Molecular basis of the kidney filtration barrier : Role of the nephrin protein complex

The kidney filtration barrier consists of fenestrated endothelial cell layer, glomerular basement membrane and slit diaphragm (SD), the specialized junction between glomerular viscelar epithelial cells (podocytes). Podocyte injury is associated with the development of proteinuria, and if not reversed the injury will lead to permanent deterioration of the glomerular filter. The early events are characterized by disruption of the integrity of the SD, but the molecular pathways involved are not fully understood. Congenital nephrotic syndrome of the Finnish type (CNF) is caused by mutations in NPHS1, the gene encoding the SD protein nephrin. Lack of nephrin results in loss of the SD and massive proteinuria beginning before birth. Furthermore, nephrin expression is decreased in acquired human kidney diseases including diabetic nephropathy. This highlights the importance of nephrin and consequently SD in regulating the kidney filtration function. However, the precise molecular mechanism of how nephrin is involved in the formation of the SD is unknown. This thesis work aimed at clarifying the role of nephrin and its interaction partners in the formation of the SD. The purpose was to identify novel proteins that associate with nephrin in order to define the essential molecular complex required for the establishment of the SD. The aim was also to decipher the role of novel nephrin interacting proteins in podocytes. Nephrin binds to nephrin-like proteins Neph1 and Neph2, and to adherens junction protein P-cadherin. These interactions have been suggested to play a role in the formation of the SD. In this thesis work, we identified densin as a novel interaction partner for nephrin. Densin was localized to the SD and it was shown to bind to adherens junction protein beta-catenin. Furthermore, densin was shown to behave in a similar fashion as adherens junction proteins in cell-cell contacts. These results indicate that densin may play a role in cell adhesion and, therefore, may contribute to the formation of the SD together with nephrin and adherens junction proteins. Nephrin was also shown to bind to Neph3, which has been previously localized to the SD. Neph3 and Neph1 were shown to induce cell adhesion alone, whereas nephrin needed to trans-interact with Neph1 or Neph3 from the opposite cell surface in order to make cell-cell contacts. This was associated with the decreased tyrosine phosphorylation of nephrin. These data extend the current knowledge of the molecular composition of the nephrin protein complex at the SD and also provide novel insights of how the SD may be formed. This thesis work also showed that densin was up-regulated in the podocytes of CNF patients. Neph3 was up-regulated in nephrin deficient mouse kidneys, which share similar podocyte alterations and lack of the SD as observed in CNF patients podocytes. These data suggest that densin and Neph3 may have a role in the formation of morphological alterations in podocytes detected in CNF patients. Furthermore, this thesis work showed that deletion of beta-catenin specifically from adult mouse podocytes protected the mice from the development of adriamycin-induced podocyte injury and proteinuria compared to wild-type mice. These results show that beta-catenin play a role in the adriamycin induced podocyte injury. Podocyte injury is a hallmark in many kidney diseases and the changes observed in the podocytes of CNF patient share characteristics with injured podocytes observed in chronic kidney diseases. Therefore, the results obtained in this thesis work suggest that densin, Neph3 and beta-catenin participate in the molecular pathways which result in morphological alterations commonly detected in injured podocytes in kidney diseases.

Effects of 17α-ethinyl estradiol on the mating system of the sand goby (Pomatoschistus minutus)

In aquatic environments, endocrine disrupting chemicals (EDCs) that interfere with the endocrinology of males and females form a threat to the maintenance of populations. EDCs are a diverse group of natural and manmade chemicals that already at very low concentrations (at nanogram levels) can have severe effects on reproduction by individuals, e.g. complete sex reversal, feminisation of males, impaired reproduction even resulting in near extinction of populations. With regard to fish, despite the extensive literature on physiological effects of EDCs, very little is known about potential population-level effects. In this thesis, I examined how 17α-ethinyl estradiol (EE2), a synthetic estrogen used in oral contraceptive pills, affects the reproductive behaviour of a marine fish, the sand goby (Pomatoschistus minutus). The aims were fourfold. First, I investigated how exposure to EE2 affects courtship and parental care of sand goby males. Secondly, I looked at effects on the mating system and sexual selection. In the third study, I observed the effects of exposure in a social context where exposed males had to compete with non-exposed males for resources and mates. Finally, I studied the effects of exposure on male-male competition and male aggressive behaviour. This work revealed that EE2 exposure impairs the ability of males to acquire and defend a nest, as well as diminishes the attractiveness of males to females by decreasing their courtship and aggressive behaviour. These effects are harmful for a male whose reproductive success is determined by the ability to compete for limited resources and to attract mates. Furthermore, this thesis showed that selection on male size was relaxed after EE2 exposure and male size had a smaller effect on mating success. These effects can be of a profound nature as they interfere with sexual selection, and may in the long run lead to the loss of traits maintained through sexual selection. The thesis shows that an exposure to environmentally relevant levels of EE2 clearly reduces the chances of individuals to reproduce successfully. Furthermore, it strongly suggests that several types of biomarkers should be used to detect and assess the effects of EDC exposure because severe behavioural effects can sometimes be seen before effects are detectable at the molecular or morphometric level. Behavioural assays should be considered an important complementary tool for the standard ecotoxicological assays because observed behavioural changes have direct and negative effects on fitness, while the connection between changes in molecular expression and fitness may be less obvious.

Context-dependent mate choice in the sand goby, Pomatoschistus minutus

The results presented in this thesis show that all females of a given population do not necessarily choose similar mating partners. Specifically, partner preferences of a fish, the sand goby (Pomatoschistus minutus), varied among individual females and depended on the social context at the time of choice. I also show that females assess multiple mate choice cues simultaneously; partner preferences were based more strongly on an interaction effect between different choice cues than on any individual cue. Furthermore, I found that preferred matings involved fitness benefits in the form of increased offspring success, but these benefits were not significantly affected by mate compatibility. Hence, mate choice for partner compatibility does not appear to be an important determinant of the observed variation in female mate preferences in this species. The context-dependency of female mating preferences revealed is relevant to how genetic variation in sexually selected traits might be maintained: as the mating success of a certain male type varies according to the choice context, directional sexual selection on male traits is shown to be less intense than generally thought making for a slower loss of genetic variation in these traits. Mating preferences of sand gobies were assessed by giving females a binary choice between males that differed in body size and/or other focus traits. These association preferences were found to be sexually motivated, repeatable and to correspond to actual mating decisions.

Voittoa tavoittelemattoman organisaation brandin rakentaminen. Case Slow Food

Tämän pro gradu-tutkielman tavoitteena oli kartoittaa voittoa tavoittelemattoman Slow Food-organisaation brandiin liitettyjä mielikuvia ja niitä tekijöitä, joilla brandin tavoitekuvaa tulisi jatkossa rakentaa. Koska nykyisin brandi ei ole enää pelkkien liikeyritysten yksityisomaisuutta, tutkimuksen teoreettinen asemointi perustui yhtäältä organisaation tunnettuuden kasvattamiseen brandin rakentamisen avulla ja toisaalta voittoa tavoittelemattoman toimintaympäristön erityispiirteiden näkökulmaan. Case-osion avulla haluttiin tarkastella globalisaation ja elintarviketalouden keskittymiselle vaihtoehtoisen, paikallisuuteen perustuvan ruokajärjestelmän vaikutuksia, mahdollisuuksia ja haasteita. Tutkimuksen empiirinen osuus muodostui laadullisesta tapaustutkimuksesta, jonka kohteena oli Slow Food-organisaatio. Tutkimus suoritettiin haastattelemalla kahtatoista Slow Food-organisaation sidosryhmien edustajaa Italian Piemontessa henkilökohtaisin haastatteluin, joiden pohjana oli brandi identiteetin elementeistä luodut kaksi teemaa: mielleyhtymiin ja arvoväittämään liittyvät tekijät. Aineiston analysoinnissa käytettiin teemoittelua ja sisällönanalyysia. Tutkimuksen lähestymistapa oli abduktiivinen. Tulosten mukaan Slow Food-brandi identifioituu sidosryhmilleen ensisijaisesti luotettavan ruoan kautta, johon katsottiin kuuluvan aidot, puhtaat raaka-aineet ja paikallisesti tuotetun ja jalostamattoman ruoan hyvä jäljitettävyys. Myös tiettyyn paikkaan ja arvoihin sidottujen kokemusten ja odostusten täyttymisen kautta saatu sosiaalinen ja kulttuurinen jatkuvuus nimettiin brandin vahvuudeksi. Lisäksi vastuullisen tuottamisen ja kuluttamisen sekä aitojen makujen ja laadukkaan ruokavalion edistäminen korostuivat jonkin verran vastauksissa. Näillä perusvahvuuksilla toivottiin brandin identiteettiä rakennettavan myös tulevaisuudessa. Haastateltavien mielestä Slow Food-organisaatio erilaistaa itsensä muista ensisijaisesti lupaamalla yksilölle korkealuokkaisia, aitoja tuotteita ja palveluja, joilla on henkilökohtaisten hyötyjen lisäksi positiivisia seuraamuksia myös oman alueen työllisyyden ja talouden kehitykseen. Organisaation tulisi myös jatkossa luoda uskottavuutta toimintaansa eritoten sen kautta, että brandi lunastaa lupauksensa laadusta ja aitoudesta. Toisaalta erottautuakseen muista lähi- ja luomuruokabrandeista Slow Food-brandin arvoväittämässä tulisi tulevaisuudessa selkeämmin korostua myös toiminnan voittoa tavoittelemattomuus ja brandiin liittyvä elämyksellisyys. Sidosryhmien kiinnostusta ja brandin lupauksen tehoa alentaviksi tekijöiksi nimettiin yleinen ruoan arvostuksen väheneminen sekä Slow Food-tuotteiden ja -palvelujen korkea hinta, huono saatavuus ja näkyvyys. Keskeisimpinä ratkaisuina pidettiin kuluttajien informatiivisen tiedon lisäämisen ohella pientuottajien verkostoitumista ja uusia yhteistyön muotoja kuten tuottajien yhteisiä jakeluketjuja ja paikallismyymälöitä. Näiden lisäksi brandin tunnistettavuuden parantaminen ja aktiivisempi tarjonta kyltityksin, tuotteiden pysyvän läsnäolon ja vähittäiskaupan mukaantulon kautta sekä eräät muut markkinoinnin osa-alueet kuten sosiaalinen media, Internet ja kansainväliset ruoka-alan tapahtumat nimettiin brandin lupausta kirkastaviksi elementeiksi. Pientuotannon lisääminen verotuksen kohdentumisen muutoksilla nähtiin keinoksi pudottaa paikallisen ruoan hintaa.Toisaalta korkeaa hintaa perusteltiin laadukkaaseen imagoon liittyvien argumenttien lisäksi luonnollisen kysynnän kasvulla, jota arveltiin saatavan tulevaisuudessa suurista ikäluokista, yhä kasvavasta koulutus- ja tulotasosta sekä valveutuneemmista kuluttajista.