Alterations in Notch signaling in neoplastic lesions of the human cervix.

The development of cancer is a cellular process that reflects and is partly driven by alterations in cell determination. Mutations in various molecules responsible for cell determination have been identified as being oncogenic, but little is known about the involvement of normal cell fate-determining mechanisms in the oncogenic process. The Notch pathway defines an evolutionarily conserved, general cell interaction mechanism that controls fundamental aspects of cell determination during vertebrate and invertebrate development. We have explored the involvement of the human Notch pathway in human cervical tissues, which define a cellular environment where cell fate changes take place and where neoplastic conditions have been well characterized. Our evidence suggests that Notch expression is associated with cell populations that are undergoing cell fate changes and that Notch activity can be used to monitor cell fate abnormalities in cervical as well as other epithelial neoplasias.

Substrate specifity of protein kinases and computational prediction of substrates

To ensure signalling fidelity, kinases must act only on a defined subset of cellular targets. Appreciating the basis for this substrate specificity is essential for understanding the role of an individual protein kinase in a particular cellular process. The specificity in the cell is determined by a combination of peptide specificity of the kinase (the molecular recognition of the sequence surrounding the phosphorylation site), substrate recruitment and phosphatase activity. Peptide specificity plays a crucial role and depends on the complementarity between the kinase and the substrate and therefore on their three-dimensional structures. Methods for experimental identification of kinase substrates and characterization of specificity are expensive and laborious, therefore, computational approaches are being developed to reduce the amount of experimental work required in substrate identification. We discuss the structural basis of substrate specificity of protein kinases and review the experimental and computational methods used to obtain specificity information. (c) 2005 Elsevier B.V. All rights reserved.

Transglutaminase 2 is involved in autophagosome maturation

Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but it is enhanced in response to stress, e.g. nutrient deprivation, hypoxia, mitochondrial dysfunction and infection. "Tissue" transglutaminase (TG2) accumulates, both in vivo and in vitro, to high levels in cells under stressful conditions. Therefore, in this study, we investigated whether TG2 could also play a role in the autophagic process. To this end, we used TG2 knockout mice and cell lines in which the enzyme was either absent or overexpressed. The ablation of TG2 protein both in vivo and in vitro, resulted in an evident accumulation of microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) on pre-autophagic vesicles, suggesting a marked induction of autophagy. By contrast, the formation of the acidic vesicular organelles in the same cells was very limited, indicating an impairment of the final maturation of autophagolysosomes. In fact, the treatment of TG2 proficient cells with NH4Cl, to inhibit lysosomal activity, led to a marked accumulation of LC3 II and damaged mitochondria similar to what we observed in TG2-deficient cells. These data indicate a role for TG2-mediated post-translational modifications of proteins in the maturation of autophagosomes accompanied by the accumulation of many damaged mitochondria.

Inhibition of nonsense-mediated mRNA decay by the cytoplasmic poly(A)-binding protein

The expression of a gene from transcription of the DNA into pre-messenger RNA (pre-mRNA) over translation of messenger RNA (mRNA) into protein is constantly monitored for errors. This quality control is necessary to guarantee successful gene expression. One quality control mechanism important to this thesis is called nonsense-mediated mRNA decay (NMD). NMD is a cellular process that eliminates mRNA transcripts harboring premature translation termination codons (PTCs). Furthermore, NMD is known to regulate certain transcripts with long 3′ UTRs. However, some mRNA transcripts are known to evade NMD. The mechanism of NMD activation has been subjected to many studies whereas NMD evasion or suppression still remains rather elusive. It has previously been shown that the cytoplasmic poly(A)-binding protein (PABPC1) is able to suppress NMD of certain transcripts. In this study I show that PABPC1 is able to suppress NMD of a long 3′ UTR-carrying reporter when tethered immediately downstream of the termination codon. I further am able to show the importance of the interaction between PABPC1 and eIF4G for NMD suppression, whereas the interaction between PABPC1 and eRF3a seems dispensable. These results indicate an involvement of efficient translation termination and potentially ribosome recycling in NMD suppression. I am able to show that if PABPC1 is too far removed from the terminating ribosome NMD is activated. After showing the importance of PABPC1 recruitment directly downstream of a terminating ribosome in NMD suppression, I am further able to demonstrate several different methods by which PABPC1 can be recruited. Fold-back of the poly(A)-tail mediated by two interacting proteins on opposite ends of a 3′ UTR manages to bring PABPC1 bound to the poly(A)-tail into close proximity of the terminating ribosome and therefore suppress NMD. Furthermore, small PAM2 peptides that are known to interact with the MLLE domain of PABPC1 are able to strongly suppress NMD initiated by either a long 3′ UTR or an EJC. I am also able to show the NMD antagonizing power of recruited PABPC1 for the known endogenous NMD target β-globin PTC39, which is responsible for the disease β-thalassemia. This shows the potential medical implications and application of suppressing NMD by recruiting PABPC1 into close proximity of a terminating ribosome.

Towards the prediction of essential genes by integration of network topology, cellular localization and biological process information

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cellular damages in the Allium cepa test system, caused by BTEX mixture prior and after biodegradation process

Petroleum and derivatives have been considered one of the main environmental contaminants. Among petroleum derivatives, the volatile organic compounds benzene, toluene, ethylbenzene and xylene (BTEX) represent a major concern due to their toxicity and easy accumulation in groundwater. Biodegradation methods seem to be suitable tools for the clean-up of BTEX contaminants from groundwater. Genotoxic and mutagenic potential of BTEX prior and after biodegradation process was evaluated through analyses of chromosomal aberrations and MN test in meristematic and F 1 root cells using the Allium cepa test system. Seeds of A. cepa were germinated into five concentrations of BTEX, non-biodegraded and biodegraded, in ultra-pure water (negative control), in MMS 4×10 -4M (positive control) and in culture medium used in the biodegradation (blank biodegradation control). Results showed a significant frequency of both chromosomal and nuclear aberrations. The micronucleus (MN) frequency in meristematic cells was significant for most of tested samples. However, MN was not present in significant levels in the F 1 cells, suggesting that there was no permanent damage for the meristematic cell. The BTEX effects were significantly reduced in the biodegraded samples when compared to the respective non-biodegraded concentrations. Therefore, in this study, the biodegradation process showed to be a reliable and effective alternative to treat BTEX-contaminated waters. Based on our results and available data, the BTEX toxicity could also be related to a synergistic effect of its compounds. © 2011 Elsevier Ltd.

Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels.

Voltage-gated sodium channels (Nav) are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the Nav. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the Nav by shifting the voltage-dependence of steady state activation toward more negative potentials.

Processing of polycaprolactone and polycaprolactone-based copolymers into 3D scaffolds and their cellular responses

Synthetic polymers have attracted much attention in tissue engineering due to their ability to modulate biomechanical properties. This study investigated the feasibility of processing poly(varepsilon-caprolactone) (PCL) homopolymer, PCL-poly(ethylene glycol) (PEG) diblock, and PCL-PEG-PCL triblock copolymers into three-dimensional porous scaffolds. Properties of the various polymers were investigated by dynamic thermal analysis. The scaffolds were manufactured using the desktop robot-based rapid prototyping technique. Gross morphology and internal three-dimensional structure of scaffolds were identified by scanning electron microscopy and micro-computed tomography, which showed excellent fusion at the filament junctions, high uniformity, and complete interconnectivity of pore networks. The influences of process parameters on scaffolds' morphological and mechanical characteristics were studied. Data confirmed that the process parameters directly influenced the pore size, porosity, and, consequently, the mechanical properties of the scaffolds. The in vitro cell culture study was performed to investigate the influence of polymer nature and scaffold architecture on the adhesion of the cells onto the scaffolds using rabbit smooth muscle cells. Light, scanning electron, and confocal laser microscopy showed cell adhesion, proliferation, and extracellular matrix formation on the surface as well as inside the structure of both scaffold groups. The completely interconnected and highly regular honeycomb-like pore morphology supported bridging of the pores via cell-to-cell contact as well as production of extracellular matrix at later time points. The results indicated that the incorporation of hydrophilic PEG into hydrophobic PCL enhanced the overall hydrophilicity and cell culture performance of PCL-PEG copolymer. However, the scaffold architecture did not significantly influence the cell culture performance in this study.

Structural and cellular features in metaphyseal and diaphyseal periosteum of osteoporotic rats

Despite the important physiological role of periosteum in the pathogenesis and treatment of osteoporosis, little is known about the structural and cellular characteristics of periosteum in osteoporosis. To study the structural and cellular differences in both diaphyseal and metaphyseal periosteum of osteoporotic rats, samples from the right femur of osteoporotic and normal female Lewis rats were collected and tissue sections were stained with hematoxylin and eosin, antibodies or staining kit against tartrate resistant acid phosphatase (TRAP), alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF), von Willebrand (vWF), tyrosine hydroxylase (TH) and calcitonin gene-related peptide (CGRP). The results showed that the osteoporotic rats had much thicker and more cellular cambial layer of metaphyseal periosteum compared with other periosteal areas and normal rats (P\0.001). The number of TRAP? osteoclasts in bone resorption pits, VEGF? cells and the degree of vascularization were found to be greater in the cambial layer of metaphyseal periosteum of osteoporotic rats (P\0.05), while no significant difference was detected in the number of ALP? cells between the two groups. Sympathetic nerve fibers identified by TH staining were predominantly located in the cambial layer of metaphyseal periosteum of osteoporotic rats. No obvious difference in the expression of CGRP between the two groups was found. In conclusion, periosteum may play an important role in the cortical bone resorption in osteoporotic rats and this pathological process may be regulated by the sympathetic nervous system.

An approximate Bayesian computation approach for estimating parameters of complex environmental processes in a cellular automata

Modelling an environmental process involves creating a model structure and parameterising the model with appropriate values to accurately represent the process. Determining accurate parameter values for environmental systems can be challenging. Existing methods for parameter estimation typically make assumptions regarding the form of the Likelihood, and will often ignore any uncertainty around estimated values. This can be problematic, however, particularly in complex problems where Likelihoods may be intractable. In this paper we demonstrate an Approximate Bayesian Computational method for the estimation of parameters of a stochastic CA. We use as an example a CA constructed to simulate a range expansion such as might occur after a biological invasion, making parameter estimates using only count data such as could be gathered from field observations. We demonstrate ABC is a highly useful method for parameter estimation, with accurate estimates of parameters that are important for the management of invasive species such as the intrinsic rate of increase and the point in a landscape where a species has invaded. We also show that the method is capable of estimating the probability of long distance dispersal, a characteristic of biological invasions that is very influential in determining spread rates but has until now proved difficult to estimate accurately.

Numerical investigation of plant tissue porosity and its influence on cellular level shrinkage during drying

Dried plant food products are increasing in demand in the consumer market, leading to continuing research to develop better products and processing techniques. Plant materials are porous structures, which undergo large deformations during drying. For any given food material, porosity and other cellular parameters have a direct influence on the level of shrinkage and deformation characteristics during drying, which involve complex mechanisms. In order to better understand such mechanisms and their interrelationships, numerical modelling can be used as a tool. In contrast to conventional grid-based modelling techniques, it is considered that meshfree methods may have a higher potential for modelling large deformations of multiphase problem domains. This work uses a meshfree based microscale plant tissue drying model, which was recently developed by the authors. Here, the effects of porosity have been newly accounted for in the model with the objective of studying porosity development during drying and its influence on shrinkage at the cellular level. For simplicity, only open pores are modelled and in order to investigate the influence of different cellular parameters, both apple and grape tissues were used in the study. The simulation results indicated that the porosity negatively influences shrinkage during drying and the porosity decreases as the moisture content reduces (when open pores are considered). Also, there is a clear difference in the deformations of cells, tissues and pores, which is mainly influenced by the cell wall contraction effects during drying.

Numerical investigation of case hardening of plant tissue during dying and its influence on the cellular level shrinkage

Dried plant food materials are one of the major contributors to the global food industry. Widening the fundamental understanding on different mechanisms of food material alterations during drying assists the development of novel dried food products and processing techniques. In this regard, case hardening is an important phenomenon, commonly observed during the drying processes of plant food materials, which significantly influences the product quality and process performance. In this work, a recent meshfree-based numerical model of the authors is further improved and used to simulate the influence of case hardening on shrinkage characteristics of plant tissues during drying. In order to model fluid and wall mechanisms in each cell, Smoothed Particle Hydrodynamics (SPH) and the Discrete Element Method (DEM) are used. The model is fundamentally more capable of simulating large deformation of multiphase materials, when compared with conventional grid-based modelling techniques such as Finite Element Methods (FEM) or Finite Difference Methods (FDM). Case hardening is implemented by maintaining distinct moisture levels in the different cell layers of a given tissue. In order to compare and investigate different factors influencing tissue deformations under case hardening, four different plant tissue varieties (apple, potato, carrot and grape) are studied. The simulation results indicate that the inner cells of any given tissue undergo limited shrinkage and cell wall wrinkling compared to the case hardened outer cell layers of the tissues. When comparing unique deformation characteristics of the different tissues, irrespective of the normalised moisture content, the cell size, cell fluid turgor pressure and cell wall characteristics influence the tissue response to case hardening.

A hybrid cellular automata model of multicellular tumour spheroid growth in hypoxic microenvironment

A three-dimensional hybrid cellular automata (CA) model is developed to study the dynamic process of multicellular tumour spheroid (MTS) growth by introducing hypoxia as an important microenvironment factor which influences cell migration and cell phenotype expression. The model enables us to examine the effects of different hypoxic environments on the growth history of the MTS and to study the dynamic interactions between MTS growth and chemical environments. The results include the spatial distribution of different phenotypes of tumour cells and associated oxygen concentration distributions under hypoxic conditions. The discussion of the model system responses to the varied hypoxic conditions reveals that the improvement of the resistance of tumour cells to a hypoxic environment may be important in the tumour normalization therapy.

Cellular Mechanisms of Sleep Homeostasis : Studies on Brain Energy Metabolism and Aging

Sleep is governed by a homeostatic process in which the duration and quality of previous wake regulate the subsequent sleep. Active wakefulness is characterized with high frequency cortical oscillations and depends on stimulating influence of the arousal systems, such as the cholinergic basal forebrain (BF), while cessation of the activity in the arousal systems is required for slow wave sleep (SWS) to occur. The site-specific accumulation of adenosine (a by-product of ATP breakdown) in the BF during prolonged waking /sleep deprivation (SD) is known to induce sleep, thus coupling energy demand to sleep promotion. The adenosine release in the BF is accompanied with increases in extracellular lactate and nitric oxide (NO) levels. This thesis was aimed at further understanding the cellular processes by which the BF is involved in sleep-wake regulation and how these processes are affected by aging. The BF function was studied simultaneously at three levels of organization: 1) locally at a cellular level by measuring energy metabolites 2) globally at a cortical level (the out-put area of the BF) by measuring EEG oscillations and 3) at a behavioral level by studying changes in vigilance states. Study I showed that wake-promoting BF activation, particularly with glutamate receptor agonist N-methyl-D-aspatate (NMDA), increased extracellular adenosine and lactate levels and led to a homeostatic increase in the subsequent sleep. Blocking NMDA activation during SD reduced the high frequency (HF) EEG theta (7-9 Hz) power and attenuated the subsequent sleep. In aging, activation of the BF during SD or experimentally with NMDA (studies III, IV), did not induce lactate or adenosine release and the increases in the HF EEG theta power during SD and SWS during the subsequent sleep were attenuated as compared to the young. These findings implicate that increased or continuous BF activity is important for active wake maintenance during SD as well as for the generation of homeostatic sleep pressure, and that in aging these mechanisms are impaired. Study II found that induction of the inducible NO synthase (iNOS) during SD is accompanied with activation of the AMP-activated protein kinase (AMPK) in the BF. Because decreased cellular energy charge is the most common cause for AMPK activation, this finding implicates that the BF is selectively sensitive to the metabolic demands of SD as increases were not found in the cortex. In aging (study III), iNOS expression and extracellular levels of NO and adenosine were not significantly increased during SD in the BF. Furthermore, infusion of NO donor into the BF did not lead to sleep promotion as it did in the young. These findings indicated that the NO (and adenosine) mediated sleep induction is impaired in aging and that it could at least partly be due to the reduced sensitivity of the BF to sleep-inducing factors. Taken together, these findings show that reduced sleep promotion by the BF contributes to the attenuated homeostatic sleep response in aging.

Cellular Cholesterol Accumulation and Egress from Endosomal Compartments

One of the most important factors determining the development of atherosclerosis is the amount of LDL particles in the circulation. In general, LDL particles are clinically regarded as “bad cholesterol” since these particles get entrapped within the vascular wall, leading to atherosclerosis. Circulating HDL particles are conversely regarded as “good cholesterol” because of their ability to transport cholesterol from peripheral tissues to the liver for secretion as bile salts. Once inside the artery wall LDL particles are engulfed by macrophages, resulting in macrophage foam cells. If the macrophage foam cells are not able to efflux the cholesterol back into the bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly dependent on the ABCA1 transporter (ATP-binding cassette transporter A1) which transfers cellular cholesterol to extracellular apoA-I (apolipoprotein A-I) particles, leading to the generation of nascent HDL particles. The process of atherosclerotic plaque development is therefore to a large extent a cellular one, in which the capacity of the macrophages in handling the excessive cholesterol load determines the progression of lesion development. In this work we have studied the cellular mechanisms that regulate the trafficking of LDL-derived cholesterol from endosomal compartments to other parts of the cell. As a basis for the study we have utilized cells from patients with Niemann-Pick type C disease, a genetic disorder resulting from mutations in the NPC1 and NPC2 genes. In these cells, cholesterol is entrapped within the endosomal compartment, and is not available for efflux. By identifying proteins that bypass the cholesterol trafficking defect, we were able to identify the small GTPase Rab8 as an important protein involved in ABCA1 dependent cholesterol efflux. In the study, we show that Rab8 regulates cholesterol efflux in human macrophages by facilitating intracellular cholesterol transport, as well as by regulating the plasma membrane availability of ABCA1. Collectively, these results give new insight in to atherosclerotic lesion development and intracellular cholesterol processing.