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Lactobacillus casei es una bacteria del ácido láctico de interés aplicado por su uso como cultivo iniciador en la industria alimentaria y por el carácter probiótico de algunas cepas. Como probiótico, L. casei debe sobrevivir a las condiciones de producción industrial y a su paso por el tracto gastrointestinal manteniendo sus propiedades. Para ello, L. casei posee rutas de reconocimiento de señales ambientales específicas y convierten esta información en una respuesta fisiológica adecuada. Un mecanismo comúnmente encontrado en bacterias para la transducción de señal son los sistemas de dos componentes (Two Components Systems, TCS). Los TCS están formados por una proteína sensora con actividad histidina quinasa (HK) y un regulador de respuesta (RR). La detección de un estímulo específico por la proteína sensora induce su autofosforilación y la transferencia del fosfato al regulador de respuesta, produciéndose la activación del mismo. Los sistemas de dos componentes median la respuesta adaptativa a una amplia gama de estímulos ambientales en bacterias. En el laboratorio de Bacterias Lácticas del Instituto de Agroquímica y Tecnología de Alimentos se ha iniciado el estudio de los TCS codificados por L. casei BL23 dentro del cual se incluye el presente proyecto de tesis doctoral. / Revilla Guarinos, A. (2014). Characterization of Two Component Systems of Lactobacillus casei BL23 and their involvement in stress response [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/43589

Cell polarisation is a key biological process crucial for the functioning of essentially all cells. Regulation of cell polarity is achieved through various processes determined by both internal and external factors. An example of the latter is that cell polarity can be disrupted or lost as a consequence of a variety of external stresses. When facing such stresses, cells adapt to unfavourable conditions by activating a range of molecular signalling pathways, collectively termed ‘stress response’. Despite the connections between external stress and cell polarity, whether stress-response signalling regulates cell polarisation and what the molecular basis for such regulation remains an open question. The fission yeast Schizosaccharomyces pombe presents an excellent biological platform to study the complexity of cell polarity regulation on a systematic level. This study is aimed at understanding the functional relationship between stress-response signalling and maintenance of cell polarity in this model organism. The findings presented in this thesis set the basis for establishing a functional link between the activation of the S.pombe stress-response pathway and the activity of the master regulator of cell polarity- the Rho GTPase Cdc42. Here, I describe experiments that identify an active involvement of the stress-response mitogen-activated kinase (MAPK) Sty1 in the dispersal of active Cdc42 from the sites of growth. This new role for Sty1 occurs independently from its involvement in transcription regulation and other previously identified signalling pathways involving Sty1. Furthermore, I also find that Sty1’s involvement in Cdc42 regulation has direct implications for fission yeast physiology as it is essential for the maintenance of cellular quiescence upon nitrogen starvation. This thesis also focuses on identifying the targets of Sty1 orchestrating the active Cdc42 disruption. Here, I describe a candidate-based approach, where I investigate the role of proteins from the Cdc42 regulatory network during Sty1 activation. Additionally, I present a global phospho-proteomics approach to identify novel targets of Sty1 and offer preliminary findings which might explain Sty1’s involvement in Cdc42 regulation.

The bacterium Streptococcus sanguinis is a primary member of the human oral microflora and also has been recognized as a key player in the bacterial colonization of the mouth. It is considered the most common viridians streptococcal species implicated in infective endocarditis. In all kingdoms of life, ATP binding cassette (ABC) transporters are essential to many cellular functions. Sequencing of the SK36 genome provided the opportunity to study ABC transporter mutants and their relationship with acidity of the oral environment. Despite numerous studies that have focused on carbohydrate uptake systems in closely related streptococcal species such as S. mutans, S. pneumonia and S. pyogenes, the mechanism of the response of these ABC transporters to acidic conditions in S. sanguinis is still unknown. The capability of S. sanguinis to adapt in these harsh environments suggests this bacterium is capable of responding to various environmental stimuli. The purpose of this study was to examine ABC mutants to identify functions that contribute to acid tolerance in S. sanguinis. This study demonstrates that two acid-sensitive mutant genes, SSA_1507 and SSA_1508, identify genes involved in acid tolerance. The two mutants grew on different sugars and none of them showed a defect in sugar utilization at acid pH. We couldn’t recognize any significant differences in sugar uptake for the two acid sensitive mutants or in mutants of their neighboring genes. Thus, the observed acid sensitivity is not due to a failure to take up any of the common sugars tested. The cytoplasmic pH of S. sanguinis was studied with the fluorescent pH indicator (BCECF) and SK36 was observed to have a wider pH range than either of the two acid-sensitive mutants SSA_1507 or SSA_1508. In these two mutants, intracellular pH was not as well maintained. At all pH values tested, the mutants displayed a lower intracellular pH than the wild type. These observations indicate that the cell membrane of these two mutants is unable to protect the interior components from adverse effects of higher pH values and lower pH values, and prove that these two mutant genes SSA_1507 and SSA_1508 are unable to grow in lower pH values. These results support a role for these ABC transporters in proton pump or export and indicate that the mutants are less able to pump out protons.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Michael Kanost / Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC₅₀ of 12 μM, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta. Biophysical analysis showed that diapausin-1 binds to the β-1,3 glucan component of the S. cerevisiae cell wall. A second insect peptide investigated in this project was M.sexta stress-response peptide 1(SRP1), an immune-related peptide upregulated under different stress conditions including immune-challenge. Preliminary results for NMR structure determination are presented. Most of the amino acid residue spin systems were assigned, and we determined the connectivities of many amino residues as a first step to solve the NMR structure. The circular dichroism spectrum of SRP1 indicates that the peptide lacks alpha-helical structure and may contain beta strands and turns.

Thesis advisor: Mary F. Roberts / Crystallization studies in presence of organic osmolytes were conducted to better understand the specific mechanism of compatible solute binding to the inositol monophosphatase of Archaeoglobus fulgidus. The synthesis of a-diglycerol phosphate, one of the natural osmolytes of A. fulgidus, was also completed for kinetic testing of its I-1-Pase thermoprotective properties and for crystallization trials. / Thesis (MS) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The maintenance of both the psychological and physiological health of captive animals is a key priority of modern zoos. Recognising that characteristics of the captive environment have the potential to decrease animal welfare, methods for quantifying and assessing welfare have been developed as part of the process for improving animal welfare. Traditionally, observations of animal behaviour and quantifying time budgets in relation to those of the animals’ wild counterparts have been utilised to assess animal welfare. Hormonal assays have also been implemented to quantify the physiological stress response of animals in captivity and identify the extent of stress being experienced. Each of these methods focuses on a different indicator of animal welfare, is quantified in different ways and provides a different perspective on the welfare of the animals. Given the limited time and financial budgets available to zoos and animal carers, identifying the most appropriate method of welfare assessment would be advantageous in helping to secure the best possible health of captive animals and to maximise their value in captivity. This thesis implemented both behavioural observations and hormonal assays to identify the strengths and weaknesses of each methodology, and make recommendations for future research. The study involved a group of four Bornean orangutans (Pongo pygmaeus) housed at Twycross Zoo. Behavioural observations involved continuous group sampling and the development of an ethogram to record a comprehensive account of orangutan activity over the course of a 12 week enrichment programme. Simultaneous to these observations, faecal samples were collected from each orangutan and processed via Enzyme Immunoassay (EIA) to quantify levels of faecal glucocorticoid metabolites (fGCM) in each sample. While recognising the recent developments in ecological analytical methods, the capacity for extending network analysis beyond the application to social networks, and its use as a welfare assessment tool were explored. Behavioural and space-use networks were developed using data from a second study of the orangutans housed at Twycross Zoo. The flexibility of network analysis in visually representing different data types allowed for the intuitive representation of complex behavioural data. Further research investigated the use of network metrics in providing deeper insights into animal behaviour and space use patterns. In addition, bipartite networks were assessed for their potential to detect and show patterns in the relationships between two sets of behavioural data. Each of the methods used had a number of strengths and weaknesses, but importantly each contributed a different perspective in the assessment of behaviour patterns and welfare, suggesting that an integrated approach to behaviour studies utilising several methods would be ideal. Cost and logistic constraints make this unlikely in most cases. However, the thesis ends with a look to the future and the recognition that the current rapid development of technology for use in animal behaviour studies, coupled with equally rapid development of analytical techniques, may help to dramatically increase the amount of information gained from the average animal behaviour study in the future. Such improvements have never been more urgent, with the requirement for understanding animal behaviour in light of current extinction rates within the context of habitat destruction and climate change. It is hoped that this thesis will make a contribution to improving future animal behaviour and welfare studies by providing an assessment of both traditional methods of study as well as demonstrating the use and potential of new ways of applying network analysis within such studies.

Caloric restriction (CR) extends lifespan from yeast to mammals. In budding yeast, inhibition of the conserved TOR and/or PKA pathways has been shown to mediate lifespan extension by CR partly through the activation of stress response. However, how the stress response is regulated at the systems level is poorly understood. In this study, by using fluorescent reporters whose expression is dependent on the transcription factors Msn2/4 and Gis1, two separate screenings were conducted to reveal novel regulators of the stress response induced by starvation. A 'focused' screening on the 272 'signalling' mutants revealed that, apart from the previously identified Rim15, Yak1 and Mck1 kinases, the SNF1/AMPK complex, the cell wall integrity (CWI) pathway and a number of cell cycle regulators are necessary to elicit appropriate stress response. The chronological lifespan (CLS) of these signalling mutants correlates well with the amount of accumulated storage carbohydrates but poorly with transition-phase cell cycle status. Subsequent analyses reveal that the levels of intracellular reactive oxygen species are controlled by Rim15, Yak1 and Mck1. Furthermore, CLS extension enabled by tor1 deletion is dependent on the above three kinases. These data suggest that the signalling pathways (SNF1 and CWI) and the kinases downstream of TOR/PKA (Rim15, Yak1 and Mck1) coordinate the metabolic reprogramming (to accumulate storage carbohydrates) and the activation of anti-oxidant defence systems (to control ROS levels) to extend chronological lifespan. A 'genome-wide' screening of a haploid deletion library indicates that less than 10% of the non-essential genes are implicated in the regulation of starvation-induced stress response. Gene ontology analysis suggests that they can be grouped into major clusters including mitochondrial function, r-RNA processing, DNA damage and repair, transcription from RNA polymerase and cell cycle regulation. Further phenotypic assays confirm the previous observation that CLS extension is mostly correlated with the accumulation of storage carbohydrates. Compromised expression of stress response reporters is confirmed by FACS in a variety of mitochondrial mutants, suggesting that mitochondrial respiration also plays a key role in the activation of stress response. Put together, the above findings indicate that stress response and metabolic reprogramming induced by glucose starvation are coordinated by multiple signalling pathways and the activation of mitochondrial respiration is essential to both cellular processes and to CLS extension.

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Adolescence is an important period in the life cycle for which to study stress, due to the many involved developmental changes that require adaptation. This adaptation can be very stressful and result in pathology. Stress is defined as a "process" involving a continual transaction between stressors in the environment, mediating variables, and stress responses. The Stress-Response Scale for Adolescents (SRSA) was developed to measure self-perceived stress responses of those between the ages of 14 to 20. The SRSA's development involved three studies. Study 1 involved item selection, scale construction, item reduction, and estimations of internal consistency and validity. Truthfulness items were developed to determine the honesty of responses. Study 2 tested the ability of the SRSA, through roe-enactment methodology, to distinguish those in a high-stress condition versus those in a low-stress condition. Study 3, again with the use of role-enactment methodology, tested the potential of the SRSA to detect changes in stress-response levels when individuals were taken from a low-stress to a high-stress condition and vice versa. The final SRSA includes 32 stress-response and six truthfulness items. Initially, factor analysis of the SRSA revealed a high loading of gender on the primary factor. Separate forms were created for males and females. Repeat factor analyses of items in the two forms revealed four factors each for males and females but were of questionable utility due to high intercorrelations. All sections of the SRSA should be used for most purposes. Internal consistency estimates of the SRSA are .96 (2 < .05) for females and .94 (2 < .05) for males. Validity estimates are all in the expected direction and range from .25 to .79 for both males and females. The truthfulness items have a coefficient alpha of .82 for females and .77 for males, with validity estimates ranging from .34 for females to .14 (25 < .05) for males. Studies 2 and 3 revealed that the SRSA does have the potential of differentiating between those in different stress conditions and also of detecting stress-response changes. It was concluded that the SRSA, although in preliminary form, has the potential of assessing the stress response in adolescents as long as the discussed weaknesses, such as small sample size and nonrandomization, are taken into account.