相关宽度Г。本文报道了19F+51V耗散反应激发函数振荡结构的实验研究结果，用能级部分重叠模型对角动量相干引起的截面涨落、能量自关联函数进行了计算分析。实验中采用ΔE-E粒子鉴别方法和飞行时间TOF测量技术队102.25Mev~109.5Mev19F+51V反应类弹产物同时进行电荷和质量鉴别。首次在各个元素、同量异位素（质量数A为常数）和同位素的耗散反应激发函数中观察到振荡结构，并进一步证实了反应产物的各个出射道之间存在着相关。检验了用小角度弹性散射计数做相对归一对激发函数振荡结构研究可能造成的影响。分别采用能量自关联函数方法和谱密度方法提取了各个激发函数的能量相关宽度Г，其值大小为~350kev，并与出射道的电荷数Z、质量数A和中质比N/Z有很大的依赖关系，表明出射产物与入射弹核的差别越打所需的反应时间久越长。首次得到了Г随N/Z值变化的趋势，Г随N/Z的分布为Gauss型，通过分析分布的宽度得到其大小随相互作用时间的增长而线性增大的结果，并进一步提取了电荷扩散系数，证实了反应系统已达到电荷平衡。Г的数值随出射角的增大有减小的趋势。双核系统的转动造成了Г随出射角的变化关系，实验提取的双核系统平均角速度发生了较强的阻尼。用能级部分重叠模型在适当的精度内对激发函数和能力自关联函数进行了模拟。计算分析说明入射道的动能大多数转化为双核系统的转动能，只有较少部分转化为双核系统的内禀激发能，双核系统被激发到能级密度不太大的区域，能级之间的部分重叠引起截面的振荡行为。入射道角动量的相互干涉、双核系统能级的部分重叠和出射道的相互关联使得耗散反应的激发函数表现出其特有的规律性。

SSC120KW高频发射机改进可行性方案研究

我所SSC120KW高频发射机是HIRFL（Heavy Ion Research Facility, LanZhou）的一个重要组成部分，长期的调机以及运行经验表明：两台发射机存在调机程序复杂运行不稳定、运行维护费用高等缺点。本论文讨论一种改进方案，并且介绍了大功率放大器的设计方法。 该方案采用国产电子管TH537作为功率放大管，槽路电感固定，采用一个可变电容调谐，另一个可变电容调整负载。槽路结构简单，调整方便，同时槽路元件较现在方案少，能节省建造和维护费用。论文中详细介绍了电子管特性的计算，槽路得设计方法，并对中和与消除寄生振荡的方法作了扼要的介绍。

HIRFL束流相位测量系统的研究和改进

兰州重离子研究装置（Heavy Ion Research Facility at LanZhou,HIRFL）是由一台1.7m扇聚焦回旋加速器（SFC）与一台能量常数K=450的分离扇回旋加速器（SSC）组成的加速器系统。束流相位测量系统式束流诊断系统中的一个重要部分，对等时场优化等具有十分重要的作用。HIRFL束流中心相位测量系统于1985年完成了桌面实验，但由于测量精度低，现场抗干扰能力差，一直未能投入使用。 本课题的目的就是找出原系统存在的问题，逐一解决，以便提高其可靠性与测量精度，达到设计要求。 在通过一系列的电子学部分改进和SSC中心相位探针改造之后，于1995年7月第一次测出了SSC中心束流相位。此后，逐步完善改进电子学硬件部分，同时全新设计了系统控制软件，提高了在SFC和SSC上束流相位的测量精度，终于使该系统达到了测量精度为±2.75°~±1.5°的水平。 本论文第一、二章阐述了束流中心相位测量原理和HIRFL束流中心相位测量系统的工作原理，这是本工作的基础和出发点。 在本论文的第三章中，分析了原系统中存在的主要问题。实践使用中可以看出原系统灵敏度低，抗干扰能力差，可靠性差，测量精度低。为了定量判断系统存在的问题，我们设计了自检系统。利用自检系统我们测出原系统测量精度为±6°，且检测出原系统sin，cos正交输出异常。同时测量了原系统多路开关串话量，大多数道与道之间高于最低要求的-40dB，最差只有-20dB，证明存在严重的道间干扰。 本文的第四章中，针对原系统的可靠性差和精度低的两个问题，采取了硬件与软件两方面的各种措施，对系统加以改进。首先，为了提高系统的可靠性，必须提高系统抗干扰能力。为此，我们进行了两个方面的工作，一是根据我们现有条件自行设计了一种新的电缆电子学长度校正方法，大大减少了电缆间相差（小于0.3°），从而有效地提高了系统的抗干扰能力。这种方法不但可以用来校正相同介质电缆，而且可以用来校正不同介质电缆的电子学长度。二是设计了新的信号预选器，其串话量达到约-70dB，并完善了电磁屏蔽，使其完全达到了设计要求。在改进硬件的同时，为了提高可靠性，重新设计了系统控制软件。新的软件测量数据可靠，漏报概率为10-3，操作简便直观，并易于发展。其次，我们工作的重点是提高测量精度。根据自检结果，我们采取了如下措施： （1） 通过对自检数据进行分析，并与理论分析比较，发现问题主要存在于90°移相电路中。而其后的检测证实了这一点。重新调整90°移相电路，并对90°电缆相移进行了精确的校正，从原81.5°校正为90.6°，从而使系统的精度从±6°提高到±4°。 （2） 通过自检数据和理论分析发现鉴相器存在输出增益不平衡，在解决问题之后使系统测量精度达到了±2.75°~±1.5°。 在本文的第五章中，对加速器运行时的中心束流相位测量结果进行了详细分析。结果证明，测量数据可靠，能正确反映出磁场变化情况，测量重复误差达到了±0.5°，从而说明改进后的中心束流相位测量系统性能良好，达到了设计指标。

SFC控制系统软件设计

兰州重离子研究装置（Heavy Ion ReSearch Facility at Lanzhou, HIRFL）是我们研究所得一个大型实验装置，它包括SFC和SSC两个加速器和两条束运线。本论文比较系统地介绍了HIRFL束流诊断系统的改造和SFC分布式控制系统的设计。 在第一章中，简单介绍了国际加速器控制系统的现状和HIRFL控制系统中存在的问题。在第二章一般性地阐述了描述束流品质的各个参数，这些参数的测量原理以及测量这些参数的装置。本论文的第三章详细叙述了HIRFL束流诊断系统的改造方法、过程和结果，结果准确可靠，人机界面非常友好，给调束带来很大的方便。第四章介绍了计算机网络的基本概念，描述了在选用TCP/IP协议的条件下，利用Socket（套接字）实现Windows环境下的实时网络通信的具体过程和步骤，其中参与通信的双方是以客户机和服务器的形式存在的。第五章讲述了SFC分布式控制系统的实现，并在实时网络通信的基础上完成了ECR源扫谱程序和I/O级的网络通信程序。 论文的最后一章，介绍了对HIRFL束运线进行优化控制的一个设想，利用系统辨识的方法可以得到束运线的数学模型，并提供了自适应控制的实现细节，这也是作者对实现HIRFL优化控制的一个愿望。

Recent developments in intelligent biomedical polymers

Intelligent polymers or stimuli-responsive polymers may exhibit distinct transitions in physical-chemical properties, including conformation, polarity, phase structure and chemical composition in response to changes in environmental stimuli. Due to their unique 'intelligent' characteristics, stimuli-sensitive polymers have found a wide variety of applications in biomedical and nanotechnological fields. This review focuses on the recent developments in biomedical application of intelligent polymer systems, such as intelligent hydrogel systems, intelligent drug delivery systems and intelligent molecular recognition systems. Also, the possible future directions for the application of these intelligent polymer systems in the biomedical field are presented.

Carboxyl-modified single-walled carbon nanotubes selectively induce human telomeric i-motif formation

As the leading nanodevice candidate, single-walled carbon nano-tubes (SWNTs) have potential therapeutic applications in gene therapy and novel drug delivery. We found that SWNTs can inhibit DNA duplex association and selectively induce human telomeric i-motif DNA formation by binding to the 5'-end major groove under physiological conditions or even at pH 8.0. SWNT binding to telomeric DNA was studied by UV melting, NMR, S1 nuclease cleavage, CD, and competitive FRET methods. These results suggest that SWNTs might have the intriguing potential to modulate human telomeric DNA structures in vivo, like biologically relevant B-A and B-Z DNA transitions, which is of great interest for drug design and cancer therapy.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>10(13) different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.

Zebrafish thymidylate synthase binds to its own mRNA in vitro and in vivo

Thymidylate synthase (TS), an essential enzyme for DNA de novo synthesis, is a critical therapeutic target in cancer therapy. Previous study has shown that TS was able to bind to its own mRNA in human and E.coli, resulting in translational repression. Zebrafish is the best animal model for vertebrate study. In order to study the regulatory mechanism of zebrafish TS, the enzyme were expressed in E. coli BL21 (DE3) and it was purified to homogeneity. Electrophoretic mobility shift assay (EMSA) was used to detect the interaction of zebrafish TS protein and its own TS transcript in vitro and the results showed that zebrafish TS could bound with its own mRNA specifically. Further study revealed that zebrafish TS was able to interact with its own mRNA in vivo using immunoprecipitation : RT-PCR technique. The results provide evidence that zebrafish may be developed as an useful model for studying the anti-metabolism agents.

Nanomedicinas para o Cancro

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

Optimisation of immune effector function within the tumour microenvironment

Cancer represents a leading of cause of death in the developed world, inflicting tremendous suffering and plundering billions from health budgets. The traditional treatment approaches of surgery, radiotherapy and chemotherapy have achieved little in terms of cure for this deadly disease. Instead, life is prolonged for many, with dubious quality of life, only for disease to reappear with the inevitable fatal outcome. “Blue sky” thinking is required to tackle this disease and improve outcomes. The realisation and acceptance of the intrinsic role of the immune system in cancer pathogenesis, pathophysiology and treatment represented such a “blue sky” thought. Moreover, the embracement of immunotherapy, the concept of targeting immune cells rather than the tumour cells themselves, represents a paradigm shift in the approach to cancer therapy. The harnessing of immunotherapy demands radical and innovative therapeutic endeavours – endeavours such as gene and cell therapies and RNA interference, which two decades ago existed as mere concepts. This thesis straddles the frontiers of fundamental tumour immunobiology and novel therapeutic discovery, design and delivery. The work undertaken focused on two distinct immune cell populations known to undermine the immune response to cancer – suppressive T cells and macrophages. Novel RNAi mediators were designed, validated and incorporated into clinically relevant gene therapy vectors – involving a traditional lentiviral vector approach, and a novel bacterial vector strategy. Chapter 2 deals with the design of novel RNAi mediators against FOXP3 – a crucial regulator of the immunosuppressive regulatory T cell population. Two mediators were tested and validated. The superior mediator was taken forward as part of work in chapter 3. Chapter 3 deals with transposing the RNA sequence from chapter 2 into a DNA-based construct and subsequent incorporation into a lentiviral-based vector system. The lentiviral vector was shown to mediate gene delivery in vitro and functional RNAi was achieved against FOXP3. Proof of gene delivery was further confirmed in vivo in tumour-bearing animals. Chapter 4 focuses on a different immune cell population – tumour-associated macrophages. Non-invasive bacteria were explored as a specific means of delivering gene therapy to this phagocytic cell type. Proof of delivery was shown in vitro and in vivo. Moreover, in vivo delivery of a gene by this method achieved the desired immune response in terms of cytokine profile. Overall, the data presented here advance exploration within the field of cancer immunotherapy, introduce novel delivery and therapeutic strategies, and demonstrate pre-clinically the potential for such novel anti-cancer therapies.

Fertility preservation: Successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin's disease

Cryopreservation of ovarian tissue is now offered as an experimental procedure to preserve the fertility of young patients with a high risk for premature ovarian failure resulting from cancer therapy. This is the only available option to preserve the fertility of prepubertal patients treated with gonadotoxic chemotherapy. At present, thousands of patients all over the world have undergone this procedure with the hope of later restoring their fertility. Although the efficiency of the transplantation of cryopreserved ovarian tissue to restore ovarian function has been established, reports of pregnancy are still very scarce. Here, we describe the second published full-term spontaneous pregnancy after an orthotopic and heterotopic transplantation of cryopreserved ovarian tissue in a 31-year-old woman previously treated by conditioning therapy for bone marrow transplantation for Hodgkin's disease. This birth gives compelling evidence for the graft origin of the gamete and confirms the efficacy of ovarian tissue transplantation in restoring human natural fertility after oncological treatment. This case report stresses the importance of proposing the ovarian tissue cryopreservation procedure to all young patients who require potentially sterilizing treatment, with all alternative options to preserve fertility being duly taken into consideration.

Portable, Fiber-Based, Diffuse Reflection Spectroscopy (DRS) Systems for Estimating Tissue Optical Properties.

Steady-state diffuse reflection spectroscopy is a well-studied optical technique that can provide a noninvasive and quantitative method for characterizing the absorption and scattering properties of biological tissues. Here, we compare three fiber-based diffuse reflection spectroscopy systems that were assembled to create a light-weight, portable, and robust optical spectrometer that could be easily translated for repeated and reliable use in mobile settings. The three systems were built using a broadband light source and a compact, commercially available spectrograph. We tested two different light sources and two spectrographs (manufactured by two different vendors). The assembled systems were characterized by their signal-to-noise ratios, the source-intensity drifts, and detector linearity. We quantified the performance of these instruments in extracting optical properties from diffuse reflectance spectra in tissue-mimicking liquid phantoms with well-controlled optical absorption and scattering coefficients. We show that all assembled systems were able to extract the optical absorption and scattering properties with errors less than 10%, while providing greater than ten-fold decrease in footprint and cost (relative to a previously well-characterized and widely used commercial system). Finally, we demonstrate the use of these small systems to measure optical biomarkers in vivo in a small-animal model cancer therapy study. We show that optical measurements from the simple portable system provide estimates of tumor oxygen saturation similar to those detected using the commercial system in murine tumor models of head and neck cancer.

Hepatocyte growth factor (HGF) protects c-met-expressing Burkitt''s lymphoma cell lines from apoptotic death induced by DNA damaging agents

The relative sensitivity of neoplastic cells to DNA damaging agents is a key factor in cancer therapy. In this paper, we show that pretreatment of Burkitt's lymphoma cell lines expressing the c-met protooncogene with hepatocyte growth factor (HGF) protects them from death induced by DNA damaging agents commonly used in tumour therapy. This protection was observed in assays based on morphological assessment of apoptotic cells and DNA fragmentation assays. The protection was dose- and time-dependent — maximal protection requiring pre-incubation with 100 ng/ml HGF for 48 h. Western blotting analysis and flow cytometric studies revealed that HGF inhibited doxorubicin- and etoposide-induced decreases in the levels of the anti-apoptotic proteins Bcl-XL, and to a lesser extent Bcl-2, without inducing changes in the pro-apoptotic Bax protein. Overall, these studies suggest that the accumulation of HGF within the microenvironment of neoplastic cells may contribute to the development of a chemoresistant phenotype.