Ameerika populatsioonide genoomne portree

Väitekirja elektrooniline versioon ei sisalda publikatsiooneAmeerika populatsioonide evolutsiooni on käsitlenud mitmed multidistsiplinaarsed uuringud. Meie teadmised Ameerika maailmajao geneetilise mitmekesisuse kujunemisest on endiselt ebatäielikud, ehkki geneetilised uuringud lisavad sel teemal pidevalt uusi detaile. Uute tehnoloogiate nagu järgmise põlvkonna sekveneerimine (NGS) väljaarendamine koos teiste tehniliste edasiminekutega avavad võimaluse eraldada ja analüüsida DNA-d iidsetest proovidest, tehes "iidsest genoomikast" (aDNA) ühe paljudest põhilistest tööriistadest meie esivanemate mineviku mõistmiseks. Veelgi enam, need tehnoloogiad on tohutult suurendanud genoomsete andmete hulka kogu maailmast, sealhulgas Ameerika mandritelt. Ehkki Ameerika maailmajagu oli viimane, milleni meie sapiens’i esivanemad jõudsid, on selle geneetilise varieeruvuse protsessid olnud väga keerukad. Nende uuringud on rohkem kui kolme kümnendi jooksul olnud paljude geneetikaalaste teadustööde teemaks. Algul domineerisid Ameerika populatsioonide populatsioonigeneetilistes uuringutes uniparentaalsed geneetilised süsteemid, alustades mitokondriaalse DNA-ga (mtDNA) ja peagi kaasates Y-kromosoomi (chrY) analüüsi. Viimasest selgus, et põlisameeriklaste kaks chrY asutajahaplogruppi olid tõenäoliselt hg C ja hg Q, mida leiti vastavalt umbes 5% ja 75% põlisameerika meestest. Kuid nende haplogruppide resolutsioon ei paranenud oluliselt enne kui mõne aasta eest. Selle doktoritöö esimese publikatsiooni (Ref I) eesmärgiks on uurida Ameerika maailmajao geneetilist ajalugu meeste perspektiivist, lahates suure täpsusastmega üleameerikalist haplogruppi Q, ning koostada kõikehõlmav ja detailne haplogrupp Q ja selle alamliinide fülogeograafia. Uniparentaalseid geneetilisi süsteeme võib pidada kaheks lookuseks, mida kasutatakse inimese ajaloo nais- ja meesperspektiivi mõistmiseks. Nad saavad kirjeldada ainult kaht esivanemat neist tuhandetest, kes on seotud tänapäeva populatsioonide geneetilise pärandi kujundamisega. Olulisem arv esivanemaid on genoomis esindatud autosomaalsetes markerites. Seega on autosomaalsed markerid hädavajalikud Ameerika maailmajao populatsioonide liikumiste ajastuse ja dünaamika mõistmiseks. Tänu arheoloogilistele ja geneetilistele tõenditele tunnistatakse nüüdseks, et esimesed Põhja-Ameerikasse jõudnud inimesed tulid Siberist, ületades pärast hilist jääaega Beringi maakitsuse. Algsetele asulakohtadele järgnesid ulatuslikud inimeste ränded, mis jõudsid Lõuna-Ameerika lõunaossa suhteliselt kiiresti, juba ~15 000 aastat tagasi. Mitu hiljutist uuringut on selle teema kohta uut informatsiooni andnud, rekonstrueerides Ameerika maailmajao erinevate piirkondade põliselanike rühmade genoomset ajalugu, kuid Isthmo-Colombia piirkond on seni puudu. Seega rakendab selle doktoritöö teine publikatsioon (Ref II) nii iidse kui ka tänapäeva DNA andmete analüüsi, et rekonstrueerida Isthmo-Colombia piirkonna genoomset ajalugu. Selle eesmärgiks on teha kindlaks Panama põlispopulatsioonide genoomne taust, et hinnata maakitsuse sisest varieeruvust ja selgitada Kolumbuse-eelsete ameeriklaste genoomset ajalugu, hinnates Isthmo-Colombia piirkonna sidemeid ülejäänud Ameerika maailmajaoga. Lisaks esialgsetele rännetele pärinevad Ameerika populatsioonid mitmest segunemisest, alates koloniseerimisest ja Atlandi orjakaubandusest. Peale selle toimus viimase kahe sajandi jooksul palju rändelaineid, millele järgnes kohalik segunemine, ning nende mõju on suuresti uurimata. Selle doktoritöö kolmas publikatsioon (Ref III) uurib, kuidas hilisemad ränded kujundasid segunenud Ameerika populatsioonide genoomset tausta. Täpsemalt on selle uuringu eesmärgiks rekonstrueerida kõrgel lahutusastmel põlvnemise komponendid, anda hinnang segunemise ajale, uurida erinevate mandrite põlvnemise demograafilist evolutsiooni pärast segunemist ning hinnata soost sõltuva geenivoolu dünaamika ulatust ja tugevust segunenud Ameerika populatsioonides. Käesoleva doktoritöö peamised tulemused ja järeldused on järgmised: • Tehti kindlaks ja dateeriti kõrge resolutsiooniga haplogrupp Q fülogeneesipuu, mis annab uut informatsiooni oma Euraasia ja Ameerika harude geograafilise jaotuse kohta tänapäeva ja iidsetes proovides. • Esimest korda tuvastati kaks eristuvat Y-kromosoomi liini, mis peegeldavad hiljutistes genoomsetes uuringutes varem kirjeldatud kaht peamist põlvnemiskomponenti (SNA ja NNA). Nende liinide lahknemine toimus tõenäoliselt Beringi maakitsuse idaosas enne Ameerika maailmajakku sisenemist, milleks kasutati kaht teed: ranniku (SNA, Q-Z780/Q-M848) ja sisemaa teed (NNA, Q-Y4276). Sinna jõudnuna segunesid need kaks põlvnemiskomponenti Põhja-Ameerikas tõenäoliselt väga vara, millele viitab iidne Kennewicki mees, kelle tuumagenoom kuulub SNA komponenti (Q-M848), kuid mtDNA haplogrupp on NNA-st (X2a). • Avastati SNA liinide kaks märkimisväärset ekspansiooni Meso- ja Lõuna-Ameerikas, üks umbes 15 000 aastat tagasi, kohe pärast esmaasustamist, ja teine 3000 aastat tagasi pärast klimaatilisi muutusi ja kohalikke kultuurilisi nihkeid. • Panama sees tuvastati märkimisväärne geneetiline struktuur, mis kattus üldjoontes käesolevas uuringus analüüsitud mineviku ja praeguste põliselanike rühmadega. Need rühmad on ka tuhandeid aastaid suguluses olnud, eriti Kariibi mere piirkonnas Panama lääne- ja Costa Rica kaguosa piiril. Ida-Panama põliselanike rühmade vahel ning Emberá ja hispaanlaste-eelsete panamalaste vahel, kes elasid Vana Panamat ümbritsevas piirkonnas enne kontakti eurooplastega, leiti vähem geneetilisi sarnasusi. • Ameerika maailmajao iidsete põliselanike seas avastati varem kirjeldamata põlvnemiskomponent. See komponent esineb ainult selles piirkonnas ning on tuvastatav iidsetes hispaanlaste-eelsetes indiviidides ja inimestes, kes ise identifitseerivad end tänapäeva põliselanike, Aafrika ja latiino-põliselanike rühmade järglastena. See jõudis Panama maakitsusele rohkem kui 10 000 aastat tagasi, levis varases Holotseenis lokaalselt ning jättis tänapäevani püsivaid genoomseid jälgi, eriti Guna rahva hulgas. • Euroopa geneetiline panus Ameerika populatsioonidesse peegeldab kolonisatsiooni aegset geopoliitilist olukorda. Avastati mitu sekundaarset Euroopa allikat, mis panustasid arvestatavasse osassse Ameerika populatsioonidest, nt Itaalia Brasiilias ja Argentiinas, Kesk-Euroopa Brasiilias. Tuletati Aafrika allikate eristuv panus Ameerika populatsioonidesse. • Segunemise ajad langevad kokku rändelainetega Euroopast ja peegeldavad ekspluateeritud Aafrika piirkondade muutumist ajas. • Segunemise demograafilise mõju analüüsist selgub üldine languse ja taastumise muster mitmes uuritavas populatsioonis, mis vastab koloniaalajastu algusele ja lõpule. Kuid Peruud ja Mehhikot iseloomustavad erinevad demograafilised trajektoorid. • Soost sõltuva segunemise dünaamika analüüs viitab sellele, et tänapäeva populatsioonidesse on panustanud rohkem Ameerika naisi kui mehi. Vastupidiselt oli Euroopa meeste panus olulisem kui samalt mandrilt pärinevate naiste oma. Sellele vastandlikult ilmnes mõnes populatsioonis, kuid mitte kõigis, tõendeid suuremast naiste panusest, mis on osaliselt vastuolus ajalooliste andmetega Aafrika päritolust.The evolution of American populations has been the subject of several multidisciplinary studies. Our knowledge regarding the formation of the genetic diversity of the Americas is still incomplete, although genetic studies are constantly adding new details on this topic. The development of new technologies, such as Next Generation Sequencing (NGS), together with other technical improvements, lead to the possibility of extracting and analysing DNA from ancient specimens, making "ancient genomic" (aDNA) one of the many fundamental tools to understand our ancestor's past. Moreover, these technologies enormously increased the number of worldwide genomic data, including those from the Americas. Although the Americas were the last continents to be reached by our sapiens ancestors, their genetic variation processes have been extremely complex. Their studies have been the topic of many genetic surveys for more than three decades. In the beginning, uniparental systems dominated the population genetics research of American populations. It started with mitochondrial DNA (mtDNA) and soon included the Y chromosome (chrY) analysis. The latter revealed that the two founding Native American chrY haplogroups probably were Hg C and Hg Q, accounting for about 5% and 75% of Native American males, respectively. However, the resolution of these haplogroups did not undergo substantial improvements until a few years ago. The first publication included in this dissertation (Ref I) aims to investigate from a male perspective the genetic history of the Americas through a fine dissection of the Pan-American haplogroup Q and to reconstruct a comprehensive and detailed haplogroup Q phylogeography and that of its sub-lineages. The uniparental systems could be considered as two loci that are used to understand the female and male perspective of human history. They can describe only two ancestors of the thousands involved in shaping the genetic legacy of modern populations. The genomic representation of a more significant number of ancestors is encrypted in the autosomal markers. Therefore, autosomal markers are crucial to understanding the timing and the dynamics of population movements in the Americas. Thanks to archaeological and genetic evidence, it is now accepted that the first people arriving in North America came from Siberia, passing through Beringia after late Glacial times. Initial settlements were followed by widespread people movements that reached southern South America relatively fast, as early as ~15 thousand years ago. Several recent studies have provided new information about this subject, reconstructing the genomic history of indigenous groups from different regions of the Americas, but the Isthmo-Colombian area is still lacking. Hence, the second publication of this thesis (Ref II) employed both ancient and modern DNA data analysis to reconstruct the genomic history of the Isthmo-Colombian area. It aims to define the genomic background of Panamanian indigenous populations to evaluate the intra-Isthmus variability and shed light on pre-Columbian Americans' genomic history assessing the connection between the Isthmo-Colombian area and the rest of the Americas. Besides the first migrations, American populations result from several admixture events since the colonial era and the Atlantic slave trade. Moreover, many waves of migration followed by local admixture occurred in the last two centuries, the impact of which has been largely unexplored. The third reference in this thesis (Ref III) explores how more recent migrations shaped the genomic background of admixed American populations. In particular, this study aims to reconstruct the fine-scale ancestry composition, estimate the time of admixture, examine the demographic evolution of different continental ancestries after the admixture and assess the extent and magnitude of sex-biased gene-flow dynamics in admixed American populations. The main results and conclusions of this research thesis are the following: • A high-resolution haplogroup Q phylogeny that presents new insights into its Eurasian and American branches' geographic distribution in modern and ancient samples was ascertained and dated. • For the first time, two distinct Y chromosome lineages reflecting the two main ancestral components (SNA and NNA) earlier described by recent genomic studies were observed. The differentiation of these lineages probably occurred in eastern Beringia before entering the Americas through two routes: the coastal (SNA, Q-Z780/Q-M848) and the internal route (NNA, Q-Y4276). Once there, these two ancestral components probably admixed very early in North America, as suggested by the ancient Kennewick nuclear genome belonging to SNA (Q-M848) yet carrying an NNA mtDNA haplogroup (X2a). • Two significant expansions of the SNA lineages in Meso- and South America, one around 15 kya, early after the first peopling, and another at 3 kya, following climatic changes and local cultural shifts, were revealed. • A remarkable genomic structure within Panama was identified, mainly overlapping with past and present Indigenous groups analysed in this study. These groups also show relatedness, especially in the Caribbean region on the border between western Panama and southeastern Costa Rica over thousands of years. Fewer genetic similarities were identified between the Indigenous groups located in eastern Panama and between the Emberá and the pre-Hispanic Panamanians who lived in the area around Old Panama before European contact. • A previously undescribed ancestry among ancient Indigenous peoples of the Americas was revealed. This ancestry is unique to the region and detectable in the ancient pre-Hispanic individuals and the self-identified descendants of current Indigenous, African and Hispano-Indigenous groups. It reached the Panama land bridge over 10 thousand years ago, expanded locally during the early Holocene, and left genomic traces up to the present day, especially among the Guna. • The European genetic contribution in American populations mirrors the geopolitical situation during colonisation. Several European secondary sources contributing to a substantial proportion of American populations were revealed, e.g. Italy in Brazil and Argentina, Central Europe in Brazil. A differential contribution of African sources among American populations was inferred. • Times of admixture are concordant with migration waves from Europe and reflect differences in African areas exploited through time. • The investigation of the demographic impact of admixture reveals a general decline and recovery pattern in several populations under study corresponding to the beginning and the end of the Colonial Era. However, Peru and Mexico are characterised by different demographic trajectories. • The analysis of sex-biased admixture dynamics suggests that a higher number of American females than males have contributed to the modern populations. In contrast, European males had a more significant contribution than females from the same continent. In contrast, some populations, but not all, showed evidence for a higher female contribution, partially conflicting with historical records for African ancestry.https://www.ester.ee/record=b545015

Novel loci and Mapuche genetic ancestry are associated with pubertal growth traits in Chilean boys

Puberty is a complex developmental process that varies considerably among individuals and populations. Genetic factors explain a large proportion of the variability of several pubertal traits. Recent genome-wide association studies (GWAS) have identified hundreds of variants involved in traits that result from body growth, like adult height. However, they do not capture many genetic loci involved in growth changes over distinct growth phases. Further, such GWAS have been mostly performed in Europeans, but it is unknown how these findings relate to other continental populations. In this study, we analyzed the genetic basis of three pubertal traits; namely, peak height velocity (PV), age at PV (APV) and height at APV (HAPV). We analyzed a cohort of 904 admixed Chilean children and adolescents with European and Mapuche Native American ancestries. Height was measured on roughly a 6−month basis from childhood to adolescence between 2006 and 2019. We predict that, in average, HAPV is 4.3 cm higher in European than in Mapuche adolescents (P = 0.042), and APV is 0.73 years later in European compared with Mapuche adolescents (P = 0.023). Further, by performing a GWAS on 774, 433 single-nucleotide polymorphisms, we identified a genetic signal harboring 3 linked variants significantly associated with PV in boys (P <5×10−8). This signal has never been associated with growth-related traits

Analysis of the human Y-chromosome haplogroup Q characterizes ancient population movements in Eurasia and the Americas

Background: Recent genome studies of modern and ancient samples have proposed that Native Americans derive from a subset of the Eurasian gene pool carried to America by an ancestral Beringian population, from which two well-differentiated components originated and subsequently mixed in different proportion during their spread in the Americas. To assess the timing, places of origin and extent of admixture between these components, we performed an analysis of the Y-chromosome haplogroup Q, which is the only Pan-American haplogroup and accounts for virtually all Native American Y chromosomes in Mesoamerica and South America. Results: Our analyses of 1.5 Mb of 152 Y chromosomes, 34 re-sequenced in this work, support a "coastal and inland routes scenario" for the first entrance of modern humans in North America. We show a major phase of male population growth in the Americas after 15 thousand years ago (kya), followed by a period of constant population size from 8 to 3 kya, after which a secondary sign of growth was registered. The estimated dates of the first expansion in Mesoamerica and the Isthmo-Colombian Area, mainly revealed by haplogroup Q-Z780, suggest an entrance in South America prior to 15 kya. During the global constant population size phase, local South American hints of growth were registered by different Q-M848 sub-clades. These expansion events, which started during the Holocene with the improvement of climatic conditions, can be ascribed to multiple cultural changes rather than a steady population growth and a single cohesive culture diffusion as it occurred in Europe. Conclusions: We established and dated a detailed haplogroup Q phylogeny that provides new insights into the geographic distribution of its Eurasian and American branches in modern and ancient samples

Creating Artificial Human Genomes Using Generative Models

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Assessing temporal and geographic contacts across the Adriatic Sea through the analysis of genome-wide data from Southern Italy

AbstractSouthern Italy was characterised by a complex prehistory that started with different Palaeolithic cultures, later followed by the Neolithic transition and the demic dispersal from the Pontic-Caspian Steppe during the Bronze Age. Archaeological and historical evidence points to demic and cultural influences between Southern Italians and the Balkans, starting with the initial Palaeolithic occupation until historical and modern times. To shed light on the dynamics of these contacts, we analysed a genome-wide SNP dataset of more than 700 individuals from the South Mediterranean area (102 from Southern Italy), combined with ancient DNA from neighbouring areas. Our findings revealed high affinities of South-Eastern Italians with modern Eastern Peloponnesians, and a closer affinity of ancient Greek genomes with those from specific regions of South Italy than modern Greek genomes. The higher similarity could be associated with the presence of a Bronze Age component ultimately originating from the Caucasus and characterised by high frequencies of Iranian and Anatolian Neolithic ancestries. Furthermore, to reveal possible signals of natural selection, we looked for extremely differentiated allele frequencies among Northern and Southern Italy, uncovering putatively adapted SNPs in genes involved in alcohol metabolism, nevi features and immunological traits, such as ALDH2, NID1 and CBLB

Continental-scale genomic analysis suggests shared post-admixture adaptation in the Americas

We thank the people working at the High Performance Computing Center of the University of Tartu for the help and support provided. We thank Marco Rosario Capodiferro for useful discussions. This work was supported by the European Union through the European Regional Development Fund (Project No. 20142020.4.01.16-0030 to LO, MMe, FM; Project No. 2014-2020.4.01.160271 to RF; Project No. 2014-2020.4.01.16-0125 to RF; Project No. 2014-2020.4.01.16-0024 to DM, LP). This work was supported by the Estonian Research Council grant PUT (PRG243) (to RF, MMe, LP). This work was supported by institutional research funding IUT (IUT24-1) of the EstonianMinistry of Education and Research (to TK). This research was supported by the European Union through Horizon 2020 grant no. 810645 (to MMe). This research was supported by the European Union through the Horizon 2020 research and innovation programme under grant no 810645 and through the European Regional Development Fund project no. MOBEC008 to MMo.American populations are one of the most interesting examples of recently admixed groups, where ancestral components from three major continental human groups (Africans, Eurasians and Native Americans) have admixed within the last 15 generations. Recently, several genetic surveys focusing on thousands of individuals shed light on the geography, chronology and relevance of these events. However, even though gene f low could drive adaptive evolution, it is unclear whether and how natural selection acted on the resulting genetic variation in the Americas. In this study, we analysed the patterns of local ancestry of genomic fragments in genome-wide data for ∼6000 admixed individuals from 10 American countries. In doing so, we identified regions characterized by a divergent ancestry profile (DAP), in which a significant over or under ancestral representation is evident. Our results highlighted a series of genomic regions with DAPs associated with immune system response and relevant medical traits, with the longest DAP region encompassing the human leukocyte antigen locus. Furthermore, we found that DAP regions are enriched in genes linked to cancer-related traits and autoimmune diseases. Then, analysing the biological impact of these regions, we showed that natural selection could have acted preferentially towards variants located in coding and non-coding transcripts and characterized by a high deleteriousness score. Taken together, our analyses suggest that shared patterns of post admixture adaptation occurred at a continental scale in the Americas, affecting more often functional and impactful genomic variants.European Commission 2014-2020.4.01.16-0030 2014-2020.4.01.16-0271 2014-2020.4.01.16-0125 2014-2020.4.01.16-0024 MOBEC008Estonian Research Council grant PUT PRG243institutional research funding IUT of the Estonian Ministry of Education and Research IUT24-1European Union through Horizon 2020 grant 81064

Evaluating the Impact of Sex-Biased Genetic Admixture in the Americas through the Analysis of Haplotype Data

A general imbalance in the proportion of disembarked males and females in the Americas has been documented during the Trans-Atlantic Slave Trade and the Colonial Era and, although less prominent, more recently. This imbalance may have left a signature on the genomes of modern-day populations characterised by high levels of admixture. The analysis of the uniparental systems and the evaluation of continental proportion ratio of autosomal and X chromosomes revealed a general sex imbalance towards males for European and females for African and Indigenous American ancestries. However, the consistency and degree of this imbalance are variable, suggesting that other factors, such as cultural and social practices, may have played a role in shaping it. Moreover, very few investigations have evaluated the sex imbalance using haplotype data, containing more critical information than genotypes. Here, we analysed genome-wide data for more than 5000 admixed American individuals to assess the presence, direction and magnitude of sex-biased admixture in the Americas. For this purpose, we applied two haplotype-based approaches, ELAI and NNLS, and we compared them with a genotype-based method, ADMIXTURE. In doing so, besides a general agreement between methods, we unravelled that the post-colonial admixture dynamics show higher complexity than previously described

A Chromosome-Painting Based Pipeline to Infer Local Ancestry under Limited Source Availability

Contemporary individuals are the combination of genetic fragments inherited from ancestors belonging to multiple populations, as the result of migration and admixture. Isolating and characterizing these layers are crucial to the understanding of the genetic history of a given population. Ancestry deconvolution approaches make use of a large amount of source individuals, therefore constraining theperformanceofLocalAncestryInferenceswhenonlyfewgenomesareavailablefromagivenpopulation.HerewepresentWINC, a local ancestry framework derived from the combination of ChromoPainter and NNLS approaches, as a method to retrieve local genetic assignments when only a few reference individuals are available. The framework is aided by a score assignment based on source differentiation to maximize the amount of sequences retrieved and is capable of retrieving accurate ancestry assignments when only two individuals for source populations are used

Creating artificial human genomes using generative neural networks

Generative models have shown breakthroughs in a wide spectrum of domains due to recent advancements in machine learning algorithms and increased computational power. Despite these impressive achievements, the ability of generative models to create realistic synthetic data is still under-exploited in genetics and absent from population genetics. Yet a known limitation in the field is the reduced access to many genetic databases due to concerns about violations of individual privacy, although they would provide a rich resource for data mining and integration towards advancing genetic studies. In this study, we demonstrated that deep generative adversarial networks (GANs) and restricted Boltzmann machines (RBMs) can be trained to learn the complex distributions of real genomic datasets and generate novel high-quality artificial genomes (AGs) with none to little privacy loss. We show that our generated AGs replicate characteristics of the source dataset such as allele frequencies, linkage disequilibrium, pairwise haplotype distances and population structure. Moreover, they can also inherit complex features such as signals of selection. To illustrate the promising outcomes of our method, we showed that imputation quality for low frequency alleles can be improved by data augmentation to reference panels with AGs and that the RBM latent space provides a relevant encoding of the data, hence allowing further exploration of the reference dataset and features for solving supervised tasks. Generative models and AGs have the potential to become valuable assets in genetic studies by providing a rich yet compact representation of existing genomes and high-quality, easy-access and anonymous alternatives for private databases

TIME- AND DOSE-DEPENDENT EFFECTS OF CHRONIC WOUND FLUID ON HUMAN ADULT DERMAL FIBROBLASTS

BACKGROUND Wound healing is a biologic process that is altered in patients affected by chronic venous ulcers. The wound microenvironment is reflected in the chronic wound fluid (CWF), an exudate containing serum components and tissue-derived proteins. OBJECTIVES We investigated the effects of increasing doses of CWF collected from patients suffering from chronic venous ulcers on human adult dermal fibroblasts cultured in vitro and the relationship among CWF effects and treatment length. METHODS Fibroblasts were treated with 60, 240, and 720 mg/mL CWF for 3 and 7 days. We evaluated cell proliferation and viability by MTT and Trypan blue assay, cell morphology by light microscopy, F-actin microfilaments organization by tetramethylrhodamine B isothiocyanate-conjugated phalloidin, a-smooth muscle actin expression by immunofluorescence, and senescence-associated b-galactosidase activity. RESULTS CWF induced an increase in cell proliferation in the first 3 days of treatment. In contrast, at 7 days, a strong decrease in cell viability was observed. These changes were related to a cytoskeletal F-actin reorganization and not to fibroblast-myofibroblast differentiation nor to changes in cellular senescence. CONCLUSIONS This study shows a dose-dependent and biphasic effect of CWF on dermal fibroblasts, suggesting that a continuous exposure to chronic wounds microenvironment may induce late cellular dysfunctions possibly involved in the delayed wound healing