Transcriptomic regulation of seasonal coat color change in hares

Color molts from summer brown to winter white coats have evolved in several species to maintain camouflage year-round in environments with seasonal snow. Despite the eco-evolutionary relevance of this key phenological adaptation, its molecular regulation has only recently begun to be addressed. Here, we analyze skin transcription changes during the autumn molt of the mountain hare (Lepus timidus) and integrate the results with an established model of gene regulation across the spring molt of the closely related snowshoe hare (L. americanus). We quantified differences in gene expression among three stages of molt progression-"brown" (early molt), "intermediate," and "white" (late molt). We found 632 differentially expressed genes, with a major pulse of expression early in the molt, followed by a milder one in late molt. The functional makeup of differentially expressed genes anchored the sampled molt stages to the developmental timeline of the hair growth cycle, associating anagen to early molt and the transition to catagen to late molt. The progression of color change was characterized by differential expression of genes involved in pigmentation, circadian, and behavioral regulation. We found significant overlap between differentially expressed genes across the seasonal molts of mountain and snowshoe hares, particularly at molt onset, suggesting conservatism of gene regulation across species and seasons. However, some discrepancies suggest seasonal differences in melanocyte differentiation and the integration of nutritional cues. Our established regulatory model of seasonal coat color molt provides an important mechanistic context to study the functional architecture and evolution of this crucial seasonal adaptation

Development of high-throughput yeast-cell-based bioreporter assays for specific monitoring of bisphenol A and chemical testing of endocrine disrupting compounds

Chemicalization of the modern society has become a topic of debate in the past few decades. Especially chemicals that affect the human reproduction and hormonal system, the so-called endocrine disrupting compounds, have raised concern in public and regulatory agencies. There is a growing need for suitable testing methods to screen endocrine disrupting potential of new and old chemicals. While the European Union chemical legislation REACH has increased the need of chemical testing methods, one of its targets is also to decrease the use of animals in these tests. It has been proposed that inexpensive high throughput in vitro assays could be used for initial screening of chemicals for further testing with other methods. In addition to chemical testing, environmental monitoring of endocrine disrupting compounds is important to assess the level of exposure and possible adverse effects of chemicals on humans and wildlife. Chemical analysis methods used in environmental monitoring are sensitive, but they are also laborious, expensive, and require specialized instruments. Consequently, robust biological methods have become valuable tools to measure endocrine disrupting potency of chemicals and environmental samples. For this purpose, several Saccharomyces cerevisiae yeast cell-based bioreporters utilizing different nuclear receptors have been developed. Yeast-cell-based bioreporter assays have several advantages in environmental analytics. In addition to being inexpensive, they are particularly useful in determining the bioavailability of contaminants. Yeast is also very tolerant towards toxicity of different sample matrices. Yeast nuclear receptor bioreporter assays have been used to determine the total hormonal activity of samples containing unknown mixture of chemicals. However, these assays cannot identify the chemicals in the sample, and thus, monitoring of a single chemical has not been possible. Many cell-based assays have already been adapted to high throughput screening plate formats of 384 and 1536 wells and even higher. However, nearly all yeast nuclear receptor bioreporter assays are still performed in 96-well plates. Consequently, yeast bioreporter assays should be adapted to automated liquid handling and high density well plates to enable screening of large chemical libraries and high number of samples. In this thesis study, a yeast nuclear receptor bioreporter assay for specific detection of a single chemical, bisphenol A (BPA) was developed. The creation of the BPA-targeted receptor included application of a oligonucleotide-based mutation method and a positive-negative genetic selection method for human estrogen receptor α (publication I). Chemical specificity of the BPA-targeted receptor (BPA-R) bioreporter assay was characterized, and its use demonstrated with chemical mixture and waste water samples (publication II). In addition, the existing battery of yeast bioreporters was adapted to automated liquid handling and high density 384 and 1536 well plates to meet the requirements of high throughput screening (publication III). Finally, a new yeast-based bioreporter utilizing a chimeric human retinoid X receptor was constructed and characterized (publication IV). This bioreporter can be used to measure organotin compounds such as tributyl tin in environmental samples.Kemikaalit ovat osa modernia maailmaamme. Lähes kaikkien käyttämiemme tuotteiden - jopa elintarvikkeiden - valmistuksessa on käytetty erilaisia kemikaaleja takaamaan niiden haluttu koostumus, laatu ja turvallisuus. Kemikaalien turvallisuus ja ihmisten elinympäristön kemikalisoituminen on kuitenkin herättänyt viime vuosi-kymmeninä huolta niin suuressa yleisössä kuin viranomaisissakin. Erityisesti salakavalalta tuntuvat hormonitoimintaan vaikuttavat kemikaalit ovat huolestuttaneet ihmisiä. Näiden ns. hormonihäritsijäkemikaalien on epäilty aiheuttavan mm. lisääntymis¬terveydellisiä, hermostollisia ja aineenvaihdunnan ongelmia. Tällaisia kemikaaleja ovat esim. torjunta-aineet, dioksiinit, monet palonestoaineet, muovi-teollisuuden käyttämät ftalaatit ja bisfenoli A sekä orgaaniset tinayhdisteet. Euroopan Unionin kemikaaliasetus REACH tähtää kohti turvallisempaa kemikaalien käyttöä. Sen tarkoituksena on laajentaa kemikaalien erilaisten vaikutusten testaamista, saattaa testien teettäminen kemikaalien valmistajien vastuulle sekä parantaa käyttö-turvallisuuden tiedonkulkua kemikaalien tuottajilta niiden käyttäjille. Kemikaalien testaus¬vaatimusten lisääntyessä myös uusien testausmenetelmien tarve kasvaa. Eläinkokeiden sijaan REACH pyrkii suosimaan tehokkaampia ja eettisempiä menetelmiä, kuten in vitro eli ns. koeputkessa tehtäviä solu- ja kudospohjaisia määrityksiä. Hormonihäiritsijäkemikaalien testauksessa sieniin kuuluva tavallinen leivinhiiva (lat. Saccharomyces cerevisiae) on osoittautunut hyväksi työkaluksi. Eläinsoluihin verrattuna hiivaa on edullista ja helppoa viljellä ja muokata. Vaikka hiivasoluilta puuttuu eläin¬solujen tumareseptoreihin perustuva hormonien viestinvälitysketju, tämä ketju voidaan saada toimimaan myös hiivassa. Monet ihmisen hormonireseptorit, kuten estrogeeni¬reseptori, on onnistuneesti siirretty hiivaan. Kun hiiva altistetaan reseptoriin sitoutuvalle kemikaalille, reseptori aktivoituu ja tuottaa solussa ulkoisesti havaittavan signaalin. Tätä signaalia mittaamalla voidaan arvioida kemikaalin mahdollista vaikutusta kyseiseen hormonireseptoriin ja sen säätelemiin viestinvälitysketjuihin. Yksittäisten kemikaalien vaikutusten testaamisen lisäksi hiivasolumenetelmät soveltuvat myös erilaisten tuntemattomien ja puhdistamattomien näytteiden analysointiin. Tällaisia näytteitä ovat esim. ympäristö- ja vesinäytteet tai vaikkapa elintarvike¬näytteet. Hiivan avulla voidaan siis seurata ja arvioida ympäristön ja ihmisten mahdollista altistumista hormonaalisesti aktiivisille yhdisteille ja niiden seoksille. Tässä väitöskirjatyössäni olen vienyt eteenpäin olemassa olevia ja kehittänyt uusia hiivasoluihin perustuvia hormonaalisten kemikaalien testausmenetelmiä. Julkaisussa (I) suunnittelin menetelmän ihmisen estrogeenireseptorin rakenteen muokkaamiseen, ja sovelsin menetelmää kehittääkseni reseptorin, joka aktivoituu vain yhden valitun kemikaalin, bisfenoli A:n, sitoutuessa siihen. Julkaisussa (II) tätä muokattua reseptoria käytettiin spesifisen bisfenoli A:ta mittaavan hiivamenetelmän kehittämiseen. Tätä menetelmää voidaan soveltaa bisfenoli A:n mittaamiseen erilaisissa näytteissä, ja arvioimaan altistusta tälle kemikaalille eri lähteistä. Julkaisussa (III) siirsin olemassa olevien käsin pipetoitavien hiivamenetelmien käytön perinteisestä 96-kuoppa-levymittauksesta pipetointirobotin avulla 384- ja 1536-kuoppalevyillä tehtäviin määrityksiin. Menetelmä mahdollistaa tehokkaamman analysoinnin ja laajempien näyte-määrien mittaamisen. Julkaisussa (IV) suunnittelin ja olin mukana toteuttamassa uutta hiivamenetelmää, jonka avulla voidaan mitata orgaanisia tinayhdisteitä. Menetelmän avulla voidaan mitata ja arvioida tinayhdisteiden biosaatavuutta ja sedimentin saastuneisuuden astetta esim. laivaväylillä

Quantitative Immunology for Physicists

The adaptive immune system is a dynamical, self-organized multiscale system that protects vertebrates from both pathogens and internal irregularities, such as tumours. For these reason it fascinates physicists, yet the multitude of different cells, molecules and sub-systems is often also petrifying. Despite this complexity, as experiments on different scales of the adaptive immune system become more quantitative, many physicists have made both theoretical and experimental contributions that help predict the behaviour of ensembles of cells and molecules that participate in an immune response. Here we review some recent contributions with an emphasis on quantitative questions and methodologies. We also provide a more general methods section that presents some of the wide array of theoretical tools used in the field.Comment: 78 page revie

An Artificial Immune System Strategy for Robust Chemical Spectra Classification via Distributed Heterogeneous Sensors

The timely detection and classification of chemical and biological agents in a wartime environment is a critical component of force protection in hostile areas. Moreover, the possibility of toxic agent use in heavily populated civilian areas has risen dramatically in recent months. This thesis effort proposes a strategy for identifying such agents vis distributed sensors in an Artificial Immune System (AIS) network. The system may be used to complement electronic nose ( E-nose ) research being conducted in part by the Air Force Research Laboratory Sensors Directorate. In addition, the proposed strategy may facilitate fulfillment of a recent mandate by the President of the United States to the Office of Homeland Defense for the provision of a system that protects civilian populations from chemical and biological agents. The proposed system is composed of networked sensors and nodes, communicating via wireless or wired connections. Measurements are continually taken via dispersed, redundant, and heterogeneous sensors strategically placed in high threat areas. These sensors continually measure and classify air or liquid samples, alerting personnel when toxic agents are detected. Detection is based upon the Biological Immune System (BIS) model of antigens and antibodies, and alerts are generated when a measured sample is determined to be a valid toxic agent (antigen). Agent signatures (antibodies) are continually distributed throughout the system to adapt to changes in the environment or to new antigens. Antibody features are determined via data mining techniques in order to improve system performance and classification capabilities. Genetic algorithms (GAs) are critical part of the process, namely in antibody generation and feature subset selection calculations. Demonstrated results validate the utility of the proposed distributed AIS model for robust chemical spectra recognition

Exploring the Impact of PQN-75 and GLH-1/Vasa on Germline Development, Maintenance, and GSC Reprogramming Using Caenorhabditis elegans as a Model

This thesis combines research on PQN-75 expression, functional motifs of GLH-1/Vasa, and germ granule components in Caenorhabditis elegans to provide a comprehensive understanding of germline development, maintenance, and reprogramming, while also examining the role of pharyngeal gland cells in stress resistance and thermotolerance. In C. elegans, pharyngeal gland cells secrete mucin-like proteins, such as PQN-75, with similarities to human PRB2. The expression of PQN-75 in gland cells confers stress resistance and thermotolerance but does not affect fertility, instead it plays a role in the organism\u27s ability to adapt to varying environmental conditions. While, GLH-1/Vasa, an ATP-dependent DEAD-box helicase, plays a critical role in safeguarding the germline by regulating translation and amplifying piwi-interacting RNAs. To elucidate the functions of GLH-1 and its role in germline development, CRISPR/Cas9 technology was employed to investigate its functional motifs in C. elegans by analyzing 28 endogenous mutant alleles. Results demonstrate that helicase activity is essential for GLH-1\u27s association with P granules, and removing glycine-rich repeats diminishes P-granule interactions at the nuclear periphery. Additional, mass spectrometry reveals an affinity between GLH-1 and three structurally conserved PCI complexes, along with a reciprocal aversion for assembled ribosomes and the 26S proteasome. Suggesting that P granules compartmentalize the cytoplasm to exclude large protein assemblies, effectively shielding associated transcripts from translation, contributing to germline maintenance. Germ granules are essential for maintaining germline integrity and stem cell totipotency. Depletion of core germ granule components in C. elegans leads to germ cell reprogramming and sterility. To better understand the initiation of somatic reprogramming and the role of GLH-1 in this process, total mRNA (transcriptome) and polysome-associated mRNA (translatome) changes in a precision full-length deletion of glh-1 where examined. Here two significant changes were observed: first, GLH-1 suppresses the expression of neuropeptide-encoding transcripts, suggesting a role in repressing somatic reprogramming and maintaining germline integrity; second, GLH-1 promotes Major Sperm Proteins levels, repressing spermatogenic expression during oogenesis and promoting MSP expression to drive spermiogenesis and sperm motility, highlighting its importance in fertility. Our findings contribute to understanding the roles of PQN-75 and GLH-1/Vasa in C. elegans germline development, maintenance, and germline stem cell reprogramming, while also shedding light on the organism\u27s stress resistance and thermotolerance mechanisms. With broader implications identifying early stem cell reprogramming processes and provides a platform for future research on germline biology in C. elegans

An artificial development model for cell pattern generation

La formation de structures cellulaires a un rôle crucial dans le développement tant artificiel que naturel. Cette thèse présente un modèle de développement artificiel pour la génération de structures cellulaires basé sur le paradigme des automates cellulaires (AC). La croissance cellulaire est contrôlée par un génome comportant un réseau de régulation artificiel (RRA) et une série de gènes structurels. Ce génome a subi une évolution par algorithme génétique (AG) afin de produire des structures cellulaires en 2D grâce à l'activation et inhibition sélective des gènes. De plus des gradients morphogénétiques ont été utilisés pour fournir aux cellules une information de position permettant de contraindre leur reproduction. Après évolution d'un génome par algorithme génétique, une cellule unique est placée au milieu de la grille de l’AC où sa reproduction, contrôlée par le RRA, produit une structure cellulaire cible. Le modèle a été appliqué avec succès au problème classique de génération de la structure d’un drapeau français (French flag problem).Cell pattern formation has a crucial role in both artificial and natural development. This thesis presents an artificial development model for cell pattern generation based on the cellular automata (CA) paradigm. Cellular growth is controlled by a genome consisting of an artificial regulatory network (ARN) and a series of structural genes. The genome was evolved by a genetic algorithm (GA) in order to produce 2D cell patterns through the selective activation and inhibition of genes. Morphogenetic gradients were used to provide cells with positional information that constrained cellular replication. After a genome was evolved, a single cell in the middle of the CA lattice was allowed to reproduce controlled by the ARN until a cell pattern was formed. The model was applied to the canonical problem of growing a French flag pattern.

Characterization of the Circadian Properties of Runt-related Transcription Factor 2 (Runx2) and its Role in the Suprachiasmatic Nucleus

Circadian rhythms orchestrate physiological, behavioral and cognitive processes in order to anticipate and adapt organisms to key environmental cues. These endogenously driven oscillations are generated by a network of interlocked auto-regulatory transcriptional-translational feedback loops driven forward by the Bmal1/Clock heterodimer transcription factor. Given the ubiquitous and dynamic quality of circadian rhythms, the identification of factors involved in the coordination and regulation of the endogenous oscillations is central in broadening our understanding of biological timing systems. In an examination of gene expression in the mammalian central circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), revealed a previously unreported rhythmic expression of runt-related transcription factor 2 (Runx2). The goal of the research described in the present dissertation was to determine the mechanistic basis of rhythmic Runx2 expression as well as determine whether Runx2 interacts with the core molecular machinery of the circadian clock. First, rhythmic gene expression Runx2 in the SCN, olfactory blub (OB) and paraventricular nucleus (PVN) of adult mice was demonstrated using quantitative real-time polymerase chain reaction and immunohistochemistry served to show rhythmic Runx2 protein expression in the SCN. Second, using SCN tissues from Bmal-/- mice or by transiently silencing BMAL1 expression in vitro it was shown that Runx2 expression is dependent on a functional core molecular clock network, namely the transcriptional activity of Bmal1. Chromatin immunoprecipitation was used to demonstrate that BMAL1 directly interacted with its putative binding sites within the RUNX2 promoter suggesting that the core clock machinery directly regulates its rhythmic gene expression. Next, Runx2 haplodeficient mice and transient silencing of RUNX2 expression in vitro revealed that decreases in Runx2 expression leads to a dampened amplitude of rhythmic Bmal1 expression and a lengthening of the period of molecular as well as running wheel behavioral rhythms. Finally, RUNX2 was found to interact with a putative binding site identified in the BMAL1 promoter suggesting that the influence of RUNX2 on the amplitude of BMAL1 expression was at least in part based on direct regulation at the gene level. Together, these findings serve to establish the foundational framework of the reciprocal relationship between the Runx2 transcription factor and the molecular network underlying circadian rhythms

The Role of CDCA7 in Akt-mediated Myc-Dependent Apoptosis and Proliferation

CDCA7, or cell division cycle associated protein A7, was described in 2001 by Prescott and colleagues as a target of Myc-dependent transcriptional regulation (Prescott et al., 2001). We have identified CDCA7 as associating with the transcription factor Myc and is the target of phosphorylation by the prosurvival serine/threonine kinase Akt. Phosphorylation by Akt at threonine 163 disrupts CDCA7 association with Myc, promotes binding to 14-3-3 and sequestration in the cytoplasm. Coexpression of CDCA7 and Myc in fibroblasts potentiates Myc-dependent apoptosis upon serum withdrawal. In contrast, knockdown of CDCA7 by shRNA abrogated Myc-dependent apoptosis. Myc induced transformation of fibroblasts was reduced in the presence of CDCA7 and significantly inhibited by the expression of the non-Myc binding mutant (156-187) CDCA7. We have shown that CDCA7 enhances the activation of an E-box in a Myc-binding dependent manner. CDCA7 increases Myc occupancy of the proapoptotic BAX promoter, elevates BAX and Cyclin B1 mRNA levels while reducing p15INK4B mRNA levels. This data points to a novel mechanism which implicates Akt phosphorylation of CDCA7 as participating in the dual signal model of Myc of function and thus affecting Myc-dependent growth and transformation. In this study, we have also shown that expression of CDCA7 reduces proliferation rates and shifts cell cycle distribution towards G2/M phase and that phosphorylation of CDCA7 at T163 occurs strictly in G2/M. CDCA7 phosphorylated at threonine 163 colocalizes with the centrosomal protein marker -Tubulin and activated Akt (phospho-serine 473) in mitotic cells. Finally, we have shown that CDCA7 co-associates with monomers of itself which is dependent on amino acids 187-234, adding to the possible mechanisms by which CDCA7 function may be regulated