| 1. |
- Vicente Carrillo, Alejandro, 1989-
(författare)
-
Sperm Membrane Channels, Receptors and Kinematics : Using boar spermatozoa for drug toxicity screening
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.
|
|
| 2. |
- Atikuzzaman, Mohammad, 1977-
(författare)
-
Seminal Influence on the Oviduct : Mating and/or semen components induce gene expression changes in the pre-ovulatory functional sperm reservoir in poultry and pigs
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Internal fertilization occurs in birds and eutherian mammals. Foetal development, however, is either extra- respectively intra-corpore (egg vs uterus). In these animal classes, the female genital tract stores ejaculated spermatozoa into a restricted oviductal segment; the functional pre-ovulatory sperm reservoir, where they survive until ovulation/s occur. Paradoxically, this immunologically foreign sperm suspension in seminal fluid/plasma, often microbiologically contaminated, ought to be promptly eliminated by the female local immune defence which, instead, tolerates its presence. The female immune tolerance is presumably signalled via a biochemical interplay of spermatozoa, as well as the peptides and proteins of the extracellular seminal fluid, with female epithelial and immune cells. Such interplay can result in gene expression shifts in the sperm reservoir in relation to variations in fertility. To further aid our understanding of the underlying mechanisms, this thesis studied the proteome of the seminal fluid (using 2D SDS-PAGE and mass spectrometry) including cytokine content (using Luminex and/or ELISA) of healthy, sexually mature and fertile boars and cocks. As well, gene expression changes (using cDNA microarray) in the oviductal sperm reservoirs of sexually-mature females, mated or artificially infused with homologous sperm-free seminal fluid/plasma were studied. Pigs were of commercial, fertility-selected modern breeds (Landrace), while chicken belonged to the ancestor Red Junglefowl (RJF, low egg laying-capacity), a selected egg-layer White Leghorn (WL) and of their Advanced Intercross Line (AIL). Ejaculates were manually collected as single sample in cocks or as the sperm-rich fraction [SRF] and the post- SRF fraction in boars to harvest seminal fluid/plasma for proteome/cytokine and infusion-studies. Oviducts were retrieved for gene-expression analyses via microarray immediately post-mortem (chicken) or at surgery (pig), 24 h after mating or genital infusion. In pigs, the protein-rich seminal plasma showed the highest amounts of cytokines [interferon-γ, interferon gamma-induced protein 10 (IP-10/CXCL10), macrophage derived chemokine (MDC/CCL22), growth-regulated oncogene (GRO/CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemo-attractant protein-1 (MCP-1/ CCL2), interleukin (IL)-6, IL-8/CXCL8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-β1-3) in the larger, protein-rich and sperm-poor post-SRF, indicating its main immune signalling influence. Chicken showed also a plethora of seminal fluid proteins with serum albumin and ovotransferrin being conserved through selection/evolution. However, they showed fewer cytokines than pigs, as the anti-inflammatory/immune-modulatory TGF-β2 or the pro-inflammatory CXCL10. The RJF contained fewer immune system process proteins and lacked TGF-β2 compared to WL and AIL, suggesting selection for increased fertility could be associated with higher expression of immune-regulating peptides/proteins. The oviductal sperm reservoir reacted in vivo to semen exposure. In chicken, mating significantly changed the expression of immune-modulatory and pH-regulatory genes in AIL. Moreover, modern fertile pigs (Landrace) and chicken (WL), albeit being taxonomically distant, shared gene functions for preservation of viable sperm in the oviduct. Mating or SP/SF-infusion were able to change the expression of comparable genes involved in pH-regulation (SLC16A2, SLC4A9, SLC13A1, SLC35F1, ATP8B3, ATP13A3) or immune-modulation (IFIT5, IFI16, MMP27, ADAMTS3, MMP3, MMP12). The results of the thesis demonstrate that both mating and components of the sperm-free seminal fluid/plasma elicit gene expression changes in the pre-ovulatory female sperm reservoir of chickens and pigs, some conserved over domestication and fertility-selection.
|
|
| 3. |
- Bednarska, Olga, 1973-
(författare)
-
Peripheral and Central Mechanisms in Irritable Bowel Syndrome : in search of links
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Irritable bowel syndrome (IBS) is a chronic visceral pain disorder with female predominance, characterized by recurrent abdominal pain and disturbed bowel habits in the absence of an identifiable organic cause. This prevalent and debilitating disease, which accounts for a substantial economic and individual burden, lacks exact diagnostic tools and effective treatment, since its pathophysiology remains uncertain. The bidirectional and multilayered brain-gut axis is a well-established disease model, however, the interactions between central and peripheral mechanisms along the brain-gut axis remain incompletely understood. One of the welldescribed triggering factors, yet accounting for only a fraction of IBS prevalence, is bacterial gastroenteritis that affects mucosal barrier function. Altered gut microbiota composition as well as disturbed intestinal mucosal barrier function and its neuroimmune regulation have been reported in IBS, however, the impact of live bacteria, neither commensal nor pathogenic, on intestinal barrier has not been studied yet. Furthermore, abnormal central processing of visceral sensations and psychological factors such as maladaptive coping have previously been suggested as centrally-mediated pathophysiological mechanisms of importance in IBS. Brain imaging studies have demonstrated an imbalance in descending pain modulatory networks and alterations in brain regions associated with interoceptive awareness and pain processing and modulation, particularly in anterior insula (aINS), although biochemical changes putatively underlying these central alterations remain poorly understood. Most importantly, however, possible associations between these documented changes on central and peripheral levels, which may as complex interactions contribute to disease onset and chronification of symptoms, are widely unknown.This thesis aimed to investigate the peripheral and central mechanisms in women with IBS compared to female healthy controls (HC) and to explore possible mutual associations between these mechanisms.In Paper I, we studied paracellular permeability and passage of live bacteria, both commensal and pathogenic through colonic biopsies mounted in Ussing chambers. We explored the regulation of the mucosal barrier function by mast cells and the neuropeptide vasoactive intestinal polypeptide (VIP) as well as a correlation between mucosal permeability and gastrointestinal and psychological symptoms. We observed increased paracellular permeability and the passage of commensal and pathogenic live bacteria in patients with IBS compared with HC, which was diminished by blocking the VIP receptors as well as after stabilizing mast cells in both groups. Moreover, higher paracellular permeability was associated with less somatic and psychological symptoms in patients.In Paper II, we aimed to determine the association between colonic mucosa paracellular permeability and structural and resting state functional brain connectivity. We demonstrated different patterns of associations between mucosa permeability and functional and structural brain connectivity in IBS patients compared to HC. Specifically, lower paracellular permeability in IBS, similar to the levels detected in HC, was associated with more severe IBS symptoms and increased functional and structural connectivity between intrinsic brain resting state network and descending pain modulation brain regions. Our findings further suggested that this association between mucosa permeability and functional brain connectivity was mainly mediated by coping strategies.In Paper III, we investigated putative alterations in excitatory and inhibitory neurotransmission of aINS, as the brain’s key node of the salience network crucially involved in cognitive control, in IBS patients relative to HC and addressed possible connections with both symptoms and psychological factors. We found decreased concentrations of the excitatory neurotransmitter Glx in bilateral aINS in IBS patients compared to HC, while inhibitory neurotransmitter GABA+ levels were comparable. Further, we demonstrated hemisphere-specific associations between abdominal pain, coping and aINS excitatory neurotransmitter concentration.In conclusion, this thesis broadens the knowledge on peripheral and central mechanisms in IBS and presents novel findings that bring together the ends of brain-gut axis. Our results depict association between mucosal permeability, IBS symptoms and functional and structural connectivity engaging brain regions involved in emotion and pain modulation as well as underlying neurotransmitter alterations.
|
|
| 4. |
- Henriksson, Hanna, 1977-
(författare)
-
Development of body composition and its relationship with physical activity in healthy Swedish children : A longitudinal study until 4.5 years of age including evaluation of methods to assess physical activity and energy intake
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Childhood obesity according to the World Health Organization is one of the most serious public health challenges of the 21st century. The proportion of childhood obesity is high both globally and in Sweden. This is of great concern since obese children tend to stay obese in adulthood. In order to develop strategies to prevent early childhood obesity more knowledge is needed regarding factors explaining why children become overweight and obese. Preventive strategies require accurate and easy-to-use methods to assess physical activity in response to energy expenditure as well as energy intake in young children, but such methods are largely lacking or have shown limited accuracy. The aims of this thesis were: 1) to describe the longitudinal development of body composition from 1 week to 4.5 years of age; 2) to study relationships between measures of body composition and the physical activity level (PAL) at 1.5 and 3 years of age; 3) to evaluate if heart rate recording and movement registration using Actiheart can capture variations in total energy expenditure (TEE) and activity energy expenditure (AEE) at 1.5 and 3 years; 4) to evaluate the potential of a 7-day activity diary to assess PAL at 1.5 and 3 years of age; 5) to evaluate a new tool (TECH) using mobile phones for assessing energy intake at 3 years of age.Healthy children were investigated at 1 and 12 weeks (n=44), at 1.5 (n=44), 3 (n=33) and 4.5 (n=26) years of age. Body composition was measured using air-displacement plethysmography at 1 and 12 weeks and at 4.5 years of age. At 1.5 and 3 years, body composition, TEE, PAL and AEE were assessed using the doubly labelled water method and indirect calorimetry. Heart rate and movements were recorded using Actiheart (four days) and physical activities were registered using the 7-day diary. Energy intake was assessed using TECH during one complete 24-hour period.Average percentage of total body fat (TBF) and average fat mass index (FMI) were higher (+3 to +81 %), while fat-free mass index (FFMI) was slightly lower (-2 to -9 %), in children in the study from 12 weeks until 4.5 years of age when compared to corresponding reference values. A relationship between TBF% and PAL was found both at 1.5 and 3 years of age. At 3 years, but not at 1.5 years, this could be explained by a relationship between PAL and FFMI. Actiheart recordings explained a significant but small fraction (8%) of the variation in free-living TEE at 1.5 and 3 years, and in AEE (6 %) at 3 years, above that explained by body composition variables. At 1.5 and 3 years of age, PAL estimated by means of the activity diary using metabolic equivalent (MET) values by Ainsworth et al. was not significantly different from reference PAL, but the accuracy for individuals was low. Average energy intake assessed by TECH was not significantly different from TEE. However, the accuracy for individuals was poor.The results of this thesis suggest that 1) The higher body fatness of the children in the study compared to the corresponding reference values may indicate the presence of a secular trend in body composition development characterized by a high body fatness. 2) Body fatness might counteract physical activity at 1.5 years of age when the capacity to perform physical activity is limited, but not at 3 years of age when such a capacity has been developed. 3) Actiheart recordings explained a significant but small fraction of the variation in TEE at 1.5 and 3 years, and in AEE at 3 years of age, above that explained by body composition variables. 4) The activity diary and TECH produced mean values in agreement with reference PAL and TEE, respectively, but the accuracy for individual children was low.In conclusion, the results of this thesis suggest the presence of a secular trend in body composition development in healthy Swedish children, from infancy up to 4.5 years of age, which is characterized by a high body fatness. Methods to assess physical activity and energy intake at 1.5 and 3 years of age provided some promising results on a group level, although further research is needed to increase the accuracy of these methods in individual children.
|
|
| 5. |
- Hilborn, Erik, 1988-
(författare)
-
The role of the androgen receptor and hydroxysteroid 17β dehydrogenase in breast cancer : Impact on tamoxifen treatment
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The healthy breast is a tissue composed of centrally located milk producing glands connected to the nipple by ducts, surrounded by fat tissue and connective tissue. The growth of the breast is primarily mediated by the estrogens, while the androgens mediate tissue homeostasis and protect against growth signals. In breast cancer, the cells of the glands or ducts undergo malignant transformation, and start proliferating in an uncontrollable fashion. Breast cancer is the most common malignancy in women, and it is estimated that 10% of all women will be diagnosed with breast cancer during their life-time. The primary classification of breast cancer is based mainly on the expression of the estrogen receptor, and 70-80% of breast cancers are estrogen receptor positive, and are classified as luminal. The remaining breast cancers are classified into HER2 positive or triple negative breast cancer. Out of all breast cancers, ~80% are androgen receptor positive. This varies in different subtypes, however, with the highest expression in luminal and lowest expression in triple negative breast cancers. The role of androgen receptor varies depending on subtype. It is considered tissue-protective in luminal breast cancer, while it’s role in HER2 positive and triple negative breast cancers is less defined, but is generally considered to be associated with worse outcome. The primary treatment for breast cancer is surgery, followed by chemotherapy and/or radiotherapy to reduce the risk of recurrence. Treatment is also subtype specific, and luminal breast cancers in premenopausalwomen are treated using the estrogen receptor blocker (antagonist) tamoxifen, which blocks estrogen signaling. In postmenopausal women, luminal breast cancers are treated using tamoxifen or aromatase inhibitors, which prevent the formation of estrogen. The knowledge of which patient will respond and who will develop treatment resistance is of great importance, and the development of markers which can be analyzed prior to treatment in order to reduce the risk of unwanted side effects or complications is the focus of a large body of research. One of the primary goals of this thesis was to establish biomarkers for prognosis and tamoxifen treatment in breast cancer, and paper I, paper II and paper III address this aim.Steroid hormones, including estrogens and androgens, are normally synthesized from cholesterol in the adrenal gland, as well as in gender specific tissues such as ovaries in women or the testis or prostate in men. This synthesis takes place as a number of enzymatic conversions, mediated by several different enzymes, and the expression of these enzymes determines the final product of this conversion. In the adrenal gland, testis and prostate, androgens are the end-product, while the ovaries synthesize estrogens. These hormones are transported through the circulation, and upon reaching their target tissues, they mediate their effect. The impact of the steroids on their destination tissue is dependent on their relative concentration and exposure time, which in turn is dependent on the amount in the circulation, but also on the presence of local steroid converting enzymes, which are present in most tissues. The enzymes of the hydroxysteroid 17β dehydrogenase family are present in most tissues, primarily the oxidative member hydroxysteroid 17β dehydrogenase type 2, which facilitate the conversion of estrogens and androgens to the less active forms, thus protecting the tissues from their effect. In breast cancer, the reductive form, hydroxysteroid 17β dehydrogenase type 1 is often up-regulated, and mediates increased activation of estrogens, resulting in increased estrogen signaling, which results in increased proliferation and growth. The second goal of this thesis was to further study the role of hydroxysteroid 17β dehydrogenase enzymes in breast cancer, and paper I and paper IV address different aspects of their role in breast cancer.Following reduction of the expression of hydroxysteroid 17β dehydrogenase type 14, an oxidative member of the family, in breast cancer, the expression of C-X-C ligand 10 was found to be altered. In paper I, in order to determine the role of C-X-C ligand 10 and C-X-C receptor 3 in breast cancer, their expression was quantified using immunohistochemistry in breast cancer patients randomized to tamoxifen or no endocrine treatment irrespectively of estrogen receptor status. The expression of C-XC ligand 10 and C-X-C receptor 3 was found to be associated with increased tamoxifen treatment benefit in the estrogen receptor positive group of patients, indicating that they could be useful markers for determining which patient would respond well to this treatment. Further, C-X-C receptor 3 expression was associated with worse outcome in patients who did not receive tamoxifen, and could be a potential target for inhibitors in order to improve patient outcome. The role of the androgen receptor in breast cancer was evaluated. In paper II the expression was quantified using immunohistochemistry in the same cohort as in paper I. We show that in patients with estrogen receptor negative tumors, the androgen receptor is associated with worse outcome. In patients with high tumoral androgen receptor expression, tamoxifen signaling results in significant improvement in outcome, despite lack of the estrogen receptor. The opposite was observed in patients without tumoral androgen receptor expression, and tamoxifen treatment was associated with adverse outcome. Similar findings were made in the triple negative cases. In the luminal cases, the androgen receptor does not provide further information pertaining to outcome. In paper III we evaluated the role of mutations in the androgen receptor in the cohort of estrogen receptor-negative and androgen receptorpositive cases from paper II. The role of mutations in the androgen receptor appear to have a modest role in regard to patient outcome, but rs17302090 appear associated with tamoxifen treatment benefit. The modulation of the members of the hydroxysteroid 17β dehydrogenase in breast cancer is associated with changes in the local steroid balance, and has been associated with worse outcome and changes in the response to tamoxifen. Further, the inhibition of hydroxysteroid 17β dehydrogenase type 1 has been proposed as an alternate treatment for breast cancer, but no inhibitors are currently used in the clinic. In paper IV, we evaluated several different mechanisms by which the expression of hydroxysteroid 17β dehydrogenase type 1 and type 2 are modulated in breast cancer. We show that the most potent estrogen estradiol, in an estrogen receptor dependent fashion, can result in decreased hydroxysteroid 17β dehydrogenase type 1 expression, and a short term reduction in type 2 expression or long term increased type 2 expression. We also show that the most potent androgen, dihydrotestosterone, can increase hydroxysteroid 17β dehydrogenase type 2 expression, but has limited impact on hydroxysteroid 17β dehydrogenase type 1. Further, we show that a number of genes involved in breast cancer, and microRNA are involved in modulating the expression of the hydroxysteroid 17β dehydrogenase type 1 and type 2 in breast cancer. These findings could potentially be used as an alternative to inhibitors, and help modulate the steroidal balance in target tissue.
|
|
| 6. |
- Kernell, Kristina
(författare)
-
Cardiac disease in pregnancy and consequences for reproductive outcomes, comorbidity and survival
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- BackgroundAdvances in medical treatment during the last 50 years have resulted in more individuals with congenital heart disease (CHD) and Marfan syndrome reaching childbearing age. The substantial physiological changes during pregnancy result in a high-risk situation, and pregnancy is a major concern in women with these conditions.AimsTo describe the socio-demographic characteristics, birth characteristics and reproductive patterns of individuals with CHD and women with Marfan syndrome.To investigate obstetric and neonatal outcomes in the firstborn children of individuals with CHD and women with Marfan syndrome.To study long-term cardiovascular outcomes after childbirth in women with Marfan´syndrome.MethodsThe studies are population-based register studies. The study population in the first paper included all women born between 1973 and 1983 who were alive and resident in Sweden at the age of 13 (494 692 women, of whom 2 216 were women with CHD). In the second paper, the same definition of the study population was chosen, except that it involved all men born between 1973 and 1983 (522 216 men, of whom 2 689 men with CHD). The third and fourth papers involved a study population of all Swedish women born between 1973 and 1993 who were still living in Sweden at age 13. This population consisted of 1 017 538 women, 273 of whom had been diagnosed with Marfan syndrome.Results and conclusionsThe individuals studied were more often born preterm, and were small-for-gestational age babies. They were more likely to have been born by cesarean section. In women with CHD, these characteristics were repeated in their firstborn children. No increased risks were found in children of men with CHD or in children of women with Marfan syndrome. There was no increased risk of aortic dissection in women with Marfan syndrome during pregnancy compared to women with Marfan syndrome who did not give birth. Higher frequencies of cardiac arrhythmia and valvular heart disease were found after childbirth in women with Marfan syndrome. Pregnancy in women with CHD is a high-risk situation associated with increased risk of adverse neonatal outcomes for the expected child. Pregnancy in women without CHD, but where the father has CHD is not so associated with increased risk of adverse obstetric or neonatal outcomes. Pregnancy in women with Marfan syndrome is not associated with adverse outcomes for the expected child.
|
|
| 7. |
- Lönnqvist, Susanna, 1986-
(författare)
-
Applications of human skin in vitro
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chronic wounds are a substantial problem in today’s health care and place significant strains on the patient. Successful modelling of the wound healing process is pivotal for the advancement of wound treatment research. Wound healing is a dynamic and multifactorial process involving all constituents of the skin. The progression from haemostasis and inflammation to proliferation of epidermal keratinocytes and dermal fibroblasts, and final scar maturation can be halted and result in a chronic wound that fails to re-epithelialise. The wound healing process constitutes an example of dynamic reciprocity in tissue where cellular changes take place on cues from the extracellular matrix and vice versa when tissue homeostasis is disturbed. The extracellular matrix provides a structural context for the resident cells and the epidermal keratinocytes, and a functioning interplay between the two tissue compartments is crucial for successful wound healing to take place. Work included in this thesis has applied viable human full thickness skin in vitro to investigate the re-epithelialisation process and barrier function of intact skin.The use of full thickness skin in vitro can take into account the contextual aspect of the process where the epidermal keratinocytes are activated and obtain a migratory phenotype, and are continuously dependent on the cues from the extracellular matrix and support of the dermis. When utilising skin for studies on re-epithelialisation, circular standardised full thickness wounds were created and cultured for up to four weeks in tissue culture. In paper I, the organisation of a thick neoepidermis was investigated in the in vitro wound healing model when resident cells were provided with a porous suspended three dimensional gelatin scaffold. In paper II we investigated the use of a fluorescent staining conventionally used for proliferation studies to facilitate the tracing of transplanted epidermal cells in in vitro wounds, in order to improve and expand the use of the model. In paper III the model was utilised to investigate the treatment approach of acidification of wounds to evaluate the suitability of such intervention in regards to keratinocyte function and re-epithelialisation. Studies on re-epithelialisation with the aid of the in vitro wound healing model provided insight in neoepidermal structure with porous gelatin scaffolding in the wound, a novel methodological approach to tracing cells and response to constrained wound healing environment. In paper IV, intact human skin was evaluated for modelling the cytotoxic response after exposure to a known irritant compound. To study barrier function, intact skin was exposed to irritants by restricting exposure topically, and full thickness skin in vitro was found suitable for modelling cytotoxicity responses. Employing human full thickness skin in vitro makes use of the actual target tissue of interest with epidermal and dermal cells, and full barrier function.
|
|
| 8. |
- Nyman, Erika, 1976-
(författare)
-
Guided Regeneration of the Human Skin : in vitro and in vivo studies
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Every day and in all parts of the world, humans experience different grades of wounding and tissue loss of the skin, thus initiating one of the most complex biological processes. Acute and chronic wounds, as well as the additional problem of skin scarring, involve not only great suffering for the patient but also extensive health care costs for the society. Although the wound-healing process is a wellstudied field much knowledge must be gained to unlock the door to regenerative pathways in humans.Epidermis heals by complete regeneration, but dermal and full thickness injuries heal with fibrosis and scar formation. In Papers I and II, we studied whether dermal scarring could be turned into regeneration by using two different types of threedimensional dermal scaffolds. In Paper I, we studied a solid scaffold made of poly(urethane urea), initially in vitro then followed by in vivo studies. In Paper II, we intradermally injected a liquid three-dimensional scaffold consisting of porous gelatin spheres in human healthy volunteers. Both materials showed ingrowth of functional fibroblasts and blood vessels and appeared to stimulate regeneration while slowly degrading. This finding could be of significant clinical importance, for example in burn wound care or after cancer surgery.In Papers III and IV, we wanted to study the effects of amniotic fluid and hyaluronic acid on adult wound healing, because early fetal wounds re-epithelialize rapidly and naturally heal dermis by regeneration without the need of a dermal scaffold. Amniotic fluid, naturally rich in hyaluronic acid, induced an accelerated reepithelialization of adult human wounds in vitro, and hyaluronic acid seemed to be important for this effect. Stimulation with exogenous hyaluronic acid in vivo induced accelerated re-epithelialization and an altered protein expression in healthy human volunteers. The inflammatory phase of wound healing, as measured by tissue viability imaging, was not affected by hyaluronic acid. Elucidating the effects of amniotic fluid and hyaluronic acid on the wound-healing process may allow improved treatment of wounds with impaired healing.Studies on finding new dermal scaffolds and studies on the positive effect of amniotic fluid or hyaluronic acid on the wound-healing process are two different ways of gaining insight that may lead to regeneration and improved wound healing for the patient.
|
|
| 9. |
- Åkerman, Linda, 1983-
(författare)
-
Aspects of the Pre-Diabetic Period in Type 1 Diabetes
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Type 1 diabetes (T1D) is an autoimmune disease characterized by insulin deficiency, due to immune-mediated destruction of beta cells. Current knowledge regarding the period preceding disease onset comes, to a large extent, from studying risk cohorts based on relatives of T1D-patients, as they have an increased disease risk. Among T1D patients in general, however, few have the disease in their immediate family. It is therefore important to study risk cohorts from the general population as well. An ongoing autoimmune reaction can often be seen in the blood long before disease onset, by detection of autoantibodies directed towards beta cell antigens. By autoantibody screening among participants in the ABIS (All Babies in the South-east of Sweden) cohort, we could identify a group of children from the general population with increased risk for T1D, positive for multiple autoantibodies. They were enrolled in a 2-year prospective follow-up aiming to characterize the prediabetic period and to identify factors indicative of progression/non-progression to T1D. We assessed glucose homeostasis and autoantibody titers over time, and searched for risk-biomarkers by analyzing the expression of immune-related genes (Th1-Th2-Th3) in peripheral blood mononuclear cells (PBMC) from these children, in comparison to healthy children and newly diagnosed T1D patients. In the same groups we also compared serum micro RNA (miRNA) profiles, knowing that miRNA molecules have desirable biomarker properties. We found that two specific autoantibodies, IA2A and ZnT8A, were detected at higher concentrations in risk-individuals who progressed to overt T1D during or after the follow-up period, compared to those who still have not. We also observed disturbed glucose homeostasis long before onset in the progressors, but it was seen among those who remain symptom free as well. Further, we found support for the possible role of insulin resistance as an accelerator of the disease process. For gene expression and serum miRNA, few differences were observed between risk-individuals and healthy children overall. However, for PBMC gene expression and serum miRNA both, there were associations to beta cell function and glucose homeostasis, and for miRNA also to islet autoantibodies. Although specific profiles for prediction of disease onset or identification of risk-individuals could not be found, these results are interesting and deserve to be evaluated further. As part of another sub-study within ABIS, the effects of physical activity on glucose homeostasis were assessed in healthy schoolchildren. The level of physical activity, measured by pedometers, was related to insulin resistance and beta cell-stress, and decreased physical activity was associated with increased insulin resistance and load on the insulin-producing beta cells, already at school-age.
|
|
