Accommodation and intraocular pressure

The relationship between accommodation and intraocular pressure (lOP) has not been addressed as a research question for over 20 years, when measurement of both of these parameters was less advanced than today. Hence the central aim of this thesis was to evaluate the effects of accommodation on lOP. The instrument of choice throughout this thesis was the Pulsair EasyEye non-contact tonometer (NCT) due principally to its slim-line design which allowed the measurement of lOP in one eye and simultaneous stimulation of accommodation in the other eye. A second reason for using the Pulsair EasyEye NCT was that through collaboration with the manufacturers (Keeler, UK) the instrument's operational technology was made accessible. Hence, the principle components underpinning non-contact lOP measures of 0.1mmHg resolution (an order of magnitude greater than other methods) were made available. The relationship between the pressure-output and corneal response has been termed the pressure-response relationship, aspects of which have been shown to be related to ocular biometric parameters. Further, analysis of the components of the pressure-response relationship together with high-speed photography of the cornea during tonometry has enhanced our understanding of the derivation of an lOP measure with the Pulsair EasyEye NCT. The NCT samples the corneal response to the pressure pulse over a 19 ms cycle photoelectronically, but computes the subject's lOP using the data collected in the first 2.34 ms. The relatively instantaneous nature of the lOP measurement renders the measures susceptible to variations in the steady-state lOP caused by the respiratory and cardiac cycles. As such, the variance associated with these cycles was minimised by synchronising the lOP measures with the cardiac trace and maintaining a constant pace respiratory cycle at 15 breathes/minute. It is apparent that synchronising the lOP measures with the peak, middle or trough of the cardiac trace significantly reduced the spread of consecutive measures. Of the 3 locations investigated, synchronisation with the middle location demonstrated the least variance (coeflicient of variation = 9.1%) and a strong correlation (r = 0.90, p = <0.001) with lOP values obtained with Goldmann contact tonometry (n = 50). Accordingly lOP measures synchronised with the middle location of the cardiac cycle were taken in the RE while the LE fixated low (L; zero D), intermediate (I; 1.50 D) and high (H; 4 D) accommodation targets, Quasi-continuous measures of accommodation responses were obtained during the lOP measurement period using the portable infrared Grand Seiko FR-5000 autorefractor. The lOP reduced between L and I accommodative levels by approximately 0.61 mmHg (p <0.00 I). No significant reduction in IOP between L and H accommodation levels was elicited (p = 0.65) (n = 40). The relationship between accommodation and lOP was characterised by substantial inter-subject variations. Myopes demonstrated a tendency to show a reduction in IOP with accommodation which was significant only with I accommodation levels when measured with the NCT (r = 0.50, p = 0.01). However, the relationship between myopia and lOP change with accommodation reached significance for both I (r = 0.61, p= 0.003) and H (r = 0.531, p= 0.0 1) accommodation levels when measured with the Ocular blood Flow Analyser (OBFA). Investigation of the effects of accommodation on the parameters measured by the OBFA demonstrated that with H accommodation levels the pulse amplitude (PA) and pulse rate (PR) responses differed between myopes and emmetropes (PA: p = 0.03; PR: p = 0.004). As thc axial length increased there was a tendency for the pulsatile ocular blood flow (POBF) to reduce with accommodation, which was significant only with H accommodation levels (r = 0.38, p = 0.02). It is proposed that emmetropes arc able to regulate the POBF responses to changes in ocular perfusion pressure caused by changes in lOP with I (r = 0.77, p <0.001) and H (r = 0.73, p = 0.001) accommodation levels. However, thc relationship between lOP and POBF changes in the myopes was not correlated for both I (r = 0.33, p = 0.20) and H (r = 0.05, p = 0.85) accommodation levels. The thesis presents new data on the relationships between accommodation, lOP and parameters of the OBFA,: and provides evidence for possible lOP and choroidal blood flow regulatory mechanisms. Further the data highlight possible deficits in the vascular regulation of the myopic eye during accommodation, which may play a putative role in the aetiology of myopia development.

Improved production of industrially relevant recombinant proteins: application of multi-factorial design of experiments and scalable process modelling

The work described in this thesis focuses on the use of a design-of-experiments approach in a multi-well mini-bioreactor to enable the rapid establishments of high yielding production phase conditions in yeast, which is an increasingly popular host system in both academic and industrial laboratories. Using green fluorescent protein secreted from the yeast, Pichia pastoris, a scalable predictive model of protein yield per cell was derived from 13 sets of conditions each with three factors (temperature, pH and dissolved oxygen) at 3 levels and was directly transferable to a 7 L bioreactor. This was in clear contrast to the situation in shake flasks, where the process parameters cannot be tightly controlled. By further optimisating both the accumulation of cell density in batch and improving the fed-batch induction regime, additional yield improvement was found to be additive to the per cell yield of the model. A separate study also demonstrated that improving biomass improved product yield in a second yeast species, Saccharomyces cerevisiae. Investigations of cell wall hydrophobicity in high cell density P. pastoris cultures indicated that cell wall hydrophobin (protein) compositional changes with growth phase becoming more hydrophobic in log growth than in lag or stationary phases. This is possibly due to an increased occurrence of proteins associated with cell division. Finally, the modelling approach was validated in mammalian cells, showing its flexibility and robustness. In summary, the strategy presented in this thesis has the benefit of reducing process development time in recombinant protein production, directly from bench to bioreactor.

The effect of antifoam addition on protein production yields

Pichia pastoris is a widely used host for recombinant protein production. The foaming associated with culturing it on a large scale is commonly prevented by the addition of chemical antifoaming agents or "antifoams." Unexpectedly, the addition of a range of antifoams to both shake flask and bioreactor cultures of P. pastoris has been shown to alter the total yield of the recombinant protein being produced. Possible explanations for this are that the presence of the antifoam increases the total amount of protein being produced and secreted per cell or that it increases the density of the culture. Antifoaming agents may therefore have specific effects on the growth and yield characteristics of recombinant cultures, in addition to their primary action as de-foamers.

Recombinant protein production in yeast:methods and protocols

In the last few years, significant advances have been made in understanding how a yeast cell responds to the stress of producing a recombinant protein, and how this information can be used to engineer improved host strains. The molecular biology of the expression vector, through the choice of promoter, tag and codon optimization of the target gene, is also a key determinant of a high-yielding protein production experiment. Recombinant Protein Production in Yeast: Methods and Protocols examines the process of preparation of expression vectors, transformation to generate high-yielding clones, optimization of experimental conditions to maximize yields, scale-up to bioreactor formats and disruption of yeast cells to enable the isolation of the recombinant protein prior to purification. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

Improving recombinant human adenosine A2A receptor production in yeast

Over 50% of clinically-marketed drugs target membrane proteins; in particular G protein-coupled receptors (GPCRs). GPCRs are vital to living cells, performing an active role in many processes, making them integral to drug development. In nature, GPCRs are not sufficiently abundant for research and their structural integrity is often lost during extraction from cell membranes. The objectives of this thesis were to increase recombinant yield of the GPCR, human adenosine A2A receptor (hA2AR) by investigating bioprocess conditions in large-scale Pichia pastoris and small-scale Saccharomyces cerevisiae cultivations. Extraction of hA2AR from membranes using novel polymers was also investigated. An increased yield of hA2AR from P. pastoris was achieved by investigating the methanol feeding regime. Slow, exponential feed during induction (μlow) was compared to a faster, exponential feed (μhigh) in 35 L pilot-scale bioreactors. Overall hA2AR yields were increased for the μlow cultivation (536.4pmol g-1) compared to the μhigh148.1 pmol g-1. hA2AR levels were maintained in cytotoxic methanol conditions and unexpectedly, pre-induction levels of hA2AR were detected. Small-scale bioreactor work showed that Design of Experiments (DoE) could be applied to screen for bioprocess conditions to give optimal hA2AR yields. Optimal conditions were retrieved for S. cerevisiae using a d-optimal screen and response surface methodology. The conditions were 22°C, pH 6.0, 30% DO without dimethyl sulphoxide. A polynomial equation was generated to predict hA2AR yields if conditions varied. Regarding the extraction, poly (maleic anhydride-styrene) or PMAS was successful in solubilising hA2AR from P. pastoris membranes compared with dodcecyl-β-D-maltoside (DDM) detergent. Variants of PMAS worked well as solubilising agents with either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or cholesteryl hemisuccinate (CHS). Moreover, esterification of PMAS improved solubilisation, suggesting that increased hydrophobicity stabilises hA2AR during extraction. Overall, hA2AR yields were improved in both, P. pastoris and S. cerevisiae and the use of novel polymers for efficient extraction was achieved.

Automated prediction of deterioration of infants in paediatric intensive care using SpO2

Acute life-threatening events are mostly predictable in adults and children. Despite real-time monitoring these events still occur at a rate of 4%. This paper describes an automated prediction system based on the feature space embedding and time series forecasting methods of the SpO2 signal; a pulsatile signal synchronised with heart beat. We develop an age-independent index of abnormality that distinguishes patient-specific normal to abnormal physiology transitions. Two different methods were used to distinguish between normal and abnormal physiological trends based on SpO2 behaviour. The abnormality index derived by each method is compared against the current gold standard of clinical prediction of critical deterioration. Copyright © 2013 Inderscience Enterprises Ltd.

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Background - Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results - Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions - The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.

A potentially scalable method for the harvesting of hMSCs from microcarriers

The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will necessitate large-scale culture techniques such as the expansion of cells on microcarriers in bioreactors. Whilst much research investigating hMSC culture on microcarriers has focused on growth, much less involves their harvesting for passaging or as a step towards cryopreservation and storage. A successful new harvesting method has recently been outlined for cells grown on SoloHill microcarriers in a 5L bioreactor [1]. Here, this new method is set out in detail, harvesting being defined as a two-step process involving cell 'detachment' from the microcarriers' surface followed by the 'separation' of the two entities. The new detachment method is based on theoretical concepts originally developed for secondary nucleation due to agitation. Based on this theory, it is suggested that a short period (here 7min) of intense agitation in the presence of a suitable enzyme should detach the cells from the relatively large microcarriers. In addition, once detached, the cells should not be damaged because they are smaller than the Kolmogorov microscale. Detachment was then successfully achieved for hMSCs from two different donors using microcarrier/cell suspensions up to 100mL in a spinner flask. In both cases, harvesting was completed by separating cells from microcarriers using a Steriflip® vacuum filter. The overall harvesting efficiency was >95% and after harvesting, the cells maintained all the attributes expected of hMSC cells. The underlying theoretical concepts suggest that the method is scalable and this aspect is discussed too. © 2014 The Authors.

Mixing theory for culture and harvest in bioreactors of human mesenchymal stem cells on microcarriers

The use of human mesenchymal stem cells (hMSCs) in regenerative medicine is a potential major advance for the treatment of many medical conditions, especially with the use of allogeneic therapies where the cells from a single donor can be used to treat ailments in many patients. Such cells must be grown attached to surfaces and for large scale production, it is shown that stirred bioreactors containing ~200 μm particles (microcarriers) can provide such a surface. It is also shown that the just suspended condition, agitator speed NJS, provides a satisfactory condition for cell growth by minimizing the specific energy dissipation rate, εT, in the bioreactor whilst still meeting the oxygen demand of the cells. For the cells to be used for therapeutic purposes, they must be detached from the microcarriers before being cryopreserved. A strategy based on a short period (~7 min) of very high εT, based on theories of secondary nucleation, is effective at removing >99% cells. Once removed, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. This approach is shown to be successful for culture and detachment in 4 types of stirred bioreactors from 15 mL to 5 L.