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Theses

Below please find the PhD theses that have been completed under supervision of Johan Jakobsson.
To go to a specific year directly, please click on the year here: 2017, 2015, 2014, 2013

2020

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Daniela Grassi: Development and applications of in vitro models to study human brain evolution and disease
Author: Daniela Grassi
Supervisor: Johan Jakobsson
Department: Experimental Medical Science
Date of defense: 2020-04-23
Place of defense:  Segerfalksalen, Wallenberg Neuroscience Center, Lund  
Opponent: Jens Hjerling-Leffler
Date of publication: 2020
No of pages: 170
Type of document: PhD Thesis
Language: English
ISBN: 978-91-7619-902-2 / ISSN: 1652-8220

Abstract

Neuroscience focusing on human development and disease has long been hampered due to ethical rea¬sons, low tissue availability, and low translatability from animal models. To circumvent these obstacles, we have developed two methods for the investigation of human neural cells in culture. Firstly, we present a robust 2-week protocol for the differentiation of human pluripotent stem cells (PSCs) into forebrain neural progenitor cells. Furthermore, we have used this protocol to differentiate PSCs from humans and chimpanzees. We show that human and chimpanzee cells differentiate in a similar man¬ner and that the difference in interspecies protein abundance is higher than transcript-level differences, suggesting that post-transcriptional mechanisms play a role in the difference between human and chim¬panzee brain development. Secondly, we have developed an all-in-one vector-based strategy to convert adult human dermal fibroblasts directly from Huntington’s disease (HD) patients and control individuals into induced neurons (iNs). After 4 weeks of conversion, we performed global analyses of RNA and protein levels by RNA-sequencing and mass spectrometry. In line with our previous results, we found that there are marked differences between HD patients and controls at the protein level but not at the transcriptional level. Taken together, our results suggest that post-transcriptional mechanisms play an important role in the brain both during development and in the adult brain.


2019

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Per Ludvik Brattås: The Epigenetic Impact of Transposable Elements in Human Brain development
Author: Per Ludvik Brattås
Supervisor: Johan Jakobsson
Department: Experimental Medical Science
Date of defense: 2019-04-26
Place of defense:  Segerfalksalen, Wallenberg Neuroscience Center, Lund  
Opponent: Associate Professor Molly Hammell
Date of publication: 2019
No of pages: 207
Type of document: PhD Thesis
Language: English
ISBN: 978-91-7619-763-9 / ISSN: 1652-8220

Abstract

Transposable elements (TEs) are mobile genetic sequences that have colonized more than half of the human genome. Their capacity to mobilize and insert themselves into new loci poses a constant threat to our genome integrity and frequently causes disease. Conversely, it is becoming increasingly evident that TEs can serve as regulatory elements for protein-coding genes, and represent key drivers in the evolution of gene regulatory networks. TEs are particularly active during brain development, and they are aberrantly expressed in a multitude of psychiatric and neurological conditions.
Nevertheless, the underlying mechanisms and functional consequences of these events remain largely unclear. Clarification of the role and impact of TEs in human brain development is crucial to better understand healthy human brain development and the various mechanisms underlying a wide range of brain disorders. In this thesis, I investigate epigenetic mechanisms acting on TEs in human brain development, as well as how TEs influence gene regulatory networks in both the healthy and diseased human brain.
Herein, we demonstrate that both heterochromatin and DNA methylation is required to repress specific types of TEs in human neural progenitor cells. Removal of these epigenetic marks activates not only TEs, but also neighboring genes implicated in brain development, neurological conditions and cancer, revealing an impact of TEs in gene regulatory networks in human brain development. We further reveal how TEs have established evolutionarily conserved miRNA-networks that act in the human cerebral cortex, as well how they underlie cause species-specific modulation of neuronal genes in human and chimpanzee forebrain development.
Overall, the work of this thesis expands our understanding of how TEs are controlled in healthy brain development, and how dysregulation of this control could lead to disease. Furthermore, we identify several mechanisms in which TEs influence gene regulatory networks, possibly contributing to the evolution of the human brain.

 


2017

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Rebecca Petri: The Regulatory Role of microRNAs in the Mouse and Human Brain
Author: Rebecca Petri
Supervisor: Johan Jakobsson
Department: Experimental Medical Science
Date of defense: 2017-10-27
Place of defense:  Segerfalksalen, Wallenberg Neuroscience Center, Lund  
Opponent: Jörgen Kjems, PhD
Date of publication: 2017
No of pages: 152
Type of document: PhD Thesis
Language: English
ISBN: 978-91-7619-526-0 / ISSN: 1652-8220

Abstract

microRNAs (miRNAs) arc 20-24 nucleotides small, single-stranded, non-coding RNAs.  They as­ sociate with Argonaute (AGO) proteins and exert their function by inhibition or degradation of mes­ senger RNAs. A single miRNA can target hundreds of genes and one gene can be targeted by several miRNAs, hereby giving rise to a complex post-transcriptional network. 1n the brain, hundreds of miRNAs are expressed and several are implicated in the regulation of important neuronal functions and neurodegenerative diseases.  However, many uncertainties remain concerning the regulatory role of miRNAs in the brain.

The first part of this thesis is focused on the role of miRNs in adult neurogenesis. We show that miR-125 controls functional integration of adult-born interneurons into the olfactory bulb (OB) (pa­ per 1) and that lct-7 is the most abundant miRNA in newborn OB interneurons (paper 2). Moreover, we demonstrate that let-7 controls radial migration of newborn neurons through positive regulation of neuronal autophagy, thereby revealing a novel link between miRNAs and autophagy in adult neu­ rogenesis. This finding led us to explore whether autophagy and miRNA regulation arc also linked in neurodegenerative diseases, such as Huntington's disease (HD), where autophagy is commonly impaired. We show that in several models of HD and in post-mortem brain tissue of HD patients, im­ paired autophagy leads to the accumulation of AG02. ThisAG02 accumulation results in increased levels of miRNAs with severe consequences on post-transcriptional regulation (paper 3). Finally, the work in this thesis revealed transposable elements as important sources for miRNAs and their target sites in both the developing and adult human brain (paper 4). Together, the studies in this thesis extend our current knowledge on the role of miRNs in the brain, and ultimately provide an insight into how disturbances in miRNA regulation can have severe consequences on  neuronal functions.


2015

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Liana Fasching: Transposable Elements in Neural Progenitor Cells
Author: Liana Fasching
Supervisor: Johan Jakobsson
Department: Experimental Medical Science
Date of defense: 2015-10-23
Place of defense:  Segerfalksalen, Wallenberg Neuroscience Center, Lund  
Opponent: Joshua Dubnau, PhD
Date of publication: 2015
No of pages: 116
Type of document: PhD Thesis
Language: English
ISBN: 978-91-7619-186-6 / ISSN: 1652-8220

 

Abstract

More than 90% of DNA does not code for proteins and for a long time these sequences were referred  to as "junk DNX' due to  their unknown  purpose. With the advent of  new technologies it is now known, that the non-coding part of the genome is of great importance for regulating gene expression and is therefore indispensable.

Transposable elements comprise about 50% of the genome and co-exist as symbionts regulated by epigenetic mechanisms - a highly defined machinery that controls gene expression and is mandatory for a proper development and maintenance of an organism. Although transposable elements are associated with diseases, their role in fine-tuning the host gene expression becomes more and more evident, which seems to justify the positive selection during evolution.

A transposable element called Line-1 was found to be active in neural progenitor cells and in the brain. Several studies report Line-1 transcription and frequent retrotransposition during normal brain development, with further evidence that Line-1 induced retrotransposition can influence neuronal gene expression. Today, there is few published data focusing on epigenetic regulation of transposable elements in neural progenitor cells.

In this thesis, I identify TRIM28 as key regulator of certain groups of transposable elements in mouse and human neural progenitor cells. This feature is unique compared to other somatic tissues, where DNA-methylation is prevalent. Here I demonstrate, that transposable elements MMERVK10C and IAP1 in mouse neural progenitor cells are repressed by the establishment of H3K9me3-associated heterochromatin. De­ repressed MMERVK10C and IAP1 furthermore activate nearby genes and generate long non-coding RNAs. Homozygous TRIM28 knockout is lethal, while mice with mono-allelic TRIM28 expression are characterised with a distinct behavioural phenotype.

Moreover we are also able to show that TRIM28 is regulating a fraction of young Alu-elements in human neural progenitor cells, which is not the case in human embryonic stem cells. Furthermore, we report that transcribed Alu-elements influence gene expression of close-by  genes.

Studying pluripotent cells revealed that TRIM28 modulates transposable elements 111 mouse embryonic stem cells. Activation of transposable elements upon TRIM28 depletion induces changes in gene expression of close-by genes and causes alteration of the repressive chromatin mark H3K9me3  at transposable element loci. Upregulated genes were shown to have bivalent promoters, characterised by H3K4me3 and H3K27mc3 and lay close to H3K9me3 regulated transposable elements. These findings in mouse embryonic stem cells are highly relevant for the interpretation of my studies in neural progenitor cells.

Taken together, this thesis demonstrates that the regulation of transposable elements in mouse and human neural progenitor cells is distinct compared to previous reports regarding somatic tissues. These results provide novel insights into why the brain has developed into such a complex organ with so many different cell types.


2014

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Malin Åkerblom: The Role of miRNA in Neurogenesis and Cell Specification

Author: Malin Åkerblom
Supervisor: Johan Jakobsson
Department: Experimental Medical Science
Date of defense: 2014-04-25
Place of defense:  Segerfalksalen, Wallenberg Neuroscience Center, Lund  
Opponent: Prof. Sebastian Jessberger
Date of publication: 2014
No of pages: 137
Type of document: PhD Thesis
Language: English
ISBN: 978-91-87651-69-4 / ISSN: 1652-8220

Abstract

Only a few decades ago, it was generally believed that gene expression was controlled in a uni­ directional way, i.e. DNA was transcribed into RNA, which simply acted as a messenger molecule used to produce the protein that executed cellular functions. It was not until the 1970-SO's that RNA interference was proclaimed as a gene regulatory process, and now it is increasingly evident that non­ coding RNA has a large impact on regulating and fine-tuning gene expression. microRNA (miRNA) constitute one of the largest classes of non-coding RNA. They are endogenously expressed, 19-23 nucleotides long and act by inhibiting or degrading mRNA . rniRNA control vital cellular and biologic processes, and importantly for tl1e work in tl1is thesis, they ;ire implicated in regulating neuronal de­velopment and neurogenesis. Neurogenesis, tl1e generation of new neurons, is primarily restricted to two niches of the adult brain. The molecular knowledge of how adult neurogenesis is regulated, and how different cell types are specified, is of great importance to understand how tl1e brain functions in health and disease. In my thesis, I have been studying the involvement of miRNA in adult neuro­ genesis and in cell type specification.

Using transgenic miRNA sensor mice, I have investigated the activity of tl1ree different miRNA expressed in the brain; mil-124, miR-9 and miR-125. The analyses of the sensor mice have revealed new information about the expression  of  tl1ese miRNA in the brain, presented  in papers I-III.

In tl1e first study, we demonstrated that miR-124 is a neuronal fate determinant in tl1e postnatal subventricular zone, balancing neurogenesis and gliogenesis. In the second study, we found tl1at miR- 9 is specifically absent from resident microglia in tl1e brain, and tint miR-9-regulated vectors can be used to visualise and isolate this cell type. In the third study, we showed that miR-125 is expressed in most cells of the brain, except  for the interneurons in  the olfactory bulb tint are generated during development, suggesting tl1at absence of an otl1erwise broadly expressed miRNA is a mechanism  to achieve neuronal  subtype specification.

Thus, I have used novel tools and techniques to study the activity of miRNA in the brain and to modulate tl1e expression of individual miRNA in different cell types. I provide new insights into the important roles of  rniRNA in neurogenesis  and in cell specification.


2013

 

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Rohit Sachdeva: Visualization and Manipulation of microRNA in Neural Cells
Author: Rohit Sachdeva
Supervisor: Johan Jakobsson
Department: Experimental Medical Science
Date of defense: 2013-09-06
Place of defense:  Segerfalksalen, Wallenberg Neuroscience Center, Lund   
Opponent: Eva Hedlund, PhD
Date of publication: 2013
No of pages: 128
Type of document: PhD Thesis
Language: English
ISBN: 978-91-87449-55-0 / ISSN: 1652-8220

Abstract

microRNA (miR NA) are small non-coding RNA, 21-23 n ucleotides long. miR NA provide a new layer of regulatory control over gene expression progra ms. It is increasingly evident that miR NA a re cell type and tissue specific. Ma ny mi R NAs have been identified to be highly abu nda nt in certain regions of the brain. It has also been shown that alterations  in levels of specific mi RNAs have implications in various neurodegenerative disorders. However, the role of individ ual miR NA remains poorly u nderstood.

There a re various methods that a re used to visualize miRNA, depending on type of sample, resolution and throughput. There is cu rrently no gold standard for transcriptional profiling of miR NA and the use of independent tech niques to verify results is preferable. This thesis takes a look at the various tech niques available to look at miR NA expression profile, providing insight into advantages, disadvantages, complexities and cha llenges with each tech nique. To study the function of miR NA it is also essential to regulate its expression. The thesis highlights various methods used by researchers to downregulate or overexpress miR NA.

This thesis comprises of three papers where I have tried to evolve tools to visualize dis­ tinct miR NA profile to differentiate between cells, and manipulate miRNA expression in vitro and in vivo. I n paper I, I have used mi R NA-regulated vectors to differentiate between embry­ onic stem cells and brain specific cells. This enabled me to sort ou t neu ral cells a nd trans­ plant them in a Parkinson disease mouse model. Using this method I was able to red uce the frequency of tumor  formation  post transplantation.  [1]

It has been shown that mi RNA-124 (miR-124) plays a crucial role in establishing and maintaining a neuronal transcription network. In paper II, I used a similar miRNA-regulated vector to look specifically at miR-124 expression in the brain. Here I also used tools to ma­ nipulate the expression of mi R-124. I was able to demonstrate that miR-124 is a neu ronal fate determinant in the subventricula r zone  [2]. Finally in paper  III, I  showed, using miR- 9 regulated vectors that miR-9 is not expressed in microglia. Thereby I developed  a novel system by which one can specifically target microglia. This system could be used to target genetic modifications to resident microglia in the rodent brain  [3].

Overall, further u ndersta nding of biogenesis a nd fu nctionality of this exceptiona l gene regulator will in turn enha nce tech niques used to study.them. Hopefully, in the future this leads to opportu nities to safely pu rsue mi RNA as therapeutic strategies.