1.
Lundberg, Stina
(författare)
Examining Female Resilience to Early Environmental Influences : Short- and long-term consequences on behaviour, HPA axis activity and alcohol intake after prolonged maternal separation
2017
Licentiatavhandling (övrigt vetenskapligt/konstnärligt) abstract
Early-life experiences are an important factor influencing further development of the individual. Adverse experiences early in life, such as various kinds of abuse or neglect, are types of early-life stress that can adversely affect an individuals health, as well as contribute to the development of an array of disorders later in life. Most prominent is the increased risk for psychiatric disorders, primarily depression, anxiety-related and substance use disorders. Many of the implicated disorders also exhibit sex-dependent differences in prevalence and severity. Thus, it is important to consider sex-dependent effects when modeling early-life stress and its consequences. A common animal model for early-life stress is prolonged maternal separation (MS). MS is an umbrella term for different manipulations of the early environment of rodent pups. In this thesis, a prolonged MS condition with separation of rat litters from their dams for six hours per day during the first three weeks of life (MS360) was used. In male offspring MS360 have been associated with early-life stress and negative effects apparent during both adolescence and adulthood. The literature regarding female offspring is not as substantial as for the males, but it seems that females’ exhibit less pronounced or no effect after prolonged MS independent of separation time. In addition, the studies that have examined female offspring have done so in adulthood and thus, short-term consequences of prolonged MS possibly present during adolescence have not been investigated. The aim of this thesis is to provide a broad investigation into the consequences of prolonged MS in female offspring, in both adolescence and adulthood. As stated above, MS360 was used as the adverse rearing condition in this thesis. As control, daily short MS (15 min; MS15) was used; this ensured that all animals were handled equally, except for the length of separation. Any detected differences are thus due to the length of separation only. Three categories of assessments were used to evaluate short- and long-term consequences: 1) hypothalamus-pituitary-adrenal (HPA) axis assessments, 2) behavioral assessments and 3) assessment of voluntary alcohol consumption. HPA axis reactivity was assessed in adolescent and adult offspring by blood sampling before and after challenge. HPA activity was also evaluated after long-term alcohol consumption by measurement of the fecal corticosterone content. Behavior was assessed in adolescence by registration of social play behavior and in adulthood by generation of behavioral profiles in the multivariate concentric square fieldTM (MCSF). Alcohol consumption was evaluated using the modified intermittent alcohol access schedule with the two- (20% alcohol) and three- bottle (5% and 20% alcohol) free-choice paradigms. Female offspring did not differ depending on rearing condition in HPA reactivity in adolescence or adulthood. However, after the long-term alcohol intake, MS360 females had increased levels of corticosterone in their feces compared to MS15 females. No difference was detected in adolescent social play among female offspring and only a minor alteration was detected in the adult behavioral profile, where MS360 females had increased risk assessment compared to MS15 females. No effect of rearing condition was seen during the two-bottle choice paradigm of alcohol intake, while whole- group differences over time were discovered. Alcohol intake and preference were highest the first week of access and directly after a two-week deprivation period, apart from those time-points, intake and preference were maintained on a stable level. In the three-bottle choice, an interaction with rearing condition was revealed for the total alcohol preference, however this only translated to a minor group-dependent difference. In conclusion, females reared under a prolonged MS paradigm exhibited no or only minor basal changes in HPA axis reactivity, behavior and alcohol consumption. However, after long-term alcohol intake females subjected to prolonged MS had increased corticosterone excretion into feces. That differences only emerge after long-term perturbation can be a sign that females have higher buffering capabilities than males after early-life adversity, as modeled through prolonged MS, and thus require additional challenges before consequences become apparent. This thesis highlights the importance of considering sex when studying the impact of early-life stress, and that the choice of animal model needs to be considered carefully in relation to the research question posed.
2.
Aslam, Muhammad
(författare)
The fruit fly Drosophila melanogaster GSTE6 and E7; characterization, immobilization and transgenic overexpression
2014
Licentiatavhandling (övrigt vetenskapligt/konstnärligt) abstract
Glutathione transferases (GSTs) are multifunctional enzymes that are universally distributed in most eukaryotes and prokaryotes. They play a pivotal role in the metabolism and detoxication of numerous endogenous and exogenous electrophiles by conjugating them with ubiquitous tripeptide glutathione. In this study we have immobilized two heterologously expressed and purified Epsilon-class enzymes, GSTE6 and GSTE7, from of Drosophila melanogaster on nanoporous alumina membranes. The membranes were derivatized with 3-aminopropyl-triethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzymes in solution. The stability of the immobilized enzymes was virtually identical to that for the enzymes in solution and no leakage of enzyme from the matrix could be observed.Additionally, we have investigated the catalytic activities of GSTE7 with organic isothiocyanates (ITCs). These reactive compounds are strong electrophilic molecules produced in plants by the hydrolysis of glucosinolates and exert toxicity in biological tissues. Our in vitro studies, showed high catalytic activity of GSTE7 towards ITCs. We have then explored the in vivo effect of phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC) in transgenic fruit flies overexpressing GSTE7. A concentration of 0.25 mM PEITC in standard fly food was shown to be toxic to flies and significantly shortened the lifespan. We noticed that overexpression of GSTE7 could protect females from the initial acute toxic effects, but had no positive effect on long term exposure. The effect on males seems to be the opposite to that of females, where a higher mortality was seen in fly males overexpressing GST E7 after one week of exposure. On the other hand 1mM concentration of AITC showed no toxic effects, but dramatically reduced the oviposition activity of wild-type flies in comparison to the transgenic flies.
3.
Attoff, Kristina, 1985-
(författare)
In vitro developmental neurotoxicity of acrylamide
2016
Licentiatavhandling (övrigt vetenskapligt/konstnärligt) abstract
The number of children with neurodevelopmental disorders is increasing worldwide which makes it a public concern. Exposure to environmental chemicals has been reported as a source of developmental neurotoxicity. There is also an increase in the number of chemicals reaching the global market each year and currently there are thousands of substances that have not yet been tested for developmental neurotoxicity. The current developmental neurotoxicity testing guidelines are time consuming, expensive, require a lot of animals and have relatively low sensitivity understanding for the mechanisms of toxicology. The field of developmental neurotoxicity testing is in need of a paradigm shift to the use of alternative in vitro methods capable of testing and screening large number of substances. The next generation developmental neurotoxicity testing will consist of both in silico and in vitro testing that has to be used in a combined fashion so that it will generate a more rapid and efficient toxicity testing. The methods need to be standardized between laboratories so that reproducible data can be obtained. Simple endpoints will simply not be enough for in vitro developmental neurotoxicity testing models. Rather, a battery of more refined endpoints that pinpoints the specific toxicity of a compound, discriminate between different neural subpopulations and different stages of neural differentiation is crucial for success. The use of mRNA biomarkers could be a good example of such an endpoint, and have been suggested to be valuable in detecting developmental neurotoxicity. This thesis will give a broad overview of different alternative in vitro models for developmental neurotoxicity. Developmental neurotoxicity of acrylamide was investigated by using selected cell models and endpoints. Acrylamide is a well-known neurotoxic compound and most people get exposed to the compound by food consumption and from environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed and the risk for adverse effects in the developing nervous system is overwhelming. The neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as indicators for developmental neurotoxicity. The reduced neurite outgrowth in the SH-SY5Y cell model occurred at up to seven orders of magnitude lower than what have been previously shown for different neural cell systems. Acrylamide also affected the differentiation process in both neurons and glia cells in the C17.2 cell line. We show that acrylamide attenuated neural differentiation at seven orders of magnitude lower concentrations than the estimated plasma concentration of free acrylamide in the fetus. The fact that low concentrations seem to delay the differentiation process in both cell lines, raises cause for an alarm for developmental neurotoxicity induced by acrylamide.
4.
Danielsson, Marie
(författare)
Development of a new protective group for tryptophan
2010
Licentiatavhandling (övrigt vetenskapligt/konstnärligt) abstract
A new protective group for the indole nitrogen of the amino acid tryptophan, the Boc-sarcosinoyl-sarcosinolyl (Boc-Sar-Sar) is described. This protective group shows good stability to the reaction conditions used in solid phase peptide synthesis and is incorporated into the peptide as FmocTrp(BocSarSar)OH. When the peptide is cleaved from the resin with trifluoroacetic acid, the Boc group is simultaneously cleaved and N-terminal nitrogen in the Sar-Sar group is exposed. At low pH this amino group is protonated and thereby the peptide will have higher solubility in aqueous solution. High solubility of the peptide is an important factor for the purification of peptides with reversed phase HPLC. During HPLC purification, the protonated Sar-Sar group will remain on the peptide. When the purified peptide is exposed to pH 7,4 the Sar-Sar group will be cleaved and the peptide will be obtained in its fully deprotected form. This new protective group was used for the synthesis of the antimicrobial peptide gramicidin A, an extremely hydrophobic peptide with no ionizable groups and only a few exposed polar functional groups.
5.
Granholm, Linnea
(författare)
Neurobiological Consequences of Social Conditions and Alcohol Exposure in Adolescent rats
2015
Licentiatavhandling (övrigt vetenskapligt/konstnärligt) abstract
Adolescence represents a time of extensive reorganisation and maturation of brain circuits involved in emotions, motivation and cognition and it is a period particular sensitive for external stimuli. External stimuli can be both socio-environmental factors and exposure to exogenous compounds such as drugs of abuse (e.g. alcohol). If these stimuli are of an adverse nature the probability of develop neuropsychiatric diseases or addiction is increased. To study the neurobiological consequences of adverse events during adolescence animal models are crucial since they give the opportunity of providing an environment where the exposure of the stimuli is controlled and also enable a detailed analysis of the effects in the brain. The overall aim of in this thesis was to investigate how environmental factors, social conditions or alcohol exposure, during adolescence affect the brain and/or drug-taking in rats. Rats are very sensitive for dis- turbances in their social conditions and to induce an adverse social environment, early adolescent rats where single-housed for either a short or prolonged time. A short period of single housing induced an acute stress response and increased levels of nociceptin/orphanin FQ in brain areas associated with stress. Prolonged single housing reduced the levels of Met-enkephalin-Arg6Phe7 in several brain areas. Rats exposed to alcohol during adolescence had an altered dopamine response in dorsal striatum after an am- phetamine challenge but displayed similar amphetamine intake-behaviour as water controls. However, animals exposed to a combination of adolescent alcohol exposure and subsequent amphetamine intake had a more efficient removal of dopamine in dorsal striatum after an amphetamine challenge. This thesis demonstrates how two different environmental stimuli are able to alter the neurobiology in adolescent rats. The results further support the notion that environmental conditions are of importance for normal brain maturation and provide new evidence that endogenous opioids are severely affected by social dis- turbances during adolescence. Furthermore, additional information is provided to the existing literature of how alcohol exposure during adolescence affects dopamine dynamics and drug-taking behaviour. In the literature, the majority of the studies of adolescent alcohol exposure have focused on the nucleus accum- bens, a brain area important in the processing of rewards. The results herein provide evidence that dorsal striatum, a brain area involved in the transition into habitual drug use is also affected by adolescent alco- hol exposure. An altered drug response in dorsal striatum may affect habit formation and contribute to a heightened susceptibility for high drug consumption later in life.
6.
Guterstam, Peter, 1977-
(författare)
Cell-penetrating peptides, novel synthetic nucleic acids, and regulation of gene function : Reconnaissance for designing functional conjugates
2008
Licentiatavhandling (övrigt vetenskapligt/konstnärligt) abstract
Our genome operates by sending instructions, conveyed by mRNA, for the manufacture of proteins from chromosomal DNA in the nucleus of the cell to the protein synthesizing machinery in the cytoplasm. Alternative splicing is a natural process in which a single gene can encode multiple related proteins. During RNA splicing, introns are selectively removed resulting in alternatively spliced gene products. Alternatively spliced protein products can have very different biological effects, such that one protein isoform is disease-related while another isoform is desirable. Splice switching opens the door to new drug targets, and antisense oligonucleotides (asONs), designed to switch splicing, are effective drug candidates. Cellular uptake of oligonucleotides(ONs) is poor, therefore utilization of cell-penetrating peptides (CPPs), well recognized for intracellular cargo delivery, is a promising approach to overcome this essential issue. Most CPPs are internalized by endocytosis, although the mechanisms involved remain controversial. Here, evaluation of CPP-mediated ON delivery over cellular membranes has been performed. A protocol that allows for convenient assessment of CPP-mediated cellular uptake and characterization of corresponding internalization routes is established. The protocol is based on both fluorometric uptake measurements and a functional splice-switching assay, which in itself is based on biological activity of conveyed ONs. Additionally, splice switching ONs (SSOs) have been optimized for high efficiency and specificity. Data suggest that SSO activity is improved for chimeric phosphorothioate SSOs containing locked nucleic acid (LNA) monomers. It is striking that the LNA monomers in such chimeric constructs give rise to low mismatch discrimination of target pre-mRNA, which highlight the necessity to optimize sequences to minimize risk for off-target effects. The results are important for up-coming work aimed at developing compounds consisting of peptides and novel synthetic nucleic acids, making these entities winning allies in the competition to develop therapeutics regulating protein expression patterns.
7.
Hjorth, Johannes, 1978-
(författare)
Information processing in the Striatum : a computational study
2006
Licentiatavhandling (övrigt vetenskapligt/konstnärligt) abstract
The basal ganglia form an important structure centrally placed in the brain. They receive input from motor, associative and limbic areas, and produce output mainly to the thalamus and the brain stem. The basal ganglia have been implied in cognitive and motor functions. One way to understand the basal ganglia is to take a look at the diseases that affect them. Both Parkinson's disease and Huntington's disease with their motor problems are results of malfunctioning basal ganglia. There are also indications that these diseases affect cognitive functions. Drug addiction is another example that involves this structure, which is also important for motivation and selection of behaviour. In this licentiate thesis I am laying the groundwork for a detailed model of the striatum, which is the input stage of the basal ganglia. The striatum receives glutamatergic input from the cortex and thalamus, as well as dopaminergic input from substantia nigra. The majority of the neurons in the striatum are medium spiny (MS) projection neurons that project mainly to globus pallidus but also to other neurons in the striatum and to both dopamine producing and GABAergic neurons in substantia nigra. In addition to the MS neurons there are fast spiking (FS) interneurons that are in a position to regulate the firing of the MS neurons. These FS neurons are few, but connected into large networks through electrical synapses that could synchronise their effect. By forming strong inhibitory synapses on the MS neurons the FS neurons have a powerful influence on the striatal output. The inhibitory output of the basal ganglia on the thalamus is believed to keep prepared motor commands on hold, but once one of them is disinhibited, then the selected motor command is executed. This disinhibition is initiated in the striatum by the MS neurons. Both MS and FS neurons are active during so called up-states, which are periods of elevated cortical input to striatum. Here I have studied the FS neurons and their ability to detect such up-states. This is important because FS neurons can delay spikes in MS neurons and the time between up-state onset and the first spike in the MS neurons is correlated with the amount of calcium entering the MS neuron, which in turn might have implications for plasticity and learning of new behaviours. The effect of different combinations of electrical couplings between two FS neurons has been tested, where the location, number and strength of these gap junctions have been varied. I studied both the ability of the FS neurons to fire action potentials during the up-state, and the synchronisation between neighbouring FS neurons due to electrical coupling. I found that both proximal and distal gap junctions synchronised the firing, but the distal gap junctions did not have the same temporal precision. The ability of the FS neurons to detect an up-state was affected by whether the neighbouring FS neuron also received up-state input or not. This effect was more pronounced for distal gap junctions than proximal ones, due to a stronger shunting effect of distal gap junctions when the dendrites were synaptically activated. We have also performed initial stochastic simulations of the Ca2+-calmodulin-dependent protein kinase II (CaMKII). The purpose here is to build the knowledge as well as the tools necessary for biochemical simulations of intracellular processes that are important for plasticity in the MS neurons. The simulated biochemical pathways will then be integrated into an existing model of a full MS neuron. Another venue to explore is to build striatal network models consisting of MS and FS neurons and using experimental data of the striatal microcircuitry. With these different approaches we will improve our understanding of striatal information processing.
8.
Hoeber, Jan, 1986-
(författare)
Generation of functional neural progenitors for spinal cord transplantation
2015
Licentiatavhandling (övrigt vetenskapligt/konstnärligt) abstract
Dorsal root avulsion injuries result in permanent impairment of sensory functions due to the disconnection of the peripheral nervous system from the spinal cord. Strategies aiming for the functional reconnection of sensory neurons with their targets in the spinal cord dorsal horn need to overcome the axonal growth non-permissive glial scar and provide a growth promoting environment. Stem cell therapy holds great promise in the context of root avulsion injuries as it combines the potential to provide a permissive and axonal growth attractive environment and the ability to form a neuronal relay between sensory neurons and spinal cord targets. In the first study, we show that human embryonic stem cell derived spinal cord neural progenitors(hNPs) restore sensorimotor functions in a model of dorsal root avulsion injury. The observed recovery of sensory functions was mediated by hNP cells forming growth permissive gates inthe glial scar that allowed spinal ingrowth of regenerating sensory axons. In the second study,we show that also human spinal cord neural stem/progenitor cells (hscNSPC) promote sensory axon ingrowth by the formation of a growth permissive tissue bridge that interferes with the spinal cord glial scar. Further, we tested whether this effect can be enhanced by combinatorial application of growth factors peptide mimetics. Interestingly, both hscNSPC and growth factor peptide mimetics alone but not in combination promote sensory regeneration. The observed failure of regeneration is likely caused by the reduced migration of hscNSPC when transplanted together with growth factor mimetics resulting in their inability to provide a continuous tissuebridge into the spinal cord. In the last study, we show first approaches to provide molecular tools that allow testing functional integration of stem cell derived neurons into the spinal cord.These tools are a prerequisite to test whether stem cells can also act as neuronal relays in the observed sensory regeneration events. In conclusion, this thesis provides first evidence that sensory regeneration is possible after dorsal root avulsion injury. This can be achieved by transplantation of human stem cell derived neuronal cells and to a certain degree by growth factor peptide mimetics.
9.
Jacobsen, Kristin, 1982-
(författare)
Processing of the APP family by the α-secretases ADAM10 and TACE
2010
Licentiatavhandling (övrigt vetenskapligt/konstnärligt) abstract
Alzheimer’s disease (AD) is a progressive neurodegenerative disease, which is characterized by formation of amyloid plaques in the brain. The major constituent of these plaques is the hydrophobic peptide Aβ. Aβ accumulation is considered to be the main cause of the pathology seen in AD brains. Aβ is produced through sequential cleavage of the amyloid precursor protein (APP). APP can be processed by two different enzymatic pathways. Formation of Aβ requires cleavage of APP by β- and γ-secretase. However, most proteolytic processing of APP does not result in Aβ formation. Instead, APP is mainly cleaved by α-secretase, which not only precludes formation of the toxic Aβ peptide but also generates the neuroprotective sAPPα fragment. Increasing the α-secretase processing of APP is thereby a potential therapeutic strategy for AD. APP is a member of a conserved gene family, also including the APP-like proteins-1 and -2 (APLP1 and APLP2). The APP family members have essential and overlapping functions and have been reported to be processed in a similar way by the same enzymes. The processing of all APP family members is increased in response to several stimuli, including retinoic acid (RA) and insulin-like growth factor-1 (IGF-1), which also induce a shift towards α-secretase processing. The aim of this thesis was to investigate the mechanisms and signaling involved in induced α-secretase processing of the APP family. The main α-secretase candidates are ADAM10 and TACE. In this thesis we wanted to study the effects on expression levels of ADAM10 and TACE during RA treatment. We also wanted to investigate the mechanism behind IGF-1-induced processing of APP and APLP2. We found that both ADAM10 and TACE are up-regulated in response to RA, but that the signaling pathways involved differed between the two enzymes. Similarly, we showed that IGF-1-induced processing of APLP2, but not of APP, is dependent on PKC. Furthermore, we showed that ADAM10 is the main α-secretase for APP, whereas TACE cleaves APLP2 in response to IGF-1. We conclude that although APP and APLP2 proteolytic processing are induced by the same stimuli, the processing is dependent on different signaling pathways and processing enzymes, which in turn are differentially regulated.
10.
Mastinu, Enzo, 1987
(författare)
Embedded Controller for Artificial Limbs
2017
Licentiatavhandling (övrigt vetenskapligt/konstnärligt) abstract
Promising developments are currently ongoing worldwide in the field of neuroprosthetics and artificial limb control. It is now possible to chronically connect a robotic limb to bone, nerves and muscles of a human being, and use the signals sourced from these connections to enable movements in the artificial limb. It is also possible to surgically redirect a nerve, deprived from its original target muscle due to amputation, to a new target in order to restore the original motor functionality. Intelligent signal processing algorithms can now utilize the bioelectric signals gathered from remaining muscles on the stump to decode the motor intention of the amputee, providing an intuitive control interface. Unfortunately for patients, clinical implementations still lag behind the advancements of research, and the conventional solutions for amputees remained basically unchanged since decades. More efforts are therefore needed from researchers to close the gap between scientific publications and hospital practices.The ultimate focus of this thesis is set on the intuitive control of a prosthetic upper limb. It was developed an embedded system capable of prosthetic control via the processing of bioelectric signals and pattern recognition algorithms. It includes a neurostimulator to provide direct neural feedback modulated by sensory information from artificial sensors. The system was designed towards clinical implementation and its functionality was proven by amputee subjects in daily life. It also constitutes a research platform to monitor prosthesis usage and training, machine learning based control algorithms, and neural stimulation paradigms.
