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.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required. © 2007 Wiley Periodicals, Inc.

Setting up a bioreactor for recombinant protein production in yeast

Scale-up from shake flasks to bioreactors allows for the more reproducible, high-yielding production of recombinant proteins in yeast. The ability to control growth conditions through real-time monitoring facilitates further optimization of the process. The setup of a 3-L stirred-tank bioreactor for such an application is described. © 2012 Springer Science+business Media, LLC.

Hybrid Fe3O4 /GaAs (100) structure for spintronics

Fe3O4 GaAs hybrid structures have been studied using reflection high-energy electron diffraction (RHEED), x-ray photoelectron spectroscopy (XPS), x-ray magnetic circular dichroism (XMCD), and low-temperature vibrating-sample magnetometry (VSM). The samples were prepared by oxidizing epitaxial Fe thin films in a partial pressure of 5× 10-5 mbar of oxygen at 500 K for 180 s. Clear RHEED patterns were observed, suggesting the epitaxial growth of Fe oxides with a cubic structure. The XPS spectra show that the oxides were Fe3O4 rather than γ- Fe2O3, as there were no shake-up satellites between the two Fe 2p peaks. This was further confirmed by the XMCD measurements, which show ferromagnetic coupling between the Fe cations, with no evidence of intermixing at the interface. The VSM measurements show that the films have a magnetic uniaxial anisotropy and a "quick" saturation property, with the easy axes along the [011] direction. This detailed study offers further insight into the structure, interface, and magnetic properties of this hybrid Fe3O4 GaAs (100) structure as a promising system for spintronic application. © 2005 American Institute of Physics.

[Poster] Friends of the Island

This poster presentation will exhibit the ongoing design work within two small islands adjacent to Diner Key Marina gifted by the City of Miami to the non-profit organization, Shake-A-Leg. This organization works to blur the lines between access, recreation, and skill, redefining what is possible. Abled and disabled children, adolescents, and young adults go to Shake-A-Leg to participate in a myriad of activities such as sailing, kayaking, swimming, painting, and socializing in these islands regardless of their inadequate walkways and installations. The users are people from different social statuses, physical and mental abilities. This poster will exhibit the design proposal to enhance the islands’ infrastructure to provide seamless access to all its visitors through the lush habitat in these islands. The proposed program challenges the disabilities of the person and helps them to mediate and transform them into new possibilities and abilities. The program proposes ten components which include a nature trail, restrooms, dinning center, art studio, and observation deck. The design of each space draws from the sailing boat to create solutions were the user will explore, participate and enjoy. The location encourages a feeling of challenge but at the same time it is a place for relief and freedom, allowing them to discover the diversity through experimentation and interaction with the environment. This poster presents design principles that value equal access and experience, celebrating the differences among users and the environment, blurring the lines between nature and access.

Musical commonplace book : manuscript, [approximately 1790-approximately 1810]

Contains songs, partly from English operas, and instrumental music.

Negotiating queer and religious identities in higher education: queering ‘progression’ in the ‘university experience’

This article addresses the negotiation of ‘queer religious’ student identities in UK higher education. The ‘university experience’ has generally been characterised as a period of intense transformation and self-exploration, with complex and overlapping personal and social influences significantly shaping educational spaces, subjects and subjectivities. Engaging with ideas about progressive tolerance and becoming, often contrasted against ‘backwards’ religious homophobia as a sentiment/space/subject ‘outside’ education, this article follows the experiences and expectations of queer Christian students. In asking whether notions of ‘queering higher education’ (Rumens 2014 Rumens, N. 2014. “Queer Business: Towards Queering the Purpose of the Business School.” In The Entrepreneurial University: Public Engagements, Intersecting Impacts, edited by Y. Taylor, 82–104. Basingstoke: Palgrave Macmillan.) ‘fit’ with queer-identifying religious youth, the article explores how educational experiences are narrated and made sense of as ‘progressive’. Educational transitions allow (some) sexual-religious subjects to negotiate identities more freely, albeit with ongoing constraints. Yet perceptions of what, where and who is deemed ‘progressive’ and ‘backwards’ with regard to sexuality and religion need to be met with caution, where the ‘university experience’ can shape and shake sexual-religious identity.

„Welch werthvolle und kunstreich geschmiedete kleine Sache.“

Richard Schaukal (1874-1942) wurde als Dichter ignoriert, als Übersetzer geschätzt und als Literaturkritiker geachtet. Es ist erstaunlich, dass dieses Tätigkeitsfeld unvollständig aufgearbeitet ist, zumal bedeutende Exponenten der Zeit wie Thomas Mann, Hermann Hesse oder Rainer Maria Rilke Schaukal um Besprechungen ihrer Werke baten. Er verfasste für zahlreiche Publikationsorgane meist unbequeme Essays, die sich − mehr noch als seine dichterischen Arbeiten − in den Diskurs der kulturkonservativen kritischen Moderne einschrieben. Mit Blick auf seine frühe Phase als Literaturkritiker werden Richard Schaukals ästhetische Positionen, Einflüsse und Funktionen untersucht. Abschließend wird sein Aufsatz über Ferdinand von Saar (1899) einer exemplarischen Analyse unterzogen.

“Niemand zeugt für den Zeugen”. Temporalidad y memoria en la poesía de Paul Celan

El artículo analiza cómo Paul Celan, utilizando recursos arreferenciales y antimiméticos propios de la poésie pure y la poesía absoluta, desarrolla un modelo poético en el que la temporalidad y la memoria son determinantes. En el nuevo modelo, el lenguaje no constituye una realidad autónoma e inmanente; antes bien, se concreta en una acción dialógica y remite a una realidad extralingüística. El mismo texto poético configura un espacio del recuerdo, se erige en un lugar de memoria que, a través de las sedimentaciones históricas que arrastra el lenguaje, da testimonio de lo ocurrido.

Large Numerical Models in Continuous Hybrid Simulation

Hybrid simulation is a technique that combines experimental and numerical testing and has been used for the last decades in the fields of aerospace, civil and mechanical engineering. During this time, most of the research has focused on developing algorithms and the necessary technology, including but not limited to, error minimisation techniques, phase lag compensation and faster hydraulic cylinders. However, one of the main shortcomings in hybrid simulation that has pre- vented its widespread use is the size of the numerical models and the effect that higher frequencies may have on the stability and accuracy of the simulation. The first chapter in this document provides an overview of the hybrid simulation method and the different hybrid simulation schemes, and the corresponding time integration algorithms, that are more commonly used in this field. The scope of this thesis is presented in more detail in chapter 2: a substructure algorithm, the Substep Force Feedback (Subfeed), is adapted in order to fulfil the necessary requirements in terms of speed. The effects of more complex models on the Subfeed are also studied in detail, and the improvements made are validated experimentally. Chapters 3 and 4 detail the methodologies that have been used in order to accomplish the objectives mentioned in the previous lines, listing the different cases of study and detailing the hardware and software used to experimentally validate them. The third chapter contains a brief introduction to a project, the DFG Subshake, whose data have been used as a starting point for the developments that are shown later in this thesis. The results obtained are presented in chapters 5 and 6, with the first of them focusing on purely numerical simulations while the second of them is more oriented towards a more practical application including experimental real-time hybrid simulation tests with large numerical models. Following the discussion of the developments in this thesis is a list of hardware and software requirements that have to be met in order to apply the methods described in this document, and they can be found in chapter 7. The last chapter, chapter 8, of this thesis focuses on conclusions and achievements extracted from the results, namely: the adaptation of the hybrid simulation algorithm Subfeed to be used in conjunction with large numerical models, the study of the effect of high frequencies on the substructure algorithm and experimental real-time hybrid simulation tests with vibrating subsystems using large numerical models and shake tables. A brief discussion of possible future research activities can be found in the concluding chapter.

Design Without Borders: A Multi-Everything Masters

Design is increasingly a global activity: addressing issues that challenge and affect people and populations other than our own, involving stakeholders from many cultures, realized through borderless networks of knowledge, services, materials, manufacturing and distribution. There is an appetite among graduates, especially in design and engineering, to broaden horizons and raise ambitions, to tackle big issues through innovation to bring about life-changing or world-changing impact. Employers demand such thinkers and doers: culturally attuned, multidisciplinary and T-shaped, unafraid to shake things up. In 2013, twelve postgraduates embarked on a new joint Masters course in London; students from eight different nations, studying together in three capital cities over two years. This programme is a collaboration between four centres of academic excellence in UK, USA and Japan; these students soon become its first graduating cohort, having experienced differing teaching styles, perspectives and specialisms around design, technology and innovation from four world-class institutions; immersion in three very different cultures; collaboration with students and faculty from many diverse disciplines and cultures; forming friendships and networks spanning the globe. This paper outlines the rationale and philosophy of the course, the challenges in its realisation and development so far, and its likely future evolution.

An examination of the techniques used to capture mangrove crabs, Ucides cordatus, in the Mamanguape River estuary, Northeastern Brazil, with implications for management.

The present research, undertaken in a mangrove swamp in northeastern Brazil (Mamanguape River Estuary), examined the factors that led to the overwhelming acceptance of the tangle-netting technique by crab harvesters in detriment to the now illegal tamping technique. Both techniques are the only ones currently used at our study site and in many other areas in Brazil, despite being prohibited by law. Data were collected through direct observations to determine capture efficiency, productivity, daily production, selectivity, and harvesting effort, and through interviews with crab harvesters, focusing on their perceptions of the capture techniques, the conditions of crab stocks and the sales price of a dozen crabs. Our results indicated that the two capture techniques did not significantly differ in terms of their efficiency or productivity, but daily production rates differed significantly, being greater using tangle-netting. The tangle-netting permits a greater harvesting effort (6 hours and 34 min) compared to tamping (4 hours and 19 min). Tangle-netting is also less selective than tamping indicated by the larger number of captured smaller specimens, including females. This results in a lower average sales price for a dozen crabs caught by tangle-netting (US$ 0.95) compared to tamping (US$ 1.02). The greater daily production of crab harvesters using the tangle-netting technique nevertheless increased their net gain, explaining their preference for this method, Given that tangle-netting results in greater harvesting pressure but lower selectivity compared to tamping, it may potentially be less sustainable. All of the crab harvesters interviewed having more than 20 years of experience (n = 34) stated they perceived that stocks of U. cordatus had become reduced over the last 20 years, together with average crab sizes. It is now important to examine the structure of the local U. cordatus population and to assess its fishery to allow evaluating whether the illegal, but prominent tangle-netting and tamping mangrove crab capture techniques are sustainable or not. We further suggest improving the dialogue between decision makers and fishermen, which barely exists to date, to initiate a discussion about possible ways of resolving the current situation of illegality of the fishermen. This will be key to achieving effective sustainable co-management of this important natural mangrove forest resource.

Optimization of lipase production from yeasts strains isolated from olive mil wastewater

Dissertação submetida à Universidade de Lisboa, Faculdade de Ciências para a obtenção do Grau de Mestre em Microbiologia Aplicada.

Ability of Gordonia alkanivorans strain 1B for high added value carotenoids production

Currently, carotenoids are valuable bioactive molecules for several industries, such as chemical, pharmaceutical, food and cosmetics, due to their multiple benefits as natural colorants, antioxidants and vitamin precursors. Hence, the increasing interest on these high added-value products has led to the search of alternatives, more cost-effective and with better yields, towards their industrial production. Indeed, microbial metabolism offers a promising option for carotenoids production. Herein it is shown the potential of the dibenzothiophene desulfurizing bacterium Gordonia alkanivorans strain 1B as a high carotenoid-producer microorganism. The novel carotenoids, produced under different culture conditions, were extracted with DMSO and then further analyzed both through spectrophotometry and HPLC. When grown in glucose-sulfate-light, strain 1B was able of achieving 2015 g carotenoids per g DCW in shake-flask assays, with about 60% corresponding to lutein, canthaxanthin and astaxanthin. Further optimization studies open a new focus of research aiming to get a hyper pigment-producer strain that may be applied towards different industrial sectors.