biologia molecular, nicolas usuga

1. Nicolás Usuga Taborda
School of Medicine/ 3º semester/ Universidad Pontificia Bolivariana

2. INTRODUCTION
RIP 1: This gene encodes a member of
the receptor-interacting protein (RIP)
family of serine/threonine protein kinases.
The encoded protein plays a role in
inflammation and cell death in response
to tissue damage, pathogen recognition,
and as part of developmental regulation.
RIP 3: The product of this gene is a
member of the receptor-interacting protein
(RIP) family of serine/threonine protein
kinases. It is a component of the tumor
necrosis factor (TNF) receptor-I signaling
complex, and can induce apoptosis and
weakly activate the NF-kappaB
transcription factor
Tributyltin (TBT) is the active ingredient of
many products that act as biocides
against a broad range of organisms. It is
primarily used as an antifoulant paint
additive on ship and boat hulls, docks,
fishnets, and buoys to discourage the
growth of marine organisms such as
barnacles, bacteria, tubeworms, mussels
and algae, it is also used in agriculture as
a pesticide for microorganisms.

3. TOXICITY AND RIP1/RIP3 REGULATION.
The toxicity of TBT
• On immune system and its immunosuppressive effects have been extensively
reported. TBT exposure can cause thymus atrophy, depletion of T-lymphocytes in
spleen and lymph nodes, decreased macrophage and NK cell activity, and
diminished T-helper 2 cell polarization. However, the molecular mechanisms by
which TBT exerts these immunotoxic effects are still not clear. Previous studies
proposed that TBT induced thymus atrophy through inhibiting cell proliferation or
inducing apoptosis in immune cells, other studies show that TBT activates induced
necrosis mechanisms as another way of cellular suicide.
RIP1/RIP3 regulation
• TBT inducesthe occurrence of necrotic cell death in macrophages through RIP1-
RIP3 pathway. Inhibition or genetic deletion of RIP1-RIP3 pathway was able to
rescue the cell death induced by TBT. Pretreatment with RIP1 inhibitor or RIP3
deficiency significantly protected the mice from TBT-induced mortality and
alleviated the tissue damage as well as immunosuppressive effect induced by TBT
• RIP1 and RIP3 also contribute to the toxicity of TBT in vivo

4. OBJECTIVE
• In this study, we have
investigated the cytotoxic
effects of TBT and explored
the underlying molecular
mechanisms both in vitro
and in vivo.

5. MATERIALES Y
MÉTODOS
INMUNOFLUORESENCIA:
• Fundamento = Distribución de un antígeno en un
grupo de células utilizando la especificidad de los
anticuerpos por dicho antígeno para dirigir
marcadores fluorescentes a una molécula diana
• Para que= se utilizo para determinar si las células
eran apoptóticas( verdes) o necróticas ( verde y rojo)
esto se pudo ver por el lavado previo que le hicieron
a las células con Annexin V-FITC/PI

6. MATERIALES Y
MÉTODOS
RNAi:
• Fundamento = Desactivación de genes específicos in
vitro, para estudiar a profundidad la función de estos,
se le conoce como silenciamiento genético.
• Para que= se utilizo para suprimir la expresión del gen
rip1 en macrófagos y ver como estos reaccionaban al
estar expuestos al tbt, se midió el ldh del sobrenadante
y se obtuvo la secuencia de arni-rip1

7. MATERIALES Y
MÉTODOS
WESTERN BLOT
• Fundamento= Técnica analítica usada para detectar
una proteína especifica en función de su peso
molecular mediante el uso de anticuerpos
• Para que: se utilizó para evidenciar como actuaban
algunos anticuerpos al se incubados y lavados en
tbs-t, Se usó un sistema de sustrato quimio
luminiscente de KPL para la detección final.

8. MATERIALES Y
MÉTODOS
HISTOLOGIA
• Fundamento= Se utilizó la hematoxilina eosina (HE),
la hematoxilina es de naturaleza básica, tiene afinidad
por lo acido y pinta de coloración morado, la eosina
es de naturaleza acida, afinidad por lo básico, pinta
de color rosado.
• Para que= se recogieron tejidos del hígado e intestino
delgado para mirar como habían reaccionado a la
exposición a la cual fueron sometidos al TBT

9. RESULTS
• En el control no hay fluorescencia de
ninguno de los 2 reactivos
• En tbt 0,6--> hay reacción, confirmando
que la tbt activa ambos procesos,
apoptosis y necrosis
• En tbt 1.0, aumento el PI y bajo la
annexine, indicando que las
concentraciones altas de tbt estimulan
necrosis y las bajas apoptosis

10. • Cuando se le agrega el tbt o el nec-1 el peso
molecular del rip1 aumento de 75kD a 80kD
• El aumento de las concentraciones de TBT aumento la
fosforilacion del rip1 en el macrófago, estimulando el
proceso de necrosidad
• Cuando se añadió nec-1( inhibidos de rip1), se mermo
la fosforilacion del rip1, bajando la tasa de necrosis
• El uso de Scr y siRip1 (ambos son ARNi para el RIP1)
disminuyo la expresión del RIP1, de esta manera la
tasa de necrosis del macrófago disminuye.
RESULTS

11. • En las 2 imágenes se comprueba que los tejidos a los que se les indujo el nec-1 tuvieron menos taño
tisular en comparación con los que fueron tratados solo con TBT, los ratones sobrevivieron mas con
nec-1
RESULTS

12. RESULTS

13. AUTHOR CONTRIBUTION OR
Nakano et al. Analysis of TBT cytotoxicity using macrophage might provide worth- while
insights into the mechanism of TBT-induced immunotoxicity. Previous studies
have shown that TBT can reduce macrophage activity and induce cell death
in macrophages
Stridh et al. It has been accepted that TBT induces apoptosis in different types of cells,
necrosis has also been detected during TBT treatment by several studies
Stridh et al. found that low concentration of TBT triggers apoptosis whereas
high concentrations cause necrosis in Jurkat cells
Takagi et al. Studied the acute toxicity of TBT in Syrian golden hamsters and found that
about 90% of female hamsters died in two weeks after 225 mg kg 1 of
TBT oral administration

14. CONCLUSION
• The mechanisms of functioning of the rip1 and rip3 genes are
essential for the management of some toxicities in the clinic,
now it is known that inactivating these the risk that the toxicity
is lethal decreases in large percentage
• Tributyltin is used regularly by people to carry out their
daily activities of agriculture or fishing, taking advantage of
the different functions it has as a pesticide or biocide, so it
is very important that we understand the mechanisms by
which this substance can become toxic to humans , we must
understand what mechanisms can diminish the effect of this
pesticide and that genes or proteins are involved in the
whole process of the disease, in order to have the certainty
that our patients can receive the help the help they need to
survive.