Nanoparticle Induced Cell Magneto-Rotation for the Multiplexed Monitoring of Morphology, Stress and Drug Sensitivity of Suspended Single Cancer Cells.

The metastatic process of a cancer relies on the transformation of some of the primary tumor cells into cells capable of migrating through the Extra-Cellular Matrix (ECM), surrounding the tumor, into the bloodstream and the lymph nodes, and then settle in distant tissue, growing new secondary tumors. By identifying, characterizing and quantifying these cells, the progression of cancer in a patient during therapy can be more accurately assessed. Here we describe the development of a new method for quantitative real time monitoring of cell size and morphology, on single live suspended cancer cells, unconfined in three dimensions. The enabling cell magnetorotation (CM) method is made possible by nanoparticle induced cell magnetization. Using a rotating magnetic field, the magnetically labeled cells are actively rotated, then imaged, using a high definition CCD camera. Under proper conditions, the rotation period of a magnetic object is proportional to its shape factor. We demonstrate first that the rotational period, when measured in real-time, can serve to track cellular response to drugs, cytotoxic agents and other chemical stimuli. In addition, while cells are rotated, they exhibit very specific morphological activities, even without a chemical stimulus. Described also is how to multiplex the CM method, to image several dozens to several thousands of cells simultaneously, and using morphology to classify cells into different phenotypic categories, with each phenotype being correlated with malignancy level. The intrinsic tumor heterogeneity, at the cellular level, can be visualized with relationship graphs. Shown is the ability to monitor cell morphological changes over long periods of time, in real time, in order to detect the metastatic potential for heterogeneous populations of cancer cells, using tools from statistical analysis methods. The method relies on unsupervised Machine Learning algorithms which do not require human inputs. Overall it is demonstrated that the CM method can be used as a diagnostic tool to evaluate the phenotypical heterogeneity in a cell population in general, and in a cancer cell population in particular. This fast and high throughput method promises to efficiently assess the efficacy of personalized therapeutic strategies.PHDApplied PhysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111434/1/relbez_1.pd

Alternative methods for regulatory toxicology – a state-of-the-art review

This state-of-the art review is based on the final report of a project carried out by the European Commission’s Joint Research Centre (JRC) for the European Chemicals Agency (ECHA). The aim of the project was to review the state of the science of non-standard methods that are available for assessing the toxicological and ecotoxicological properties of chemicals. Non-standard methods refer to alternatives to animal experiments, such as in vitro tests and computational models, as well as animal methods that are not covered by current regulatory guidelines. This report therefore reviews the current scientific status of non-standard methods for a range of human health and ecotoxicological endpoints, and provides a commentary on the mechanistic basis and regulatory applicability of these methods. For completeness, and to provide context, currently accepted (standard) methods are also summarised. In particular, the following human health endpoints are covered: a) skin irritation and corrosion; b) serious eye damage and eye irritation; c) skin sensitisation; d) acute systemic toxicity; e) repeat dose toxicity; f) genotoxicity and mutagenicity; g) carcinogenicity; h) reproductive toxicity (including effects on development and fertility); i) endocrine disruption relevant to human health; and j) toxicokinetics. In relation to ecotoxicological endpoints, the report focuses on non-standard methods for acute and chronic fish toxicity. While specific reference is made to the information needs of REACH, the Biocidal Products Regulation and the Classification, Labelling and Packaging Regulation, this review is also expected to be informative in relation to the possible use of alternative and non-standard methods in other sectors, such as cosmetics and plant protection products.JRC.I.5-Systems Toxicolog

Defining The Effect Of Environmental Perturbation On The Male Germline

Periconceptional environment, according to the Developmental Origins of Health and Disease (DOHaD) theory, influences offspring phenotype, primarily via epigenetic mechanisms. Although the paternal component in humans is poorly understood, both maternal and paternal peri-conceptional environment are now believed to contribute to this phenomenon. Manipulation of the early embryo for treating human infertility, is suspected of contributing to offspring abnormalities through epigenetic mechanisms. To directly address the effects of common assisted reproductive technology procedures on the offspring epigenome, the DNA methylation profiles of newborns conceived naturally, or through the use of intrauterine insemination (IUI), or in vitro fertilization (IVF) using Fresh or Cryopreserved (Frozen) embryo transfer, were compared. In addition to a reduction of epigenetic aberrations in the IVF conceptions using cryopreservation, metastable epialleles also exhibited altered methylation with fertility status. ART, embryo nutrition, and fertility status are thus suggested to have a lasting epigenetic effect of on the developing embryo. While the paternal contribution to the human embryo is uncertain, sperm deliver a collection of proteins and RNA to the zygote. To identify the entire cadre of intergenic spermatozoal RNAs, RNA Element (RE) discovery algorithm (REDa) was developed and applied to a spectrum of germline, embryonic, and somatic tissues. This highlighted extensive transcription throughout the human genome and yielded previously unidentified human RNAs. Human spermatogenesis was found to exhibit extensive intergenic transcription and pervasive repetitive sequence expression. By analyzing the collection of novel and annotated spermatozoal RNAs in sperm samples from the Mesalamine and Reproductive Health Study (MARS), the effect of endocrine disruptor exposure on human sperm RNA profiles was determined. Sperm RNA profiles among men and their relationship to di-butyl phthalate (DBP) was longitudinally assessed across binary (high or background) DBP crossover exposures. Numerous changes in the composition of sperm RNA elements were detected during the acute and recovery phases, which suggest that exposure to, or removal from high DBP, produces effects that require longer than one spermatogenic cycle to resolve, if at all. Overall, chronic phthalate exposure influences the male germline, and acts on the dynamic RNA expression during human spermiogenesis

Discovery and characterization of genetic variants associated with extreme longevity

Over the last decade, there have been multiple genome-wide association studies (GWASs) of human extreme longevity (EL). However, only a limited number of genetic variants have been identified as significant, and only few of these variants have been replicated in independent studies. There are two possible reasons for this limitation. First, genetic variants might have a varying effect on EL in different populations, and GWAS applied to a dataset as a whole may not pinpoint such differences. Second, EL is a very rare trait in a population, and rare and uncommon variants might be important factors in explaining its heritability but GWASs have focused on the analyses of variants that are relatively common in the population. In this dissertation, I present three projects that address these issues. First, I propose PopCluster: an algorithm that automatically discovers subsets of individuals in which the genetic effects of a variant are statistically different. PopCluster provides a simple framework to directly analyze genotype data without prior knowledge of subjects ethnicities. Second, I investigate ethnic-specific effects of APOE alleles on EL in Europeans. APOE is a well-studied gene with multiple effects on aging and longevity. The gene has 3 alleles: e2, e3 and e4, whose frequencies vary by ethnicity. I identify several ethnically different clusters in which the effect of the e2 and e4 alleles on EL changes substantially. Furthermore, I investigate the interaction of APOE alleles with the country of residence. Results of this analysis suggest possible interaction of this gene with dietary habits or other environmental factors. For the third project, I perform a GWAS of rare variants and EL in a case-control dataset with median age of cases 104 years old. I analyze 4.5 million high-imputation quality rare SNPs imputed with HRC panel with minor allele frequency < 0.05. The analysis replicates all previous genome-wide level significant SNPs and identifies a few more potential targets. Additionally, I use serum protein data available for a subset of subjects and find significant pQTLs which have potential functional role. Based on these analyses, both genetic and environmental factors appear to be important factors for EL.2020-07-31T00:00:00

Impaired Mitochondrial Bioenergetics under Pathological Conditions

Mitochondria are the powerhouses of cells; however, mitochondrial dysfunction causes energy depletion and cell death in a variety of diseases. Altered oxidative phosphorylation and ion homeostasis are associated with ROS production resulting from the disassembly of respiratory supercomplexes and the disruption of electron transfer chains. In pathological conditions, the dysregulation of mitochondrial homeostasis promotes Ca2+ overload in the matrix and ROS accumulation, which induces the mitochondrial permeability transition pore formation responsible for mitochondrial morphological changes linked to membrane dynamics, and ultimately, cell death. Finally, studies on the impaired mitochondrial bioenergetics in pathology could provide molecular tools to counteract diseases associated with mitochondrial dysfunction

Life Sciences Program Tasks and Bibliography

This document includes information on all peer reviewed projects funded by the Office of Life and Microgravity Sciences and Applications, Life Sciences Division during fiscal year 1995. Additionally, this inaugural edition of the Task Book includes information for FY 1994 programs. This document will be published annually and made available to scientists in the space life sciences field both as a hard copy and as an interactive Internet web pag

Life Sciences Program Tasks and Bibliography for FY 1996

This document includes information on all peer reviewed projects funded by the Office of Life and Microgravity Sciences and Applications, Life Sciences Division during fiscal year 1996. This document will be published annually and made available to scientists in the space life sciences field both as a hard copy and as an interactive Internet web page

Elucidation of the occurrence of extracolonic cancers in Lynch syndrome

Includes abstract.Includes bibliographical references (leaves 126-147).Lynch Syndrome, also known as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) (OMIM #120435), is a familial disorder resulting from mutations within DNA MMR genes. Effective surveillance, diagnosis and treatment of the disorder is complicated due to the phenotypic and genetic heterogeneity of lynch syndrome, which exhibits an autosomal dominant mode of inheritance. Determine the molecular pathology of extracolonic cancers in Lynch syndrome and to elucidate whether or not the occurrence of these extracolonic cancers are a direct result of the mismatch repair deficiency. First, a modifier study was performed assessing the effect of a variant within the DNA MMR gene hMLH1 in a cohort of individuals predisposed to Lynch syndrome in order to examine a potential epistatic effect in the gene. In order to obtain a genetic signature of Lynch associated tumours, germline DNA and corresponding tumour DNA was isolated from Lynch syndrome patients. The genetic material was assessed via a panel of microsatellite rich genes and MS-MLPA. Finally, in silico analyses were undertaken assessing microarray data from microsatellite unstable colorectal and endometrial cancers to characterise novel candidate genes. The modifier study did not prove fruitful as no association was found between the hMLH1 promoter variant and site of cancer in individuals predisposed to Lynch syndrome. An association was observed heterozygous and homozygous variant genotypes and an increased risk of colorectal cancer, regardless of predisposing mutation (p = 0.000181). Two tumour suppressor genes; HIC1 and TIMP3, were found to be methylated in the tumour samples in the germline/tumour tissue study. This study also showed instability of the Erβ gene in the majority of tumour samples. Bioinformatic analysis utilising existing microarray data resulted in common under-expression of four genes and common overexpression in three genes in microsatellite unstable colorectal and endometrial cancers. Further investigation into the modifier study and elucidation of a genetic signature in MMR deficient, MSI cancers. The results obtained in this study contribute to the increasing body of knowledge in the field and the various stages of malignancies should be assessed for a more informative result. Genetic and functional studies should be performed on the information garnered from the bioinformatics analysis. Overall evaluation and molecular classification of Lynch syndrome tumours may guide better diagnosis, surveillance and treatment of those at risk