Browsing by Subject "immunohistochemistry"

As the most common mental disorder, anxiety disorders present a major burden to healthcare worldwide and a challenging problem to overcome for the ones suffering from it. Recently, researchers have started to recognize that the relationship between sleep and anxiety disorders is bidirectional; disturbed sleep is a potential risk factor for the progression of anxiety and anxiety can lead to sleep disturbances. However, the neural mechanisms underlying anxiety and sleep problems are still poorly recognized. In this study, we used a chronic sleep fragmentation (SF) paradigm to investigate how disturbed sleep alters anxiety-like behavior in mice and what are the potential underlying neuronal mechanisms. This model was chosen because we wanted to focus on a common form of disturbed sleep in humans rather than total sleep deprivation. We measured anxiety-like behavior in the light-dark box and open field tests right after the 2-week SF period and again after a week of recovery. Additionally, we performed immunohistochemical analysis to study prolonged cell activity (transcription factor ∆FosB), parvalbumin (PV) interneurons and perineuronal net (PNN) structures in the medial prefrontal cortex (mPFC) of the mice. Changes in mPFC activity and related brain areas are associated to anxiety in humans and anxiety-like behavior in rodents alike. Similarly, changes in PV interneurons and PNNs, that regulates PV cell function, are associated to anxiety-like behavior. However, PV interneurons and PNNs have not been previously studied in a setting that combines sleep fragmentation and anxiety-like behavior. We found that chronic SF increases anxiety-like behavior in female mice and that this effect persists at least for a week. Conversely, we did not observe significant increase in anxiety-like behavior in male mice. Both female and male mice showed decrease in ∆FosB in the mPFC suggesting that SF treated mice had lower overall levels of cell activity. Similarly, we found that SF treated mice had decreased PV interneuron intensity in both sexes which could indicate changes in the cell activity. However, the pattern of changes in the IHC results was not identical in males and females. Based on the IHC results, we suggest that SF affects neuronal processes in both sexes but the disparity in them could explain the difference in the behavioral effect. This thesis shows that disturbed sleep can lead to increased anxiety-like behavior in rodent models and recognizes potential targets to study the mechanisms behind the phenomena.

Uterine leiomyomas are common smooth muscle tumours, with a prevalence as high as 80%. Even though they are benign, they present severe symptoms such as heavy menstrual bleeding, pelvic pain and reproductive dysfunction. Uterine leiomyomas can be classified to conventional tumours and leiomyoma variants based on their histopathology. The tumours usually harbour one of the three driver alterations: MED12 mutations, HMGA2 overexpression or biallelic FH inactivation. Known risk factors for leiomyoma development are African ancestry, family history and age. Uterine leiomyomas are most typically treated by surgery, through either uterus preserving myomectomy or by definitive hysterectomy. This Master’s thesis is continuation of a study from Äyräväinen et al. 2020, a retrospective study of 234 patients undergoing myomectomy at Helsinki University Hospital during 2009-2014. The aim of this study was to analyse how many of these patients had developed recurrent leiomyomas and how often the tumours in subsequent operations were potentially clonally related. In addition, clinical characteristics associated with the operations were analysed. In total 18% of these patients had recurrent operations, leading to the screening of 77 individual uterine leiomyomas from 32 patients. The mutational statuses were studied systematically with molecular screening using Sanger sequencing and immunohistochemistry. Altogether 33 tumours from 21 patients were found to have identical mutational status with a tumour from the original study. Of these tumours, 14 had a MED12 mutation. All the MED12 mutations were found in exon two affecting either codons 44 or 36. Six tumours had HMGA2 overexpression, and eight tumours were FH deficient. Five tumours were triple negative for all studied alterations. Whereas 81% of the patients had had two removal operations, the rest of them had had three to five operations. The years between operations ranged from performing them on the same year to performing them ten years apart. Even though most of the recurrent tumours were sporadic, almost half (43%) of them had identical mutations, suggesting that though uterine leiomyomas usually arise independently, some might be clonally related. The mutational distribution was different in the recurrent tumours than in uterine leiomyomas in general, indicating that in addition to germline predisposition, the driver related characteristics might also contribute to the potential of recurrence and to the likelihood of developing clonal lesions. Tumours harbouring MED12 abnormalities were the least probable to be clonally related. The tumours showing identical HMGA2 overexpression were likely clonally related. The number of identical FH deficient ULs was high, but not unexpected, since all the patients harbouring the mutation in the recurrent tumours had HLRCC, and therefore having a predisposition. Most surprisingly, all patients with recurrent triple negative tumours had identical mutation statuses in the recurrent tumours, which points to previously unknown clonal development of these lesions. Most of the patients with more than two surgeries had recurrent mutations, suggesting that multiple surgeries might indicate the development of clonally related tumours. However, further research is required to confirm the clonal relationships and to investigate the pathological nature of the tumours with different driver alterations.

Pleomorfinen adenooma (PA) on yleisin sylkirauhasten hyvänlaatuinen kasvain. Hoitamattomana niihin kehittyy maligniteetti noin 5-10% tapauksista. Malignisoituneista kasvaimista yleisin on carcinoma ex pleomorphic adenoma (CxPA). Tämän lisäksi niillä on taipumus uusiutua, recurrent pleomorphic adenoma (RPA) riittämättömän kirurgisen hoidon yhteydessä, mutta uusiutumia tavataan kuitenkin myös adekvaatin kirurgisen hoidon jälkeen. Uusiutuvat kasvaimet aiheuttavat hoidollisia haasteita. Tutkimuksemme tavoitteena oli selvittää ja tutkia mahdollisia histopatologisia muutoksia, sekä solusyklin säätelyyn liittyvän cyclin D1 proteiinin ja solujen proliferaatioon vaikuttavan Ki-67 proteiinin roolia liittyen pleomorfisen adenooman uusiutumiseen ja malignisoitumiseen. Cyclin D1 ja Ki-67 proteiinin ilmentymistä tutkittiin immunohistokemiallisin menetelmin. Ki-67 proteiinin ilmentymistä tutkittiin tutkimuksessamme patologiassa yleisesti käytetyn MIB-1 vasta-aineen avulla. Aineistona tutkimuksessamme käytettiin retrospektiivisesti kerättyä potilasaineistoa (n=65) ja potilasaineiston perusteella laadittiin tutkittavat kasvainryhmät. Tutkittavat kasvainryhmät koostuivat tavallisista hyvänlaatuisista PA kasvaimista (n=26), uusiutuneista kasvaimista (n=20) sekä maligneista CxPA kasvaimista (n=19). Uusiutuneiden kasvainten ryhmä jaettiin lisäksi alaryhmiksi, jotka koostuivat uusiutuneiden PA kasvainten primääreistä kasvamista (PA-prim) sekä primäärejä kasvaimia seuranneista uusiutuneista kasvaimista (PA-rec). Vertailimme kasvainryhmien välisiä eroavaisuuksia kasvainten kliinisten tietojen, histopatologisten ominaisuuksien sekä cyclin D1 ja MIB-1 ilmentymisen osalta. Tutkimuksemme perusteella MIB-1 ilmentyminen oli kohonnut CxPA ja PA-rec kasvaimissa, mutta ei PA-prim kasvaimissa. Tämä viittaa mahdollisesti siihen, että MIB-1 ilmentymisen eroavaisuudet näissä kasvaimissa voivat kehittyä vasta primäärin kasvaimen jälkeen. Cyclin D1 ilmentymisessä sekä histopatologiassa ei havaittu merkitseviä eroavaisuuksia ryhmien välillä.

Chronic psychosocial stress is a major risk factor for anxiety disorders, but the molecular background is still poorly known. Chronic social defeat stress (CSDS) is a mouse model simulating the psychosocial stress that humans face in their life. In CSDS, the examined mice are confronted by an aggressor mouse daily for 10 days, leading to defeat behavior and predisposing to anxiety-like symptoms. Some individuals develop these symptoms (susceptible) whereas others do not (resilient). Chronic stress has been shown to alter myelin-related gene transcription and myelin microstructure. Myelin is a membranous component around axons increasing the velocity of action potentials, and it is produced by oligodendrocytes (OLs). In this study, I investigated if CSDS affects the number of OLs or the size of the myelinated area (estimating the amount of myelin) in two inbred mouse strains that differ in their innate level of anxiety: the non-anxious C57BL/6NCrl (B6) and anxious DBA/2NCrl (D2). I studied three brain regions previously associated with anxiety: the medial prefrontal cortex (mPFC), bed nucleus of stria terminalis (BNST) and ventral hippocampus (vHP). The mice used in this study were previously exposed to CSDS and divided into resilient or susceptible phenotypes, and their brains were collected together with control mice. I performed two immunohistochemical staining experiments to calculate the number of OLs and to measure the myelinated area. I used anti-CNPase for OL cell counts and BlackGold II to stain myelin. I manually calculated the number of OLs using CNPase and cell morphology as markers. I built a macro to measure the BlackGold II stained myelinated area. I also measured the thickness of the corpus callosum (CC, major white matter tract) using the CNPase stained images to examine if the thickness is affected by CSDS. I observed a strain and region-specific effect of chronic stress in the BNST; B6 resilient mice had more OLs than susceptible mice whereas no differences were seen in the D2 strain, or other B6 brain regions. The size of the myelinated area did not differ between the phenotypes in either strain. Moreover, there was no significant correlation between the myelinated area and OL cell number. The CC thickness did not differ between the phenotypes. My findings indicate that myelin and OLs are affected by stress in a region specific manner and possibly contribute to the stress-resilient behavior. The response is genetic background-dependent, as I saw differences in B6 mice but not in D2 mice. Because CC thickness did not differ between the phenotypes, we suggest that CSDS does not induce extensive white matter atrophy in the mice brain. The mechanism underlying this dynamic myelin plasticity during stress requires more investigation, but this study provides evidence that alterations in OLs associate with chronic stress.

Prolyl oligopeptidase (POP, prolyl endopeptidase, EC 3.4.21.26) is a serine-type peptidase (family S9 of clan SC) hydrolyzing peptides shorter than 30 amino acids. POP has been found in various mammalian and bacterial sources and it is widely distributed throughout different organisms. In human and rat, POP enzyme activity has been detected in most tissues, with the highest activity found mostly in the brain. POP has gained scientific interest as being involved in the hydrolyzis of many bioactive peptides connected with learning and memory functions, and also with neurodegenerative disorders. In drug or lesion induced amnesia models and in aged rodents, POP inhibitors have been able to revert memory loss. POP may have a fuction in IP3 signaling and it may be a possible target of mood stabilizing substances. POP may also have a role in protein trafficking, sorting and secretion. The role of POP during ontogeny has not yet been resolved. POP enzyme activity and expression have shown fluctuation during development. Specially high enzyme activities have been measured in the brain during early development. Reduced neuronal proliferation and differentation in presence of POP inhibitor have been reported. Nuclear POP has been observed in proliferating peripheral tissues and in cell cultures at the early stage of development. Also, POP coding mRNA is abundantly expressed during brain ontogeny and the highest levels of expression are associated with proliferative germinal matrices. This observation indicates a special role for POP in the regulation of neurogenesis during development. For the experimental part, the study was undertaken to investigate the expression and distribution of POP protein and enzymatic activity of POP in developing rat brain (from embryonic day 14 to post natal day 7) using immunohistochemistry, POP enzyme activity measurements and western blot-analysis. The aim was also to find in vivo confirmation of the nuclear colocalization of POP during early brain ontogeny. For immunohistochemistry, cryosections from the brains of the fetuses/rats were made and stained using specific antibody for POP and fluorescent markers for POP and nuclei. The enzyme activity assay was based on the fluorescence of 7- amino-4-methylcoumarin (AMC) generated from the fluorogenic substrate succinyl-glycyl-prolyl-7-amino-4-methylcoumarin (Suc-Gly-Pro-AMC) by POP. The amounts of POP protein and the specifity of POP antibody in rat embryos was confirmed by western blot analysis. We observed that enzymatic activity of POP is highest at embryonic day 18 while the protein amounts reach their peak at birth. POP was widely present throughout the developmental stages from embryonic day 14 to parturition day, although the POP-immunoreactivity varied abundantly. At embryonic days 14 and 18 notably amounts of POP was distributed at proliferative germinal zones. Furthermore, POP was located in the nucleus early in the development but is transferred to cytosol before birth. At P0 and P7 the POP-immunoreactivity was also widely observed, but the amount of POP was notably reduced at P7. POP was present in cytosol and in intercellular space, but no nuclear POP was observed. These findings support the idea of POP being involved in specific brain functions, such as neuronal proliferation and differentation. Our results in vivo confirm the previous cell culture results supporting the role of POP in neurogenesis. Moreover, an inconsistency of POP protein amounts and enzymatic activity late in the development suggests a strong regulation of POP activity and a possible non-hydrolytic role at that stage.

Introduction: A significant barrier to medical diagnostics in low-resource environments is the lack of medical care and equipment. Here we present a low-cost, cloud-connected digital microscope for applications at the point-of-care. We evaluate the performance of the device in the digital assessment of estrogen receptor-alpha (ER) expression in breast cancer samples. Studies suggest computer-assisted analysis of tumor samples digitized with whole slide-scanners may be comparable to manual scoring, here we study whether similar results can be obtained with the device presented. Materials and methods: A total of 170 samples of hu-man breast carcinoma, immunostained for ER expression, were digitized with a high-end slide-scanner and the point-of-care microscope. Corresponding regions from the samples were extracted, and ER status was determined visually and digitally. Samples were classified as ER negative (<1% ER positivity) or positive, and further into weakly (1-10% positivity) and strongly positive. Interobserver agreement (Cohen’s kappa) was measured and correla-tion coefficients (Pearson’s product-momentum) were calculated for comparison of the meth-ods. Results: Correlation and interobserver agreement (r = 0.98, p < 0.001, kappa = 0.84, CI95% = 0.75-0.94) were strong in the results from both devices. Concordance of the point-of-care microscope and the manual scoring was good (r = 0.94, p < 0.001, kappa = 0.71, CI95% = 0.61-0.80), and comparable to the concordance between the slide scanner and man-ual scoring (r = 0.93, p < 0.001, kappa = 0.69, CI95% = 0.60-0.78). Fourteen (8%) discrepant cases between manual and device-based scoring were present with the slide scanner, and 16 (9%) with the point-of-care microscope, all representing samples of low ER expression. Con-clusions: Tumor ER status can be accurately quantified with a low-cost imaging device and digital image-analysis, with results comparable to conventional computer-assisted or manual scoring. This technology could potentially be expanded for other histopathological applications at the point-of-care.

SerpinE2 is a serine protease inhibitor (serpin) family protein that inhibits several extracellular proteases, such as thrombin, urokinase-type plasminogen activator and trypsin. Proteases and their inhibitors are often involved in cancer. SerpinE2 transcripts are upregulated in several cancers and found to predict poor prognosis of cancer patients. However, such studies regarding protein levels of serpinE2 are scarce. In this study, serpinE2 protein was analysed in three urological cancers, with patient groups that address the greatest needs for clinical biomarkers. The major aim of this study was to examine the association of serpinE2 staining with patient survival and clinicopathological features in prostate, urinary bladder and kidney cancers, and to evaluate its usability as an immunohistochemical biomarker. Tissue microarray slides from cancer patient tissues were stained immunohistochemically for serpinE2. The staining intensity was scored with four-point scale from 0 (no staining) to 3 (very intensive staining). Prostate and kidney cancer patients had been treated surgically and some of the cancers had relapsed after the surgery. In bladder cancer, association of serpinE2 with treatment response to neoadjuvant chemotherapy was evaluated. SerpinE2 expression was also measured in two prostate cancer cell lines with quantitative PCR and Western blotting. The serpinE2 staining was observed both in cancer cells and epithelial structures of benign tissues. The results showed that cancer tissue serpinE2 is not associated with relapse, treatment response or survival in prostate and bladder cancer patients. However, serpinE2 staining was more pronounced in prostate cancer tissues compared with benign tissues adjacent to cancer, and, surprisingly, the staining in such benign tissues was stronger in tissues from patients who developed metastases after surgery as compared to those without detectable metastases during 10.3-year (median) follow-up (p = 0.017). In addition, higher serpinE2 staining intensity was observed in higher grade bladder cancers (p = 0.034). In kidney cancer, on the other hand, serpinE2 staining intensity was significantly lower in patients whose cancer relapsed (p = 0.048), and high intensity predicted favourable disease-specific survival (p = 0.013). To conclude, serpinE2 is worth of further investigation in urological cancers. In prostate cancer, the possible field effect of cancer on serpinE2 in adjacent benign tissues could be examined more closely. In kidney cancer, the impact of serpinE2 on patient survival was inverse compared to transcript data in the Cancer Genome Atlas/the Human Protein Atlas database, and most other cancers. Thus, further validation studies need to be performed, and if the results hold true, serpinE2 staining could be used as part of a prognostic model predicting kidney cancer-specific survival.

Rare, yet aggressive Merkel cell carcinoma (MCC) is commonly found on skin of the elderly or immunocompromised. Merkel cell polyomavirus (MCPyV) is the leading cause of carcinogenesis in most MCC tumors. Cluster of differentiation 47 (CD47) protein found on the surface of normal cell’s is commonly upregulated in many cancers, for its ability to prevent the phagocytosis by macrophages. Immunohistochemical staining of CD47 was performed using 161 MCC tumor samples. The expression intensity was correlated statistically with tumor and patient related parameters. CD47 expression was widely expressed in MCC tumor cells. The expression did not correlate with survival. Among markers, PD-L1 and CD8 correlated with CD47. Due to the small amount of samples, random variation may affect our results. However, high expression levels of CD47 were shown in MCC, serving as a potential oncotarget in the treatment of this malignancy.

Zebrafish (Danio rerio) is a vertebrate model organism. It is suited for many phases of drug development process like toxicological studies. The major advantage of using zebrafish is the possibility to conduct high-throughput screens on a whole vertebrate animal. However, there is not as much knowledge about zebrafish as there is about other model organisms. Therefore there might be differences between zebrafish and humans that affect the use of zebrafish as a model in the drug development process. The purpose of this thesis was to characterize the structure of the zebrafish oxytocin system and assess the role of oxytocin on zebrafish behaviour. In humans defects in the oxytocin system have been linked to many psychiatric disorders like autism. If the mammalian and zebrafish oxytocin systems resembled each other functionally and structurally, it would enable the use of zebrafish as a model when studying the role of oxytocin in pathophysiology of diseases and also in oxytocin system related drug development. The structure and development of zebrafish oxytocin system was studied by staining adult zebrafish brain cryosections and larval brains with antibodies made against mammalian oxytocin. The specificity of the antibodies to recognize zebrafish oxytocin was determined by absorption and cross-reactivity controls. The role of oxytocin on zebrafish locomotion was studied by inhibiting the splicing of oxytocin messenger RNA with morpholino oligonucleotides (MOs). The MOs were used to address the relevance of the model in pharmacology, since the zebrafish oxytocin receptors have not been expressed and pharmacologically characterized. In zebrafish oxytocin was produced in the cells of the preoptic nucleus. There were thick oxytocin fibers towards the pituitary and also thinner fibers into areas in the telencephalon, diencephalon, mesencephalon and rhombencephalon. One of the MOs was able to inhibit the production of oxytocin with a dose that did not cause morphological abnormalities. The MO reduced the locomotor activity of the fish, but the specificity of the MO has to be determined. The structure of the zebrafish oxytocin system resembles mammalian oxytocin system in terms of the location of oxytocin cells and fiber projections. Therefore zebrafish seems a suitable model organism for oxytocin research. However, the structure of the zebrafish oxytocin receptor system and the effect of oxytocin on other behavioural aspects have to be determined in order to further evaluate the applicability of zebrafish for oxytocin research.