Élesztő sejtciklusának modellezése = Modeling the yeast cell cycle

Az ötéves kutatás a hasadó élesztő sejtciklusával kapcsolatos kérdésekre irányult. Az alkalmazott vizsgálati eljárások háromfélék voltak: (1) mikroszkópos filmek analízise, (2) matematikai modellezés, (3) modellillesztés statisztikai módszerek segítségével. Az élesztősejtek átmérőjének ciklus alatti vizsgálata során megállapítottuk, hogy az szinte minden esetben állandónak tekinthető ugyan, de a wee1-6 mutánsok tenyészetében néhány sejt jelentősen vastagodik ciklusa során. Utóbbi jelenséget az ezekre a sejtekre egyébként is jellemző spontán diploidizációval magyarázhatjuk. Sikerült kidolgozni a hasadó élesztő mitózisos sejtciklusának egy olyan determinisztikus, differenciálegyenletekből álló matematikai modelljét, amely képes leírni bizonyos mutánsok kísérletesen tapasztalt endoreplikációs ciklusait, más mutánsok esetében pedig a mitózisos katasztrófa jelenségét. A cdc25 vagy a wee1 gént túltermelő törzsek fenotípusát szintén nagy pontossággal tudtuk szimulálni. Modellünknek elkészült egy olyan sztochasztikus változata, amely a szaporodó tenyészet sejtjei közötti különbségeket is figyelembe veszi, hipotézisünk szerint a sejtmag méretének változása révén. Végezetül kidolgoztunk egy statisztikai módszereken alapuló modell szelekciós eljárást, amelynek segítségével eldönthető, hogy egy sejt növekedése exponenciális vagy (bi)lineáris függvénnyel írható-e le. | The aim of this five-year long scientific project was to investigate different aspects of the regulation of the fission yeast cell cycle. The applied methods were threefold, namely (i) analysis of microscopic films, (ii) mathematical modelling, and (iii) model fitting to experimental cell length data. (i) By studying cell diameter during whole cycles on time-lapse films, we have found that it is almost exclusively constant. However, in some individual wee1-6 mutant cells, the diameter significantly increased. We hypothesised that this phenomenon is probably connected to spontaneous diploidisation of these cells. (ii) We have developed a deterministic mathematical model of the fission yeast cell cycle, consisting of ordinary differential equations. It is able to describe endoreplication cycles of certain cell cycle mutants, mitotic catastrophe in others, as well as the phenotypes of cells overproducing either the cdc25 or the wee1 gene. A stochastic version of this model describes scattering in cell size and cycle time in a fission yeast population; this is based on differences in nuclear volume among the cells. (iii) We have also developed some statistically aided model selection criteria, which discriminates between exponential and (bi)linear growth patterns of individual cells

Studies within Fragment-Based Drug Discovery: Library Synthesis and Hit-to-Lead Optimisation

This thesis reports two projects aimed at addressing challenges within fragment-based drug discovery. The first project describes efforts towards utilising synthetic methodology to address deficiencies within fragment screening collections. This involved the development of a modular, robust and scalable route to access α,α-disubstituted amino ester building blocks, which in turn were derivatised to allow the rapid assembly of five (a total of eight in collaboration) spirocyclic scaffolds. Importantly, this library was structurally diverse, comprising three (a total of six in collaboration) pharmacophore-like heterocycles and carbocycles. Moreover, numerous three-dimensional exit vectors were incorporated within each core spirocycle, and the ability of these handles to effect a diverse set of chemical modifications was exemplified through the generation of 16 (a total of 21 in collaboration) examples. Computational studies highlighted the excellent physicochemical and 3D properties of the library, as well as the broad coverage of underexplored chemical space that was achieved. This library was also screened for antibacterial activity in a phenotypic assay against the clinically relevant bacterial strains, Pseudomonas aeruginosa and Staphylococcus aureus. The second project examined the inhibition of propionate detoxification mechanisms in bacteria as an attractive strategy for the development of antibacterial agents. A fragment screening campaign against 2-methylcitrate synthase (PrpC) from Pseudomonas aeruginosa identified several hit compounds based on an indole unit. Synthetic efforts were undertaken to elaborate these fragment hits to increase potency. The adopted strategy focused on growing the indole fragment towards the nearby oxaloacetate binding pocket and occupying it with a fragment mimicking its natural substrate. This approach yielded a compound with an in vitro half maximal inhibitory concentration of 130 µM against the enzymatic activity of PrpC

Size control in growing yeast and mammalian cells

BACKGROUND: In a recent publication it was claimed that cultured mammalian cells, in contrast to yeasts, maintain a constant size distribution in the population without a size checkpoint. This inference may be challengeable. RESULTS: (1) It is argued that "weak" size control implies the existence of a checkpoint, and unfortunately the technique used by Conlon and Raff might obscure such a weak mechanism. (2) Previous investigations of size control in yeasts have shown that individual cell data, rather than means and variances of cell populations, are prerequisites for reliable interpretation. (3) No experimental data so far obtained suggest that in any cell culture a linear growth pattern in cell mass can maintain size homeostasis on its own without size control. (4) Studies on fission yeast mutants indicate that the molecular mechanisms of size control vary with genetic background, implying that no single mechanism is likely to apply to any cell type, including cultured mammalian cells, under all conditions. CONCLUSION: The claim that cultured mammalian cells maintain size homeostasis without a checkpoint needs to be re-evaluated by measurements on individual cells

Optimizing periodicity and polymodality in noise-induced genetic oscillators

Many cellular functions are based on the rhythmic organization of biological processes into self-repeating cascades of events. Some of these periodic processes, such as the cell cycles of several species, exhibit conspicuous irregularities in the form of period skippings, which lead to polymodal distributions of cycle lengths. A recently proposed mechanism that accounts for this quantized behavior is the stabilization of a Hopf-unstable state by molecular noise. Here we investigate the effect of varying noise in a model system, namely an excitable activator-repressor genetic circuit, that displays this noise-induced stabilization effect. Our results show that an optimal noise level enhances the regularity (coherence) of the cycles, in a form of coherence resonance. Similar noise levels also optimize the multimodal nature of the cycle lengths. Together, these results illustrate how molecular noise within a minimal gene regulatory motif confers robust generation of polymodal patterns of periodicity.Comment: 9 pages, 6 figure

DEVELOPING A MATHEMATICAL MODEL FOR THE FISSION YEAST CELL CYCLE: SIMULATING MUTANTS OVEREXPRESSING EITHER CDC25 OR WEE1

During the last decade several mathematical models were constructed to describe the fission yeast cell cycle. In these models, fluctuations of MPF activity were responsible for cell cycle transitions, and they successfully explained the behaviour of wild-type fission yeast cells and many cell division cycle mutants as well. However, the mutants involved in these models were mainly loss-of-function mutants (either temperature-sensitive point mutants or gene deletion ones). By contrast, the phenotypes of several gene overproducing (op) mutants have been published during the last twenty years, like those of cdc25op and wee1op cells (in the case of the latter one, even the effects of different overexpression levels are known). Since Wee1 and Cdc25 is a kinase-phosphatase pair, regulating MPF activity and as a consequence, timing mitotic onset in fission yeast, a detailed mathematical model of the fission yeast cell cycle should be able to simulate these overexpression mutants. Within the framework of this paper, a formerly published model was tested for these mutants. In order to describe properly the behaviour of cdc25op and wee1op mutants, some alterations had to be made in the original model, both in the parameter values and in the equations. If these corrections have been involved, the newly developed model also maintained its capability to explain the phenotypes of all those mutants, for which the original model was made. Furthermore, the model predicts the phenotypes of two mutants not yet constructed by geneticists

Spirocycles as Rigidified sp3-Rich Scaffolds for a Fragment Collection.

Novel divergent methodology to access sp3-rich spirocyclic fragments is reported. First, a robust modular synthesis of bis-alkene amino ester building blocks was developed. Three different carbocycles and six heterocycles were then constructed to assemble eight spirocycles. Importantly, strategic exit vectors were incorporated within each scaffold to aid fragment growth and were elaborated via chemical modifications. Finally, computational methods demonstrate higher levels of rigidity, three-dimensionality, and structural diversity of the library compared to a commercial collection

The time-profile of cell growth in fission yeast: model selection criteria favoring bilinear models over exponential ones

BACKGROUND: There is considerable controversy concerning the exact growth profile of size parameters during the cell cycle. Linear, exponential and bilinear models are commonly considered, and the same model may not apply for all species. Selection of the most adequate model to describe a given data-set requires the use of quantitative model selection criteria, such as the partial (sequential) F-test, the Akaike information criterion and the Schwarz Bayesian information criterion, which are suitable for comparing differently parameterized models in terms of the quality and robustness of the fit but have not yet been used in cell growth-profile studies. RESULTS: Length increase data from representative individual fission yeast (Schizosaccharomyces pombe) cells measured on time-lapse films have been reanalyzed using these model selection criteria. To fit the data, an extended version of a recently introduced linearized biexponential (LinBiExp) model was developed, which makes possible a smooth, continuously differentiable transition between two linear segments and, hence, allows fully parametrized bilinear fittings. Despite relatively small differences, essentially all the quantitative selection criteria considered here indicated that the bilinear model was somewhat more adequate than the exponential model for fitting these fission yeast data. CONCLUSION: A general quantitative framework was introduced to judge the adequacy of bilinear versus exponential models in the description of growth time-profiles. For single cell growth, because of the relatively limited data-range, the statistical evidence is not strong enough to favor one model clearly over the other and to settle the bilinear versus exponential dispute. Nevertheless, for the present individual cell growth data for fission yeast, the bilinear model seems more adequate according to all metrics, especially in the case of wee1Δ cells

Designer Gene Networks: Towards Fundamental Cellular Control

The engineered control of cellular function through the design of synthetic genetic networks is becoming plausible. Here we show how a naturally occurring network can be used as a parts list for artificial network design, and how model formulation leads to computational and analytical approaches relevant to nonlinear dynamics and statistical physics.Comment: 35 pages, 8 figure