<p>\abstract</p><p>Transcription and translation describe the flow of genetic information from DNA to mRNA to protein. Recent studies show that at a single cell level, these processes are stochastic, which results in the variation of the number of mRNA and proteins even under identical environmental conditions. Because the number of mRNA and protein in each single cell are actually very small, these variations can be crucial for cellular function in diverse contexts, such as development, stress response, immunological and nervous system function. Most studies examine the origin and effects of stochastic gene expression using computer simulations. My goal is to develop a theoretical framework to study activity-dependent gene expression using simplified models that capture essential features. </p><p>I have examined the dynamics of stochastic gene regulation in three contexts. First, I examine how fluctuations in promoter accessibility lead to "bursty" transcription, during which genes are turned "on" or "off" stochastically. I describe a mathematical formalism to represent bursty gene expression in a coarse-grained manner as a Markov process and derive a master equation for the time evolution of the probability distribution of the number of mRNA molecules. This allows us to examine how transcript number responds to time varying stimuli. This model forms a basic building block for understanding the signal transmission and noise of the transcription process to time varying inputs as would be sensed by cells in dynamic environments. In addition to synthesis, gene expression is subject to additional modes of regulation. One such mechanism that controls transcript numbers is by microRNAs (miRNAs), which pair with target mRNAs to repress protein production following transcription. Although hundreds of miRNAs have been identified in mammalian genomes, the function of miRNA-based repression in the context of gene regulation networks still remains unclear. I explore the functional roles of feedback regulation by miRNAs and show that protein fluctuations strongly depend on the mode of miRNA-mediated repression. I discuss the functional implications of protein fluctuations arising from miRNA-mediated repression on gene regulatory networks. Finally, I examine the impact of fluctuations on alternative splicing, which is a major source for proteomic complexity in higher eukaryotes. Although the proteins regulating alternative splicing have been extensively studied, little is known about how noise arising from the stochastic nature of alternative splicing contributes to the entire gene expression process. I explore the functional roles and noise properties of alternative splicing, focusing on the case of exon skipping and intron retention. I show that while the overall counts of the mRNAs of the two isoforms are independent and Poisson distributed, diffusion and binding of the splicing factors contributes to the variance in the abundance of the isoforms. </p><p>Noise in gene expression may be of particular relevance in the nervous system. Environmental stimuli drive the rapid remodeling of neural circuitry in part by inducing the activation of genes to make proteins that modify neuronal excitability and connectivity, ultimately influencing higher order brain function. Finally, I examine the implications of our studies for activity dependent gene expression in the nervous system.</p> / Dissertation

Posttraumatic stress disorder (PTSD) is an anxiety disorder that may result from an exposure to a severely traumatic life-event. It is characterised by a delayed onset of psychological and physical symptoms including re-experiencing the event, avoidance of reminders associated with the trauma, increased autonomic arousal and distinct memory deficits. This disorder is also characterised by a maladaptive hypothalamic-pituitary-adrenal (HPA)-axis response and altered monoamine concentrations in the hippocampus and pre-frontal cortex. The Time Dependent Sensitization (TDS) model is a putative animal model of PTSD that is based on the concept of repeated trauma, using three acute stressors (TS) of intense severity followed by a mild situational reminder (RS) on day 7 subsequent to the acute stressors. The aims of this study were to determine if the Triple Stressor (TS) induces stress and if the situational reminder (RS) is necessary for the maintenance of the stress response over time and whether these two stress responses are qualitatively and quantitively different. This was done to further validate the TDS model and to characterize the development and progression of the stress-related pathology of PTSD. Methods used were High Performance Liquid Chromatography (HPLC) with electrochemical detection (biochemical correlates) for quantifying the monoamines dopamine (DA), noradrenaline (NA) and serotonin (5-HT) concentrations in the hippocampus and pre-frontal cortex (PFC); radio immuno assay (RIA) for the determination of plasma corticosterone concentrations (neuroendocrine parameter) and the use of the Elevated Plus Maze (EPM) to detect anxiety-like behaviour (behavioural analyses). The study was subdivided into an Acute and Re-Stress study (n = 10). In the Acute Study rats were exposed to TS as the only stressor. Group 1 was sacrificed immediately after TS, Group 2 was sacrificed 3 days post TS and Group 3 on day 7 post TS. In the Re-Stress Study both TS and RS were used as stressors. Group 4 was sacrificed immediately after the situational reminder, Group 5 was sacrificed 3 days post RS and Group 6 on day 7 post RS. A group of unstressed rats were used as Control. The results of this study found corticosterone concentrations elevated immediately after the TS (p<0.05). Exposure to the RS resulted in a profound hypocortisolism (p<0.05). These results indicate a possible disturbance in the regulation of the HPA-axis, which manifests as an enhanced negative feed-back upon re-introduction of the stressful situation. Changes in MA concentrations were evident. Although no definite fixed trend is apparent in this study, it is evident that the TDS model does induce monoamine dysregulation. Hippocampal NA. DA and 5-HT concentrations were noted to be elevated on day 7 post TS (p<0.05). On day 7 post RS only hippocampal 5HT was decreased significantly (p<0.05). Behavioural analyses indicate that stress related anxiety was not sustained after the TS but 7 days after the exposure to the RS rats were most anxious (p<0.05). The results confirm that the TDS model does induce PTSD-like symptoms in rats and that the situational reminder (RS) is necessary for the maintenance of the stress response. This model may be useful in the investigation of future experimental pharmacological interventions in the management of PTSD. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

Posttraumatic stress disorder (PTSD) is classified as an anxiety disorder and the characteristic symptoms (re-experiencing, avoidance as well as numbing of general responsiveness and hyperarousal) of this disorder develop in response to a traumatic event. The disorder is characterised by hypothalamic-pituitary-adrenal (HPA) axis abnormalities linked with changes in cortisol moreover, the hippocampus and cortex also play a role in the neurobiology. With regard to the neurochemistry of this disorder it is known that gamma amino butyric acid (GABA) is involved however, the precise role of GABA in PTSD and how stress changes GABA concentrations in the brain are still not fully understood. Another aspect regarding PTSD that has not been clearly defined is the treatment of PTSD. Classic anxiolytics such as diazepam is expected to relieve the anxiety linked with PTSD. Studies with this group of drugs have however not produced the concrete evidence needed to establish it as a treatment of choice for PTSD and subsequently other classes of drugs have been investigated as possible treatment options for PTSD. Among these is lamotrigine, which in a clinical study was found to be effective in alleviating symptoms of PTSD. Moreover, a possible pharmacokinetic-pharmacodynamic relationship for each of these drugs has also not been elucidated. In order to elude on some of these uncertainties, an animal model of PTSD, time dependent sensitisation (TDS), was used. GABA levels in the rat hippocampus and frontal cortex were determined at two different time intervals following the TDS procedure (1 day and 7 days post re-stress). High performance liquid chromatography (HPLC) with electrochemical (EC) detection was used to determine gamma amino butyric acid (GABA) concentrations. To investigate the possible anxiolytic effects of diazepam and lamotrigine in this model, as well as a possible pharmacokinetic-pharmacodynamic relationship for each drug, pharmacokinetic profiles for both drugs were established in order to find the times of peak and trough levels of each drug. Blood samples were collected at different time intervals after drug administration either from the tail vein of rats (lamotrigine) or directly from the heart (diazepam). Subsequently, drug concentrations at each time interval were determined by means of HPLC with ultraviolet (UV) detection. The behaviour of rats was analysed using the elevated plus-maze (EPM) at peak or trough concentrations of the drugs and this was performed after either acute administration of the drug, or after a 14 day chronic treatment regime. GABA levels in the hippocampus were not found to change statistically significantly in response to stress at either 1 day or 7 days post re-stress. In the frontal cortex, however, GABA levels increased in response to stress at 1 day post re-stress, with a statistically insignificant, but strong trend towards an increase, at 7 days post re-stress. With regard to the pharmacokinetic profiles, the peak concentration of diazepam was found to occur at 60 minutes, with lamotrigine's peak at 120 minutes. The behavioural studies indicated that acute treatment with diazepam 3 mg/kg resulted in a statistically significant increase in both ratio open arm entries and ratio time spent in the open arms at peak level of the drug. After acute treatment with diazepam 3 mg/kg a statistically significant decrease in ratio time spent in open arms was also found when the ratio time spent in open arms at peak level of the drug and the ratio time spent in open arms at trough level of the drug was compared. In response to chronic treatment with diazepam 3 mg/kg for 14 days, test animals exhibited an increase in the ratio open arm entries at trough level of the drug, with a statistically insignificant yet definite trend towards an increase at peak level. Acute treatment with lamotrigine 10 mg/kg resulted in no statistically significant change in EPM parameters. In response to chronic treatment, however, a statistically significant increase was found in ratio time spent in open arms at peak level of the drug, with a statistically insignificant trend towards an increase at trough level. From the results of this study, we may therefore conclude that GABA-levels in the brain are definitely affected, but in different ways, following TDS-stress. A pharmacokinetic-pharmacodynamic relationship between the drugs' levels and aversive behaviour could also be established. Furthermore it appears that more sustained anxiolytic effects are evident following chronic treatment with both drugs than with acute administration of these drugs. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2006

Posttraumatic stress disorder (PTSD) is an anxiety disorder that may follow exposure to severe emotional trauma and presents with various symptoms of anxiety, hyperarousal and cognitive anomalies. Interestingly, only 10-30% of an exposed population will go on to develop full-blown PTSD. Cholinergic neurotransmission is implicated in anxiety as well as other typical manifestations of PTSD, particularly cognitive changes. The frontal cortex and hippocampus regulate and in turn are affected by stress, and have also been implicated in the underlying neuropathology of PTSD. These areas are densely innervated by cholinergic neurons originating from the basal forebrain. In this study, the time dependent sensitization (TDS) model was used to induce symptoms of PTSD in animals. The study was designed to determine the long-term effects of an intense, prolonged aversive procedure on central muscarinic acetylcholine receptor (mAChR) characteristics and the correlation if any of those findings to cognitive aspects and general arousal as characteristics associated with PTSD. In order to achieve this goal, male Sprague-Dawley rats were exposed to the TDS stress paradigm with behavioral/neuro-receptor assessments performed on day 7 post re-stress (duration of each experiment in whole is 14 days). Acoustic startle reflex (ASR) was used to determine emotional state (hyperarousal), while the conditioned taste aversion (CTA) paradigm was implemented in order to assess aversive memory. Muscarinic receptor binding studies were performed in the frontal cortex and hippocampus. Moreover, both the stress-exposed and control animals were pre-tested in the acoustic startle chamber in order to attempt to separate stress sensitive from stress-resilient animals based on predetermined ASR criteria. The ASR niodel was previously validated in our laboratory, while the CTA model was validated in this project before application. In the CTA model, an i.p. injection with lithium chloride (LiCl) (associated with digestive malaise), was used as unconditioned stimulus (US) and was paired with a saccharinlcyclamate drinking solution as conditioned stimulus (CS) to induce aversion to the novel taste (CS) when presented in the absence of the US. Population data of animals tested in the ASR experiment indicated no statistical significant difference between stressed and control animals. However, when each animal was assessed individually, 22.5 % of the exposed population displayed all increase above the predetermined criteria of 35 % in startle response, indicating a state of heightened arousal. In contrast, only 4.2 O h of control animals (no stress) displayed an increase in arousal based on the above mentioned criteria. Muscarinic receptor densities (Bm,) in the total population of animals exposed to stress showed a statistical significant increase in both the hippocampus and frontal cortex when compared to controls, with no changes in & values observed in either one of the areas. In the CTA experiment, TDS stress was implemented as US paired with a saccharinlcyclamate drinking solution as CS. An acute session of prolonged stress (as used in the TDS model) effectively induced aversion to a novel taste and a subsequent reminder of the stress (restress) paired with the CS sustained the acquire adversive memory. Furthermore, LiCl was reintroduced as US in order to assess the effect of prior exposure to two types of stress (acute and TDS) on subsequently acquired CTA memory. Prior exposure to acute stress had no significant effect on subsequently acquired aversive memory when measured either 3- or 7 days post-conditioning (CS-US). Stress-restress (TDS) exposure, however, indicated a significant decrease in aversive memory from 3- to 7 days post-conditioning (CS-US) as well as a significant decrease in aversive memory between the control- and the TDS group 7 days post-conditioning. The mAChR density (B,,) in the frontal cortex; but not in the hippocampus, was elevated at the same point in time (7 days post CS-US pairing) that CTA memory was impaired following TDS stress (stress-restress). Ultimately, these data support an association between altered cholinergic receptors and hyperarousallanxiety in an animal model of PTSD. The data also support the phenomenon of individual susceptibility to stress in animals that parallels that observed in humans exposed to severe trauma. Impaired aversive memory (CTA) is a consequence of prior exposure to TDS stress, but not acute stress, and is likewise mediated by an altered central cholinergic transmission displayed as an increase in mAChRs in the frontal cortex. The lack of studies regarding the influence of the cholinergic system in PTSD related behavior earns ,this project value as inimitable PTSD research. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2007.

The purpose of this study is to evaluate the effects and importance of different cross–section representations and energy group structures for steady state and transient analysis. More energy groups may be more accurate, but the calculation becomes much more expensive, hence a balance between accuracy and calculation effort must be find. This study is aimed at comparing a multi–group energy dynamics code, MGT (Multi–group TINTE) with TINTE (TIme Dependent Neutronics and TEmperatures). TINTE’s original version (version 204d) only distinguishes between two energy group structures, namely thermal and fast region with a polynomial reconstruction of cross–sections pre–calculated as a function of different conditions and temperatures. MGT is a TINTE derivative that has been developed, allowing a variable number of broad energy groups. The MGT code will be benchmarked against the OECD PBMR coupled neutronics/thermal hydraulics transient benchmark: the PBMR–400 core design. This comparative study reveals the variations in the results when using two different methods for cross–section generation and multi–group energy structure. Inputs and results received from PBMR (Pty) Ltd. were used to do the comparison. A comparison was done between two–group TINTE and the equivalent two energy groups in MGT as well as between 4, 6 and 8 energy groups in MGT with the different cross–section generation methods, namely inline spectrum– and tabulated cross–section method. The characteristics that are compared are reactor power, moderation– and maximum fuel temperatures and k–effective (only steady state case). This study revealed that a balance between accuracy and calculation effort can be met by using a 4–group energy group structure. A larger part of the available increase in accuracy can be obtained with 4–groups, at the cost of only a small increase in CPU time. The changing of the group structures in the steady state case from 2 to 8 groups has a greater influence on the variation in the results than the cross–section generation method that was used to obtain the results. In the case of a transient calculation, the cross–section generation method has a greater influence on the variation in the results than on the steady state case and has a similar effect to the number of energy groups. / Thesis (M.Ing. (Nuclear Engineering))--North-West University, Potchefstroom Campus, 2011.

The purpose of this study is to evaluate the effects and importance of different cross–section representations and energy group structures for steady state and transient analysis. More energy groups may be more accurate, but the calculation becomes much more expensive, hence a balance between accuracy and calculation effort must be find. This study is aimed at comparing a multi–group energy dynamics code, MGT (Multi–group TINTE) with TINTE (TIme Dependent Neutronics and TEmperatures). TINTE’s original version (version 204d) only distinguishes between two energy group structures, namely thermal and fast region with a polynomial reconstruction of cross–sections pre–calculated as a function of different conditions and temperatures. MGT is a TINTE derivative that has been developed, allowing a variable number of broad energy groups. The MGT code will be benchmarked against the OECD PBMR coupled neutronics/thermal hydraulics transient benchmark: the PBMR–400 core design. This comparative study reveals the variations in the results when using two different methods for cross–section generation and multi–group energy structure. Inputs and results received from PBMR (Pty) Ltd. were used to do the comparison. A comparison was done between two–group TINTE and the equivalent two energy groups in MGT as well as between 4, 6 and 8 energy groups in MGT with the different cross–section generation methods, namely inline spectrum– and tabulated cross–section method. The characteristics that are compared are reactor power, moderation– and maximum fuel temperatures and k–effective (only steady state case). This study revealed that a balance between accuracy and calculation effort can be met by using a 4–group energy group structure. A larger part of the available increase in accuracy can be obtained with 4–groups, at the cost of only a small increase in CPU time. The changing of the group structures in the steady state case from 2 to 8 groups has a greater influence on the variation in the results than the cross–section generation method that was used to obtain the results. In the case of a transient calculation, the cross–section generation method has a greater influence on the variation in the results than on the steady state case and has a similar effect to the number of energy groups. / Thesis (M.Ing. (Nuclear Engineering))--North-West University, Potchefstroom Campus, 2011.

The nature of the ground state and the first (lowest) singlet excited state geometrical conformations and electronic transitions in the aromatic five-membered heterocyclic oligomers &ndash / oligothiophenes (nT), oligofurans (nF), and oligopyrroles (nP)- containing up to six monomer units (total of 18 molecules) were explored using several computational methodologies. Geometry optimizations were carried out at Austin Model 1 (AM1), Restricted Hartree-Fock (RHF/6-31G*), and Density Functional Theory (DFT, B3LYP/6-31G*) levels for the ground-state conformations of these structurally well-defined heterocyclic oligomers. The Configuration Interaction Singles (CIS) method with the 6-31G* basis set was chosen in computation of the optimal geometry of the lowest singlet excited state. Lowest singlet excitation S1&szlig / S0 energies were calculated using the Zerner&rsquo / s Intermediate Neglect of Differential Overlap for Spectroscopy (ZINDO/S), CIS (CIS/6-31G*), and Time-Dependent DFT (TDDFT/6-31G* and TDDFT/6-31+G*) methods. In computation of the emission S1&agrave / S0 energies, we have employed all methods above except ZINDO/S. In investigation of geometries of the ground and lowest singlet excited state, we compared the bond length alternation (BLA) parameters, Dri in the conjugated backbone of the oligomers. Saturation of the geometrical parameters at the center of oligomers was observed after a certain chain length. Among all methodologies used in computation of excitation (S1&szlig / S0) and emission (S1&agrave / S0) energies, TDDFT results showed the best agreement with experimental data. Fits of computed and experimental excitation energies to an exponential function using the least squares method enabled us to predict Effective Conjugation Length (ECL) values. We obtained the ECLs of 17 (17), 16 (15), and 14 (13) monomer units for polythiophene (PTh), polyfuran (PFu), and polypyrrole (PPr), which have very good agreement with the results obtained from the fits of experimental data (the values in parentheses).

Viele Problemstellungen der Naturwissenschaften führen zur Betrachtung von nichtlinearen Wellengleichungen. Dabei ist von großem Interesse, ob zu vorgegebenen kleinen Daten Lösungen eindeutig existieren und ob diese stetig von den Daten abhängen. Hilfsmittel für diese Probleme sind Aussagen über lineare Wellengleichungen. In der vorliegenden Arbeit werden lineare Klein-Gordon Gleichungen, also Wellengleichungen mit Potentialterm, mit zeitabhängiger Masse bzgl. des Verhaltens ihrer Lösungen untersucht. Von speziellem Interesse sind Resultate mit Bezug auf verallgemeinerte Energieerhaltung und sogenannte Lp – Lq decay-Abschätzungen. Aus der Arbeit geht hervor, dass man eine Klassifizierung für Gleichungen mit fallendem Masseterm finden kann. Für Gleichungen vom Wellentyp ist der Einfluss des Potentialterms gering und die Lösungen verhalten sich wie Lösungen der Wellengleichung. Dem gegenüber stehen Gleichungen vom Klein-Gordon-Typ mit erkennbarem Einfluss des Masseterms. Ausgangspunkt für die Klassifizierung ist das kritische Verhalten der Lösungen einer skaleninvarianten Gleichung mit speziellem Masseterm.

The time-dependent Schrödinger equation (TDSE) models the quantum nature of molecular processes.  Numerical simulations based on the TDSE help in understanding and predicting the outcome of chemical reactions. This thesis is dedicated to the derivation and analysis of efficient and reliable simulation tools for the TDSE, with a particular focus on models for the interaction of molecules with time-dependent electromagnetic fields. Various time propagators are compared for this setting and an efficient fourth-order commutator-free Magnus-Lanczos propagator is derived. For the Lanczos method, several communication-reducing variants are studied for an implementation on clusters of multi-core processors. Global error estimation for the Magnus propagator is devised using a posteriori error estimation theory. In doing so, the self-adjointness of the linear Schrödinger equation is exploited to avoid solving an adjoint equation. Efficiency and effectiveness of the estimate are demonstrated for both bounded and unbounded states. The temporal approximation is combined with adaptive spectral elements in space. Lagrange elements based on Gauss-Lobatto nodes are employed to avoid nondiagonal mass matrices and ill-conditioning at high order. A matrix-free implementation for the evaluation of the spectral element operators is presented. The framework uses hybrid parallelism and enables significant computational speed-up as well as the solution of larger problems compared to traditional implementations relying on sparse matrices. As an alternative to grid-based methods, radial basis functions in a Galerkin setting are proposed and analyzed. It is found that considerably higher accuracy can be obtained with the same number of basis functions compared to the Fourier method. Another direction of research presented in this thesis is a new algorithm for quantum optimal control: The field is optimized in the frequency domain where the dimensionality of the optimization problem can drastically be reduced. In this way, it becomes feasible to use a quasi-Newton method to solve the problem. / eSSENCE

COSTA, Camila Ferreira. Nearest Neighbors with Operating Time Constraints and Optimal Sequenced Route Queries in Time-Dependent Road Networks. 2014. 75 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências, Departamento de Computação, Fortaleza-CE, 2014. / Submitted by guaracy araujo (guaraa3355@gmail.com) on 2016-06-06T19:14:12Z No. of bitstreams: 1 2014_dis_cfcosta.pdf: 2126584 bytes, checksum: a2635ed2f82226579173a9e49d960c00 (MD5) / Approved for entry into archive by guaracy araujo (guaraa3355@gmail.com) on 2016-06-06T19:18:16Z (GMT) No. of bitstreams: 1 2014_dis_cfcosta.pdf: 2126584 bytes, checksum: a2635ed2f82226579173a9e49d960c00 (MD5) / Made available in DSpace on 2016-06-06T19:18:16Z (GMT). No. of bitstreams: 1 2014_dis_cfcosta.pdf: 2126584 bytes, checksum: a2635ed2f82226579173a9e49d960c00 (MD5) Previous issue date: 2014 / In this thesis we study the problems of processing a variation of nearest neighbors and of routing planning queries in time-dependent road networks, i.e., one where travel time along each edge is a function of the departure time. We first study the problem of finding the k points of interest (POIs), for example, museums or restaurants, in which a user can start to be served in the minimum amount of time, accounting for both the travel time to the POI and the waiting time there, if it is closed. Previous works have proposed solutions to answer k-nearest neighbor queries considering the time dependency of the network but not the operating times of the points of interest. We propose and discuss three solutions to this type of query which are based on the previously proposed incremental network expansion and use the A* search algorithm equipped with suitable heuristic functions. We also present experimental results comparing the number of disk access required in each solution with respect to a few different parameters. In the second query, we aim at finding the optimal route that connects a origin to a destination and passes through a number of POIs in a specific sequence imposed on the categories of the POIs. Previous works have addressed this problem, but they do not consider the time dependency of the network. We propose an optimal sequenced route query algorithm which performs an incremental network expansion adopting an A* search. Furthermore, as an OSR query on road network tends to re-expand an extremely large number of nodes, we propose a scheme to reduce the re-expansions. For comparison purposes, we also present a baseline solution which was obtained by extending the previously proposed progressive neighbor exploration algorithm to cope with the time-dependent problem. We performed experiments in synthetic networks comparing the proposed solutions according to the number of expanded vertices in the search and the processing time of the queries. / Nesta dissertação nós estudamos os problemas de processar uma variação de consulta de vizinhos mais próximos e de planejamento de rotas em redes viárias dependentes do tempo. Diferentemente de redes convencionais, onde o custo de deslocamento de um ponto a outro é geralmente dado pela distância física entre esses dois pontos, uma rede dependente do tempo representa de forma mais realista o custo de realizar esse deslocamento, considerando o histórico das condições de tráfego. Mais especificamente, o tempo que um objeto móvel leva para percorrer uma via em tal rede depende do tempo de partida. Por exemplo, o tempo para se deslocar de um ponto a outro em grandes centros durante os horários de pico, quando o tráfego é intenso e as ruas estão congestionadas, é muito maior do que em horários normais. Dentro do contexto apresentado, primeiramente nós estudamos o problema de encontrar k pontos de interesse, como por exemplo, museus ou restaurantes, nos quais um usuário pode começar a ser servido o mais rápido possível. Em outras palavras, nós buscamos minimizar a soma do tempo de viagem até um ponto de interesse mais o tempo de espera até que ele abra, caso esteja fechado. Trabalhos anteriores tratam do problema de encontrar os k vizinhos mais próximos em redes dependentes do tempo, porém, eles não levam em consideração o horário de funcionamento dos pontos de interesse. Desta forma, a consulta abordada nesses trabalhos pode retornar pontos de interesse que estão mais próximos do usuário, considerando um dado tempo de partida, mas que podem demorar para abrir, fazendo com que o usuário espere por muito tempo. Nós propomos e discutimos três soluções para essa consulta que são baseadas em um algoritmo de expansão incremental da rede previamente proposto na literatura e usam o algoritmo de busca A* equipado com funções heurísticas adequadas para cada solução. Com o uso do algoritmo A*, nós visamos reduzir o percentual da rede avaliado na busca, evitando expandir vértices que oferecem uma baixa probabilidade de alcançar nosso objetivo. Também apresentamos resultados experimentais que comparam o número de acessos ao disco exigido em cada solução em relação a alguns parâmetros diferentes e que indicam em que casos deve-se optar por cada solução. Na segunda consulta, nós visamos encontrar a rota ótima que conecta uma dada origem a um dado destino e que passa por uma série de pontos de interesse pertencentes a categorias determinadas pelo usuário em uma certa ordem também especificada pelo usuário. Esse tipo de consulta é conhecida como OSR, do inglês, Optimal Sequenced Route, na literatura. Como exemplo, considere que alguém está indo do trabalho para casa e no seu caminho deseja passar em um banco para sacar dinheiro e depois ir a um restaurante para jantar. Embora existam vários bancos e restaurantes em uma cidade, uma consulta OSR deve procurar pelo banco e pelo restaurante que minimizam o custo da viagem do trabalho para casa. Trabalhos anteriores propuseram soluções para consultas OSR em redes com arestas de custo fixo, mas nenhum deles considerou que esse custo pode variar de acordo com o tempo de partida. Nós propomos uma solução ótima para esse problema que, assim como as abordagens propostas para o problema anterior, expande a rede incrementalmente e usa o algoritmo A* para guiar essa expansão. Além disso, como uma consulta OSR em redes viárias tende a re-expandir um número muito grande de vértices, nós incorporamos à essa solução um esquema para reduzir o número de re-expansões. Nós também apresentamos resultados experimentais que mostram a eficiência dessa solução em comparação com uma solução de base que foi obtida a partir da estensão de um algoritmo anteriormente proposto na literatura. Todos os experimentos foram realizados em redes sintéticas.