趋磁细菌AMB-1培养条件优化及磁小体变化过程研究

趋磁细菌(Magnetotactic bacteria)的研究是国际微生物学研究热点之一。趋磁细菌体内含有纳米单磁畴的氧化铁/硫化铁(Fe3O4或Fe3S4)晶体，称为磁小体。由于趋磁细菌营养条件要求苛刻，在环境中需要微好氧条件，且营养类型属于化能自养，使得培养趋磁细菌时常遇到问题。 本研究首先通过正交试验优化趋磁细菌AMB-1菌株培养条件，在培养条件铁源为奎尼酸铁0.02 mmol/L，装瓶量75% ，pH值6.7，温度25 ℃时，AMB-1 OD600达到0.440(1.166×109 cells/ml)。同时运用磁收集传代法，使带有磁小体的AMB-1细胞比例占95％以上(Cmag值稳定在1.9-2.0)。 在AMB-1具有较好的生物量，同时又具有较好的含磁小体细胞比例后，研究磁小体的变化过程。通过透射电镜观察磁小体变化过程，发现培养24 h细菌体内已有较小晶体形成(平均27 nm，n=188)且沿长轴分布；48 h晶体长大(平均43 nm，n=203)且形成分段链沿长轴排列；72 h晶体进一步成熟(平均50 nm，n=191)仍以分段链沿长轴排列；随后细菌逐渐衰亡磁小体变小，168 h可见部分自溶细菌中仍有磁小体链(平均37 nm，n=186)；192 h细菌自溶磁小体链(平均33 nm，n=184)分散到环境中。 通过透射电镜在细胞水平上研究趋磁细菌细胞分裂时发现，磁小体在细菌分裂时采用两种分离方式：一种为磁小体分配到两个子细胞；另一种为磁小体只分配到一个子细胞。无磁小体的子细胞，在随后的生长过程又分为两种情况：一种为细胞逐渐产生磁小体，另一种为不再产生磁小体。这种现象的发现，解释了随着传代次数的增多，细菌磁性有所下降的原因(Cmag值降低)。 在对趋磁细菌磁小体合成机制的研究中，常使用基因敲除的办法获得缺陷型，并与野生型对比进行研究。但是，利用基因敲除获得缺陷型不仅操作繁琐并且所得缺陷型不稳定。本研究利用特殊的磁富集传代法，先将带有磁小体的菌体收集并连续传代，筛选获得了高磁菌株；利用这种方法，收集不含磁小体的菌体并连续传代，筛选获得了无磁菌株。 趋磁细菌磁小体在医疗、环保等领域具有广阔应用价值，但是目前由于趋磁细菌难以大规模培养，并且磁小体纯化存在成本高等原因，将磁小体真正实际应用尚有一段距离。通过研究磁小体在趋磁细菌中的变化过程发现，AMB-1菌株在培养192 h后自溶，并且磁小体随着细胞的破碎释放到环境中去。

Biosensors using photonic crystal fibres

In this thesis, we present the unique properties of hollow-core photonic crystal fibres (HC-PCFs) for sensing applications in terms of viscosity detection and DNA sensing using a special poly(ethylene) glycol (PEGDA) hydrogel. The low loss HC-PCFs ensure a long interaction length between the sample and the optical signals. Thus in this thesis, we report the characterisation of filled HC-PCFs and the development of a selective filling process. For the first time, we report the investigation of a new viscometer device, and a new device for DNA sensing development, and also the chemical process for hydrogel growth was adapted to the fibres. By combining HC-PCFs with the hydrogel we enable 3D volumetric sample confinement within the HC-PCF, further increasing the interaction between the sample and the optical signal. However, the hydrogel has a large influence on the guidance properties of the HC-PCF and the HC-PCF has a strong influence on the growth process for the hydrogel itself. When we integrate the hydrogel and HC-PCFs we detect concentration levels as low as 400 nM of labelled DNA. However, using our technology for fluorescence detection we can achieve results two orders of magnitude better than those previously reported.

High speed optical communication systems: From modulation formats to radically new fibres

High volumes of data traffic along with bandwidth hungry applications, such as cloud computing and video on demand, is driving the core optical communication links closer and closer to their maximum capacity. The research community has clearly identifying the coming approach of the nonlinear Shannon limit for standard single mode fibre [1,2]. It is in this context that the work on modulation formats, contained in Chapter 3 of this thesis, was undertaken. The work investigates the proposed energy-efficient four-dimensional modulation formats. The work begins by studying a new visualisation technique for four dimensional modulation formats, akin to constellation diagrams. The work then carries out one of the first implementations of one such modulation format, polarisation-switched quadrature phase-shift keying (PS-QPSK). This thesis also studies two potential next-generation fibres, few-mode and hollow-core photonic band-gap fibre. Chapter 4 studies ways to experimentally quantify the nonlinearities in few-mode fibre and assess the potential benefits and limitations of such fibres. It carries out detailed experiments to measure the effects of stimulated Brillouin scattering, self-phase modulation and four-wave mixing and compares the results to numerical models, along with capacity limit calculations. Chapter 5 investigates hollow-core photonic band-gap fibre, where such fibres are predicted to have a low-loss minima at a wavelength of 2μm. To benefit from this potential low loss window requires the development of telecoms grade subsystems and components. The chapter will outline some of the development and characterisation of these components. The world's first wavelength division multiplexed (WDM) subsystem directly implemented at 2μm is presented along with WDM transmission over hollow-core photonic band-gap fibre at 2μm. References: [1]P. P. Mitra, J. B. Stark, Nature, 411, 1027-1030, 2001 [2] A. D. Ellis et al., JLT, 28, 423-433, 2010.

CYSTATIN RELATED EPIDIDYMAL SPERMATOGENIC PROTEIN RESIDES IN THE OUTER DENSE FIBRES AND LIKELY TRANSIENTLY ASSOCIATES WITH THE SURFACE OF EPIDIDYMAL MOUSE SPERMATOZOA

Cystatin Related Epididymal Spermatogenic protein (CRES) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as non-glycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES is enigmatic and dependent on localization of its isoforms, which is the objective of this study. The initial approach was to investigate testicular and epididymal origins of these isoforms by immunohistochemistry and immunogold cytochemistry. To further pinpoint CRES localization we then selectively extracted and fractionated epididymal spermatozoa in order to find by immunoblotting which sperm fractions contained CRES isoforms. Immunohistochemical analysis of mouse spermatogenesis showed that CRES was expressed in the tail cytoplasm of elongating spermatids from step 9-16, with a pattern reminiscent of outer dense fibre (ODF) proteins. Ultrastructural immunocytochemistry revealed that the immunogold label was concentrated over growing ODFs and mitochondrial sheath in the testes which persisted in spermatozoa through the epididymis. Sequential extractions of isolated sperm tails with Triton X-100-dithiothreitol (DTT) to remove the mitochondrial sheath, whose extract contained an unrelated 66 kDa immunoreactive band, followed by either sodium dodecyl sulfate (SDS)-DTT or urea-DTT to solubilise accessory fibres of the tail revealed a 14 kDa immunoreactive band associated with the ODF. In addition, Western blots revealed glycosylated and non-glycosylated CRES isoforms in nonyl phenoxylpolyethoxylethanol (NP40) extracts of the caput, but not cauda, sperm. Immunohistochemical analysis of the caput and cauda epithelium showed that CRES is secreted by the Golgi apparatus of the ii initial segment, fills the proximal caput lumen, and disappears by mid caput. Western blots of caput and cauda tissue and luminal fluid revealed 14 and 19 kDa immunoreactive bands in caput tissues and luminal fluid, but not in the cauda. This study concludes that there are two origins of CRES, one arising in the testis and the other in the epididymis. Testicular CRES is ionically and covalently associated with the ODF while epididymal CRES is detergent soluble and is most likely associated temporarily with the surface of caput epididymal sperm.

The application of colloidal gas aphrons in the recovery of fine cellulose fibres from paper mill wastewater

Colloidal gas aphrons (CGAs) are micron-sized gas bubbles of 25–30 µm in diameter produced by a high-speed stirrer in a vessel containing dilute surfactant solution. These bubbles, because of their small size, exhibit some colloidal properties. In this work, CGAs were used to separate fine fibres from a lean slurry of cellulosic pulp in a flotation column. The pulp fibres were recovered as foamate from the top. Sodium dodecyl sulphate at a concentration of 2.0 kg/m3 was used as a surfactant to generate the CGAs in a spinning disc apparatus. The results indicated that up to 70% flotation efficiency could be obtained within a short column height of 0.3–0.35 m. This technique can be applied to recover fine cellulosic pulp from paper-machine backwater.