Life Sciences Institute of New Jersey (LSINJ), Belle Mead, NJ (Nallaseth, F.S.)

Genetics (Somatic Cell, Mitochondrial, Yeast and Mouse (Epigenetics), Biochemistry and {Molecular, Stem/Cellular, Developmental, Cancer, Chromosome/Genome, Structural, Reproductive (Sex Determination and Gametogenesis) and Evolutionary (Speciation, Protein/Nucleotide Divergence)} Biology, Neurobiology, Virology and Microbiology (Microbial - Evolution, Ecology, Genetics).

(Introduction follows affiliations)

Affiliations

(1) Member

Complex Biological Systems Alliance (CBSA)

(3) The AAT Project Mentor

The AAT Project.com/mentors/ferez-nallaseth.php

The AAT Academy

America's Amazing Teens (AAT) - Discovering Brilliance

The AAT project.com/aat-project-mentors.php

Introduction (Life Sciences Institute of New Jersey)

The central focus of the lab has been the regulation of Y chromosome biology using Testis Determination as a reporter for putative mutations with the application of an array of methods for the recovery of rearranged Y sequences. Our work is conceptually based on well established principles in the relationship between the Evolutionary Genetics of Speciation, the Genetic regulation of Chromosome/Genome Biology and the Developmental Genetics of Sex Determination and has qualified fundamental principles governing these sub-fields of the Life Sciences. Our experimental approach is based on the core Bateson-Haldane-Muller-Dobzhansky principles that de novo Speciation either in laboratory crosses or in natural Hybrid Zones results from mutations and that heterogametic rather than homogametic sexes are inviable or sterile. Mutations in turn result from the disruption of regulation of Chromosome Biology at the Genotypic level and are manifested as dysfunctional Sex Determination and Spermatogenesis (among other) Phenotypes which are readily detected due to their non-lethality and effects in altering Sex Ratios. Furthermore, the high rates of mutation and adaptability of the Sex Determining pathway i.e. its Evolutionary Plasticity, are reflected in the wide range and diversity of regulatory mechanisms operating at the Molecular, Cellular and Organismal levels. Ongoing work includes establishing: (a) the sequence lability of the Y chromosome and the Sex Determining Sry locus either over Evolutionary and Generational time spans or within individual mice, (b) the Structural and Epigenetic basis of XY female sex reversal regulated by feral mouse Y chromosomes that are unstable in Genomes of specific inbred strains, (c) Genetic modulation of the instability of the Sry locus and the Y chromosome, (d) determination of the threshold of Sry(+) Pre-coelomic/Pre-Sertoli cells required for commitment to Sertoli cell/Testicular Chord lineages and (e) by single cell/molecule analyses any contributions of SF1-SRY-HMG-SOX9/Sry secondary structure complex formation as by-products of DNA replication to the Developmentally /Physiologically relevant regulation of Testis Determining functions of Sry at its native Y linked locus in Pre-coelomic and Sertoli cells of day 10 - day 12.5 Embryos. Current results on Sex Determination identify Sox9 which is downstream from and induced by the 'Master Switch' Sry in the Testis Determining pathway, as the pivotal gene on which upstream male and female pathway regulatory genes act and equilibrate to initiate and enforce choices of Testicular or Ovarian pathways of Development. However, nothing in the data excludes the upstream role of Sry, the contributions of its highly unstable Inverted Repeat (IR) structure to up and down regulation of Transcription and thus its surviving counter-selection in Evolution. The contributions of these secondary structures may include the obligatory spike upto maximal rates of up-regulation and levels of expression, followed by sharp down regulation and suppression of expression that lead to the stringent Kinetic, Temporal and Spatial constraints on the transcription of Sry required for Testis Determination. These functions are suggested by a range of results in the literature, and even if they made relatively small contributions to Testis Determination and Development, their effects would be magnified on an Evolutionary scale by offsetting the high degree of instability and selecting for the IR structure of Sry. Such amplified Evolutionary selection and fixation of small effects of Gene structure on Expression and Phenotype have been reported with, the Ped gene regulating Blastomere number/size in mice and both rDNA magnification-reduction at the Bobbed locus and X:A ratios setting of Transcription rates of tra/tra2/Sxl and the subsequent Splicing cascades responsible for Sex Determination in Fruit Flies (Synopsis, Select Publications and Abstracts, PubMed). Epigenetics and Evolution of the Y chromosome, Sxr region and the Sry locus in Speciation are touched on in this exchange of comments on LinkedIn (Epigenetics of Sex Reversal - Evolutionary Implications. LinkedIn Comments-9.25.2013.pdf).

The processes of Replication, Recombination and Repair of DNA and Segregation and Condensation of Chromosomes constitute Chromosome Biology. Work done in Model Organisms in classical Genetics has established that Chromosome and Genome Biology are under stringent Chromosome and Locus specific Genetic regulation. Work done in Yeast has shown that a DNA integrity network comprising of 875 genes with diverse functions maintains the Genome. Genome guardians such as p53, p21, Homologous Recombinational Repair (HRR), Replication-Restart and Mutator/Repair Gene products, whose functions have been worked out in mammalian Cell Lines and lower Eukaryotes such as Yeast or Prokaryotes such as E.coli, are members of such networks. Genes, including those with specialized roles in Development and in disease states, are substrates for such a network and either the fixation of mutations in these Genes, or the induction of Chromosomal aberrations such as Translocations and Aneuploidies in Loci spanning them, reflects the failure(s) of surveillance, maintenance and repair functions of this network. The mouse is the only mammal with Classical and Molecular Genetics sufficiently well developed, including the availability of Consomic, Recombinant Inbred and Collaborative Cross strains, high density SNPs and Haplotype Map Intervals, to allow the eventual establishment of an equivalent mammalian DNA integrity network in all 200 cell types representing the Physiologically relevant context of a Developing organism. Assays for the de-regulation of, Hotspots/Coldspots of DNA recombination (PR1, VNTR, Micro- and Mini- satellite Sequences, SNPs), Interchromosomal /Intrachromosomal Recombination in repeated sequences (PR1, VNTR, Micro- and Mini- satellite Sequences, SNPs), double Holliday Junction/Stem Loop intermediates of Recombination, Immunoglobulin/MHC rearrangements, Telomeres, Centromeres, Repair functions, Mutator induction, Chromosome Synapsis and Synaptonemal complexes, Condensation/Heterochromatinisation, DNA replication initiation and timing, Fragile Sites (Replication Slow Zones), DNA amplifications, Tandem Repeat Array expansions, DNA replication-restart-repair, Insulators/Locus Control Regions/Enhancers/Silencers, X chromosome inactivation transcripts (Xist)/X chromosome controlling elements (Xce) and micro RNA (miRNA) coding sequences are some of the myriad Genomic targets that could serve as reporters for the de-regulation of Chromosome Biology induced by appropriate combinations of Genetic Backgrounds, with the eventual systematization of the regulation of this network.

We and others have initiated this undertaking by applying the Bateson-Haldane-Muller-Dobdzhansky Principles as well as advantage of the magnitude of Evolutionary Diversity of Regulatory and Structural Alleles regulating mammalian Chromosome/Genome Biology and the power of Mouse and Yeast Genetics.Furthermore, Evolutionary Geneticists among others have shown that in Natural Hybrid Zones and Interspecific Matings of Mice in the Lab deregulates fundamental mechanisms regulating Chromosome Biology and presents a barrier to introgression. Some of the putative end products resulting from the deregulation of, Y chromosome biology and loci of, the Major Histocompatibility Complex (MHC), t complex, Hybrid Sterility (Hst) and Hybrid Breakdown complexes in specific Inbred Strains, feral mice or their various combinations; Genome wide Phylogenetic Disequilibria have also been documented. However, the larger endeavor of systematizing the regulation of mammalian Chromosome/Genome Biology in Homeostasis and Development requires the long term commitment of personnel and resources which remains the responsibility of senior levels of the Scientific community. Without this systematization the pathways for generation, progression and fixation of mutations, which are the necessary if not sufficient cause of most disease states, will remain refractory to analyses and modern medicine will be restricted to its current approach of perpetuating the management of their consequences (pathologies, Biology, symptoms and syndromes) rather than preempting them as causes. As powerful, efficient and of high resolution as they are in analyzing development regulated by single copy Genes, contemporary methods such as various ablations ('Knockouts', 'Knockins', CRISPR/Cas9) of Genes and Transgenes are restricted to the analysis of these downstream Biologies/Pathologies and to a small fraction of the Genome governing Differentiation or Developmental states, often either lacking or having similar Phenotypes especially when regulating Chromosome Biology and requiring drug selection protocols with unknown effects on Chromatin and the Cell. CRISPR/Cas9 can neither excise mutations in multi copy sequences e.g. mitochondrial DNA in cardiomyopathies nor make relevant excisions of primary mutations that have become largely irrelevant after unleashing irreversible pathologies driven by secondary cascades as in some cancers e.g. mutations, dys-regulated miRNA, epitomics, expression, etc.. as e.g. in Neurodegeneration and Cancer pathologies. They neither reproduce endogenous mutational pathways in Homeostasis and Development nor confer the ability to control the regulators that preempt mutations. Therefore, these contemporary methods simply cannot replace the systematization of the regulation of Chromosome/Genome Biology in Homeostasis and Development by exploiting Evolutionary Diversity and the complementary classical tools that would eventually emerge from it, in analyzing, the maintenance of the Genome, its role(s) in various disease states and the functions of multi-copy Genes in both Development and disease states. Similarly, as powerful as Genomic Biomarkers and Genomic Fingerprints (GWAS, SNP Haplotypes in Cancer Genome Atlases, etc..) are in predicting diseases and possible therapies, they are by-products of dysfunctions in the regulation of Chromosome/Genome Biology. Therefore, neither OMICS (Bio-markers, CRISPR/Cas9) nor Genomic Fingerprints/Cancer Genomics have the eventual preemptive capacity that would be derived by systematizing the regulation of Chromosome/Genome Biology and elucidating the pathways and mechanisms causing disease mutations in Homeostasis and Development. This approach would yield lines of mice in which these mutational pathways could be induced or repressed at will and when coupled with Classical (Synteny, Orthology, Model Organisms, Human Genetics and Pathologies, etc..) and Contemporary (miRNA, Microenvironment, Genomics, Proteomics, Epigenomics, Transcriptomics, Systems, etc..) Analytical approaches lead to the eventual preemption of these disease states. This logic also applies to Stem Cell/Cell Replacement Therapies (for Parkinson's Disease, Diabetes and Pancreatic Islet Cells, etc...) which as powerful as they are will not pre-empt de novo cases.Furthermore, contemporary methods such as OMICS, CRISPR/Cas9 excision, Biomarkers, Stem Cell Therapy, etc.. although remarkably powerful and relevant for now, do belong to retrospective interventions and will do absolutely nothing to forestall new mutations with their burgeoning human and economic costs. Evolutionary Genetics, and its near infinite supply of variants, when coupled with contemporary methods would readily allow both the systematization AND CONTROL of these networks and thus the preemption of disease causing mutations. Pre-emption is the ONLY way that some intractable diseases can be cured! A combinatorial approach is the only way to bring about a Paradigm Shift in the Biomedical Sciences from 'perpetuating after the fact' management of consequences (Pathologies, etc...) of disease states to preempting ('before the fact') their Mutational causes in 4000 to 10000 monogenic diseases (excluding translocations, aneuploidies, mitochondrial mutations and multifactorial causes) costing hundreds of trillions of dollars per year. Not to mention the Human costs which in Neurodegenerative diseases and Cancers are more ghastly and dehumanizing than any Death Camp would have the ken to inflict!

Our work on mechanisms of, Viral/Chromosomal DNA replication in the laboratory of Mel DePamphilis, DNA recombination in the laboratories of Steven J. Brill and Gerry Smith and z DNA formation/Strand Transfer activity with Alex Rich were drawn on as models for the regulation of Y chromosome biology. Work on the Demethylation of specific sites in CYP17 regulatory sequences during the Senescence of cells of the Adrenal Cortex in Peter Hornsby's laboratory, the interaction of Chromatin Remodelling and Replication proteins, NAP, SET, TAF and PP2A with Leukemogenic fusion proteins retaining the A/T-hooks domain of HRX, in Doug Tkachuk's laboratory and the analysis of Neocentromeres in Peter Warburton's laboratory also contributed to our appreciation of the conceptual basis of Chromosome Biology.

The above work has direct implications for mechanisms in Physiologically and Developmentally relevant, regulation of Chromosome and Genome Biology, Sex Determination, Gametogenesis, Evolution and Speciation and the causal and mutational basis of 4000 to 10000 disease states (as different as Cancer(s), Sickle Cell Anemia, Schizophrenia, Alzheimer's Disease, Fragile X Syndrome and Huntington's chorea) that occur at a frequency of 1 in 200 births (Department of Energy, Genome Project, URL: www.ornl.gov/sci/techresources/Human_Genome/medicine/assist.shtml; Many rare mutations may underpin diseases, Nicholas Wade, Published May 17, 2012, http://www.nytimes.com/2012/05/18/science/many-rare-mutationsmay underpindiseases.html?ref=nicholaswade;Same Genetic Basis Found in 5 Types of Mental Disorders, By GINA KOLATA Published: February 28, 2013, http://www.nytimes.com/2013/03/01/health/study-finds-genetic-risk-factors-shared-by-5-psychiatric-disorders.html?_r=0). In the Biomedical Sciences, Cancer remains the paradigm for the paradox of perpetuating management of consequences (pathologies, symptoms, syndromes, etc..) to the exclusion of understanding, systematizing pathways and preempting mutational causes of disease states. In fact the estimated costs for treatments of Cancer(s) alone were $895 billion in 2008 and are $286 billion/anum for de novo cases ( Cancer cost 'crisis' warning from oncologists, James Gallagher, 26 September 2011, http://www.bbc.co.uk/news/health-15032862 and Delivering affordable cancer care in high-income countries, Richard Sullivan MD et al, The Lancet Oncology, 12(10): 933-980 (2011), http://www.thelancet.com/journals/lanonc/article/PIIS1470-2045(11)70141-3/fulltext#article_upsell) and for all disease states amount to tens of trillions of dollars annually. With Oncologists raising questions about the very usefulness in treating Cancer(s) of the premise of early detection by approaches such as the application of Biomarkers (Considering When It Might Be Best Not to Know About Cancer, GINA KOLATA, NYT, Published: October 29, 2011, http://www.nytimes.com/2011/10/30/health/cancer-screening-may-be-more-popular-than-useful.html and A Decade Later, Genetic Map Yields Few New Cures, NICHOLAS WADE, Published: June 12, 2010, http://www.nytimes.com/2010/06/13/health/research/13genome.html?pagewanted=all) and mixed results being obtained with both traditional Chemotherapy as well as with sophisticated Biologically targeted Cancer therapies (http://www.nytimes.com/2010/06/15/health/15canc.html?pagewanted=all, Therapies for Cancer Bring Hope and Failure By ANDREW POLLACK, Published: June 14, 2010;Chemo 'undermines itself' through rogue response, http://www.bbc.co.uk/news/health-19111700, BBC, Published 5 August 2012 and in http://www.natureasia.com/en/research/highlight/7516#.UEYu7yIix5Y, Peter Nelson MD et al, Nature Medicine, 6th August 2012; http://www.nytimes.com/2012/09/10/health/research/for-a-lung-cancer-drug-treatment-may-be-within-reach.html, Cancer Study Points to Tighter Pairing of Drugs and Patients, By GINA KOLATA, Published: September 9, 2012) these approaches, at best, are interim solutions. In fact it has been surmised from relapse rates that 3000 cells/Metastasis are programmed to circumvent all current therapies i.e. are "fait accompli" making the management of Cancer symptoms a short term therapeutic recourse of limited prognosis (Bert Vogelstein, M.D., http://www.eurekalert.org/pub_releases/2012-07/gsoa-ftg070312.php). Furthermore, it has been asserted by the Nobel Laureates, Dr. Carlo Croce and Dr. Phil Sharp, among others, that the necessary albeit insufficient, initiating step in Cancer is the induction of a mutation(s) in sequences encoding, small RNAs, micro RNAs (miRNAs), Tumor Suppressors and Proto-oncogenes. The specification of Cancer Stem Cells, Epigenetic Modifications, circumvention of Immune-surveillance by primary growths and Circulating Tumor Cells (CTCs), secondary Genomic rearrangements in Metastasis/acquisition of drug resistance and differential expression at the interface of pathologically malignant and normal tissues (detected with Laser Capture Dissection dependent MS/MS/TOF) are some of the subsequent steps driving Oncogenesis. These data make the case for initiating the potential preemption of these mutations by the systematizing the Genetic regulation of mammalian Chromosome/Genome Biology in Homeostasis and Development, even more compelling. Finally, our work also has implications for the instability of the Genome, for a practical recourse in recovering Genomic Biomarkers and building representative consensus sequences of the Genome especially from current single molecule sequencing approaches despite this instability. To reiterate, a combinatorial approach is the only way to bring about a Paradigm Shift in the Biomedical Sciences from 'perpetuating after the fact' management of consequences (Pathologies, etc...) of disease states to preempting ('before the fact') their Mutational causes in 4000 to 10000 monogenic diseases costing hundreds of trillions of dollars per year. Not to mention the Human costs which in Neurodegenerative diseases and Cancers are more ghastly and dehumanizing than any Death Camp would have the ken to inflict!

Past and ongoing collaborations include, (1) the function of the Collagen cleaving enzyme Human cathepsin K with Dieter Bromme, (2) the secretion of proteins from Yeast (P. pastoris) with Steve Anderson, (3) transcriptional and Redox state maintenance functions of Peptide Methionine Sulfoxide Reductase with Herb Weissbach, (4) the analysis of instability of the sex determining mouse Sry locus with Marty Tracey, Lee Ann Schein, Dale Woodbury, Boris Umylny, Steve Tuske and Regina Felder-Gibbions, (5) Sex reversal and Y chromosome sequence Evolution with J. Barry Whitney III, Mike Dewey, Jeff Ceci, Z. Sheng Guo, In-Seob Han and Bob DeLisio (6) the transcriptional response in the Neurophysiological resistance of the Turtle (T. scripta) Brain under conditions of Oxygen deprivation with Marty Tracey and Peter Lutz and (7) contributions of Stem cell and Differentiated components to the Morphogenesis of Human skin in 3D aggregates with Ramsey Foty, Dale Woodbury and Bozena Michniak-Kohn.

Current areas of work

(1)Y chromosome Evolution, Speciation, Sequence instability/modifications, Sex Determination and Gametogenesis in the Mouse

(2) Regulation of Mammalian Chromosome/Genome Biology in Homeostasis and Development with the exploitation of Evolutionary diversity of Regulatory and Structural Alleles and Orthologs in Yeast/Mouse

(3) Cancer Biology and mechanisms in item #s 2, 4, 5, 6 and 7 in initiating and driving Oncogenesis

(4) Hypoxia - Anoxia resistance of the Turtle (T.scripta) Brain, Comparative Evolutionary, Functional Genomics and Phylogeny, Yeast (mitochondrial rho0/petite, protein export) mutants, Mouse strains with behavioral and cognitive disabilities and Turtle Neurobiology in the analysis of Ischemia, Neurodegeneration (e.g. Alzheimer's Disease), Sexual Dimorphism of the Brain and other Neurobiological functions such as cognitive dysfunctions. Hypoxia in Vasculogenesis and Angiogenesis.

(5) Sorting and Fusion mechanisms in the interactions of Stem cells, Dermal cells and Keratinocytes in the Morphogenesis of Human Skin in 3D as per the Differential Adhesion Hypothesis (DAH)

(6) Structural and functional characterization of Human Cathepsin K and Collagen in Homeostasis and Disease states

(7) Protein design and secretion in Yeast (P.pastoris)

(8)