Bachelor in Biology at University of Innsbruck
In her Biology undergraduate degree at Uni Innsbruck Theresa chose two focus topics: zoology and molecular biology. There she got the chance to rotate through several research groups doing laboratory practical courses, among those
- a Basic Laboratory Techniques practical course in which she learned the basics of chemical calculations, weighing and pipetting, cell lysis methods, photometric protein and nucleic acid determination, handling acids, bases and buffers, cryoscopy, filtration, centrifugation and chromatographic methods.
- a Basic Microbiology practical course in which different culture media were prepared, microorganisms were extracted from various samples and sterile culturing conditions were trained.
- a Basic Molecular Biology practical course taught me basic PCR techniques, DNA extraction and purification methods, TA-cloning, transformation, plasmid preparation, analytical DNA digests and gel electrophoresis.
- a Molecular Cell Biology practical course in the group of David Teis at Medical University Innsbruck, in which we investigated mutations in (MVB) pathway genes. For this we generated yeast knock-out and knock-in strains by PCR-based homologous recombination, did mating type tests and tetrad dissection and created double mutants through mating. We transfected yeast with GFP-tagged contructs of the proteins of interest and performed fluorescence widefield microscopy. We also transformed MVB pathway genes into E.coli, performed IPTG protein expression and purification, FPLC as well as coomassie staining and SDS-PAGE.
- a Cell Physiology practical course with Adolf Sandbichler at the Institute of Zoology at University of Innsbruck, in which we were working with wildtype and CLOCK mutant zebrafish embryonic fibroblast cell lines, cultured under both light/dark cycle conditions or constant dark conditions. For this we used a Bionas 2500 Analyzing System to determine acidification, oxygen consumption and cell adhesion. Among cell cuture passaging and cell counting through tryptan blue staining, we did Phalloidin (F-actin), Hoechst (nuclei) and Mitotracker (mitochondria) stainings and analyzed cell morphology using laser scanning confocal microscopy.
- a Genomics practical course with Norbert Polacek (now in Bern) at Medical University Innsbruck, in which we cultured organisms from all three domains of life, bacteria, archea and eucaryotes, and purified their genomic DNA. We isolated 70S ribosomes from T.aquaticus through sucrose gradient centrifugation and tested the ribosomal activity through a puromycin reaction and their GTPase activity through the detection of radioactively labelled GTP on a thin layer chromatography .
- several practical courses on Advanced Molecular Biology Techniques and Developmental Biology in the group of Dirk Meyer at the Institute for Molecular Biology at University of Innsbruck. Working with Dirk and his team greatly contributed to my fascination with genetics and with the amazing zebrafish model organism. It was in these practical courses that became very familiar with basic and advanced molecular cloning techniques, both ligation- and recombination-based, PCR and in vitro transcription. I learned how to use microinjection, in situ hybridization as well as proliferation and apoptosis assays to scrutinize the embryonic development of zebrafish.
In these practical trainings I fell in love with the zebrafish model and picked Dirk Meyer’s lab for my Bachelor thesis work. Here, I subcloned the genetically encoded calcium indicators GCaMP4 and GCaMP4.1 under the control of pancreatic beta-cell specific promoters of the hb9/mnx1 and ins genes using Gateway cloning. I then co-injected the constructs with Tol2 recombinase into fertilized zebrafish eggs to generate stable transgenic zebrafish lines in which the insulin release in pancreatic beta-cells (which is coupled to high cyosolic calcium levels) can be monitored with fluorescence microscopy.
Master in Molecular Medicine at Trinity College Dublin and Master thesis at the Institute for Neuroscience at Karolinska Institute Stockholm
In her Masters degree in Molecular Medicine at Trinity College Dublin Theresa deepened her understanding of Human Genetics, Molecular Mechnisms of Human Disease, Oncology as well as Immunology and Infectious Biology. In practical courses she performed ELISAs, Flow Cytometry, Western Blots and High Content Screening Analysis using the IN Cell Analyzer 1000.
Theresa did her Master thesis with Dagmar Galter at Lars Olson’s group in the Instititue for Neuroscience of the Karolinska Institute in Stockholm. Here she scutinized the role of MAGI proteins during mammalian neuronal development through in situ hybridization and studied a genetic variant of MAGI1 from a bipolar patient in cultured lymphocytes using qPCR.
PhD in Molecular Medicine at the Developmental Biology Department at University of Freiburg
For her PhD, Theresa chose the lab of Wolfgang Driever at the University of Freiburg. Wolfgang was a key figure in developing zebrafish as an embryological model organism and saved his spot in the textbooks for performing the large-scale screens that identified countless genetic factors in vertebrate embryonic development. Shortly after the publication of no less than 27 parallel articles in the legendary 1996 issue of Development, Wolfgang moved back from Boston to Freiburg to scrutinize the development of the nervous system with a particular focus on the dopaminergic system.
Theresa joined his lab in 2012 shortly after RNA-Seq data suggested Brain specific Homeobox (Bsx) as a developemental determinant of the dopaminergic system that has previously not being described in this context. It was around the same time that first Transcription Activator-like Effector Nucleases (TALENs) and shortly after Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) revolutionized molecular biology. Particularly for zebrafish scientists these targeted nucleases changed everything – and catalyzed the turn from forward genetics to reverse genetics. For the first time, we could knock out genes of our choice in zebrafish.
Thus, Theresa used TALENs to generate a stable zebrafish line in which the bsx gene has been mutated in a way that renders the Bsx protein unfunctional. While waiting for the stable line to grow and breed, Theresa performed double fluorescent in situ hybridization and was able to show that the bsx gene was indeed expressed in cells that also expressed enzymes involved in dopamine biosynthesis. However, when analyzing zebarfish from the bsx mutant line by immunohistochemistry, it appeared that the dopaminergic neurons all developped normally even in the absence of the Bsx protein.
This was a setback. And Theresa was wondering if not for dopaminergic neuron developemnt, what is the Bsx protein good for? At one day serendipity struck and Theresa – almost accidentylly found that genes encoding enzymes in the melatonin biosynthesis pathway of the pineal gland are missing in bsx mutant embryos. Thorough epistasis anaylsis of mutliple transcripts, some of which Theresa overexpressed through mRNA injection or transgenic constructs and some of which she knocked down by morpholino injection, Theresa was able to position Bsx in a developmental network that controls pineal gland development.
There is, however, another region of the brain in which the bsx gene is highly expressed: the hypothalamus. And after some tedious search, Theresa was able to find several neuropeptidergic and neuromodulatory cells in the hypothalamus to be affected by the loss of the Bsx protein. While trying to name the different regions in whci cells were affected, Theresa found that neuroanatomy of the embryonic hypothalamus was quite a mess. It took countless double fluorescent in situ hybridizations and hours at the confocal laser scanning microscope to stain, image and compare markers for different subregions within the hypothalamus.
Finally, Theresa was able to present a map of the zebrafish embryonic hypothalamus. After that, describing the areas in which neuronal populations were affected by Bsx loss, was just a diligent but routine piece of work.
Postdoc at the Pediatric Genetics unit at University Hospital Freiburg
Theresa’s expertise in molecular cloning, microinjection, transgenesis, targeted nuclease-mediated genetic knock-out generation, staining, imaging and animal handling, led to a job offer from Ekkehart Lausch. Ekkehart leads the pediatric genetics section at the University hospital in Freiburg and specializes on congenital skeletal malformations. In Ekkehart’s group, Theresa worked on a genetic model for ochondrodontoplasia and achondrogenesis through genetic alterations of the thyroid hormone receptor interactor 11 (trip11) gene in zebrafish.
