Mutations as molecular tools : The metabolic-rate dependent molecular clock and DNA barcoding of allied species

Mutation and recombination are the fundamental processes leading to genetic variation in natural populations. This variation forms the raw material for evolution through natural selection and drift. Therefore, studying mutation rates may reveal information about evolutionary histories as well as phylogenetic interrelationships of organisms. In this thesis two molecular tools, DNA barcoding and the molecular clock were examined. In the first part, the efficiency of mutations to delineate closely related species was tested and the implications for conservation practices were assessed. The second part investigated the proposition that a constant mutation rate exists within invertebrates, in form of a metabolic-rate dependent molecular clock, which can be applied to accurately date speciation events. DNA barcoding aspires to be an efficient technique to not only distinguish between species but also reveal population-level variation solely relying on mutations found on a short stretch of a single gene. In this thesis barcoding was applied to discriminate between Hylochares populations from Russian Karelia and new Hylochares findings from the greater Helsinki region in Finland. Although barcoding failed to delineate the two reproductively isolated groups, their distinct morphological features and differing life-history traits led to their classification as two closely related, although separate species. The lack of genetic differentiation appears to be due to a recent divergence event not yet reflected in the beetles molecular make-up. Thus, the Russian Hylochares was described as a new species. The Finnish species, previously considered as locally extinct, was recognized as endangered. Even if, due to their identical genetic make-up, the populations had been regarded as conspecific, conservation strategies based on prior knowledge from Russia would not have guaranteed the survival of the Finnish beetle. Therefore, new conservation actions based on detailed studies of the biology and life-history of the Finnish Hylochares were conducted to protect this endemic rarity in Finland. The idea behind the strict molecular clock is that mutation rates are constant over evolutionary time and may thus be used to infer species divergence dates. However, one of the most recent theories argues that a strict clock does not tick per unit of time but that it has a constant substitution rate per unit of mass-specific metabolic energy. Therefore, according to this hypothesis, molecular clocks have to be recalibrated taking body size and temperature into account. This thesis tested the temperature effect on mutation rates in equally sized invertebrates. For the first dataset (family Eucnemidae, Coleoptera) the phylogenetic interrelationships and evolutionary history of the genus Arrhipis had to be inferred before the influence of temperature on substitution rates could be studied. Further, a second, larger invertebrate dataset (family Syrphidae, Diptera) was employed. Several methodological approaches, a number of genes and multiple molecular clock models revealed that there was no consistent relationship between temperature and mutation rate for the taxa under study. Thus, the body size effect, observed in vertebrates but controversial for invertebrates, rather than temperature may be the underlying driving force behind the metabolic-rate dependent molecular clock. Therefore, the metabolic-rate dependent molecular clock does not hold for the here studied invertebrate groups. This thesis emphasizes that molecular techniques relying on mutation rates have to be applied with caution. Whereas they may work satisfactorily under certain conditions for specific taxa, they may fail for others. The molecular clock as well as DNA barcoding should incorporate all the information and data available to obtain comprehensive estimations of the existing biodiversity and its evolutionary history.Mutaatiot ja geneettinen rekombinaatio ovat luonnonpopulaatioiden perinnöllisen muuntelun perusta. Evoluutio muovaa tätä ainesta luonnonvalinnan ja ajautumisen kautta. On ilmeistä, että mutaatiofrekvenssin tutkiminen valaisee niin evoluution kulkua kuin eliöiden sukulaisuussuhteita. Tässä väitöskirjassa tutkittiin kahta molekyylibiologien työkalua, DNA-viivakoodia ja molekyylikelloa. Työn ensimmäisessä osassa tarkasteltiin eri geenialueiden kykyä luonnehtia lähisukuisia lajeja ja tämän kysymyksen merkitystä luonnonsuojelubiologiassa. Työn toisessa osassa tutkittiin hypoteesia, jonka mukaan hyönteisten mutaatiofrekvenssi noudattaa molekyylikelloa. Kohteena oli erityisesti hypoteesi, jonka mukaan molekyylikellon tarkkuutta voi parantaa huomioimalla eri lajien lämpötila. DNA-viivakoodauksella otaksutaan voitavan erottaa lajeja toisistaan tietyn geenialueen avulla. Tutkimuksessa verrattiin Suomesta hiljattain uudelleen löydetyn kovakuoriaisen, haapasepikän ja lajin venäläisten yksilöiden geenialueita keskenään. Kaikki yksilöt osoittautuivat identtisiksi jokaisen tutkitun geenialueen suhteen. Toisaalta suomalaiset ja venälaiset yksilöt erosivat toisistaan niin elintapojensa kuin rakenteensa suhteen. Suomessa laji kehittyi kovapuisissa halavissa ja mustuvapajuissa, Venäjällä suurissa lahoavissa haavoissa. Suomalaiset yksilöt olivat myös hiukan hoikkarakenteisempia kuin venäläiset ja molempien muotojen koiraiden sukupuolielimet olivat selvästi toisistaan eroavat. Näillä perusteilla Suomesta kuvattu laji piti vanhan tieteellisen nimensä, Hylochares cruentatus (Gyllenhal), mutta suomeksi se ristittiin uudelleen halavasepikäksi. Haapasepikän nimi jätettiin maamme rajojen ulkopuolella elävälle lajille, joka kuvattiin tieteelle uutena nimellä Hylochares populi Brüstle -- Muona. Haapasepikän biologiaa ei tunnettu Suomesta, koska siitä tiedettiin vain kaksi hyvin vanhaa löytöä. Lajin katsottiin hävinneen maastamme suurten haapojen myötä, elihän se tällaisissa puissa Venäjän Karjalassa. Havainnot Suomessa osoittivat, että olettamus oli virheellinen. Kyse oli kahdesta eri lajista, joiden elintavat erosivat toisistaan selvästi. Luonnonsuojelubiologian kannalta tämä oli tärkeä havainto. DNA-viivakoodin tutkiminen ei pystynyt osoittamaan kahden lajin olemassaoloa. Aiempi päättely Karjalan havaintojen kelpoisuudesta Suomen lajin suojeluun oli myös virheellinen. Halavasepikkä on ilmeisesti hyvin hiljan eriytynyt haapasepikän sisarlaji, joka tunnetaan vain Suomesta. Kaikki vanhat havainnot Suomen ulkopuolelta koskevat haapasepikkää. Halavasepikkä on saimaannorpan kaltainen, kotoperäinen suomalainen eläin. Molekyylikellon ajatuksena on, että mutaatiot kertyvät tietylle geenialueelle vakionopeudella ja näin makromolekyylien erojen perusteella voidaan päätellä evolutiivisten haarautumien ikä. Tätä kellon hypoteesia on tarkennettu eri tavoin myöhemmin. Sen sijaan, että kello tikittää vakionopeudella, otaksutaan sen noudattavan hienovaraisempia sääntöjä. Hiljan esitetyn hypoteesin mukaan kello ei tikitä vain aikayksikköä kohden. Ajan ohella mutaatiot kertyvät suhteessa eliön lämpötilasta ja massasta riippuvaan aineenvaihduntaenergiaan. Tämän hypoteesin mukaan molekyylikello tulee kalibroida huomioiden eliön paino ja lämpötila. Tässä tutkimuksen osassa selvitettiin lämpötilan vaikutusta mutaatiofrekvenssiin tasakokoisilla lajeilla. Käytössä oli kaksi eri aineistoa. Ensimmäinen näistä oli pantrooppinen sepiköiden heimoon kuuluva a kovakuoriaissuku Arrhipis. Ennen kellohypoteesin tutkimista myös Arrhipis-lajien sukulaisuussuhteet selvitettiin. Toinen aineisto koostui kukkakärpäsistä, joiden fylogenia oli selvitetty aiemmin. Molempia aineistoja tutkittiin eri analyysimenetelmin, usean geenin suhteen ja kokeillen useaa kellohypoteesia. Tulokset osoittivat, että lämpötilalla ja mutaatiofrekvenssillä ei ollut yhteyttä tutkituilla eliöillä. Näin ollen lämmöstä riippuvaa aineenvaihduntaenergia-hypoteesin mukaista molekyylikelloa ei havaittu. Tulosten mukaan on suhtauduttava varovaisuudella kelloon perustuviin molekyylibiologisiin menetelmiin. Ne voivat toimia tietyissä tilanteissa tietyillä eliöillä, mutta pettää toisessa tilanteessa. Sovellettaessa molekyylikelloa ja DNA-viivakoodausta monimuotoisuuden ja evolutiivisen historian tutkimuksessa on syytä käyttää mahdollisimman laajasti saatavilla olevaa muuta tietoa ja välttää yksinkertaistettuja ratkaisuja

Peeping over Galileo's shoulders: Laying the foundations of heliocentrism in elementary school

In this work, an adaptation of the first observation attempted by Galileo to demonstrate the heliocentric hypothesis to be correct is developed. The objective of the proposal is to let students transiting the last year of the primary school confront by themselves the same contradicting scenario that put Galileo at crossroads during the second decade of the 17th century. Results of its implementation on two courses encompassing 32 students with ages 11-12 are reported and first impressions of this pedagogical approach are analyzed.Fil: Pincelli, Michelina Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Física del Sur. Universidad Nacional del Sur. Departamento de Física. Instituto de Física del Sur; ArgentinaFil: Brustle, Marina. Colegio Victoria Ocampo; ArgentinaFil: Formichella, Maria del Carmen. Colegio Victoria Ocampo; ArgentinaFil: Perez Millán, Cecilia Andrea. Universidad Nacional del Sur. Departamento de Física; ArgentinaFil: Palmieri, Natalia. Colegio Victoria Ocampo; ArgentinaFil: Otranto, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Física del Sur. Universidad Nacional del Sur. Departamento de Física. Instituto de Física del Sur; Argentin

Evaluación de la actividad del aceite esencial de Geranium maculatum (Geraniaceae) en el áfido Brevycorine brassicae (Hemiptera: Aphididae)

El áfido Brevycorine brassicae se caracteriza por ser especialista de la familia Brassicaceae y es considerado uno de los másperjudiciales en plantas del género Brassica como la colza. Frente a los tradicionales insecticidas de síntesis, se buscanalternativas ecológicas para su control promoviéndose el uso de aceites vegetales. Los objetivos del trabajo fueronevaluar la repelencia y el efecto sobre la supervivencia y fecundidad del aceite esencial de Geranium maculatum sobre B.brassicae. Para evaluar la repelencia la arena experimental consistió en dos hojas de colza una de las cuales se trató conlas soluciones del aceite y la otra con emulsificante. Luego de la evaporación del emulsificante se liberaron en el centrode la arena experimental diez áfidos adultos. Se prepararon soluciones madre disolviendo los aceites esenciales en 1 mlde una solución de etanol y Tween-20 (0,012: 10). Se realizaron tres réplicas. El número de áfidos presentes en cada hoja seregistró a las 24 horas y a las 48 horas y se calculó un índice de repelencia. Los datos se analizaron mediante ANOVA y DMS(p < 0,05). Para evaluar los efectos sobre la supervivencia y la fecundidad, hojas de colza se trataron con las soluciones delaceite esencial. Como control se utilizaron hojas tratadas con el emulsificante. Luego de la evaporación del emulsificante,las hojas se colocaron en cajas de Petri con agar al 1 % junto con una hembra adulta. La caja se cerró en su parte superiorcon malla antiáfidos. Diariamente se registró el número de ninfas paridas y el número de adultos vivos. A las 24 horas elaceite esencial produjo repelencia a todas las concentraciones evaluadas (DMS, p˂ < 0,05). A la mayor concentración el aceitegeneró un 86 % de repelencia. Al evaluar la fecundidad se observó que el aceite produjo una disminución en el número dedescendientes. A las 24 horas, el aceite redujo en un 53 % el número de ninfas paridas en comparación con el control. A partirdel tercer día el aceite esencial redujo la fecundidad de B. brassicae en un 60 %. El aceite de G. maculatum podría proteger elcultivo de colza repeliendo y afectando la reproducción del áfido B. brassicae.Fil: Sanchez Chopa, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Brustle, Carlos Manuel. Universidad Nacional del Sur. Departamento de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Bizet Turovsky, Jorge Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Descamps, Lilian Renée. Universidad Nacional del Sur. Departamento de Agronomía; Argentin

Repellent activity of Eucalyptus globulus Labill (Myrtaceae) and Mentha x piperita L. (Lamiaceae) essentials oils against Acyrthosiphon pisum Harris (Hemiptera: Aphididae)

En la región semiárida pampeana Acyrthosiphon pisum Harris afecta la implantación de las pasturas de alfalfa reduciendoel stand de plantas. La principal forma de control es por medio de insecticidas sintéticos. En distintas partes delmundo el uso desmedido de los mismos ha favorecido el desarrollo de individuos resistentes. Un nuevo enfoque demanejo en la actualidad es el uso de aceites esenciales que presentan múltiples sitios de acción. Los aceites esencialesestán constituidos principalmente por monoterpenos, alcoholes, ésteres y fenoles y pueden actuar como insecticidas,atrayentes, repelentes y/o disuasorios de la alimentación. El objetivo del presente trabajo fue evaluar el efecto repelentede los aceites esenciales de Eucalyptus globulus y Mentha x piperita en adultos de A. pisum. Hojas de alfalfa se sumergierondurante 10 segundos en diferentes soluciones de los aceites esenciales. Las hojas se dejaron secar durante 1 hora atemperatura controlada. Se colocaron dos hojas en forma alterna, una tratada con la solución del aceite esencial y otratratada con el emulsificante solo. En el centro se liberaron 10 adultos. Se realizaron tres réplicas por concentración.El número de áfidos presentes sobre las hojas se registró a las 24 horas y 48 horas. Se calculó un índice de repelencia(IR) = C/(C+T), donde C es el número de áfidos en las hojas control y T es el número de áfidos en las hojas tratadas. Losresultados fueron analizados mediante ANOVA y test de diferencias mínimas significativas (DMS p < 0,05). A las 24 horasambos aceites esenciales resultaron repelentes a las concentraciones de 7 y 3,5 % p/v. A las 48 horas el aceite esencialde M. x piperita mantuvo la repelencia para ambas concentraciones mientras que el aceite de E. globulus solo registrórepelencia para la concentración del 7 % p/v. Estos resultados indicarían que los aceites esenciales de Mentha x piperita yEucalyptus globulus podrían ser utilizados como repelentes de Acyrthosiphon pisum durante la implantación de la pasturade alfalfa.In semiarid Pampa`s of Argentine, Acyrthosiphon pisum Harris affects seedlings of alfalfa reducing the stand of plants. This pest is controlled with synthetic insecticides. Frequent and often excessive of chemical application had resulted in the development of resistant individuals. Therefore, an ecofriendly approach, using essential oils is an alternative in the control of A. pisum. Essential oils are composed of monoterpenes, alcohols, esters and phenols. They possess insecticide and repellent properties, and deterrent effects on feeding behavior against insect pests. The aim of this paper was to study the repellent effect of the essential oils of Eucalyptus globulus and Mentha x piperita on A. pisum adults. Deterrent activity was assessed using leaf choice tests. Excised alfalfa leaves were dipped for 10 s in essential oil emulsions. To evaluate repellency an experimental arena consisted of two leaves, one treated with the test solution and one dipped in the control solution was used. Ten adult aphids were released in the center of the experimental arena. Three replicates were prepared for each treatment. After 24 h and 48 h, the number of aphids on each leaf was recorded. The deterrency index (DI) was calculated as C/(C+T), where C is the number of aphids on control leaves and T is the number of aphids on treated leaves. Data were analysed by ANOVA and Fisher`s PLSD test. At 24 h essential oils of E. globulus and M. piperita showed deterrent effect at the highest concentrations (p < 0.05). At 48 h after treatment, M. x piperita was repellent at concentrations of 7 % w/v and 3,5 % w/v (P < 0.05). E. globulus show repellent at 7 % (w/v) but no repellence was found at 3,5 % (w/v). These results would indicate that the essential oils of Mentha x piperita and Eucalyptus globulus could be used as repellents for seedling protection in the presence of Acyrthosiphon pisum.Fil: Descamps, Lilian Renée. Universidad Nacional del Sur. Departamento de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Bizet Turovsky, Jorge Alejandro Jose. Universidad Nacional del Sur. Departamento de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Brustle, Carlos Manuel. Universidad Nacional del Sur. Departamento de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Sánchez Chopa, Federico. Universidad Nacional del Sur. Departamento de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentin

Promoting remyelination through cell transplantation therapies in a model of viral-induced neurodegenerative disease.

Multiple sclerosis (MS) is a central nervous system (CNS) disease characterized by chronic neuroinflammation, demyelination, and axonal damage. Infiltration of activated lymphocytes and myeloid cells are thought to be primarily responsible for white matter damage and axonopathy. Several United States Food and Drug Administration-approved therapies exist that impede activated lymphocytes from entering the CNS thereby limiting new lesion formation in patients with relapse-remitting forms of MS. However, a significant challenge within the field of MS research is to develop effective and sustained therapies that allow for axonal protection and remyelination. In recent years, there has been increasing evidence that some kinds of stem cells and their derivatives seem to be able to mute neuroinflammation as well as promote remyelination and axonal integrity. Intracranial infection of mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in immune-mediated demyelination and axonopathy, making this an excellent model to interrogate the therapeutic potential of stem cell derivatives in evoking remyelination. This review provides a succinct overview of our recent findings using intraspinal injection of mouse CNS neural progenitor cells and human neural precursors into JHMV-infected mice. JHMV-infected mice receiving these cells display extensive remyelination associated with axonal sparing. In addition, we discuss possible mechanisms associated with sustained clinical recovery. Developmental Dynamics 248:43-52, 2019. © 2018 Wiley Periodicals, Inc

The role of machine learning in developing non-magnetic resonance imaging based biomarkers for multiple sclerosis: a systematic review

Background Multiple sclerosis (MS) is a neurological condition whose symptoms, severity, and progression over time vary enormously among individuals. Ideally, each person living with MS should be provided with an accurate prognosis at the time of diagnosis, precision in initial and subsequent treatment decisions, and improved timeliness in detecting the need to reassess treatment regimens. To manage these three components, discovering an accurate, objective measure of overall disease severity is essential. Machine learning (ML) algorithms can contribute to finding such a clinically useful biomarker of MS through their ability to search and analyze datasets about potential biomarkers at scale. Our aim was to conduct a systematic review to determine how, and in what way, ML has been applied to the study of MS biomarkers on data from sources other than magnetic resonance imaging. Methods Systematic searches through eight databases were conducted for literature published in 2014-2020 on MS and specified ML algorithms. Results Of the 1, 052 returned papers, 66 met the inclusion criteria. All included papers addressed developing classifiers for MS identification or measuring its progression, typically, using hold-out evaluation on subsets of fewer than 200 participants with MS. These classifiers focused on biomarkers of MS, ranging from those derived from omics and phenotypical data (34.5% clinical, 33.3% biological, 23.0% physiological, and 9.2% drug response). Algorithmic choices were dependent on both the amount of data available for supervised ML (91.5%; 49.2% classification and 42.3% regression) and the requirement to be able to justify the resulting decision-making principles in healthcare settings. Therefore, algorithms based on decision trees and support vector machines were commonly used, and the maximum average performance of 89.9% AUC was found in random forests comparing with other ML algorithms. Conclusions ML is applicable to determining how candidate biomarkers perform in the assessment of disease severity. However, applying ML research to develop decision aids to help clinicians optimize treatment strategies and analyze treatment responses in individual patients calls for creating appropriate data resources and shared experimental protocols. They should target proceeding from segregated classification of signals or natural language to both holistic analyses across data modalities and clinically-meaningful differentiation of disease.</p

Obtaining Consent for Future Research with Induced Pluripotent Cells: Opportunities and Challenges

Human induced pluripotent stem cells have revolutionized stem cell research. However, their great potential also raises important concerns about consent for future research