The adoption of complex systems thinking to biology has gained momentum over the past decades as scientists have become more aware of the value of considering whole systems rather than individual components. This thesis is an exploration of complexity science tools in the context of modern biology at the cellular level. Three biological systems are introduced and a novel application of complex systems theory applied to each of them. Firstly, a mathematical modelling approach is applied to the problem of understanding how memories are formed through the process of synaptic plasticity. The approach is shown to be able to accurately predict the behaviour of a synapse, allowing it to be used to inform future experiments. Secondly, a novel statistical approach is applied to the problem of predicting the coiled-coil protein structure based on amino acid sequence alone. The implementation and benchmarking of Spiricoil is described, demonstrating that it outperforms the current leading techniques in the field as well as providing comprehensive evolutionary information about coiled coils to the field for the first time. Finally, a network-based predictor is produced with the aim of predicting the correct biological factors required to turn humans cells from one type to another. A system called Mogrify is developed that can integrate gene expression and interaction data in order to produce predictions of reprogramming factors that until now would have only been possible through experimentation. Each of these projects represents a novel contribution to their field and this thesis as a whole provides a model for how complex systems thinking can be used to better understand biological systems. iii

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Dans le thymus, la médulla constitue un compartiment essentiel pour la délétion clonale et la génération de lymphocytes T régulateurs (Tregs). En retour, les thymocytes CD4+ contrôlent le développement des cellules épithéliales thymiques médullaires (CETms). Ces échanges bidirectionnels sont appelés « crosstalk ». Il a été montré que durant le crosstalk, les thymocytes CD4+ surexpriment la lymphotoxine $alpha$ (LT$alpha$) qui contrôle l’organisation de la médulla. Durant ma thèse, j’ai étudié le rôle de la LT$alpha$ dans la : (1) régénération thymique après greffe de moelle osseuse (GMO), (2) migration des cellules présentatrices d’antigènes (Ag) (CPAs) dans le thymus et (3) fonction suppressive des Tregs. Le projet 1 a montré que la LT$alpha$, régulée par RANKL, est surexprimée dans les cellules inductrices de tissus lymphoïdes (LTi) après GMO et est critique pour la régénération du thymus. Par ailleurs, le projet 2 a montré que la LT$alpha$ limite la migration des CPAs dans le thymus pour contrôler la délétion clonale. De façon intéressante, les Tregs expriment fortement la LT$alpha$. Le projet 3 a montré que les Tregs Lta-/- présentent une signature hyper-suppressive et que le transfert de ces cellules protège de l'inflammation intestinale et atténue le développement de l’auto-immunité. Enfin, le dernier projet a démontré que les thymocytes CD4+ via les interactions Ag-spécifiques induisent le développement et la fonction des CETms. En absence de ces interactions, les cellules T sont auto-réactives et induisent de l’auto-immunité, indiquant qu’elles sont essentielles à l’induction de la tolérance centrale. / In the thymus, medulla constitutes an essential compartment for clonal deletion and the generation of regulatory T lymphocytes (Tregs). Reciprocally, CD4+ thymocytes control the development of medullary thymic epithelial cells (mTECs). These bi-directional interactions are referred to as "crosstalk". It has been shown that this crosstalk induces the expression of lymphotoxin $alpha$ (LT$alpha$) in autoreactive CD4+ thymocytes, which controls the medulla organization. During my thesis, I studied the role of LT$alpha$ in: (1) thymic regeneration after bone marrow transplantation (BMT), (2) migration of antigen (Ag) presenting cells (APCs) in the thymus and (3) suppressive functions of Tregs. Project 1 has shown that LT$alpha$, regulated by RANKL, is overexpressed in lymphoid tissue inducer cells (LTi) after BMT and is critical for thymic regeneration. Furthermore, projet 2 has demonstrated that LT⍺ limits the migration of thymic APCs to fine-tune clonal deletion. Interestingly, Tregs strongly express LTα. Project 3 has demonstrated that Lta-/- Tregs exhibit a highly suppressive signature and that the transfer of these cells protects from intestinal inflammation and attenuates the development of autoimmunity. Finally, the last project has revealed that CD4+ thymocytes via Ag-specific interactions induce the development and function of mTECs. In absence of these interactions, T cells are autoreactive and induce autoimmunity, indicating that they are essential for the induction of T-cell tolerance.

Les cellules dendritiques (CD) sont bien établies comme étant le groupe de cellules le plus efficace pour réguler le système immunitaire. L'augmentation de la synthèse protéique fait partie des changements biologiques lors de l'activation, donc sa régulation est une priorité. On a signalé que la réponse des protéines dépliées (RPD) était liée à la réponse immunitaire. Ce mécanisme permet de contrarier les situations où il y a accumulation de protéines mal pliées dans le réticulum endoplasmique (RE). Elle fait partie d'une réponse intégrée au stress (RSI) centrée sur la régulation de la phosphorylation du facteur eIF2α, qui bloque la synthèse protéique. Ce mécanisme déclenche un programme spécifique d'expression génique qui dépend du facteur de transcription ATF4 qui a de multiples cibles comme GADD34 at CHOP. Nous avons montré qu'un niveau élevé de p-eIF2α est acquis pendant le développement de CD in vitro, et est également présent dans les CD splénique. Néanmoins, ce phénotype ne conduit pas à une arrestation translationnelle. De plus, nous avons montré que lors de la détection bactérienne, la maturation des CDs nécessite une déphosphorylation eIF2α, afin d'augmenter la synthèse et la maturation des protéines. Nos résultats suggèrent que l'expression de GADD34 est déterminée par la signalisation TLR4 et non par la voie RSI. Les résultats obtenus suggèrent que les niveaux élevés de p-eIF2α dans les CD à l'état d'équilibre sont partiellement à l'état activé du PERK. Ce phénotype ne semble pas altérer la production de molécules co-stimulantes, ni de cytokines pro-inflammatoires, mais il a une influence sur l'appareil lysosomal et sur la vitesse de migration in vitro. / Dendritic cells (DCs) are the most effective group of cells regulating the immune system. Increased protein synthesis is a major biological change ongoing upon activation, thus its regulation is key to respond to the maturation process requirements. The unfolded protein response (UPR), has been reported to be related to the immune response. This mechanism responds to the accumulation of unfolded of misfolded proteins in the endoplasmic reticulum (ER), leading to a stress situation. It is part of the integrated stress response (ISR) centered in the regulation of the phosphorylation of eIF2α, that blocks protein synthesis initiation. Furthermore, it triggers a specific gene expression program through the transcription factor ATF4 that has multiple targets like GADD34 and CHOP. We showed that high levels of p-eIF2α are acquired during the development of DCs in vitro, and is also present in steady-state splenic DCs, and unexpectedly is not associated with a global translation arrest. Further, we showed that upon TLR4 stimulation with LPS, DC maturation requires eIF2α dephosphorylation, in order to increase protein synthesis and maturate. Our results suggest that the expression of GADD34 is driven by TLR4 downstream signaling and not by ATF4. The results obtained suggest that high levels of p-eIF2α in steady-state DCs are partially due to the activated status of PERK. This phenotype doesn’t seem to impair the production of co-stimulatory molecules, neither pro-inflammatory cytokines, however it has an influence in the lysosomal machinery and DC migration in vitro.

The broad aspect of the growth of vatiola virus in human polls has been considered from a biological point of view. A general discussion and review of the literature on the subject of smallpox and the forms it which the disease exists today summarises the background against which this present study was undertaken. The experimental work embodies a comparison of the behaviour of variola major and variola minor strains in human cells, bearing in mind the possibility of finding new markers for the biological characterisation of variola virus strains. The comparison was made by investigating the growth patterns of the International Reference Strains of variola major (Harvey) end Minor (Butler). The progress of cytopathic effect and production of infections virus, complement-fixing antigen, diffusible antigen and haemagglutinin, and the development of virus DNA and antigen in situ in cells infected by the two variola strains were compared. Particular attention was paid to the effect of incubation temperatures ranging from optimal to non-permissive for virus growth (35° - 41° C) upon the synthesis of virus products.

The work described was done with larvae and adults of virginoparae of the green peach aphid, Myzus persicae Sulz. and the cabbage aphid, Brvicoryne brassicae L. on Brussels sprouts, Brassica oleracea L. var. bullata gemmifera. The responses of the aphids to three leaf ages of the plants subjected to differing levels of intermittent water strain are described and compared for each aphid species in terms of 1) the selection of the leaves and plants by settling alatae, 2) the preferences of the apterae for leaves of different age and water status, and 3) the fecundity, reproductive rate, rate of development, longevity, reproductive life, post-reproductive life and proportion of alatae in the progeny of apterae on leaves of different age and water status. The suitability of the different leaves and plants as hosts for the two aphid species is assessed. The responses of the aphids to three leaf ages of plants subjected to no water strain or to a single period of increasing water strain are described and compared for each aphid species in terms of the survival, reproductive rate and restlessness of apterae. A relationship is demonstrated between the water content of the growing medium of the plants and the larvi position of aphids feeding on the plants. The methods of penetration and the destinations of the stylets and salivary sheaths of the two aphid species in the leaves of the plants are described. Preliminary experiments are described in which aphids were fed on artificial liquid diets under controlled pressures. The possible causes of the responses of the aphids to leaves of different age on plants of different water status are examined particularly by comparing the two aphid species. The possible economic importance of the responses of aphids to water strain in plants is discussed with reference to aphid control.

Group-specific polysaccharide was extracted from group A streptococci by various methods and after purification it was adsorbed on to polystyrene latex particles, which were used in an agglutination test to detect group-specific antibody in hyperimmune rabbit sera, The most pure and serologically specific extracts were obtained by extraction with formamide. Formamide-extracted polysaccharides of groups A-var, B, C, G and L were also adsorbed on to latex particles. Inter-group cross-agglutination reactions corresponded to known chemical relationships between group polysaccharides. Latex agglutination tests were used to detect group .A and A-var antibodies in human sera. Antibodies were found in 90% of patients suspected of suffering from streptococcal disease but in less than 40% of controls. In paired sera from patients with known streptococcal infections, initial antibody titres were high in most cases. M-proteins from group A streptococci belonging to a number of types were extracted with hot acid and precipitated with 30% and with 60% saturated ammonium sulphate. The precipitates were designated P30 and P60 respectively. The supernatant (S60) was further purified by precipitation at pH 5.5 and washing with absolute alcohol. Immunological tests showed that purified S60 fraction was more type-specific than P60 fraction. Absorption of rabbit hyperimmune M antisera with S60 fraction removed the specific bactericidal activity. The purified S60 was used in latex agglutination tests to detect type-specific M antibodies in human sera. Sera from patients suspected of suffering from streptococcal infection and from control persons showed no significant difference in the number of serotypes for which antibody was present in high titre. Most of the paired sera from patients who had suffered from infection with group A streptococci of known serotype showed a significant rise in antibody titre to the infecting serotype. Rises in titre to heterologous serotypes also occurred frequently, but showed no constant pattern. This thesis is a report of full-time research undertaken by the author in the Cross-infection Reference Laboratory, Central Public Health Laboratory, Colindale, London, N.W.9, and in the Wright-Fleming Institute, St. Mary's Hospital Medical School, London, W.2 from April, 1966 to August, 1968. An account of a part of this work is in preparation and will be submitted for publication as fo11ows: Erwa, H.H., Maxted, W.R. & Brighton, W.D. (1968) "A latex agglutination test for the measurement of antibodies to group-specific streptococcal polysaccharides."

The course of the chemical inactivation of coliphage T6r was followed by a standardised plaque counting technique. The antimicrobial agents used were cetrimide, chloramine, formaldehyde and phenol. Dilution of the reaction mixture was used to prevent the residual virus inhibitory effect of phenol, while the residual action of the other three compounds was stopped by the use of suitable neutralising agents. The effects on the kinetics of inactivation of concentration of antimicrobial agent and of temperature were examined to compare the agents tested. The concentration exponent and temperature coefficient of chloramine and formaldehyde were alike but were different from both cetrimide and phenol. The course of inactivation was influenced more by the medium in which the phage was suspended than the medium in which it was cultivated. The effect of the presence of various extraneous materials in the reaction mixture was also investigated. In general the presence of extraneous matter reduced the activity of cetrimide, chloramine and phenol, whilst the activity of formaldehyde appeared to be enhanced. The stability of phase surviving inactivation from reaction mixtures containing peptone was examined. In the presence of peptone, formaldehyde inactivation seemed partly reversible. This phenomena was not observed with the other agents tested. Comparison with previously postulated modes of action of these antimicrobial agents suggests that formaldehyde inactivates the phage by damaging a portion of the tail structure. The other agents were thought to affect the other protein components of the phage. The action of chloramine was attributed to denaturation of the protein shell with the subsequent loss of DNA. The action of cetrimide may be due to lysis of the phage proteins and it is likely that phenol has a similar mode of action.