Nonequilibrium work methods for determining potentials of mean force (PMF) w(z) have recently gained popularity as an alternative to standard equilibrium based methods. Introduced by Kosztin et al., the forward-reverse (FR) method is a bidirectional work method in that it requires the work to be sampled in both forward and reverse directions along the reaction coordinate z. This bidirectional sampling leads to much faster convergence than other nonequilibrium methods such as the Jarzynski equality, and the calculation itself is extremely simple, making the FR method an attractive way of determining the PMF. Presented here is an extension to the FR method that deals with sampling problems along essentially irreversible reaction coordinates. By oscillating a particle as it is steered along a reaction coordinate, both forward and reverse work samples are obtained as the particle progresses. Dubbed the oscillating forward-reverse (OFR) method, this new method overcomes the issue of irreversibility that is present in numerous soft-matter and biological systems, particularly in the stretching or unfolding of proteins. The data analysis of the OFR method is non-trivial however, and to this end a software package named the ‘OFR Analysis Tool’ has been created. This software performs all of the complicated analysis necessary, as well as a complete error analysis that considers correlations in the data, thus streamlining the use of the OFR method for potential end users. Another attractive feature of the FR method is that the dissipative work is collected at the same time as the free energy changes, making it possible to also calculate local diffusion coefficients, D(z), from the same simulation as the PMF through the Stokes-Nernst-Einstein relation Fdrag = −γv, with γ = kB T /D. While working with the OFR method, however, the D(z) results never matched known values or those obtained through other methods, including the mean square displacement (or Einstein) method. After a reformulation of the procedure to obtain D(z), i.e. by including the correct path length and particle speeds, results were obtained that were much closer to the correct values. The results however showed very little variation over the length of the reaction coordinate, even when D(z) was known to vary drastically. It seemed that the highly variable and noncontinuous velocity function of the particle being steered through the “stiff-spring” method was incompatible with the macroscopic definition of the drag coefficient, γ. The drag coefficient requires at most a slowly varying velocity so that the assumption of a linearly related dissipative work remains valid at all times. To address this, a new dynamic constraint steering protocol (DCP) was developed to replace the previously used “stiff-spring” method, now referred to as a dynamic restraint protocol (DRP). We present here the results for diffusion in bulk water, and both the PMF and diffusion results from the permeation of a water molecule through a DPPC membrane. We also consider the issue of ergodicity and sampling, and propose that to obtain an accurate w(z) (and D(z)) from even a moderately complex system, the final result should be a weighted average obtained from numerous pulls. An additional utility of the FR and OFR methods is that the permeability across lipid bilayers can be calculated from w(z) and D(z) using the inhomogeneous solubility-diffusion (ISD) model. As tests, the permeability was first calculated for H2O and O2 through DPPC. From the simulations, the permeability coefficients for H2O were found to be 0.129 ± 0.075 cm/s and 0.141 ± 0.043 cm/s, at 323 K and 350 K respectively, while the permeability coefficients for O2 were 114 ± 40 cm/s and 101 ± 27 cm/s, again at 323 K and 350 K respectively. As a final, more challenging system, the permeability of tyramine – a positively charged trace amine at physiological pH – was calculated. The final value of P = 0.89 ± 0.24 Ang/ns is over two orders of magnitude lower than that obtained from experiment (22 ± 4 Ang/ns), although it is clear that the permeability as calculated through the ISD is extremely sensitive to the PMF, as scaling the PMF by ∼ 20% allowed the simulation and experimental values to agree within uncertainty. With accurate predictions for free energies and permeabilities, the OFR method could potentially be used for many valuable endeavors such as rational drug design.

Connectomics – the study of neural connectivity – is primarily concerned with the mapping and characterisation of wired synaptic links; however, it is well established that long-distance chemical signalling via extrasynaptic volume transmission is also critical to brain function. As these interactions are not visible in the physical structure of the nervous system, current approaches to connectomics are unable to capture them. This work addresses the problem of missing extrasynaptic interactions by demonstrating for the first time that whole-animal volume transmission networks can be mapped from gene expression and ligand-receptor interaction data, and analysed as part of the connectome. Complete networks are presented for the monoamine systems of Caenorhabditis elegans, along with a representative sample of selected neuropeptide systems. A network analysis of the synaptic (wired) and extrasynaptic (wireless) connectomes is presented which reveals complex topological properties, including extrasynaptic rich-club organisation with interconnected hubs distinct from those in the synaptic and gap junction networks, and highly significant multilink motifs pinpointing locations in the network where aminergic and neuropeptide signalling is likely to modulate synaptic activity. Thus, the neuronal connectome can be modelled as a multiplex network with synaptic, gap junction, and neuromodulatory layers representing inter-neuronal interactions with different dynamics and polarity. This represents a prototype for understanding how extrasynaptic signalling can be integrated into connectomics research, and provides a novel dataset for the development of multilayer network algorithms.

The aim of this thesis was to determine the content of biogenic amines (BA)and polyamines (PA)in selected types of cheese and the influence of storage time on the content of biogenic amines.

The consumption of fermented goods has been related with outbreaks of foods intoxications, like these biogenic amines have been associated to the consummations of these products. The aims went to assess the potential of amines formation in dry fermented sausage added or not with starter and fiber, determination of amines formation in cooked salami added or not with organic acids. The dry fermented sausages were fabricated and cased in artificial collagen no eatable gut then, ripening under controlled conditions of temperature, moisture and air stream. The cooked salamis were added with organic acids (lactic and citric), cased in artificial collagen no eatable gut and cooked. The products were according to the microbiologic available legal patterns. The histamine reached amounts of 145,61; 93,38 and 130,10 mg·kg-1 and the tyramine reached amounts of 67,05; 70,28 and 71,87 mg·kg-1 for the dry fermented sausages treatments: control, starter and starter and fiber respectively. The histamine in the cooked salami treatments: control, lactic acid and citric acid reached amounts of 48,06; 55,37 and 49,16 mg·kg-1 and the tyramine reached amounts of 11,00; 11,06 and 11,35 mg·kg-1 respectively. There were no differences at the level tested (p<0,05). In conclude the starter did not avoid the amine formation, attributed to the polluting bacterium found already in the raw materials. The temperature and the organic acids did not influence in the final amounts of amines in the cooked salami. The taste assess was not influenced by the presence of amines. The addition of glucose and sucrose avoided larger amines amounts in the fermented salami and the wheat fiber did not interfere in the metabolism of the microorganisms. / O consumo de alimentos fermentados tem sido relacionado com surtos de intoxicações alimentares e as aminas biogênicas são associadas ao consumo destes produtos. Os objetivos foram avaliar o potencial de formação de aminas em salame tipo Italiano, adicionado ou não, de cultura starter e fibra; determinar a formação de aminas no salame cozido, adicionado ou não de ácidos orgânicos. Os salames foram elaborados e embutidos em tripa artificial de colágeno não comestível. Maturados sob condições de temperatura, umidade e velocidade do ar controlados. Na fabricação do salame cozido, foram adicionados ácidos orgânicos (láctico e cítrico), embutidos em tripa artificial de colágeno não comestível e as peças cozidas. Os produtos se apresentaram conforme os padrões microbiológicos vigentes. A histamina alcançou valores de 145,61; 93,38 e 130,10 mg·kg-1 e a tiramina valores de 67,05; 70,28 e 71,87 mg·kg-1 para os tratamentos do salame controle, starter e starter e fibra respectivamente. A histamina alcançou valores de 48,06; 55,37 e 49,16 mg·kg-1 e a tiramina valores de 11,00; 11,06 e 11,35 mg·kg-1 para os tratamentos do salame cozido controle, ácido láctico e ácido cítrico respectivamente. Não houve diferenças ao nível testado (p<0,05). Conclui-se que o starter comercial não evitou a formação de aminas, atribuída às bactérias contaminantes encontradas já nas matérias-primas. A temperatura e os ácidos orgânicos não influenciaram nos níveis finais de aminas no salame cozido. A análise sensorial não foi influenciada pela presença das aminas. A adição de glicose e sacarose evitou maiores acúmulos de aminas no salame fermentado e a fibra de trigo não interferiu no metabolismo dos microrganismos.

An animal’s ability to navigate through its natural environment is critical to its survival. Navigation can be slow and methodical such as an annual migration, or purely reactive such as an escape response. How sensory input is translated into a fast behavioral output to execute goal oriented locomotion remains elusive. In this dissertation, I aimed to investigate escape response behavior in the nematode C. elegans. It has been shown that the biogenic amine tyramine is essential for the escape response. A tyramine-gated chloride channel, LGC-55, has been revealed to modulate suppression of head oscillations and reversal behavior in response to touch. Here, I discovered key modulators of the tyraminergic signaling pathway through forward and reverse genetic screens using exogenous tyramine drug plates. ser-2, a tyramine activated G protein-coupled receptor mutant, was partially resistant to the paralytic effects of exogenous tyramine on body movements, indicating a role in locomotion behavior. Further analysis revealed that ser-2 is asymmetrically expressed in the VD GABAergic motor neurons, and that SER-2 inhibits neurotransmitter release along the ventral nerve cord. Although overall locomotion was normal in ser-2 mutants, they failed to execute omega turns by fully contracting the ventral musculature. Omega turns allow the animal to reverse and completely change directions away from a predator during the escape response. Furthermore, my studies developed an assay to investigate instantaneous velocity changes during the escape response using machine based vision. We sought to determine how an animal accelerates in response to a mechanical stimulus, and subsequently decelerates to a basal locomotion rate. Mutant analysis using this assay revealed roles for both dopamine and tyramine signaling. During my doctoral work, I have further established the importance for tyramine in the nematode, as I have demonstrated two additional roles for tyramine in modulating escape response behavior in C. elegans.

Despite the current understanding that sensorimotor circuits function through the action of transmitters and modulators, we have a limited understanding of how the nervous system directs the flow of information necessary to orchestrate complex behaviors. In this dissertation, I aimed to uncover how the nervous system coordinates these behaviors using the escape response of the soil nematode, Caenorhabditis elegans, as a paradigm. C. elegans exhibits a robust escape behavior in response to touch. The worm typically moves forward in a sinusoidal pattern, which is accompanied by exploratory head movements. During escape, the worm quickly retreats by moving backward from the point of stimulus while suppressing its head movements. It was previously shown that the biogenic amine tyramine played an important role in modulating the suppression of these head movmemetns in response to touch. We identified a novel tyramine-gated chloride channel, LGC-55, whose activation by tyramine coordinates motor programs essential for escape. Furthermore, we found that changing the electrical nature of a synapse within the neural circuit for escape behavior can reverse its behavioral output, indicating that the C. elegans connectome is established independent of the nature of synaptic activity or behavioral output. Finally, we characterized a unique mutant, zf35 , which is hyperactive in reversal behavior. This mutant was identified as a gain of function allele of the C. elegans P/Q/N-type voltage-gated calcium channel, UNC-2. Taken together, this work defines tyramine as a genuine neurotransmitter and completes the neural circuit that controls the initial phases of the C. elegans escape response. Additionally, this research further advances the understanding of how the interactions between transmitters and ion channels can precisely regulate neural circuit activity in the execution of a complex behavior.

The thesis deals with the use and effectiveness of some less common methods of conservation of fish meat. The formation of biogenic amines in meat is observed in connection with the non-traditional preservative methods. Amines can serve as indicators of protein degradation. The quality of fish was considered in connection with the increasing content of selected biogenic amines (putrescine, cadaverine, spermidine, spermine, 2-fenylathylamine, histamine, tyramine and tryptamine). Ultra performance liquid chromatography (UPLC) was used as the method for determination of biogenic amines. Amines were derivatized by dansylchloride before their UPLC separation. The fish flesh was vacuum-packed. Samples were stored for several weeks in a thermostat at the selected storage temperature after the application of selected preservative technique. Beta-irradiation and high hydrostatic pressure were used for the preservation of fish flesh. Control samples were not exposed to the â-irradiation and high pressure. The organoleptic properties were studied for all samples (smell/odor, insight and shape). Beta-irradiation was applied to fish meat of common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). Both these species of freshwater fish are economically significant. Carp and trout are the species being mostly consumed in the Czech Republic. Fish meat is considered to be provided the flesh is fresh. A testing series of samples was created with common carp to determine the appropriate dose of â-irradiation. The maximum permissible dose of irradiation for fish meat is 3 kGy. Fish samples were exposed this dose in the first experiment. The dose of irradiation was reduced in following experiments based on the experience from the initial experiment. The doses of 0.25, 0.5, 0.75, 1.0 and 2.0 kGy were applied to rainbow trout. The value of 0.75 kGy of â-irradiation or higher (1.0, 2.0 and 3.0 kGy) prolonged the shelf life of fish meat, which was stored for three months (98 days). Prolonging of the shelf life of fish meat to approximately 98 days at 3.5 °C is redundant from technical point of view. For that reason lower doses 0.25, 0.5 and 0.75 kGy were tested in more detail in the repeated experiment with carp meat. Lower doses of â-irradiation are considered to be more acceptable and-at the same time-sufficiently effective for delaying the beginning of degradation processes. 6 High hydrostatic pressure was applied to meat of common carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss) and pike (Exos lucius). Pike is another very popular kind of freshwater fish. Pike flesh is very tasty, but in spite of this, pike is not so much popular among consumers compared to carp and trout. The cause is its high price. Samples of pike were stored at standard temperature 3.5 °C and also at higher temperature 12 °C (unlike experiments with â-irradiation). Lower temperature of storage (3.5 °C) followed the conditions of storing of fish meat in industrial refrigeration facilities and households. The high pressure might not be sufficient for preservation at higher storage temperatures. This assumption was based on available information. Samples were treated by high pressure and stored at both 3.5 °C and 12 °C to verify this assumption. Higher temperature simulated either failure of refrigeration equipment or unsuitable store temperature of meat. In all species selected freshwater fish two levels of high pressure were applied ? 300 and 500 MPa. Both levels had significantly reduced the formation of biogenic amines, especially in samples stored at 3.5 °C. At this temperature, the effect of 300 and 500 MPa delayed start of degradation processes in fish meat by 3?4 weeks. At 12 °C and 500 MPa, high pressure extended the sustainability of meat by no more than one week. 500 MPa is effective treatment at the lower temperature of 3.5 °C. High pressure is not reliable preservative techniques at higher temperature.

Universidade Federal do Cearà / O presente trabalho relata o estudo quÃmico dos constituintes volÃteis das folhas e frutos de Myrcia sp., coletados no municÃpio de Amontada-CE em marÃo de 2005 e a obtenÃÃo de fraÃÃes e substÃncias provenientes do decocto das folhas frescas e do extrato etanÃlico das folhas secas de Cissus verticillata, bem como o estudo concomitante do seu potencial farmacolÃgico como hipoglicemiante atravÃs de testes realizados com a fraÃÃo solÃvel em metanol, fraÃÃo rica em tiramina e com a tiramina, obtidos de C. verticillata. Os Ãleos essenciais de Myrcea sp. foram analisados por cromatografia gÃs-lÃquido acoplada à espectrometria de massa (CGL/EM) e, quantitativamente, atravÃs do uso de CGL acoplada a detector do tipo FID. A anÃlise do Ãleo essencial das folhas de Myrcia sp. (GAOFOLHAS), permitiu a identificaÃÃo de treze componentes: d-elemeno, b-elemeno, trans-cariofileno, a-humuleno, b-chamigreno, germacreno D, b-selineno, a-guaieno, a-selineno, a-Z-bisaboleno, d-cadineno, epi-a-murolol e a-cadinol. O Ãleo essencial dos frutos de Myrcia sp. (GAOFRUTOS) permitiu a identificaÃÃo de onze componentes: d-elemeno, b-elemeno, trans-cariofileno, a-guaieno, a-humuleno, germacreno D, b-selineno, a-selineno, germacreno A, d-cadineno e germacreno B. O estudo fitoquÃmico do decocto de C. verticillata resultou na fraÃÃo rica em tiramina (CVFDSM-F19-24) que apÃs fracionamento cromatogrÃfico permitiu o isolamento da substÃncia tiramina, inÃdita no gÃnero Cissus. Os testes realizados com esta fraÃÃo e com a tiramina, em ratos com diabetes aloxan-induzida, mostraram reduÃÃo na glicemia, colesterol total, triglicÃrides e nÃveis de VLDL e aumento nos nÃveis de HDL. O fracionamento cromatogrÃfico da fraÃÃo hexÃnica, proveniente do extrato etanÃlico das folhas secas de C. verticillata, permitiu o isolamento dos esterÃides, b-sitosterol e b-sitosterol-glicosilado. A caracterizaÃÃo estrutural das substÃncias isoladas de C. verticillata foi realizada atravÃs do uso de tÃcnicas espectroscÃpicas, tais como, infravermelho e RMN de 1H e 13C, incluindo tÃcnicas uni e bidimensionais (HMBC e HMQC), bem como a comparaÃÃo com dados descritos na literatura. / The present work reports on the volatile constituents from leaves and fruits of Myrcia sp. collected in Amontada County-Cearà State, in march/2006. It also reports on the phytochemical analysis of the decoction solution from fresh leaves, and the ethanol extract of dried leaves of Cissus verticillata in a concomitant study of its pharmacological potential as hipoglycemiant. GLC/MS and GLC/FID analysis of the essential oil from leaves (GAOFOLHAS) allowed the identification of 13 components: d-elemene, b-elemene, trans-caryophyllene, a-humulene, b-chamigrene, germacrene D, b-selinene, a-guaiene, a-selinene, a-Z-bisabolene, d-cadinene, epi-a-murolol and a-cadinol, while the essential oil from fruits (GAOFRUTOS) allowed the identification of 11 components: d-elemene, b-elemene, trans-cariophylene, a-guaiene, a-humulene, germacrene D, b-selinene, a-selinene, germacrene A, d-cadinene and germacrene B. The silica-gel chromatography analysis of the decoction solution of fresh leaves of C. verticillata allowed the separation of a fraction rich in tyramine, and from it, pure tyramine. Pharmacological tests on rats with aloxan-induced glycemia with both the tyramine rich fraction and pure tyramine allowed reduction on the glycemia levels, as well as for total cholesterol, triglycerids and VLDL, but HDL increasing. Silica-gel chromatography analyses of the ethanol extract from dried leaves of C. verticillata allowed the isolation of b-sitosterol and its glucoside derivative. Structure determination of all substances of C. verticillata was accomplished by means of spectroscopic techniques such as IR, 1H and 13C NMR, including uni and bi-dimensional pulse sequences (HMBC and HMQC) ad comparison with data from the literature.